Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of
Carrying Capacity for Aquaculture,
Modeling Frameworks for
Determination of
JOAO G. FERREIRA1, JON GRANT2, DAVID W.
VERNER-JEFFREYS3, NICK G. H. TAYLOR3
1 whether directly or indirectly. Such representations,
Departamento de Ciências e Engenharia do Ambiente,
Faculdade de Ciências e Tecnologia Universidade Nova
de Lisboa, Monte Caparica, Portugal
2 underlying variables and processes, in order to
Department of Oceanography, Dalhousie University,
Halifax, NS, Canada
3
Cefas Weymouth Laboratory, Weymouth, Dorset, UK
Article Outline
Glossary
Definition of the Subject and Its Importance
Introduction
Modeling Frameworks for Carrying Capacity
Future Directions
Acknowledgments
Bibliography
Glossary
Dynamic models Models that incorporate a time-
varying element. Such models may have no spatial
dimension or extend to modeling spatial variation
in all three dimensions.
Integrated multi-trophic aquaculture (IMTA)
Cocultivation of aquatic species at different trophic
levels, such as salmon, mussels, and kelp or scallop
and sea cucumber, in order to maximize yield and
minimize the environmental footprint.
Modeling framework Models are a representation of
reality, which they aim to reproduce in terms of
generality, realism, accuracy, and simplicity. As a
rule, generality and simplicity are maximized.
A model framework usually consists of two or
more models, which are combined to address dif-
ferent levels of complexity.
Research models Models that are more detailed, usu-
ally complex to develop, implement, and apply.
Aimed mainly at the scientist, rather than the
manager.
P. Christou et al. (eds.), Sustainable Food Production, DOI 10.1007/978-1-4614-5797-8,
# Springer Science+Business Media New York 2013
Originally published in
Robert A. Meyers (ed.) Encyclopedia of Sustainability Science and Technology,
417
Screening models Models that use a limited set of
inputs, and provide highly aggregated outputs,
such as an index of suitability or an environmental
score card.
Virtual technology for aquaculture Any artificial rep-
resentation of ecosystems that support aquaculture,
exemplified by mathematical models, are designed
to help measure, understand, and predict the
inform an Ecosystem Approach to Aquaculture.
Definition of the Subject and Its Importance
Aquaculture, defined simply as the cultivation of
aquatic organisms, has many similarities to agriculture,
most notably that it is based on the interaction between
humans and other elements of the natural system,
converting the latter (at least in part) into a managed
system.
In parts of the world, such as Southeast Asia, the
distinction between the two types of cultivation
becomes increasingly fuzzy, especially if they take
place on land. Cocultivation of rice and tilapia in
paddy fields [1] or the combination of penaeid shrimp
(e.g., the whiteleg shrimp Penaeus vannamei) and water
spinach (Ipomoea aquatica) in earthen ponds is com-
mon, as are many other combinations (Fig. 1). Carry-
ing capacity in such intensive systems, whether in
monoculture or in integrated multi-trophic aquacul-
ture (IMTA), might at first glance seem equivalent to
assimilative capacity.
Aquaculture in open water, whether in reservoirs,
lakes, or coastal systems, must take into account the
complexities of water circulation, together with the
harmonization of different uses. In the context of
organically extractive open-water culture, Bacher et al.
[2] and Smaal et al. [3] defined carrying capacity as:
The standing stock at which the annual production
of the marketable cohort is maximized.
Although this definition was proposed for bivalve
shellfish culture, it is sufficiently broad to be relevant
for production in open freshwater, coastal, and off-
shore environments, as well as in land-based systems
using ponds or raceways. However, production carry-
ing capacity needs to be further qualified, because in
# 2012, DOI 10.1007/978-1-4419-0851-3
418 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of
Carrying Capacity for Aquaculture, Modeling
Frameworks for Determination of. Figure 1
Pigs might fly? Cocultivation of pigs and fish such as carp or
rohu in India http://harfish.gov.in/technology.htm
economic terms the maximization of annual produc-
tion is not the objective function, and brings with it
increased environmental costs. Commercial produc-
tion must seek to achieve optimal profit, well before
the inflection point in the production function, where
total physical product (TPP) maximizes income (e.g.,
[4, 5]).
This production-oriented view of carrying capacity
for aquaculture, whether in terms of assimilative capac-
ity for fed aquaculture such as finfish or shrimp or with
respect to food depletion in the case of shellfish such as
oysters, clams, or abalone, has been expanded over the
last decade into a four pillar approach based on phys-
ical, production, ecological, and social carrying capac-
ity [6, 7]. These pillars encompass the three elements of
sustainability, viz., planet, people, and profit.
The terms carrying capacity and assimilative capac-
ity are frequently associated with models of aquacul-
ture impacts. Because these terms attempt to define the
limits of sustainable aquaculture, predictive capability
is highly sought (Fig. 2).
This chapter reviews the state of the art in modeling
frameworks that assist with that prediction and sup-
port proactive management of aquaculture.
Introduction
The establishment of aquaculture activities in different
geographical areas has historically been a bottom-up
process, without any systemic planning or definition of
a zoning framework. This is seen throughout the world,
from the development of salmon cage culture in
Scottish lochs to the incremental destruction of man-
groves for construction of shrimp farms (Fig. 3).
This approach to licensing (or in many cases just to
development) has been based on space availability and
limits to production rather than on any environmental
criteria, and has led to undesirable ecosystem effects,
including habitat destruction both on land and in
open waters, coastal eutrophication through increased
nutrient loading from land, organic enrichment of
sediments, loss of benthic biodiversity, and major out-
breaks of disease.
In the last decade, better regulatory frameworks have
led to a more stringent approach to licensing, most
notably in the European Union, the United States, and
Canada. The European Union’s Marine Strategy Frame-
work Directive (EU MSFD – EC [80]), together with
guidelines for an Ecosystem Approach to Aquaculture
(EAA – [8]), highlights the ecological component and
aims to optimize production without compromising
ecosystem services. Part of the challenge of determining
carrying capacity is the quantification of negative exter-
nalities as a first step toward improved management.
In Brazil, for instance, where aquaculture has grown
very rapidly over the past decade (Fig. 4), environmen-
tal permitting of new tilapia farms in reservoirs is
determined through the application of the Dillon and
Rigler [9] phosphorus loading model, a rather simplis-
tic view of carrying capacity.
A maximum limit of 30 mg L–1 of P has been
established for reservoir waters, 5 mg L–1 of which is
reserved for fish farming, to allow for multiple uses
including cattle ranching, sugar cane production,
urban discharges, and the natural background.
Fish farms are licensed sequentially, based on the
contribution to P loading of their declared production.
Although this approach does address carrying
capacity at the system scale (i.e., DP = 5 mg L–1), it
does not consider any spatial or temporal variation,
nor does it account for factors such as organic enrich-
ment, disease, or impacts on biodiversity – all of which
may be linked.
Whereas production and social (e.g., tourism)
impacts are often local, ecological impacts of aquacul-
ture can have far broader scales, as can large-scale
effects on aquaculture, such as advection of harmful
algae from offshore [10]. Sustainability is “easier to
plan than to retrofit,” [10] which makes a case for the
 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of 419

140 3.0

120 2.5
TPP

2.0

APP and MPP (no units)

100

1.5
TPP (ton TFW)

80 APP
1.0
60
0.5
Optimisation
40 MPP
VMP = MPP.Po 0.0
VMP = Pi 90 ton
20 For Pi = 15% Po, MPP = 0.15 −0.5

Stage I Stage II Stage III

0 −1.0
0 50 100 150
Seed (ton TFW)

Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of. Figure 2
Marginal analysis indicates that the seed that provides maximum profit (red arrow) falls well to the left of the maximum
production, shown by the dotted line. Production beyond the optimal profit point adds no value and potentially
increases environmental pressure (Results from the FARM model, Adapted from [4])

analysis of carrying capacity at the system scale
(e.g., Nobre et al. [11]), i.e., defining and quantifying
the overall potential for different types of aquaculture
prior to local-scale assessment (e.g., [12]) of new
operations. Simulation models of varying degrees of
complexity must play a role in the determination of
carrying capacity for aquaculture, often combined as
model frameworks, given the range of time and space
scales and the number of processes involved.
Over the next decades, the growth of aquaculture
will take place in developing nations [13–15], which
makes it paramount that the digital divide, which is
already considerable (as is the legal divide; see [16]),
does not become wider.
Simulation technology that can support planning
should be close to the production centers and be able to
deal with data-poor environments and limited compu-
tational access and skills.
Modeling Frameworks for Carrying Capacity
The concept of carrying capacity in aquaculture, based
on four pillars of sustainability, has been adapted
in Fig. 5 to include governance [16, 17]. This is consid-
ered more relevant than the physical element, which in
many respects is encapsulated in the production pillar.
Governance, on the other hand, is clearly missing from
the original model of Inglis et al. [6], and the quality of
balanced regulation, stakeholder involvement, and
community-based management [18] often constitutes
the difference between sustainable aquaculture and an
environmental time bomb.
The social (here used in the context of social oppo-
sition to visual or other impacts of aquaculture devel-
opment, such as conflict with leisure areas) pillar is at
the forefront of decision-making for aquaculture in the
EU, the US, and Canada and can frequently be identi-
fied as the single most important criterion for carrying
capacity assessment and site selection (Fig. 5). By con-
trast, in Asia and other parts of the world where food
production is the paramount concern, licensing criteria
are more frequently based on the production pillar,
with ecological considerations assuming less relevance.
In China and Southeast Asia, the social component acts
as a driver for increased aquaculture, for reasons of
economy and food security. Governance is not usually
420 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of

November 19,1999

Gulf of
Fonseca

January 6,1987

Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of. Figure 3
Expansion of shrimp ponds over a 13-year period in Estero Real, Nicaragua [83]

limited by a lack of legal instruments [16] but often by
their adequacy and acceptance by stakeholders.
Two of the pillars illustrated in Fig. 5, production
and ecology, are amenable to mathematical modeling,
and two are not. This does not mean that those math-
ematical models will be entirely successful in describing
growth, environmental effects, and particularly ecosys-
tem responses, but they do make a significant contri-
bution to the improved evaluation of carrying capacity.
Part of the difficulty lies with our understanding of the
relevant processes, parameters, and rates, part with
other factors, such as the lack of a paradigm in ecology
to support prediction. For instance, in the EU MSFD,
as in other legal instruments, there are complex
descriptors of ecosystem health such as biodiversity,
and the scientific community struggles to establish
meaningful classification systems and their relation-
ship to anthropogenic pressures.
 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of 421

21% annualized
140000
growth

120000
Annual production (ton)

100000

80000

60000

40000

20000

0
1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009

Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of. Figure 4
Production of farmed tilapia in Brazil (MPA, personal communication, 2011)

There are also interactions with the human compo- US, Europe, Types of carrying Southeast Asia,
nent of cultivation that constitute simulation chal- Canada capacity China
lenges. Culture practice, for instance, is widely Highest Production
variable and can generally only be modeled in average
terms [19].
Issues related to disease, which fall squarely between Ecological
production and ecology and are extremely difficult to
model, are a huge challenge in aquaculture and
often include a significant element of poor governance, Governance
such as relaying of infected animals to hitherto
uncontaminated areas.
Rearing large numbers of animals of the same spe- Social Highest
cies in close proximity to each other favors the estab-
lishment and spread of infectious diseases within those Carrying Capacity for Aquaculture, Modeling
farmed populations [84]. Disease can affect both sur- Frameworks for Determination of. Figure 5
vival and growth of animals. Outbreaks of highly viru- The relative importance of the four pillars of carrying
lent bacterial and viral diseases can result in high capacity (Adapted from [17])
mortality of an affected farm stock. For example, out-
breaks of white spot syndrome virus (WSSV) in farmed
whiteleg shrimp can result in greater than 60% mor- that do not result in any significant mortality or mor-
tality, with an attendant dramatic impact on the farms bidity, but still result in the product being downgraded
and regions dependent on farming this species. or rejected by processors after harvest, imposing signif-
Additionally, with ever increasing demands from icant economic costs on the farmer.
consumers for high-quality products, the effects that The ecological effects of disease in aquaculture can
disease can have on product quality are of increasing also be profound. This includes spread of pathogens
importance. For example, tilapia that have survived from farmed fish to wild fish and vice versa [86].
outbreaks of Francisella often have unsightly lesions Disease agents that affect aquaculture species generally
in the fillets. There are also examples of diseases, have their origins in wild aquatic animals [86]. Many
such as Red Mark Syndrome in rainbow trout [85], systems (e.g., salmonid netcage and bivalve culture)
422 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of
involve rearing the farmed animals in relatively open
systems where the reared animals are in direct contact
or close proximity with wild animals. Water is an ideal
medium for the dissemination of many pathogens,
leading to a high risk of transfer. For instance, in
Norway and Scotland, exchange of sea lice
Lepeophtheirus salmonis between farmed Atlantic
salmon and wild salmonids has been implicated in
causing significant declines in populations of wild
Atlantic salmon and sea trout.
In order to examine modeling frameworks, the
following sections review the definitions of, and dis-
tinctions between, the pillars of carrying capacity that
are more amenable to mathematical modeling.
Assimilative Capacity and Carrying Capacity
Assimilative capacity is sometimes considered a sub-
class of the more general term carrying capacity, but
other more specific definitions have been applied. We
make a distinction as detailed below. Assimilative
capacity refers to the ability of biological systems in
the water column or sediments to process organic
matter, nutrients, therapeutants, or contaminants
without alteration of ecosystem state or function.
Carrying capacity refers to the biomass of cultured
organisms that can be supported without altering sys-
tem state or function measured by water or sediment
quality standards and processes [20, 21]. The latter is
thus determined by aquacultured biomass [22], where
the former is independent of aquaculture and deter-
mined by biological properties of the habitat.
The application of standards to models of impact
takes two forms:
1. Absolute criteria determined by regulatory bodies
such as the Water Framework Directive of the EU
(EU WFD – EC [79])
2. Relative standards compared to reference condi-
tions or the range of variation observed in the
environment
In both cases, there is an attempt to use these values
as sustainability criteria. In the case of absolute stan-
dards, the background of the environmental conditions
is not considered. For example, naturally eutrophic
waters may show higher nutrient levels, with no rela-
tionship to aquaculture. This is particularly true of
chlorophyll impacted by shellfish depletion. It may be
more realistic to consider depletion in the context of
the range of values observed in the environment as
a means of establishing whether aquaculture signals
can be detected against background noise. This type
of standard has been applied to shellfish depletion of
chlorophyll by Filgueira and Grant [20] and to shellfish
biodeposits by Grant et al. [23].
Tett et al. [22] formalize both carrying capacity and
assimilative capacity as the result of dose–response
curves, couched in the terminology of DPSIR
(Drivers-Pressure-State-Impact-Response; see also
[24]; Fig. 6), where pressure is the farmed biomass
and state is the system response modeled as
a water quality standard. By comparing physical and
geochemical rate processes, a net balance is deter-
mined, whereby the quantity of interest displays an
increase (e.g., ammonia) or decrease (e.g., dissolved
oxygen) relative to a water quality standard. When
two of these processes are balanced, an index is created,
constituting some of the earliest models in this research
area, sometimes referred to as screening models.
A further distinction is that assimilative capacity be
a function only of biological processing. Tett et al. [22]
use the net results of water quality models which
include advection–diffusion as well as biological
processing to define assimilative capacity. Following
bioenergetic lexicon, assimilation is a metabolic pro-
cess involving digestion and/or decomposition, but
excluding physical exchange.
There has been some decoupling of the measure-
ments used to characterize aquaculture impacts in sed-
iments (redox, sulfides; [25]) with models that are
based on oxygen fluxes. Brigolin et al. [26] used
a more classical early diagenesis model to examine
coupled redox reactions at fish farm sites. This is one
of the few cases where the field measurements used in
regulation could be compared to model output includ-
ing total sulfides and seabed oxygen consumption.
In terms of benthic impacts, assimilative capacity
as the ability of sediments to accept some degree of
organic enrichment without generating anoxic crises is
well grounded conceptually. A comparison of benthic
carbon supply via fish feed and fecal input to oxygen
demand of the sediment forms the basis of some the
original models of benthic impact [27] including
the long-standing Norwegian MOM approach [21].
 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of 423

State indicator State change indicator Uncertainty in knowledge

e.g. maximum of relationship
e.g. chlorophyll,
oxygen chlorophyll, change in
Uncertainty in knowledge 'balance of organisms'
Impact Impact
of relationship
Relationship
'safe' region - change between
below threshold, pressure and
allowing state change
for uncertainty
Maximum
tolerable
change Maximum
tolerable
'safe' region - state change
above threshold, Relationship
allowing between
for uncertainty pressure
and
state change

e.g. bivalve e.g. waste

Pressure indicator Pressure indicator
production loading
Maximum safe pressure Maximum safe pressure
(carrying capacity) (assimilative capacity)

Management aims to keep pressure within ecosystem's

sustainable capacity

Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of. Figure 6
Conceptual framework of assimilative and carrying capacities [22]

This concept was widely applied to multiple salmon Limit of organic matter loading
sites by Morrisey et al. [28]. De Gaetano et al. [78] Acceptable Aquaculture
Concentration or Rate

produced a more sophisticated version of the model -S)

S
for Mediterranean fish culture. AV
e(
lfid
The intermediate disturbance hypothesis (e.g., Su
[29]) provides a functional form to the model by Ox
suggesting a parabolic response of benthic diversity yg
Standard value en
Up
and activity to organic loading. Stimulation of aerobic tak
e
demand occurs at low enrichment, peaking at interme-
diate levels and declining at high levels (Fig. 7; [30]). Organic matter loading
The latter authors define “acceptable aquaculture”
Carrying Capacity for Aquaculture, Modeling
as keeping sediment oxygen demand at or below the
Frameworks for Determination of. Figure 7
peak. They produced a numerical model which
Acceptable limits for organic matter loading from fish
included a 3D circulation component, organic deposi-
farms and the relationship to benthic oxygen uptake and
tion, and decomposition based on a sediment diagen-
sediment sulfide content [30]
esis model using oxygen fluxes and anaerobic
processes. Output is in the form of mapped values of
organic loading based on fish stocking density that
produces maximum aerobic oxygen concentration loading based on the capacity of the benthos to absorb
(Fig. 8). organic matter while maintaining oxic conditions. In
The map shows that farms within the inner parts of addition, inclusion of a spatial diffusion–advection
the inlet have less permissible organic sedimentation model allows incorporation of nonlocal processes and
due to reduced flushing. We suggest that this example is provides mapped output. Results are inherently inclu-
the appropriate approach to assimilative capacity since sive of far-field effects and include interaction of mul-
it models system function and its response to organic tiple farms. Although the paper did not have the
424 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of
20 60 100
10 40 80
Fish Farming site
Carrying Capacity for Aquaculture, Modeling
Frameworks for Determination of. Figure 8
Isopleths of the limit values of the organic matter loading
rate (mmolO2/cm2/day) in Gokasho Bay, estimated by
a numerical model [30]
context of marine spatial planning, it is clearly appli-
cable in terms of both the approach and the results. The
comprehensive nature of this study and its faithfulness
to the concept of assimilative capacity make it note-
worthy in the literature.
Models of assimilative capacity, like those for shell-
fish carrying capacity, differ in spatial resolution. The
original index models are 1-box, 0D models treating
any body of water as a single basin. Physical exchange is
thus averaged over the entire domain. This is often
problematic since estuaries or other embayments are
places with strong gradients in flushing. In addition,
aquaculture sites are averaged in this scheme, so no
questions regarding optimal location can be addressed.
One hybrid approach which solves some of these
problems is the use of a full circulation model whose
output is applied to local models. In this case, at least
the physics is location specific, and subregions of the
system may be considered without the 0D averaging.
Lee et al. [31] applied this approach to yellowtail
culture in Hong Kong Harbour by comparing oxygen
delivery to fish oxygen consumption and determining
net oxygen concentration relative to water quality
standards for different levels of stocked biomass.
Similar schemes have been used for finfish culture in
Scotland [22].
Models for Finfish, Shrimp, and Bivalve Culture
Models for Open Water Culture Models for organic
loading by finfish include submodels of circulation,
particle transport, and benthic response [21]. Models
may also include a phytoplankton-nutrients-
zooplankton (PNZ) component, simulating
trophodynamics in the water column. This may be
necessary due to the importance of phytoplankton
and the microbial loop as nutrient processors [22].
For either finfish or shellfish, some version of a farm
production or bioenergetics model is essential to esti-
mate waste outputs from cultured animals. Benthic
models applied to aquaculture are typically diagenetic,
aimed at resolving sediment decomposition and nutri-
ent regeneration [78].
For finfish, benthic deposition of farm wastes and
consequent impacts are the primary emphasis; since
fish are not dependent on the environment for food,
trophodynamics are less relevant. Similarly, the models
are usually localized since much of the waste material
remains near the cage site (e.g., DEPOMOD). None-
theless, there is concern that wastes reach the far field
and produce negative benthic impacts. Despite this
potential, far-field models of finfish cage culture are
uncommon. Some early examples used 3D circulation
models to examine waste dispersal over kilometer
scales [87].
In a more recent example, Symonds [32] compared
near-field models such as DEPOMOD to a far-field
model based on a 3D circulation. He found that the
dependence of near-field models on limited current
meter observations was subject to the noise and uncer-
tainty inherent in those measurements. In addition, the
far-field model had the potential for bidirectional
transport of waste, whereas the near-field model had
permanent escape from its limited domain causing
potential underestimation of benthic impacts. In addi-
tion, the far-field model allows consideration of mul-
tiple farms and their interaction. Skogen et al. [33] used
a 3D circulation model coupled to a full ecosystem
model to study the effects of fish farms on far-field
oxygen, nutrients, and primary production, conclud-
ing that eutrophication in the far field of a Norwegian
fjord was not enhanced by the farm.
The goal of most bivalve shellfish models is to
understand seston depletion as a limiting food resource
 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of
for farmed animals. This requires a trophodynamic
model which includes primary production, bivalve
grazing, and advection–diffusion. Because farmed
shellfish feed on phytoplankton from distant sources,
recent examples involve models which include the far
field. Nevertheless, there are many examples of 1-box,
0D models in which the physics and biology are
averaged over the basin. Environmental impact of
biodeposition to the benthos has been considered in
several models including spatial examples [30, 34], but
this is less frequently addressed compared to seston
depletion. The opposite is true for field studies where
benthic impacts are emphasized, and seston depletion
is rarely addressed due to the difficulty in observing it.
Because seston depletion occurs first at a farm scale,
models of the depletion process have also been created
at the local scale, prominently the FARM model [4].
Far-field models of seston depletion are increasingly
common [20, 35].
It may be concluded that models of finfish aqua-
culture impacts would benefit from more spatial real-
ism and far-field content, as well as further emphasis
on assimilative capacity. Similarly, shellfish models
would benefit from inclusion of benthic impact pre-
diction in association with existing focus on seston
depletion. The development of assimilative capacity
models would be identical for both finfish and shell-
fish culture. This also places the context to that of
ecosystem function. The present context of faunal
indices based on carbon deposition in local models is
important [36, 37], but is predicted on the basis of
somewhat tenuous empirical relationships which are
a departure from the more quantitative nature of the
models.
Models for Land-Based Culture Models that simu-
late land-based culture taking place in ponds, tanks, or
raceways use many of the biogeochemical features
described above, but the physics is simplified to
a reactor type of system and serves to determine
water exchange and effluent loading to adjacent water
bodies. Such models are cheaper to develop and
implement than the examples given for open waters
(here including lakes and estuaries, where water cir-
culation should also be accounted for). According to
the latest figures from FAO, over 70% of freshwater
aquaculture in China takes place in ponds,
425
corresponding to an area the size of New Jersey and
an annual production of over 15 million tons [88].
This number is triple the total aquaculture produc-
tion of America, Europe, and Africa combined, which
suggests that substantial emphasis should be placed on
models that can assist with site selection, optimization
of carrying capacity, and evaluation of negative envi-
ronmental externalities of pond culture.
Various examples of this type of approach exist
(e.g., [38, 77]. Figure 9 shows mass balance results
obtained with the Pond Aquaculture Management
and Development (POND) model [39], which simu-
lates the production and environmental effects of
shrimp, fish, and bivalves cultivated in ponds, in
monoculture or IMTA.
The model was run for a site at 23oN, with a daily
water renewal of 3% of the pond volume, and esti-
mates an environmental discharge of about 45 kg of
nitrogen (mostly dissolved, but also as algae), roughly
14 population equivalents (PEQ) per year for the 90-
day cultivation cycle. The cost of offsetting these emis-
sions is over 500 USD [40]. In developing countries,
these waste costs are not internalized but rather
imposed on to the environment, contradicting the
first principle of the ecosystem approach to aquacul-
ture [8] that:
" Aquaculture should be developed in the context of
ecosystem functions and services (including biodiver-
sity) with no degradation of these beyond their
resilience.
By contrast, pond production in the United States
already requires a National Pollutant Discharge Elimi-
nation System (NPDES) permit [41]. Often, this means
that large agro-industrial companies from developed
countries price-leverage the lack of environmental reg-
ulation and/or implementation in the developing
world.
Models of this kind can be coupled with a range of
other models to provide a decision support framework.
POND uses the well-tested Assessment of Estuarine
Trophic Status (ASSETS) model [42], providing
a color-coded assessment (Fig. 9) of the degradation
of water quality over the culture cycle.
Models for Integrated Multi-trophic Aquaculture
IMTA has been practised in Asia for thousands of
426 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of

Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of. Figure 9
Mass balance of whiteleg shrimp (Penaeus vannamei) culture, including production and environmental externalities for
a 1-ha pond

years. In the fifth century B.C., the Chinese production, optimizing resources, and reducing envi-
aquaculturalist Fan Lee [81] wrote: ronmental waste [14]. Despite this oriental wisdom,
multi-trophic aquaculture is still rare in North America
" You construct a pond out of six mou of land. In the
and Europe, although commercial interest is growing
pond you build nine islands. Place into the pond plenty
rapidly. This is reflected in research (e.g., [43, 44]), with
of aquatic plants that are folded over several times.
the annual number of scientific publications on IMTA
Then collect twenty gravid carp that are three chih in
doubling from 2007 to 2010 (SCOPUS).
length and four male carp that are also three chih
The combinations of species, relevant proportions,
in length. Introduce these carp into the pond
and culture practice are key to successful IMTA. There
during the early part of the second moon of the year.
is traditional knowledge in China and other parts of
Leave the water undisturbed, and the fish will spawn.
Asia on what works best, but advances in mathematical
During the fourth moon, introduce into the pond one
modeling can make a substantial contribution by quan-
turtle, during the sixth moon, two turtles: during the
tifying energy flows, production, and environmental
eighth moon, three turtles. The turtles are heavenly
externalities ([45], Nobre et al. [11], [16]).
guards, guarding against the invasion of flying
Figure 10 shows a simulation of IMTA for a
predators.
shrimp farm, with cocultivation of the Pacific oyster
A substantial proportion of Asian aquaculture Crassostrea gigas at different densities, using the POND
currently takes place in cocultivation, improving model. As the oyster density in the ponds increases,
 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of 427

Worse Better
ASSETS Chl a
ASSETS DO
ASSETS grade
NPP (kg chl a), Oyster ind. wt. (g), NH4 outflow (kg) 90 600

80
500
70

NPP (kg chl a) 400

Oyster harvest (kg)

60

50 Individual oyster wt (g)

300
Ammonia outflow (kg)
40
Oyster harvest (kg)
30 200

20
100
10

0 0
0 10 20 30 40 50
Oyster density (seed m−2, 10% of pond)

Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of. Figure 10
Analysis of IMTA production and externalities by means of the POND model

the net primary production (NPP) of phytoplankton Models for Disease Spread and Control In deter-
is reduced due to top-down control, although mining the carrying capacity of aquaculture opera-
NH4+ increases due to bivalve excretion. The model is tions, it is important to ensure aquaculture
not simulating macroalgae or other aquatic plants, production practices and systems within a farm, man-
which if added would significantly reduce the output aged region, or zone are resilient to the effects of dis-
of ammonia. ease. Modeling provides a means of investigating the
The ASSETS score is best at the higher oyster den- interactions occurring among the four pillars and the
sity, but at a density of 10 oysters m–2, the shrimp spread and establishment of pathogens; however, to
culture cycle would yield a harvestable oyster biomass date, most models have ignored the influence of society
of over 500 kg to provide an annualized extra income on aquatic disease. Many different models have been
of over 10,000 USD. The removal of algae and detritus derived to investigate the spread and impact of partic-
by the filter-feeding bivalves corresponds to three ular pathogens: In aquatic systems, two of the most
PEQ per year, about 15% of the discharge. At the common approaches are (1) compartment-based
higher densities, the bivalves are performing a models and (2) network models.
bioextractive function and do not reach market size Compartment-based models assume that individ-
in a short cycle due to food depletion. A more detailed uals transition through a series of states from suscep-
analysis can be made by simulating oyster growth tible (S) to infected (I), and potentially back, or
continuously over multiple cycles of shrimp culture. through a series of other states such as resistant (R).
Such interactions can also be modeled for other com- These models are often referred to as SIR models [46]
binations, for example, tilapia and shrimp or salmon and are usually based on continuous time, and there-
and mussels. fore use differential equations; however, stochastic and
428 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of
discrete time approaches are also used. SIR models do
not track individuals but assume the population fol-
lows a set of behavior rules, and that at each point in
time, a proportion of the population leaves one state to
enter another according to these rules. In aquatic sys-
tems, these models are generally used to track disease
through a population of animals, but they have also
been applied to look at spread through a population of
sites. Simple implementations are often analytically
tractable, allowing conditions under which thresholds
and equilibria occur to be found without the need to
run simulations.
One of the key pieces of information that can be
derived from these models is a maximum (suscepti-
ble) host carrying capacity for which a pathogen can-
not persist. Such carrying capacities used in the
context of pathogens are often referred to as a critical
threshold (NT) and may be useful to wildlife and farm
managers when attempting to control or prevent dis-
ease. This threshold is however largely dependent on
the assumptions made regarding the way a pathogen is
transmitted, and obviously does not apply if transmis-
sion is not dependent on host density, but is frequency
based (as is often assumed to be the case when model-
ing sexually transmitted infections). It is therefore
important that the correct form of transmission is
used in a disease model to avoid false inference. The
most commonly used form of model assumes that
transmission occurs by contact between animals or
sites, that the number of contacts is density dependent,
and that mixing between individuals occurs equally
and at random. This is often referred to as a “density-
dependent transmission” or “pseudo mass action”
model [47, 48].
Under these assumptions, one method by which NT
can be derived is by determining the conditions
under which the basic reproductive number, R0, is
equal to 1. In simple terms, R0 is the number of
secondary infections that arise if an infected indi-
vidual enters a wholly susceptible population. If
greater than 1, the pathogen will establish; if less
than 1, it will die out. R0 is governed by the total
population density, the period over which an
infected animal sheds pathogen, and the transmis-
sion rate b (the rate of contacts between individuals
and that the probability that given a contact, infec-
tion occurs). Both environmental conditions and
management/husbandry decisions can influence this
number, and therefore the critical threshold for
establishment. Understanding the influence of each
of the four pillars of carrying capacity on R0 is
therefore critical in order to predict and manipulate
NT. Omori and Adams [49] illustrate the application of
compartment-based models to assess the influence of
the ecology and production pillars on the dynamics
of Koi herpesvirus in farmed carp populations
(Cyprinus carpio). Their approach showed that temper-
ature and on-farm production processes used in
conjunction with this could be used to immunize
a population, preventing clinical disease expression.
The assumption of random mixing of a population
is often not reasonable, as in reality some individuals
will be more social or isolated than others, and
individuals tend to have discrete groups of contacts
that may or may not be connected to other groups
within a population. Under these circumstances, the
assumption of random mixing can lead to substantial
overprediction of the epidemic process. Social network
analysis (SNA) and modeling approaches provide
a means of incorporating the contacts that occur
between individuals, and therefore the consequence
this may have on pathogen spread. In the case of
agriculture and aquaculture, these models tend to be
based at the level of the site, rather than the animal
in question.
A major advantage of this modeling approach
is that much epidemiologically useful information
can be obtained merely by examining the network
properties, without parameterizing for a particular
disease. For example, in order to develop generic sur-
veillance and control strategies, it may be useful
to identify:
● Clusters of connected individuals within the net-
work and whether it is possible to make them epi-
demiologically distinct through the removal of
a few connections.
● Long chains of connection that join the network
throughout and thus allow disease spread.
● Super-spreaders, which are individuals that contact
a high number of other individuals and can thus
rapidly spread pathogens.
● Super-sinks, which are individuals that receive from
a lot of other individuals. Though they may not
 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of
spread a pathogen, they are most likely to receive
one and may therefore be useful for surveillance
purposes.
Many statistics can be generated to summarize dif-
ferent properties of a network. Although R0 can also be
estimated, other statistics such as the degree of central-
ity or clustering coefficient may provide more useful
means of assessing the likelihood of spread through
a network. Green et al. [50] demonstrated the applica-
tion of such statistics using SNA applied to the Scottish
network of trout and salmon farms. The analysis
showed how much transmission was likely to occur in
the network as it stood but also used a variety of
methods to remove the most influential connections
to reduce transmission.
In addition to examining the network properties,
stochastic simulations can be run over the network. In
such simulations, the network is randomly seeded with
infected individuals and, at each subsequent time
point, connected individuals change state from suscep-
tible to infected at a given probability and given the
likelihood of a contact. This approach is often useful
when evaluating rates of spread and the effectiveness of
control strategies but also has the potential to be used
as a real-time tool during an outbreak, if it can be
parameterized for the pathogen of interest. Further
useful information may be gained from these models
by making network models spatially explicit, as this
facilitates the designations of control zones.
Werkman et al. [51] used stochastic network simu-
lations to good effect to determine the efficacy of dif-
ferent fallowing strategies in eradicating disease from
areas producing different amounts of fish. Thrush and
Peeler [52] used a simulation of movements over the
English and Welsh trout industry network to investi-
gate the potential spread of Gyrodactylus salaris. This
demonstrated that in 95% of the simulations run, less
than 63 of 193 river catchments would be at risk of
getting the pathogen in the 12 months following intro-
duction. Jonkers et al. [53] developed this model fur-
ther into a strategic tool that could be used to evaluate
the effectiveness of different control and surveillance
efforts on disease spread.
One of the major limitations of network approaches
is that they rely on knowledge of the complete network
of connections and assume that this remains stable over
429
time. Missing connections or changes to the network
with time could lead to misdirected efforts. For many
aquatic systems, reliable network data are not available
or are difficult to compile. Under these circumstances,
simpler compartment-based approaches may still be of
value in informing control policies. One such applica-
tion was demonstrated by Taylor et al. [54] to evaluate
the effectiveness of different control options in reduc-
ing the spread of Koi herpesvirus in the UK between
sites holding carp.
Most models applied to aquatic systems to date
tend to be based at either the level of the site or animal,
with few attempting to combine the two approaches.
There is, however, substantial scope to develop future
models that incorporate multiple levels that account
for transmission between individual animals in a unit,
the influence this has on the transmission between
units on a farm, and the subsequent effect on
between-farm spread (Fig. 11).
Where attempts to link these two levels together have
been made, useful results have been obtained. Hydrody-
namic models have been applied to look at the spread of
sea lice between Norwegian salmon cages in fjord sys-
tems [55]. These models track the number of lice gen-
erated and monitor their dispersal and decay over time
and space to see whether they reach and infect other
sites. Green [56] combined compartment-based and
network approaches to incorporate the influence of
within-site processes on network spread and found
that differences in site biomass influenced the rate of
between-site transmission if density-dependent trans-
mission was assumed. One of the major advantages of
models that link the within-site epidemic with
between-site spread is that they allow for the conse-
quences of different detection (or action) rates to be
assessed, allowing more efficient resource planning.
The primary goal of all of the approaches described
above is often to apply changes to the system to under-
stand their influence on pathogen spread. In applica-
tion to aquaculture systems, this may be conducted to
establish how best to maximize NT (either in the num-
ber of animals produced within a site or the number of
sites in an area) and thus increase carrying capacity.
Generally speaking, NT can be increased by reducing
the amount of connectivity between individuals
(restrictions in movements), changing their suscepti-
bility to disease (e.g., by vaccination) or reducing the
430 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of

Animal
Trestle
INDEX CASE Farm

Relaying
Fish: escapes and migration
Hydrodynamic connectivity

Example for three oyster farms

Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of. Figure 11
Network models working at different scales in time and space

period over which an individual is able to transmit
a pathogen (e.g., through good surveillance and rapid
culling). Other important disease management tools
that have or could be evaluated by the modeling
approaches described above include:
● The use of management areas in which aquaculture
activities such as harvesting or treatment are coor-
dinated between all farms in an area
● Fallowing of sites between production cycles to allow
pathogens present to die out and potentially reduce
the infection pressure other sites are exposed to
● Year class separation, where only one age class is
present at a time causing all animals to be harvested
prior to stocking new animals
● Removal of high-risk contacts between sites that are
likely to spread disease widely
● Biomass limits to reduce the maximum amount of
pathogen that could be discharged from a site
● Minimum distances between sites to reduce trans-
mission via hydrographic connectivity
Building a Framework
Screening Models and Research Models The models
reviewed above address different components of aqua-
culture carrying capacity, focusing mainly on
production and environmental effects, including dis-
ease aspects. Additionally, they consider different scales
in time and space and range from statistical models to
spatially discrete representations, which may (e.g.,
hydrodynamic models) or may not (GIS) be time vary-
ing. It is useful in this context to distinguish between
screening models and research models.
Screening models typically use a limited set of
inputs and provide highly aggregated outputs, such as
an index of suitability or an environmental scorecard.
Examples include a comparison of ammonia excretion
by caged salmon compared to tidal flushing [57] and
the production of biodeposits compared to tidal
removal for suspended mussel culture [23].
Models of this type (e.g., FARM, [4]; POND; Fig. 9)
are easy to use by the management community and
provide a quick diagnosis for a specific site, or a generic
overview comparison for multiple areas.
Models that are complex to apply (and usually
lengthy and complex to develop and implement) may
be termed research models and are of limited practical
use in day to day management. Partly, this is due to
the knowledge required for parameterization, volumes
of data involved, and substantial requirements in
processing and interpreting results. This does not
mean that the results of such models do not have
 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of
a clear application for management, but operating
them may be beyond the scope of many institutions.
The concept of a single overarching model, able to
provide answers across a range of space and time, has
been shown repeatedly to be unsound. Just as software
suffers from feature creep [58, 59], stand-alone models
tend to become increasingly overparameterized, partly
in an effort to better match reality and partly in
an attempt to solve per se what should really be
approached with a combination of models.
A more promising alternative is to combine GIS,
dynamic models, network models, and remote sensing
tools as appropriate to deal with questions that range
from the impact of a HAB event on a salmon cage at the
scale of days to the economic success of 10 years of
mussel farming.
An increase in model complexity does not neces-
sarily equate to increased accuracy [60], whether in
physical or biological models. In both cases, the scale
at which predictions may be made is limited by our
incapacity to accurately predict the weather. As
a consequence, model resolution and accuracy are lim-
ited by key drivers such as river flows, salinity patterns,
and water and air temperature, which impact meta-
bolic rates, algal blooms, turbidity, spawning, or larval
dispersal.
Models in the Context of Integrated Coastal Zone
Management Carrying capacity models for aquacul-
ture are useful in their predictive capability and there-
fore in management of both cultured biomass as well as
location. Spatially resolved models are particularly
notable in this context. Within the catchment, this
may be addressed by means of GIS (see, e.g., [61])
and can be enhanced through a combination of
dynamic simulations and spatially resolved models.
Aguilar-Manjarrez and Nath [89] performed an
extended analysis of the potential for fish aquaculture
in Africa, based on GIS models, using a resolution of
3 arc minutes (25 km2 at the equator). For small-scale
operations, suitability was based on water require-
ments, soil and terrain, availability of feed inputs, and
farmgate sales projections. This analysis was also car-
ried out for larger, commercial farming and concluded
that for the three species considered (Nile tilapia,
African catfish, and carp), about 23% of the area of
continental Africa scored very suitable for both types of
431
farming. These authors did not explicitly consider envi-
ronmental effects of fish farming, probably because
that analysis is best performed regionally or locally, as
part of detailed site selection, but the modeling tools
for addressing these impacts are available today.
GIS models have also been combined with remote
sensing to assess aquaculture opportunities, for exam-
ple, for crab and shrimp in the Khulna region of
Bangladesh [62]. In this case, the focus was on gross
production, economic output, and employment, and
discussed species suitability with respect to factors such
as salinity distribution and freshwater availability. Once
again, the environmental externalities of the different
types of culture were not included but can be modeled
to provide a more complete picture for decision sup-
port, including externality costs.
Figure 12 shows the Jaguaribe estuary, in the state of
Ceará, northeast Brazil. As part of the application of the
ASSETS model to determine the eutrophication status
of the estuary (Eschrique and Braga, personal commu-
nication, 2011), the nitrogen loading from 1,200 ha of
shrimp farms, located between the city of Aracati and
the dunes fringing the beach, was determined using
POND.
The contribution of shrimp farming to the nitrogen
load was estimated to be of the order of 60 t year–1,
roughly equivalent to 20% of the total loading, or
about 15,000 PEQ.
Screening models of this kind are simple to use and
help water managers in developing countries to address
the challenge of multiple uses and nutrient sources to
the coastal zone and to make better decisions with
respect to site selection and waste treatment. They can
also be included in a more general catchment modeling
approach, combining their outputs with hydrological
models such as the Soil and Water Assessment Tool
(SWAT) model (e.g., Nobre et al. [11]).
In open water, including semi-enclosed systems
such as estuaries and bays, because spatially resolved
models incorporate physical circulation, they are
potentially useful for other aspects of coastal zone
management.
There are of course a variety of possibly impactful
activities in the coastal zone including eutrophication,
fisheries and fish processing, contaminant input,
resource extraction, and transportation. There are
inevitably competing uses of the water and bottom,
432 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of

Jaguaribe
estuary 7 km
Fortim

Atlantic
Ocean

Beach and
fringing dunes

Shrimp
ponds

Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of. Figure 12
The Jaguaribe estuary, in Ceará, NE Brazil. Shrimp farms occupy an area of about 12 km2 and discharge the effluent into
the estuary

and making predictions about aquaculture in isola-
tion is insufficient. Water and sediment quality stan-
dards applied over large spatial scales help to apply
objectives that may be seen as ecosystem-based man-
agement. The implementation of these objectives is
achieved through marine spatial planning. The impli-
cation of planning is that predictions can be made to
anticipate overlaps, conflicts, and cumulative impacts
of various activities.
Although models exist for these other activities,
particularly those acting through eutrophication, the
models need to interact coherently, and their outputs
should be tied together in a way that is useful for
management decisions. Two essential features of this
integration are physical models that unify the transport
of materials common to almost every process in the
ocean and GIS to maintain data layers. The physical
model establishes the spatial domain which can be
either conserved, collapsed, or expanded within the
GIS. At present, models run within GIS are necessarily
simple and based on averaged values. For example, the
AKVAVIS tool developed in Norway [63] utilizes a GIS
with calculation of shellfish growth as well as fish farm
effects on water quality at any location based on an
inline model using depth, temperature, and other spa-
tially located characteristics within the GIS [63].
 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of
Tironi et al. [24] utilized an open source circulation
model to predict far-field deposition for Chilean
salmon culture. An essential feature of their work was
a GIS interface used to depict other coastal activities in
light of hydrodynamics and dispersal of farm waste.
Nobre et al. [11]) combined models of watersheds,
aquaculture, and eutrophication to look at mitigation
strategies for improving nutrification in Chinese bays.
In marine systems with aquaculture but lacking
other activities (e.g., some Norwegian fjords), the cul-
ture model may form the basis of the decision support
system. In Lysefjord (southern Norway), a pump was
used to transport water to depth where it enhanced
diffusion of nutrient from below the thermocline to
the euphotic zone [90]. The increase in primary pro-
duction was used as a food source for cultured mussels.
A model was employed to determine the best location
for the upweller as well as the mussels in order to
benefit from increased primary production, balancing
the increase in new production with mussel removal to
maintain chlorophyll within its natural limits. This
example is of interest because the ecosystem was truly
managed in terms of bottom-up nutrient supply, new
production, grazing in terms of mussel culture, and
marine spatial planning optimized for the extent and
level of shellfish culture.
As more types of activities are added to decision
support tools, one approach is to use GIS as a wrapper
for model outputs produced as layers. Decision sup-
port comes from ancillary software with features such
as portrayal of alternative land uses, exclusion of
protected areas, weighting of valued features and hab-
itats, and economic analyses. Several initiatives have
been undertaken in this vein, with the incorporation
of simulation models mostly in the initial stages of
progress. Examples which have freely available ArcGIS
extensions include NatureServe Vista (natureserve.org)
and Marine InVEST (naturalcapitalproject.org). There
are different emphases in the various projects, includ-
ing conservation, community engagement, ecosystem
services, and land use, in addition to water quality
issues.
Among these, Marine InVEST seeks to maintain
ecosystem services in the context of activities such as
fisheries, aquaculture, renewable energy, and recreation
[64]. Input information ranging from oceanographic
data to species distribution is used in various models to
433
consider outputs in terms of ecosystem services pro-
vided, traditional model results (e.g., water quality),
and socioeconomic valuation (Fig. 13).
We note the importance of models other than those
based on eutrophication and water quality. For exam-
ple, some of the same spatial data used to model aqua-
culture impacts can be used to predict the location of
sea grasses or species at risk [65, 66]. Protected areas or
buffer zones may then become part of the plan. In this
way, critical habitat and biodiversity are also consid-
ered along with aquaculture siting. Moreover, these
decision-support frameworks can be used in public
forums to incorporate community input, as has been
done with protected area planning via MARXAN GIS
[67]. In practice, these are few examples of models used
to this extent, but it provides a very useful management
shell in which to consider aquaculture submodels.
The Other 50% of the Problem At present, two
major components of carrying capacity, the social and
governance aspects, are not amenable to mathematical
modeling (Fig. 5); they are nevertheless fundamental
areas for aquaculture management.
The social pillar, from the perspective of social
acceptance of aquaculture and, in particular, of the
definition of limits to industry growth, has been
discussed, for example, by Byron et al. [18]. Stake-
holder dialogue, understanding of terms and concepts,
and the simple fact that opinions can be voiced during
the decision-making process are major contributors
to generate consensus. The tools employed include
questionnaires, meetings, and presentations of differ-
ent sides of the issue. Simulation models can help
inform those discussions by, for example, providing
quantitative data on development scenarios but social
positions often have a strong emotional component.
The governance pillar plays a major role in the assess-
ment of carrying capacity. Table 1 shows key aspects of
the human interaction in aquaculture and highlights
the consequences of poor practices and governance.
The issues identified have consequences that vary in
severity, from a reduction in revenue and profit to
major outbreaks of disease and the loss of aquaculture
resources across large areas.
Because the effects of human mismanagement can
be so far reaching, it is important to discuss to what
extent modeling frameworks can be used to assist the
434 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of

Optimization
SPATIAL MODELS

Digital maps & Circulation

Spatio-temporal
data layers

n
sto
Se
nts
u trie Biogeochemistry
N Output maps:
yll Culture production
r o ph Ecosystem services
l o
Ch Economic returns

Boundary & Aquaculture

Initial Conditions
Decision su
ppor t

Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of. Figure 13
Models for different purposes are linked through input data layers that are used to build scenarios about management
actions (e.g., restoration of eelgrass, increase in aquaculture) and climate change (e.g., sea-level rise, water temperature)

farmer and the water manager. The eight topics in
Table 1 fall under the heading of governance (here
taken to include self-regulation by farmers and farmers’
associations, as well as legal instruments, policy,
and implementation), but only three (dark blue)
can benefit directly from recommendations from sim-
ulation models, addressing stocking density and feed-
ing practices.
Five of the issues identified introduce disease-
related problems, and whereas procedures such as
relaying, or seed import from contaminated sources,
are strictly within the remit of good governance,
models can to some extent assist in predicting the
likelihood (i.e., risk), spread, and establishment of dis-
eases, if present within an aquaculture area.
Regulations enacted to control spread of diseases
can directly mandate what species and culture prac-
tices, stocking densities, etc., are permitted within
a zone/region. Firstly, this would include planning
applications assessments that would consider, among
other factors, how siting a farm may potentially
adversely affect other aquaculture operations located
in that area, as well as potential effects on wild animal
populations. In some cases, e.g., in Chile, regulations
have been enacted that specify minimum distances
between farms.
There is also an increasing move by some large-scale
industries, as part of developing common codes of
practice, to manage farms to minimize the effects of
disease on production. These codes can include
restricting stocking densities, production on farms,
specifying minimum allowable distances between
farms, and introduction of mandatory fallowing
periods between production cycles. All these are mea-
sures that would theoretically constrain carrying capac-
ity, at least in the short term, but might well increase
the overall sustainability of the industry in the zone or
region in the longer term. Although these may not be
enforced by government regulations when first devel-
oped (e.g., they are often “voluntary codes”), those
signing up may be entering legally binding agreements.
These codes may then be used as the basis to develop
more formal regulatory frameworks in the future at the
local, national, and even supranational level.
 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of 435

Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of. Table 1 Key issues in culture practice,
time frames, and potential consequences (color coding reflects the extent to which models can be applied: black –
substantial; dark red – reasonable; light red – inapplicable)
Time
No Topic frame Issues/consequences Examples
1. Species Prior to Imported exotics, disease, proliferation Pacific oyster, now considered invasive in
selection cultivation the Netherlands; Perkinsus (dermo) and
MSX, U.S. East Coast
2. Seed Start of Disease from imports, stable broodstock Herpes virus spreading in oysters across
purchase cultivation Europe
3. Relaying Variable Disease spread Transmission of Herpes in oysters across
Europe, ISA in Chilean salmon
4. Stocking Critical Overstocking can lead to: mass mortalities Whitespot Syndrome Virus (WSSV) in
(farm or periods due to dissolved oxygen depletion, other Penaeid shrimp, Perkinsus in clams
pond scale) environmental factors, and stress-related
disease outbreaks
5. System- Months to If carrying capacity is significantly Marennes-Oléron, France, longer oyster
wide years exceeded: harvest reductions, disease culture cycles; Sacca di Goro, Italy, mass
carrying outbreaks, economic hardship, system mortality of Manila clam; Qinshan, Fujian
capacity collapse, long recovery cycles, long-term province, China, 50,000 fish cages (yellow
loss of resource croaker), mass mortality
6. Feeding Culture Ecologically damaging practices with Fertilization of intertidal/subtidal areas in
practice cycle ecosystem consequences, e.g., overfeeding China to promote seaweed growth,
of caged fish and benthic hypoxia increasing yields of commercial products
(seaweed, gastropods); use of juvenile fish
as fish meal for cage culture in parts of Asia
7. Spatial Culture If inappropriate, e.g., by combining year Clam culture in southern Portugal
distribution cycle classes in shellfish, more labor-intensive,
of culture greater impacts on the ecosystem, e.g.,
through sediment reworking
8. Lease – Fragmented/inexistent lease structure, Pond culture in Thailand (average
structure smallholdings governance issues, due to freshwater pond: 0.28 ha); Clam culture in
multiple stakeholders southern Portugal (average lease: 0.15–
0.5 ha)

Regulation can also influence the carrying capacity
of a zone or region both directly and indirectly. In
particular, to help control the spread of diseases
between countries, the OIE aquatic animal code [91]
lists a number of infectious aquatic animal diseases that
are generally considered untreatable, pose a significant
threat to aquaculture and/or wild fish populations, and
are not widely distributed (e.g., are exotic to most
countries where the species they affect are farmed).
Countries and regions also have laws and regulations
to prevent the entry and, where pathogens do gain
entry, specify measures for their control and eradica-
tion. For instance, Directive 2006/88/EC lays down, for
European Member States, the required minimum ani-
mal health requirements, disease prevention, and con-
trol measures for aquaculture animals and products.
Eradication of a disease from a country or zone may
involve extensive depopulation of affected farms and
not restocking them until it is confirmed that the risk
from disease is significantly reduced. This has obvious
short- and long-term consequences on the carrying
capacity of the affected species in the affected areas.
436 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of
For example, following detection of the notifiable viral
disease Infectious Salmon Anemia (ISA) on an Atlantic
salmon farm in Chile in 2007, the disease spread
throughout the salmon industry [92]. The effects were
dramatic. It is estimated that salmon production in
2010 was little more than 98,000 t, down from
386,000 t in 2006 [93]. With the 1.5–2.5-year produc-
tion cycle for salmon, these effects will be felt for years
to come, with estimated smolt release in 2009 only
10% what it was in 2007 [94]. It is estimated that,
compared to production in 2007, levels will be reduced
cumulatively by at least 700,000 tons during the period
2009–2011, with their value reduced by more than two
billion USD.
Disease controls can restrict the types of aquacul-
ture activity that are permitted, or are practically pos-
sible, within a zone or region. Here, the constraints to
carrying capacity are often opportunity costs, with fish
not reared in a particular area because of potential
disease risks. The effects can be quite subtle. For
instance, in the UK, controls in place to restrict the
spread of bacterial kidney disease are considered by
some sectors of the rainbow trout farming industry to
adversely affect their operations at the expense of the
larger Atlantic salmon industry that has generally
supported the maintenance of these controls. This
would also include the effects of restrictions placed on
the movements of live fish and ova between countries
that limit the opportunities to farm those species in
other countries.
Finally, societal acceptance of aquaculture activi-
ties may be influenced by disease risk concerns, par-
ticularly to wild fish stocks. This is perhaps best
illustrated by sea lice spread to wild fish. There is
additional public concern as to the effects on the
environment of use of chemical treatments. In partic-
ular, there is strong resistance to fish farming in many
remote and pristine environments due to fears they
may adversely affect local ecosystems. Increased public
awareness of welfare issues in aquaculture may also lead
to practices that reduce the incidence of disease as
a consequence.
Blueprint for a Model System
There are considerable challenges in selecting and com-
bining different types of models, each of which plays
a particular role, for carrying capacity assessment.
A number of models are available, many of which
reviewed above, but the level of usability varies widely,
as does the capacity of one model to “speak” to another.
This comes about through the “one tool” syndrome: If
your only tool is a hammer, all your problems are nails.
One tool, i.e., one model, may be fine for a particular
level of analysis, but it will be incapable of doing all
the work.
For instance, a local-scale model such as
DEPOMOD [36], MOM [68], or FARM [4] will need
current velocity fields supplied either by field measure-
ments or by other types of models. Equally, it will not
provide a robust answer with respect to system-scale
carrying capacity. Even at the farm scale, such models
do not consider the interactions among farms,
although the siting of a new farm in a region will
inevitably affect environmental conditions and pro-
duction in existing farms. These changes will then
feed back, so that the original farm-scale assessment
will change because the model input data will change.
Figure 14 illustrates this difference for mussel cul-
ture in Killary Harbour, Ireland, by comparing two
models at differing scales [35]. EcoWin2000 (E2K) is
an ecological model applied at the system scale,
whereas FARM simulates production and environmen-
tal carrying capacity at the local scale. Both models can
be used to perform a marginal analysis [4] to determine
stocking densities that lead to optimal profitability.
This is extremely useful for licensing purposes since
farms often maximize income rather than profit (i.e.,
aim for the highest TPP). It can be seen that for
a coastal or semi-enclosed system, there appear to be
significant differences between the results for the sys-
tem-scale model, where the dotted line indicating
highest TPP (which exceeds the seeding density of
maximum profit) occurs at a density 7–8 times greater
than the present situation. FARM, however, which
deals only with the local scale, determines the end of
stage 2, i.e., highest TPP, as around X15 density. This is
because (1) E2K runs multiple production cycles, typ-
ically for periods of 10–20 years, i.e., the ecosystem
model reports what is actually harvested, whereas
FARM reports what is harvestable over one cycle, and
(2) FARM does not account for interactions among
farms, whereas E2K considers the farms in the whole
water body.
 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of 437

EcoWin2000 TPP FARM TPP

EcoWin2000 APP FARM APP
60 18
16
50

TPP (ton·ha−1·y−1)
14
40 12

APP (ratio)
Stage 3E2K
10
30 Stage 3FARM
8
20 6
4
10
2
0 0
0 5 10 15 20
Ratio of seeding (x present-day)

Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of. Figure 14
Impacts of different mussel-stocking densities on total physical product (TPP, harvestable biomass) and average
physical product (APP, the ratio between seeded and harvested biomass) as simulated by EcoWin2000 (system scale,
per unit of cultivated area) and FARM (single farm) for Killary Harbour, Ireland (Adapted from [35])

The following set of properties is desirable when
assembling a modeling system to address aquaculture
carrying capacity:
1. No single model solves all problems. Overparame-
terization and code bloat only make matters worse.
2. Models should only be as complex as the problem
requires. In other words, as simple as possible.
Increased computational power is no excuse for
unnecessary complexity.
3. Models should be able to work independently, pre-
sent value in doing so, and add further value when
working in conjunction with other models.
4. Models should define exactly what problems they
can address, as part of the overall questions for an
ecosystem, rather than the opposite.
5. Any model in the system must be able to receive
input from data or from other models and be able
to supply outputs in a form that can be easily used
by other models.
6. Different models are appropriate for different scales
in space and time. Carrying capacity assessment
may require scales as short as a tidal cycle (e.g., for
intertidal culture of clams) and as long as a decade
(e.g., for coupling ecological models with economic
models).
7. Models share a challenge with field sampling with
respect to the conversion of data (measured or
modeled) into information that is useful for man-
agers; the use of screening models, or other
approaches that help to distil data into meaningful
information, is a vital component of any system.
Figure 15 shows an example of such a multi-model
framework for investigating the role of bioextraction by
oysters, clams, and ribbed mussels as part of a nutrient
control strategy for Long Island Sound. The various
models chosen conform to the seven properties
described above.
Previous applications of this kind of framework
include the SMILE project in Northern Ireland [19] and
the SPEAR project in China ([69], Nobre et al. [11]).
A different kind of multi-model framework has
been developed by Kapetsky et al. [70], through the
application of various types of remotely sensed
data in a GIS system, incorporating variables such
as sea surface temperature, current speed, and chloro-
phyll a. In the example shown in Fig. 16, current
speeds of 0.1–1 m s–1 are combined with suitable
depth ranges for fish cages and shellfish longlines.
High current speeds are problematic for both culture
structures, due to excessive hydrodynamism, and fin-
fish production, due to higher metabolic costs and
lower yields; very low current speeds may cause
sediment organic enrichment and quality loss for cer-
tain species.
438 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of

Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of. Figure 15
Modeling framework: the REServ shellfish bioextraction project, Long Island Sound, USA (NOAA/EPA REServ Project)

Quantification of suitable areas indicates that
123 countries have at least 100 km2 within their exclu-
sive economic zone that meet these criteria.
For an annual production of 1 kg m 2 year–1, this
corresponds to 107 t year–1 of aquatic products. This
analysis has been further refined by considering dis-
tance to port, a key economic consideration for off-
shore aquaculture, which will considerably reduce this
estimate. The same applies to other factors, such as
marine protected areas. An assessment of spatial poten-
tial (a component of production carrying capacity) was
executed at a finer scale for selected areas in Canada,
Chile, Ireland, and Norway (Fig. 17). For a number of
specific locations (farms) in these four countries, the
time required for growing an adult salmon was then
determined using a dynamic model [71], forced by
remotely sensed sea surface temperature. In this case,
the model end point was a particular fish biomass,
rather than a specific cultivation period.
It is easy to see how this kind of coupling can be
leveraged, for example, by introducing the effect of
current speeds on metabolic requirements, using eco-
nomic models to analyze the trade-offs between bio-
mass and market price (lower yield, higher product
quality), and using models such as FARM to quantify
environmental effects, considering factors such as
current speed and depth of site, both of which are
readily available.
It is clear from this discussion that different model-
ing products and services are required for different
markets. Water managers or planners may for instance
be concerned with bay-scale carrying capacity, taking
into account the multi-sectorial context of aquaculture,
i.e., integrating ecological models with marine spatial
planning. Those types of models will help inform deci-
sions on the conservation of wild populations and
biodiversity and also provide support for licensing,
legislative compliance, and source apportionment for
managing pressures.
On the other hand, a farmer will probably be more
focused on optimization of production, improvement
of feed conversion ratios, and business profits.
 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of 439

Current speed in Exclusive Econonmic Zones

Current speed 10 - 100 cm/s High Seas Countries mariculture

Current speed >100 cm/s, <10 cm/s, or no data Countries no mariculture

Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of. Figure 16
World ocean areas suitable for offshore aquaculture (From [70])

Areas with Potential for Atlantic Salmon and Locations of Atlantic Salmon Farms in the Gulf of Corcovado, Chile

Temperatures, depths and currents speeds apt Temperatures apt; depths and currents Mariculture Landlocked areas
Temperatures and depths apt; no currents speed coerage speeds not apt in Econmic Zone No mariculture Outside Economic Zone/No coverage
Atlantic salmon farms

0 100 km

Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of. Figure 17
Areas in Chiloé Island, Chile, with temperature apt for Atlantic salmon mariculture (22–32 C) and depths (25–100 m) and
current speeds (10–100 cm/s) apt for submerged cages (From [70])
440 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of
But increasingly, the environmental footprint of the
farm, together with potential positive impacts, such as
the role of shellfish in reducing eutrophication symp-
toms, will be of interest. In the European Union, with
legislative requirements for Good Ecological Status by
2015 in all EU water bodies and Good Environmental
Status in all marine waters by 2020, some of the model-
ing approaches and tools described above will
undoubtedly be in demand to perform self-assessments
and to resolve conflicts.
As models of disease risks evolve, and become better
integrated with other types of models, these too will be
in demand for siting decisions.
Some of these models will be products, some will
be services, and some will be a combination of both.
The ways in which those markets develop and the
future directions in which the science and technol-
ogy may evolve will be discussed in the final section of
this entry.
Future Directions
Support for an Ecosystem Approach to Aquaculture
Application of the EAA on a worldwide scale requires
the harmonization of (1) environmental, (2) social,
and (3) multi-sectorial planning objectives [8].
These three principles and their relative weights
differ substantially across world regions, and it is not
feasible from a social and political standpoint to
establish uniform compliance with respect to limits
and thresholds.
The only solution is to define appropriate
approaches, which within particular world regions
define a gradient in relative terms, assessing EAA in
terms of the principles stated above. The three princi-
ples of EAA can be mapped onto the four pillars of
carrying capacity and illustrated as the overlap of these
[16]. The importance of each theme represented will
vary regionally and will evolve through time based on
societal cues.
It is clear that aquaculture in Europe and North
America is more closely aligned with EAA, and the
present effort in marine spatial planning will only rein-
force that. It is also clear that aquaculture production
in developed countries will grow little, and that con-
sumer demand in these regions is satisfied in large part
through imports (Fig. 18).
In the United States, 84% of aquatic products are
presently imported, of which 50% are from aquacul-
ture. This has resulted in a nine-billion-USD seafood
trade deficit [82]. Table 2 presents a summary of the
main issues that are presently considered in aquacul-
ture carrying capacity and site selection, together with
what may constitute future components for assess-
ment. Models play a key role.
Since developing nations supply the bulk of farmed
aquatic products, will continue to do so, and will prob-
ably increase their contribution, it is critical that state-
of-the-art tools are available to ensure sustainability in
the countries where they are most needed.
Which developing countries and which types of
culture should be priorities for the application of
such models? From an EAA perspective, those that
have the highest impact on the environment are the
most promising candidates. Kapetsky et al. [73] used
FAO production statistics at country environment level
(freshwater, brackish water and marine) to estimate the
intensity at which aquaculture was practised in each of
those environments. A knowledge of the species being
cultured can reveal the production systems and their
associated impacts in a very general way. The approach
outlined above can be refined to focus more closely on
modeling needs by considering the potential impacts
by species and culture systems in countries in which
production data by species are reported.
Dissemination of models can be passive (e.g.,
packages freely accessible via the Internet) or active
(training courses and workshops by region or by coun-
try). In both cases, it is essential to establish the
technical capacity, level of interest, and financial com-
mitment of the audience and the status of the Internet
as a communications and data pipeline for technical
support in each country. The focus should not be on
developing countries alone because (1) virtual technol-
ogy specialists in developed countries may be in a
position to aid dissemination and (2) companies
established in developed countries often have aquacul-
ture operations in developing countries, and could
therefore also help to bridge the gap.
Looking into the Crystal Ball
The Thematic Review “Virtual Tools for Aquaculture,”
presented at the FAO Global Aquaculture Conference
 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of 441

Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of. Figure 18
Trade flows of aquatic products into Europe [72]

Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of. Table 2 Novel management
approaches (Adapted from [10])
Topic
Feed based (cage, pond)
Shellfish farming
Integrated multi-trophic aquaculture Optimize production
Now
Site selection based on holding
capacity (cages), wastewater
minimization (ponds)
Large areas
Focus on production and social
carrying capacity NIMBY, (Not in my
backyard) NIMTO (Not in my term in
office)
Reduce negative externalities
Tomorrow
Integrated model systems, risks, welfare,
and disease. Holistic indicators
LCA: inefficiencies and eco-labeling
Mechanistic and statistical models
Data assimilation models
Economic sustainability, ecology and
economics, disease
Coupled GIS expert systems including
xenobiotics, HAB, etc.
Model uncertainties in yield
Early warning
Integrated coastal zone management
Simulate species combinations
Full economic assessment
Combine GIS, remote sensing, and
modeling
442 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of
in 2010 [10], encapsulates much of our thought on the
future of models in aquaculture. The final points in this
entry are drawn from that review.
The aquaculture industry is going to be affected by
many different issues and trends over the coming years,
often operating concurrently, sometimes in unexpected
ways, and producing changes in the industry that may
be very rapid indeed. Mathematical models will play an
important role in addressing many of these, particu-
larly in the following areas:
1. Information exchange and networking are going
to accelerate the use of virtual technology and
decision-making for problem solving to support
industry growth. Web-based access to real-time
information will further accelerate this growth.
2. Links between industry and research centers will
become more effective responding to objective-led
demand for virtual technology-driven RTD.
3. Strategic alliances will need to be reinforced or
created for the implementation of virtual technol-
ogy for aquaculture in developing countries, for
example, FAO and WorldFish Center are working
in many of the same target countries, and this could
facilitate the transfer of research outcomes on vir-
tual technology to end users. The same applies to
collaborative research with third countries medi-
ated, for example, by the European Union, the
United States of America, and Canada.
4. Many virtual technology tools will need to be more
production and management oriented. And even if
attractive and promising, these tools will have to
be adapted to local realities and conditions to
really become useful (and used) in the future.
This requires a compromise with respect to ease of
use, data requirements, and scientific complexity.
Many such tools will evolve from service to prod-
uct, requiring academic developers to accept a loss
of control in conditions of application, as a natural
trade-off (and inherent risk) of product maturity.
A number of key thematic and technical areas
where models for aquaculture are currently incipient,
and expected to develop strongly in the next decade or
so, have been identified below. In all the examples,
such models will contribute to an overall modeling
framework, by integrating and complementing existing
tools.
Disease Disease in cultivated aquatic species is
a major source of concern, yet disease modeling is still
relatively underutilized in these systems though its
worth has clearly been demonstrated in several studies.
However, while epidemiological theory predicts that
host density thresholds may be an important part of
host–parasite dynamics, clear empirical examples are
rare, as much in aquaculture as in other studied agri-
cultural systems. It is suggested that this is not evidence
that host thresholds do not exist, rather that statistical
difficulties arise with confounding factors or inade-
quate data. Evidence here is afforded from much bet-
ter-understood and more data-rich systems, for
example, human measles [95–97], where there is
compelling evidence on the effects of host density on
the likelihood of disease outbreaks taking place within
populations.
One of the major limitations in aquatic systems,
however, is that the routes by which pathogens are
transmitted and their relative contributions to an epi-
demic are often not understood. If transmission is
incorrectly specified, there can be substantial conse-
quences to estimates of NT. Additional experimental
and field data are required to identify and quantify
transmission routes for many aquatic animal patho-
gens, if prediction of the effects of host density on
disease incidence is to be made through modeling.
To facilitate this, increasing emphasis is required on
the use of real-time data acquisition combined
with models for real-time analysis and short-term pre-
diction of animal welfare. It is expected that such sys-
tems will become cheaper and more generalized, and
that some of the indicators and trends will find appli-
cation at longer timescales, albeit by means of
a probabilistic approach.
A further potential limitation of the models cur-
rently used is their inability to predict across and link
scales from the processes occurring within a tank to
the transmission across and then between sites.
Methods of investigating disease processes through
these metapopulation approaches should be investi-
gated further.
To date, only a few models have been developed to
simulate pathogenic infections of shellfish with respect
to physiology, for example, Powell et al. (1996) for the
American oyster C. virginica, but with widespread con-
cerns about relaying, susceptibility, and mortality,
 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of 443

models focusing on a more mechanistic approach will

undoubtedly appear over the next decade.

Harmful Algal Blooms This is another area where

little predictive capacity exists, except in the short term
through the use of operational oceanography, relying
on bloom identification and tracking. Management is
at present reactive, and modeling of appearance and
development of such blooms is in its infancy, due to the
lack of an appropriate paradigm. Sensors such as
targeted RNA probes (e.g., [74]), integrating handheld
devices, or potentially deployed in situ and used in
a networked framework will both help in early detec-
tion and management and contribute to the under-
standing of the underlying triggers. Considerable
developments are also expected in remote sensing algo-
rithms able to discriminate (at least) between HAB and
nontoxic blooms (S. Bernard, personal communica- Carrying Capacity for Aquaculture, Modeling
tion, 2010). Frameworks for Determination of. Figure 19
QR code for farmed shellfish, readable on any smart phone

Certification and Traceability The arrays of sensors

that can be deployed at the farm scale to enable coupled
monitoring and modeling are important for both
product certification and traceability. The number,
reliability, and accuracy of underwater sensors will
increase, and the cost will decrease, both with techno-
logical developments and market growth.
Real-time data acquisition and interpretation will
make it possible for consumers to visualize the whole
“cradle to grave” cycle of an aquaculture product. For
instance, a batch of oysters may be “bar coded” to
reveal the origin of seed and the entire environmental
interaction over the culture period, including metadata
and measured data on water quality, HAB events, con-
dition (meat ratio) of the animals, and impact on their
environment, for example, in terms of reduction of
eutrophication symptoms through the indirect
removal of nitrogen and phosphorus, or conversely
the addition of particulate organic material due to
biodeposition. Such sensors will typically be queried
at a sub-hourly frequency, particularly if they are also
used for welfare monitoring; this will easily allow
importers, health inspectors, or consumers to perform
verification and certification, and will provide an
important contribution to both food safety and envi-
ronmental awareness.
For the farmer, the existence of this kind of inte-
grated coding will also help improve various aspects of
culture practice and increase attractiveness of the busi-
ness model to the key sector of insurance. For the
consumer, such data will need to be presented in
a comprehensible format, for example, in the form of
a few indicators (Fig. 19).
Modeling with Data Scarcity Good data are required
to support acceptable model predictions. The acquisi-
tion of high-quality data, with appropriate spatial and
temporal resolution, is expensive and often beyond
the scope of developing countries, except on a fairly
limited scale. This, together with an often fragmented
approach to the study of interacting ecosystems, in
many cases driven by institutional barriers, presents
a challenge to the application of models.
Improved mechanisms for data access, particularly
for remotely sensed data, together with models that
deal with uncertainty and risk, will contribute to con-
version of sparse data into more meaningful informa-
tion – although such an approach may be considered
unsuitable in parts of the developed world, in many
countries it will be a much better basis for decisions
444 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of

than the options that are presently used. In addition, it
will promote a “virtuous cycle” toward more informed
decision support and promote the use of better data
and more sophisticated models, as the data become
available to drive them.
A wide variety of models will benefit from this
development because many models developed and
tested for temperate systems need adjustment with
respect to parameters, thresholds, and equations,
when applied to tropical and subtropical systems.
Information Technology The last 5 years have seen
a huge leap in various areas of distributed computing,
all of which are expected to develop significantly in the
coming years.
Three examples are presented here:
1. Web 2.0 now provides a large diversity of commu-
nity- and corporation-based resources. Currently,
over 7,000 items exist for aquaculture on YouTube –
at the time of presentation of the VITAL Thematic
Review [10] there were 1,800. For aquaculture
modeling, that number has also quadrupled since
2010 and now stands at 75.
2. There is a strong trend toward the development and
use of Software as a Service (SAAS), deployed on the
web and competing with traditional desktop appli-
cations. This is incipient in the aquaculture world
but can be seen, for example, in the WinShell
application (http://longline.co.uk/winshell), which
allows users to simulate individual shellfish growth
on line. Central to the development of this kind of
application are Rich Internet Applications (RIA),
which provide a full user experience and are an area
of rapid growth [75]
3. Mobile computing is increasingly ubiquitous, and it
is now possible to use models on many handheld
devices, as illustrated in Fig. 20, which shows a tide
prediction model for Mumbai, India. This kind of
model might be used to help predict the yield in
intertidal culture of bivalve shellfish.
The trend toward the increasing use of such devices,
including for various real-time applications in aqua-
culture management, will increase. In parallel, the
stand-alone server is rapidly being replaced by cloud
computing, which will tend to make the circulation of
data both easier and cheaper. Both elements will con-
tribute to bridge the information divide between richer
and poorer nations.
Computer access, literacy, and internet connectivity
are significant barriers to entry for the population in
the rural areas in developing countries, where aquacul-
ture takes place. Over the coming decade, many aqua-
culture farmers will have their first contact with
the World Wide Web by means of smart phones.

Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of. Figure 20
Tidal prediction for the port of Mumbai using the Oceanus21 smart phone app (http://longline.co.uk/oceanus21). Ports
can be automatically selected based on GPS coordinates and phone location
 Carrying Capacity for Aquaculture, Modeling Frameworks for Determination of
Cell phones are ubiquitous even in remote areas and are
much better adapted to local language and alphabet,
which constitute other barriers in accessing technology.
The future of aquaculture is promising. It needs to
be, given the world population growth expected in the
next decades. Simulation models, particularly when
used within appropriate frameworks, show enormous
potential to inform and guide the future development
of aquaculture, toward a world which is more socially
responsible, more equitable, and more sustainable.
Acknowledgments
We are grateful to the EU FP7 COEXIST project, and to
FAO, for the opportunity to develop many of the ideas
presented here. We would like to thank B. Costa-Pierce
for the opportunity to contribute to this volume, all
our coauthors in the Virtual Tools for Aquaculture FAO
report presented at the FAO Global Aquaculture
Conference 2010, E.S. Braga and S. Eschrique for data
on the Jaguaribe estuary, Ceará, Brazil, and S.B. Bricker
and L. Ramos for comments on the draft. We thank
H. Mildmay-White for design work on figures.
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Carrying Capacity for Sustainable
Bivalve Aquaculture
CHRISTOPHER W. MCKINDSEY
Ocean and Environmental Sciences Division, Fisheries
and Oceans Canada, Institut Maurice-Lamontagne
Fisheries and Oceans Canada, Mont-Joli, Québec,
Canada
Article Outline
Glossary
Definition of the Subject
Introduction
Functional Categories of “Carrying Capacity”
Decision Framework to Determine Carrying Capacity
Knowledge Gaps and Future Directions
Bibliography
Glossary
Benthic Pertaining to the sea floor.
Carrying capacity The intensity of a practice that
a given environment can sustain indefinitely given
the availability of various necessities in that envi-
ronment and the various pressures on them.
Ecosystem approach to aquaculture (EAA) A strategy
to integrate aquaculture into context of the wider
ecosystem such that it promotes sustainable devel-
opment, equity, and resilience of interlinked social
and ecological systems.
Ecosystem-based management (EBM) A process that
highlights the need to use the best available knowl-
edge about the marine ecosystem to manage marine
resources, with an emphasis on maintaining eco-
system service functions.
Integrated coastal zone management (ICZM) A pro-
cess for managing coastal zones that uses an inte-
grated approach and considers all aspects of the
coastal zones, including biological, geographical,
and political boundaries, to achieve sustainability.
Model A simplified description, conceptual or mathe-
matical, of a system or process, to assist in calcula-
tions, predictions, and understanding.
Pelagic Pertaining to the water column.
P. Christou et al. (eds.), Sustainable Food Production, DOI 10.1007/978-1-4614-5797-8,
# Springer Science+Business Media New York 2013
Originally published in
Robert A. Meyers (ed.) Encyclopedia of Sustainability Science and Technology,
Carrying Capacity for Sustainable Bivalve Aquaculture 449
Definition of the Subject
Bivalve aquaculture is one of the fastest growing sec-
tors of the food industry, raising concerns about the
influence of the activity on the environment. This is
true at two levels: First, farmers must make sure that
the bivalves that they raise do not deplete resources in
a given area to such an extent that bivalve production
is decreased. Second, society in general wishes that
such activities have an acceptable impact on the envi-
ronment and are sustainable. There is also intense
competition for space and its use in many coastal
zones, making siting of many farms contentious.
Thus, many organizations have stressed the impor-
tance of determining the carrying capacity of different
areas for bivalve culture. There are a number of ways
that “carrying capacity” may be defined, including
physical, production, ecological, and social, and the
first three categories are to lesser or greater degrees
related to social expectations and standards.
A number of methods have been developed to calcu-
late these different categories of carrying capacity for
bivalve culture. This entry outlines some advances to
estimate the different categories of carrying capacity
and suggests a framework that may be followed to
encourage the development of a sustainable bivalve
aquaculture industry. An emphasis is placed on
the latter two categories as these are the ones for
which knowledge is the most lacking, are arguably the
most complex, and for which advances are the most
pressing.
Introduction
Aquaculture is the fastest growing sector of the food
industry. Since the 1970s, production in the sector has
increased at a rate of about 7% per year and by 2008
accounted for 43% of the total annual fisheries produc-
tion of 160 million tons and projections by the UN [1]
suggest that this production will increase greatly in the
future. This increase in production has raised concerns
about the impacts of the activity on local environ-
ments, (e.g., [2]) and much work has been focused on
understanding the interactions between aquaculture
and the ecosystem, (e.g., [3, 4]). Although concerns
were initially largely directed at the influence of finfish
cage culture on the environment (e.g., nutrient
# 2012, DOI 10.1007/978-1-4419-0851-3
450 Carrying Capacity for Sustainable Bivalve Aquaculture
loading, disease issues, and interactions due to
escapes), concerns have also been raised about the
influence of farmed bivalves.
As for aquaculture in general, production of farmed
bivalves has increased greatly over the past few years
and as of 2008, 11.7 million tons of the total 13.5 mil-
lion tons of worldwide bivalve captures was from aqua-
culture production. This includes the culture of over
4 million tons each of clams and oysters and about
1.5 million tons each of scallops and mussels. The
greatest concerns relating to farmed bivalves include
enhanced localized biodeposition [5], food depletion
in the water column due to bivalve grazing [6], alter-
ation of nutrient and oxygen fluxes [7], and the transfer
of disease and hitchhiking species [8]. Recent reviews
on the environmental interactions associated with
bivalve culture have been done for oysters [9], clams
[10], and mussels [11].
Increasingly, regulators and other groups are
looking to science to determine whether sites have
reached their carrying capacity for bivalve culture.
Moreover, a number of standards – performance stan-
dards, best management practices, certification stan-
dards, etc. – have been or are being developed to ensure
that bivalve production is being done in a manner
that is consistent with a global environmental ethic
[12–14]. Some of these codes have been developed
by industry groups and are voluntary in nature, some
have been developed and are used by public authorities
and regulatory agencies, and others are being devel-
oped by a variety of organizations (e.g., buyers,
nongovernmental organizations, marketing groups)
as a means of informing consumers about a product
with the goal of influencing farm practices through
consumer choice and market forces [15]. In all cases,
the idea is that such criteria will encourage the devel-
opment of a sustainable bivalve aquaculture industry
such that the carrying capacity of a given area has not
been exceeded and impacts are minimized. Recently,
the National Research Council [15] suggested that per-
formance standards based on the carrying capacity of
sites be developed and implemented at the ecosystem
level. Similarly, the FAO [16] has suggested that an
Ecosystem Approach to Aquaculture (EAA) be
followed to ensure the “integration of the activity
within the wider ecosystem in such a way that it pro-
motes sustainable development, equity, and resilience
of interlinked social and ecological systems.”
Many advances have been made in the estimation of
carrying capacity for bivalve culture over the past few
years. The broad aim of this entry is thus to update an
earlier paper on the subject [17] and to discuss
a number of ideas that have been developed in the
interim. It focuses on the outgrowing stage of bivalve
culture as this stage is arguably the most important in
terms of its interactions with the environment.
Many other activities related to bivalve culture exist
(see list below) and these are discussed by McKindsey
et al. [17].
Abbreviated selection of activities related to bivalve
culture that may influence the ecological carrying
capacity of coastal areas. (Modified from McKindsey
et al. [17].)
1. Seed collection
(a) Dredging
(i) Disturbance of benthic communities,
especially the removal of long-living
species
(ii) Removal of juveniles from wild
populations of target species
(iii) Collection of non-target species
(iv) Suspension of sediments
(v) Depletion of food resources for other
species
(vi) Release of H2S and reduction of dissolved
oxygen in the water due to oxygen-
consuming substances, release of
nutrients
(b) Artificial collectors
(i) Removal of juveniles from wild popula-
tion of target species
(ii) Increasing target and non-target species
recruitment success
(iii) Alteration of the hydrodynamic regimes
(iv) Acting as FAD
(v) Risk of entanglement for large vertebrates
(e.g., marine mammals, sea birds, turtles,
sharks)
(vi) Foci for nuisance species
(c) Hatcheries
(i) Chemical pollution (e.g., pharmaceuticals)
(ii) Genetic selection
(iii) Spread of diseases

(d) Importation
(i) Introduction of alien species
(ii) Genetic pollution
(iii) Spread of diseases
2. Ongrowing
(a) Effects common to all techniques
(i) Organic enrichment of seafloor
(ii) Providing reef-like structures
(iii) Alteration of hydrodynamic regime (cur-
rent speed, turbulence)
(iv) Food web effects: competition with other
filter feeders, increasing recycling speed
of nutrients, removal of eggs and larvae
of fish and benthic organisms
(v) Spawning: release of mussel larvae
(vi) Providing food for predators of bivalves
(vii) Control of predators and pests
(b) Bottom culture
(i) Activities to prepare the culture plots,
e.g., dredging for predator removal
(ii) Placement of protective structures (net-
ting, pipes)
(iii) Removal of associated organisms by
dredging and relaying
(iv) Competition for space with wild benthos
organisms
(c) Artificial structures for suspended and off-
bottom culture (trestles, poles, rafts, longlines)
(i) Acting as artificial reef or FAD
(attraction/displacement or enhancement
of animals)
(ii) Risk of entanglement for large vertebrates
(e.g., marine mammals, sea birds, turtles,
sharks)
(iii) Foci for nuisance species
3. Harvesting
(a) Effects common to all techniques
(i) Removal of biomass, nutrients
(ii) Removal of filtration capacity
(iii) Removal of non-target species
(iv) Competition with predators
(b) Dredging
(i) Disturbance of benthic communities,
especially removal of long-living species
(ii) Suspension of sediments
(iii) Release of H2S and decrease of diss-
olved oxygen in the water due to
Carrying Capacity for Sustainable Bivalve Aquaculture 451
oxygen-consuming substances, release of
nutrients
(c) Collection of off-bottom structures
4. Processing
(a) Dumping of by-catch
(b) Relaying near auction houses
(c) Depurating
(d) Dumping of shells
(e) Effluents from processing plant
(f) Spread of alien species or diseases
As a first step, it is important to understand what is
meant by “carrying capacity.” A number of definitions
have been suggested that consider a variety of physical,
biological, and social criteria. In this entry, the four
functional categories of “carrying capacity” as outlined
by Inglis et al. [18] and McKindsey et al. [17] are used:
1. Physical carrying capacity – the total area of marine
farms that can be accommodated in the available
physical space.
2. Production carrying capacity – the stocking density
of bivalves at which harvests are maximized.
3. Ecological carrying capacity – the stocking or farm
density above which unacceptable ecological
impacts become apparent.
4. Social carrying capacity – the level of farm develop-
ment above which unacceptable social impacts are
manifested.
The specific aims of this entry are to (1) provide an
overview and update of different categories of carrying
capacity, (2) give a more in-depth review of factors
that could be considered for the determination of eco-
logical and social carrying capacity as these categories
are the least developed conceptually and in terms of
modeling, (3) outline a decision framework for incor-
porating all four categories of bivalve carrying capacity
into the determination of the overall carrying capacity
of a given area for bivalve culture, and (4) outline
research to address knowledge gaps for carrying capac-
ity studies.
Functional Categories of “Carrying Capacity”
Physical Carrying Capacity
The physical carrying capacity of a site (embayment,
inlet, offshore area, etc.) is simply the geographic area
452 Carrying Capacity for Sustainable Bivalve Aquaculture
in which conditions are suitable for the production of
a given species using a given method. It is a function of
the overlap of the requirements of the species being
harvested and the physical resources (e.g., depth, sub-
strate type, salinity, hydrodynamics) in the site. The
physical carrying capacity of a site may differ greatly
between species and culture methods being employed.
For example, an embayment may have an extensive
area that is suitable for on-bottom clam culture but
relatively little area that is appropriate for suspended
oyster culture. Likewise, modification of an area (e.g.,
by the addition of a species-specific appropriate sub-
strate) may extend the physical carrying capacity of
an area.
Traditionally, the physical carrying capacity of sites
was determined by building knowledge of an area using
hydrographic charts as the base layer, adding other
appropriate layers (e.g., temperature and salinity)
as available, and analyzing the layers using formal
or ad hoc Geographic Information Systems (GIS),
(e.g., [19, 20]). With the expansion of culture sites in
more remote regions where such information may not
be readily available, the use of remote sensing methods
to estimate various physical parameters (e.g., depth,
salinity, temperature, as well as chlorophyll levels and
related biological parameters that are needed to esti-
mate production carrying capacity – see further) may
be used in combination with GIS to facilitate site selec-
tion [21]. Although more of a social factor (see fur-
ther), an additional advantage of using GIS for
aquaculture site selection is that it can be used within
a coastal management framework to include other
activities so as to avoid user conflicts [22, 23].
Production Carrying Capacity
The production carrying capacity of a site is the stock-
ing density at which harvests are maximized. Given
that trade-offs between bivalve growth rates, market
tastes, and economic returns, etc., would likely
encourage selection of a specific bivalve size and type
(see further), production carrying capacity is often
not necessarily the greatest biomass. This category of
carrying capacity is what most people think about
when they consider “carrying capacity” and is the
best studied. The production carrying capacity of
a given site is strongly related to hydrodynamic and
food regimes and its physical carrying capacity.
Indeed, various “habitat suitability models” have
been developed that combine both categories of data
to predict the best areas for bivalve production, (e.g.,
[24]). Models to predict production carrying capacity
have three main components: (1) a hydrodynamic
model that transports food, nutrients, and other
wastes; (2) a biogeochemical component that describes
processes that influence food production and con-
sumption; and (3) a physiological component that
determines the rate of food consumption and growth
of the farmed bivalves [25].
Bivalve culture systems are hierarchical, with indi-
vidual bivalves and their associated fouling organisms
nested within culture units (socks, cages and stacks of
cages, pearl nets and strings of pearl nets, etc.), these
being nested within culture gear (longlines or rafts),
which are nested within farms, and so on [26–28]. An
understanding of processes operating at each scale as
well as the relations between these scales is needed to
predict hydrodynamics and how this influences bio-
geochemical and physiological processes within culture
systems and to understand cascading effects on the
greater ecosystem.
In its simplest form, the production carrying capac-
ity of a given location is a function of the available food
resources and the rate at which they are renewed via
in situ production and/or flushing relative to their rate
of removal (filtration) by the farmed bivalves. This type
of approach has been developed for mussel culture in
eastern Canada [29]. However, such models commonly
under- or overestimate flushing and a number of
authors have suggested that their use be avoided for
production carrying capacity studies, (e.g., [30]).
Despite this, a number of groups have advocated cal-
culating the ratio of clearance time by farmed bivalves
to flushing rate or water residence times (as calculated
by such methods) to evaluate the sustainability of sites
for bivalve culture [31] and as a rough first step in
evaluating whether carrying capacity is potentially
exceeded for ecological certification standards [32]. In
the latter case, if the ratio between clearance and
renewal times is less than unity, then further analyses
based on primary productivity are required.
More encompassing approaches to calculating pro-
duction carrying capacity based on hydrodynamic and
mathematical models that include spatially and

+ hydrodynamics
+ temperature, salinity, etc.
Excretion
Dissolved N / P
Dissolved Si Flagellates
Detrital Si
Detrital N / P
Benthic plants
Oxic sediments
NO3− NO2−
Nitrification
Si(OH)4/ PO4
NO3− NO2−
Denitrification
Carrying Capacity for Sustainable Bivalve Aquaculture. Figure 1
Conceptual diagram of bivalve aquaculture interactions in coastal ecosystems considered in production carrying
capacity estimations. Only main pathways are indicated
temporally explicit feedbacks between the farmed
bivalves, phytoplankton, zooplankton, detritus, and
physical (e.g., temperature, nutrients, see Fig. 1)
parameters are becoming the standard. Models vary
greatly in complexity. Although a simple box model
for a location may be sufficient for some situations, it is
likely unsuitable for embayments with some degree of
environmental heterogeneity [33]. The next level of
complexity is a simple 1-D hydrodynamic transport
model, (e.g., [34]), which may be expanded upon to
include vertical transport for suspended culture, (e.g.,
[35]). More complex models include multiple coupled
boxes within an area driven by a 2-D hydrodynamic
model (with tidal and meteorological forcings). The
most complex models, fully refined 3-D finite-element
hydrodynamic models, generally provide a much more
accurate estimation of hydrodynamics and may be used
to drive the biological models. Historically, computa-
tional power has limited the broad application of com-
plex models [36] but this is no longer the case as
powerful computers are now readily available [25].
Carrying Capacity for Sustainable Bivalve Aquaculture 453
Harvest N2
Water column
Diatoms
Zooplankton / filterers
(benthic-pelagic)
Farmed bivalves
(benthic-pelagic)
Biodeposition / mortality
NH4+ Detrital N / P / Si Refractory
material
N2
Anoxic sediments
However, increased development time and sampling
requirements to validate more complex models make
the use of simpler box models a good approximation
for understanding the hydrodynamics of an area and
slow ecological processes [37] and for carrying capacity
modeling [38].
Production carrying capacity models require data
on biogeochemical components which include a large
number of variables related to the flux of nutrients and
primary production and interactions between different
food sources for the farmed bivalves. This includes
nutrient fluxes due to excretion by the farmed bivalves
and associated organisms and organic matter in
trapped sediments in culture structures [39–41], nutri-
ent uptake by and production of phytoplankton
[42–44], zooplankton and detritus components, and
a benthic component that includes biological and
chemical processes [45–47]. This latter submodel is of
particular importance in shallow areas [33]. Figure 1
gives an example of the types of processes that are
included within such models.
454 Carrying Capacity for Sustainable Bivalve Aquaculture
Bivalves graze on and may impact plankton and
other suspended components in the water column
[48]. These components and environmental factors
(e.g., temperature) in turn greatly influence the growth
of the farmed bivalves. Models to predict the physio-
logical response and growth of bivalves vary greatly in
their complexity. Models may be divided into statistical
and more mechanistic approaches that include bioen-
ergetics [49, 50]. Today, many studies use an approach
based on dynamic energy budget (DEB) models that
were developed by Kooijman, (e.g., [51–53]). In these
models, energy budgets are partitioned into core pro-
cesses: structural volume, reserves, and a reproductive
buffer, and forced by food and physical parameters.
Examples include work on a variety of bivalves, (e.g.,
[25, 54–56]) and have been incorporated into general
models for bivalve production carrying capacity [35,
57]. A generalized set of DEB models for a few of the
most commonly farmed bivalve species has been
assembled and is available (http://www.shellsim.com).
Production carrying capacity models based on this
latter suite of components have been successfully used
in a number of studies, (e.g., [35]). Although these
types of models are attractive in that they include
logical mechanistic processes, their complexity makes
them difficult to apply at times and requires substantial
ground truthing and simpler models will often yield
acceptable results.
Ultimately, the different sub-models must be put
together to estimate production carrying capacity. An
important advance in modeling production carrying
capacity is the development of Graphical User Interface
(GUI) modeling environments that allow users to link
different sub-models fairly easily through a visual envi-
ronment rather than with text commands. An example
of this is the use of object-oriented modeling environ-
ment, as promoted for the FARM model developed by
Ferreira et al. [35, 57]. This model links together
various components forced by a 1-D hydrodynamic
model, or 1-D with horizontal mixing for off-bottom
bivalve culture, to estimate production carrying capac-
ity and eutrophication assessment (see further) at the
farm-scale based on a limited number of parameters. It
may also be used to guide the selection of growing sites,
culture layouts, production densities, farmed species,
and to maximize production or economic returns. The
model has been shown to be useful in a number of
locations and for a number of species [57] and is
available as a client–server application (www.
farmscale.org; www.longline.co.uk/winshell). Simile
[58] is an object-based GUI modeling environment
that has been used extensively by Grant and colleagues
[25, 33, 38, 54, 59]. This environment is well suited to
constructing carrying capacity models for bivalve cul-
ture because of its inherent ability to represent spatial
elements and specify hydrodynamic connections
between them and, because of its GUI environment, it
is transferable to nonexperts [38].
Models for production carrying capacity usually
consider only the farmed bivalves as consumers of
plankton. However, in some situations, particularly
shallow sites with large densities of natural bivalves,
grazing by natural bivalves may also exert a large pres-
sure on plankton communities [60–62]. Thus Sequeira
et al. [63] have recently developed a model that
includes natural benthic communities as a forcing
function on plankton communities and suggest that
these populations may significantly reduce production
carrying capacity. Similarly, Cugier et al. [64]
found that wild native and exotic filter-feeding organ-
ism had a greater effect on the control of primary
productivity than did the farmed bivalves in Mont
Saint Michel Bay, France (Fig. 2). The biomass of
filter-feeding fouling organisms, particularly tuni-
cates, on farmed bivalves and infrastructure may be
considerable, at times greater than that of the farmed
bivalves [65]. Given that they may have similar
grazing rates to that of farmed bivalves [66], these
should be included within production carrying
capacity models where fouling is great. This has been
attempted for the Thau Lagoon [67] and the
Oosterschelde Estuary [68].
Ecological Carrying Capacity
The definition the “ecological carrying capacity” of
a system is greatly dependent on social values as what
is considered to be an “unacceptable” impact is depen-
dent on the values of a given society. A society or
their representatives must select which components
(e.g., species or habitats) of a given area are important
and set acceptable limits of change for each. Often,
information on specific parameters may not be avail-
able and managers may choose to consider components
 Carrying Capacity for Sustainable Bivalve Aquaculture 455

20.0 100
15.6 92
12.1 83
9.4 75
7.2 66
5.5 58
4.2 49
3.1 41
2.2 32
1.6 24
1.0 15
0.6 7
0.3 −2
a 0.0 b −10

150 100
138 92
125 83
113 75
101 66
88 58
76 49
64 41
52 32
39 24
27 15
15 7
2 −2

c −10 d −10

280 400
258 368
235 337
213 305
191 274
168 242
146 211
124 179
102 148
79 116
57 85
35 53
12 22
e −10 f −10

Carrying Capacity for Sustainable Bivalve Aquaculture. Figure 2

Simulated annual maximum chlorophyll a for the reference case (A – current situation with all filter feeders included) and
differences with the reference case (in%) for five scenarios of with different filter feeders removed, (B – without exotic
slipper-limpets, C – without farmed mussels, D – without farmed oysters, E – without native filter feeders, F – with no filter
feeders in the bay). Because of the wide range of differences between the scenarios, the scale bar is not the same for each
sub-figure (From Cugier et al. [64])
456 Carrying Capacity for Sustainable Bivalve Aquaculture
for which information is available or more easily
obtained. As pointed out by McKindsey et al. [17],
this may be a logical choice given that components
that have a high societal value are also likely to have
been studied.
While production carrying capacity focuses on the
farmed bivalves themselves and the organisms that sup-
port their production, ecological carrying capacity also
includes other organisms and habitats in the ecosystem.
There are three main categories of ecological carrying
capacity: (1) that related to the pelagic habitat; (2) that
related to the benthic habitat; and (3) that which
employs ecosystem function approaches. The first cate-
gory is largely related to plankton depletion due to graz-
ing by farmed bivalves. The second category is related to
increased sedimentation within culture sites due to
biodeposition by the farmed bivalves. The third category
considers both these issues and changes in biomass and
energy flow in a system. Ecological carrying capacity is
strongly related to both the physical carrying capacity
and the production carrying capacity of a site, particu-
larly with respect to that related to the pelagic habitat.
Models to estimate the production carrying capac-
ity of a site contribute to determining the ecological
carrying capacity of a site for organisms that are depen-
dent on the delivery of food from the water column.
Presumably, if the farmed filter-feeding bivalves impact
themselves by overgrazing the available resources, then
they are also likely impacting other organisms that feed
in a similar manner [69]. Indeed, some recent produc-
tion carrying capacity models explicitly included ben-
thic filter-feeding organisms [63, 64] and some work
has suggested that overgrazing by farmed bivalves may
impact other suspension feeders in the surrounding
ecosystem [70]. The calculation of carrying capacity
for other planktivores in the water column may be
similarly evaluated. Sedimentation rates to the bottom
are a function of the plankton communities in an area
(e.g., [71]). Thus, as has been shown for natural sys-
tems with bivalves [72, 73], depletion of the water
column via grazing by farmed bivalves may influence
sedimentation rates within a given area and thereby
benthic communities, potentially enhancing differ-
ences between benthic communities within and outside
of culture sites [11].
With respect to the benthic habitat, research has
largely focused on the impact of increased
sedimentation due to biodeposit production by
farmed bivalves on infaunal communities [9–11].
Research on the ecological carrying capacity of ben-
thic habitats has likewise been focused on predicting
how increased sedimentation due to biodeposit pro-
duction by farmed bivalves influences infaunal com-
munities. Grant et al. [74] predicted benthic loading
rates from biodeposits from farmed mussels (Mytilus
edulis) at a bay scale as a balance between biodeposit
production, as calculated using a simple physiological
model, and flushing, as calculated based on a simple
tidal prism model (validated using a finite-element
2-D hydrodynamic model and field work). Although
there was some variation between modeled and
observed biodeposition for both studied bays, the
authors suggest that the approach is valuable as
a screening tool to develop relative ranking of different
systems and identify potential issues. A number of
approaches have also been evaluated that couple spa-
tially explicit hydrodynamic-dependent particle
tracking models to predict flux of biodeposits from
farmed bivalves to the bottom to predict benthic load-
ing footprints and associated benthic community
changes. A simple approach is to modify the existing
DEPOMOD model, which was developed to this end
for finfish cage culture but for which individual bivalve
culture structures (e.g., mussel longlines, see Fig. 3) or
groups of structures in a system may be modeled [75].
However, this approach has a number of limitations.
First, the model assumes a homogenous flow field, an
assumption that is unlikely to be true for extensive
culture sites. Second, the module for resuspension
of sedimented biodeposits is not fully developed
and remains one of the greatest sources of uncertainty
for biodeposition/impact models [74]. Although
resuspension may be negligible in areas with weak
currents [75], Giles [76] used a more advanced hydro-
dynamic model to drive a biodeposit dispersion model
and stressed the importance of this aspect for
predicting the dispersal of biodeposits in areas with
strong currents. A number of recent studies have
started to address this issue and supply data to better
parameterize this module, (e.g., [75, 77, 78]). Although
general relationships exist for benthic organic loading
and infaunal community structure [79], predictive
dose-dependent relationships between organic loading
from bivalve biodeposits and faunal responses are
 Carrying Capacity for Sustainable Bivalve Aquaculture 457

Mussels only Tenore et al. [82] used a mass-balance approach to

100
examine the influence of bivalve culture as a part of
1 a coastal ecosystem but specifically to predict ecologi-
2
75
cal carrying capacity. Gibbs [83] used food web analysis
Northing (m)

g m−2d−1
to estimate the level of bivalve culture that could
3

4
50
develop before it dominated the energy flow in
16

3
a marine system and impacted fisheries resources.

2
25 14
4

5 More recent work has used Ecopath with Ecosim [84]

12 to determine the trophic functioning of various areas
0
a 0 25 50 75 100 125 150 that include bivalve culture [85–88]. These models
10
differ considerably in their complexity (i.e., number
Mussels and Ciona intestinals 8 of trophic groups included) and have generally found
100 6 that bivalve culture promotes short energy pathways
with high trophic efficiency and energy cycling. How-
2 4
75 10 12 86 4 ever, the aim of these studies was not specifically to
Northing (m)

2 determine the ecological carrying capacities of the

50 studied areas. Research [69] to evaluate the carrying
0
capacity of an area for mussel culture in New Zealand
25 using Ecopath found that the ecological carrying
4

6 8
0 that would not significantly change the major
0 25 50 75 100 125
b Easting (m)
Carrying Capacity for Sustainable Bivalve Aquaculture.
Figure 3
Modeled biodeposition footprint (g m 2 d 1) within an
idealized suspended mussel farm with (a) mussels only and
(b) mussels fouled with Ciona intestinalis. Biodeposition
rates were modeled for five backlines (white lines in figure)
measuring 100 m each based on currents measured during
a 24 h period in St. Marys Bay, eastern Canada (Modified
from McKindsey et al. [65])
largely lacking, making predictions difficult [80]. Sim-
ilarly, there is no universal metric available to describe
benthic responses although a large number of indices
have been developed, some showing good relationships
with aquaculture [81]. As noted above, the biomass of
tunicates and other fouling organisms on farmed
bivalves and infrastructure may be considerable, and
these likely contribute greatly to organic loading to the
sea bottom in some cases [65, see Fig. 3] and thus must
be considered when determining the biodeposition-
related ecological carrying capacity of an area for the
benthic habitat.
capacity of the area (defined as the level of culture
150
energy fluxes or structure of the food web) was much
less than the production carrying capacity (defined as
the level of production at which the ecosystem collapses
down to a nutrient–phytoplankton–culture–detritus-
dominated system) of the area. More recent research
in the eastern United States based on the same
approach but for coastal lagoons with oysters also
found that the stocking density calculated for ecologi-
cal carrying capacity was less than that calculated for
production carrying capacity [89]. Although the orig-
inal version of Ecopath was limited in its applicability
because it could not be used to simulate changes to
flows with time, the new version does not assume
a steady state. Rather, it bases the parameterization on
an assumption of mass balance over an arbitrary
period, often a whole year, and biomasses of any
given trophic group need not be at equilibrium.
A component of Ecopath, Ecosim, accepts time series
data for different trophic groups and may be appropri-
ate to evaluate different management (i.e., stocking and
harvesting) options. These approaches have some lim-
itations. Perhaps the most important for bivalve cul-
ture is that the models are not spatially explicit. Thus,
the model may not be used to identify near-field and
far-field effects and exchanges between areas are
assumed to be instantaneous. This is not logical in
458 Carrying Capacity for Sustainable Bivalve Aquaculture

complex coastal areas where most bivalve culture is Social Carrying Capacity
done. This issue has been addressed to some extent
The social carrying capacity of a site is the most com-
with the development of Ecospace, a dynamic, spatial
plex of all types of carrying capacity to determine. It
version of Ecopath that incorporates the key elements
depends on not only the three above categories of
of Ecosim [84]. This review found no studies that use
carrying capacity but also on trade-offs between stake-
either Ecosim or Ecospace to estimate ecological carry-
holders to meet the demands of both the population
ing capacity for bivalve culture; information to do this
(socioeconomic factors such as traditional fisheries,
is usually not available and when such spatially explicit
employment in other sectors, and recreational use)
temporal data are not available, Ecopath provides
and the environment (protected habitats, species,
a standardized methodology for developing a model
etc.) [22, 31, 83, 93–95]. Moreover, social issues often
[89]. As it is, data on many specific biological variables
inform other categories of carrying capacity (see fur-
(life history values, interactions, etc.) are lacking
ther in “Decision Framework” section). This category
for most systems. If default values are used, these
is at the heart of Marine Ecosystem-Based Management
must not be simply accepted without critical evaluation
(EBM) and Integrated Coastal Zone Management
[90]. Even when data is available to guide the determi-
(ICZM) and must be fully developed so that responsi-
nation of variable values, many must be adjusted to
ble management decisions may be made [96]. That
make the model balance [69]. Perhaps most impor-
being said, the techniques for determining the social
tantly, Ecopath uses a largely top-down mass-balance
carrying capacity of a site are the least developed of all
approach and thus poorly represents bottom-up effects
carrying capacity categories [97].
[91], a situation that will be problematic for bivalve
Despite these limitations, a number of criteria
culture given that it largely impacts nutrients and lower
are common to each of the different methods: represen-
trophic levels.
tativeness, independence, and involvement/buy-in of the
In sum, most potential measures of ecological car-
groups involved in the process, and transparency [98].
rying capacity consider only a single or a restrained
One method that has been developed to manage
number of ecosystem components [92]. Little research
natural resources is the concept of “limits of acceptable
has been directed at understanding the impact of
change,” or LAC. Although originally developed to
biodeposition (of biodeposits and of farmed bivalves
define the limit of recreational activities in terrestrial
and associated organisms) on the productivity or
wilderness areas, it may also be applicable to a wider
sustainability of benthic infaunal communities or
range of natural resource management issues [99, 100].
the communities of larger invertebrates and fishes
More recently, it has been applied to bivalve aquacul-
that may prefer to associate with culture sites to
ture in New Zealand [101]. It was recognized that LAC
profit from the biodeposition. Likewise, little research
was not a tool to determine the level of bivalve culture
has addressed the impact of the modification/addition
that was ecologically sustainable. Rather, it provided an
of physical structure associated with bivalve culture
adaptive management framework to prevent signifi-
(i.e., both the infrastructure and the farmed product,
cant negative effects due to the activity. Through
as well as removal of seagrass, etc.) [11]. The addition
a collaborative process, LAC provides a framework to
of structure may attract and create suitable habitat
identify indicators of change, setting levels of changes
for a large variety of organisms, such as fish and deca-
of indicators that are acceptable, and identifying man-
pods, but also of fouling organisms that may otherwise
agement responses that are to be undertaken if these
not have appropriate habitat in a given area. In con-
levels are exceeded. The selected indicators must be
trast, the removal of some features for bivalve culture
relevant and practical to measure. Although in the
may repel other organisms. Thus, flexible approaches
New Zealand example ecological indicators were
to evaluating ecological carrying capacity need to be
selected (spatial extent of phytoplankton depletion,
developed to incorporate our evolving understanding
also done recently in eastern Canada [25]), the
of the functioning of marine ecosystems and their
approach could also be used for other types of
interactions with bivalve culture [17, 89].

indicators derived from other categories of carrying
capacity evaluations. The important aspect is that the
selected indicators are chosen in a collaborative way
[102], including input and discussions with members
of the public, environmental managers, scientists,
members of the industry, groups with conflicting inter-
ests, etc. In addition to the process being representative,
it must also be transparent and adaptive so that new
information may be easily included in the decision-
making process. Indeed, much work has shown that
collaborative efforts between all stakeholders are essen-
tial to ensuring satisfaction in the consultation process
and developing policy [103].
A recent example of this is the evaluation of the
ecological carrying capacity of lagoons in Rhode Island,
eastern United States, for oyster culture [97]. This
involved the development of the Working Group on
Aquaculture Regulations (WGAR), which guided and
oversaw the development of a mass-balance modeling
approach to calculate both the ecological and social
carrying capacities of the area for oyster culture.
Whereas it is usually left up to the modelers to deter-
mine what constitutes an acceptable or an unacceptable
impact [97], it was considered that the process would
be much more transparent and inclusive by including
input from all stakeholders, following the criteria
outlined by Soto et al. [104] stating that an ecosystem
approach to aquaculture (EAA) should:
1. Be developed in the context of ecosystem functions
and services with no degradation of these beyond
their resilience capacity
2. Improve human well-being and equity for all rele-
vant stakeholders
3. Be developed in the context of (and integrated to)
other relevant sectors
The above strategies to evaluate the social carrying
capacity of locations may be considered as social-
ecological systems (SESs) where all of the potential
stakeholders participate. Walker et al. [105] outline
four general steps in the process:
1. Stakeholder-led development of a conceptual
model of the system, including its history and pre-
liminary assessments of the drivers of key ecosystem
goods and services.
Carrying Capacity for Sustainable Bivalve Aquaculture 459
2. Identification of the range of drivers of the system,
stakeholder visions for the future, and contrasting
possible future policies, to identify a limited set of
future scenarios.
3. Identify the resilience of the system by examining
the results from i and ii. This is generally done
through the development of models of the system’s
dynamics to identify important attributes that
affect resilience.
4. Stakeholder evaluation of the process and outcomes
in terms of policy and management implications.
Ostrom [106] recently developed a framework to
evaluate the sustainability of management strategies for
SESs. It is a nested approach with four first-level core
subsystems. In bivalve culture systems, these may
include (1) resources systems – a given embayment or
other logical management area; (2) resource units –
such as phytoplankton biomass or physical space;
(3) governance systems – the specific rules related to
culture activities that manage resource use; and
(4) users – the various groups that use the resource
for sustenance, recreation, etc. Each core subsystem is
made up of a series of secondary subsystems and inter-
acts with other core subsystems. One of the main find-
ings was that, although many researchers have
predicted that users of a system will not self-organize
to create a sustainable SES and thus regulation by
governments is needed, this prediction was not
supported when stakeholders were enabled to discuss
management options. Monitoring and enforcement
were other key components that determined the suc-
cess of the studied SESs.
A further method to determine the social carrying
capacity of a site is by attaching a monetary value to the
different categories of impact due to aquaculture [15].
However, this is a complex undertaking and must
include both positive and negative effects of the
operations [107]. Moreover, acceptability of different
effects is quite variable among locations and groups,
further complicating the goal of attaining consensus for
different variables.
The use of fuzzy expert systems has also been advo-
cated to determine the social carrying capacity of an
area for bivalve culture [17]. This approach has been
shown to be useful when data types are disparate and
460 Carrying Capacity for Sustainable Bivalve Aquaculture

uncertainties are great or simply unknown. In short,
instead of stating that a given level of production is
acceptable or unacceptable, a given level is treated as
being, say, 50% acceptable. It is rather more like dealing
with varying shades of gray rather than cases that are
black and white. This has the important effect of induc-
ing less conflict between stakeholders on issues that
may be contentious.
In sum, methods to determine social carrying
capacity for bivalve culture remain poorly developed.
In general, few methods have been developed specifi-
cally for aquaculture although methods developed
for other sectors may be adapted. Analysis of the
attempts to use SESs in other systems has shown that
some approaches work; such approaches may be
looked to for determining social carrying capacity for
bivalve culture.
Decision Framework to Determine Carrying
Capacity
A hierarchical approach to determine the carrying
capacity of an area for bivalve culture is proposed
(Fig. 4). The first level, the physical carrying capacity,
relates to the available natural conditions and the needs
of the operation and bivalves to be cultured. The
second level, the production carrying capacity, is
a function of the supply of food to the farmed bivalve
in an area and is determined using modeling efforts.
At the third level, the ecological carrying capacity of an
area is estimated using modeling or by following
a logical framework to evaluate the range of possible
outcomes for production estimates varying between
none (and/or the current level) and the maximum
calculated production carrying capacity; there is
little use to go beyond this point as it is assumed that
this level will not be surpassed knowingly. Finally,
managers weigh and balance the different scenarios
based on the outcomes from each of the preceding
categories of carrying capacity and make a decision as
to what level of productivity is acceptable – the social
carrying capacity.
It must be understood that the three first categories
of carrying capacity are a function of social values. For
example, a given coastal area in which bivalve culture
may be done is often valued for myriad competing
activities, such as water quality, visual aesthetics,

Water depth
1 Currents
Physical carrying capacity
Temperature
etc...

Plankton
2
guidance / feedback

Detritus
Production carrying capacity
Nutrients
etc...

Community structure
3 DEPOMOD
Ecological carrying capacity
Mass balance models
etc...

Fisheries / Recreation1
Social carrying capacity Economics2
Valued species / habitats3
etc...

Carrying Capacity for Sustainable Bivalve Aquaculture. Figure 4

Hierarchical structure to determine carrying capacity of a given area. Note that social carrying capacity provides
guidance to choosing pertinent response variables and on establishing limits for these. Superscripts indicate examples
of the type of information that informs the selection of response variables for other carrying capacity categories
(Modified from McKindsey et al. [17])

competition for limited space, other species, etc. For
the first category, physical carrying capacity, competi-
tion for physical space may be direct or indirect. For
example, direct competition may result as there may be
multiple demands to use the same area for different
purposes, such as bivalve culture as well as fishing or
recreational boating. Indirect competition may arise
because of visual aesthetics or the not-in-my-backyard
factor where waterfront owners or users do not want
their views affected by aquaculture installations. Pro-
duction carrying capacity is likely rarely maximized
because of economic costs related to production.
Thus, Nobre et al. [108] developed a modeling
approach that considers both ecological and economic
aspects and their interactions to maximize profit and
EAA as focusing aquaculture management on maxi-
mizing output is likely economically inefficient and
carries unnecessary ecological risks. In fact, this
approach addresses all four categories of carrying
capacity with an emphasis on maximizing economic
returns. The ecological carrying capacity of an area
is also clearly a function of social values as what is
valued varies among populations (e.g., specific bird
and fish populations, water clarity, eelgrass, or specific
rare habitats).
Consultation with stakeholders throughout the
process of defining the carrying capacity for a given
area will identify the appropriate (given the social
values of the population) response variables or indices
to be examined [97]. Ideally, the scientists should then
be able to select suitable tools from a toolbox (e.g.,
models, GIS, and comparisons with previous studies)
to predict the form of the response curves of the
selected response variables to a range of production-
level scenarios. It is important to note that although it
is the role of scientists to describe the form of the
responses to a range of production-level scenarios
(see Fig. 5), their role ends once they have done this.
It is then up to managers, in consultation with the
various stakeholders, to use all available scientific infor-
mation to inform regulations and policy [17, 97].
Again, this should be done within the context of EBM
and ICZM, given that there is likely a paucity of infor-
mation about a number of factors that are needed to
make informed judgments [96]. Thus managers will
likely have to rely on instinct, local knowledge, extrap-
olation from studies done elsewhere, contributions
Carrying Capacity for Sustainable Bivalve Aquaculture 461
response curve
response variable
acceptable level of
acceptable level of production
response variable
production level
Carrying Capacity for Sustainable Bivalve Aquaculture.
Figure 5
Hypothetical response curve of an environmental variable
under the influence of varying levels of bivalve culture
production. The dotted line indicates the level of the
indicator that has been determined to be acceptable and
the dashed line the corresponding level of production
(Modified from McKindsey et al. [17])
from various stakeholders, etc., to make management
and policy decisions. Even faced with an absence of
some types of information, this does not remove the
logic of the hierarchical nature of the decision frame-
work outlined above. Indeed, it could be argued that
this is precisely when the process is most useful and
should be followed using all available information – so
that an unbiased view of the situation may be formed
and thus promoting appropriate management deci-
sions. As pointed out by McKindsey et al. [17], failure
to follow the process (e.g., by stating at the outset of the
process that certain types of development or develop-
ments in certain areas are not permitted) will likely
result in otherwise feasible bivalve culture installations
not being initiated. It is also contrary to the notion of
effective and transparent processes in the establishment
of ICZM or EBM.
Knowledge Gaps and Future Directions
This entry concludes with a brief discussion of knowl-
edge gaps and directions for future research to better
estimate the carrying capacity of areas for sustainable
bivalve culture, except for the estimation of physical
carrying capacity for which methods are fairly well
developed and are currently used extensively to this end.
462 Carrying Capacity for Sustainable Bivalve Aquaculture
While it is generally true that “existing models must
be made spatially explicit” [17], this is currently being
done. However, models to this end require much infor-
mation that is often not easily available. So, perhaps
a better knowledge gap to address is to determine when
such models are needed and when can general models
be used.
Similarly to the first point, although temporal var-
iation must be included in models to consider seeding
and harvesting activities, some generalized models to
this end have already been developed [35, 57, 108, 109].
These should be expanded upon and evaluated in sev-
eral locations to fully assess their generality.
Far-field impacts for the benthic environment
need to be evaluated. For example, is sedimentation
outside of culture sites decreased due to grazing and, if
so, what is the influence of this on the benthic environ-
ment? How does this influence our interpretation of
“impacts” on the benthic environment within farm sites?
Work is needed on how nonlinear or unexpected
results may be incorporated into carrying capacity
models. For example, how can the provision of bivalve
culture-related structure in the environment – which
may increase the abundance of fouling organisms and
thus filtration capacity or attract various other organ-
isms – be included in carrying capacity estimates?
Methods need to be developed to include both
“positive” and “negative” effects into the decision-making
process. For example, while suspended bivalve culture
may increase organic loading locally, it may also mitigate
the effects of eutrophication (and models have been
developed to assess this, e.g., 35) or increase the abun-
dance of commercially important species locally [11].
Despite the growing prevalence of certification
standards and ecolabeling for aquaculture products, it
is not clear that such processes are improving condi-
tions in the field [14]. Although such standards are
often elaborated within a multi-stakeholder frame-
work, it is not clear under which conditions they are
necessary and how the three previous points are con-
sidered in their development or application.
Given that many groups promote including the
notions of reversibility or resilience in the definition
of carrying capacity, (e.g., [16]), surprisingly little work
has actually addressed this for the benthic environment
[but see 110]. Work on evaluating the resilience of
benthic systems to bivalve culture is needed.
Appropriate management tools, such as methods
for combining disparate data types, need to be devel-
oped to incorporate the information needed to esti-
mate various aspects of carrying capacity to aid in
decision making and policy development.
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 Commercialisation of GM Crops: Comparison of Regulatory Frameworks 467

Commercialisation of GM Crops: on 29 January 2000 and entered into force on

11 September 2003 (for full text, see http://bch.
Comparison of Regulatory cbd.int/protocol/text/).
Frameworks Convention on biological diversity (CBD) The objec-
WENDY CRAIG1, SIVA REDDY VANGA2, tives of this Convention are the conservation of
JORGE CABRERA MEDAGLIA3,4 biological diversity, the sustainable use of its com-
1 ponents, and the fair and equitable sharing of the
Biosafety Unit, International Centre for Genetic
Engineering and Biotechnology (ICGEB), Trieste, Italy benefits arising out of the utilization of genetic
2 resources, including by appropriate access to
ICGEB, Plant Biology: Plant Transformation Group,
New Delhi, India genetic resources and by appropriate transfer of
3 relevant technologies, taking into account all rights
Centre for International Sustainable Development
Law, Lead Counsel on Biodiversity and Biosafety, over those resources and to technologies, and by
Montreal, Canada appropriate funding (for full text, see http://www.
4 cbd.int/convention/text/).
University of Costa Rica, San Rafael de Heredia,
Costa Rica Convention/protocol/treaty A treaty is an agreement
in written form between nation-states (or interna-
tional agencies, such as the United Nations,
Article Outline
that have been given treaty-making capacity by
Glossary the states that created them) that is intended to
Definition of the Subject establish a relationship governed by International
Introduction Law. It may be contained in a single instrument or
Argentina in two or more related instruments such as an
Canada exchange of diplomatic notes. Various terms have
China been used for such an agreement, including treaty,
India convention, protocol, declaration, charter, cove-
The Philippines nant, pact, act, statute, exchange of notes, agree-
South Africa ment, modus vivendi (“manner of living”
United States of America (USA) or practical compromise), and understanding.
International Obligations Relevant to the Biosafety The particular designation does not affect the
Frameworks agreement’s legal character.
A Comparative Analysis of Regulatory Processes in Genetically modified/genetically engineered/trans-
All Study Countries genic organisms Organisms, such as plants, ani-
Conclusion and Future Directions mals, and microorganisms (with the exception of
Disclaimer human beings), in which the genetic material
Bibliography (DNA) has been altered in such a way that does
not occur naturally by mating and/or natural
recombination (The terms “genetically modified”
Glossary
(GM), “transgenic,” “genetically engineered” (GE),
Cartagena protocol on biosafety (CPB) The Carta- and “living modified” (LM) are used in different
gena Protocol on Biosafety to the Convention on legal instruments around the world. It is useful
Biological Diversity is an international agreement (and deliberate) in this document to essentially
which aims to ensure the safe handling, transport, use them interchangeably).
and use of living modified organisms resulting from Living modified organism (LMO) Any living organ-
modern biotechnology that may have adverse ism that possesses a novel combination of genetic
effects on biological diversity, taking also into material obtained through the use of modern bio-
account risks to human health. It was adopted technology (according to the CPB).

P. Christou et al. (eds.), Sustainable Food Production, DOI 10.1007/978-1-4614-5797-8,

# Springer Science+Business Media New York 2013

Originally published in
Robert A. Meyers (ed.) Encyclopedia of Sustainability Science and Technology, # 2012, DOI 10.1007/978-1-4419-0851-3
468 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
Modern biotechnology The application of [1] in vitro
nucleic acid techniques, including recombinant
deoxyribonucleic acid (DNA) and direct introduc-
tion of nucleic acid into cells or organelles, or [2]
fusion of cells beyond the taxonomic family, that
overcome natural physiological reproductive or
recombination barriers and that are not tech-
niques used in traditional breeding and selection
(according to the CPB).
Definition of the Subject
This contribution describes and compares the regula-
tion of GMOs and the underpinning legislative frame-
works in selected countries from around the world. It
also includes a description of the relevant international
agreements related to biosafety and a description of the
main characteristics and attributes of a modern bio-
safety regulatory framework in this area.
Introduction
The rapid development and deployment of modern
biotechnology in the last decades have made biosafety
a critical issue. Although modern biotechnology has
the potential of benefiting agricultural interests in
developing countries as well as overall human wel-
fare, living modified organisms (LMOs) resulting
from modern biotechnology or genetically modified
organisms (GMOs) remain a source of concern with
regard to the conservation and sustainable use of bio-
diversity, as well as to human health. The perceived
risks, which relate to the release of GMOs into the
environment as well as the placement of GMOs onto
the market, have as much to do with social values as
scientific concerns. For example, social concerns may
require the labeling of genetically modified (GM) food
and feed, the mitigation of socioeconomic impacts, and
the demonstrated potential for the co-existence of
organic, conventional, and GM farming [1].
Global use of GM crops is growing rapidly, increas-
ing approximately 87-fold from 1996 to 2010 to
148 million hectares under cultivation by 15.4 million
farmers in 29 different countries [2]. Furthermore, the
estimated value of the global GM crop market in
2010 grew to US$11.2 billion, while the value of
harvested products was estimated at US$150 billion
[2]. Although the majority (52%) of GM crops are
still grown in industrialized countries, developing
countries are rapidly approaching parity and, due to
their high rates of adoption, are soon expected to grow
the majority of GM crops [2]. By 2009, the major
biotech crops had achieved high levels of market pen-
etration: 77% of soybean, 49% of cotton, 26% of maize,
and 21% of oilseed rape grown globally in 2009 were
GM varieties [3].
Despite expected high benefit-cost ratios from
biotechnology, only a few developing countries, such
as Brazil and Argentina, have had high uptake rates
(over 20 million hectares) [2] of GM crops, with uptake
typically concentrated in crops that are exported to
developed country markets. Few others (mainly
India and China) have started exploring their own
national research capability in biotechnology. In the
vast majority of developing countries, both investment
in biotechnology research and development and
the transfer to farmers of transgenic crops already
being marketed have been generally low. This in part
reflects a lack of transparent regulatory capacity
necessary in dealing with risks associated with bio-
technology as well as in addressing the issues of prop-
erty rights development and protection that are
essential to promoting innovative research. This is
particularly important because of the high cost of
undertaking the initial research and development in
biotechnology [4].
As GM technologies are very recent and fast devel-
oping, most governments are trying to keep pace by
developing regulatory policies that reflect consumer
demands and preferences affecting GM agricultural
products. Almost all developed countries require prod-
ucts derived from GM sources to be assessed both for
their safety as foods and for their environmental
impacts. However, there are considerable differences
in the approaches taken by different countries. In the
United States, analysis and approval mechanisms for
GM foods have been subsumed into existing regula-
tions governing the release of new foods, plants, and
pesticides, whereas in the European Union, regulation
of GM products requires considerable separate scrutiny
(see chapter “▶ Commercialisation of GM Crops:
Comparison of Regulatory Frameworks” by Devos
et al.). Countries worldwide are in different stages of
policy development, with the majority of the develop-
ing countries still in the infant stage [4]. In setting up
 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
domestic legislation, developing countries seem to be
paying increasing attention to international trade
concerns [5].
India along with Argentina, South Africa, and
others constitute a group of developing countries that
aspire to develop domestic biotechnology through
national public R&D and/or by creating incentives for
the participation of multinationals as sources of tech-
nology. They each have relatively liberal regulations as
well as more explicit regulatory institutional arrange-
ments [6]. Those developing countries with well-
developed public agricultural research and extension
systems (such as India) are well placed to benefit
promptly from the new biotechnology by working in
partnership or in parallel with private biotechnology
and seed companies. Approving investments in those
activities by the private sector – and the overall invest-
ment climate – will allow the process of adaptation and
adoption to move forward. The experiences in India,
China, and South Africa all indicate that rapid and
widespread adoption is then possible, including by
small farmers [7].
The biosafety frameworks of the described coun-
tries were selected based, primarily, on considering the
following criteria: level of production and commercial-
ization of GMOs, investments made and political
commitment with the research and development par-
ticularly in the field of GMOs, regional leadership in
the adoption of GMOs and in the elaboration and
implementation of biosafety legal frameworks, and the
existence of a functional biosafety framework in place.
Argentina
Argentina is a major producer of agricultural products
and the third largest producer of soybeans. Initially, the
harvested area of soybean was 36,000 ha (59,000 mt) in
1970, increasing to 5.98 million hectares in 1995/1996
(12.43 mmt). The country has historically been the
earliest and most aggressive adopter of GM crops in
Latin America, first planting glyphosate-tolerant soy-
beans in 1996, which sparked a further expansion of soy
production and which is now in excess of 14 million
hectares, of which at least 98% is GM [8]. This rate of
adoption is far higher, and much faster, than that in the
USA, which was the first country to introduce this
technology [9]. In addition, Argentina also grows
469
significant quantities of GM corn (Bt and glufosinate
ammonium tolerant) and Bt cotton, comprising 40%
and 20% of overall production in 2009 for these two
crops, respectively [3, 10]. Available estimates place
accumulated benefits (extra income which would
have not been generated in the absence of the technol-
ogy) until the year 2001/2002 at approximately US$5.2
billion in the case of soybeans, about US$400 million
for Bt maize, and approximately US$40 million for Bt
cotton [7]. Argentina is now third only to the United
States and Brazil in terms of the area planted with
transgenic crops (22.9 million hectares) [2] and is
thus a very important player in the international
arena. Notably, Argentina signed, but has yet to ratify,
the Cartagena Protocol on Biosafety (CPB, hereinafter
referred to as “the Protocol”) of the Convention of
Biological Diversity in May 2000. Argentina is currently
undergoing a consultation process, analyzing and
debating with all the involved sectors the position the
country will take in this respect [8].
Regulatory Oversight in Argentina
Argentina was one of the first countries to establish
a system of regulatory oversight for GMOs [11]. It
has instituted regulatory measures for the safe devel-
opment and application of biotechnology in general
and GMOs in particular. It chose to develop the
policy on biosafety within the context of trade-related
issues [12] and as such has policies, procedures, and
institutional arrangements to regulate the develop-
ment, importation, and export of GMO products.
The Argentine biosafety system is based on guidelines,
not on legislation. Argentina’s legislative framework for
regulating GMOs is based on the existing agricultural
regulatory system (e.g., for plant protection chemicals)
supplemented with GM crop-specific regulations
established to specify conditions for environmental
release or to assess food safety. The non-statutory
guidelines include standards for facilities and
practices designed to prevent the unintended release
of a GMO, conditions of isolation, monitoring field
trials, and standards for risk assessment for conducting
the environmental release. This approach gives the
system flexibility and allows for changes needed
to keep up with scientific advances. One disadvantage,
however, is that compliance with guidelines is
470 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
not legally enforceable; there is no way to prosecute
offenders in the rare cases of non-compliance that have
occurred [13].
Similar to the USA and Canada (see below), Argen-
tine biosafety regulation follows a product-based
approach which results in several agencies, all within
the Agriculture Directorate of the Secretariat of Agri-
culture, Livestock, Fisheries and Food (SAGPyA), man-
dated to regulate GM crops and products. The National
Advisory Commission on Agricultural Biotechnology
(CONABIA) is the lead agency in charge of regulating
GM crops and was established in 1991 by Resolution
124/91 (later expanded by Resolution 669/93) of the
SAGPyA to provide advice and oversee the implemen-
tation of biosafety regulations [14]. CONABIA’s juris-
diction and procedures were established in Resolutions
656/92, 837/93, and 289/97 (later replaced with 39/03)
[13]. Resolution 39/03 is part of the general regulatory
system governing the existing agricultural regulations
in Argentina related to plant protection (Decree-Law of
Agricultural Production Health Defense 6704/66 and
its amendments), seed and phytogenetic creations
(Seed and Phytogenetic Creations Law 20.247/73 and
its regulatory decree), and animal health (Law of
Veterinarian Products, and Supervision of Creation
and Commercialisation 13.636/49).
CONABIA is a multidisciplinary and inter-
institutional organization with advisory duties and
comprises representatives from the public sector, aca-
demia, and private sector organizations related to agri-
cultural biotechnology. Its main responsibility is to
assess the potential environmental impact of the intro-
duction of GMOs in Argentine agriculture [8]. The
Commission handles applications for laboratory and
greenhouse testing, field trials, and governs the
“flexibility status” of release conditions (unconfined
release, usually large-scale, for regulatory purposes or
off-season seed multiplication) of GM plants [13].
Resolution 60/2007 provides a differentiated treatment
for the authorization of the breeding of parental
material which contains transgenic events already
approved for commercialization. Furthermore, it
advises SAGPyA on the issuance of necessary licenses
and authorizations for experimentation and/or envi-
ronmental release of GM microorganisms, as well as
GMO-derived or GMO-containing products (although
the final decision is made by SAGPyA) [6]. In order to
obtain the appropriate marketing license, varieties
must also comply with requirements stipulated by the
National Service of Health and Agrofood Quality
(SENASA) [15]. SENASA’s jurisdiction concerning the
oversight of GMO-derived food was established in
Resolution 289/87, while Resolution 511/98 including
Annexes established the food-safety review criteria
[13]. The latter was based on FAO and WHO docu-
ments, as well as on relevant regulations from Australia,
Canada, the EU, Japan, and the US, but has since been
replaced by Resolution 412/2002.
A key part of the GMO regulatory process consists
of verifying that the commercial approval will not have
a negative impact on Argentina’s foreign trade. This
specific assessment is carried out under Resolution
39/03 by the National Bureau of Agrifood Markets
(NBMA), and it includes an analysis of the current
status of regulatory systems and public acceptance in
the importing countries. The National Seed Institute
(ex-INASE) is responsible for ensuring that all the
necessary requirements for registration in the National
Registry of Cultivars have been established. Ex-INASE
plays a further role in the biosafety system by receiving
and logging applications for GMO field trials. Applica-
tions containing confidential business information are
kept secure at INASE’s offices. Agency personnel per-
form field test site inspections, checking for compliance
with the biosafety requirements set by CONABIA [13].
During 2001, the SAGPyA actively cooperated
with members of the Argentine Congress in drafting
a biosafety law. This draft represented a major
improvement on the current situation, since it clearly
set forth a conceptual framework, as well as issues and
instances to be considered as participants in risk
analysis procedures. But due to the institutional and
economic crisis that broke out on December 2001, the
draft was never discussed in Congress, and there is no
evidence that it will be discussed in the near future [8].
Commercialization of GM Crops: Argentine
Approval Process
When an organization intends to obtain an authoriza-
tion for commercialization of a GM crop in Argentina,
it has to pass reviews by the three regulatory agencies.
Briefly, CONABIA should determine that the environ-
mental impact of the large-scale release of the GM crop
 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
will not significantly differ from that of its non-
modified counterpart; SENASA’s Technical Advisory
Committee on the Use of GMOs should determine
that the derived foods are safe for human and animal
consumption, and the DNMA should determine that
the release will not have an undesired impact on the
country’s international trade [16].
The prerequisite for entering the commercial eval-
uation process is that authorizations for experimenta-
tion and/or release into the environment of the specific
GM crop have been previously granted [16]. After at
least one release into the environment has been
approved and the safety of the GM crop has been
demonstrated, the applicant can apply for a
“flexibilization” permit which allows future releases
by simply providing notification of the location, area,
sowing date, and intended harvest date [11]. Flexibility
status conditions are granted for the following
purposes [13]:
● For providing testing material
● For export
● For off-season seed multiplication (not for use in
Argentina)
● For tests, which need to be presented at later stage
(e.g., variety registration)
● For pre-commercial seed multiplication for
a pending variety registration
The deregulation of field testing conditions is
dependent on the results of the biosafety assessment
conducted by CONABIA with regard to the criteria laid
down in Resolution 131/98. These include the charac-
terization of the GMO (recipient organism, genetic
modification, insert, donor organisms, phenotypic
characterization, potential environmental interactions
of GMO) and the impacts expected from the produc-
tion of the GM crop at commercial scale (environ-
mental effects and impact on human health) [16]. If
SAGPyA, on the recommendation of CONABIA,
authorizes “flexibility status” release conditions for
the GM crop in question, the applicant only needs to
submit information on the area to be sown, the date of
sowing, the site of release and the harvest date [16]. The
flexibility status of a GM crop allows large-scale plant-
ing, but not planting for commercial purpose.
The second step to commercialization is the evalu-
ation of the safety of the GM crop for human
471
consumption and feed. This evaluation is carried out
by SENASA under Resolution 412/2002 [17]. In the
third step of the commercialization process, the
NBMA assesses the impact of the GM crop in question
on export market security. It does this by analyzing the
status (if any) of the specific event in the destination
markets and, as a result, whether the addition of this
event to Argentina’s export supply might represent
a potential barrier to the access to these markets.
After completion of all of the steps mentioned
above, CONABIA’s Office of Technical Coordination
compiles all pertinent information and prepares
a “Project of Resolution” on the basis of its own,
SENASA’s, and DNMA’s assessments and submits it to
the SAGPyA, which takes the final decision on approval
or denial of the commercialization request [13]. Should
the GM crop be authorized for open cultivation, it
must also be registered in the National Registry of
Cultivars, a process overseen by ex-INASE. For those
GM crops expressing either herbicide tolerance or
insect resistance, they require a pesticide approval
from SENASA prior to their commercial use [13].
Canada
Regulatory Oversight in Canada
In line with a similar approach adopted in the United
States (see below), the regulatory framework
established in Canada is based on the extension of the
existing regulations to GMOs [18]. However, in con-
trast to all other countries, Canada relies on the concept
of novelty to trigger regulatory oversight, thereby
enabling the regulation of a wider array of novel seeds
or food [19], and includes those produced by conven-
tional breeding, mutagenesis, or rDNA techniques.
Directive 94–08, first published in 1994, defines
these as “plants containing traits not present in
plants of the same species already existing as a stable
population” [20].
The Canadian Environmental Protection Act
(CEPA) of 1988 formally recognizes biotechnology as
a manufacturing process for products potentially pos-
ing environmental risks, and therefore, the act requires
environmental assessments. CEPA, however, embraces
a product-based approach to biotechnology, an
approach explicitly defined in the 1993 Regulatory
Framework for Biotechnology [21]. In accordance
472 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
with this framework, policy-makers proceeded to the
amendment of a series of regulations (below) contigu-
ous to the laws governing the products of biotechnol-
ogy. These amendments were mostly aimed at
inscribing a trigger (the novelty of the trait) launching
the risk evaluation process for the products of
biotechnology.
The Canadian approach is based on an agreement
between the Canadian Federal agencies in 1993 that was
renewed in 1998. The responsibility for regulating
plants with novel traits (PNTs), including GM plants,
is shared between the Canadian Food Inspection
Agency (CFIA) and Health Canada [18]. The CFIA
operates under the authority of the Seed Act, the
Plant Protection Act, the Feeds Act, the Fertilizer Act,
and the Health of Animals Act. It also shares some
responsibilities with Environment Canada under the
Canadian Environmental Protection Act, and with
Health Canada under the Pest Control Products Act,
and the Food and Drugs Act. The Canadian Environ-
mental Protection Act is an umbrella legislation
intended to serve as a regulatory “safety net” for any
biotechnological products not currently regulated by
another federal act. The Department of Fisheries
and Oceans regulates aquatic organisms under the
Fisheries Act.
In 1997, the CFIA took over the risk management of
novel seeds and feeds from Agriculture and Agri-food
Canada. The agency regulates novel plants following
assessment criteria provided by Directive 94–08. In
particular, the CFIA is responsible for the regulations
and guidelines dealing with cultivating PNTs, assessing
their impact on the environment and biodiversity.
Canadian authorities state that “all plants derived
through genetic engineering have been considered
novel, and as such have undergone a full, comprehen-
sive, and rigorous safety assessment prior to release
into the environment” [22]. In addition, the agency is
also in charge of ensuring livestock feed safety, along
with the responsibility for the regulation of seeds,
veterinary biologics, and fertilizers. Furthermore, the
CFIA develops standards related to the packaging,
labeling, and advertising of foods and handles all
inspection and enforcement duties [23].
Many GM crops are destined, in whole or in specific
parts, for the human food supply system. For this
reason, they must not only obtain CFIA approval but
must also be assessed by Health Canada. It is within the
jurisdiction of Health Canada to regulate GM foods
according to the Food and Drugs Act under Division 28
of Part B of Food and Drug Regulations (Novel Food).
As with seeds, the trigger for pre-market safety assess-
ments is novelty, and as such, Health Canada treats as
novel food all those derived from GMOs “whether it
is a micro-organism, a plant or an animal, such that
it exhibits characteristics that were not previously
observed, no longer fall within the anticipated range
or no longer exhibits characteristics that were
previously observed, for that plant, animal or micro-
organism” [23].
Environment Canada is only responsible for the
environmental assessment of GMOs used in industrial
processes. Although the Canadian Environmental Act
requires environmental risk assessments for GMOs, the
responsibility for conducting assessments relevant to
novel plants, feeds, and food rests with the CFIA,
PMRA, and Health Canada. Notably, CFIA, as the
agency most involved in the environmental risk assess-
ment of GMOs, is the responsibility of the agriculture
minister whose mandate is to promote agricultural
development.
Commercialization of GM Crops: Canadian
Approval Process
Before crops with novel traits may be authorized for
unconfined release, they must be fully assessed for
environmental safety by the CFIA. In meeting the
extensive information requirements for these applica-
tions to the CFIA, applicants will have conducted
experiments at the earlier confined release stage.
These experiments are expected to contribute scientif-
ically robust data to address the key criteria of environ-
mental safety assessments. The applicant is required to
provide the Plant Biosafety Office (PBO) at the CFIA
with extensive high-quality, statistically sound data
and/or valid scientific rationale to demonstrate the
environmental safety of the PNT. This information
initiates a review and decision for authorization of the
release. CFIA officials also use pertinent information
generated from the Agency’s own research, either
conducted in-house or contracted out, on specific key
environmental areas [18].
The unconfined release assessment by the PBO
focuses on real or potential interactions of the PNTs
 Commercialisation of GM Crops: Comparison of Regulatory Frameworks 473

with the wider agricultural and ecological environ- China

ment, using “substantial equivalence” as the basis for
China has become one of the Asian leaders in biotech-
these assessments. Evaluations consider the unique
nology and has dedicated substantial economic, scien-
combination of species and traits, using standard
tific, and technological resources to R&D. Since the
descriptions of each species known as biology docu-
1980s, ministries and relevant government agencies in
ments as a baseline for comparison. If the PBO con-
China have been investing significantly in agro-
cludes that there is minimal potential for significant
biotechnology research and have established more
negative environmental impact of the PNT relative to
than 150 laboratories, resulting in the largest plant
its unmodified counterpart, an unconfined environ-
biotechnology capacity outside of North America
mental release may be authorized. In some cases, the
[25]. The government has allocated research budgets
PBO may authorize an unconfined release with condi-
to biosafety and management, and nearly all biotech-
tions, such as a requirement that the applicant ensures
nology research programs have expanded their scope
that users of an insect-resistant PNT deploy methods
into biosafety issues [5]. The commitment to sustain
to delay development of resistance among insect
biosafety after project closure is demonstrated by its
populations. Note that for species that may be used
growing budget to support agricultural research in
for food or feed, developers of PNTs must also seek
biosafety over the last few years. From an initial budget
approvals from Health Canada for human food use and
of slightly over US$ 80,000 in 1999, China now spends
from the CFIA Feed Section for livestock feed use [19].
about US$ 3 million annually on agricultural biosafety-
The starting point for the safety assessment of novel
related activities [10].
foods is also based on “substantial equivalence,” where
A wide variety of crops and traits has passed
the novel food is evaluated relative to conventional
through China’s biosafety system and are now planted
counterparts that have a history of safe use [23]. Health
commercially, while many others remain at the field
Canada has 45 days to decide whether the product is
trial stage, including many varieties with adaptation-
safe or to request additional information to pursue the
related traits developed by Chinese institutes and com-
risk analysis, even to the extent of involving experi-
panies [26]. In contrast to the other countries described
ments [23].
in this chapter, most of these crops have been devel-
All imported PNTs (or products derived from
oped predominantly by public sector laboratories in
them) require a prior import permit, being subject
China. The biosafety regulatory system in China has
to the CFIA regulatory review under the Plant Protec-
also reviewed a large number of applications since it
tion Act and Regulations. Pest risk assessments (PRAs)
was formally set up in the late 1990s. The government
are conducted by the Plant Health Division in order
received 1,044 applications for field trials or commer-
to evaluate the potential capability of PNTs to pose
cial release, and 777 of these were approved [27, 28].
a pest risk to the agricultural and forestry environ-
These applications predominantly covered 60 crops, as
ment. Those commodities determined not to pose
well as several animals and a large number of microor-
a plant pest risk are now no longer required to have
ganisms [28]. Varieties of cotton, tomatoes, phytase
an import permit. Additional exempted commodities
maize, insect-resistant rice, and sweet and chili peppers
include: PNTs with prior approval; PNTs (or products
have all been approved for commercial planting [3].
derived from them) that are incapable of sexual or
asexual propagation, i.e., have been processed in
Regulatory Oversight in China
some way to render them non-viable, such as by
grinding or freezing; and plants further developed China has adopted a policy that promotes research and
from exempted PNTs, or considered substantially development of biotechnology, while at the same time,
equivalent to them provided that the intended use is retaining control over research in genetic engineering.
similar, and that the plants do not display any addi- In the early 1990s, China had already implemented
tional novel traits, do not contain novel genetic ele- a very pragmatic approach to GM crop regulation.
ments, and have not been subject to inter-specific Regulations were basically product-based with special
breeding [24]. attention given to the economic interest of a given
474 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
application. By 1993, China had already established its
first biosafety regulation, namely the “Safety Adminis-
tration Regulation on Genetic Engineering” issued by
the Ministry of Science and Technology (MOST). This
instrument required relevant ministries to draft and
issue corresponding biosafety regulations on biological
engineering (i.e., the Ministry of Agriculture (MOA)
for agriculture and the Ministry of Public Health for
food safety) and established general principles, safety
categories, risk assessment and risk management pro-
cedures, application and approval mechanisms, and
legal responsibilities [29]. It was followed in 1996
with the “Safety Administration Implementation Reg-
ulation on Agricultural Biological Genetic Engineer-
ing” by the MOA [12]. This was an explicit regulatory
regime for the risk assessment and management of
agricultural products of genetic engineering. Labeling
was not part of this regulation, nor was any restriction
imposed on imports or exports of GM products. The
regulation did control GMOs for research and com-
mercial production, as well as establishing the National
Agricultural GMO Biosafety Committee (Biosafety
Committee) to provide the MOA with expert advice
on biosafety regulations.
Criticism of GM crops on environmental, food-
safety, and ethical grounds, however, led to some
significant changes in the Chinese legal framework on
agro-biotechnology [5]. In 2001, the State Council
decreed a new and general rule on biosafety, with the
aim of protecting human, animal, and plant health and
the environment. This new “Regulations on Safety of
Agricultural Genetically Modified Organisms” replaced
the 1993 Regulation issued by MOST. The 2001 regu-
lations provide the MOA with overall national author-
ity to oversee the use of GM crops, whereas the 31
provincial biosafety management offices are responsi-
ble for the supervision and administration of biosafety
in their respective areas [30]. The 2001 regulations
meet the generally accepted risk assessment procedures
outlined in the relevant international instruments
and also stipulate a comparative risk assessment
approach, in which a GM crop is compared with the
corresponding non-transgenic crop for environmental/
ecological safety and food safety.
To implement this Regulation, the MOA issued
three implementation regulations including Imple-
mentation Regulations on Safety Assessment of
Agricultural Genetically Modified Organisms, which
provided the legal basis and technical guidelines in
GM crops risk assessment in China [31]. These new
regulations primarily concerned Biosafety Evaluation,
Import Safety, and Labeling and included several
important changes to existing procedures and details
of regulatory responsibilities after commercialization.
The changes included an extra pre-production trial
stage prior to commercial approval, new processing
regulations for GM products, labeling requirements
for marketing, new export and import regulations for
GMOs and GMO products, and local and provincial-
level GMO monitoring guidelines [29]. Specifically, the
Regulation on Biosafety Evaluation establishes proce-
dures for handling applications for GM cultivation and
sets up an advisory body, the National Biosafety Com-
mittee (NBC), and a decision-making body, the Office
of Agricultural Genetic Engineering Biosafety Admin-
istration (OGEBA), under the MOA to handle applica-
tions. Applicants must provide information on risk
assessment, and GMOs are classified into four classes
depending on their potential danger to human and
animal health and to the environment. The Regulation
establishes the requirements that should be met to
obtain authorization to import GMOs and will vary
according to the intended purposes of the imports,
i.e., research, release into the environment, or processing.
In response to representations from GM-producing
countries, China agreed to allow trade to continue as
normal until the new Regulation on Safety of Imports
entered into force on 20 April 2004 [32, 34]. The
Ministry of Public Health (MPH) is responsible for
food-safety management of biotechnology products
(processed products based on GMOs) and promul-
gated its first regulation on GMO food safety in April
2002, to take effect after July 2002.
China’s policy on GM regulation is now under the
responsibility of an agency which was established by the
State Council, the name of which has been variously
reported as either the Joint Monitoring and Manage-
ment Commission [12] or the Allied Ministerial
Meeting [29]. It has a multi-stakeholder membership
comprising the highest representatives from ministries
like Agriculture, Health, Commerce, Science and Tech-
nology, the National Development and Reform Com-
mission, the National Inspection and Quarantine
Agency, and the State Environmental Protection
 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
Administration. It is responsible for the coordination
of key issues related to the biosafety of agricultural
GMOs, the examination and approval of the applica-
tions for GMO commercialization, determining the list
of GMOs for labeling, and establishing import or
export policies for agricultural GMOs and their prod-
ucts. In addition, under the new regulation, foreign
investment in biotechnology has been prohibited [5].
The NBC remains the major player in the process of
biosafety management. Currently, the NBC is com-
posed of 56 members who come from different admin-
istrative departments, academic institutions, etc.
They are experts in biological research, production,
processing, inspection and quarantine, public health,
and environmental protection with respect to agricul-
tural GMOs [31]. The committee meets twice each year
to evaluate all biosafety assessment applications related
to experimental research, field trials, environmental
release, pre-production trials, and commercialization
of agricultural GMOs. It makes recommendations to
the OGEBA based on the results of its biosafety assess-
ments. OGEBA is responsible for the final approval of
decisions, as well as handling routine work and daily
operations [29]. In 2005, all 31 provinces in China
established biosafety management offices. These bio-
safety management offices collect local statistics on and
monitor the performance of research and commercial-
ization of agricultural biotechnology in their provinces
and assess and approve (or disapprove) all applications
of GM-related research, field trials, and commerciali-
zation in their provinces. Only those cases that are
approved by provincial biosafety management offices
are submitted to the NBC for further assessment [29].
In May 2007, the National Development and
Reform Commission in China announced that it had
approved the establishment of a National Biosafety
Research Centre. To be completed by 2009, the Centre
will manage agricultural and biological-related issues.
It is to house several research departments, including
laboratories for high-risk plant pathogens, insects, and
plants, as well as units for agriculture-related informa-
tion analysis and quarantine facilities. The Centre will
be supervised by the Plant Protection Institute of the
Chinese Academy of Agricultural Sciences.
In 2009, a major development in China was the
commercialization of transgenic Bt rice, which reached
pre-production trials but was still pending final
475
commercialization approval for several years.
Influenced in part by opposition to the technology in
Europe and to some extent Japan, the final approval of
many crops stalled at the level of China’s inter-
ministerial committee even as research and field trials
continued apace. The discovery by Greenpeace that
some transgenic seed was planted by farmers without
authorization caused international debates about
China’s biosafety system and may have contributed to
regulatory approval delays [33].
Commercialization of GM Crops: Chinese Approval
System
If the product is for cultivation, applications must be
authorized before the first import of a specific GMO
can take place and must be accompanied by a safety
assessment carried out in the country of origin of the
GM material [5]. For permanent approval of each
imported GM product, compulsory field trials are car-
ried out in China in order to re-assess safety within the
Chinese context [35]. Generally, the practice in China is
to use a comparative risk assessment approach, in
which the transgenic crop is compared with the
corresponding non-transgenic crop in ecological risk
assessment and hazard identification in transgenic
foods [36, 37]. Although there are no official guidelines
in China for risk assessment on food derived from
transgenic crops, the assessments carried out so far on
nutrition, toxicity, and allergenicity generally followed
the relevant Codex principles and guidelines [31].
In China, agricultural GMOs also need to satisfy the
procedures governing the release of new seed varieties.
These procedures are governed by the Seed Law in
China. Only agricultural GMOs that have previously
obtained a biosafety certificate are eligible to be classi-
fied as a new seed variety in accordance with the Seed
Law and relevant regulations. After the GMO has
passed seed variety testing and received the permission
for production, it is eligible to enter into the chain of
production and marketing [31].
India
In India, a wide variety of crops have been field trialed,
but most have not yet been commercialized. The first
approval for the commercial production of any GM
crop in India occurred in March 2002 when the Indian
476 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
competent authority approved three varieties of
Bacillus thuringiensis (Bt) cotton (MECH 12, MECH
162, and MECH 184 expressing the cry1Ac gene for
insect resistance) amid widespread protests by anti-
GM activists. This was followed by a significant
increase in the availability of Bt cotton (currently 809
hybrid varieties) [38] better suited to Indian cultiva-
tion. By 2003, more than 34 genes were being tested in
a wide variety of crops, including cotton, rice, mustard,
maize, potatoes, eggplant, tomatoes, pigeon pea, and
cabbage [29]. Most of the varieties initially introduced
included insect-resistant cotton varieties as well as
some crops modified with herbicide tolerance. Several
Bt crop varieties have passed through the field trial
stage and have received approval for commercial
planting. The primary technology used in India origi-
nated in the Monsanto Company, which partnered
with Maharashtra Hybrids Company (MAHYCO) to
develop transgenic hybrid Bt cotton for sale in India.
Once some Bt varieties had been approved for com-
mercial planting, it was discovered that 800,000 ha of
unapproved BT cotton had been planted, weakening
confidence in the biosafety system [29]. The latest
GM crop awaiting regulatory approval for commercial
release is Bt brinjal (known also as aubergine or
eggplant), which received positive assessments by the
regulatory bodies based on years of field trials [3] but
was refused authorization by the Environment Minis-
ter at the last stage of the commercialization process.
Regulatory Oversight in India
With the signing of the CPB in 2001, India became
committed to introducing structures and procedures
commensurate with the conditions laid down in the
CPB agreement – one of the main guiding principles
for India when dealing with products derived from
agricultural biotechnology. This commitment pro-
vided India with the incentive to strengthen its bio-
safety capacity and have relevant institutional
mechanisms at hand to enable the proficient dealing
of GMOs. As the CPB places due importance to
national legislations, provided it is developed in accor-
dance with the former, the existing domestic policy on
GMOs was required to be fine-tuned and amended
wherever necessary. The goal of the Indian regulatory
system is therefore to ensure that their GM crops pose
no major risk to food safety, environmental safety, or
agricultural production and that there are no adverse
economic impacts on farmers [29]. As such, the
Government of India has adopted a policy of careful
assessment of the benefits and risks of GMOs at various
stages of their development and field release to ensure
biosafety [39].
The existing regulatory framework takes the form
of rules and guidelines and is based upon three specific
provisions of the Environment Protection Act of 1986
(EPA). These are sections 6, 8, and 25. While Section 6
of the Act empowers the Central Government to make
rules on procedures, safeguards, prohibition, and
restrictions for handling of hazardous substances,
Section 8 of the Act prohibits a person from handling
hazardous substances, except in accordance with
procedures and after complying with safeguards.
Section 25 of the EPA empowers the Central Govern-
ment to lay down rules regarding procedures and safe-
guards for handling hazardous substances. Thus, the
biosafety rules in India are statutory in nature as they
originate from the EPA. These provisions of the EPA led
to the adoption of the 1989 Rules for the Manufacture,
Use, Import, Export and Storage of Hazardous Micro
organisms Genetically Engineered Organisms or Cells
(“1989 Rules”) [39, 40].
In 1994, the Department of Biotechnology revised
its earlier guidelines of 1990, entitled “Revised Guide-
lines for Safety in Biotechnology.” These revised guide-
lines aimed at regulating large-scale production and the
deliberate release of GMOs, plants, animals, and prod-
ucts into the environment and shipment and importa-
tion of GMOs for laboratory research [6]. By 2002,
an array of legislation likely to impact biosafety
regulations had come into existence. This included
the National Biodiversity Act 2002 (NBA) and the
Protection of Plant Varieties and Farmers’ Rights Act
2001 (PPVFR), the latter of which derived from
a broad-based consultation with a view to incorporate
a form of farmers’ rights into the national plant variety
rights legislation. The biosafety rules have since been
supplemented by the Biotechnology Safety Guidelines
issued by the Department of Biotechnology (DBT).
These guidelines have been issued in pursuance of
Rule 4[2] of the Biosafety Rules, which require manuals
of guidelines to be brought out by the Review
Committee on Genetic Manipulation [40].
 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
Therefore, the Indian biosafety regulatory frame-
work, comprising the 1989 Rules and the 1990, 1994,
and 1998 DBT guidelines, covers the entire spectrum of
activities relating to GMOs. This includes “research
involving GMOs, as well as genetic transformations of
green plants, recombinant DNA (rDNA) technology in
vaccine development, and large-scale production and
deliberate/accidental release into the environment of
organisms, plants, animals and products derived from
rDNA technology.” Production facilities such as distill-
eries and tanneries that use GMOs are also covered. In
India, the risk assessment and regulatory approval for
releases of GMOs and GM products are mandatory.
The concept of “biosafety” used in the regulations is
a broad one, covering the health safety of humans and
livestock, environmental safety (ecology and biodiver-
sity), and economic impact. The first two safety aspects
dominate the regulations, while economic impact is
given less prominence.
Two nodal agencies, the Ministry of Environment
and Forests (MoEF) and the DBT at the Ministry of
Science and Technology, are responsible for the imple-
mentation of the regulations [39]. The life cycle of
a GM product features four domains, pre-research,
research, release, and post-release, and is characterized
by the presence of six competent authorities [41, 42].
The Recombinant DNA Advisory Committee (RDAC)
is in the pre-research domain as it triggers research
through its initial approval mechanisms. The Review
Committee on Genetic Manipulation (RCGM) resides
in the DBT and functions in the research domain,
closely monitoring the process of research and experi-
mental releases. It requests food biosafety, environmen-
tal impact, and agronomic data from applicants who
wish to do research or conduct field trials and will give
permits to import GM material for research. Pursuant
to Rule 4 [2] of the 1989 “Rules,” the RCGM is also
required to produce manuals of guidelines. The RCGM
is primarily made up of scientists (including agricul-
tural scientists) and can request people with specialized
knowledge to review cases. It has a Monitoring cum
Evaluation Committee (MEC) that monitors limited
and large-scale field trials of GM crops and is primarily
made up of agricultural scientists. Commercial pro-
duction of GM crops, large-scale field trials of GM
crops, and the imports of GM commercial products
and GM-derived products (e.g., foodstuffs, ingredients
477
in foodstuffs, and additives including processing aids
containing or consisting of GMOs) come under the
authority of the Genetic Engineering Approval Com-
mittee (GEAC) at the MoEF. The committee members
are primarily bureaucrats representing different minis-
tries, and they draw on the scientific expertise of each
ministry. Additional to these national committees are
the State Biotechnology Coordination Committee
(SBCC) and the District Level Biotechnology Commit-
tee (DLC), who, along with the MEC, basically occupy
the post-release domain, although they also contribute
to the research domain activities through data provi-
sioning to the RCGM. Completing the regulatory
apparatus are the Institutional Biosafety Committees
(IBSC) which undertake the monitoring and imple-
mentation of safeguards at the R&D sites, under the
close supervision of the RCGM, the SBCC, and the
DLC. IBSCs must be established in any public or
private institute using rDNA in their research and
comprise scientists from their respective institutes
and a member from the DBT. There are more than
230 IBCs in India, of which 70 deal with agricultural
biotechnology. They can approve contained research at
institutes unless the research uses a particularly hazard-
ous gene or technique which will require specific
approval from the RCGM [29]. In general, these
authorities are vested with non-overlapping responsi-
bilities [39, 43].
Under the Constitution of India, it is not the central
Government of India but the state governments that
exercise formal authority over agriculture. Thus, while
the national government may take the initiative in the
policy arena and formulate policies concerning agricul-
tural biotechnology and GM crops (in R&D as well as
commercialization), as well as being where the deci-
sion-making process resides, the agreement and active
cooperation of state governments are indispensable for
their implementation [44].
The multitude of rules and regulations underline
the complexities involved in biosafety as it cuts across
ministries and agencies and does not merely govern
environmental issues. Most of these regulations deal
with GMOs in seclusion without referring to a
common agency or secretariat to deal with the risks
that are associated with the organism [46], resulting in
the biosafety regulations being subjected to criticism
both by industry and civil society groups. While
478 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
industry associations consider these regulations as
affecting their growth, civil society groups consider
biosafety regulations as not being strong enough to
check the introduction of potentially harmful biotech-
nology products. Since 2004, there have been serious
discussions in India on re-engineering the structure of
biosafety regulations. The primary objective of the
exercise is to cut down red tape and ensure greater
transparency in decision-making. Calls have come for
the replacement of the present regulatory system (with
its dispersed, unclear, and confusing mandates; respon-
sibilities; and powers) by a new, single, integrated, and
professionally led authority, the National Biotechnol-
ogy Regulatory Authority (NBRA), with a comprehen-
sive mandate and a wide range of responsibilities, with
the power to implement the regulatory regime with
speed and efficiency [40, 44] and to help the assessment
of risks and benefits associated with GM crops in
a credible and transparent manner [47]. In May 2007,
it was announced that the NBRA will be fully functional
in 2 years time and will be administered by the DBT to
expedite the application of biotechnology in the agri-
culture, veterinary, and medicine sectors [48].
The current amendments or changes that have
favored the industry relate to changes in the 1998
revised guidelines for research in transgenic plants,
whereupon a relaxation was permitted regarding the
concept of deliberate release. This amendment, by
conferring powers to the RCGM to permit limited
conduct of field trials in multi-locations, was at vari-
ance with the 1989 Rules that prohibited deliberate or
unintentional release for experimental purposes, except
where the GEAC approved it as a special case. The
distinction between small-scale and large-scale releases
brought about by the changed guidelines was unusual
and was designed to ensure the control of the DBT and
the RCGM over initial field testing of transgenic crops.
An amendment was made by the DBT in September
1999 conferring rights to the RCGM to approve small
experimental field trials for research, limited to a total
area of 20 acres in multi-locations with any one loca-
tion not exceeding 1 acre. Through this amendment,
the DBT removed small experimental trials for
research from the deliberate release clause of the 1989
Rules [40].
The changes that have been made to accommodate
civil society concerns are basically twofold. The first
relates to the formation of the MEC by the DBT in 1998
in order to closely and objectively monitor private
sector biosafety data and through the mandatory
involvement of state-level agricultural university scien-
tists. The second change, which was induced by the Bt
cotton controversy in India, has been the introduction
of allergenicity tests of transgenic seeds, leaves, and
vegetables on rodents, rabbits, guinea pigs, and goats
in the 1998 version of the Biotechnology Safety Guide-
lines [42]. This precautionary step is viewed by the
industry as having contributed to the delay in the
regulatory approval for Bt cotton [40]. Additional reg-
ulations have recently been added to the PPVFR Act of
2001, requiring applicants to provide relevant GEAC
clearances and approvals for registering transgenic
varieties, as well as an affidavit stating that the “Termi-
nator” Technology or the Genetic Use Restriction
Technology is not involved [52]. Notably, the deci-
sion-making circle does not include the participation
of industry, civil society, or consumer groups. While
the 1989 Rules explicitly say that the RCGM, the GEAC,
the SBCCs, and the DLCs may co-opt other members/
experts as necessary, they neither include nor exclude
representatives of NGOs and the private sector. In
practice, however, these non-governmental stake-
holders have been excluded [44]. However, following
the “Terminator” controversy, the National Bureau of
Plant Genetic Resources is now mandated by the gov-
ernment of India to develop probes to detect the pres-
ence of terminator genes in imported material,
highlighting how the force of public opinion can still
shape biosafety rules in India.
To keep up with the rapid pace of developments in
plant biotechnology, especially GMOs, the Indian reg-
ulatory system revised its existing guidelines in 2008.
These were to provide greater clarity on data require-
ments and include: Standard Operating Procedures
(SOPs) for Confined Field Trials of Regulated, Genet-
ically Engineered (GE) Plants [49], Guidelines for the
Safety Assessment of Foods Derived from Genetically
Engineered Plants [50], and Protocols for Food and
Feed Safety Assessment of GE crops [51]. Guidelines
for Institutional Biosafety Committees (IBSCs) are also
currently under review.
While the overall regulatory system remains
unchanged, a notable difference is the classification of
all GM field trials into two categories, based on size.
 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
The RCGM, operating in the DBT, is now the regula-
tory authority for Biosafety Research Level I (BRLI)
trials. BRLI trials are limited in size to no more than
1 acre (0.4 ha) per location and a maximum cumulative
total of 20 acres (8.1 ha) for all locations for each plant
species/construct combination (e.g., one or more
events originating from the transformation of a plant
species with the same genetic construct), per applicant,
per crop season. The GEAC, operating in the MoEF, is
now the regulatory authority for Biosafety Research
Level II (BRLII) trials. BRLII trials are limited in size
to no more than 2.5 acres (1 ha) per location, and the
number of locations is decided on a case-by-case basis
for each plant species/construct combination, per
applicant, per crop season.
Members of the MEC, SBCCs, and DLCs and mon-
itoring teams of SAUs have the authority to inspect and
monitor confined field trials at the time of planting,
during the growing and harvesting season, and during
the period of post-harvest land-use restriction for com-
pliance with the terms and conditions of authorization.
Commercialization of GM Crops: Indian Approval
Process
The approval process in India begins with the submis-
sion of an application regarding a new LMO event with
potential benefits over the conventional variety/hybrid
in terms of economic benefit to the farmer and/or the
environment. The developer is required to follow a set
procedure that involves providing all the necessary
information specified by the regulatory body. Such an
application is reviewed by the RCGM which, in the first
instance, may recommend various limited contained or
open field trials to be undertaken in order to generate
specific biosafety data which may be lacking in the
original submission. Once the full dossier of informa-
tion from experiments undertaken under confined
conditions is submitted, the RCGM will then ascertain
as to whether the LMO presents any immediate adverse
effects, either to humans, animals, and the environ-
ment (including the likely impact of large-scale culti-
vation on biodiversity). If considered as presenting
minimal risk, the RCGM may permit large-scale open
field trials to be conducted to generate data concerned
with the agronomic performance of the LMO. Once
more, possible adverse impacts on the environment,
479
including on non-target organisms, are evaluated. The
unconfined, open field trials are conducted either by
the applicant or by the ICAR, involving their institutes/
State Agriculture Universities, and are monitored by
the MEC. The MEC reports their observations directly
to the RCGM and the GEAC. Based on the biosafety
data and the field performance, the RCGM may rec-
ommend the case to the GEAC for further evaluation.
The GEAC will consider all the data provided and may
ask the company to furnish additional data or repeat
the trials in multi-locations during the next season.
Based on the overall recorded benefits, the GEAC can
approve the commercialization of the GM crop for
a limited period and in a specified geographical zone.
Data collected during this period will form the basis of
the review undertaken by the GEAC for any extension
or expansion to the set conditions of commercializa-
tion. Any adverse impact on human, animals, and
environment derived from such large-scale cultivation
is required to be immediately notified by any individual
or organization directly to the GEAC.
All commercial authorizations are for a limited
period, requiring renewal after the expiry period. Fur-
thermore, approval is conditional upon the observing
and collecting of relevant information on the risks, if
any, arising from the commercial use of the GMOs and
products thereof [42]. A key addition in the 1998
guidelines is the requirement to generate data on com-
parative economic benefits of a modified plant. Thus,
the 1998 guidelines call for a demonstration that
a transgenic crop is both “environmentally safe and
economically viable.” An agronomic evaluation of the
transgenic crop to determine economic advantage to
farmers is seen as an integral component of the trans-
genic crop approval process, along with the biosafety
evaluation [42]. Thus, when the government granted
permission for large-scale field testing of transgenic
cotton in India in July 2000 (the first crop to receive
such approval), mandatory data to be generated by the
applicant included “cost of transgenic seed, projected
demand, and the area to be covered under transgenic
cotton cultivation” [45].
The Philippines
The Philippines’ National Agenda for Sustainable
Development for the twenty-first Century (PA 21)
480 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
provides the policy framework of the country’s strategy
for sustainable development. In 2001, the Presidential
Policy Statement on Modern Biotechnology [53]
reiterated the government policy of promoting the
safe and responsible use of modern biotechnology
and its products as one of several means to achieve
and sustain food security, equitable access to health
services, sustainable and safe environment, and indus-
try development. The Philippine government formally
funds biotechnology as part of the annual budget for
agricultural R&D through legislation [54]. In Decem-
ber 2002, the Philippines became the first country in
Asia to commercialize a GM crop for use as food, feed,
or for processing [55] when the Department of
Agriculture (DA) approved the Bt corn MON810 for
import and propagation [56]. By summer 2005, the
Philippines had approved 19 different LMOs for direct
use as food, feed, or propagation [57], while in 2010,
this had increased to 53 (when stacked events are also
included).
Regulatory Oversight in the Philippines
The Filipino biotechnology regulatory system was
established as a result of the recommendations from
the scientists asking the national government to for-
mulate a national policy on biosafety and create
a technical body to draft guidelines to ensure that
experiments using GMOs do not pose unacceptable
risks to human health and the environment [12]. The
Philippines has a body of policies aimed at regulating
the development, importation, transfer, and use of
GMOs. The first guidelines for biosafety were promul-
gated in October 1990 as Executive Order (EO) 430,
which established the National Committee on Bio-
safety of the Philippines (NCBP). The NCBP was
established to “oversee the compliance with policies
and guidelines in all institutions, public or private, as
well as to coordinate with the appropriate national
bodies that have regulatory powers over any violations”
[58]. At present, the NCBP is concerned with contained
use (confined laboratory and greenhouse experiments
on the regulated article), and its primary function is to
identify and evaluate potential hazards involved in
initiating genetic modification experiments and rec-
ommend measures to minimize risks [59]. Additional
guidelines were developed and published by the NCBP
and the Department of Science and Technology in
1991, 1998, and 2002 before being incorporated into
the National Biosafety Framework, which was finalized
in 2004 and issued as EO 514 in April 2006 [5, 12]. The
rules and regulations for the import and release into the
environment of plants and plant products derived from
the use of modern biotechnology are set out in Admin-
istrative Order no. 8, Series of 2002 of the Philippine
Department of Agriculture (AO 8) [35, 55]. The fol-
lowing year, the DA issued Memorandum Circular
No. 8, which outlined the import requirements for
biotech products. This was quickly followed by the
issuance of Memorandum Circulars 11 and 12 in
August 2003, which further clarified the import rules
for biotech products for direct use as seed, food, feed,
or for further processing [57]. Importers of GM
plants for contained use, field testing, and propagation
(or commercial planting), as well as GMOs for direct
use as food, feed, and processing, are required to
obtain an approval permit [15, 55] which stipulates
that the performance of the GM crop and its effect on
the environment as well as human and animal health
have been positively assessed [11].
The decision-making process is vested in multiple
national competent authorities (NCAs) after consul-
tation with other agencies and/or with a multi-
stakeholder advisory body. The Department of
Agriculture (DA) is the competent national authority
responsible for biosafety decisions concerning plants
and plant products derived from modern biotechnol-
ogy, fisheries and other aquatic resources, domesticated
animals and biological products used for animal hus-
bandry or veterinary purposes, and biological agents
used for biocontrol. It is the government institution
with mandatory responsibility for GM crop field
releases and commercialization. Likewise, the Depart-
ment of Science and Technology is responsible for
research and development, the Department of Health
for pharmaceuticals which are not addressed by other
relevant international agreements or organizations,
and the Department of Environment and Natural
Resources concerning regulated organisms intended
for bioremediation, the improvement of forest genetic
resources, and wildlife genetic resources, and appli-
cations of modern biotechnology with potential
impact on the conservation and sustainable use of
biodiversity [12].
 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
Commercialization of GM Crops: Filipino Approval
Process
In consultation with the NCBP, the Bureau of Plant and
Industry (BPI) of the DA is responsible for the granting
of permits issued under AO 8 and are classified
according to the intended use of the regulated article:
(a) importation for contained use, (b) field testing,
(c) release for propagation, and (d) importation for
direct use as food or feed or for processing [61]. Appli-
cations for import must be accompanied by a certificate
from the competent authority in the country of origin
stating that the regulated article has been locally
approved and a notification in accordance with interna-
tional obligations. Local applications must be supported
with the necessary technical and scientific dossiers,
a public information sheet (PIS), and a certificate from
the BPI stating that the regulated article has undergone
satisfactory field testing in the Philippines. The AO
8 policy for commercial propagation stipulates that no
regulated article will be released unless (a) field testing
showed that the GM crop will not pose any significant
risks to the environment, (b) food and/or feed safety
studies showed that the GM crop will not pose any
significant risks to human and animal health, and
(c) a permit for propagation has been secured from the
DA. If the GM crop has transgene-derived pesticidal
properties, it must also be duly registered with the
Fertilizer and Pesticide Authority (FPA) [61]. Upon
receipt of an application, the BPI has 5 days to process
and evaluate all of the documentation to ensure that it
is sufficient in form and substance. If it is found to be
defective, then the applicant is given a 60-day grace
period to correct or provide further necessary informa-
tion. Only a complete application will be accepted for
evaluation by a multi-stakeholder advisory body, the
results of which must be reported to the BPI within
30 days of acceptance. For the duration of the evalua-
tion process, the reviewers remain anonymous to both
the public and the BPI. The Scientific and Technical
Review Panel (STRP) and the Bureau of Agriculture
and Fisheries Products Standards (BAFPS) evaluate all
applications; the STRP comprises of at least three
experts from a roster of independent scientists and
particularly evaluates the risk assessment and risk man-
agement strategies outlined by the applicant, whereas
the BAFPS will make a determination of compliance
481
with food-safety standards. Additionally, the Fertilizer
and Pesticide Authority (FPA) and the Bureau of Ani-
mal Industry (BAI) will also evaluate applications of
those regulated articles which are also pest-resistant
plants or to be used as feed, respectively. Concurrently,
the applicant must carry out a public consultation by
publishing the PIS in two newspapers of general circu-
lation and inviting the public to make comments
directly to the BPI within 60 days of posting the notice.
A decision, together with any agreed permit conditions,
is made within 120 days of publication of the notice
[62]. Approved products are then included in the reg-
istry for direct use maintained by the BPI. Once in the
registry, for imported articles, the applicant is no lon-
ger required to secure an import permit for succeeding
shipments. However, a notification of shipment to
BPI is required within 15 days before its arrival at
a Philippine port [57].
South Africa
South Africa is a biotechnology leader in Africa and is
involved in sophisticated biotechnology activities,
including those pertaining to the development and
commercialization of GMOs [6, 63]. In 2000, the
South African government began to focus on, and
substantially increased, its research support for bio-
technology. This led to the adoption of the 2001
National Biotechnology Strategy (NBS), a policy
framework to create incentives for the biotechnology
sector [63], involving several government departments
[64]. The NBS commits more than US$300 million per
year from government to finance a variety of biotech-
nology initiatives [6].
South Africa is among only three African countries
in which GM crops are commercially grown [2]. In
2006, the commercial release of insect-resistant (Bt)
cotton and maize; herbicide-tolerant (RR) soybeans,
cotton, and maize; and cotton with the “stacked gene”
(Bt and RR) had been approved. At the time, it was
estimated that these GM crops accounted for the culti-
vation of 30.5% of yellow maize, 28.8% of white maize,
59% of soybean, and 90% of cotton in South Africa
[65]. The total area of commercialized transgenic crops
increased in 2010 to 2.2 million hectares [2], which is
mostly due to white and yellow maize, followed by RR
soybeans and insect-resistant cotton. Giving an insight
482 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
into potential future commercial releases, several vari-
eties of these crops were also in field trials as of 2009,
along with additional new crops. Between January and
September 2009, the number of field trial permits
issued totaled 267 [3]. Maize topped the list with
222 approvals, followed by 24 permits for cotton, 15
for vaccines, 3 for soybeans, and one each for sugar
cane, sorghum, and table grapes. The traits associated
in these approvals included drought tolerance and her-
bicide tolerance in maize, herbicide tolerance in cotton,
biofortified sorghum, fungus resistance in table grape,
alternative sugar production pathways in sugar cane,
and cassava with altered starch content.
Regulatory Oversight in South Africa
Concerns regarding the commercial release of GMOs
led to South Africa enacting legislation to regulate
the development, importation, and application of
GMOs. The Genetically Modified Organism Act 1997
(GMO Act) was passed in 1997, and it was subsequently
modified in 2006 to bring it in line with the CPB [66].
Regulations for its implementation were initially
adopted in 1999, and then, amended regulations took
effect in February 2010. The formal structures for the
implementation of this act include an Executive
Council, which reviews applications for GMO work;
a scientific advisory committee (South African Com-
mittee for Genetic Experimentation [SAGENE]);
a registrar to administer the GMO Act; and an inspec-
torate to monitor function. According to the legisla-
tion, no person may import or export from South
Africa, or develop, produce, use, release, or distribute
any GMO in South Africa, other than under a permit
for undertaking such an activity [67]. Such a permit is
to be issued after a technical assessment and risk anal-
ysis report has been submitted by the applicant and has
been approved by the Executive Council. This Council
is responsible for making regulatory decisions and is
comprised of ten members: one representative from
each of eight government ministries (Agriculture,
Science and Technology, Health, Environmental Affairs
and Tourism, Trade and Industry, Labour, Water
Affairs and Forestry, and the Department of Arts and
Culture), the chair of SAGENE who provides scientific
and technical analysis of risk assessment data, and the
GMO Registrar [68]. The GMO Regulations provide
that an applicant shall notify the public of any pro-
posed release of GMOs prior to the application for
a permit for such release. Public notifications shall be
in the form of a standard notice published in the
printed media informing the public of the intended
release. It is worth noting, however, that the first field
trials were allowed in 1994, and since 1997, several
multinational companies have been permitted to
grow and import GMOs even before the GMO Act
was belatedly implemented in November 1999 [63, 69].
Commercialization of GM Crops: South African
Approval Process
Commercial activities concerning GMOs all require
a permit, including those for: import, export,
contained use (including development, production,
distribution, transport, but not those under contain-
ment levels 1 & 2, i.e., in the laboratory or growth
chamber), deliberate release of GMOs into the envi-
ronment (trial and general release), and commodity
clearance [70]. All applications must be submitted to
the GMO Registrar at the DA, along with a copy of the
public notice and proof (newspaper clippings) in order
for the application to be processed. The public notice
allows interested parties to submit comments or objec-
tions in connection with the intended release to the
Registrar within 30 days after the date of the notifica-
tion [71]. The Registrar then undertakes an initial review
of the application to determine compliance with
the provisions of the GMO Act. If the application is not
compliant, the application is referred back to
the applicant. Once compliant, the application is
forwarded to a committee (expertise nominated by
SAGENE chairperson) formed under SAGENE to con-
duct a review of the proposed activity. The review
includes an evaluation of the risk assessment data,
including food safety (if applicable), submitted in the
application. Conclusions of the assessment are detailed
in a recommendation report, which is sent to the
Registrar on completion of the review. At this stage, the
application can be referred back to the applicant to
address any concerns raised or to supply additional
information, and the response returned to the commit-
tee. Once all concerns have been addressed, the commit-
tee makes a recommendation on the application. The
recommendation document, public input, and a copy of
 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
the application is forwarded to the Executive Council
for consideration, who will also take into account
the socioeconomical impact that the GMO may have.
The Council then submits its decision in writing to the
Registrar. Should the Council raise any concerns, the
Registrar will once again refer the application back to
the applicant for clarification. Based on the information
received from the applicant and the assessment done by
the Council, the application will be approved or rejected.
If the application is approved, the Council authorizes the
Registrar to issue a permit to the applicant. This permit
will be accompanied by specific containment conditions
as prescribed by the Council. If the application was
rejected, the Registrar will communicate the decision
back to the applicant with reasons for the rejection
[70]. Regulations of the Department of Health of
2004 provides for the labeling of foodstuff with genet-
ically modified ingredients that are significantly differ-
ent to the non-GMO ingredients. The Consumer
Protection Act of 2008 also addresses this issue.
United States of America (USA)
In the USA, GM crops have been sold since 1994 and in
2006 were already planted on 54.6 million hectares
(soybean, maize, cotton, canola, squash, papaya, and
alfalfa), confirming the USA’s role as the world leader in
agro-biotechnology [10]. The regulatory system in the
USA relative to biotechnology products is rather
different from the one put in place in the EU, and
the discrepancies mainly reflecting the different
approaches taken by the governmental authorities, cit-
izens, and firms toward GMOs and GM food, especially
in the initial years of the biotechnology revolution.
In the USA, agricultural biotechnology politics has
been dominated by a strong and cohesive coalition of
pro-biotechnology upstream and downstream pro-
ducers and farmers. Lower public outrage has made
mobilization of NGOs in the United States difficult
and, in combination with a less-favorable institutional
environment (notably, centralized regulatory policy-
making), has resulted largely in their exclusion from
agri-biotech policy-making [72].
Regulatory Oversight in the USA
Taking the approach that GM products are essentially
an extension of conventional products, the US
483
Government has made use of existing laws to ensure
the safety of GM products [15]. US regulatory author-
ities operate under the assumption that the fact that
a plant has been genetically modified is less important
than the specific effects of the modification [4]; there-
fore, the regulation focuses on the characteristics of the
products rather than the way in which the product was
produced [35]. The current system was delineated by
the White House Office of Science and Technology
Policy under the 1986 Coordinated Framework for
Regulation of Biotechnology [73]. It is still the key
document for regulating gene technology in the United
States and provides the basis for the regulation of crop
varieties produced by rDNA techniques. Under the
Framework, the US Department of Agriculture
(USDA), the Food and Drug Agency (USFDA), and
the Environmental Protection Agency (USEPA) that
were responsible for regulatory oversight of certain
product categories or for certain product uses are also
responsible for evaluating those same kinds of products
developed using genetic engineering techniques. Trans-
portation, growing (including field testing), and prop-
agation of GM crops are governed by the USDA’s
Animal and Plant Health Inspection Service (APHIS)
under the Federal Plant Pest Act 1996 (FPPA) and the
National Environmental Policy Act 1969. Specifically,
the APHIS has two responsibilities: deciding on which
GM seeds to oversee, so-called regulated articles, and
which GM seeds are safe enough to be free from the
agency’s oversight, so-called deregulated articles.
Deciding on GM seeds to be regulated depends on
“familiarity,” gained from direct experience through
field testing under regulated conditions. Any eventual
deregulation, that is, exempting a GMO from the
oversight of APHIS, involves a petition process
whereby an advertisement is published in the US Fed-
eral Register, and a period to comment is provided to
the public [74].
If a GM plant is not intended for human consump-
tion and is not modified to contain a pesticide, the
USDA is the leading agency. For plants genetically
modified to produce their own pest protection,
APHIS coordinates its evaluation with the USEPA.
Pest-resistant GM crops fall under the authority of
the USEPA under the Federal Insecticide, Fungicide,
and Rodenticide Act 1996 (FIFRA) and the Toxic Sub-
stances Control Act 1976. They are subject to a strict
484 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
testing regime, where producers must submit testing
data to the USEPA who determines the quantity of
pesticidal substances that may be present in food
[75]. In fact, industry is required to obtain an Experi-
mental Use Permit (EUP) from the USEPA to test any
pest-resistant plant in a field larger than 10 acres. Once
the permit is granted, firms are expected to consult the
USEPA on the details of the field experiment. While the
APHIS analyzes data on the source of the new gene,
the nature of the pesticidal substance produced, differ-
ences with its natural equivalent, effects on non-target
organisms, and environmental fate, the USEPA focuses
on the toxicology, the digestive fate, and the potential
allergenicity of the toxin. In line with the product-
based approach, the USEPA does not assess the GM
plant per se but the toxin produced by the plant. As for
any pesticides, the USEPA subjects these toxins to
a registration process. Pest-resistant plants whose
toxin falls under this process are mostly of the Bt
variety. Following this registration logic, if the toxin
produced by a GM plant has been approved previously
as a regular pesticide, a new registration is not required.
The USEPA has been requesting for years new regula-
tions to obtain a wider role in the assessment and the
management of GM plants, but thus far with only
limited success [11, 72, 74]. The US Food and Drug
Administration (USFDA) regulates food applications
of GM crops and relies on existing laws that hold food
manufacturers responsible for food safety. Of the three
agencies, the USFDA has had the most influence on
biotechnology policy because most biotechnology
products on the American market are health care or
food products [72]. In 1999, public meetings were
held by the agency with the aim of sharing its experi-
ence regarding GM foods and soliciting views on
whether its policies and procedures should be
modified. Public comments indicated considerable
public support for a mandatory and more transparent
process [4].
Commercialization of GM Crops: US Approval
Process
The APHIS oversees the confined and unconfined
release of transgenic plants as well as any importation
and interstate movement under the FPPA. In addition
to the FPPA, the USDA issued rules in 1987 for the
“introduction of organisms and products altered or
produced through genetic engineering which are
plant pests or which there is reason to believe are
plant pests” [76]. By these rules, the introduction of
a crop produced by rDNA techniques into the environ-
ment is only legal with an authorization by the APHIS.
The APHIS grants a release permit after preparing an
environmental impact assessment and “Finding Of No
Significant Impact” (“FONSI”). Exempt from these
rules are experiments with plants produced by rDNA
technology in a contained environment (e.g., labora-
tory, green house).
In 1997, the USDA simplified the procedure for the
unconfined release of GM crops into the environment
by allowing the applicant to petition the APHIS for
a “determination of non-regulated status” [77]. When
receiving a petition, the APHIS prepares an environ-
mental impact assessment taking into account the fol-
lowing eligibility criteria:
● The crop must not be listed as noxious weed or
weed.
● The introduced genetic material must be stable and
characterized.
● The introduced genetic materials must not
– Result in any plant disease
– Confer an infectious entity or encode toxic
substances to non-target organisms
– Encode products intended for pharmaceutical
use
● Any plant virus-derived sequences must not pose
a significant risk for new plant virus creation.
● The GM crop must be free of known human and
animal pathogens or allergens.
After a complete petition is filed, it is published in
the Federal Register to solicit comments from the
public. Thereafter, the APHIS reviews the data, taking
into account public comments and takes a final deci-
sion, which is again announced in the Federal Register.
The issuance of a “non-regulated” status for a trans-
genic crop means that it is deregulated and can be freely
commercialized in the US (unconfined release, import,
interstate movement), except if it contains a pesticidal
substance. In that case, an additional “plant pesticide”
approval by the USEPA is required.
The responsibility of the USEPA is to evaluate the
risks of GM crops “producing their own pesticide” for
 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
human consumption under the FIFRA. The evaluation
process is held to the same standards as those for
pesticides applied to plants. To be registered under
the FIFRA, a pesticide must not cause “unreasonable
adverse effects” on the environment and on human
health [78]. Transgenic insect- and virus-resistant
plants fall under the jurisdiction of the USEPA; how-
ever, viral coat proteins are normally exempted from
the requirements as the USEPA considers these proteins
as “low-risk applications” based on the principle of
familiarity and their ubiquitous presence in the food
supply. Today, Bt toxins, one viral coat protein, and the
potato leaf roll virus protein are registered as pesticides
and supervised by the USEPA. The agency evaluates the
risks of these “plant-incorporated protectants” by tak-
ing into account the following criteria: toxicological
effects, effects on non-target organisms, insect resis-
tance management, and persistence of the substance
in the environment. The evaluation process lasts
approximately 1 year. If adverse effects of insect- or
virus-resistant plants are observed after commerciali-
zation, the USEPA has the legal power to amend
existing registrations. Moreover, the USEPA may
impose new measures such as new pest resistance
schemes [79].
Besides the pesticide registration under FIFRA,
Section 408 of the FFDCA requires the USEPA to deter-
mine tolerance limits for substances used as pesticides
on and in food and feed [78, 80]. “Nucleic acids that are
part of a plant-incorporated protectant” are exempted
from this requirement because the USEPA considers
them as “safe” [80]. Once approvals from the APHIS,
and from the USEPA when pesticidal substances are
used, have been granted, it is legal to commercialize
the GM plant or derived product in the USA. However,
applicants normally engage in a voluntary consultation
process with the USFDA before marketing of the
GM plant or derived products. This policy is now
under review.
International Obligations Relevant to the
Biosafety Frameworks
Countries do not have complete discretion when decid-
ing how to set up their biosafety regulatory system as
there are several international treaties and agreements
that relate to biosafety. If a country is bound by any or
485
all of those international agreements, then their bio-
safety regulatory system must be compliant with those
obligations [81].
Cartagena Protocol on Biosafety
The conclusion of the CPB was broadly recognized as
a step forward in providing an international regulatory
framework to reconcile trade with environmental pro-
tection by creating an enabling environment for the
environmentally sound application of biotechnology
[82]. The Convention on Biological Diversity (CBD)
[83] in Article 19 [3] states that:
" The Parties shall consider the need for and modalities
of a protocol setting out appropriate procedures,
including, in particular, advance informed agreement,
in the field of the safe transfer, handling and use of and
living modified organism resulting from biotechnology
that may have adverse effect on the conservation and
sustainable use of biological diversity.
In 1995, the second Conference of the Parties
(COP) to the CBD began the consideration of the
need for and modalities of such a protocol. COP Deci-
sion II/5 commenced the negotiations for the protocol
by launching an open-ended ad hoc Working Group on
Biosafety [1]. Meeting six times between 1996 and
1999, the Working Group concluded its work with the
submission of a draft protocol for consideration by the
first extraordinary meeting of the COP, convened with
the purpose of adopting a protocol on biosafety to the
CBD. The result of this first extraordinary meeting,
which took place in two separate meetings in 1999
and 2000, was the adoption of the Protocol [84]. In
accordance with its Article 36, the Protocol was opened
for signature by States and regional economic integra-
tion organizations from 15 to 26 May 2000 and
remained open for signature from 5 June 2000 to
4 June 2001. By that date, the Protocol had received
103 signatures. The Protocol entered into force on
11 September 2003, 90 days after receipt of the 50th
instrument of ratification [85]. There are presently
160 parties to the Protocol [86], with the COP to the
CBD serving as the meeting of the Parties to the Pro-
tocol (COP-MOP), the Protocol’s governing body.
Since the coming into force of the Protocol, the COP-
MOP has met five times. The fifth meeting of the
486 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
COP-MOP took place from 11 to 15 October 2010 in
Nagoya, Japan, and it approved a Supplementary Pro-
tocol on Liability and Redress to the Cartagena Proto-
col on Biosafety [87].
The Protocol’s scope is the “transboundary move-
ment, transit, handling, and use of all living modified
organisms that may have adverse effects on the conser-
vation and sustainable use of biological diversity,
taking also into account risks to human health” [82].
To ensure the safe transfer, handling, and use of GMOs,
the Protocol sets up two separate procedures. The first
time that a GMO is to be intentionally introduced into
the environment, the Protocol sets up an Advance
Informed Agreement (AIA) procedure. That procedure
requires that the exporter of the GMO provides a notice
with detailed information about the GMO to the
importing country. The importing country then
reviews the information, conducts a risk assessment,
and decides, based on the risk assessment results,
whether to approve or reject the GMO. The second
procedure set up by the Protocol is for GMOs to be
used for food or feed or for processing (such as corn,
soybeans, wheat, or other grains that will be fed directly
to humans or animals or used for processing). For
those GMOs, the AIA procedure is not required.
Instead, the Protocol establishes a simpler system that
reflects the decreased likelihood that those GMOs will
affect the importing country’s biodiversity. Before the
GMO can be exported to another country, the only
requirement is that the safety decision in the exporting
country is communicated to other countries through
the Biosafety Clearing-House. For LMOs used for other
purposes, such as LMOs used in the laboratory, the
Protocol leaves any regulation to the discretion of the
individual country. The Protocol also does not cover
products derived from LMOs, such as processed foods
that have ingredients that came from LMOs. Although
the Protocol comprehensively covers many issues, there
are a few remaining to be addressed by the individual
Party when establishing their biosafety regulatory
regime [81].
In addition to the CPB, other relevant international
agreements exist. Under international law, countries
shall comply with all treaties to which they are parties,
provided that the provisions of these treaties are not
contradictory (principle of accumulation of interna-
tional obligations). According to the principle of
integration contained in the Rio Declaration on Envi-
ronment and Development (Principle 4) and the 2002
Plan of Implementation of the World Summit on Sus-
tainable Development (Paragraph 92), environmental
treaties and trade goals shall mutually support each
other. In fact, the various agreements and instruments
on the topic of the development and use of agro-
biotechnology are all closely interrelated and need to
be carefully considered in the decision-making process,
both individually and collectively. This regulatory and
public policy background consists of relevant interna-
tional instruments and processes, such as: the entry
into force of the CPB, the Codex Alimentarius Guide-
lines on Food Safety and Labelling of GMOs, and the
World Trade Organization (WTO) Agreements on
Sanitary and Phytosanitary Measures and Technical
Barriers to Trade. Table 1 presents a summary of the
main international instruments and processes of
relevance for biosafety.
A Comparative Analysis of Regulatory Processes
in All Study Countries
Attributes of a Modern Regulatory Biosafety System
Although the development of regulatory regimes is
essentially based on national specific practices, legal
systems, public management strategies, and local
socioeconomic considerations, national regulatory
biosafety frameworks will generally always consider
the following elements [110–112]:
● Biosafety policy, occasionally as part of a broader
biotechnology or biodiversity policy
● Regulatory policy
● A mechanism to handle requests and permits
related to LMO use, in particular, notifications
required by the CPB (i.e., an administrative system)
● A mechanism for monitoring and inspections
● A system that allows public participation and
information
According to Jaffe [113]:
" The purposes of a national biosafety regulatory system
are to scientifically assess the safety of genetically
engineered (GE) organisms to humans and the envi-
ronment, manage any potential risks, and authorize the
development and marketing of safe GE organisms and
 Commercialisation of GM Crops: Comparison of Regulatory Frameworks 487

Commercialisation of GM Crops: Comparison of Regulatory Frameworks. Table 1 Relevant international instruments

and processes (Taken from [88])

Agreement, declaration,
or process Content
Rio Declaration on The precautionary principle is a fundamental instrument for the safe use of biotechnology.
Environment and This principle is contained in Principle 15, which sets forth that: “In order to protect the
Development environment, the precautionary approach shall be widely applied by States according to their
capabilities. Where there are threats of serious or irreversible damage, lack of full scientific
certainty shall not be used as a reason for postponing cost-effective measures to prevent
environmental degradation”.
United Nations Environment Enacted in Cairo in 1995, these guidelines may assist governments, intergovernmental
Program – International organizations, private organizations, and others to strengthen capacities and exchange
Technical Guidelines for biosafety information.
Safety in Biotechnology
The guidelines are based on the following principles: (a) identification of hazards; (b) risk
assessment, taking into account the probability of any hazards arising and the potential
consequences of such hazards; and (c) risk management, applying adequate management
strategies which include developing procedures and methods to minimize risks and their
consequences, or making decisions not to proceed. Such management strategies shall be
proportional to the risk assessment results.
World Trade Organization The WTO Agreement recognizes the goal of sustainable development, considering that
(WTO) Agreement free trade shall protect and preserve the environment [89]. There are three WTO
Agreements which can be associated with GMOs: The Agreement on the Application of
Sanitary and Phytosanitary Measures (SPS; [90]), the Agreement on Technical Barriers to
Trade (TBT; [91]), and the Agreement on Trade-Related Aspects of Intellectual Property
Rights (TRIPS; [92]). It is also important to understand the regulations and procedures that
govern Dispute Settlement Understanding (DSU) in the WTO context [93].
Agreement on Technical The TBT Agreement is relevant to biotechnological products as it applies to technical
Barriers to Trade (TBT) regulations and rules, including requirements for packaging and labeling [91]. The TBT
Agreement recognizes that no country shall be prevented from taking necessary measures
to ensure the quality of its exports, the prevention of deceptive practices, and the
protection of the environment and human, animal, and plant life or health. However, such
measures shall not be taken as means of arbitrary or unjustifiable discrimination between
countries, where the same conditions prevail, or as disguised restrictions on international
trade. Furthermore, such measures must otherwise comply with the provisions of the
Agreement [91].
The Agreement on the SPS rules are summarized in the following manner [90]:
Application of Sanitary and 1. Members have the right to take necessary sanitary and phytosanitary measures to
Phytosanitary Measures protect human, animal, and plant life or health, provided that such measures are not
(SPS) inconsistent with the provisions of the Agreement.
2. Any sanitary or phytosanitary measures are applied only to the extent that is necessary
to protect human, animal, and plant life or health, based on scientific principles.
Furthermore, such measures cannot be maintained in the absence of sufficient scientific
evidence of their necessity.
3. Members shall ensure that their sanitary and phytosanitary measures do not arbitrarily or
unjustifiably discriminate between members among whom identical or similar conditions
prevail. Moreover, sanitary and phytosanitary measures shall not be applied in a manner
that would constitute a disguised restriction on international trade.
488 Commercialisation of GM Crops: Comparison of Regulatory Frameworks

Commercialisation of GM Crops: Comparison of Regulatory Frameworks. Table 1 (Continued)

Agreement, declaration,
or process Content
4. Sanitary or phytosanitary measures, which are taken in accordance with the relevant
provisions of the Agreement, are presumed to comply with the members’ obligations
under GATT 1994, which relate to the use of sanitary or phytosanitary measures, in
particular, the provisions of Article XX(b) [94].
5. In principle, members shall base their sanitary or phytosanitary measures on
international standards, guidelines, or recommendations, where these exist.
6. Sanitary or phytosanitary measures which comply with international standards,
guidelines, or recommendations are deemed necessary to protect human, animal, and
plant life or health and are presumed to be consistent with the relevant provisions of GATT
1994 [94] and the SPS Agreement [90].
7. Members may introduce or maintain sanitary or phytosanitary measures which result in
a higher level of sanitary or phytosanitary protection than the level that is required by the
relevant international standards, guidelines, or recommendations, provided that there is
a scientific justification to this heightened level of protection or that this level of protection
is determined by a member country to be appropriate according to Article 5 of the SPS
Agreement regarding risk assessment and adequate protection levels [90]. However,
measures which result in a heightened level of sanitary or phytosanitary protection need
not go against any other provisions in the Agreement.
The above-mentioned standards, guidelines, and recommendations are defined as those
established by international organizations, such as the Codex Alimentarius Commission,
the Office International des Epizooties, and the Secretariat of the International Plant
Protection Convention. As for other subjects that are not within the scope of the
aforementioned organizations, they shall be regulated by “other international
organizations” identified by the WTO Committee on Sanitary and Phytosanitary Measures
as being open to the Parties’ membership.
The Codex Alimentarius Created by the World Health Organization (WHO) and the Food and Agriculture
Organization (FAO) of the United Nations, the Codex Alimentarius (http://www.
codexalimentarius.net) is the organization which is in charge of establishing regulations
related to food safety. Codex Alimentarius standards need to ensure fair trade practices in
food trade, according to WTO rules.
The Codex Alimentarius Commission established an Ad Hoc Intergovernmental Task Force
on Food Derived from Biotechnology [95] to handle issues associated with food obtained
through biotechnological processes, particularly food used for health and nutrition
purposes. In July 2003, the Codex Alimentarius Commission held a meeting during which
three risk assessment standards for food resulting from biotechnology were approved [96].
These standards establish risk assessment principles for food derived from modern
biotechnology. The principles refer to the concept of “tracing,” which is a risk assessment
tool whose meaning is the subject of an important debate. In fact, the United States of
America consider this concept to be different from traceability and limit themselves to
following only the previous and subsequent link in the LMO movement chain. The
approved standards are the following:
– Principles for the Risk Analysis of Foods Derived from Modern Biotechnology [97]
– Guideline for the Conduct of Food Safety Assessment of Foods Derived from –
Recombinant-DNA Plants [98]
– Guideline for the Conduct of Food Safety Assessment of Foods Produced Using
Recombinant-DNA Micro-organisms [99]
 Commercialisation of GM Crops: Comparison of Regulatory Frameworks 489

Commercialisation of GM Crops: Comparison of Regulatory Frameworks. Table 1 (Continued)

Agreement, declaration,
or process Content
In 2008, the Ad Hoc Intergovernmental Task Force on Food Derived from Biotechnology
completed a Guideline for the Conduct of Food Safety Assessment of Foods Derived from
Recombinant-DNA Animals [100]. At its 31st session, the Commission noted that the Task
Force had completed its work, one year ahead of schedule, and agreed to its dissolution
[101].
Likewise, the Codex Committee on Food Labelling is working on draft guidelines for the
Labelling of Food and Food Ingredients Obtained through Certain Techniques of Genetic
Modification/Genetic Engineering, as well as on a Draft Amendment to the General
Standard for the Labelling of Pre-packaged Foods (in order to address the issue of
genetically modified foods) [102]. Finally, in 2007, the Codex Alimentarius Commission
adopted the “Working Principles for Risk Analysis for Food Safety for Application by
Governments” that were proposed by the Codex Committee on General Principles [103].
These principles allude to the precautionary approach. For more information on Codex
activities, see www.codexalimentarius.net.
International Plant The IPPC (https://www.ippc.int/) aims to prevent the international spread and introduction
Protection Convention of pests of plants and plant products and to promote appropriate measures for their
(IPPC) control. The Convention was reviewed in 1997 and entered into force in 2005 [104].
A Commission Working Group studied plant pest risks associated to LMO issues and an
international standard (ISPM No.11 pest risk analysis for quarantine pests, including
analysis of environmental risk and LMOs) [105]. This text intends to protect plants and
ecosystems from LMO-related risks. According to the text, such protection measures
should be cost effective, nondiscriminatory, and feasible and should not limit basic trade
needs.
World Organization for The World Organization for Animal Health, also known as the Office International des
Animal Health (OIE) Epizooties (OIE; http://www.oie.int/), is an intergovernmental organization created by the
International Agreement signed on 25 January 1924. OIE’s mission is to ensure the
transparency of animal health conditions worldwide. Member countries promise to declare
animal diseases detected on their territory. In addition, the OIE is in charge of safeguarding
international trade through the elaboration of health regulations destined to be applied to
international transboundary movements of animals and animal products. The WTO
recognizes such regulations as international reference rules.
Regional International In 2000, the Regional International Organization for Plant Protection and Animal Health
Organization for Plant (OIRSA; http://www.oirsa.org) produced a Regional Guideline on the Safety of Plant
Protection and Animal Biotechnology [106]. In fact, the Guideline is based on the CPB and essentially regulates the
Health (OIRSA) same issues as the Protocol. However, in view of its nature, the guideline also aims to
harmonize the various regional laws and practices pertaining to the issue of biosafety.
Central American The Central American Commission for Environment and Development (CCAD), which is the
Commission for environmental authority of the Central American Integration System (SICA), through its
Environment and biodiversity program and territorial legal system, prepared a Central American Protocol on
Development (CCAD) the Safety of Modern Biotechnology. A series of technical consultations was undertaken
and led to the adoption of a draft Protocol, which was then approved by the Central
American Ministries of Environment in 2002. Although the Central American Protocol is
based on the CPB, some of its provisions go beyond the scope of the CPB. In fact,
provisions regarding labeling, documentation, liability, contained use, and transit, as well
as various biosafety principles, have been included in the Central American Protocol in an
attempt to avoid the inconsistencies and ambiguities which resulted from the multilateral
negotiations that lead up to the adoption of the CPB.
490 Commercialisation of GM Crops: Comparison of Regulatory Frameworks

Commercialisation of GM Crops: Comparison of Regulatory Frameworks. Table 1 (Continued)

Agreement, declaration,
or process Content
The Inter-American Institute As requested by the Ministries of Agriculture in 2002, IICA, in collaboration with the OIRSA
for Cooperation in and the Tropical Agricultural Research and Higher Education Centre (CATIE), was put in
Agriculture (IICA) charge of preparing a regional biosafety regulatory framework, in response to the lack of
regulations in certain countries that are receiving, or may potentially receive, requests for
field trials or trade in LMOs. A group comprising of various agencies was formed, along
with a consultative process, which led to the preparation of a Regulatory Framework Draft
for Living Modified Organisms for Agricultural and Livestock Use in Central American
Countries. This initiative intends to promote a model regulation on agricultural
biotechnology that is to be adapted by each country, according to its particular needs and
situation. There is hope that this regulation will be enacted in a similar fashion by the
various nations, thereby triggering a process of harmonization of regulatory frameworks in
the region.
Other relevant agreements – CGRFA draft Code of Conduct on Biotechnology [107]
and declarations – United Nations Model Regulations on the Transport of Dangerous Goods (UN
Recommendations) [108]
– The Aarhus Convention on Access to Information, Public Participation in Decision-
making and Access to Justice in Environmental Matters, which contains specific guidelines
on public participation in LMO-related decision-making processes [109]. The Convention
entered into force in 2001. Although it was enacted by the United Nations Economic
Commission for Europe, the convention is open to other nations. The Convention was
amended in 2005 in order to set out more precise provisions on the deliberate release of
genetically modified organisms, but the amendment has not yet entered into force. The
amendment will enter into force once it has been ratified by at least three-quarters of the
Parties. In September 2007, the amendment had only been ratified by four countries. The
third meeting of the Parties was held in Riga, Latvia, on 11–13 June 2008. The meeting
adopted the Riga Declaration and a strategic plan for the Convention, resolved the issue of
how to calculate ratification of amendments, and renewed the mandates of task forces
dealing with access to justice, electronic information tools, and public participation in
international forums.

their products. To develop such a regulatory system,
a government can use existing laws or develop new
laws. Any national biosafety regulatory system that is
proposed, however, must be functional, protective,
and also comply with international trade standards
that are evolving in recognition of the growing impor-
tance of GE organisms in world affairs.
A national biosafety regulatory system is a regula-
tory regime responsible for assessing and managing the
full range of potential risks that might be posed by
a GMO and its products. It addresses potential risks
to the environment and biological diversity as well as
any food/feed risks or other safety-related issues involv-
ing GMOs (e.g., worker health, drug safety, etc.).
A protective biosafety regulatory system ensures that
any risks from GMOs are managed and allows safe
GMOs to be developed, marketed, and utilized for
their intended purpose. Such a system, however, must
also be functional, which means that it should be
understandable, workable, equitable, fair, adaptive,
and enforceable [114].
Existing biosafety regulatory systems from around
the world reflect, among other things, the type of
government in the country, the politics of the country,
the country’s view on the relative safety of GMOs, and
the country’s regulation of food, agriculture, and envi-
ronmental issues. Establishing those systems required
balancing numerous goals and trading off different
interests. Through an analysis and comparison of
 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
different existing biosafety regulatory systems, how-
ever, one can identify key characteristics and compo-
nents that are generally important to a functional and
protective biosafety regulatory system [113]. Incorpo-
rating each of those characteristics and components in
a functional and protective biosafety regulatory system
involves problem solving because there can be tensions
between the different characteristics.
Jaffe [113] indicates the following attributes of
a system:
● Comprehensive.
● Adequate legal authority to subject each GMO to
a food-safety and environmental risk assessment
approval.
● A clear safety standard.
● Proportionate risk-based reviews.
● Transparent and understandable.
● Participatory.
● Post-approval oversight. A biosafety regulatory
system does not stop its oversight once a GMO
has been approved for a confined field trial or for
a commercial release.
● Flexible and adaptable.
● Efficient, workable, and fair.
Decision-makers are facing an important challenge
because biotechnology and biosafety fields are ever
evolving at a dramatic pace. In this context, overly
precise and detailed regulatory frameworks can easily
become obsolete in a short period of time. In order to
avoid forever having to enact new legislation and pol-
icies, it is preferable to develop a regulatory framework
that takes on the form of a general guide, thus regulat-
ing more specific biosafety aspects by way of by-laws
and other regulations. In fact, this alternative allows
more efficient regulation of future situations but has
the effect of conferring the power of regulating specific
aspects of biotechnology to the executive branch of
government and other administrative institutions,
instead of to the Parliament [115].
General Comments on Evolution of Biosafety
Regulatory Systems
The nature of GM crop regulations around the world
has as much to do with social and political values as
with concerns about health and safety. Consumers’
491
growing awareness of their rights and farmers’ increas-
ing fear of dependence on multinational companies are
symptoms of a deeper concern about values and prior-
ities, the type of environment that people want, the role
of biodiversity, their tolerance to risk, and the price that
people are prepared to pay for regulation. Therefore,
the regulations in the study countries were formed, or
amended, during this period of heightened public
awareness and are a reflection of how the various gov-
ernments have attempted, or not, to address these
concerns. A comprehensive discussion of regulatory
requirements for GM crops at the national and inter-
national levels is a broader topic than can be covered
here, and previous studies have addressed them in
detail [5, 116].
In general terms of regulatory systems, several
approaches have been considered worldwide with
respect to the safe use of modern biotechnology:
(a) certain countries decided to apply preexistent
plant or animal health regulations to GMO-related
issues or to incorporate biosafety provisions into
plant and animal health protection laws; (b) other
countries decided to adopt specific biosafety regula-
tions that need to be enacted by the Parliament, the
executive power (through agreements or resolutions),
or public sector institutions; (c) certain countries
decided to mention biosafety in their environmental
regulations; (d) some countries decided to apply seeds,
pest control, and plant or animal regulations to mod-
ern biotechnology, enacting but a few coordination
rules and specific provisions associated to biosafety.
The original trend of agricultural biosafety regula-
tions focusing on transgenic plants continues to exist in
some countries but is in the process of being replaced
by a broader focus that comprises biosafety regulation
of animals, microorganisms, fish, and forest species.
This progressive expansion of biosafety toward new
areas results from the need to regulate research and
eventual trade of other types of organisms created
through genetic engineering. The CPB, whose scope
extends beyond the issue of plants, and the subsequently
developed regulatory frameworks implementing its
provisions, brought on a gradual increase in the
number and variety of organisms and activities being
regulated. In addition, health authorities have
increased participation in biosafety advisory commis-
sions and committees.
492 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
In view of the complexity of biosafety issues, par-
ticularly issues of risk assessment and risk manage-
ment, and several other issues associated to the
introduction of GMOs into the environment, almost
all of the countries have established a committee or
a national commission to assist decision-makers in
the development of biosafety regulations. The compo-
sition of these committees and commissions varies
considerably in the region, mainly with respect to the
inclusion of the productive sector, consumers, and
non-governmental organizations. However, the trend
points toward the inclusion of these organizations,
even though such an inclusion can potentially lead to
conflicts of interest. Currently, these committees or
commissions are for the most part directly connected
to the regulatory authorities in place. Nevertheless,
independent national commissions (commissions that
are not connected to the regulatory authorities) were
created in some countries and were assigned political
and coordinative functions, leaving the issue of
technical recommendations to be dealt with by other
sector-based authorities established by each country’s
competent Ministry or Secretariat.
The entry into force of the CPB had the effect of
introducing the Ministries of Environment to the bio-
safety debate. For the reasons mentioned in the previ-
ous paragraph, the Ministries of Agriculture have
traditionally been the authorities involved in the deci-
sion-making process regarding GMOs. Gradually,
environmental authorities started demanding and
assuming an active role in the regulation of GMOs in
view of their mandate to protect the environment. This
trend has been strengthened by the incorporation of
biosafety provisions into environmental laws and
regulations. In some cases, GMOs are even subject to
environmental impact assessments (at least based on
a literal interpretation of the pertinent legal provisions).
Comparison of Approval Systems
In Asia, the only major GM crops approved for com-
mercial release are Bt cotton, which is grown commer-
cially in China, India, and Indonesia, and GM corn
recently approved in the Philippines. To date, no
Asian government has given official permission to
plant GM soybeans or rice.
In the context of GM crops, the concept of “bio-
safety” is, in principle, a broad one, covering three
areas: the health safety of humans and livestock, the
safety of the environment (i.e., ecology and biodiver-
sity), and socioeconomic safety (i.e., the economic and
social impact on farmers, consumers, and different
social classes, as well as on trade and economy in
general) [44]. While the biosafety regulations in force
in industrialized countries (e.g., Canada, the European
Union, and the USA) address only the health and
environmental risks and exclude socioeconomic con-
siderations, the regulations in developing countries
(e.g., India, Argentina, SA, and the Philippines) tend
to include all three areas.
Countries have responded differently to the oppor-
tunities presented by GM crops and the potential risks
associated with them (Table 2). The composition of the
“trade off ” between potential benefits and risks in each
case depends upon whether a government adopts
a permissive, precautionary, or prohibitive policy
approach to GM crops. Three basic conditions may
thus trigger application of protective measures: uncer-
tainty, risk, and lack of proof of direct causal link [5].
As major agricultural exporters, Argentina, Canada,
and the USA have each adopted a permissive attitude
very early on, widely authorizing most GM products
for production and consumption, thereby benefiting
from lower production costs and greater export profits.
Regulators in India, Europe, and the Philippines, on the
other hand, have taken a more cautious approach based
on guaranteeing a very low level of risk to human
health and the environment. They have therefore
imposed strict control measures on approval and mar-
keting of GMOs and GM products [5]. While China
had initially moved quickly on the approval of GM
crops for environmental and commercial releases, the
approval process has slowed considerably since 2000,
and strict regulations have been implemented for GMO
imports [15].
Further differences are obvious. Process-based reg-
ulation is the rule in almost all countries that have
developed national biosafety regulatory systems. Even
in countries employing a product-focused RA process,
the scope of regulatory oversight is defined by the
process of genetic modification. Canada is the only
country in which regulatory oversight is triggered
solely by the novelty of the trait(s) expressed by plants,
irrespective of the means by which the novel traits were
introduced – an approach that is most consistent with
 Commercialisation of GM Crops: Comparison of Regulatory Frameworks 493

Commercialisation of GM Crops: Comparison of Regulatory Frameworks. Table 2 Main characteristics of

commercialization approval systems in study countries
Country/region
Element of approval system Argentina Canada China Europe India Philippines S. Africa USA
Biosafety/GMO-specific law/ X X X ✓ X X ✓ X
act
Regulatory trigger: process (A) B Ba B A A A A B
or product (B)
Responsible ministry or A A,H A A or Eb E A A, Ec
government department:
agriculture (A), environment
(E), or health (H)
ERA committee composition: A,C, G,P G A,G A G A,G A,G G
academia (A), commercial (C),
government (G), or public (P)
representatives
Obligatory domestic field ✓ Xd ✓ Xd ✓ ✓e
testing
Obligatory prior approval in X ✓ X ✓ ✓
export country
Compulsory compliance with ✓ ✓ ✓ ✓ ✓ ✓ ✓ Xf
food-safety requirements
Socioeconomic impacts ✓ X Xg X ✓ ✓ X
considered
Compulsory variety ✓ ✓ ✓ ✓
registration
Mandatory post-market X ✓ ✓ X
monitoringh
Time- or spatially restricted ✓ ✓ ✓ X
authorization
Public consultation (days) 30 30i 60 30 ✓
a
In contrast to all other countries, Canada relies on the concept of novelty to trigger regulatory oversight and has declared that all plants
derived through genetic modification are considered novel
b
The European Commission sends its draft approval to the Council of Ministers (agricultural or environmental ministers), which has three
months to reject or adopt it. If they do not act within this time, the Commission may adopt its own decision and authorize the new GM
product
c
For those GM plants producing their own pesticide, the evaluation is coordinated between APHIS and the USEPA
d
Where data from field studies on other continents are supplied, the applicant should submit a reasoned argument that the data is
applicable to domestic conditions
e
For local applications only, not for applications for import
f
Developers of GM crops engage in a voluntary, but recommended, consultation process with the USFDA (voluntary pre-market review).
This process is currently under review
g
Taking socioeconomic considerations into account during the risk assessment process is not legally required in China [30]
h
✓ – Mandatory requirement for approved post-marketing monitoring plans and reporting. X – No specific approval requirement, but the
developer is expected to monitor for existing and emerging risks that may be associated with its product and notify the regulatory
authorities whenever new information is uncovered
i
Thirty-day time period is provided for public consultation after the formal approval
494 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
the scientific principle that the risks associated with
GM crops are not inherently different than those
posed by more conventional crops [117, 118]. Indeed,
the US National Research Council has explicitly
recommended using objective compositional changes,
not breeding method, as the basis for regulatory scru-
tiny and even then, only “when warranted” [119].
India, Argentina, Canada, most EU countries, and
South Africa have all used non-statutory guidelines to
manage the environmental impact of GM crops before
promulgating new acts or regulations. There is no
evidence that this approach has ever compromised
environmental safety. India is the exception among
the study countries in locating its biosafety decision-
making authority solely within the ministry responsible
for the environment, while the Ministry of Agriculture
predominates among the remaining study countries.
As a rule, environment ministries have a more
precautious or preventative approach to introducing
new technologies, as compared to those with the
responsibility for agriculture. Different structural
approaches are used to secure the necessary scientific
advice for the decision-making process. The EU has
implemented a system of expert advisory committees,
while others, such as India, Canada, and the USA, rely
primarily on scientists and professionals working
within government departments and agencies. Other
countries, e.g., Argentina, China, and South Africa,
have a combination of both. Only India and the EU
mandate post-market monitoring in attempts to gauge
the impacts of the introduction of GM crops over
the long-term as well as larger spatial scales. Other
countries may address this indirectly by authorizing
time-limited or geographically limited introductions
(e.g., Canada), whereas the USA does neither.
Conclusion and Future Directions
Governments have an important role in ensuring that
novel foods are safe for human consumption and that
novel agricultural inputs do not cause major negative
impacts on the environment and long-term agricul-
tural production. The adoption of biosafety regulatory
frameworks is a challenging task since decision-
makers are faced with numerous difficulties, such as
ever-evolving technology, which can quickly render
specific regulations obsolete. Countries and policy-
makers are responding to these various problems with
different legislative and policy-based strategies. As seen
above, most countries, with the notable exception of
the USA, consider GM crops to be novel foods, regard-
less of the characteristics of their final product. Hence,
new laws and institutions to regulate potential bio-
safety and food-safety issues have and continue to be
established, requiring that GM products be approved
before they may be grown in, consumed in, or
imported into a country. Concurrently, public opinion
in many parts of the world still regards the use of GM
crops as controversial. Concerns about new risks have
led biosafety, food-safety, and labeling regulations to
become complex and costly, with no tiered mecha-
nisms in place to regulate the various and different
GMOs based upon the level of risk presented and the
amount of regulatory experience gained with similar
products. As a result, regulation has evolved which
implicitly assumes that all GMOs have the ability to
present the same (high) risk unless proven otherwise,
requiring the over-production of data of questionable
value to decision-making. This regulatory position has
become a real threat to the future development of GM
crops in the non-corporate and public sectors, espe-
cially for those subsistence crops involving tolerance to
abiotic stress and higher nutrient contents being spe-
cifically developed for the benefit of farmers and con-
sumers in the developing world. Should regulatory
safety standards be set to an impossibly high threshold
(e.g., at zero risk), these GM crops are unlikely to be
approved in those countries who stand to gain most
from their potential benefits.
The requirements of setting up effective and effi-
cient regulations and legislative systems pertaining to
products of rDNA technology inevitably involve addi-
tional costs, e.g., the development and maintenance of
institutions, procedures, and management tools, costs
which many developing countries cannot afford. Even
should developing countries decide to form their reg-
ulatory frameworks by adapting regulatory guidance
already implemented elsewhere, cost sharing still
carries a financial burden. Once a system is in place,
other relevant costs include the cost of compliance with
biosafety regulations and risk management conditions,
as well as the economic, environmental, and health
costs related to delayed access to new technologies
and products and their associated benefits. The variety
 Commercialisation of GM Crops: Comparison of Regulatory Frameworks
and disparity of potential frameworks call for a nor-
malization of information requirements and, wherever
synergies and cooperation mechanisms are promoted,
a harmonization of the scientific and technical aspects
of regulatory oversight at the national and subregional
level. As such, an expanded use of internationally
accepted consensus can promote the acceptance of
regional approaches to regulation. Examples include
OECD documents on scientific aspects of risk assess-
ment, as well as guidelines issued by international stan-
dard-setting bodies such as the International Plant
Protection Convention and the Codex Alimentarius.
Programs that create networks on regional and subre-
gional levels may also facilitate the acquisition and dis-
semination of biosafety expertise at reduced costs.
Providing useful, relevant information is a worthwhile
task, but it can also prove expensive to provide training
on how best to utilize such information in decision-
making. It is therefore imperative that external funding
streams continue to be directed toward supporting the
establishment of effective regulatory systems in develop-
ing countries that respond to national needs and poli-
cies, until such time that they become self-sustainable.
Disclaimer
Opinions and views expressed in this chapter are
strictly those of the authors and may not necessarily
represent those of the organizations where the authors
are currently employed.
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 Crop Breeding for Sustainable Agriculture, Genomics Interventions in 501

Crop Breeding for Sustainable Gene pyramiding Gene pyramiding is a process of

Agriculture, Genomics
Interventions in
PAWAN L. KULWAL1, MAHENDAR THUDI2
RAJEEV K. VARSHNEY2,3,4
1 Genome-wide selection or genomic selection is
State Level Biotechnology Centre, Mahatma Phule
Agricultural University, Rahuri, Maharashtra, India
2 for complex traits in elite genotypes by utilizing
Centre of Excellence in Genomics (CEG),
International Crops Research Institute for
the Semi-Arid Tropics (ICRISAT), Patancheru,
Hyderabad, AP, India
3 focused on few individual genes or few QTLs to
Genomics Towards Gene Discovery Sub programme,
Generation Challenge Programme (GCP), Generation
Challenge Programme (GCP) c/o CIMMYT, Mexico,
DF, Mexico
4 Genomics-assisted breeding (GAB) Genomics-
School of Plant Biology, Faculty of Natural and
Agricultural Sciences, The University of Western
Australia, Crawley, WA, Australia
Article Outline
Glossary
Definition of the Subject
Introduction and Importance of Sustainable Agriculture
Contribution of Plant Genomics Technologies to Crop
Breeding
Some Modern Breeding Approaches
Challenges in Adoption of Genomics Technologies
Future Directions
Acknowledgments
Bibliography
Glossary
Association mapping Association mapping is a high-
resolution method for mapping quantitative trait
loci (QTLs) or gene(s) for traits of interest based on
linkage disequilibrium (LD) and holds great prom-
ise for the dissection of complex genetic traits.
Back cross (BC) Back cross is a cross of the F1 with
either of the parental genotype and the resultant
progeny is called BC1. The progeny of the cross
between BC1 and the recurrent parent is called
as BC2.
accumulating the favorable genes/alleles from dif-
ferent genotypes into an elite/commercial cultivar.
Gene pyramiding is often performed through
marker-assisted selection (MAS).
Genome-wide selection or genomic selection (GS)
a concept for accelerating genetic gain especially
genomic information and estimating their breeding
values in breeding strategies. GS is becoming very
popular over marker-assisted selection that was
improve genotypes, especially when recent
advances in genomic technologies have drastically
reduced the cost on marker genotyping.
assisted breeding is a holistic approach, where
genomics technologies including molecular
markers, trasncriptomics, metabolomics, proteo-
mics, bioinformatics, and phenomics are integrated
with conventional breeding strategies for breeding
crop plants resistant/tolerant to biotic and abiotic
stresses or improved for quality and yield.
Haplotype Haplotype is a set of alleles of closely linked
loci on a chromosome that tend to be inherited
together.
Linkage disequilibrium (LD) Linkage disequilibrium
is a nonrandom association of alleles at different
loci, describing the condition with non-equal
(increased or reduced) frequency of the haplotypes
in a population at random combination of alleles at
different loci. LD is not the same as linkage,
although tight linkage may generate high levels of
LD between alleles.
Marker-assisted selection (MAS) Marker-assisted
selection is a process of indirect selection for
improving the traits of interest by employing mor-
phological, biochemical, or DNA-based markers.
DNA-based markers/molecular markers, in the
recent past, were proven to be the markers of choice
for MAS.
Narrow genetic base Narrow genetic base does fre-
quently exists in modern crop cultivars or breeding
lines due to the continuous use of small number of
elite genotypes in breeding programs. In fact, it is

P. Christou et al. (eds.), Sustainable Food Production, DOI 10.1007/978-1-4614-5797-8,

# Springer Science+Business Media New York 2013

Originally published in
Robert A. Meyers (ed.) Encyclopedia of Sustainability Science and Technology, # 2012, DOI 10.1007/978-1-4419-0851-3
502 Crop Breeding for Sustainable Agriculture, Genomics Interventions in
a serious obstacle to sustain and improve crop
productivity due to rapid vulnerability of geneti-
cally uniform cultivars to emerging biotic and
abiotic stresses.
Next-generation sequencing (NGS) technologies
Next-generation sequencing (NGS) technologies
include various novel sequencing technologies for
example 454/FLX (Roche Inc.), ABI SOLiD
(Applied Biosystems), Solexa (Illumina Inc.), etc.,
that have surpassed traditional Sanger sequencing
in through-put and in cost-effectiveness for gener-
ating large-scale sequence data.
Polygenes Polygenes are a group of non-allelic genes,
each having a small quantitative effect, that together
produce a wide range of phenotypic variation.
Quantitative trait loci (QTLs) Quantitative trait loci
are the loci or regions in the genome that contribute
towards conferring tolerance to abiotic stresses
(e.g., drought, salinity) or resistance to biotic
stresses (e.g., fungal, bacterial, viral diseases) or
improving agronomic traits (e.g., yield, quality)
which are generally controlled by polygenes and
greatly depend on gene  environmental (G  E)
interactions.
Sustainable agriculture Sustainable agriculture refers
to efficient agricultural production while
maintaining the environment, farm profitability,
and prosperity of farming communities.
Sustainable development Sustainable development is
defined as balancing the fulfillment of human needs
with the protection of the environment so that
these needs can be met not only at the present
time, but also in the future.
Definition of the Subject
There has been significant improvement in production
and productivity of important cereal crops globally
as a consequence of the “Green Revolution” and
other initiatives [1]. However, today the stage has
reached that the available traditional methods of crop
improvement are not sufficient to provide enough and
staple food grains to the constantly growing world
population [2]. This situation is projected to be worse
by the year 2050, especially in context of climate change
[3]. In other words, the conventional plant breeding
practices may not be able to achieve the sustainability
in today’s agriculture.
It is under such circumstances that advances in
plant genomics research are opening up a new era in
plant breeding, where the linkage of genes to specific
traits will lead to more efficient and predictable breed-
ing programs in future. Several initiatives have been
started towards use of genomics technologies in num-
ber of crop plants to ensure the sustainable production
of healthy and safe crops and the results are encourag-
ing. It is therefore expected that the genomics will be
the integral part of the agricultural/plant breeding
practices in future for improving crop productivity
leading to achieve food security and sustainable
production.
Introduction and Importance of Sustainable
Agriculture
The goal of agricultural science is to increase crop
productivity coupled with the quality of the products,
and maintain the environment [1]. Food security is
a growing concern worldwide, and more than 1 billion
people are estimated to lack sufficient dietary energy
availability [2]. The issue of “food security” has become
so important that prominent scientific journals,
including Science, have also published a special issue
on this subject recently (February 12, 2010 issue). With
the current rate of growth, the global population is
likely to plateau at some 9 billion people by roughly
the middle of this century [3]. With this ever-
increasing human population and amidst the fear of
shrinking resources in terms of cultivable area, irriga-
tion resources, newly emerging insect pests, stagnated
yields, etc., it has become difficult to maintain agricul-
tural sustainability. In order to make today’s agricul-
ture sustainable, it is necessary that plant breeders
adopt innovative technologies that can increase
the efficiency of selection with more precision [4].
Under such circumstances, molecular approaches
including modern genomics and genetic engineering
technologies have emerged as powerful tools to assure
rapid and precise selection for the trait(s) of interest.
Maintaining effective and environmentally friendly
agricultural practices is a necessary prerequisite for
maintaining sustainability.
 Crop Breeding for Sustainable Agriculture, Genomics Interventions in 503

Plant genomics is a rapidly developing field, which Contribution of Plant Genomics Technologies
is radically improving our understanding of plant biol- to Crop Breeding
ogy by making available novel tools for the improve-
Plant genomics technologies have contributed
ment of plant properties relevant to sustainable
immensely in today’s agriculture which has led to
agricultural production. Recent advances in high-
better understanding of how plants function, and
throughput genomics technologies, including that of
how they respond to the environment. This has also
next-generation sequencing and high-throughput
helped in achieving targeted objectives in breeding pro-
genotyping, have helped immensely in understanding
grams to improve the performance and productivity of
the functions and regulation of genes in crop plants [5].
crops. The DNA-based molecular markers have facili-
The ever-increasing availability of genome sequences in
tated smarter and knowledge-based breeding, by
crop plants have facilitated greatly the development of
enabling early-generation selection for key traits, thus
genomic resources that will allow us to address biolog-
reducing the need for extensive field selection. Besides
ical functions and a number of basic processes relevant
this, the molecular tools can effectively be used for the
to crop production leading to sustainable agriculture.
characterization, conservation, and use of genetic
One of the myths linked to sustainable agriculture
resources.
means going back to past techniques/farm practices,
Recent advances made in the area of molecular
which were followed by our ancestors. In fact, sustain-
biology and bioinformatics offer substantial opportu-
able agriculture can be achieved by combining some of
nities for enhancing the effectiveness of classical plant
the wisdom of past practices with careful use of current
breeding programs. These tools can be integrated into
technology, including the vast array of information
breeding work in order to analyze efficiently high num-
technologies now available. Sustainable agriculture is
bers of crosses at the early seedling stage. This approach
a key element of sustainable development and is essen-
is known as “genomics-assisted breeding” [6]. Through
tial to the future well-being of the human race and the
this approach, both the phenotype and the genotype of
planet. A compelling need exists for restorative and
new varieties can be analyzed and the performance of
sustainable agriculture to help address the pressing
new specific introgressed traits can be predicted. The
trends of population, climate, energy, water, soil, and
goals of the integration of these technologies in classical
food. Sustainable agriculture needs to be economically
breeding are to create genotype-to-phenotype trait
viable, environmentally sound, and socially acceptable.
knowledge for breeding objectives and to use this
In other words, it is a system of agricultural production
knowledge in product development and deployment
that, over the long term, will: (1) satisfy human food,
for the resource poor farmer.
feed, and fiber needs; (2) enhance the environmental
For successful utilization of genomics-assisted
quality and the natural resource base upon which the
breeding approach in a crop, availability of basic
agricultural economy depends; (3) make the most effi-
molecular tools, such as molecular markers, genetic
cient use of available technologies, nonrenewable
maps, etc., is a prerequisite. Among molecular markers,
resources, and on-farm resources, and integrate,
though a variety of molecular markers such as restric-
where appropriate, natural biological cycles and con-
tion fragment length polymorphism (RFLP), randomly
trols; (4) sustain the economic viability of farm opera-
amplified polymorphic DNA (RAPD), microsatellite
tions; and (5) enhance the quality of life for farmers
or simple sequence repeat (SSR), amplified fragment
and society as a whole.
length polymorphism (AFLP), and single nucleotide
There are various components of sustainable agri-
polymorphism (SNP) markers have been developed
culture, which include technological interventions, and
in a range of crops, SSR and SNP markers have emerged
environmental and socio-economic factors. As the fac-
as the markers of choice [7, 8]. Because of advent of
tors related to socio-economics and environments have
NGS technologies [5] and high-throughput
been discussed in a number of reviews earlier, in this
genotyping platforms, SNP marker system and array-
article, we focus on the interventions of plant genomics
based genotyping platforms are becoming more
technologies in crop breeding.
504 Crop Breeding for Sustainable Agriculture, Genomics Interventions in
popular [9, 10]. An overview on availability of genomic
resources in some selected important crop species is
shown in Table 1. It is evident that cereal crops, espe-
cially rice, maize, wheat, barley, etc., are on top in terms
of availability of genomic resources (see [11]). Genome
sequences have already become available for several
crop species, including rice (http://rgp.dna.affrc.go.jp/
IRGSP/), sorghum [12], and maize (http://gbrowse.
maizegdb.org/cgi-bin/gbrowse/maize/). Recent invest-
ments coupled with advances in genomics technologies
have contributed towards developing a good resource
of genomics tools in legumes as well [13, 14].
Some Modern Breeding Approaches
The availability of genomic resources in almost all
important crops combined with information on
pedigrees as well as optimized methods of precise
phenotyping make it possible to undertake genomics-
assisted breeding approaches for crop improvement.
In fact, some molecular breeding approaches like
advanced backcross QTL (AB-QTL) analysis and
marker-assisted selection (MAS) have been successfully
employed in several crops, leading to improved culti-
vars, some other approaches such as marker-assisted
recurrent selection (MARS) or genomics selection (GS)
are being used in several crops [15, 16].
Marker-Assisted Selection (MAS)
There are three major steps involved in MAS: (1) identi-
fication of molecular marker(s) associated with trait(s)
of interest to breeders, (2) validation of identified
marker(s) in the genetic background of the targeted
genotypes to be improved, and (3) marker-assisted
backcrossing (MABC) to transfer the QTL/gene from
the donor genotype into the targeted genotype. In
context of marker-trait association, linkage mapping
has been extensively used for identifying the markers
associated with a trait of interest in a range of crops
including cereals, legumes, horticultural crops,
etc. These studies have been reviewed in detail in sev-
eral reviews [14, 17] and books [18]. Although hun-
dreds of studies have been undertaken, only a few
studies were taken further to marker validation and
MABC. This may be attributed to (1) identification of
few markers associated with small-effect QTLs,
(2) non-validation of markers in elite genotypes, and
(3) slow adoption of markers by breeders in their
breeding programs. Recent advances in association
genetics, however, offer opportunities to overcome
the first two constraints.
Association mapping (AM) is considered an alter-
native strategy to linkage mapping for identifying
marker-trait associations and has been used extensively
in human and animal systems. AM has a number of
advantages over linkage mapping, including the poten-
tial for increased QTL resolution and an increased sam-
pling of molecular variation (for reviews see [19, 20]).
AM involves studying a natural population rather than
the offspring of crosses, and associations in natural
populations are typically on a much finer scale because
they reflect historical recombination events. Several
examples on marker-trait association using AM are
available [21]; however, there is a need for optimization
of more advanced analytical tools in the area of associ-
ation genetics [22]. It is anticipated that because of
reduction in costs on marker genotyping [10], AM
will be extensively used for trait mapping in the future.
Once the markers associated with a trait of interest
are identified through linkage mapping or AM, the next
step is to use these markers in the breeding programs.
In this context, the selection of one or a few genes
(QTLs) through molecular markers using backcrossing
is a very efficient technique [23, 24]. Important advan-
tages of MAS are that it can be effectively utilized for
traits with low heritability; for gene pyramiding, selec-
tion can be made at seedling stage; and, above all, there
are no issues involving GE crops [25]. Although use of
markers in breeding programs through MABC is
a common practice in the private sector [26], MAS is
in routine use in wheat and barley breeding programs
in Australia [8, 27–29] and USA (www.maswheat.
ucdavis.edu; http://barleycap.cfans.umn.edu/). Never-
theless, there are several success stories in many crops
including wheat, rice, barley, maize, soybean, etc., where
MAS has successfully been utilized to develop superior
lines/varieties/hybrids for improving quality, resistance to
diseases or tolerance to abiotic stresses. For example,
Gupta et al. [17] has recently summarized success stories
of molecular breeding in wheat.
A widely discussed success story of molecular
breeding is the introgression of the FR13A Sub1 locus
conferring resistance against submergence in an Asian
rice cultivar, Swarna [30, 31] that can confer tolerance
 Crop Breeding for Sustainable Agriculture, Genomics Interventions in 505

Crop Breeding for Sustainable Agriculture, Genomics Interventions in. Table 1 Genomic resources among selected
cereals and legumes
Genome
Molecular markers Molecular maps (Genetic/QTL map/ Transcript data and sequence
Crop plant (SSRs and SNPs) comparative/physical maps) expression profiling data
Rice ++++a ++++b,c ++++d ++++e
Maize ++++f ++++b,g,h ++++d ++++i
Wheat +++j +++b,k,l +++d ++m
Sorghum +++n ++++b,o +++d ++++p
Barley +++q +++r +++d,s +++t
Soybean ++++u,v +++w,x,y +++z ++++aa
Groundnut ++bb +cc +z
Cowpea +++bb ++cc +z
Common ++bb ++cc +z
bean
Chickpea +++bb ++cc +z
Pigeonpea +++bb
+, Very few; ++, Few; +++, Moderate; ++++, Abundant
a
http://www.gramene.org/markers/index.html
b
http://www.gramene.org/cmap/
c
http://www.gramene.org/db/qtl/qtl_display?query=&search_field=&species=Oryza+sativa&submit=Submit
d
http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summary.html; http://compbio.dfci.harvard.edu/tgi/plant.html
e
http://www.gramene.org/Oryza_sativa/Info/Index
f
http://www.maizegdb.org/probe.php
g
http://www.maizegdb.org/map.php
h
http://www.gramene.org/db/qtl/qtl_display?query=*&search_field=trait_name&species=Zea+mays+subsp.+mays&submit=Submit
i
http://www.maizesequence.org/Zea_mays/Info/Index
j
http://wheat.pw.usda.gov/cgi-bin/graingenes/browse.cgi?class=marker
k
http://wheat.pw.usda.gov/GG2/maps.shtml#wheat
l
http://wheat.pw.usda.gov/cgi-bin/graingenes/quickquery.cgi?query=qtls&arg1=*
m
http://wheat.pw.usda.gov/cgi-bin/graingenes/search.cgi?class=sequence
n
http://www.gramene.org/db/markers/marker_view?
marker_name=*&marker_type_id=&taxonomy=sorghum&action=marker_search&x=0&y=0
o
http://www.gramene.org/db/cmap/map_set_info?species_acc=sorghum&map_type_acc=-1
p
Paterson et al. [12]
q
http://wheat.pw.usda.gov/GG2/Barley/
r
http://wheat.pw.usda.gov/GG2/maps.shtml#barley
s
http://wheat.pw.usda.gov/cgi-bin/graingenes/browse.cgi?class=sequence&query=barley1_*
t
http://www.public.iastate.edu/imagefpc/IBSC%20Webpage/IBSC%20Template-home.html
u
http://soybeanbreederstoolbox.org/
v
http://soybase.org/BARCSOYSSR/index.php
w
http://lis.comparative-legumes.org/cgi-bin/cmap/viewer?changeMenu=1
x
http://soybeanbreederstoolbox.org/search/search_results.php?category=QTLName&search_term=
y
http://soybeanphysicalmap.org/
z
http://lis.comparative-legumes.org/lis/lis_summary.html?page_type=transcript
aa
http://www.phytozome.net/cgi-bin/gbrowse/soybean/?name=Gm09
bb
See Varshney et al. [13]
cc
See Varshney et al. [14] marker assisted recurrent selection (MARS) or genomic selection (GS) are being used in several crops [15, 16].
506 Crop Breeding for Sustainable Agriculture, Genomics Interventions in
up to 2 weeks of complete submergence. This has
offered great relief to the large number of Asian farmers
whose rice land is located in deltas and low-lying areas
that are at risk from flooding during the monsoon
season every year. Some selected examples of molecular
breeding in rice and wheat (adopted from [17]) are
summarized in Table 2.
Advanced Backcross QTL (AB-QTL) Analysis
Although MAS has been quite successful, it has always
been a difficult task to tackle linkage drag, especially
when a QTL or a gene is to be introgressed from wild/
exotic species. Furthermore, in MAS, QTL/gene discov-
ery and variety development are two separate processes.
To deal with this problem and to harness the potential
of the wild/unadapted germplasm in breeding pro-
grams, a new approach referred as advanced backcross
QTL (AB-QTL) analysis was proposed by Tanksley and
Nelson [32]. AB-QTL aims at simultaneous detection
and transfer of useful QTLs from the wild/unadapted
relatives to a popular cultivar for improvement of
a trait. In this context, a superior cultivar/variety is
crossed with a wild species leading to the production
of a backcross population (BC2, BC3), and molecular
markers are used to monitor the transfer of QTLs by
conventional backcrossing. The advanced backcross
approach has already been successfully utilized in dif-
ferent crops, including tomato [59], rice [60, 61], bar-
ley [62], and wheat [63]. It is anticipated that the use of
AB-QTL will be accelerated in a range of crops for
improving important traits such as disease resistance
as well as yield traits.
Marker-Assisted Recurrent Selection (MARS)
In the majority of traits of interest, quantitative varia-
tion is controlled by many QTLs, each with minor
effect. Moreover, minor QTLs show an inconsistent
QTL effect in different environments and over different
seasons. Even when the effect of these minor QTLs is
consistent, their introgression into the desired geno-
type through MABC becomes extremely difficult as
a larger number of progenies are required to select
appropriate lines. In such cases, MARS has been pro-
posed for pyramiding of superior alleles at different
loci/QTLs in a single genotype [64, 65].
It was demonstrated in recent studies that the
response of MARS is larger when prior knowledge of
the QTLs exists and the response decreases as the knowl-
edge of the number of minor QTL associated with the
trait decreases [66]. In sweet corn, MARS was employed
to fix six marker loci in two different F2 populations
which showed an increase in the frequency of marker
allele from 0.50 to 0.80 [64]. Similarly, in a separate
study, enrichment of rust resistance gene (Lr34/Yr18)
with an increase in frequency from 0.25 to 0.60 was
reported in wheat BC1 through MARS [28]. MARS can
be utilized effectively for selection of traits associated
with multiple QTLs by increasing the frequency of
favorable QTLs or marker alleles. Several companies
are using MARS in their maize, soybean, etc., breeding
programs [66, 67]. Recently, some institutes like Interna-
tional Crops Research Institute for the Semi-Arid Tropics
(ICRISAT), the French Centre for International Agricul-
tural Research (CIRAD), and the University of California-
Riverside, USA, have also initiated MARS programs in
chickpea (PM Gaur, personal communication 2010), sor-
ghum (J-F Rami, personal communication 2010), cowpea
(J Ehlers, personal communication 2010), etc., for
pyramiding favorable drought-tolerant alleles.
Genome-Wide or Genomic Selection (GS)
Although MAS has been practiced for the improve-
ment of quantitative traits, it has its own limitations.
Therefore, in addition to MARS, Genomic Selection
can be used to pyramid favorable alleles for minor
effect QTLs at the whole genome level [68, 69]. Geno-
mic selection predicts the breeding values of lines in
a population by analyzing their phenotypes and high-
density marker scores. A key to the success of GS is that,
unlike MABC or MARS, it calculates the marker effects
across the entire genome that explains the entire pheno-
typic variation. In simple terms, genome-wide selection
refers to marker-based selection without significance
testing and without identifying of a subset of markers
associated with the trait [68]. The genome-wide marker
data (marker loci or haplotypes) available or generated
on the progeny lines, therefore, are used to calculate
genomic estimated breeding values (GEBV) as the sum
of the effects of all QTLs across the genome, thereby
potentially exploiting all the genetic variance for a trait
[68, 69]. The GEBVs are calculated for every individual
 Crop Breeding for Sustainable Agriculture, Genomics Interventions in 507

Crop Breeding for Sustainable Agriculture, Genomics Interventions in. Table 2 Some examples of improved cultivars
or varieties developed through marker-assisted introgression of important genes/QTLs in rice and wheat

Variety
Genes/QTL developed/
Crop introgressed Function released Reference
Rice GBSS Unique cooking and processing Cadet and [33]
quality traits including amylose Jacinto
content
Xa33t Bacterial blight resistance BC3F2 [34]
Xa21 Bacterial blight resistance Zhongyou 6 [35]
and Zhongyou
1176
Sub1 Submergence tolerance BC3F2 [30]
Sub1 Submergence tolerance Samba [36]
Mashuri-Sub1
IR64-Sub1
TDK1-Sub1
CR1009-Sub1
BR11-Sub1
SUB1QTL Submergence tolerance Sub1 [37]
introgression
lines
Piz-5+Xa21 Blast and bacterial blight BC4F2 [38]
resistance
Xa4+xa5 and Bacterial blight resistance Angke and [39]
Xa4+Xa7 Conde
xa7 and Xa21 Bacterial blight resistance Zhenshan97  [40]
Minghui 63
xa13 and Bacterial blight resistance, strong Pusa1460, [41]
xa21 aroma IET18990
xa13 and Bacterial blight resistance Improved Pusa [42]
Xa21 RH1
xa5, xa13 and Bacterial blight resistance Improved [43]
Xa21 PR106
Xa5, xa13 and Bacterial blight resistance BC3F2 [44]
xa21
Xa5, Xa13 Bacterial blight resistance IET19046 http://www.drricar.org/four_varieites.htm
and Xa21
Xa4, xa8, Bacterial blight resistance BC1F3 [45]
xa13 and
Xa21
Xa4, Xa5, Improved bacterial blight Pusa1526–04– http://www.iari.res.in/?q = node/233
Xa13 and resistance 25
Xa21
508 Crop Breeding for Sustainable Agriculture, Genomics Interventions in

Crop Breeding for Sustainable Agriculture, Genomics Interventions in. Table 2 (Continued)

Variety
Genes/QTL developed/
Crop introgressed Function released Reference
– Bacterial blight resistance Xieyou218 [46]
QTL Drought-tolerant aerobic rice MAS946–1 www.hindu.com/2007/11/17/stories/
2007111752560500.htm
qSALTOL and Enhanced salt and submergence F6 http://open.irri.org/sabrao/images/
qSUB1 tolerance stories/conference/site/papers/
apb09final00098.pdf
QTL Improved performance under Birsa http://claria13.securesites.net/News/
drought VikasDhan111 releases/2009/may/26018.htm
(PY 84)
Wheat QPhs.ccsu- Preharvest sprouting tolerance BC3F3 [47]
3A.1 and Lr24 and leaf rust resistance
+Lr28
Lr47 Resistance to leaf rust BIOINTA2004 [48]
Gpc-B1 High grain protein content Lillian [49]
Qfhs.ndsu- Resistance to fusarium head blight Bena [50]
3AS
Sm1 Resistance to the insect orange Goodeve [51]
blossom wheat midge
Stb4 Resistance to Septoria Kern Cited from [17]
Wsm-1 Resistance to wheat streak mosaic Mace [52]
virus (WSMV)
Yr15 Seedling stripe rust BC3F2:3 [53]
Qss.msub-3BL Resistance to wheat stem sawfly McNeal, http://www.wheatworld.org/pdf/
Reeder, Hank dubcovsky.pdf
Bdv2 Resistance to yellow dwarf virus Above, http://www.wheatworld.org/pdf/
Avalanche, dubcovsky.pdf
Ankor
Yr17 and Lr37 Stripe rust and leaf rust resistance Patwin [54]
CreX and CreY Cyst nematode resistance F3 progenies [55]
Yr36 and Gpc- Resistant to stripe rust and high Westmore [56]
B1 grain protein content
Yr17 and Yr36 Resistance to stripe rust Lassik Cited from [17]
Lr19 and Sr25 Resistant to stem rust race UG99 UC1113 [57]
(PI638741)
Yr36 and Gpc- Resistance to stripe rust, high grain Farnum http://www.ars-grin.gov/cgi-bin/npgs/
B1 protein content (WA7975) acc/display.pl?1671746
Yr15 and Gpc- Resistance to stripe rust and high Scarlet http://css.wsu.edu/Proceedings/2005/
B1 grain protein content (WA7994) 2005_Proceedings.pdf
Lr1, Lr9, Lr24, Leaf rust resistance BC1F2 [58]
Lr47
 Crop Breeding for Sustainable Agriculture, Genomics Interventions in
of the progeny based on genotyping data using a model
that was “trained” from the individuals of another
training populations having both phenotyping and
genotyping data. These GEBVs are then used to select
the progeny lines for advancement in the breeding cycle.
Thus GS provides a strategy for selection of an individ-
ual without phenotypic data by using a model to predict
the individual’s breeding value [69].
Recently, Wong and Bernardo [70] simulated the
comparative responses of phenotypic selection (PS),
MARS, and GS with small population sizes in oil
palm, and assessed the efficiency of each method in
terms of years and cost per unit gain (i.e., the time and
cost saved by these different methods over each other for
making selection). They used markers significantly
associated with the trait to calculate the marker scores
in MARS, whereas all markers (without significance
tests) to calculate the marker scores in GS. Responses
to PS and GS were consistently greater than the response
to MARS. Furthermore, with population sizes of N = 50
or 70, responses to GS were 4–25% larger than the
corresponding responses to PS, depending on the heri-
tability and number of QTLs. In terms of economics,
cost per unit gain was 26–57% lower with GS than with
PS when markers cost US $1.50 per data point, and
35–65% lower when markers cost $0.15 per data point.
Reduction in costs in sequencing and high-throughput
marker genotyping may enhance uptake of GS for crop
improvement in the future.
Challenges in Adoption of Genomics Technologies
Developing sustainable approaches to agriculture is
one of the most difficult challenges facing growers
and scientists today. Agricultural sustainability involves
successful management of resources for agriculture to
satisfy changing human needs, while maintaining or
enhancing the quality of the environment and conserv-
ing natural resources [71]. However, sustainable pro-
duction is hampered by the decline in land and soil
productivity as a result of inappropriate soil and water
management and other agricultural practices, as well as
misguided policies and frequent opposition to techno-
logical advances that have the potential to improve the
quality of life of billions of people worldwide. This is in
addition to the postulated challenges of climate change,
the number of hazardous chemicals (pesticides and
509
fungicides) that are constantly being released into the
environment and are becoming increasingly toxic to
human and animal life [71]. In recent years, the use of
promising biotechnology tools like genetic engineering
(GE) has offered potential solutions to the above prob-
lems. However, the adoption of any new technique,
particularly related to genetic engineering, remains
a policy matter and as mentioned above faces stiff
opposition many a times. In a recent review, Farre
et al. [25] addressed several of these issues and advo-
cated to overcome on the major barriers to adoption,
which are political rather than technical, for realization
of the potential of GE crops in developing countries.
It is thus obvious that the challenges facing agricul-
ture are massive, particularly with the controversies over
GE crops world over. It is clear that current methods of
food production, in both the developing as well as the
developed countries, are neither sufficient nor sustain-
able [72]. Under these circumstances, genomics inter-
ventions have great role to contribute to sustainable
agriculture. As mentioned in this article, genomics
approaches are very powerful to predict the phenotype,
with higher precision and efficiency, based on the geno-
type. A variety of approaches ranging from MAS to GS
are available to become integral part of plant breeding.
While in the past, plant breeders were hesitant to use
genetic variation existing in wild relatives of crop spe-
cies in commercial breeding programs due to the long
time it takes to recover desired phenotypes because of
linkage drag, approaches like AB-QTL, in addition to
MAS, can be successfully utilized. Availability of NGS
technologies, associated with low costs and high-
throughput, offers the opportunity to sequence either
entire or major proportion of the germplasm collection
for a species present in the gene banks around the word
to understand genome variation. In case, the genome
variation can be associated with the phenotype, which
is not trivial, it will be possible to develop the ideotype,
based on haplotype, of the variety to be developed.
Future Directions
While success stories of genomics-assisted breeding are
available in several crops, it must also be recognized
that much of the genome information generated is not
being routinely used by plant breeders, especially in
public breeding programs [26]. This may be due to
510 Crop Breeding for Sustainable Agriculture, Genomics Interventions in

Germplasm Traditional breeding

Technological innovations (wild, landraces, elite lines, varieties) (integrated with physiology,
entomology, pathology, etc.)
• NGS sequencing technologies
- 454/FLX • Selection
Improved
- Solexa/ Illumina Markers/ Modern breeding approaches - Mass selection
lines
- ABSOLiD Genes - Pedigree selection
• Marker assisted selection (MAS)
• Marker assisted recurrent selection • Hybridization
• High-throughput genotyping
Genomics (MARS)
platforms
platform • Genome-wide or genomic selection • Mutation
- Capillary electrophoresis for SSRs
(GS)
- Diversity array technologies
(DArTs)
- GoldenGate assay for SNPs
- Infinium assays for SNPs
- BeadXpress assays for SNPs Precision breeding Chance breeding

• -omics approaches
- Transcriptomics
- Metabolomics
- Proteomics
Crop improvement leading to
sustainable agriculture

Crop Breeding for Sustainable Agriculture, Genomics Interventions in. Figure 1

Schematic representation of genomics technologies for crop improvement and sustainable agriculture
In general, traditional crop improvement programs (shown in the box on the right hand side in Fig. 1) employ different
breeding strategies integrated with physiology, pathology, entomology, etc., and generate superior lines or improved
crop varieties. These approaches, however, take more time, and sometimes such breeding is referred as “chance breeding”
due to uncertainty in successes predicted in these approaches. On the other hand, genomics technologies (shown in the
box on the left hand side) such as a number of next-generation sequencing (NGS) technologies, availability of high-
throughput genotyping such as capillary electrophoresis for large-scale SSR genotyping, microarray-based DArTs,
GoldenGate/Infinium/BeadXpress assays for large-scale SNP genotyping, and a range of -omics technologies provide
candidate markers, gene(s), and QTLs to be integrated into the breeding programs by using high-throughput genomics
platforms. Integrated breeding approaches (shown in the box in the middle) such as marker-assisted selection (MAS),
marker-assisted recurrent selection (MARS), and genome-wide selection (GS) offer “precision breeding” with a great
potential, versus “chance breeding” to contribute to sustainable crop improvement

shortage of trained personnel, inadequate access to
genotyping, inappropriate phenotyping infrastructure,
unaffordable bioinformatics systems, and a lack of
experience of integrating these new technologies with
traditional breeding [4, 26]. However, recently, several
international initiatives such as the Integrated Breeding
Platform (formerly Molecular Breeding Platform,
www.mbp.genertaioncp.org), a joint initiative of The
Bill and Melinda Gates Foundation and The Genera-
tion Challenge Program, have been started so that plant
breeders especially from developing countries can have
access to many genotyping, phenotyping, as well as
information technologies to integrate their breeding
programs with modern genomics approaches.
We believe that integration of modern genomics in
combination with other cutting edge technologies in
breeding programs is invaluable for crop improvement
(Fig. 1) and will lead to sustainable agriculture for food
security, especially in developing countries.
A vital task facing the plant breeding community
today is to enhance food security in an environmentally
friendly and sustainable manner. Though genomics
interventions will not solve all the problems associated
with agricultural production leading to sustainability,
they have the potential, especially when they are used
in an integrated manner as described in Fig. 1, to
improve the breeding efficiency to address specific prob-
lems. These include increasing crop productivity, diver-
sification of crops, enhancing nutritional value of food
(biofortification), and reducing environmental impacts
of agricultural production. However, only through judi-
cious, rational, and science- and need-based exploitation
 Crop Breeding for Sustainable Agriculture, Genomics Interventions in
of genetic resources through genomic technologies
coupled with conventional plant breeding and genetic
engineering will lead to sustainable agriculture.
Acknowledgments
We thank the Generation Challenge Program (www.
generationcp.org), the Indian Council of Agriculture
Research (ICAR) and the Department of Biotechnol-
ogy (DBT) of Government of India for funding various
research projects (RKV) on genomics applications in
breeding.
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514 Crop Development Related to Temperature and Photoperiod

Crop Development Related to when the harvested part of the plant was at the opti-
mum stage. This knowledge was especially important
Temperature and Photoperiod (even vital) in medicinal plants, where the timing of
MARC MORAGUES1, GREGORY S. MCMASTER2 harvesting defines the medicinal value of the product.
1
Department of Soil and Crop Sciences, Colorado State This interest increased as groups moved from hunting
University, Fort Collins, CO, USA and gathering to agrarian societies.
2
USDA-ARS, Agricultural Systems Research Unit, Fort Crop development can be defined with the number
Collins, CO, USA and rate of appearance, growth, and senescence of
phytomers. However, that definition lacks information
Article Outline about when the switch of vegetative to reproductive
phytomers occurs, which is defined by the phenology of
Glossary
the crop. Crop development is of great importance in
Definition of the Subject
agriculture because it is the main mechanism for plants
Introduction
to escape both biotic and abiotic stresses, and adapt to
Canopy Structure: Phytomers, Phyllochron, and
the environment. At a more practical level, it affects the
Plastochron
management of the crop because cultural practices are
Regulation of Crop Development
more effective at specific stages of crop development.
Modeling Approaches
Future Directions
Bibliography Introduction

Glossary Major food, feed, and industrial crops were domesti-

Base temperature Lower temperature threshold
below which development ceases.
Epigenetics Genetic information other than DNA
sequence information.
Phenology Study of the sequence of developmental
stages of a plant and how it relates to climate.
Photoperiod sensitivity Requirement for a minimum
(or maximum) day length for reproductive phase
induction.
Phytomer Fundamental building block of plant cano-
pies. A vegetative phytomer is comprised of leaf,
node, internode, and axillary bud.
Phyllochron Rate of appearance of leaves on a shoot.
Shoot apex The tip of the shoot where usually there is
meristematic tissue producing new organs.
Thermal time Temperature response curve used to
estimate development rate.
Vernalization sensitivity Requirement for a period of
low temperatures for reproductive induction.
Definition of the Subject
Plant development, or the progression of plants
through their life cycle, has been of great interest in
human history because of the need to know and predict
cated in a few centers of origin, including the “Fertile
Crescent,” the Americas, and China. The wild relatives
of modern crops were adapted to survival in the envi-
ronment prevalent in the center of origin. Those origi-
nal crops were locally grown in the region of origin, but
some species showed a significant ability to adapt to new
environments and were spread globally with human
migrations and trade. For instance, wheat
was domesticated in the Middle East (the “Fertile Cres-
cent”) [1, 2], around 32ºN and is currently cultivated
from between 30ºN and 60ºN and from 27ºS to 40ºS.
However, it can grow beyond those limits, in lower
latitudes, in high altitudes, or even in the arctic circle.
This range of environments where wheat can grow
makes wheat one of the most plastic crops currently
grown. It requires a large range of developmental mech-
anisms to adapt to such different environments, where
photoperiods vary from 13–14 h to nearly 20 h. One of
the major accomplishments of the “Green Revolution”
was the discovery of photoperiod insensitive mutants
in wheat that could be grown at lower latitudes.
Plasticity in development is key for the adoption
of crops in a wide range of environments. Although
growth and development are related, they are different
processes. Development is the initiation and

P. Christou et al. (eds.), Sustainable Food Production, DOI 10.1007/978-1-4614-5797-8,

# Springer Science+Business Media New York 2013

Originally published in
Robert A. Meyers (ed.) Encyclopedia of Sustainability Science and Technology, # 2012, DOI 10.1007/978-1-4419-0851-3

differentiation of organs and the progression of stages
through which cells, organs, and plants go during their
life cycle, whereas growth is the change in size or weight
of the initiated organs. Biotic (e.g., genetics, weeds, and
diseases) and abiotic (e.g., temperature, light, water,
and nutrients) factors influence the initiation, growth,
and senescence of plant organs.
Since the “The Metamorphosis of Plants” by
Johann Wolfgang von Goethe, originally published in
1790 [3], there has been extensive research on how
plants proceed from germination to maturity in an
orderly and predictable manner. This research has led
to an extensive conceptual framework of plant develop-
ment resulting in many tools to predict plant develop-
ment. Plant shoots develop by forming a series of nearly
identical building blocks, called phytomers [4, 5]. The
vegetative phytomer is associated to a leaf, and
phytomers are produced in an orderly manner on
a shoot; for example, the phytomer of leaf 2 is formed
after the phytomer of leaf 1.
Phenology is the study of the plant (or animal) life
cycle and how it is influenced by seasonal and inter-
annual variations in climate. The phenology of a crop is
defined by the sequence of stages, which, in turn, define
phases. Identification of certain stages may require
examination of the shoot apex. For example, after
germination, the apical meristem produces vegetative
structures such as leaf primordia. When temperature
and photoperiod requirements are met, the shoot apex
will start initiating reproductive structures (e.g., spike-
let and floret primordia).
The rate of appearance of phytomers and changes
at the shoot apex are regulated by the genetics of the
plant, the environment, and often an interaction of
both. The main environmental drivers of plant devel-
opment are temperature and photoperiod; their effects
on phenology interact with the genetics of the plant
responses by photoperiod sensitivity genes, vernaliza-
tion genes, and earliness per se.
This entry covers how plant development is regu-
lated by temperature and photoperiod. Temperate
cereals are emphasized because they are adapted to
a wide array of environments and show a diverse set
of adaptive mechanisms regulating their development.
However, defining plant parts and how canopies are
built as well as the sequence of events throughout the
crop cycle is required before their regulation can be
Crop Development Related to Temperature and Photoperiod 515
explained. This entry starts by defining the canopy
structure and the sequence of events that define the
developmental processes of a crop, followed by the
current knowledge on how temperature (including
vernalization) and photoperiod regulate crop develop-
ment. This entry finalizes with two important parts:
modeling crop development and future directions.
Modeling crop development is twofold important;
first to understand the physiology and genetic basis of
crop development, and second to predict when key
developmental events are likely to happen as accurately
as possible.
Canopy Structure: Phytomers, Phyllochron,
and Plastochron
The phytomer is considered the basic building block
of plant canopies and is most commonly defined as
the leaf, node, internode above the node, and the
axillary bud [6]. Therefore, canopy architecture and
development is determined by the dynamic appear-
ance, growth, and abortion/senescence of phytomers
(and components of the phytomer). Phytomers origi-
nate at the shoot apex with the initiation of a leaf, and
the potential for a new shoot is formed with the pres-
ence of the axillary bud. The growth and differentiation
of each component of a phytomer will lead to their
visual appearance. For example, the internode can con-
tinue differentiating and growing, resulting in its
appearance from the leaf sheath. Tillers may appear
when the axillary bud differentiates and grows.
Naming Plant Parts
Because plant development is an orderly process, accu-
rately identifying plant parts aids in describing the
process and quantifying the developmental rate.
Several naming systems of plant parts have been
proposed, but most are quite similar. For example,
true leaves can be numbered acropetally for each
shoot with an L [7–9] (Fig. 1) beginning with the first
foliar leaf, L1 [9]. Similarly, Jewiss [7] proposed
a system for naming tillers that has been modified
and extended by many, but the modified system pro-
posed by Klepper et al. [8, 9] is increasingly being
adopted. This system uses the leaf axil number of
the parent shoot to name the tiller. The first shoot
to appear from the seed is the main stem (MS).
516 Crop Development Related to Temperature and Photoperiod

Leaf 3 MS Tiller Leaf 2 MS

(L3) (T2) Leaf 2 MS
Tiller Leaf 3 MS (L2)
(T3)

Leaf 1 MS
(L1)

Tiller Leaf 1 MS
(T1) Coleoptile Tiller
(TC or T0)
Tiller of the prophyII
on T1 (T10)

Main Stem
(MS)

Crop Development Related to Temperature and Photoperiod. Figure 1

Naming leaves and tillers of a winter wheat plant

Tillers appearing from the axils of leaves on the main
stem are primary tillers and are named with a T and
a digit corresponding to the leaf number. For example,
the tiller appearing from the first leaf (L1) on the main
stem is called T1. This system is however limited by the
potential production of tillers from the axil of leaf 10
and above, which rarely happens. Primary tillers can
produce tillers that are called secondary tillers. Second-
ary tillers can arise from the axil of the prophyll of
primary tillers and their second digit is a zero (Fig. 1).
Haun [10] proposed a numerical leaf staging system
to quantify the number of leaves appearing on the main
stem (which can be extended to any shoot):


Ln Ln
Haunstage ¼ ðn  1Þ þ ; 0< 1
Ln1 Ln1
where n is the number of leaves that have appeared on
a shoot, Ln1 is the blade length of the penultimate leaf,
and Ln is the blade length of the youngest visible leaf
extending from the sheath of the penultimate leaf.
Therefore, when a shoot is identified, it can be further
characterized using the Haun system. For example,
the Haun stage for shoots on the plant shown in
Fig. 1 is: MS (5.3), T0 (1.5), T1 (2.4), T2 (1.4), and
T3 (0.7). The Haun stage of a tiller with one leaf not
fully unfolded is arbitrarily assigned as 0.1 or 0.9 when
only the tip of the leaf is visible or most of the leaf blade
has appeared, respectively.
Similarly, the leaf and tiller naming scheme
has been extended to the wheat inflorescence.
Klepper et al. [11] defined a numerical index for the
ð1Þ
development of the inflorescence and Wilhelm and
 Crop Development Related to Temperature and Photoperiod 517

Spike Spikelet 7 (S7)

S 12

S 11
Rachilla
S 10 Lemma
S9
F1 F3 F5
S8 (C1) (C3) (C5)
S7

S6
Rachis
S5
Palea
S4 F2 F4
(C2) (C4)
S3 Glume

S2
Floret/Kernel
S1 Rachis

Peduncle

Flag leaf

Crop Development Related to Temperature and Photoperiod. Figure 2

Naming scheme for reproductive organs of spike inflorescence. Spikelet positions are denoted by the letter S and
numbered acropetally along the rachis. Florets/caryopsis positions are denoted by the letters F/C and numbered
acropetally along the rachilla (From Wilhelm and McMaster [12])

McMaster [12] extended it to uniquely identify each
plant part. Spikelets are named with an S followed by
the position from the base of the spike. Then, S1 is the
basal spikelet and S2 is the second spikelet from the
peduncle (Fig. 2). Florets are designated with an F and
numbered acropetally from the base of the rachilla.
After fertilization, the letter F designating a floret is
changed to a C for caryopsis. This system allows nam-
ing reproductive structures in grasses with one spikelet
per rachis node such as wheat.
These naming systems of leaves, tillers, spikelets,
and florets or caryopsis allow the accurate identifica-
tion of each plant part. For example, the second cary-
opsis on the third spikelet on the primary tiller from
the axil of the second leaf of a wheat plant would be
T2S3C2.
The systems for naming individual plant organs can
be easily extended to name vegetative and reproductive
wheat phytomers [13]. A phytomer would be denoted
with a “P” followed by an “L,” if it is a phytomer associ-
ated with a leaf or an “S,” if it is a reproductive phytomer
and the leaf or spikelet number. The name of the shoot
can be added to identify the tiller being described. For
example, MS PL2 is the second phytomer on the main
stem and T1 PS1 is the basal spikelet phytomer in the
spike of the first tiller.
Dynamic Appearance of Plant Organs: The
Plastochron and Phyllochron
The naming systems of plant parts described earlier are
the landmarks to describe the development and struc-
ture of a wheat plant. However, plant development is
a dynamic process that follows the formation, growth,
and senescence of phytomers and their components
resulting in a continually changing architecture.
518 Crop Development Related to Temperature and Photoperiod
The creation of the vegetative phytomer is dependent
on the initiation of the leaf primordium. Therefore, the
rate of leaf primordia initiation controls the timing of
phytomer formation. The plastochron was first defined
as the interval between the formation of two successive
internode cells of the green alga Nitella flexilis [14], as
cited in [15]. Milthorpe [16] and Esau [17] defined the
plastochron as the interval between the formations of
successive leaf primordia at the shoot apex, which is
now commonly accepted. Similarly, the phyllochron
was defined as the interval between appearances of
consecutive leaves on a shoot [18] as cited in [6].
Wilhelm and McMaster [6] further refine the definition
of phyllochron by defining appearance as “visible with-
out magnification, dissection or changing leaf display.”
The inverse of the phyllochron is termed development
rate (DR), which can be generalized as the inverse of the
time interval between two developmental events.
The relationship between the plastochron and
the phyllochron depends on the species. In wheat, leaf
primordia are produced more rapidly than they appear,
suggesting that different mechanisms are involved in
regulating each process. Leaf primordia are initiated at
the meristem on the shoot apex, where new cells are
produced very quickly. After a leaf primordium is
formed, it continues to grow in cell number and cell
size, but this growth does not happen at the shoot apex
meristem, rather at the intercalary meristem at the base
of the leaf. The amount a leaf grows until it appears
through the curl of leaves is much larger than the
growth involved in forming a leaf primordia, therefore
it is more dependent on the available resources (e.g.,
water, carbohydrates, and nutrients).
Developmental Stages and Phases: Phenology
Plants develop by the repetition of elementary building
blocks (i.e., phytomers), whose morphological, dimen-
sional, functional, and anatomical features change dur-
ing ontogeny and according to several processes called
heteroblasty, phase change, life stages, maturation,
aging, age states, or morphogenetic progression [19].
In this entry, those changes will be referred as develop-
mental stages (or simply stages) and the time between
stages will be referred as developmental phases (or
phases). It is common in the literature referring to
these stages as “growth stages.” However, there is little
growth in some developmental events such as anthesis.
The phenology of a crop is the ordered succession of
stages and phases that can have different lengths deter-
mined by internal factors (e.g., the genetics of a variety
or species) or biotic and abiotic external factors (e.g.,
diseases, temperature, light, and nutrients).
Plant development can broadly be divided into
vegetative and reproductive phases that often overlap.
The switch from vegetative to reproductive phase hap-
pens at the meristem level, which stops producing
vegetative phytomers (i.e., leaves, nodes, and inter-
nodes) to start producing reproductive phytomers
(rachis, spikelets, and florets).
Phenological Scales
Developmental stages occur in a consistent pattern in
a crop each year and numerous approaches exist that
characterize crop phenology. The most widely used
phenological scales for temperate cereals are Feekes
[20], Zadoks [21], Haun [10], and BBCH [22]. The
Feekes scale is shown in Fig. 3 and described, jointly
with the Zadoks’ decimal code, in Table 1. The BBCH
scale for cereals is mainly based on the Zadoks scale,
and the Haun scale primarily describes the develop-
ment of shoots until the last leaf is fully expanded.
Phenological scales consider basic developmental
stages like germination, emergence, tillering, stem
elongation, heading, flowering, grain filling, and phys-
iological maturity, with differences among scales pri-
marily in how much detail each stage is characterized.
Some developmental stages are not well defined, lead-
ing to confusion in measuring and reporting these
stages. For example, the beginning of stem elongation
is usually recorded as the jointing date or when the first
node is visible above ground; however, the first node is
formed when the apex is underground and is only
visible after the stem has elongated sufficiently to ele-
vate the apex and the node above ground. Likewise,
physiological maturity is defined as when the maxi-
mum dry weight is reached. In wheat, determining
physiological maturity is somewhat difficult because
there is not a clear morphological change in the plant
morphology as it happens in maize (and sunflower). In
maize, a black layer near the base of the kernel appears
when the maximum dry weight is reached. The Feekes
scale defines harvest maturity as when the grain is

Crop Development Related to Temperature and Photoperiod. Figure 3
Feekes developmental scale with the approximate timing of some shoot apex developmental events (From [13])
difficult to divide along the crease and Zadoks [21]
defines the 90% of ripeness of rice (Oryza sativa L.)
when the kernel cannot be dented with the fingernail.
These definitions likely are not precisely correlated with
maximum seed biomass. Therefore, it is now com-
monly accepted practice to assume physiological matu-
rity for temperate cereals occurs when all green color
has disappeared from the spike. This definition seems
reasonable as leaves and internodes have long since lost
all green color so that no photosynthesis occurs, and
there is no report showing retranslocation from carbo-
hydrate reserves to the grain at this time.
Regulation of Crop Development
Plant development is highly dependent on tempera-
ture, which controls the rate of development and
the switch from vegetative to reproductive states.
Both high and low temperatures may have a major
Crop Development Related to Temperature and Photoperiod 519
effect on plant development, especially controlling the
switch to the reproductive state. For example, winter
wheat requires a period of low temperatures to start
producing reproductive structures (vernalization). The
length of the cold period varies with genotype.
Besides vernalization, day length (or photoperiod)
modifies the temperature-controlled rate of develop-
ment. Photoperiod refers to the number of daylight
hours, which changes through the seasons and with lat-
itude. Photoperiod increases after the winter solstice
(December 21 in the northern hemisphere or June 21 in
the southern hemisphere) and decreases after the summer
solstice. Crops and genotypes vary in their sensitivity to
photoperiod and the minimum amount of daylight
required to switch from vegetative to reproductive phases.
Developmental Response to Temperature
The relationship between temperature and phenology
has been long recognized. Temperature is a better
520 Crop Development Related to Temperature and Photoperiod

Crop Development Related to Temperature and Photoperiod. Table 1 Description of the main developmental stages
according to Feekes [20] and Zadoks [21], and suggested measurements characteristics (Modified from [23])

Description Measurement
Stage or phase Feekes Zadoks characteristics
Germination No stage Stages: 00 – dry seed, Beginning of imbibition:
01 – beginning of Seed begins to swell
imbibition, 03 – imbibition
complete, 05 – radicle
emerged from caryopsis,
07 – coleoptile emerged
from caryopsis
Emergence Stage 1 – main shoot only Stage 09 – leaf at the tip of Beginning of emergence:
the coleoptile First true leaf emerges
through the coleoptile
and the tip is visible
above the soil surface
Tillering Stage 2 – beginning of tillering Stages 21–29 – main stem Beginning of tillering:
plus 1 to 9 tillers The first tiller is visible
Single ridge No stage No stage Shoot apex shape
changes from dome to
more elongated and leaf
primordia begin to form
a ridge around the apex
Double ridge No stage No stage Formation of double
ridges around the apex.
Bottom ridge is leaf
primordia and top ridge
is spikelet primordia
Terminal spikelet No stage No stage Apical spikelet
primordium appears and
noted by a 90o rotation
from the plane of
previous spikelets
Jointing Stage 6 – first node visible Implicit in stage 31 – first First node visible above
node detectable and the soil surface
change of plant habit from
prostrate to erect
Stem elongation Stages: 4 – change of plant Stages: 30 – pseudo-stem Beginning when the first
habit from prostrate to erect, erect, 31 to 36 first to sixth node is formed, usually
5 – pseudo-stem clearly erect, node detectable, 39 – flag below soil surface, and
6 – first node visible, 7 – second leaf ligule visible, 43 to pushed above the soil
node visible, 8 – last leaf 49 – flag leaf sheath surface
appearance, 9 – last leaf ligule swelling
visible, 10 – last leaf sheath
swelling
Flag leaf Stage 9 – last leaf ligule visible Stage 39 – Flag leaf ligule Ligule of last leaf is visible
visible and no new leaf is
emerging
 Crop Development Related to Temperature and Photoperiod 521

Crop Development Related to Temperature and Photoperiod. Table 1 (Continued)

Description Measurement
Stage or phase Feekes Zadoks characteristics
Booting Stage 10 – last leaf sheath Stages 43–49 – flag leaf Begins at flag leaf and
visible sheath swelling ends at heading
Heading Stages: 10.1 – first ears just Stages: 51 – first spikelet Begins when first spikelet
visible, 10.2 – ¼ of heading just visible, 53 – ¼ of is visible and ends when
completed, 10.3 – ½ of heading the inflorescence visible, the inflorescence is fully
completed, 10.4 – 3⁄4 of heading 55 – ½ of the inflorescence emerged
completed, 10.5 – all ears out of visible, 57 – 3⁄4 of the
the sheath inflorescence visible, and
59 – inflorescence
completely emerged
Anthesis Stages: 10.5.1 – anthesis starts, Stages: 61 – beginning of Starts when the first
10.5.2 – flowering completed to anthesis, 65 – mid-anthesis, anther is visible on an
the top of the ear, 10.5.3 – 69 – anthesis completed inflorescence and ends
flowering completed to the when no more anthers
bottom of the ear, 10.5.4 – appear on the
flowering over/kernel watery inflorescence
ripe
Grain filling Stages: 10.5.4, 11.1 – milky ripe, Stages: 71 to 77 – milk Begins at fertilization,
11.2 – mealy ripe grain, 83–87 – dough grain usually considered
anthesis and ends at
physiological maturity
Physiological maturity No Stage When all spike
components, internodes,
and leaves lose green
color
Ripening Stages: 11.3 – kernel hard, 11.4 – Stages 91–99 Ripening and dormancy
ripe for cutting

predictor of many developmental processes than calen-
dar time. Reamur [24] formalized this relationship by
creating the concept of heat units, now referred to as
thermal time. The relationship between temperature
and developmental rate is a curve with a maximum
development rate at the optimum temperature (To)
and developmental rate reaching zero at temperatures
below the base temperature (Tb) or above the maxi-
mum temperature (Tm; Fig. 4). This nonlinear relation-
ship is shown in several studies [25, 26].
Thermal time has two components: (1) the average
temperature (Ta) over some time interval (e.g., hourly,
daily), and (2) a temperature response curve describing
the effectiveness of Ta on the development rate for the
process (e.g., phyllochron, phenology). Ta is the
integral of temperature over the time period of interest;
however, in practice the average of the maximum
and minimum temperatures in the time interval is
often used:
Tmax þ Tmin
Ta ¼ ð2Þ
2
This approximation is fairly accurate, but its error
increases with deviations of from 12 h photoperiods
and if sudden changes in temperature happen within
the time interval. The time interval mostly depends on
data availability, but common intervals range from
daily to hourly time intervals, with daily the most
commonly used. There are many temperature–
response curves, which greatly diversifies the
522 Crop Development Related to Temperature and Photoperiod

Development rate (DR)

Development rate (DR)

T upper

Tb Tb

a Temperature b Temperature

To Tol Tou
Development rate (DR)

Development rate (DR)

Tb Tm Tb Tm

c Temperature d Temperature

Crop Development Related to Temperature and Photoperiod. Figure 4

Development rate as a function of temperature. Temperature response curves: (a) linear response, (b) extended linear
response with an upper temperature threshold, (c) bilinear model with two zero development temperatures (base
temperature, Tb, and maximum temperature, Tm) and an optimal temperature (To), and (d) trilinear model with two zero
development temperatures (Tb and Tm) and a range of optimal temperatures defined by optimal lower temperature (Tol)
and optimal upper temperature (Tou)

calculation of thermal time (Tt). The most simple form
is a linear relationship with the temperature at which
there is some plant development (Fig. 4a), i.e., the
difference between Ta and the temperature at which
development is zero or base temperature (Tb):
Tt ¼ Ta  Tb ; ðTt  0Þ
where thermal time is expressed as growing degree-
days (GGD,  C·days). Modifying Eq. 2 to include
a maximum development rate (Fig. 4b) is useful if
plants are grown at higher temperatures. Given that
the relationship between temperature and develop-
ment rate is not linear [25–27], further refinements to
the accumulation of thermal time can include changing
the development rate based on certain cardinal
temperatures (Fig. 4c, d). These approaches assume
development rate increases with temperatures above
Tb until an optimum temperature (To, or a range of
optimal temperatures, Tol to Tou) is reached, and then
decreases until development stops at a maximum
ð3Þ temperature (Tm). This can be approximated by two-
or three-segmented linear models, or curvilinear
models such as a quadratic curve [25] or beta distribu-
tion [27, 28].
Vernalization
The term vernalization was first used by Lysenko in
1928, but research on the need of a cold period for

winter cereals to flower started as early as 1857 [29].
Vernalization can be viewed as an adaptive mechanism
to avoid unfavorable periods for development (e.g.,
winter) and ensure flower development and subse-
quent seed growth occurs under favorable conditions
(e.g., spring and summer). Hence, vernalization syn-
chronizes plant development with seasonal climate
changes.
Commonly, genotypes requiring vernalization are
referred to as “winter” wheat (or “winter” barley) and
are normally planted late summer or early fall with
vernalization occurring during the late fall or early
winter. Conversely, “spring” genotypes are commonly
viewed as not requiring vernalization, and normally are
planted in the spring or in regions where temperatures
are often above effective vernalizing temperatures. This
well-entrenched distinction between “winter” and
“spring” genotypes does not reflect that “spring” geno-
types (1) often have at least some vernalization require-
ments, (2) reach flowering faster if experiencing
vernalizing temperatures, and (3) mask the continuum
of vernalization requirements present among all wheat
genotypes.
Effective vernalizing temperatures range from 0 C
to 10 C [30], and a few weeks of cold are usually sufficient
to promote the switch from vegetative to reproductive
phases and longer periods of cold temperatures
can shorten the time to flowering until the vernalization
response is saturated [30]. Genotypes vary in the length of
the cold period required to saturate the vernalization
response. For instance, time to flowering in wheat was
reduced in response to longer cold periods [31, 32], and
interestingly, genotypes thought to require vernalization
to flower eventually flowered without undergoing
vernalization treatment [32]. Quantifying responses to
varying periods of vernalizing temperatures in calendar
time or thermal time does not reflect the biology of the
response. A better method to quantify the effects of
vernalization would be by counting the number of
leaves produced at flowering time [33]. Using this
quantification method, vernalization reduces the time
to flowering by reducing the number of leaves being
produced rather than the phyllochron [31, 33]. That is,
the number of leaves produced at flowering increases
with shorter vernalization periods, while the
phyllochron is not affected by the duration of the
vernalization period.
Crop Development Related to Temperature and Photoperiod 523
Genetic Regulation of Vernalization Although the
mechanisms by which plants sense cold and initiate the
cellular signaling to induce flowering are not known,
the genetic regulation of vernalization is fairly well
known in cereals like wheat and barley, which benefited
of the research done on the model plant Arabidopsis
thaliana. Four major genes are involved in the expres-
sion of vernalization sensitivity in wheat and barley,
VRN-1, VRN-2, VRN-3, and VRN-4. The first three
genes have been cloned and identified [34–36]. How-
ever, these three genes do not explain the spring habit
of all varieties [35], suggesting that other genetic mech-
anisms may be involved.
The VRN-1 gene encodes the MADS box transcrip-
tion factor similar to APETALA-1, which is responsible
for meristem identity in several plants [34]. This gene is
up-regulated by vernalizing temperatures and the
degree at which it is up-regulated depends on the
length of the vernalization period. However, spring
varieties also show an up-regulation of VRN-1 during
the initiation of the reproductive phase and remain
high throughout the reproductive phase, suggesting
an involvement of VRN-1 in meristem identity but
not limited to vernalization response.
The VRN-2 gene encodes the ZCCT1 protein,
which shows high similarity to the CCT domain of
the Arabidopsis CONSTANS and CONSTANS-like
genes. VRN-2 represses flowering and is down-
regulated by vernalization [35]. The spring allele
vrn-2 in wheat has a point mutation at the CCT
domain that replaces an arginine with a tryptophan.
It has been suggested that the CCT domain may be
involved in protein–protein interactions [37], so
a mutation in this domain can alter these interactions.
The VRN-3 gene is a RAF kinase inhibitor like
protein with high homology to the Arabidopsis
FLOWERING LOCUS T (FT ) gene [36], which induces
flowering when expressed. FT is a flowering signal that
moves from leaves to stem apices.
VRN-1, VRN-2, and VRN-3 interact with each
other regulating flowering under the vernalization
pathway (Fig. 5). Before vernalization occurs, VRN-2
is expressed and represses the expression of VRN-3.
When plants are vernalized, VRN-1 is induced and it
represses the expression of VRN-2, allowing VRN-3 to
express and promote flowering. At the same time, there
is a feedback mechanism by which VRN-3 up-regulates
524 Crop Development Related to Temperature and Photoperiod
VRN-3 VRN-2
VRN-1 Vernalization
Crop Development Related to Temperature and
Photoperiod. Figure 5
Model of the vernalization pathway in temperate cereals.
Arrows mean induction (or up-regulation) of gene
expression; for example, vernalization induces the
expression of VRN-1. Bar-headed lines mean repression
(or down-regulation) of gene expression; for example,
VRN-2 represses the expression of VRN-3
VRN-1. It seems that VRN-1 is the primary target
of vernalization and is essential for flowering.
VRN-4 has been recently fine mapped and its clon-
ing is underway [38]. Identifying and including this
gene in the vernalization pathway may increase the
understanding of vernalization responses in wheat.
Photoperiod
Photoperiod can be defined as the number of hours of
light in a 24-h period, which changes throughout the
season depending on the latitude. It has been long
recognized that plants, including crops, normally
flower only when the length of the day was favorable
[39]. Crops sense the amount of light they receive daily
and respond to it by accelerating or slowing their
development. There is, however, genetic variation of
a quantitative nature in the response to photoperiod
within a crop, meaning that different varieties respond
differently to changes in the photoperiod, and some
show no response to photoperiod (or are photoperiod
insensitive). Photoperiod sensitivity is thought to be
the wild type phenotype because it is wide spread in
wild barley (Hordeum spontaneum L.) and confers
strong adaptive features in the center of origin of barley,
where there might be late spring frosts. Photoperiod
insensitivity, however, brings wide geographical adapt-
ability because plants do not require long days to
flower, and, hence, they are suitable for environments
with short seasons or latitudes where long days do not
occur. For example, in wheat, day length neutrality,
jointly with semi-dwarfism and rust resistance traits,
were used to develop the high yielding varieties of the
“green revolution” [40]. Photoperiod insensitivity pro-
motes earliness thought to be a desirable adaptive trait
for environments closer to the equator where high
temperatures and drought can be expected at the end
of the season. Therefore, earliness allows wheat and
barley varieties to escape this terminal stress.
Although the rate of development mainly responds
to temperature, when temperature is fixed, longer pho-
toperiod alters the development rate by shortening the
phyllochron [41, 42]. In Fig. 6, the appearance of leaves
over time follows a linear model when plants are grown
at a constant temperature, and the rate of appearance
(slope of the curve) increases with the photoperiod;
however, as day length increases, fewer leaves are
formed per hour of light, hence there is a reduction in
photoperiod efficiency for leaf emergence.
Different crops respond differently to photo-
period; crops can be classified as long day (LD) and
short day (SD) depending on which photoperiod
accelerates development (promotes flowering).
Temperate cereals are long-day crops because flowering
is promoted by photoperiod longer than 12–14 h, while
maize and rice are short-day crops, meaning that short
days induce the switch to reproductive phase. In wheat
and barley, increased photoperiod shortens the time to
flowering by modifying the length from emergence to
terminal spikelet initiation. The effects of photoperiod
on the duration from terminal spikelet initiation to
flowering depend on what environments they are mea-
sured (field versus controlled environments). This
reduction of time to flowering is not only due to an
accelerated phyllochron, but also by the production of
fewer leaves.
Genetic Regulation of Photoperiod Sensitivity In
temperate cereals, such as wheat and barley, sensitivity
to photoperiod is mainly regulated by Ppd genes.
Homologous genes have been identified in wheat and
barley and have the function of a pseudo response
regulator (PRR), most similar to the Arabidopsis PRR7
[43]. The PPR proteins are characterized by a pseudo
receiver domain near the amino-terminus and a CCT
domain near the carboxy-terminus [44], which makes
 Crop Development Related to Temperature and Photoperiod 525

0.28

x
0.26 y=
(a + bx)
Haun stage of main stem

0.24

Development rate
0.22

0.20

0.18

Daylength 16 h 0.16
Daylength 8 h
0.14
a Days after emergence
b 10 15 20
Daylength (h)

Crop Development Related to Temperature and Photoperiod. Figure 6

Development rate responses to changes in day length at constant temperature. (a) Leaf appearance of plants grown in
different day lengths and (b) development rate as a function of day length (Based on [41])

them distantly related to other CCT domains impor-

Ppd
tant in regulation of flowering such as CONSTANS [45]
and VRN-2 [35].
CONSTANS
The photoperiod insensitive allele (ppd-H1) in bar-
ley slightly delays the gene expression of CONSTANS
(HvCO1) that follows a circadian pattern [43] and is FT/VRN-3
a transcriptional regulator of the FT gene [46]. In
Crop Development Related to Temperature and
contrast, photoperiod insensitivity in wheat is regu-
Photoperiod. Figure 7
lated by a series of three homoeologous genes located
Model of the photoperiod pathway of flowering time.
in the colinear region on chromosome 2 group, which
Arrows mean induction (or up-regulation) of gene
seem to be upstream of the CONSTANS gene in the
expression; for example, CONSTANS is induced by the
photoperiod pathway (Fig. 7). The Ppd-1 Da allele
expression of Ppd genes
confers insensitivity to photoperiod in a semidominant
fashion, allowing wheat plants to flower regardless of
the photoperiod [47]. Sequence analysis of wheat
Coordinated Temperature and Photoperiod
varieties known to have this mutation shows that
Regulation of Crop Development
there is a 2,089 bp deletion upstream of the coding
region responsible for the photoperiod insensitivity The temperature and photoperiod regulation of crop
phenotype. Other sequence variations producing development has been described in previous parts of
nonfunctional proteins (null alleles) at the 2A and 2D this article, but both environmental factors act in coor-
genes in wheat have been observed; however their dination. It is clear that the phyllochron varies with
effects on photoperiod sensitivity are difficult to assess planting date in temperate cereals and it has been
because they might be masked by functional proteins suggested that the phyllochron is fixed by the rate of
from other homoeologous genes. These, and other change of the photoperiod at crop emergence [48].
mutations, however, may have a quantitative effect in However, in general, temperature and photoperiod
photoperiod sensitivity and flowering time [47]. change together in the field. Results from experiments
526 Crop Development Related to Temperature and Photoperiod
in controlled environments show an effect of photope-
riod on the temperature–response curve [49, 50]. The
term “thermo–photo ratio” (the degree-days divided
by day length in hours) has been used to study the
coordinated effect of temperature and photoperiod
on the phyllochron [49]. A linear relationship was
found between the phyllochron and the thermo–
photo ratio under both controlled environments and
field conditions [49]. Slafer and Rawson [51]
partitioned the photoperiod sensitivity of wheat
phenophases into different parameters, which were
affected by temperature, describing an interaction
between genotype, photoperiod, and temperature.
The effect of planting date on crop development has
been studied extensively, yet it is difficult to draw
conclusions of the coordinated effect of photoperiod
and temperature on crop development. This is because
when planting date is changed, both photoperiod and
temperature are changed and their effects are difficult
to separate.
Genetic Framework of Flowering Time Several
pathways regulate time to flowering in crops, namely,
vernalization, photoperiod, autonomous, and
gibberellic acid. The vernalization and photoperiod
pathways have been described in parts 5.2 and 5.3 and
FT/VRN-3 gene is in both pathways, thus integrating
the response to both vernalization and photoperiod
factors (Figs. 5, 7, 8).
In the ancestral form of wheat and barley, after
germination in the fall VRN-2 is highly expressed by
the long days and represses the expression of FT/
VRN-3. As winter progresses, the photoperiod
decreases and low temperatures induce the expression
of VRN-1 in the leaves, which represses the expression
of VRN-2, allowing FT to be expressed by long days in
the spring, a process regulated by photoperiod genes
Ppd and CO. Then, the protein encoded by FT/VRN-3
is translocated to the shoot apex, where it up-regulates
VRN-1, which will induce the switch to reproductive
phase. The HAP (HEME ACTIVATOR PROTEINS)
complexes may mediate the transcriptional regulation
of the CCT domain of CO and VRN-2. In plants, HAP
subunits are encoded by multiple genes that, together
with CCT domain proteins that can interact with HAP
complexes, generate a large number of molecular com-
binations. These combinations provide a flexible
Long days Vernalization
Ppd
CO VRN-2 VRN-1
HAP3
HAP5
Leaf
FT/VRN-3
FDL2
Apex
VRN-1 Flowering
Crop Development Related to Temperature and
Photoperiod. Figure 8
Model of flowering related to photoperiod and
vernalization. Thick blue arrows mean induction (or up-
regulation) of gene expression or developmental
process; for instance, photoperiod induces the
expression of VRN-2. Square headed lines indicate
repression (or down-regulation) of gene expression; for
instance, VRN-3 up-regulates VRN-1 at the apex
signaling system that can integrate responses to envi-
ronmental cues as photoperiod, vernalization, or stress
(reviewed in [52]).
In environments where temperate cereals usually
grow and were domesticated, vernalization requirements
are met long before the photoperiod is inductive of the
reproduction phase. This requires plants to “remember”
they had been vernalized. The mechanism by which
temperate crops “remember” vernalization is not
known, but it has been studied in the model plant
Arabidopsis. In addition to the genetic information car-
ried on the DNA sequence (genes and alleles), chromatin
structure is recognized as another source of genetic
information. The chromatin can be highly condensed
(heterochromatin) or more relaxed (euchromatin).
The DNA is combined with proteins called histones
and specific covalent histone modifications of histones
favor the formation of chromatin structure that
influences the level of gene expression. Other nongenetic
mechanisms of gene expression are related to DNA

methylation. The DNA methylation at the promoter
region of genes is generally related to lower levels of
gene expression, and even gene silencing. Of the two
mechanisms, it seems that histone modifications are
involved in regulating the memory of vernalization
(reviewed in [53]) in Arabidopsis. In Arabidopsis,
the FT gene is repressed by FLC, which is down-
regulated by vernalization. The stable repression of
FLC involves de-acetylation of histone 3 (H3) upstream
of FLC, methylation of H3 Lys9 and Lys27, which
allows the binding of HP1 inducing the stable silencing
of FLC.
Less known is the genetic basis of the quantitative
response to temperature (thermal–response curve).
The Earliness per se A1 (EpsmA1) gene affects time to
flowering by reducing the vegetative phase and it has
been associated with responses to temperature, proba-
bly by modifying the optimum temperature [54]. Two
candidate genes for EpsmA1 are located in the genomic
region where this gene has been located, Mot1 and
FtsH4 [55]. The Mot1 gene has features of the SNF2
family of transcriptional regulators. Other members of
this family have been related to regulation of flowering
in Arabidopsis, but the gene expression data do
not show differences between the two alternative
alleles [55]. The FstH4 is a member of the FstH
family of proteases and is homologous to the
Arabidopsis FstH4, which has been found highly
expressed in seed, and mutants in this gene show
delayed germination that is carried over the growth
cycle [55].
Modeling Approaches
For centuries, people have wanted to understand and
predict aspects of crop development, particularly phe-
nology. To do this, different conceptual, statistical, and
mathematical models have been developed. Beginning
in the 1970s, a variety of digital technologies began to
emerge, one of which was crop simulation models for
predicting growth, development, and yield. This sec-
tion presents a broad overview of these crop simulation
models, emphasizing wheat.
Many crop simulation models exist for simulating
growth, development, and yield, and they cover scales
from specific processes to the agroecosystem. Crop
simulation models are a simplified mathematical
Crop Development Related to Temperature and Photoperiod 527
representation of the plant. At the most fundamental
level [56], crop simulation models generally simulate
a trait, for example, yield (Y ), as the function of
daily growth rate (GR) that is partitioned to the
yield component (P) and integrated over a daily time
step from emergence (emerge) through physiological
maturity (maturity):
Z
Y ¼ GR  P ð4Þ
Implementing Eq. 3 in a model usually begins
assuming non-limiting conditions, thereby allowing
for potential production to be estimated. The parame-
ters can either be generic for a crop or adjusted to
a specific genotype. By incorporating environmental
variables (e.g., temperature, water, light, CO2, and
nutrients), crop simulation models can examine crop
or genotype responses across a broad environmental
range of limiting conditions, avoiding a common lim-
itation of statistical (or regression) models. Depending
on the purposes, including the role of biotic factors or
management practices may also be important.
How a model implements Eq. 3 depends on model
objectives and interests of the model developers. The
earliest crop simulation models tended to focus on the
scale of whole-plant growth and development, with
little detail on processes at lower scales. These models
use an energy- or light-driven approach to determine
the growth rate, and this approach remains popular
today. The basic approach simulates leaf area index
on a daily time step, which is used to capture energy/
sunlight and produce biomass that is then distributed
to basic plant components of leaves (providing the
feedback to the cycle), stems, roots, and seeds.
Partitioning coefficients are often used to allocate the
biomass produced, and phenology sub-models are
essential in accurately predicting the timing when
sources and sinks are present, and changing
partitioning coefficients based on developmental stage.
As crop simulation modeling progressed, certain
trends emerged. First, greater attention focused on
representing plant processes below the whole-plant
level. In general, energy- or light-driven modeling
emphasized functional physiology, particularly for
assessing energy balance and leaf functioning at the
individual organ level (e.g., [57, 58]). Second, consid-
erable research on crop development during the 1970s
528 Crop Development Related to Temperature and Photoperiod

Kernel Growth

WINTER WHEAT Floret Primordium Abortion

Floret Part Primordium Initiation

Floret Primordium Initiation

Flag Leaf
Spikelet Primordium Initiation Appears
Terminal Spikelet?
Rachis Elongation

Tiller Abortion

Internode Elongation

Tiller Bud Growth and Appearance

Peduncle Elongation
Tiller Bud Primordium Initiation

Leaf Growth and Appearance

Leaf Primordium Initiation Flag Leaf

Σ TT: 100 300 480 605 785 945 1090 1250 2200
TT: 100 180 125 180 160 145 160 750 200
200 1.7 --- ---
1.7 1.2 1.5 --- ---
# LVS: --- 1.9

S G E T1 JAN1 SR DR J B H A M HR
Growth Stage
120 GDD

Crop Development Related to Temperature and Photoperiod. Figure 9

Developmental sequence diagram of a generic winter wheat for optimal conditions. Question marks refer to
uncertainty, important cultivar variation, or conflicting reports in the literature. Time line legend is TT is the thermal
time for the interval; #LVS is number of leaves for the interval; S, sowing date; G, germination; E, seedling emergence;
TI, tiller initiation/appearance; SR, single ridge stage; DR, double ridge stage; J, jointing; B, booting; H, heading;
A, anthesis; and M, physiological maturity (From [59])

and 1980s undoubtedly spurred interest in including
this new knowledge in the models. This led to alterna-
tive modeling approaches based on more developmen-
tally driven approaches that recognized that plant
development is orderly and predictable based on basic
units (i.e., the phytomer) that dynamically appear,
grow, and senesce over time as discussed earlier in
this entry and shown in Fig. 9.
Early efforts beginning in the mid-1980s focused on
developmental concepts such as leaf appearance (the
phyllochron) and tillering that led to more accurate
representation of canopy architecture. For instance,
the AFRCWHEAT1/2 model developed in Europe
[60–62] contained detailed tillering and leaf dynamics
sub-models (e.g., appearance, growth, and senescence/
abortion), and the resulting effect on canopy LAI
was simulated and then used to estimate biomass.
Simultaneously and independently, another effort was
underway in the USA that resulted in the developmen-
tally driven MODWht3 [63] and SHOOTGRO [http://
arsagsoftware.ars.usda.gov, [59, 64–67]] models.
SHOOTGRO is slightly more developmentally detailed
for canopy processes than MODWht3, but less detailed
in the root system and simulating biomass production.

SHOOTGRO provides the foundation to simulate the
development and growth of individual phytomers (and
phytomer components as shown in Fig. 9) on each
morphologically identified shoot (main stem and til-
lers) on the median plant of up to six age classes, or
cohorts, based on time of seedling emergence.
The Sirius model has one of the most developed
and robust leaf appearance sub-models of any wheat
simulation model [68]. As with the MODWht3 and
SHOOTGRO models, the assumption used is that the
developmental “clock” from emergence to anthesis is
best represented by the rate of leaf appearance and final
number of leaves, rather than thermal time. Based on
vernalization requirement and photoperiod sensitivity
of the variety being simulated and leaf ontogeny, the
final leaf number is determined [69, 70]. This allows for
an elegant quantitative description of both spring and
winter wheat leaf appearance and integration with
developmental events.
Regardless of modeling approach and goals, the
ability to simulate genotype phenology across a broad
range of environments for major crops such as wheat
has been quite reliable. Many alternative approaches
exist for predicting phenology, and approaches differ in
input requirements and number of developmental
stages simulated. Essentially all models are based on
the thermal time approach, reflecting the importance
of temperature discussed in Section Developmental
Response to Temperature. An alternative to a strict
thermal time approach, particularly for small-grain
cereals, has been to use leaf numbers to estimate the
time interval between developmental stages. In phenol-
ogy sub-models, temperature effects are well consid-
ered, but rarely are the effects of water deficits (or
nutrient availability) considered [71]. Exceptions
include the SHOOTGRO model and PhenologyMMS
(http://arsagsoftware/ars.usda.gov).
Determining plant parameters and how to address
the genotype by environment interaction are
common concerns for all models. With the explosion
of genome mapping and molecular biology research,
opportunities for understanding and resolving these
issues are emerging [72–74]. For example, the presence
or absence of known alleles influencing a trait can be
used to determine the parameters used in the algorithm
representing the process [75] or the response to envi-
ronmental factors [76]. Clarification of gene networks
Crop Development Related to Temperature and Photoperiod 529
controlling processes such as time of flowering has
considerably advanced the understanding and simula-
tion of these processes [77].
Crop simulation modeling is increasingly benefit-
ing from the advent of object-oriented design and
programming languages such as C++, C#, and Java,
both in terms of developing and maintaining models as
well as providing greater flexibility in representing
plant processes within models. Initial object-oriented
designs tended to view the plant as a collection of
objects that equate to leaf, stem, root, and seed com-
ponents. Recent attempts have begun to incorporate
the phytomer approach of building plant canopies into
the object-oriented design that can also be scaled up, or
aggregated, into lower levels of resolution, such as the
seed component of earlier designs [78–80].
Future Directions
Crop development is regulated by environmental factors
that interact with the genetics of the plant. Many devel-
opmental responses related to temperature and photope-
riod are well known and it is possible to predict them
reasonably well at the crop level. Similarly, rapidly emerg-
ing knowledge from genomics research is helping to
provide understanding of the genetic basis of certain
aspects of crop development. Unfortunately, the quanti-
tative integration of genetic and physiological knowledge
is largely unknown, and both genetic and physiological
models would benefit from better integration of knowl-
edge. For example, the genetic basis of photoperiod and
vernalization pathways is fairly well known and as new
genomic studies are carried out, more complex models
are being built [81] showing the complexity of flowering
time. However, the genetic mechanisms described in
this entry interact with responses to other environmen-
tal factors defining a network of signaling a highly
complex response that is not fully understood.
Knowledge in crop development has greatly
benefited from research in model organisms; however
there are key differences, like the lack of the vernaliza-
tion gene FLC in temperate cereals. For example, four
vernalization genes have been described in wheat, but
only three have been cloned and located in the vernal-
ization pathway. Completing this pathway would
greatly increase the understanding of how temperate
crops respond to nonfreezing cold temperatures.
530 Crop Development Related to Temperature and Photoperiod
At the same time, the physiological response to tem-
perature has been extensively studied, and genetic dif-
ferences are well documented; however, the basis of
genetic effects is not known. It is encouraging that
new genes (e.g., Eps-1) are being identified, but the
quantitative variation of crop development, once
major processes (photoperiod and vernalization
requirements) are solved, requires the identification
of new genes, probably of small effect. For example,
a recent study in maize found no large effect QTL for
flowering time in a nested mapping population [82].
Although no major epistatic or environmental interac-
tions were found, the individual QTL effect varied
across founder lines of the population [82]. QTL or
gene effects need to be accurately determined to build
quantitative genetic models.
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Crop Diseases, Management
and Control of
DALE R. WALTERS
Crop & Soil Systems Research Group, Scottish
Agricultural College, Edinburgh, UK
Article Outline
Glossary
Definition of the Subject
Introduction: The Need for Disease Control in Crops
Controlling Crop Diseases
Future Directions
Bibliography
Glossary
Biotroph A plant pathogenic microorganism which
requires living host tissue in order to complete its
life cycle. Rust and powdery mildew fungi are
examples of biotrophs, as are viruses.
Oomycetes Also known as water molds, the Oomycetes
are a large group of terrestrial and aquatic organisms.
They superficially resemble fungi in mycelial growth
and mode of nutrition, but molecular studies
and distinct morphological characteristics place
them in the kingdom Stramenopila (or Chromista)
with brown and golden algae and diatoms.
Phytoalexin Antimicrobial substances synthesized de
novo by plants and which accumulate rapidly at
areas of infection by an incompatible pathogen.
Phytoalexins are broad spectrum in action and are
chemically diverse with different types characteris-
tic of particular plant species. They can be grouped
into several classes including terpenoids, alkaloids,
phenolics.
Saprophyte An organism, e.g., a fungus or bacterium,
that grows on and derives its nourishment from
dead or decaying organic matter.
Virulence Refers to the relative ability of a pathogenic
organism to cause disease.
Definition of the Subject
Plant diseases cause substantial crop losses every year
and, historically, have led to considerable economic
P. Christou et al. (eds.), Sustainable Food Production, DOI 10.1007/978-1-4614-5797-8,
# Springer Science+Business Media New York 2013
Originally published in
Robert A. Meyers (ed.) Encyclopedia of Sustainability Science and Technology,
Crop Diseases, Management and Control of 533
damage and human suffering. Controlling plant dis-
eases is therefore vital to maintaining crop productivity
and feeding the ever expanding human population.
Crop diseases can be controlled using a variety of
methods, notably cultural approaches, the use of resis-
tance in the plant, and the application of chemicals
(fungicides). However, the organisms that cause plant
disease (plant pathogens) are genetically adaptable and
can overcome plant resistance, and the toxic effects
of fungicides. Ensuring that crops are adequately
protected depends therefore on continually keeping
one step ahead of the pathogens by improving existing
control measures and developing new approaches. This
article provides an overview of the various methods,
traditional and novel, used to control crop diseases.
Introduction: The Need for Disease Control
in Crops
Plant disease has plagued mankind ever since the
beginnings of agriculture. Today, despite the many
advances in crop protection technology, crop diseases
continue to wreck havoc on crops, because of the
genetic adaptability of the pathogens which cause
plant disease. Crop losses at farm level can have serious
implications for growers, but crop disease can inflict
much more serious damage on a larger scale. A good
example of just how devastating crop disease can be is
the potato blight epidemic of the 1840s in Europe.
This disease, caused by the Oomycete pathogen
Phytophthora infestans, decimated crops across Europe
and in Ireland, led to the death of some one million
people and the emigration of several million more
[1, 2]. Astonishingly, today, more than 170 years later,
potato blight still poses a major problem for potato
growers across the globe.
Crop losses as a result of disease can be expressed in
various ways, such as potential losses and actual losses.
Potential loss compares yields in a system without any
form of crop protection treatment, with yields from
a system with a similar intensity of crop production,
but receiving crop protection treatments. Actual losses
are those sustained despite the use of crop protection
[3]. The efficacy of crop protection can be calculated as
the percentage of potential losses prevented. Potential
losses range from 8.5% for cotton to 21.2% for
# 2012, DOI 10.1007/978-1-4419-0851-3
534 Crop Diseases, Management and Control of
Crop Diseases, Management and Control of. Table 1
Estimated loss potential and actual losses due to patho-
gens (fungi and bacteria) in six major crops worldwide in
2001–2003 (Adapted from [3])
Crop losses (%) due to pathogens
Crop Potential Actual
Wheat 15.6 [12–20] 10.2 [5–14]
Rice 13.5 [10–15] 10.8 [7–16]
Maize 9.4 [8–13] 8.5 [4–14]
Potatoes 21.2 [20–23] 14.5 [7–24]
Soybeans 11.0 [7–16] 8.9 [3–16]
Cotton 8.5 [7–10] 7.2 [5–13]
potatoes, while actual losses range from 7.2% for cot-
ton to 14.5% for potatoes, highlighting the importance
of crop protection in reducing potential losses in all of
these crops (Table 1).
Globally, agricultural production has grown faster
than the human population over the past few decades
[4]. In most parts of the world, this has been the result,
not of increased area of cropped land, but of increased
inputs, including pesticides [4]. In the period from
1963 to 2002, cereal yields increased by 114% globally,
although the annual rate of growth fell from 3.14%
in the period 1963–1976 to 0.84% in the period
1989–2002 [4]. In the period from 1960 to 2004,
pesticide sales worldwide increased more than ten-fold
to some $30 billion [3]. However, despite this increased
pesticide use, crop losses as a result of pests, diseases,
and weeds have not fallen significantly in the past
40 years.
Controlling Crop Diseases
Crop disease can be controlled using a variety of
approaches. The first line of defense is the exclusion
of the pathogen through plant quarantine and, for
example, the use of pathogen-free propagating mate-
rial. The next line of defense is to exclude, eliminate, or
reduce pathogen inoculum. This can be achieved in
various ways, including cultural control, use of host
plant resistance, and chemical control. Finally, several
of these approaches might be used together in an
integrated program of disease control. In the sections
below, these disease control options will be examined
in more detail.
Cultural Control
Cultural control aims to prevent contact with the path-
ogen, to create environmental conditions unfavorable
to the pathogen or at least to avoid favorable condi-
tions, or to reduce the amount of pathogen inoculum
available to infect crop plants. Methods used include
host eradication, crop rotation, sanitation, irrigation,
tillage, and improving crop growth conditions, for
example, through appropriate fertilizer use. Cultural
control provides the foundation for disease control in
crops, and yet its important is often overlooked.
Host Eradication Host eradication refers to the
removal and disposal of whole infected plants. This
method is used routinely in nurseries, greenhouses,
and fields to prevent the spread of pathogens, since it
eliminates the infected plants that act as a source of
inoculum. In potato cultivation, pathogens can over-
winter in infected tubers left in the field and give rise to
infected plants (known as volunteers) in the spring.
These volunteers can acts as sources of inoculum, and
their removal from the field and subsequent destruc-
tion will reduce levels of pathogen inoculum.
If a pathogen requires two hosts to complete its life
cycle, control is possible by eradication of the less
important host. The wheat stem rust fungus, Puccinia
graminis f.sp. tritici, is a case in point. It requires two
hosts, wheat and barberry, to complete its life cycle and
until the 1950s was the most important pathogen of
wheat in the United States [5]. Since the 1950s, how-
ever, stem rust has declined in importance in the
United States, due in part to successful eradication of
its alternate host, common barberry [6].
Sanitation Sanitation refers to eliminating or reduc-
ing the amount of inoculum present by various means,
including removal of infected plant parts and plant
debris. Destroying crop residues is an important prac-
tice, but how it is performed depends upon the type of
crop and the type of pathogen. For example, burying
crop debris can destroy certain pathogens, particularly
if the residues are plowed in deeply enough, while

burning crop residue is common practice for cereal
crops in some parts of the world and will destroy
many pathogens. However, burning has some draw-
backs, particularly loss of nutrients and increased soil
erosion.
Crop Rotation Crop rotation is an ancient cultural
practice and its benefits include maintenance of soil
structure and organic matter, and a reduction in soil
erosion that is often associated with continuous row
crops [7]. The main purpose of rotating crops in con-
ventional arable rotations is to reduce the incidence of
diseases, pests, or weeds that are difficult to control
with pesticides, and for this reason, short rotations of
two to three crops are usually employed. In the United
States, for example, the majority of the maize crop is
grown on a 2–3 year rotation, while in the UK, barley
and wheat usually form the main part of the rotation,
with breaks of oilseed rape, beans, peas, or potatoes [8].
Continuous cropping with the same susceptible
host plant will result in the establishment of a soil
population of pathogenic microbes. Crop rotation
avoids this and is often associated with a reduction in
crop diseases caused by soilborne pathogens [7]. Using
nonhost or less susceptible crop plants in the rotation
can lead to a decline in specific populations of plant
pathogens in the soil and is best suited for biotrophs,
since they require the presence of the specific living
host for survival, or pathogens with low saprophytic
ability [9, 10]. Crop rotation is less suitable for con-
trolling root-inhabiting pathogens that survive sapro-
phytically or can exist for long periods in soil, e.g.,
pathogens with tough survival structures such as
Rhizoctonia solani, Sclerotinia sclerotiorum, and
Pythium spp. [11, 12].
Tillage Tillage has indirect effects on pathogen
spread and can also be used to reduce pathogen inoc-
ulum in the soil. Conventional tillage uses primary and
secondary cultivation to prepare a seed-bed for plant-
ing and results in considerable soil disturbance, while
reduced tillage uses a single cultivation, or even no
cultivation (no-tillage, zero tillage, direct drilling),
and as a result leads to minimal soil disturbance.
Minimum tillage and no-tillage practices can be
grouped together under the generic term: conservation
tillage [13].
Crop Diseases, Management and Control of 535
Tillage can bury pathogens deeper in the soil where
they are less likely to become a problem. It can alter soil
texture, aeration, temperature, moisture, and density,
and can also influence nutrient release in the soil, with
benefits to the crop [8]. Tillage also leads to clear
fluctuations in microbial activity and biomass in the
soil [14]. Reduced tillage or no-till is often associated
with higher microbial biomass and activity in upper
soil layers compared to regular tillage (plowing) [15].
This concentration of crop debris in the top layers of
the soil can promote the overwintering and survival of
numerous pathogens and has prompted concern that
increased disease and decreased yields will be the inev-
itable result of using conservation tillage practices.
Although this has proved to be the case under some
conditions, there have also been reports of decreases in
the incidence of soilborne diseases. As suggested by
Sturz et al. [13], such contradictory reports may reflect
differences in root development and soil microbial
biomass and activity under the different regimes.
Thus, conservation tillage practices can lead to patho-
gen inoculum concentrations several orders of magni-
tude greater than those found under conventional
tillage [16, 17] and, as a result, plant roots growing in
the upper soil layers might be more prone to pathogen
infection [13]. In contrast, increased microbial biomass
and activity in the top soil layers can give rise to greater
root density and root activity [18, 19], which may offset
the damaging effects of disease on yield, and might also
provide a highly competitive soil environment with
resulting disease suppressive effects [20].
Severity of tan spot in wheat was found to increase
under no-till conditions, but was reduced following
reduced tillage [21]. To control blackleg
(Leptosphaeria maculans) on canola (oilseed rape), it
is recommended that crop debris is buried in the
autumn and a nonhost crop be direct seeded the fol-
lowing spring to avoid reexposing the buried residue
[22, 23]. Recent research suggests that inoculum pro-
duction by L. maculans decreased with increasing dura-
tion of stubble burial in the field over 10 months before
stopping completely [24]. This effect may be due to the
mycobiota associated with the buried stubble, and
these workers suggest that it might be possible to
manipulate the population of saprophytic microbiota
present on oilseed rape stubble to facilitate the decline
of L. maculans [24].
536 Crop Diseases, Management and Control of
Sowing Practices
Time of Sowing Altering the time of sowing to avoid
high levels of pathogen inoculum or conditions
conducive for development of a particular disease can
lead to reduced severity of several crop diseases. For
example, in the UK, sowing winter oilseed rape in
August rather than September exposes the earlier
sown crop to inoculum from stubble of the previous
crop, resulting in more severe Alternaria infection on
pods. In contrast, the risk of infection is reduced in the
later sown crop because the stubble is buried by tillage
[25]. Late sowing may also be recommended for
autumn-sown barley crops, in order to decrease
exposure of newly emerging seedlings to inoculum of
Rhynchosporium secalis produced on previous barley
crops in the area [26].
Depth of Sowing Sowing depth can influence the risk
of infection, since the preemergence stage of the
seedling, which is usually more susceptible to pathogen
infection, is longer when seeds are sown deeper. In
Brassica rapa, for example, rapid emergence of
seedlings reduces preemergence damping-off because
the period of contact between the emerging seedlings
and R. solani in the soil is reduced [27]. Thus,
significantly higher seedling emergence was reported
for several cultivars of B. rapa sown at a depth of 1.5 cm
compared to 3.0 cm [27].
Crop Density Crop density can exert considerable
influence over disease incidence due to the ease with
which pathogen inoculum can be transferred between
closely spaced plants and alterations in crop
microclimate. In densely planted crops, temperatures
are more uniform, humidity is increased, and leaves are
wet for longer, all of which provides favorable
conditions for pathogen infection and subsequent
development. Crop density can be manipulated in
various ways, e.g., sowing, pruning, and fertilization.
Soil Amendments: Mulching, Fertilizers, and
Organic Amendments
Mulches Mulches are used to conserve organic matter
and moisture and to reduce soil erosion. A variety of
materials can be used as mulches, including straw,
manure, plastics, and paper. Mulching can lead to water
retention and nutrient enrichment in the soil and can
decrease soil temperature, all of which can influence
pathogen infection and disease development in plants.
Although mulching can reduce the spread of splash
dispersed pathogens, by altering the environment, it
could lead to increased severity of some diseases.
Further, if crop residues are used in mulching, disease
incidence could increase since the residues could be
used as a food source by a range of pathogens.
Fertilizers Adequate mineral nutrition is central to
crop production, and can also exert considerable
influence on disease development [28, 29]. Below, the
influence of nitrogen, phosphorus, potassium, calcium,
and silicon on plant disease will be dealt with briefly.
Nitrogen Using nitrogen fertilizer above recommen-
ded rates can lead to increased disease incidence and
lesion area. This has been shown for biotrophic fungal
pathogens such as powdery mildews and rusts [30, 31]
and necrotrophic pathogens such as Magnaporthe
grisea, the cause of rice blast [32]. It is commonly
thought that application of nitrogen fertilizer can
increase disease severity via effects on crop canopy
development. Thus, large canopies with high shoot
densities may be more conducive to spore transfer
and pathogen infection than sparse canopies. For
example, nitrogen has been shown to increase the
severity of Fusarium head blight in wheat, and it has
been suggested that this might be the result of
a nitrogen-induced increase in canopy size, leading to
an altered microclimate [33]. In contrast, work on
yellow rust on winter wheat suggested that the impact
of nitrogen on disease was the result of effects of
nitrogenous substances in wheat leaves on pathogen
growth, rather than effects on canopy growth and
microclimate [34].
But nitrogen is not always associated with increased
crop disease. Indeed, various studies have reported no
effect of nitrogen on disease severity (e.g., [35, 36]),
while Hoffland et al. [31] found that the effect of
nitrogen depended on the type of pathogen. Thus,
nitrogen increased susceptibility of tomato to the pow-
dery mildew pathogen Oidium lycopersicum and the
bacterial pathogen Pseudomonas syringae pv. tomato,
while it had no effect on susceptibility to the vascular
wilt pathogen Fusarium oxysporum f.sp. lycopersici [31].
In contrast, tomato plants were more susceptible to
Botrytis cinerea when grown under low nitrogen con-
ditions [37]. These results do not support the view that

nutrient-limited plants are better defended [38, 39]. It
seems that generalizing about the effects of nitrogen on
plant disease is unwise, and practically, although
manipulation or assessment of crop nitrogen status
might be used as part of disease control strategies, the
approach adopted will depend on the crop and the
pathogens from which it is most at risk [29].
Phosphorus In general, phosphorus fertilization
tended to improve plant health, with reductions in
disease recorded in 65% of cases studied by Perrenoud
[40]. Nevertheless, phosphorus fertilization increased
disease and pest problems in 28% of the cases examined
[40]. As with nitrogen, the effects of phosphorus on
plant disease might be the result of direct effects on the
pathogen, host plant metabolism, leading to effects on
pathogen food supply, and effects on plant defenses
[29]. Indeed, foliar application of phosphate salts has
been shown to induce resistance to pathogens in
a range of crop plants, including cucumber [41],
broad bean [42], grapevine [43], maize [44] and
rice [45].
Clearly, an adequate phosphorus supply is impor-
tant for crop growth and in turn, may well help to
reduce disease. However, the regime of phosphorus
fertilizer used will depend on a range of factors, includ-
ing the crop and the pathogens likely to be important.
Reuveni and Reuveni [46] suggested that foliar-applied
phosphate might be used as part of an integrated dis-
ease control program. However, grower adoption of
such an approach will depend on the existence
of other, effective disease control measures and the
economics of disease control in the particular crop.
Potassium There are many reports that potassium is
associated with disease reductions [47]. However, as
these authors point out, inadequate consideration has
been given to the effects of associated anions, nutrient
balance, and nutrient status, to allow the definitive role
of potassium to be determined. Thus, it has been
suggested that in some cases, the effects of potassium,
applied as potassium chloride fertilizer, might be due to
the chloride ion rather than potassium [48]. Further,
chloride fertilization has been shown to suppress dis-
ease in cereal crops [49].
There has been much interest in the application of
fertilizers to crop foliage, including the effects of foliar
fertilizer application for crop disease control [46, 50].
Crop Diseases, Management and Control of 537
Foliar-applied potassium chloride has been shown to
control Blumeria graminis and Septoria tritici on wheat
in field studies [51, 52], probably due to osmotic effects
on the fungal pathogens, disrupting pathogen develop-
ment and subsequent infection [52, 53].
Application of potassium to deficient soils usually
increases plant resistance to diseases [47]. This might
be partly related to the effect of potassium in increasing
epidermal cell wall thickness or disease escape as
a result of vigorous crop growth [47], although the
mechanisms by which potassium affects plant disease
are not well understood.
Calcium There are many reports that application of
calcium to soils, foliage, and fruit reduces the incidence
and severity of a range of diseases of crops, including
cereals, vegetable crops, legumes, fruit trees, as well as
postharvest diseases of tubers and fruits [54]. For
example, calcium has been shown to inhibit anthrac-
nose (caused by Colletotrichum gloeosporiodes or
C. acutatum) in apples [55] and to decrease postharvest
disease development on strawberry [56], while treat-
ment of tomato with calcium carbonate reduced fusar-
ium crown rot disease [57]. In contrast, Nam et al. [58]
could find no effect of calcium on anthracnose on
strawberry. Because calcium increases resistance of
plant cell membranes and cell walls to microbial
enzymes, increasing calcium concentrations in storage
organs could lead to enhanced resistance to pathogens
[59, 60]. However, the form in which the calcium is
applied can influence the mechanism by which calcium
affects disease. For example, the addition of lime
can affect disease by altering pH, while calcium salts
(e.g., propionate) can be directly inhibitory to patho-
gens [54]. Making general recommendations for the
use of calcium in plant disease control would be
unwise, due to the range of crops and pathogens
affected by calcium application. Instead, the appropri-
ate amount and form of calcium to apply need to be
determined for individual crop–pathogen interactions.
The dwindling availability of fungicides, together with
increasing public concern for the environment means
that the use of calcium to control plant disease, espe-
cially postharvest, is attracting increased attention.
Silicon The effects of silicon in reducing disease
severity have been known since 1940 [61]. However, it
was not until the 1980s that more detailed work was
538 Crop Diseases, Management and Control of

150
M. graminicola E. graminis
P. recondita Fusarium spp.

Change in disease (%)

100

50

−50

−100
20 30 40 60 70 80
Growth stage

Crop Diseases, Management and Control of. Figure 1

Changes in disease in plots with incorporated straw. Bars show the difference between straw-treated and control plots
as a percentage of the average in the control plots. For Mycosphaerella graminicola, the data are for severity on the top
three living leaves; for other diseases, they refer to incidence. For M.graminicola, errors bars were calculated on the
transformed scale and back-transformed; for other diseases, no transformation was needed (From [66])

carried out in this area. Thus, cucumbers grown in
nutrient solutions supplemented with silicon were
found to have significantly less powdery mildew infec-
tion than plants not receiving silicon supplementation
[62, 63]. Indeed, silicon has been shown to suppress
both foliar and soilborne pathogens in cucurbits [64]
and to reduce susceptibility of rice to various pathogens
[65]. Wheat grown in soil amended with silicon
showed reduced infection by several pathogens, includ-
ing B. graminis f.sp. tritici, S. tritici, and Oculimacula
yallundae (Fig. 1) [66, 67].
It has been suggested that the effects of silicon in
providing disease control are due to the creation of
a mechanical barrier to penetration [68]. However,
this has been disputed by studies which could find no
evidence for the creation of a physical barrier following
silicon treatment in wheat inoculated with powdery
mildew and bitter gourd and tomato inoculated with
Pythium aphanidermatum [69, 70]. Rather, several
studies have suggested that silicon activates defenses
in plants. For example, in wheat inoculated with
B. graminis f.sp. tritici, epidermal cells of silicon-treated
plants were shown to react to attempted infection with
specific defenses, including papilla formation and
callose production [71]. In the rice–M. grisea
pathosystem – silicon-mediated resistance was found
to be associated with accumulation of antimicrobial
compounds at infection sites, including diterpenoid
phytoalexins [72]. In fact, phytoalexin accumulation
occurs in silicon-mediated resistance in both dicots
and monocots and since phytoalexins are highly spe-
cific to plant species, it has been suggested that silicon
might be acting on mechanisms shared by all plant
species, e.g., those resulting in activation of plant stress
genes [73].
Organic amendments Organic amendments include
animal manure, solid wastes, and composts. Such
amendments are often used to improve soil quality,
usually by contributing to general suppressiveness
through enhanced microbial biomass and activity [7].
Organic amendments are rich in labile carbon fractions
which are an energy source for microorganisms, and
moreover, they can themselves contain antagonistic
microbes. A substantial body of data indicates that
organic materials can reduce incidence of diseases
caused by a range of plant pathogens [74, 75].
Irrigation Although an adequate water supply is vital
to crop production, irrigation can play a detrimental
rather than a beneficial role in managing plant diseases.
For example, irrigation water can spread pathogen
propagules and under dry conditions, can prevent
desiccation of such propagules, thereby effectively
increasing the level of inoculum in soil. Watering
from overhead prolongs leaf wetness, thereby

providing favorable conditions for germination and
infection by fungal spores. Overhead watering also
increases the risk of splash-dispersal of spores, thus
increasing pathogen spread. However, irrigation can
be used to reduce the level of pathogen inoculum.
Thus, the activity of microbes that destroy fungal scle-
rotia can be increased by alternate wetting and drying
of the soil. Generally, drip or trickle irrigation, which
delivers water directly to the root zone at a rate insuf-
ficient to lead to pathogen spread, is least likely to
encourage disease development.
Use of Host Resistance
Most plants are resistant to most microbes. Thus,
wheat plants are not affected by pathogens of tomato,
and vice versa. This is known as nonhost resistance.
However, every plant is attacked by its own pathogens,
e.g., barley is attacked by the barley powdery mildew
fungus, B. graminis f.sp. hordei, although it might not
be able to defend itself equally well against all patho-
gens that are able to attack it. This ability of plants to
resist attack by pathogens is genetically determined, but
the diversity of types of genetic control of this resis-
tance, and of conditions required for their expression,
has led to a bewildering number of classifications of
resistance types in plants. To complicate matters fur-
ther, in some cases, different terms used by different
authors are synonymous, while in other cases, the
terms might be based on different criteria.
Types of Resistance
Seedling and Adult Plant Resistance Seedling
resistance operates from the onset of plant growth
and is effective throughout the life of the plant. It is
generally controlled by single genes and is effective in
the absence of matching virulence in the pathogen. In
contrast, adult plant resistance (APR) covers a broad
range of resistance types all of which are not effective at
the plant’s seedling stage. APR tends to be controlled by
a number of genes, which might operate through
a wide variety of mechanisms.
Polygenic and Oligogenic Resistance Resistance
controlled by one, or at most two or three genes, is
known as oligogenic. Here, a single gene can confer
complete resistance. A gene conferring such resistance
is often referred to as a major gene. Where resistance is
Crop Diseases, Management and Control of 539
controlled by a larger number of genes, it is known as
polygenic. Genes conferring polygenic resistance are
often referred to as minor genes.
Race-specific and Race-nonspecific Resistance The
terms race-specific and race-nonspecific sought to
differentiate between resistance that was subject to
loss of effectiveness with the appearance of new
virulent strains of a pathogen and resistance that was
thought would never be lost because the pathogen was
not capable of developing virulence to it. A major
problem with these terms has been a lack of evidence
to suggest that any particular resistance was race-
nonspecific. The assumption was generally made that
genes of large effect (major or seedling genes) were
race-specific and genes of small effect (minor genes or
APR) were race-nonspecific. This has subsequently and
comprehensively been shown not to be the case in
many host–parasite systems.
Vertical and Horizontal Resistance These terms were
introduced to convey the difference between race-
specific and race-nonspecific resistance [76]. In vertical
resistance, there tends to be a high level of resistance by
the host to some races of the pathogen and a low level of
resistance to others, while in horizontal resistance,
resistance tends to be exhibited equally to all races of
the pathogen. Vertical resistance has also been denoted
as specific resistance and horizontal resistance as
general resistance. However, although the specific
nature of some types of resistance can usually be
established fairly easily, it is much more difficult to
prove that resistance is truly general, since there is
always the chance of a new pathogen race appearing
with the ability to overcome the resistance.
Qualitative and Quantitative Resistance These terms
are a rough match for seedling/APR and vertical/
horizontal resistance, with qualitative resistance
referring to a high level of resistance controlled by
a single gene and quantitative resistance being
controlled by several genes of smaller effect. The term
“quantitative” subsequently came to be used to
describe locations on chromosomes where genes of
small effect were identified through mapping studies,
hence “quantitative trait locus” or QTL. More recently
the use of the term QTL has come to be used for major
gene loci as well.
540 Crop Diseases, Management and Control of
Partial Resistance Partial resistance was originally
used in studies of the resistance in barley to leaf rust
caused by Puccinia hordei. It describes resistance that
reduced the rate of epidemic development despite
having a susceptible reaction type [77]. Resistance to
leaf rust was found to be governed by up to 6–7 minor
genes with additive effects and was correlated with
increased latency period and reduced infection
frequency, pustule size, infectious period, and spore
production. The term is now used more generally to
describe any resistance that is only partially effective in
reducing disease expression and is usually synonymous
with minor gene resistance.
Durable Resistance Durable resistance was suggested
for use by Johnson and Law [78] to refer to rust
resistance in wheat that in practice had provided
stable resistance in varieties that had been grown over
a large area for many years. The term specifically
avoided identifying the resistance with particular
phenotypes or suggesting that the resistance would
never be lost to a change in pathogen virulence.
Specific well-studied examples are the Sr2 and Rpg1
genes for partial stem rust in wheat and barley,
respectively, the genes Lr34 and Yr18 which provide
partial resistance to leaf and stripe rust in wheat
and which may in fact be the same gene and the mlo
locus which provides resistance to powdery mildew in
barley.
Genetic Engineering for Disease Resistance Using the
techniques of genetic manipulation (GM), it is now
possible to sequence and clone resistance genes from
distantly related species. These genes could then be
transferred into crop species, providing varieties with
enhanced disease resistance. For example, the gene for
resistance to bacterial blight of rice, Xa21, was
sequenced, cloned, and transformed into rice,
providing resistance against a range of pathogens
[79]. This is a good example of how transgenic
technologies could be used in a major crop and
indeed, transgenic rice lines expressing Xa21 are
currently being tested in the field. The vision is to use
GM technology to pyramid several different resistance
genes in a rice variety, providing what is hoped will be
durable resistance. However, unless there is
coordination among plant breeders and the industry
to prevent varieties being grown together, thereby
allowing the pathogen to mutate one gene at a time,
this strategy is unlikely to be successful.
One option for improving resistance to virus infec-
tion is to transform the gene that expresses the viral
coat protein into the host plant. This approach was
used to develop a line of papaya resistant to the very
damaging papaya ringspot virus (PRSV). As a result of
this work, two papaya varieties, Rainbow and SunUp,
were commercialized. Rainbow was widely planted in
Hawaii and was important in preventing the complete
destruction of the papaya industry by PRSV [80].
Deployment of Resistance in Practice Many new
crop varieties have been bred where resistance to
a particular pathogen is based on the introduction of
one gene. All too often history has shown that the
introduction of such varieties is followed, within just
a few years, by the appearance of a new race of the
pathogen, able to overcome the “new” resistance. Such
rapid breakdown of host resistance is favored by mod-
ern intensive agriculture, where crops are planted in
monoculture covering huge areas. This favors any
genetic variants of the pathogen with the ability to
infect these new varieties. Breaking this “boom-and-
bust” cycle of introducing a new resistant variety,
followed by the rapid breakdown of resistance, can be
achieved in various ways. One approach is to find
durable sources of resistance. Another is to breed for
combinations of race-specific genes in one crop variety.
This is known as pyramiding and should be quite
durable. However, it is controversial, since it might
select for complex races of the pathogen possessing
several matching virulences. Another approach is to
diversify the deployment of resistance genes. This
could involve spatial diversity across regions or fields,
or the use of multilines or mixtures of cultivars.
A multiline is a series of near-isogenic (genetically
identical) plant lines each differing in a single character,
e.g., disease resistance. The plant lines can be grown
together like a conventional crop, thereby retaining
agronomic advantages such as crop uniformity, while
confronting the pathogen with the problem of over-
coming several different genes for resistance. A number
of mechanisms have been proposed to account for
disease control in multilines: (1) The high proportion
of resistant plants in the multiline grown in the field
reduces the amount of pathogen inoculums per unit

7 Fungicide
Control
% yield increase 6
5
y = 0.716x + 1.61
4 R2 = 0.8383
0
2 3
Mixture component number
Crop Diseases, Management and Control of. Figure 2
Change in R. secalis infection of mixtures of winter barley cultivars compared with the mean of their components with
different numbers of component cultivars (From [82])
area of crop, thereby reducing the amount of infection
in the succeeding generation and the amount of inoc-
ulums produced by that generation. (2) Movement of
pathogen inoculums between susceptible plants might
be hampered by the presence of intervening resistant
plants. (3) The average distance which the pathogen
inoculums must travel in order to reach another sus-
ceptible plant is increased. (4) There might be induc-
tion of resistance, i.e., pre-inoculation with a race of the
pathogen to which the line is resistant might protect it
from a race to which it is normally susceptible [81].
However, the breeding program required for the devel-
opment of a multiline is extensive. Similar in concept,
but easier to put into practice, is the use of variety
mixtures. Here, seed of a number of genetically distinct
varieties of the crop are mixed and grown together as
a single crop. Each variety possesses a different resis-
tance gene(s), again presenting the pathogen with
a genetic puzzle. But the use of mixtures requires care-
ful choice of varieties, since they must possess similar
characteristics, such as crop height and time to
flowering. Nevertheless, mixtures have been shown to
develop less disease than would be expected in compo-
nent varieties grown alone. Mixtures can also provide
yield increases, and the magnitude of disease control
Crop Diseases, Management and Control of 541
y = 1.083x + 2.185
R2 = 0.8753
4 5 6
and effects on yield are dependent on the number of
components in the mixture (Fig. 2). The mechanisms
proposed to account for reductions in disease in mix-
tures include less efficient spread between plants and
the induction of plant resistance by incompatible
strains of the pathogen attempting to infect different
hosts in the mixture [82].
Use of Chemicals to Control Crop Disease
Chemicals continue to play an important role in crop
disease control. These chemicals act either by inhibiting
germination, growth, and multiplication of the patho-
gen, or by killing the pathogen. Although chemicals can
be used to control bacterial pathogens (bactericides)
and nematodes (nematicides), this section will concen-
trate on chemicals with activity against fungal patho-
gens (fungicides).
Fungicides can be divided into several groups
according to their mode of action. Protectant fungi-
cides protect the plant against fungal propagules (e.g.,
spores) landing on the surface of leaves, stems, or fruit,
but tend to be ineffective against established infections,
since they do not enter the plant to any extent. To be
effective therefore, protectant fungicides need to be
542 Crop Diseases, Management and Control of
applied before the fungal pathogen enters the plant.
In contrast, systemic fungicides enter the plant, and
can become generally distributed within its tissues,
thereby offering protection against fungal pathogens.
Eradicant fungicides can enter plant tissues and can kill
established infections. A number of protectant and
systemic fungicides possess eradicant properties,
while some systemic fungicides also have protectant
activity.
Protectant Fungicides Protectant fungicides can be
applied to the crop as high-volume or low-volume
sprays, or occasionally as dusts. These fungicides tend
to be broad spectrum, with activity against a range of
different fungal pathogens. As a result, it is unlikely that
pathogens would develop resistance to these chemicals.
However, because they must be applied before the
pathogen attempts to penetrate the plant, there is the
need for reliable forecasting of infection risk. Protec-
tant fungicides include the oldest and still widely used
inorganic sulfur and copper compounds. Sulfur,
applied as a dust, wettable powder, paste, or liquid, is
used to control powdery mildews, some rusts, leaf
blights, and fruit rots. Bordeaux mixture, made by
mixing copper sulfate solution with calcium oxide or
calcium hydroxide, consists mainly of colloidal
hydrated cupric hydroxide stabilized by calcium
sulfate. It has been replaced by copper oxychloride,
which can be formulated by the manufacturer and
simply diluted by the grower.
Organic sulfur compounds are an important and
versatile group of fungicides and include thiram,
ferbam, maneb, zineb, and mancozeb. These fungicides
are all derivatives of dithiocarbamic acid, and because
they are metabolized to isothiocyanate, they inactivate
the sulfhydryl groups (-SH) in amino acids and
enzymes. Thiram is used mostly for seed and bulb
treatment for vegetables, flowers, and grasses, while
ferbam is used to control foliage diseases, ornamentals,
and fruit on trees. Maneb and mancozeb belong to
a group of dithiocarbamic acid derivatives known
as the ethylenebisdithiocarbamates. Maneb is a broad-
spectrum fungicide for the control of foliage and
fruit diseases of vegetables such as tomato, potato,
and vine crops, as well as flowers, turf, and some fruit
crops. Mancozeb is formed by the addition of zinc
ion to maneb and a secondary effect of using mancozeb
is the provision of manganese and zinc to plants
deficient in these elements.
Various aromatic compounds have been developed
as fungicides and include dichloran, used as a foliar or
fruit fungicide, or as a postharvest spray for vegetables
and flowers, and chlorothalonil, which is a broad-
spectrum fungicide used on vegetables, field crops,
ornamentals, and turf.
A number of very effective protectant fungicides
belong to the rather heterogeneous group of heterocy-
clic compounds and include captan, iprodione, and
vinclozolin. Captan is used for the control of leaf
spots, blights, and fruit rots on various crops, as well
as a seed treatment. It works by inhibiting thiol-
containing enzymes in the fungal cell, and may also
react with sulfhydryl groups.
Systemic Fungicides For a systemic fungicide to be
effective, it must enter the plant and, to be translocated,
it must be reasonably water-soluble. It must also be
selective, possessing toxicity against the pathogen but
not the host plant. Most systemic fungicides are xylem-
mobile and as a result, tend to move from the base to
the top of the plant, accumulating in leaves and shoot
apices. As a result, such fungicides possess no
activity against soilborne pathogens affecting roots.
Phosphonate fungicides such as fosetyl-Al are also
phloem-mobile and so can move down the plant to
the roots, providing protection against, for example,
root rots caused by Phytophthora species.
A wide range of systemic fungicides are now used to
control plant pathogens. Unlike protectant fungicides,
most systemic fungicides are site specific, and target
only one, or just a few, specific steps in fungal metab-
olism. Although this was seen initially as a strength of
systemic fungicides, it soon proved to be a weakness,
since fungal pathogens were able to develop resistance
to the chemicals, in some cases with alarming rapidity.
Acylalanine fungicides are effective against
Oomycete pathogens such as Phytophthora and
Pythium and include the widely used fungicide,
metalaxyl. This is used as a soil or seed treatment and
also possesses some curative activity. It moves readily
from roots to shoots, although its lateral movement is
slight. Some fungal pathogens have developed resistance
to metalaxyl, and its use tends to be recommended
in conjunction with a broad-spectrum fungicide.

Benzimidazole fungicides include some well-
established and important chemicals, including beno-
myl, carbendazim, and thiabendazole. They are
converted to methyl benzimidazole carbamate (MBC,
carbendazim), which interferes with cell division.
Benzimidazoles are broad spectrum in activity. Thus,
benomyl is effective against a wide range of leaf spots,
blights, rots, scabs, seedborne, and soilborne diseases.
It can be applied to plants as a seed treatment, foliar
spray, trunk injection, root dip, or fruit dip.
Oxanthiins, such as carboxin and oxycarboxin, pos-
sess the distinction of being the first chemicals with
demonstrated systemic activity. They are active against
some smut and rust pathogens, and also against Rhi-
zoctonia. By inhibiting succinate dehydrogenase in fun-
gal mitochondria, they affect respiration.
Organophosphate fungicides, such as foestyl-Al
and phosphorous acid, are effective against Oomycete
pathogens on a range of crops. Interestingly, fosetyl-Al
has been reported to trigger plant defense mechanisms,
e.g., phytoalexin biosynthesis and accumulation.
Other systemic fungicides include the pyrimidines,
such as ethirimol and bupirimate, with activity
against powdery mildews, and the triazoles,
such as triadimefon, propiconazole, cyprodinil, and
tebuconazole. The triazoles exhibit long-acting protec-
tive and curative activities against a broad spectrum of
pathogens.
The most recently developed group of fungicides is
the strobilurins. They are based on chemicals extracted
from the wood rotting fungus Strobilurus tenacelus, and
a range of highly effective strobilurins have been devel-
oped over the years. They work by blocking electron
transfer at the site of quinol oxidation (Qo site,
hence their description as QoI fungicides), thereby
preventing ATP formation in respiration. These fungi-
cides move trans-laminarly within leaves and some also
move systemically through the vascular system. Impor-
tant strobilurins include azoxystrobin, pyraclostrobin,
and kresoxim methyl. They have broad-spectrum activ-
ity, and some also possess growth-promoting activity
on treated plants, apparently by delaying senescence
and altering plant–water relations.
Development of Resistance to Fungicides Until the
advent of systemic fungicides, fungicide resistance was
rare. The development of resistance in Pyrenophora to
Crop Diseases, Management and Control of 543
mercury used as a seed dressing, in the apple scab
fungus Venturia inaequalis to dodine, and in
Penicillium species to diphenyl compounds are
exceptions. In contrast, the use of systemic fungicides
soon led to the appearance of fungicide resistance.
Thus, the pyrimidine fungicide dimethirimol was
introduced in 1968, and by 1971, strains of the
powdery mildew fungus Sphaerotheca fuligenea were
detected on glasshouse cucumbers in the Netherlands.
Similarly, following the introduction of QoI fungicides
in the UK in 1996, resistance to powdery mildew in
wheat was first recorded in 1998.
It seems that where a single site systemic fungicide
is used intensively, there is the risk of fungal pathogens
developing resistance to it. With single site fungicides,
only a single mutation in the fungus might be required
for resistance to develop. Some of the mechanisms by
which a fungus might develop resistance following such
a mutation include: (1) detoxification of the chemical,
(2) decreased permeability of fungal cell membranes to
the fungicide, (3) reduced affinity of the fungicide to
the reactive site within the fungal cell, and (4) bypassing
a blocked reaction via an alteration in fungal
metabolism.
Given that fungicide resistance is now an accepted
fact of life in crop protection, how can it be managed
effectively? The first point to remember is that fungi-
cides to which resistance has developed can still be
useful in disease control if deployed sensibly. Strategies
to minimize the risk of fungicide resistance developing
include:
1. Reducing fungicide use
● Applying fungicides only when necessary
● Using fungicides as part of an integrated disease
control program, including, for example,
appropriate cultural control, and if available,
resistant varieties
2. Diversifying fungicide treatments
● Avoiding the repeated use of fungicides with the
same mode of action
● Instead, use mixtures of fungicides with differ-
ent modes of action
● During the growing season, alternating fungi-
cides with different modes of action
● Including a multisite fungicide in any fungicide
mixture or spray program
544 Crop Diseases, Management and Control of
Pesticides and Nontarget Organisms Pesticide use has
greatly increased the quantity and improved the quality
of food for the increasing world population. However,
as pesticide use increased, so did concern about their
adverse effects on nontarget organisms, including
humans. Increasing public concern about the
accumulation of pesticides in the environment and
the impact on nontarget organisms has led to the
introduction of rigorous regulatory processes [83,
84]. Nevertheless, there is continued concern over the
impacts of pesticides on wildlife, including invertebrate
populations, wild plants, and farmland birds [85].
These concerns have led to reviews of active
substances used in plant protection, with the resulting
withdrawal of an increasing number of crop protection
products from the market [86]. This has created
problems, and in some situations, effective control
measures are no longer available to meet all the
challenges posed by pathogens, pests, and weeds [86].
Biological Control of Plant Diseases Using
Antagonistic Microorganisms
Biological control can be defined as the control of a plant
pathogen using another living organism or organisms.
This definition includes direct and indirect effects, as
a result of either the introduction of antagonists, or
manipulation of existing microbial populations to
reduce disease. This section will deal with biological
control using antagonistic microorganisms.
Antagonistic microorganisms control plant disease
because they weaken or destroy the pathogen. They
achieve this by various mechanisms, including:
(1) directly parasitizing the pathogen, (2) producing
antibiotics or toxins that exert an effect on the patho-
gen, (3) competing for space and nutrients with the
pathogen, and (4) producing hydrolytic enzymes that
destroy components of pathogen cells.
To be effective in disease control, a biological con-
trol agent (BCA) must be able to colonize a particular
habitat or occupy a niche in sufficient numbers to
disrupt the growth and survival of the target pathogen.
For this reason, the most effective BCAs are likely to be
found in the environment in which they are to be used.
Thus, if the pathogen to be controlled infects plant
roots, the best place to look for a potential BCA
would be the rhizosphere. Finding appropriate BCAs
is one thing, but using them in practice is quite another.
For a start, as a living organism, the BCA must be
formulated in such a way as to allow it to remain viable
and survive following application [87]. Great advances
have been made in the production and formulation of
BCAs over the years, but despite the fact that 1,000 of
microorganisms have been shown to interfere with
growth and survival of plant pathogens under con-
trolled and field conditions, relatively few have been
registered and used in commercial practice. Currently,
there are more than 50 bacterial products and 50 fungal
products available commercially. The majority of both
bacterial and fungal products are sold for control of
seedborne or soilborne pathogens (Table 2), with fewer
for foliar pathogens and timber decay fungi and even
fewer for postharvest pathogens. Most contain individ-
ual microorganisms although there are some products
that contain microorganism mixtures, and some indi-
vidual microorganism strains are marketed in several
different products expanding the potential market of
a single active ingredient. Bacterial products are dom-
inated by Bacillus species reflecting their ease of growth
and production of long-lived spores mentioned earlier.
Fungal products are dominated by Trichoderma
spp. which are also easy to produce, generally have a
low toxicity, and can sporulate well.
America’s first biological fungicide seed treatment,
Kodiak (marketed by Bayer), contains spores of Bacillus
subtilis GB03 for control of Alternaria, Aspergillus,
Fusarium, and Rhizoctonia spp. that attack root systems
of a number of plants, including seed and pod vegeta-
bles, cotton, peanut, soybean, wheat, barley, and maize.
The spore concentrate can be applied through standard
slurry or mist seed treatment equipment, with fungi-
cides if required, and the bacterium colonizes the root
system providing control over the whole growing
season.
Numerous products contain strains of Trichoderma
harzianum, but one isolate, KRL-AG2 (T-22), sold by
BioWorks Inc., USA, has been used and developed for
several markets in US horticulture and agriculture in
a number of different forms. When applied to soil,
planting mixes, or turf, this BCA colonizes plant roots
and provides protection against root pathogens such as
Cylindrocarpon, Fusarium, Pythium, Rhizoctonia, and
Thielaviopsis. RootShield granules are largely targeted
at glasshouse and nursery use, and can be incorporated,
 Crop Diseases, Management and Control of 545

Crop Diseases, Management and Control of. Table 2 Examples of bacteria and fungi registered or commercially
marketed as biological control agents for control of soilborne and seedborne plant pathogens (from [87])

Target pathogen(s)/
Antagonist activity Disease/host Product name and source
BACTERIA
Agrobacterium Agrobacterium tumefaciens Ornamentals and other Norback 84-C, Galltrol-A, Nogall,
radiobacter causing root galls plants sensitive to A. Diegall, Dygall (Becker Underwood
tumefaciens root galls Pty Ltd, Australia; Bio-Care
Technology Pty Ltd. Australia; New
BiProducts, Inc. USA; AgBiChem
Inc., USA; Agbioresearch Ltd.
New Zealand)
Bacillus cereus BP01 Plant growth promotion Cotton Mepplus (MicroFlo Co. LLC, USA)
B. pumilus GB34 Fusarium spp., R. solani Soybean YieldShield concentrate; GB34
Biological Fungicide (Gustafson
LLC, USA)
Bacillus licheniformis Numerous pathogens, Ornamentals, turf EcoGuard, Green Releaf
SB3086 especially Sclerotinia (Novozymes Biologicals Inc., USA)
homeocarpa
B. subtilis Pythium damping-off Tomato Cillus, Green-all G (Greenbiotech
Co, Korea)
R. solani, Fusarium spp., Root rots and seedling Kodiak, Epic, Concentrate, Kodiak
Alternaria spp., and diseases generally HB, Quantum 4,000, System 3
Aspergillus spp. pathogens (Gustafson LLC, USA)
Fusarium spp., Verticillium Various vegetable and PHYTOVIT WG (Prophyta
spp., R. solani, and Pythium field crops Biologischer Pflanzenschutz GmbH,
spp. pathogens Germany)
B. subtilis GB03 Fusarium spp., Ornamentals, turf, dry Companion, Kodiak (Growth
Phytophthora spp., Pythium and snap bean, cotton, Products Ltd, USA; Gustafson LLC,
spp., R. solani peanut, soybean, wheat, USA; Bayer CropScience LP, USA)
and barley
B. subtilis MBI600 Alternaria spp., Aspergillus Alfalfa, dry/snap beans, HiStick N/T, Pro-mix, Subtilex;
spp., Fusarium spp., peanut, soybean, field Subtilex HB (Becker Underwood
Pythium spp., R. solani crops, turf, cotton Inc., USA; Premier Horticulture Inc,
Mexico)
B. subtilis subsp. Fusarium spp., R. solani Various vegetables and Taegro, Tae-Technical (Earth
amyloliquefaciens FZB24 ornamentals Biosciences Inc. USA)
B. subtilis, Bacillus Damping-off (bacterial) All crops Hydroguard (American Agritech,
circulans, Bacillus diseases USA)
amyloliquefaciens,
Paenibacillus polymyxa
(Mixture)
Burkholderia cepaciaa Pythium spp., Fusarium Alfalfa, beans, clover, Deny, Blue Circle (Stine Microbial
spp., R. solani, nematodes cotton, peas, wheat, Products, USA)
vegetables, and others
Pythium spp., Fusarium Maize, vegetables, Intercept (Soil Technologies Corp.,
spp., R. solani, cotton USA)
546 Crop Diseases, Management and Control of

Crop Diseases, Management and Control of. Table 2 (Continued)

Target pathogen(s)/
Antagonist activity Disease/host Product name and source
Erwinia carotovora, Bacterial soft rots Vegetables, cruciferous Biokeeper (Central Glass Co. Ltd.,
nonpathogenic plants, rice Japan)
P. polymyxa AC-1 Damping-off Cucumber NH, Topseed (Greenbiotech Co.,
Korea)
Pseudomonas sp. Growth promotion, various Vegetables, potato, Proradix (Sourcon-Padena,
seedborne and soilborne cereals, etc. Germany)
diseases
Pseudomonas Various turf grass Turf grass diseases Spotless (Turf Science laboratories
aureofaciens TX-1 pathogens Inc, USA)
Pseudomonas Drechslera spp., Septoria Cereal seedborne Cedomon (BioAgri AB, Sweden)
chlororaphis MA 342 spp., Fusarium spp. diseases
P. chlororaphis 63-28 Pythium spp. R. solani, Stem and root rots, and AtEze (Eco Soil Systems Inc., USA)
F. oxysporum wilt disease in various
crop plants
Pseudomonas fluorescens Frost damage, E. amylovora Fruit, potato, tomato, BlightBan A506 (NuFarm Inc., USA)
berries
Pseudomonas P. solanacearum P. solanacearum rots in PSSOL (Natural Plant Protection,
solanacearum, vegetables France)
nonpathogenic
P. syringae Botrytis spp., Penicillium Fruit, potato Bio-save (EcoScience Corp., USA)
spp., Mucor spp.
Streptomyces R. solani Turf Mycocide (KIBC Co., Korea)
colombiensis WYE20
Streptomyces goshikiensis R. solani Rice, turf Safegrow (KIBC Co, Korea)
WYE324
Streptomyces griseoviridis Various soilborne Vegetable and Mycostop (Verdera Oy, Finland)
K61 pathogens ornamental soilborne
diseases
Streptomyces lydicus Various soilborne Root rots in many crops, Acinovate (Natural Industries
WYCD108 pathogens turf, and ornamentals Inc. USA)
FUNGI
Aspergillus flavus AF36 A. flavus (aflatoxin +) Cotton AF36 (Arizona Cotton Research and
Protection Council, USA)
A. flavus NRRL 21882 A. flavus (aflatoxin +) Peanut Afla-guard (Circle One Global Inc,
USA)
Coniothyrium minitans Sclerotinia minor, Protected vegetable and Contans WG (Prophyta Biologischer
CON/M/91-08 S. sclerotiorum field crops Pflanzenschutz GmbH, Germany;
Sylvan Bio Products Inc, USA) and
Intercept WG (Encore Technologies,
MN, USA)
C. minitans KONI S. minor, S. sclerotiorum Glasshouse crops and KONI (Bioved Ltd.,
amenity areas Szigetszentmiklos, Hungary)
 Crop Diseases, Management and Control of 547

Crop Diseases, Management and Control of. Table 2 (Continued)

Target pathogen(s)/
Antagonist activity Disease/host Product name and source
F. oxysporum Fo47 F. oxysporum Asparagus, basil, Fusaclean (Natural Plant Protection,
carnation, cyclamen, France)
gerbera, tomato
Gliocladium catenulatum Pythium spp., R. solani, and Damping-off of Prestop Mix, Prestop WP, Primastop
J1446 numerous other pathogens vegetables, herbs, (Verdera Oy, Finland)
ornamentals, and
numerous other plants
Gliocladium (Trichoderma) Pythium ultimum, R. solani Damping-off of bedding SoilGard 12 G formerly GlioGard
virens G-21 plants, greenhouse (Certis Inc, Columbia, MD, USA)
crops, and ornamentals
Pythium oligandrum Numerous diseases Numerous crops Polyversum (Biopreparaty Ltd,
Czech Republic)
Trichoderma spp. Soilborne fungal Turf, glasshouse crops, Trich-A-Soil (Becker Underwood Pty
pathogens and field crops Ltd, Australia)
Sclerotium cepivorum, Onion Tenet (Agrimm Tecnologies,
Pyrenochaeta New Zealand)
Soilborne plant pathogens Ornamentals, fruit, turf, Trichomic (AMC Chemical, Spain)
olive, vine
Armillaria Tree seedlings Arborguard (Biodiscovery Ltd,
New Zealand)
T. harzianum T-22 Fusarium spp., Pythium Range of crops, T-22 HC, T-22 Planter Box, T-22
(KRL-AG2) spp., Cylindrocarpon spp., ornamentals, and turf Granules PlantShield HC,
Thielaviopsis spp., R. solani, RootShield drench and granules,
S. homeocarpa TurfShield, TRIANUM-P, TRIANUM-G
(Bio-Works Inc, Fairport, NY, USA;
Koppert, the Netherlands)
T. harzianum Various fungi Legumes and leaf Supresivit (Borregaard and Reitzel,
vegetables Denmark or Fytovita, Czech
Republic)
Pythium spp., R. solani Numerous crops Eco-T (Plant health Products, South
seedling diseases Africa)
T. harzianum GBF-0208 Numerous pathogens Vegetables, bulbs, turf Green-all T WP (Green Biotech Co.
Ltd., Korea)
T. harzianum + Fusarium spp., Field crops, vegetables, Trichodry, Trichoflow, Trichogrow,
Trichoderma viride Phytophthora spp., Pythium ornamental and turf Trichopel R Trichopel
spp., and R. solani (Agrimm Technologies, New
Zealand)
T. harzianum T 35 + Fusarium spp., Pythium Seedlings diseases on Root Pro and Root-Protato
T. harzianum TH315 spp., R. solani, S. rolfsii a range of crops and (Mycontrol, Hamovil, Israel)
potato
548 Crop Diseases, Management and Control of
Crop Diseases, Management and Control of. Table 2 (Continued)
Target pathogen(s)/
Antagonist activity
T. harzianum + Various root-infecting fungi Glasshouse crops
T. polysporum
T. viride Fusarium spp., Pythium
spp., R. solani
a
B. cepacia is no longer available in North America because of concerns over the potential for human pathogenicity of some strains of this
species
top-dressed, broadcast, or applied in-furrow or to
planting holes for use on flowers, bedding plants, orna-
mentals, vegetables, pome and stone fruit, trees, and
tree nuts. For agriculture, T-22 Planter Box is applied as
a coating on seeds and seed pieces, an in-furrow spray,
and as a transplant starter and T-22 HC as a broadcast
or in-furrow treatment, both for use on field and row
crops, hay and forage crops, bulbs and vegetables. In
Europe, T-22 (Koppert, the Netherlands) is available as
TRIANUM-G and TRIANUM-P, wettable granule and
wettable powder formulations. Much of the scientific
background concerning the use of this isolate has been
extensively reviewed [88].
In terms of the future, advances that can improve
quantity, quality, and shelf life of inocula would be
welcome, particularly for bacteria such as Pseudomonas
that do not form spores. In recent work, both Pseudo-
monas and Trichoderma isolates have been simulta-
neously applied to seed via drum priming and found
to survive and proliferate on roots similarly to when
applied individually [89]. This procedure may be a way
to apply and maintain multiple BCAs in a commer-
cially relevant process. Further approaches include
integration with other control strategies such as cul-
tural methods and chemical treatments [87]. A better
understanding of the natural ecology of any potential
BCA might facilitate a more rational approach to pro-
duction and use.
Induced Resistance to Control Crop Diseases
Following infection by a microbial pathogen, suscepti-
ble plants can develop an enhanced resistance to
Disease/host Product name and source
BINAB-T W P(Bio-Innovation Eftr AB,
Bredholmen, Sweden; or Svenska
Predator AB, Sweden; or Bayer,
Sweden)
Damping-off, foot rots Ecoderma (Margo Biocontrols
and collar rots of several Private Ltd., Bangalore, India)
plants
further infection [90]. This is known as induced resis-
tance and can be split broadly into two types: systemic
acquired resistance (SAR) and induced systemic resis-
tance (ISR).
In SAR plants, develop a broad-spectrum systemic
resistance to pathogen infection following a localized
infection by a necrotizing pathogen or treatment with
various agents, e.g., acibenzolar-S-methyl (ASM) or
Probenazole (Oryzemate®). SAR is associated with
increased levels of salicylic acid (SA) and with the
coordinate expression of a specific set of genes
encoding PR proteins ([91]; Fig. 3). Moreover, appli-
cation of SA or one of its functional analogues, such as
ASM, induces SAR and activates the same set of PR
genes [92]. Indeed, expression of a set of PR genes, and
PR-1 in particular, is used as a marker for SAR induc-
tion, although it is important to note that the induction
of resistance is not always accompanied by PR-1 expres-
sion [93, 94].
In contrast to SAR, ISR develops as a result of
colonization of plant roots by certain strains of plant
growth–promoting rhizobacteria (PGPR) and is medi-
ated by jasmonic acid– (JA) and ethylene (ET)-
sensitive pathways ([91]; Fig. 3). Phenotypically, ISR
is similar to SAR in that it acts unspecifically against
taxonomically different pathogens [91, 95].
The resistance responses described above can be
associated with direct activation of defenses. However,
such responses can also be associated with an ability to
“recall” previous infection, root colonization, or chem-
ical treatment. This phenomenon is known as priming
and results in plants responding more rapidly and
 Crop Diseases, Management and Control of 549

Rhizobacteria Pathogen

other
LPS flagellin signals AVR gene product

Plant receptors R gene product

JA HR
Systemically
transported
SAR signal
ET SA

Systemically
transported ISR signal

NPR1

NPR1 modulating NPR1 modulating

signal signal

TGA
NPR1 NPR1 transcription
factors

SNI

transcriptional activation transcriptional activation

defensive compounds PRs, defensive compounds

SAR

Crop Diseases, Management and Control of. Figure 3

A model for the signal transduction network controlling induced systemic resistance (ISR) mediated by plant growth–
promoting rhizobacteria and pathogen-induced systemic acquired resistance (SAR) in Arabidopsis thaliana. LPS,
lipopolysaccharide; PRs, pathogenesis-related proteins; AVR, avirulence gene product; R, resistance gene product; HR,
hypersensitive response; SA, salicylic acid; JA, jasmonic acid; ET, ethylene; NPR1, a regulatory protein involved in signaling
in SAR and ISR in A. thaliana; SNI, transcriptional repressor of SAR genes; TGA transcription factors, family of transcription
factors interacting with SA-induced NPR1 (From [91])

effectively when exposed to subsequent pathogen might be a chemical elicitor such as ASM or a challeng-
attack ([96]; Fig. 3). Usually, no changes in gene ing pathogen. Interestingly, priming of resistance is
expression or in the levels of resistance traits are detect- usually caused by agents that fully induce resistance
able in response to the priming agent alone, which when applied at higher doses [97, 98] and suggests
550 Crop Diseases, Management and Control of
that direct resistance induction and priming might
differ from one another quantitatively rather than
qualitatively.
A wide range of microbes and chemicals are known
to induce resistance and it seems likely that other forms
of induced resistance exist. Thus, it is well known that
the nonprotein amino acid, b-aminobutyric acid
(BABA), can induce resistance in a variety of crop
plants [99]. BABA-induced resistance (BABA-IR)
has been used as a model for the study of priming and
in Arabidopsis, it is based on various mechanisms.
Thus, BABA-IR against P. syringae and B. cinerea func-
tions via priming for SA-inducible defenses, while
against a different set of pathogens (Hyaloperonospora
parasitica, Plectosphaerella cucumerina and Alternaria
brassicicola), it is based on priming for resistance
through the formation of callose-rich papillae
[100–102].
Since the introduction of the first chemical resistance
activator Probenazole (registered in Japan as
Oryzemate®, Meiji Seika Kaisha Ltd) in 1975, a number
of other chemical and microbial activators have been
developed, including ASM, registered as Bion® and
Actigard® (Syngenta), Milsana® (Reynoutria
sacalinensis extract, KHH BioScience Inc. USA), Elexa®
(chitosan, SafeScience, USA), and Messenger® (harpin
protein, Eden Bioscience, USA). However, although high
levels of disease control can be achieved with plant
activators in controlled environments, their perfor-
mance under field conditions has been less impressive.
In fact, the moderate levels of disease control and high
levels of variability exhibited by plant activators in the
field have been instrumental in the very slow uptake of
induced resistance in crop production systems. In the
following paragraphs, the performance of selected plant
activators under field conditions will be discussed.
Probenazole (3-allyloxy-1,2-benziothiazole-1,1-
oxide) was first introduced for the control of rice
blast disease (Pyricularia oryzae) and bacterial blight
(Xanthomonas oryzae). It is widely used in Asia where it
is applied as a granular treatment either to paddy fields
or as a seedling box treatment. Following application,
the compound is absorbed by the roots, then systemi-
cally transferred to the rest of the plant and can control
rice blast disease for between 40 and 70 days post
application [103]. However, despite continuous use
since its introduction, there have been no reports of
pathogen insensitivity to probenzole and indeed, it still
accounted for 53% of the chemicals used for seedling
box treatments on rice in 2005 [103]. It is believed this
is because the compound is only weakly toxic to fungi
and activates disease defense systems in rice [104, 105].
A large body of data has accumulated on the effi-
cacy of ASM against a range of diseases under field
conditions [106, 107]. Most studies report disease con-
trol, although the level of control ranges from 4% to
99%. High levels of disease control were achieved on
tobacco, where infections by P. syringae pv. tabaci,
Cercospora nicotianae, and Alternaria alternata were
reduced by 99%, 91%, and 89%, respectively [108,
109]. In wheat, the crop that ASM was originally
aimed at, disease control was not so impressive, ranging
from 35% for Puccinia recondita and Septoria spp., to
77% for B. graminis f.sp. hordei [110, 111]. ASM even
increased disease levels in peanut, where infection by
Cercosporidium personatum was greater than untreated
controls by 52% [112]. Working on oilseed rape,
Liu et al. [113] found that pretreatment with ASM in
October/November decreased the number of leaf
lesions caused by the Phoma stem canker pathogen
L. maculans in the autumn/winter, as well as the
severity of stem canker in the subsequent spring/sum-
mer. In this work, reductions in numbers of leaves with
lesions were between 25% and 55% [113]. More
recently, ASM was shown to reduce infection of barley
by the leaf scald pathogen R. secalis by 45% [114].
Chitosan is a common polymer in shells of crusta-
ceans, exoskeletons of insects, and cell walls of fungi
[115]. A commercial formulation, Elexa®, contains 4%
chitosan as its active ingredient and has been shown to
protect a range of crops against pathogens. For exam-
ple, when used as a seed treatment, it reduced downy
mildew severity on pearl millet by 58%, and when used
as a foliar spray, it reduced infection by 75% [116].
When used on grapevines, eight applications of Elexa®
applied over the season reduced the incidence of downy
mildew by 50% and powdery mildew by 75% com-
pared to untreated controls [117].
A number commercially available products are
based on microbial proteins. One such is Messenger®,
which is based on the protein harpin obtained from
Erwinia amylovora [118]. Used as a crop protectant,
Messenger® has had mixed success. For example,
although it possessed good efficacy against blue mold
 Crop Diseases, Management and Control of 551

in apples [119], its efficacy against gray mold in straw- growth in plant tissue, e.g., R. secalis on barley? (3) Can
berry [120] and target spot of tomato [121] was poor. resistance inducers be used as a means of reducing fun-
In some interesting recent work, Chen et al. [122] gicide applications to crops, e.g., can resistance inducers
generated specific fragments of HpaGXooc, a harpin be applied early to reduce pathogen infection and colo-
from X. oryzae pv. oryzicola, and found that one of nization, thereby allowing less fungicide to be used?
these fragments, HpaG10–42, stimulated growth of rice What is required is research related to specific crops
plants and provided enhanced resistance to X. oryzae aimed at trying to determine how best to fit induced
pv. oryzae and M. grisea. HpaG10–42 was also shown to resistance into disease control programs. Farmers and
control bacterial blight, rice blast, and sheath blight, crop protectionists have grown accustomed to high
and to increase grain yields, under field conditions levels, or even complete, disease control. Ultimately, for
[123]. Here the level of disease control depended on induced resistance to gain more widespread acceptance
the cultivar, with greater control obtained with indica in crop protection, there will need to be a lowering of
compared to japonica cultivars. expectation in terms of levels of disease control.
ISR was first shown to be effective under field con-
ditions in the mid-1990s, when application of PGPR as Other Approaches to Controlling Crop Diseases
a seed treatment followed by soil drench application led
Biofumigation Biofumigation involves the suppres-
to a reduction in severity of bacterial wilt [124], and
sion of plant pests and diseases by biocidal hydrolysis
control of bacterial angular leaf spot and anthracnose
products, most notably the isothiocyanates released by
[125]. Subsequent research by Raupach and Kloepper
glucosinolate (GSL)-containing plants in soil. It can
[126] showed that treatment of cucumber seed with
involve GSL-containing plants as rotation crops, or
PGPR led to increased plant growth and control of
intercrops by incorporating fresh plant material as
angular leaf spot and anthracnose. Field experiments
green manure, or by utilizing processed plant products
in Thailand in 2001 and 2002 studied the effects
high in GSLs, e.g., seed meals. Strategies for the field
of PGPR, used alone or as mixtures, on control of
implementation of biofumigation are described in
southern blight of tomato caused by Sclerotium rolfsii,
detail by Kirkegaard [128].
anthracnose of long cayenne pepper caused by
Colletotrichum gloeospoiroides, and mosaic disease of
Soil Solarization Soil solarization, or soil heating,
cucumber caused by cucumber mosaic virus (CMV)
harnesses solar energy in order to increase soil temper-
[127]. Mixtures of PGPR (all Bacillus spp.) were found
ature. This is commonly achieved by mulching (cover-
to suppress disease more consistently than the PGPR
ing, tarping) the soil with transparent polyethylene or
strain Bacillus pumilus IN937b, used alone.
other transparent plastic sheets. The following are a few
Induced resistance offers the prospect of durable,
of the important principles of soil solarization: (1) It
broad-spectrum disease control using the plants own
should be carried out during periods of high tempera-
resistance. However, it is plagued by inconsistency and
ture and intense solar radiation, with low levels, or no
relatively poor disease control compared with fungi-
precipitation. (2) Soil needs to be moist in order to
cides. These problems relate to the fact that induced
increase the thermal sensitivity of resting structures
resistance is a host response and as such is greatly
and improve heat conduction. (3) The mulching
influenced by genotype and environment. Unfortu-
period needs to be sufficiently long (4 weeks or
nately, the understanding of the impact of these influ-
greater) in order to achieve disease control at all desired
ences on induced resistance is poor, as is the
depths. The efficacy of soil solarization in controlling
understanding of how best to use induced resistance
crop diseases and the mechanisms involved are
in crop protection practice. Understanding in these
discussed in detail by Gamliel and Katan [129].
areas needs to improve, and answers to several impor-
tant, practical questions are required, for example:
Integrated Control of Plant Diseases
(1) Should resistance inducing agents be applied early
or late in the season? (2) Is induced resistance effective As pointed out by Agrios [130], plant disease control is
against pathogens with long periods of asymptomatic most effective when all of the relevant information
552 Crop Diseases, Management and Control of
regarding the crop, potential pathogens, previous dis-
ease history, availability of host resistance, environmen-
tal conditions, etc., are taken into account in devising the
disease control program. An integrated program of dis-
ease control aims to (1) eliminate or reduce the initial
pathogen inoculum, (2) reduce the effectiveness of the
initial pathogen inoculum, (3) maximize host plant
resistance, (4) delay the onset of disease in the plant/
crop, and (5) slow down the development and progres-
sion of secondary cycles [130].
The precise approach adopted will depend on the
crop and its disease spectrum, but would probably
include several of the following: (1) appropriate
hygiene/sanitation, e.g., using disease-free seed/plant-
ing material, (2) using appropriate cultural measures,
(3) using resistant plant varieties, (4) using appropriate
fungicides, and (5) using biologically based methods, if
available and appropriate, e.g., biological control,
induced resistance.
Control of late blight (P. infestans) on potato pro-
vides a good example of an integrated approach to
disease control (see www.endure-network.eu). For late
blight, the first step in an integrated strategy is reducing
primary sources of inoculum, such as avoiding infected
seed tubers, using certified potato seed, preventing or
reducing oospore production by controlling volunteer
potatoes, for example. The next step is variety choice,
since use of a variety expressing some resistance to late
blight offers the potential to reduce fungicide inputs as
part of an integrated strategy. Nevertheless, fungicides
play a crucial role in integrated control of late blight.
Although control measures are aimed mainly at
preventing infection, if late blight appears in a crop,
the control strategy switches to stopping or reducing
the epidemic. Effective control of late blight depends
on access to information such as phase of crop growth,
fungicide products available, dose rates, timings,
weather conditions, as well as access to tools, such as
an appropriate decision support system (DSS). The
DSS can integrate all relevant information to generate
spray recommendations, thereby increasing the efficacy
of the control strategy without increasing risk.
Future Directions
High crop yields are maintained in most developed
countries through the use of improved varieties, together
with fertilizers and pesticides. In these countries, farmers
and growers are accustomed to achieving high levels of
disease control with fungicides, although, as discussed
above, the development of fungicide resistance can erode
fungicide efficacy. Levels of disease control obtained with
many biologically based control methods are lower than
those achieved using fungicides, and moreover, many
biologically based methods tend to provide inconsistent
disease control. Thus, although induced resistance can
provide high levels of disease control on some crops, with
many crops, disease control is less impressive. Expression
of induced resistance in crop plants can also be variable,
depending, for example, on genotype and environment.
Problems also exist with variability and inconsistency
of disease control provided by some BCAs. Farmer per-
ceptions of inadequate and inconsistent disease control
will not persuade them to adopt biologically based
approaches. Minimizing the effects of these problems
clearly requires further research.
In spite of the huge effort by researchers to develop
novel biologically based solutions for disease control
(such as BCAs, plant-derived substances, induced resis-
tance agents), few products have reached the market-
place. The relatively high cost of registration, together
with the limited market size for some products, has been
identified as a major barrier. However, this problem has
been recognized by regulatory authorities, and in the
UK, for example, the Chemicals Regulation Directorate
(Pesticides) has launched a scheme that encourages
applicants to register their products. Under this scheme,
the requirements for registration can be tailored to the
product type and importantly, the application fee can be
reduced [86].
The continued ability of pathogens to overcome host
resistance genes and to develop resistance to fungicides
seriously erodes the ability to provide effective, lasting
disease control on important crops. These problems
combined with the withdrawal of active substances
from the market and increasing public concern with the
effects of pesticides on the environment create a huge
challenge for plant pathology in the future. Plant disease
control has an important role to play in efforts to feed the
world’s increasing population. However, providing effec-
tive and lasting disease control, without harming the
environment, will require not just a sensible approach to
the use of host resistance and fungicides, but also a range
of innovative approaches. In some situations, innovative

control methods might be used to complement existing
approaches. In other cases, for example for those diseases
for which no adequate control measures exist, innovative
control options might provide the only solution.
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558 Crop Plants Transformation Methods

Crop Plants Transformation Methods Concatemer A DNA molecule containing multiple

DAWEI YUAN1, SVETLANA DASHEVSKAYA1, RICHARD M.
TWYMAN2, TERESA CAPELL1, PAUL CHRISTOU3,4
1
Department of Vegetal Production and Forestry
Science, ETSEA, University of Lleida, Lleida, Spain
2
Department of Biological Sciences, University of
Warwick, Coventry, UK
3
Department de Produccio Vegetal i Ciencia Forestal,
Universitat de Lleida/ICREA, Lleida, Spain
4
Institució Catalana de Recerca i Estudis Avançats
(ICREA), Barcelona, Spain
Article Outline
Glossary
Definition of the Subject and Its Importance
Introduction
Principles and Methods of Plant Transformation
Vectors for Plant Transformation
Consequences of Nuclear Transformation
Plastid Transformation Methods
Future Directions
Bibliography
Glossary
Acetosyringone A phenolic compound that activates
the Agrobacterium tumefaciens vir genes and thus
initiates DNA transfer.
Agrobacterium-mediated transformation The intro-
duction of DNA into plants by the bacterium
Agrobacterium tumefaciens.
Bactofection Gene transfer to animal cells mediated
by bacteria.
BIBAC A binary vector for plant transformation based
on the high-capacity bacterial artificial chromo-
some (BAC).
Binary vector A plant transformation vector in two
parts, one carrying the T-DNA and one carrying the
vir genes.
Cassette A modular DNA sequence designed for por-
tability between different vectors.
Chimeric (transformed plants) Comprising cells with
different genotypes, usually a first-generation
transformant (T0) where some cells are transgenic
and others are not.
contiguous copies of the same sequence.
Conjugation DNA transfer between cells through
a specialized conduit known as a pilus.
Counterselectable marker A selectable marker gene
whose absence is required for cells to survive.
Destination vector The vector designated to receive
a DNA cassette during LR recombination in the
Gateway system.
Direct DNA transfer DNA transfer mediated by phys-
ical or chemical means rather than by bacteria or
viruses.
Entry clone The vector holding the DNA cassette
that needs to be transferred to the Destination vector
during LR recombination in the Gateway system.
Episomal A genetic entity that replicates indepen-
dently of the host chromosome, such as a plasmid
or nonintegrating virus.
Explant A piece of plant tissue transferred to culture
and propagated independently.
Filler DNA Extra DNA added at junctions during ille-
gitimate recombination, either by synthesis across a
template or random addition of nucleotides.
Gateway A proprietary cloning system based on LR
recombination, a form of site-specific recombina-
tion using the attB and attP sites of Escherichia coli
and bacteriophage l.
Gene targeting Disruption of a preselected gene by
homologous recombination with a DNA cassette.
Helper plasmid A plasmid that is not used as a cloning
vehicle, but which supplies necessary functions in
trans.
Homing endonuclease A specialized type of restric-
tion enzyme encoded by introns and inteins with
a long asymmetric recognition sequence.
Homologous recombination Recombination requir-
ing long regions of homology but no sequence
specificity.
Illegitimate recombination Recombination requiring
neither long regions of homology nor specific
sequences; it can occur by direct nonhomologous
end-joining (all sequences conserved, sometimes with
added filler DNA) or at regions of microhomology.
In planta transformation Transformation methods
that can be used with intact plants rather than tissue
explants, and which therefore require no regeneration.

P. Christou et al. (eds.), Sustainable Food Production, DOI 10.1007/978-1-4614-5797-8,

# Springer Science+Business Media New York 2013

Originally published in
Robert A. Meyers (ed.) Encyclopedia of Sustainability Science and Technology, # 2012, DOI 10.1007/978-1-4419-0851-3

Integration The insertion of one DNA sequence in the
midst of another.
Minichromosome A vector comprising the minimal
elements that allow it to function as a chromosome.
Minimal cassette The minimal sequences required to
achieve transformation and transgene expression,
typically a promoter, gene, and terminator.
Multigene transfer The simultaneous transfer of more
than one gene into a plant.
Particle bombardment A physical transformation
method based on the acceleration of DNA-coated
metal particles into intact plant cells.
Promoter The DNA sequence in front of a gene which
controls its spatiotemporal expression profiles and
whether its expression is sensitive to external
stimuli.
Reporter gene A gene whose function is to yield an
easily detectable product that can be used to mea-
sure or delimit the activity of a regulatory element
such as a promoter.
Screenable marker Another name for a reporter gene.
Selectable marker A gene whose function is to confer
on transformed cells some property that allows
them to be propagated in conditions under which
nontransformed cells either die or cannot grow
efficiently.
Southern blot (DNA blot) A method for the detection
of DNA sequences using specific labeled comple-
mentary probes.
Stable transformation Transformation followed by
integration of DNA into the host genome such
that the transgene becomes a permanent new geno-
mic locus.
Superbinary vector A binary vector in which the vir
gene functions are enhanced.
Supervirulent An Agrobacterium tumefaciens strain
with a broader host range or more efficient DNA
transfer ability than normal strains.
T-DNA The section of DNA within a binary vector
which is transferred to the host plant.
Transactivation domain The part of a transcription
factor that activates transcription.
Transient expression The expression of introduced
DNA for a short time before the DNA is diluted
and degraded.
Transfection (a) In bacteria, the transfer of phage
DNA into bacterial cells by chemical or physical
Crop Plants Transformation Methods 559
means; (b) in animals and plants, the transfer of
any DNA into cultured cells by chemical or physical
means.
Transformant A plant that has been transformed with
exogenous DNA.
Transformation (a) In bacteria, the transfer of plasmid
or genomic DNA into bacterial cells by chemical or
physical means; (b) in animal cells, the spontaneous
or induced change from a normal to an oncogenic
phenotype; (c) in plants, any means of DNA
transfer that does not involve the use of a virus.
Transgenic Containing integrated exogenous DNA in
the nuclear genome.
Transplastomic Containing integrated exogenous
DNA in the plastid genome.
Terminator The sequence following the gene that is
required to terminate transcription.
vir Gene One of several genes on binary vectors that
are required in trans to mediate T-DNA transfer.
Zinc finger A zinc-coordinating protein motif usually
associated with DNA binding, often found in
sequence-specific DNA binding proteins such as
transcription factors.
Definition of the Subject and Its Importance
The term transformation (or more properly “genetic
transformation”) was first coined to describe the natu-
ral process by which bacteria take up free DNA from
their surroundings. The term was required to distin-
guish this process from two other ubiquitous natural
gene transfer mechanisms in bacteria: conjugation
(direct transfer of DNA from cell to cell through
a connecting tube called a pilus) and transduction
(transfer of DNA between cells carried by the capsid
of a virus). All three of these natural processes have
been exploited in the laboratory as ways to introduce
exogenous DNA into bacterial cells, and in this context
the definition of transformation was later refined to
take into account the different consequences of gene
transfer depending on the origins of the transforming
DNA. The meaning of transformation was thus
restricted to the uptake of naked plasmids or genomic
DNA fragments, whereas an alternative term – trans-
fection – was coined to describe the uptake of naked
phage DNA, the distinction required because only the
latter can initiate a phage infection.
560 Crop Plants Transformation Methods
Unsurprisingly, the same terms have been adopted
to describe analogous processes in other organisms, but
the precise definitions vary due to preexisting conven-
tions. Cultured animal cells are said to undergo trans-
formation when they change from the normal
phenotype (cessation of growth when confluence is
achieved) to an oncogenic phenotype (continued
growth, formation of foci) so “transformation” is usu-
ally avoided in the context of gene transfer to prevent
ambiguity. Therefore, transfection is used to describe
the introduction of any naked DNA into animal cells
regardless of its origin. The transfer of DNA from
bacteria to animal cells (including the use of conjuga-
tion-like mechanisms) is unusual and has only recently
been adopted as a gene transfer procedure in the labo-
ratory, so a new term has been coined for this process –
bactofection. The introduction of DNA into animal
cells using a virus as a carrier is known as transduction,
just as it is in bacteria.
The terminology for plants is a hybrid of
the conventions used for bacteria and animal cells.
Transformation refers to any gene transfer process
where a virus is not used as a carrier (otherwise
transduction is the correct term, as in bacteria and
animal cells). This means that plant transformation
includes both the uptake of naked DNA (direct DNA
transfer) and the transfer of DNA by the conjugation-
like method adopted by Agrobacterium tumefaciens
and Agrobacterium rhizogenes (Agrobacterium-
mediated transformation). However, due to opera-
tional analogies with cultured animal cells, the intro-
duction of DNA into cultured plant protoplasts
is also termed transfection, so in this specific context
the terms transformation and transfection are
synonymous.
The fate of the introduced DNA depends on many
factors. If it does not contain an origin of replication
that is active in plant cells it is usually maintained in
the nucleus for a short period of time (hours to days),
eventually being diluted and degraded. If the intro-
duced DNA contains an active expression cassette,
then a product may be expressed during this time,
a process known as transient expression. In a very
small number of cells, the DNA integrates into the
genome and becomes a permanent new locus. This is
known as stable transformation. It is a rare event
(approximately one in every 10,000 cells that takes up
exogenous DNA integrates it into the genome) and
selectable marker genes are usually employed to ensure
that the small number of transformed cells can grow at
the expense of their nontransformed peers, allowing
the recovery of transgenic tissues and whole plants.
Cells, tissues, and whole plants that are stably
transformed with a particular fragment of exogenous
DNA are termed transgenic if the DNA integrates into
the nuclear genome or transplastomic if it integrates
into the plastid genome. The integrated DNA is known
as the transgene (depending on context this term can
refer to a single gene or to the integrated segment of
exogenous DNA which may contain one or more
genes).
If the introduced DNA does have an origin of rep-
lication that functions in plants, then it may be
maintained episomally (as an extrachromosomal rep-
licon). This occurs when the introduced genetic mate-
rial is part of a replication-competent recombinant
plant virus, or when it includes the components of
a plant centromere, allowing it to function as
a minichromosome. Viral vectors are generally short-
lived, so they are also used for transient expression.
However, the ability of viruses to spread systemically
means that the transgene may be expressed at very high
levels throughout the plant, and the infection may last
days or weeks. Minichromosome vectors are a relatively
new development in plants but as with equivalent
“artificial chromosome” vectors in yeast and animal
cells, the idea is that they should facilitate stable trans-
formation without the need for integration into the
genome.
Plant transformation is a fundamental component
of both basic and applied plant biology. For basic
research, transformation allows scientists to study
how genes function and allows the expression of both
endogenous genes and transgenes to be controlled.
This has increased our understanding of how plants
grow, develop, and defend themselves against pests,
diseases, and harsh environments, how photosynthesis
is controlled, and the basis of primary and secondary
metabolism. For applied research, transformation can
be used to improve the agronomic performance of
crops, making them hardier, more nutritious, more
productive, or converting them from conventional
crops into green factories producing chemical precur-
sors, novel oils, industrial enzymes, and

pharmaceuticals. Plants provide human beings with all
types of useful products: food and animal feed, fibers
and structural materials, and small molecules that can
be used as dyes, scents, and medicines. Plants have been
cultivated for these products since the dawn of history,
and for the same length of time people have sought to
improve plants by breeding them and selecting the
better-performing and most useful varieties. The limi-
tations of this approach, i.e., the fact that breeders are
restricted to the existing gene pool in each group of
sexually compatible species, and that breeding takes
a long time to achieve its goals, can be overcome by
plant transformation, thus accelerating the develop-
ment of plants with novel, beneficial traits.
Introduction
Plant transformation became a routine process in the
1980s but several attempts to transfer DNA into plant
tissues were reported in the previous 2 decades,
although stable transformation was never confirmed.
The first deliberate transformation of plant tissue with
laboratory-created recombinant DNA was achieved in
1983 when several researchers reported the introduc-
tion of recombinant plasmids, including backbone
sequences from cloning vectors and selectable marker
genes [1–3]. This marked the first creation of trans-
genic plant cells in the currently accepted meaning of
the word. In the intervening 30 years, heterologous
genes have been introduced into well over 100 different
plant species either through the use of the soil bacte-
rium A. tumefaciens or alternative strategies involving
direct DNA transfer to plant cells and tissues. In addi-
tion, many plant viruses have been developed as epi-
somal vectors, allowing high-level transient gene
expression, although because of their inability to
achieve stable transformation they are not considered
further in this article.
Principles and Methods of Plant Transformation
A fundamental difference between animals and plants
is that differentiated plant tissue shows a high degree of
developmental plasticity. Depending on the species,
isolated stem segments, leaf disks, and seed-derived
callus tissue may be able to regenerate an entire new
plant under appropriate culture conditions. For most
Crop Plants Transformation Methods 561
plant species, some form of tissue culture step is there-
fore used for the successful production of transgenic
plants after cells or small tissue explants have been
subjected to the actual transformation procedure.
However, it should be noted that whole-plant (in
planta) transformation strategies are also available in
some species, in which the need for tissue culture is
minimized or eliminated.
The ease with which plant material is manipulated
and interconverted in culture provides many opportu-
nities for the development of techniques for gene trans-
fer and the recovery of transgenic plants (Fig. 1). DNA
can be introduced into most types of plant material –
protoplasts, cell suspensions, callus, vegetative tissue
explants, gametes, seeds, zygotes, embryos, organs,
and whole plants; so, the ability to recover fertile plants
from such material is often the limiting step in plant
genetic engineering rather than the DNA transfer pro-
cess itself. It is also possible to maintain transformed
plant cell lines or tissues (e.g., root cultures) rather than
regenerating whole plants as these can often be
sustained indefinitely in culture.
Gene transfer to plants can be achieved through
three types of mechanism – viral transduction, bacte-
rial gene delivery, and chemical/physical direct DNA
transfer. Bacterial gene delivery using the soil pathogen
A. tumefaciens is the most widely used method for
dicotyledonous plants, but increasingly also for mono-
cots. Physical methods are the next most popular, espe-
cially particle bombardment for the transformation of
more recalcitrant monocots such as cereals. Chemical
transfection methods are little used these days, and are
only suitable for protoplasts. Each of the above
methods can be used to achieve either transient expres-
sion or stable transformation of the nuclear genome,
while direct gene transfer can also be used to achieve
chloroplast transformation. As stated earlier, viral
transduction does not lead to stable transformation
and virus vectors are not discussed in this article.
Principles of Agrobacterium-Mediated
Transformation
Gene transfer from bacteria to plants occurs naturally
and is responsible for crown gall disease. This is
a plant tumor that can be induced in a wide variety of
gymnosperms and dicotyledonous angiosperms by
562 Crop Plants Transformation Methods

Zygotic

transformation
embryogenesis

Inplanta
Whole plant

Seeds Organogenesis

Gametes Explant

Organogenesis
or
Somatic embryogenesis

Particle bambardment
Agrobacterium tumefaciens

Callus

Cells

electroporation
PEG/Ca2+
Protoplasts

Crop Plants Transformation Methods. Figure 1

Strategies for the transformation of higher plants [4]. The boxes show the various different targets for transformation
and how they are obtained from whole plants (black arrows). The methods used to transform each of these targets are
shown in black text. The red arrows show the routes used to obtain whole transgenic plants

inoculating wound sites with the Gram-negative soil
bacterium A. tumefaciens. The tumor produces plant
hormones responsible for the proliferation of undiffer-
entiated plant tissue, and specialized amino acid deriv-
atives known as opines that the bacteria use as a carbon
source. The entire system has therefore evolved as a way
for the bacteria to exploit plants for accommodation
and food.
Even so, the continued presence of the bacteria is
not required to maintain the tumor, indicating that
some “tumor-inducing principle” is transferred from
the bacterium to the plant at the wound site. Research
in the late 1970s identified the principle as a small
segment of DNA that is transferred to the plant genome
(hence T-DNA, for transferred DNA). The source of
the T-DNA is a large plasmid, the tumor-inducing (Ti)
plasmid, resident in the bacterium.
Agrobacterium-mediated transformation was devel-
oped as a platform technology following the dissection
and functional analysis of two key components of nat-
ural Ti-plasmids: the T-DNA, which contains DNA
sequences required in cis for DNA transfer, and the
vir (virulence) region, which contains genes whose
products are required in trans for DNA transfer.

The resulting transformation system is known as
a binary vector system because the T-DNA and vir
genes do not need to be present on the same vector.
This means that the T-DNA can be housed on a small
shuttle vector suitable for cloning in bacteria, while the
vir genes are provided on a second “helper” plasmid.
Natural T-DNA carries genes encoding enzymes for
plant hormone synthesis and enzymes for opine synthe-
sis, and these are the factors that drive the formation of
the crown gall tumor. The hormone genes are often called
oncogenes because they lead to tumor formation. How-
ever, neither the oncogenes nor the opine genes are
necessary for DNA transfer. If these genes are removed,
leaving an “empty” T-DNA cassette, the “disarmed”
T-DNA can still be transferred to the plant genome, but
the transformed cells at the wound site no longer prolif-
erate and form a tumor. All that is required for transfer is
the vir region, the T-DNA border sequences which are
targets for excision from the Ti-plasmid, and a small
number of loci on the A. tumefaciens genome. The
T-DNA border sequences are 25-bp imperfect direct
repeats which define the boundaries of the T-DNA.
An enhancer, sometimes called the overdrive sequence,
is located external to the right-hand repeat and is also
required for high-efficiency transfer [5, 6]. Disarming
the T-DNA does not improve the efficiency of trans-
formation but it does improve the efficiency of regen-
eration, since the oncogenes produce phytohormones
that interfere with normal plant development. At the
same time, however, this removes any visual confirma-
tion that transformation has taken place, and selectable
or screenable marker genes must be included in the
T-DNA to allow the identification of transformants.
The vir genes are organized into several operons.
Two of these genes, virA and virG, are constitutively
expressed at a low level and control the plant-induced
activation of the other vir genes. The VirA protein is
a kinase that spans the inner bacterial membrane, and
acts as the receptor for certain phenolic molecules that
are released by wounded plant cells. Many such com-
pounds have been characterized, but acetosyringone is
the most widely used in the laboratory to induce vir
gene expression [7]. These phenolic compounds do not
actually attract bacteria to wounded plant cells (the
bacteria are attracted by simple molecules such as
sugars and amino acids) but the vir genes are induced
after bacterial attachment. Activated VirA
Crop Plants Transformation Methods 563
transphosphorylates the VirG protein, which is a tran-
scriptional activator of the other vir genes. Further
genes on the bacterial chromosome also encode tran-
scription factors that regulate vir gene expression
(reviewed in [8, 9]). The induction of vir gene expres-
sion results in the synthesis of proteins that form
a conjugative pilus through which the T-DNA is trans-
ferred to the plant cell. The components of the pilus are
encoded by genes in the virB operon (reviewed in [10]).
DNA transfer itself is initiated by an endonuclease
formed by the products of the virD1 and virD2 genes.
This introduces either single-strand nicks or a double-
strand break at the 25-bp borders of the T-DNA,
a process enhanced by the VirC12 and VirC2 proteins,
which recognize and bind to the overdrive enhancer
element. The VirD2 protein remains covalently
attached to the processed T-DNA. Recent studies have
suggested that the type of T-DNA intermediate pro-
duced (single- or double-stranded) depends on the
type of Ti-plasmid, with double-stranded T-DNA
favored by nopaline plasmids (where the T-DNA is
a single element) and single “T-strands” favored by
octopine and succinopine plasmids, where the
T-DNA is split into noncontiguous sections. T-strands
are coated with VirE2, a single-stranded DNA-binding
protein. The whole complex, sometimes dubbed the
firecracker complex because of its proposed shape, is
then transferred through the pilus and into the plant
cell. The VirD2 protein may protect the T-DNA against
nucleases, target the DNA to the plant cell nucleus, and
help integrate it into the plant genome. The protein has
two distinct nuclear localization signals, with the
C-terminal signal thought to play the major role in
targeting the T-DNA [11]. Once in the nucleus, the
T-DNA is thought to integrate through a process of
illegitimate recombination, perhaps exploiting natu-
rally occurring chromosome breaks [12–14].
Contemporary binary vectors have all the conve-
niences of bacterial cloning vectors such as multiple
unique restriction sites in the T-DNA region to facili-
tate subcloning, the lacZ gene for blue-white screening
and a l cos site for preparing cosmid libraries.
A popular binary vector in current use is pGreen,
which is <5 kbp in size, has 18 unique restriction
sites in the T-DNA, a lacZ gene for blue-white screen-
ing of recombinants, and a selectable marker that can
be used both in bacteria and in transformed plants
564 Crop Plants Transformation Methods
[15]. The progressive reduction in size has been made
possible by removing essential genes required for rep-
lication in A. tumefaciens and transferring those genes
to the bacterium’s genome, or onto a helper plasmid.
The pGreen plasmid, for example, contains the Sa
origin of replication, which is much smaller than the
more traditional Ri and RK2 regions. Furthermore, an
essential replicase gene is housed on a second plasmid
called pSoup resident within the bacterium. All conju-
gation functions have also been removed [15].
More recent innovations have also been incorpo-
rated into binary vectors, such as the inclusion of
Gateway technology, a proprietary technology devel-
oped by Invitrogen (now part of Life Sciences,
Carlsbad, California, USA) which is based on the attB
and attP site-specific recombination sites and associ-
ated enzymes employed by bacteriophage l to integrate
into the Escherichia coli chromosome during lysogeny
(the process by which the bacteriophage genome inte-
grates into the bacterial chromosome and becomes
dormant). Under normal circumstances, the attP site
in the phage genome and the E. coli attB site
undergoes site-specific recombination catalyzed by
a phage enzyme, resulting in integration and the for-
mation of two hybrid sites flanking the prophage, attL,
and attR. To develop this as a cloning system, sequences
are prepared with flanking attB sites (typically gener-
ated using extended PCR primers) and these undergo
recombination with attP sites in a Gateway vector to
generate an Entry Clone in which the sequence is
flanked by attL sites. The Entry Clone can then be
mixed with a plant-specific Destination Vector, which
contains attR sites flanking a marker gene, such that
the cloned gene is transferred into the destination vec-
tor to replace the marker (LR recombination). A large
number of Gateway-compatible binary vectors have
been developed [16] allowing universal cloning
independent of the presence of restriction endonucle-
ase sites and the assembly of multiple genes on one
plasmid, a modification known as MultiRound
Gateway [17].
Methods of Agrobacterium-Mediated
Transformation
Many dicotyledonous plants can be transformed using
variations of the simple protocol developed by
Horsch et al. [18] in which small disks punched from
leaves are surface-sterilized and inoculated in
a medium containing A. tumefaciens harboring the
recombinant binary vector. The disks are cultured for
2 days, during which T-DNA transfer takes place, and
are then transferred to a medium containing the selec-
tion agent and carbenicillin to kill the bacteria. After 2–
4 weeks, developing shoots are excised from the callus
and transplanted to root-inducing medium, and there-
after into soil.
This leaf disk method is unsuitable for most mono-
cotyledonous plants because they lie outside the
Agrobacterium host range, and do not respond to
wounding in the same way as dicots. In order to trans-
form staple crops such as cereals, modified culture
conditions were developed involving explants
containing a high proportion of actively dividing
cells, such as embryos or apical meristems. In dicots,
cell division is often induced by wounding, whereas
wound sites in monocots tend to become lignified.
This probably explains why traditional procedures
such as the leaf disk method are inefficient in mono-
cots. Hiei et al. [19] showed that the cocultivation
of Agrobacterium and rice embryos in the presence of
100 mM acetosyringone was a critical factor for suc-
cessful transformation. Efficiency could be improved
further by using so-called “supervirulent” strains like
AGL-1, which incorporate modifications to boost gene
transfer activity, such as overexpressed virG (switching
on the expression of the other vir genes) and/or virE1,
which is a major limiting factor in T-DNA transfer
(reviewed in [20]). Komari et al. [21] used a different
strategy, in which a portion of the virulence region
from the Ti-plasmid of supervirulent strain A281 was
transferred to the T-DNA-carrying plasmid to generate
a so-called superbinary vector. The advantage of the
latter technique is that the superbinary vector can be
used in any Agrobacterium strain.
Principles of Direct DNA Transfer
Direct DNA transfer, as the name suggests, involves the
introduction of DNA directly into the cell without
using a biological carrier. This has two important
advantages over Agrobacterium-mediated transforma-
tion. First, there is no dependency on the biological
properties of the carrier, so any species and variety is

suitable for transformation. Second, there is no need to
use specialized vectors for transformation – indeed
transformation is possible without any vectors at all.
The principles of direct DNA transfer are therefore
much more straightforward than those for
Agrobacterium-mediated transformation. All that is
required is a mechanism for getting DNA into the
plant cell, and ensuring it reaches the nucleus. This
can be achieved using physical or chemical means.
Chemical Methods for Direct DNA Transfer Histor-
ically, the first direct transfer methods were chemical,
and were closely related to the (at the time) newly
devised methods for gene transfer to animal cells.
Animal cells lack a cellulose wall and are protected by
a simple plasma membrane. Therefore, the methods
devised for animal cells would not work directly on
plant cells, but were suitable for protoplasts. Gene
transfer across the protoplast membrane is promoted
by a number of chemicals, of which polyethylene glycol
has become the most widely used due to the availability
of simple transformation protocols [22]. Alternatively,
DNA uptake may be induced by electroporation [23].
Putative transformants are transferred to selective
medium, where surviving protoplasts regenerate their
cell walls and commence cell division, producing
a callus. Subsequent manipulation of the culture con-
ditions then makes it possible to induce shoot and root
development, culminating in the recovery of fertile
transgenic plants. The major limitation of protoplast
transformation is not the gene transfer process itself,
but the ability of the host species to regenerate from
protoplasts. Protoplast transformation was also the
first method developed for gene transfer to the chloro-
plast genome of higher plants (see below).
Physical Methods for Direct DNA Transfer There is
a great diversity of physical approaches for gene trans-
fer to plants, including electroporation of walled plant
cells, perforation of the cell with silicon carbide whis-
kers, microinjection, and poration with a finely focused
laser beam. In most of these cases, only transient
expression of the introduced DNA has been achieved,
although transgenic corn plants have been recovered
following whisker-mediated transformation [24]. Par-
ticle bombardment (microprojectile bombardment,
biolistics) is a more robust and reliable method for
Crop Plants Transformation Methods 565
stable transformation which has been successful for
the transformation of cereals, soybean, cotton,
phaseolus, and other recalcitrant crops. Initially,
a modified shotgun was used to accelerate small
(1–4 mm) metal particles into plant cells at a velocity
sufficient to penetrate the cell wall (
250 m/s). In the
initial test system, intact onion epidermis was
bombarded with tungsten particles coated in tobacco
mosaic virus (TMV) RNA. Three days after bombard-
ment, approximately 40% of the onion cells that
contained particles also showed evidence of TMV rep-
lication [25]. A plasmid containing the cat (chloram-
phenicol acetyltransferase) reporter gene driven by the
CaMV35S promoter was then tested to determine
whether DNA could be delivered by the same method.
Analysis of the epidermal tissue 3 days after bombard-
ment revealed high levels of transient CAT activity [26].
The stable transformation of explants from several
plant species was achieved soon after these initial
experiments. Early reports included the transformation
of soybean [27], and corn [28, 29]. The ability to stably
transform plant cells by this method offered the excit-
ing possibility of generating transgenic plants
representing species that were, at the time, intractable
to other transformation procedures. Early successes
included soybean, cotton, papaya, and tobacco
(reviewed in [30]). Particle bombardment has also
been pivotal in the development of chloroplast trans-
formation technology (see below).
There is no intrinsic limitation to the potential of
particle bombardment since DNA delivery is governed
entirely by physical parameters [31]. Many different
types of plant material have been used as transforma-
tion targets, including callus, cell suspension cultures,
and organized tissues such as immature embryos, mer-
istems, and leaves. The number of species in which
transgenic plants can be produced using variants of
particle bombardment has therefore increased dramat-
ically over the last 20 years including rice [32], wheat
[33], and oat [34], as well as many other crops
(reviewed in [30]). The original gunpowder-driven
device has been improved and modified resulting in
greater control over particle velocity and hence
greater reproducibility of transformation conditions.
An apparatus based on electric discharge has been
used for the development of variety-independent gene
transfer methods for the more recalcitrant cereals and
566 Crop Plants Transformation Methods
legumes [35], while several instruments have been
developed where particle acceleration is controlled by
pressurized gas (reviewed in [36]). Physical parameters
such as particle size and acceleration (which affect the
depth of penetration and the amount of tissue damage)
as well as the amount and conformation of the DNA used
to coat the particles must be optimized for each species
and type of explant [30, 37]. However, the nature of the
transformation target is probably the most important
single variable in the success of gene transfer.
In Planta Transformation Methods
Most transformation methods for plants require some
form of tissue culture step. This is because the funda-
mental principle of most transformation methods is
that plants are regenerated from a small number of
transformed cells that can survive under selection.
Experiments using the model dicot Arabidopsis
thaliana have led the way in the development of so-
called in planta transformation techniques, where the
need for tissue culture is minimized or eliminated
altogether. Such methods involve the introduction of
DNA, either using A. tumefaciens or direct transfer, into
intact plants [38, 39].
The procedure is carried out at an appropriate
time in the plant’s life cycle so that the DNA becomes
incorporated into cells that will contribute to the germ
line, directly into the germ cells themselves (often at
around the time of fertilization), or into the very early
plant embryo. Generally, in planta transformation
methods have a very low efficiency, so the small
size of Arabidopsis and its ability to produce over
10,000 seeds per plant is advantageous. This limitation
has so far prevented in planta techniques from being
widely adopted for crop species, although radish, pak
choi, and Medicago truncatula are exceptions [40–42].
The first in planta transformation system involved
imbibing Arabidopsis seeds overnight in an A.
tumefaciens culture, followed by germination [43].
A large number of transgenic plants containing T-
DNA insertions were recovered but in general this
technique has a low reproducibility. A more reliable
method has been described by Bechtold et al. [38] in
which the bacteria are vacuum infiltrated into
Arabidopsis flowers. An even simpler technique called
floral dip has become widely used [39]. This involves
simply dipping Arabidopsis flowers into a bacterial
suspension at the time of fertilization. In both these
methods, the transformed plants are chimeric, but give
rise to a small number of transgenic progeny. It has
been established that T-DNA is transferred into the
ovule during the transformation procedure [44].
An alternative to the direct transformation of germ
line tissue is the introduction of DNA into meristems in
planta followed by the growth of transgenic shoots. In
Arabidopsis, this has been achieved simply by severing
apical shoots at their bases and inoculating the cut
tissue with A. tumefaciens suspension [45]. Using this
procedure, transgenic plants were recovered from the
transformed shoots at a frequency of about 5%. In rice,
explanted meristem tissue has been transformed using
A. tumefaciens and particle bombardment, resulting in
the proliferation of shoots that can be regenerated into
transgenic plants. Such procedures require only
a limited amount of tissue culture.
Vectors for Plant Transformation
Components of Plant Transformation Vectors
The vectors used for plant transformation are usually
designed with four purposes in mind – the ability to
replicate in both E. coli and A. tumefaciens (i.e., shuttle
vector capability), suitability for subcloning (multiple
restriction enzyme sites and/or Gateway compatibil-
ity), the ability to confer a selectable phenotype on
transformed cells (selectable marker genes), and the
ability to drive transgene expression (an expression
cassette, consisting minimally of a promoter, site for
transgene insertion and a terminator/polyadenylation
site).
For Agrobacterium-mediated transformation, the
binary vector system comprises one transformation
vector and one helper plasmid containing the vir
genes (see above). The transformation vector contains
the T-DNA, with the selectable marker gene and
expression cassette housed within (reviewed in [46]).
The replication functions are not required for DNA
transfer and are found on the plasmid backbone, but
they are required for maintenance in A. tumefaciens
and cannot be dispensed with entirely, nor moved to
the helper plasmid because the replication functions
are required in cis. The E. coli replication functions
(generally the ColE1 origin) are not required in

A. tumefaciens but are needed for basic cloning opera-
tions prior to transformation (reviewed in [47]) and
these are also found on the plasmid backbone.
In contrast to Agrobacterium-mediated transforma-
tion, direct transfer (e.g., particle bombardment) is an
entirely physical process with no dependence on bio-
logical functions. Therefore, replication functions on
vectors used for direct DNA transfer are solely present
to facilitate cloning in E. coli. They are entirely dispens-
able for the transformation process and can indeed be
a nuisance if integrated into the plant genome since
they encourage recombination and may in some cases
promote transgene silencing. Only the expression cas-
sette and selectable marker are required in planta, and
therefore the plasmid backbones can be removed prior
to transformation, leaving the small, linear cassettes as
the substrate (reviewed in [31]). Although this is func-
tionally equivalent to the linear T-DNA which is excised
from the binary vector during Agrobacterium-mediated
transformation, it should be noted that the T-DNA
excision process is often imprecise, resulting in varying
amounts of backbone sequences being cotransferred to
the plant genome.
The Development of Binary Vectors
One of the first binary vectors for Agrobacterium-
mediated transformation was pBIN19 [48] although
this has fallen out of favor because of its low copy
number in E. coli, which makes it difficult to obtain
large amounts of DNA for cloning (Table 1). Another
disadvantage of pBIN19 is that the selectable marker is
next to the right border. Because T-DNA transfer is
directional, with the right border being transferred
first, it is better to have the marker next to the left
border to ensure that resistant plants have received
a complete (or nearly complete) copy of the T-DNA.
These two disadvantages were addressed in more recent
vectors such as pPZP and pBINPLUS, which contained
a high-copy-number origin of replication for E. coli and
a selectable marker gene at the left T-DNA border
[49, 50]. Two rare restriction sites were also provided
for cloning convenience.
As discussed above, the successful Agrobacterium-
mediated transformation of monocots rested on the
development of superbinary vectors with extra copies
of some of the vir genes to enhance transformation
Crop Plants Transformation Methods 567
efficiency. In the first instance, the virB, virC, and
virG genes were transferred from Ti-plasmid
pTiBo542 [52, 53] carried by supervirulent strains of
the bacterium, such as A281 or EHA101. Hiei et al. [19]
constructed a new superbinary vector called pTOK233
by adding the virB, virG, and virC genes of pTiBo542 to
achieve rice transformation. The T-DNA in this case
carried the nptII selectable marker under the control of
the nos promoter, the hpt selectable marker driven by
the CaMV 35S promoter and an intron-gusA fusion
gene also driven by the CaMV 35S promoter.
A comparison between the binary vector pIG121Hm
in the supervirulent strain EHA101 and the
superbinary vector pTOK233 in the regular strain
LBA4404 showed that pTOK233 was slightly more
efficient [19]. Similar combinations have been used to
achieve the transformation of many monocot species
(reviewed in [54, 55]). In corn and sorghum, efficient
transformation systems were established only with
superbinary vectors in LBA4404, whereas a standard
binary vector in a supervirulent strain was inefficient
even with improved co-culture conditions [56].
One disadvantage of the superbinary system is the
large size of all the vector components, reducing the
convenience of cloning by standard methods. There-
fore, the final construction step of a superbinary vector
involves the cointegration of an intermediate vector
such as pSB11 and an acceptor vector such as pSB1
via homologous recombination between the shared
DNA segments [21]. The intermediate vector is
a small plasmid containing the T-DNA and ColE1
origin for replication in E. coli. The acceptor vector is
an IncP plasmid, which can be replicated in E. coli and
A. tumefaciens, and carries the 14.8-kb KpnI fragment.
If a gene of interest is to be introduced into plants in
tandem with a marker gene, the two genes are first
inserted into an intermediate vector, which is intro-
duced into a strain of A. tumefaciens that carries an
acceptor vector so that the cassette is integrated into the
acceptor to generate the final superbinary vector.
This approach also facilitates the transfer of large seg-
ments of DNA with minimal rearrangement and favors
a low number of integrated copies [46].
Other improvements have been made to reduce the
frequency of vector backbone transfer. For example,
Hanson et al. [57] placed the lethal barnase gene out-
side the left T-DNA border so that any transgenic plants
568 Crop Plants Transformation Methods

Crop Plants Transformation Methods. Table 1 Development of binary vectors for Agrobacterium-mediated
transformation

Vector Category Details References

pBIN19 Binary vector Low copy number in Escherichia coli, plant resistance marker is [48]
next to the right border
pPZP Binary vector ColE1 origin of replication, plant marker is adjacent to the left [49]
border of T-DNA
pCAMBIA Binary vector Modification of pPZP [49]
pBINPLUS Binary vector Selectable marker gene at the left T-DNA border, a higher copy [50]
number in E. coli, and two rare restriction sites for easier cloning
pIG121Hm Binary vector A derivative of pBIN19 [19]
pTOK233 Superbinary vector Contains virB, virC, and virG genes from pTiBo542 [19, 51]
pTiBo542 Ti-plasmid Has virB, virC, and virG [52, 53]
pSB11 Intermediate vector of ColE1 origin of replication, multiple cloning sites within T-DNA, [21]
superbinary system replicates only in E. coli, has a fragment homologous to the
acceptor vector pSB1
pSB1 Acceptor vector of pSB11 Replicates in E. coli and Agrobacterium [21]
of superbinary system

containing sequences beyond the left border were
counterselected. Another way to reduce backbone
cotransfer is to insert additional left border sequences,
increasing the likelihood of recognition by the
corresponding Vir proteins and thus suppressing trans-
fer, as has been demonstrated in rice [58] and
Arabidopsis [59]. Improvements have also been made
to facilitate rapid and efficient subcloning. The zero
background TA cloning system [60] was developed for
Agrobacterium-mediated transformation and uses
restriction enzyme XcmI to generate 30 -T overhangs in
the linearized vector within the counterselectable
marker gene ccdB, which ensures that self-ligated vec-
tors are not propagated after transformation, but that
PCR products with 30 -A overhangs can be inserted
without further modification.
Multiple Gene Transfer (MGT)
In the early years of plant biotechnology, most trans-
genic plants contained two transgenes – one selectable
marker under the control of a constitutive promoter to
facilitate the selective propagation of transformed cells,
and a “primary transgene” or “gene of interest” which
could be under the control of any sort of promoter and
was intended to alter the plant’s phenotype in a specific
manner. MGT is now being embraced as an approach
to generate plants with more ambitious phenotypes
[61]. MGT allows goals that were once impossible to
be achieved, e.g., the import of complex metabolic
pathways, the expression of entire protein complexes,
and the development of transgenic crops simulta-
neously engineered to produce a spectrum of added-
value compounds [61, 62].
Essentially there are two MGT methods known as
the linked and unlinked cotransformation strategies. In
the linked strategy, all the different genes are linked on
the same piece of transforming DNA. This is the nor-
mal approach chosen with Agrobacterium-mediated
transformation where several genes are carried
within the same T-DNA borders. However, it is quite
possible to transform plants efficiency with (a) an
Agrobacterium strain carrying a binary vector that has
two separate T-DNAs, and (b) different Agrobacterium
strains carrying different T-DNAs, although this
becomes increasingly complex as the number of genes
increases. There are several reports of a mixed strategy
where two Agrobacterium strains carrying different
T-DNAs each with multiple genes has been used to
achieve MGT (reviewed in [63]).

MGT is more easily achieved by direct gene transfer
because there is no need to combine multiple genes
on the same length of DNA. When the genes are
supplied separately as a mixed preparation of plas-
mids, there appears to be no bias in the process of
integration. Tandem cotransformation can be under-
taken, although it becomes more cumbersome as larger
numbers of genes are required, but cotransformation
with discrete, unlinked genes is just as efficient, and the
genes tend to cointegrate at the same locus. As many as
12 transgenes have been integrated using this unlinked
cotransformation approach [64].
The main issue with linked MGT is the inconve-
nience of serial cloning. Very rare restriction sites can
help to address this challenge, as shown by Goderis
et al. [65] who developed a binary vector for MGT
incorporating 13 hexanucleotide restriction sites, six
octanucleotide restriction sites, and five sites for hom-
ing endonucleases, which are extremely rare in natural
sequences and allow unidirectional cloning. Six differ-
ent expression cassettes in auxiliary vectors with differ-
ent promoter and terminator sequences were cut with
the five different homing endonucleases plus an addi-
tional octanucleotide restriction endonuclease and
transferred into the homing endonuclease sites of
the binary vector. Modified auxiliary vectors also facil-
itate N- or C-terminal fusions to five different
autofluorescent tags (EGFP, EYFP, Citrine-YFP, ECFP,
and DsRed2) expressed from the tandem CaMV 35S
constitutive promoter [66]. Similar systems have been
developed by Thomson et al. [67], with the pUGA
vectors for direct transfer and the pUGA2 vectors for
Agrobacterium-mediated transformation. A series
of unidirectional shuttle vectors containing various
combinations of homing endonuclease sites was
constructed and used to create artificial gene clusters
in the pUGA or pUGA2 vectors, allowing the simulta-
neous transfer of up to six genes. Versatile systems now
exist that offer a large number of promoters, termina-
tors, and autofluorescent tags (reviewed in [68]).
A further strategy for linked MGT is to use the Cre/
loxP recombination system together with homing
endonucleases. In this method, two donor vectors are
needed to introduce the expression cassettes into the
acceptor vector using Cre recombinase [69]. After each
round of recombination, the unnecessary backbone
sequence from the donor vector is cleaved out using
Crop Plants Transformation Methods 569
the homing endonucleases leaving just one loxP site for
the next step. In this manner, ten transgenes were
inserted in the acceptor vector and introduced into
rice by Agrobacterium-mediated transformation.
The most recent generation of vectors are modular
and multifunctional, such as the pCLEAN-
G/pCLEAN-S dual binary vectors for Agrobacterium-
mediated transformation [70] and the pORE vector
system that can be adapted for both Agrobacterium-
mediated transformation and direct DNA transfer [71].
The pORE vector series consists of “open” vectors for
general plant transformation, “reporter” vectors for
promoter analysis, and “expression” vectors for trans-
gene expression. The sets comprise various combina-
tions of promoters (PHPL, PENTCUP2, and
PTAPADH), selectable markers (nptII and pat), and
reporter genes (gusA and smgfp), and any element can
be modified independently.
Direct DNA Transfer Using Minimal Expression
Cassettes
As discussed above, vector backbone sequences are not
required for direct DNA transfer and they may also
promote recombination within the transgenic locus
and/or act as triggers for de novo DNA methylation.
To determine whether minimal linear cassettes (pro-
moter, transgene, and terminator) could be used for
direct DNA transfer, Fu et al. [72] separated the expres-
sion cassettes for the marker genes gusA and hpt from
the parent vector, and used these as substrates for
coating the microprojectiles used in particle bom-
bardment, with the intact plasmids as a control.
They found not only that the linear cassettes were
equally efficient for transformation, but also that the
elimination of the plasmid backbone had a remarkable
positive effect on the resulting transgenic rice plants
(Fig. 2). The cassette transformants generally contained
fewer transgene copies than those transformed with
intact plasmids, and they showed stronger and more
stable expression. Further analysis of the minimal cas-
sette population showed that the transgenes were
expressed in most of the transgenic plants and that,
for all the transgenes, overall expression levels and
coexpression frequencies were higher than previously
reported for whole plasmid transformants. These
results were confirmed using the yfp (yellow
570 Crop Plants Transformation Methods

Plasmid DNA Clean fragment

Bombardment
of callus

Regeneration

Southern blot

Plasmid DNA: Clean fragment:

Complex integration patterns Simple integration patterns
Some loss of expression stability Stable transgene expression

Crop Plants Transformation Methods. Figure 2

The clean DNA transformation system compared to particle bombardment with whole plasmid DNA [4]. The
transformation strategy is shown, and two representative DNA blots are compared to demonstrate the simpler integration
patterns resulting from transformation with linear minimal cassettes

fluorescent protein) and hpt markers, with
concatemers forming only rarely in such plants [73].
Promoters Used for Plant Transformation
The promoters used in plant biotechnology are tradi-
tionally divided into three categories – constitutive
(active continuously in most or all tissues), spatiotem-
poral (tissue-specific or stage-specific activity), and
inducible (regulated by the application of an external
chemical or physical signal) [74]. Most basic transfor-
mation vectors incorporate strong constitutive pro-
moters because it is assumed that the objective is to
express the transgene(s) at the highest possible level.
Initially, the A. tumefaciens nos (nopaline synthase), ocs
(octopine synthase), and mas (mannopine synthase)
promoters were popular because they were already
present in the natural T-DNA sequences from which
the early binary vectors were developed, and had
evolved to be active in many plant species (at least
those within the A. tumefaciens host range). However,
the CaMV 35S promoter was found to be stronger and
unaffected by wounding, and its modular nature made
it easy to modify [75, 76]. The activity of the CaMV 35S
promoter can be increased by duplicating the enhancer
up to four times [77] and the enhancer can also
increase the activity of heterologous promoters to
which it is attached [78].
Although widely used, the CaMV 35S promoter has
certain limitations such as its poor performance in
monocots, its suppression by feeding nematodes, and

the intellectual property issues affecting its commercial
deployment (reviewed in [79]). For this reason, alter-
native virus promoters with similar or improved prop-
erties have been sought. Thus far, however, the only
virus promoters that have been developed into
established expression vectors are those from Cestrum
yellow leaf curling virus (CmYLCV) [80], which can be
licensed from Syngenta Biotechnology, Inc. for limited
research purposes, and from Subterranean clover stunt
virus (SCSV), which has been used to construct the
pPLEX series of expression vectors for use in both
dicots and monocots [81, 82].
Constitutive expression in monocots is usually
achieved with housekeeping promoters, particularly
those from the rice actin1 and corn ubiquitin1 genes
(reviewed in [83, 84]). In both cases, the presence of the
first intron of the gene is required for high-level expres-
sion [83, 84], and the addition of this intron to the
CaMV 35S promoter also enhances its activity in
monocots, e.g., 40-fold in corn [85]. In contrast, the
first intron of the recently characterized rice actin2 gene
contains a negative regulatory element whose removal
is required for high-level promoter activity [86].
Many different plant promoters have been
described that restrict expression to particular cells,
tissues, organs, or developmental stages, and seed-
specific promoters are probably the most diverse.
Seeds are a frequent target for genetic engineering in
plants because they can accumulate recombinant pro-
teins to levels that would be lethal in vegetative tissues
but can do so without compromising plant growth and
development; they are also a harvestable product and
thus the target for nutritional improvement. Many
promoters have been identified that target gene expres-
sion specifically to the seed, or to a particular region of
the seed such as the endosperm, embryo, or aleurone
[74, 79, 87]. Anther-specific promoters are also very
useful because they can be used to control male fertility,
an important trait in crop breeding, while fruit- and
tuber/root-specific promoters are valuable for the
nutritional improvement of fruit and root vegetable
crops, pest/disease resistance, and the use of staple
crops as factories for the production of novel proteins
and metabolites.
Inducible promoters are also highly valued in plants
because they allow transgenes to be controlled by inter-
nal and external physical or chemical cues. Many
Crop Plants Transformation Methods 571
different inducible promoters have been identified in
plants and these generally fall into three categories –
(1) those responsive to endogenous signals (plant
hormones); (2) those responsive to external physical
stimuli (abiotic and biotic stresses); and (3) those
responsive to external chemical stimuli. Such pro-
moters provide immense scope for the precise regula-
tion of transgene expression through external control,
ranging from the precise control of transgene activa-
tion/inactivation in experimental settings to the ability
to activate transgenes on an agricultural scale by the
application of chemical sprays. Examples of commonly
used promoters include those responsive to phytohor-
mones (particularly auxin, abscisic acid, gibberellin,
and ethylene), heat-shock promoters responsive to
raised temperatures, light-inducible promoters, pro-
moters induced by wounding or by exposure to elici-
tors produced by pathogens, and promoters that
respond to specific metabolites [74, 79]. Sugar respon-
sive promoters fall into the latter category and the cis-
acting elements that confer sensitivity to sugar are
particularly useful for controlling gene expression in
cultured plant cells. For example, elements from
sporamin and amylase promoters have been studied
in detail and the minimal a-amylase 3 promoter
makes the normally constitutive rice actin1 promoter
sensitive to the presence of sugar [88]. Inducible pro-
moters that respond to xenobiotic signals are also valu-
able because transgenes can be activated without
affecting endogenous genes. Martinez et al. [89] devel-
oped a hybrid system consisting of the tobacco bud-
worm ecdysone receptor ligand-binding domain fused
to the mammalian glucocorticoid receptor DNA-
binding domain and the VP16 transactivation domain.
The receptor responds to tebufenozide (an insecticide
better known by its trade name CONFIRM). Similarly,
Padidam et al. [90] have developed a system that is
based on the spruce budworm ecdysone receptor
ligand-binding domain, and responds to another com-
mon insecticide, methoxyfenozide (INTREPID).
Selectable and Screenable Markers
As stated above, selectable marker genes provide
a phenotype that allows transformed cells to be prop-
agated under conditions where nontransformed cells
cannot survive, such as in the presence of an otherwise
572 Crop Plants Transformation Methods

Crop Plants Transformation Methods. Table 2 Selectable markers used for plant transformation (Data compiled
and updated from [91–93])

Gene (Product) Source Phenotype and other comments

aad (aminoglycoside Shigella flexneri Provides resistance to trimethoprim, streptomycin, spectinomycin,
adenyltransferase) and sulphonamides. Used mainly for chloroplast transformation
bar (phosphinothricin Streptomyces Resistance to phosphinothricin (PPT), which is a component of the
acetyltransferase) hygroscopicus herbicides bialophos, Basta, and glufosinate
ble (glycopeptide-binding Streptalloteichus Resistance to the glycopeptide antibiotics bleomycin and
protein) hindustantus pheomycin (and the derivative Zeocin)
dhfr (dihydrofolate reductase) Mouse Resistance to methotrexate
sul1 (dihydropteroate Escherichia coli Resistance to sulfonamides (Asulam)
synthase)
epsps (enolpyruvylshikimate Petunia hybrida Resistance to the herbicide glyphosate
phosphate synthase)
hpt (hygromycin Klebsiella spp. Resistance to the aminoglycoside antibiotic hygromycin B
phosphotransferase)
manA (mannose-6-phosphate E. coli Ability to grow on mannose as sole carbon source
isomerase, MIP)
neo/nptII/aphII (neomycin E. coli Resistance to the aminoglycoside antibiotics neomycin, kanamycin,
phosphotransferase) and geneticin (G148)

toxic or growth-disrupting reagent (positive selection)
or in the absence of an otherwise essential nutrient
(negative selection). This is necessary to isolate the
small number of transformed cells from the over-
whelming majority of their nontransformed peers
which, without selection, would quickly outcompete
them. Although a wide range of selectable markers
has been tested in plants, only a few are used routinely
(reviewed in [47, 91]). The broadest markers are suit-
able in most plants and are expressed using the most
active constitutive promoters to ensure that all cell
types are protected under selection (the CaMV35S
promoter in dicots and the actin or ubiquitin
promoters in monocots, as discussed earlier).
Most selectable markers are described as conditional
because an external reagent must be applied to facilitate
selection, whereas others are nonconditional, i.e., they
work without any external selection reagent. The typi-
cal selectable markers used in plant transformation are
positive and conditional, and work by conferring resis-
tance to a toxic substance such as an antibiotic or
herbicide that has a very specific intracellular target
(Table 2).
Marker genes that confer antibiotic resistance orig-
inate from bacteria but have been modified to function
well in plants. The first marker to be used in plants was
neomycin phosphotransferase (nptII, aphII), which
confers resistance to the aminoglycoside antibiotics
neomycin, kanamycin, and geneticin (G148) [94].
This is probably still the most widely used marker in
the laboratory but some plants are naturally resistant to
kanamycin, and the antibiotic can also interfere with
normal development in some species. An alternative is
hygromycin phosphotransferase (hph, hpt, aphIV),
providing resistance to the antibiotic hygromycin
through the ATP-dependent phosphorylation of a 7-
hydroxyl group [95]. Other antibiotic-resistance
markers used less frequently include those conferring
resistance to bleomycin [96], gentamycin [97], and
methotrexate [98].
Marker genes that confer herbicide resistance may
originate from bacteria or plants, and those conferring
resistance to the broad-spectrum herbicides
phosphinothricin (PPT)/glufosinate and glyphosate
are used the most widely. PPT/glufosinate is a compet-
itive inhibitor of glutamine synthetase (GS), the only

enzyme that can catalyze the assimilation of ammonia
into glutamic acid in plants. Inhibition of GS therefore
results in the accumulation of toxic levels of ammonia.
The enzyme phosphinothricin N-acetyltransferase
(PAT) encoded by either the bar or pat genes
(these are genes from different microbial species)
can be used to provide PPT/glufosinate resistance in
transformed plant cells [99]. Glyphosate, the
active ingredient of Roundup, inhibits the enzyme 5-
enolpyruvylshikimate-3-phosphate synthase (EPSPS),
which is required for the synthesis of aromatic amino
acids. Glyphosate resistance can be conferred
by markers encoding a modified EPSPS that is not
affected by the herbicide, or those encoding
a bacterial enzyme that breaks down the herbicide
(glyphosate oxidoreductase, GOX) [100]. Markers pro-
viding resistance against sulfonamindes such as
Asualam [101] and chlorsulfuron [102] are also used
occasionally.
Negative selectable markers are useful because they
allow transformed cells to be selected based on the
absence of something that is necessary for
nontransformed cells to grow or regenerate efficiently.
The E. coli manA/pmi gene confers the ability to use
mannose as a sole carbon source so that only
transformed cells survive on media containing man-
nose but lacking sucrose [103]. The gene is a negative
conditional marker because mannose itself is not toxic
to nontransformed plants, but rather the absence of
sucrose (nontransformed plants grow perfectly well in
the presence of both sugars). Another example is the
A. tumefaciens ipt gene, which promotes the synthesis
of cytokinins [104]. This is a nonconditional marker
because it confers the ability to produce shoots in
growth medium lacking exogenous cytokinins, i.e.,
nothing has to be added to the medium to facilitate
selection. Simply, transformed tissues are placed on
medium lacking cytokinins and only those tissues
that produce shoots are transgenic.
The other major class of marker genes are known as
screenable, scorable, or visible markers (or reporter
genes) because rather than providing cells with a selec-
tive advantage they confer a phenotype that can easily
be detected without interfering with other cellular pro-
cesses, allowing transformed cells to be identified and
studied (Table 3). Although screenable markers have
been used for the prosaic purpose of identifying
Crop Plants Transformation Methods 573
transformed cells and manually separating them from
nontransformed cells, they tend to be used for more
sophisticated purposes, such as reporter assays and
tracing experiments. Some screenable marker genes
only provide their visual signal when provided with
a particular substrate, i.e., they are conditional (e.g.,
gusA, luc). Others have an intrinsic ability to yield
a visible signal, i.e., they are nonconditional (e.g., gfp,
DsRed), and these are the most useful since they can be
used in living organisms.
The E. coli gusA (uidA) gene encodes the enzyme
b-glucuronidase (GUS), the most widely used condi-
tional screenable marker in plants. As well as its
endogenous substrates, GUS can process a range of
chromogenic and fluorescent derivatives in a range of
assays that allow the quantification or in situ localiza-
tion of reporter gene activity. The most common
substrate for GUS histochemical staining is 5-bromo-
4-chloro-3-indolyl glucuronide (X-gluc), a clear sub-
strate that yields a blue product. Other common
substrates include p-nitrophenyl b-D-glucuronide,
which is used for spectrophotometric quantitative
assays, and 4-methylumbelliferyl-beta-D-glucuronide
(MUG), which produces a quantitative fluorescent
signal. GUS is preferred in plants over the very similar
reporter GAL (encoded by lacZ and widely used in
microbes and animals) because of its stability in plants
and its lack of toxicity and background activity. The
main disadvantage of GUS is that it cannot be conve-
niently used for in vivo imaging because plant cells must
be fixed or destroyed to visualize the reaction. Its stabil-
ity can also be problematic if the aim of an experiment is
to study fluctuations, since the longevity of the protein
can mask transient decreases in expression.
Both the disadvantages of GUS are addressed by
luciferase (LUC) an enzyme from the firefly (Photinus
pyralis) which catalyzes the ATP-dependent oxidative
decarboxylation of luciferin, producing light in the
process. LUC allows nondestructive qualitative and
quantitative assays to be carried out both in vitro and
in vivo, and because the reaction has a short half-life, it
can be used to monitor fluctuating activity. A series of
vectors that incorporate the luc gene have been devel-
oped for plants, including the LucTrap series that allow
targeted and random transcriptional and translational
fusions of a transgene with a luc gene optimized for
plant cells [106]. One drawback of luc is that it is still
574 Crop Plants Transformation Methods
Crop Plants Transformation Methods. Table 3 Screenable markers (reporter genes) used in plants (Data compiled
and updated from [83, 105])
Gene (product)
gusA (b-glucuronidase)
cat (chloramphenicol
acetyltransferase)
luc (luciferase)
Anthocyanin regulators
GFP (green fluorescent
protein)
Comments
Source: Escherichia coli gusA/uidA gene
Activity: catalyzes the hydrolysis of b-glucuronides
Assays: nonisotropic; in vitro assays are colorimetric or fluorometric; also histochemical
assay format using X-gluc
Advantages: simple, sensitive, quantitative, many assay formats available, inexpensive
Disadvantages: assays are destructive; enzyme is stable so unsuitable for studies of
downregulation
Source: E. coli Tn9
Activity: catalyzes the transfer of acetyl groups from acetyl coenzyme A to
chloramphenicol
Assays: in vitro assays only, isotropic
Advantages: simple to perform
Disadvantages: low sensitivity, expensive, low resolution in vivo, reliance on isotopic
assay format
Source: The firefly Photinus pyralis
Activity: light produced in the presence of luciferase, its substrate luciferin, oxygen, Mg2+,
and ATP
Assays: nonisotopic bioluminescent assays in vitro and in vivo
Advantages: sensitive, rapid turnover, quantitative
Disadvantages: Expensive detection equipment, limited reproducibility of some assay
formats
Source: corn (Zea mays)
Activity: induces pigmentation
Assays: visual screening for pigmented cells in vivo
Advantages: simple, inexpensive, nondestructive
Disadvantages: low sensitivity, not quantitative, background expression, adverse effects
on transgenic plants
Source: the jellyfish Aequorea victoria
Activity: intrinsic fluorescence under blue/UV light
Assays: nonisotopic, in vivo assays in live plants
Advantages: intrinsic activity (no substrate requirements), sensitivity, use in live plants;
many variants with modified absorption and emission spectra available, and different
subcellular targeting signals; several variants can be used simultaneously
Disadvantages: weak signal in some systems (this is being addressed through the use of
modified GFPs with stronger emission and reduced photobleaching); no variants that
emit in the orange-red part of the spectrum
 Crop Plants Transformation Methods 575

Crop Plants Transformation Methods. Table 3 (Continued)

Gene (product) Comments

DsRed (red fluorescent
protein)
Source: coral reef Discosoma spp.
Activity: intrinsic fluorescence under blue/UV light
Assays: nonisotopic, in vivo assays in live plants
Advantages: intrinsic activity (no substrate requirements), sensitivity, use in live plants;
many variants with modified absorption and emission spectra available, and different
subcellular targeting signals; several variants can be used simultaneously
Disadvantages: weak signal in some systems, no variants that emit in the violet-blue-
green part of the spectrum

a conditional reporter, requiring the substrate luciferin
and the presence of oxygen, ATP, and magnesium ions.
In contrast, the green fluorescent protein (GFP) from
the jellyfish Aequorea victoria is a nonconditional
reporter allowing the direct, noninvasive visualization
of fluorescence in vivo in real time merely by exposure
to blue/UV light. The original gfp gene was
nonfunctional in plants because of a cryptic splice
site, but this has been corrected and the protein has
been widely deployed as a vital marker [107]. One of
the major advantages of GFP is that its spectral qualities
can be modified by mutation, giving rise to a whole
family of derivatives with enhanced brightness, less
susceptibility to quenching, and a range of excitation/
emission wavelengths allowing different reporters to be
used in vivo simultaneously. In combination with var-
iants that allow targeting to different compartments
within the plant cell, many sophisticated forms of anal-
ysis become straightforward to implement, e.g.,
allowing the real-time monitoring of protein
processing, trafficking and protein–protein interac-
tions [108]. As well as GFP and its derivatives, other
bioluminescent proteins have also been identified,
including DsRed from Discosoma spp. This is similar
to GFP but covers a different spectral range (red GFP is
not available) but there is little background fluores-
cence and it can be visible under white light [109].
The useful properties of selectable and screenable
markers can also be combined into one protein.
For example, Ochiai-Fukuda et al. [110] developed
a fusion marker incorporating enhanced green fluores-
cent protein and blasticidin deaminase, conferring
resistance to the aminoacylnucleoside antibiotic
blasticidin S. The gfbsd marker was introduced into
rice callus and allowed the rapid and efficient selection
and visual confirmation of transformed cells.
Consequences of Nuclear Transformation
Integration of Nuclear Transgenes
As discussed above, plant transformation is a multistep
process, the first step involving the transfer of DNA
into the plant cell, which can be achieved either by
direct transfer or by Agrobacterium-mediated transfor-
mation. Once the DNA reaches the nucleus, the next
step (transgene integration) is dependent predomi-
nantly on that DNA and on factors provided by the
plant cell, although in the case of Agrobacterium-
mediated transformation it is possible that the Vir pro-
teins complexed with the T-strand may facilitate the
integration process (see below). A number of groups
have investigated the structure of genomic/T-DNA and
T-DNA/T-DNA junctions in plants and have con-
cluded that integration occurs by illegitimate recombi-
nation (see [111, 112]). A strand invasion mechanism
has been proposed (reviewed in [12]), in which the 30
end of the T-strand initiates the integration process by
hybridizing to a short region of homology in the plant
genome, the second strand being completed by primer
extension of the plant DNA. Other models suggest
conversion of the T-strand into a double-stranded
intermediate, which integrates at the site of naturally
occurring chromosome breaks via double-strand DNA
break repair. This is supported by experiments that
show transformation efficiency increases following
UV irradiation, which generates nicks and breaks in
576 Crop Plants Transformation Methods
genomic DNA. However, since T-DNA integration still
occurs in DNA repair mutants, it is possible both
mechanisms occur simultaneously albeit with different
efficiencies.
DNA repair models argue that proteins encoded by
the host plant have a much more important role in
T-DNA integration than Agrobacterium proteins, such
as VirD2, which are imported into the plant with the
T-DNA. However, since the VirD2 protein remains
covalently attached to the 50 end of the T-strand during
transfer it is also likely to influence integration [113].
In an in vitro assay, VirD2 can ligate together a cleaved
T-DNA border sequence but cannot ligate T-DNA to
other genomic targets unless plant cell extracts are also
present [114], a phenomenon supported by the iden-
tification of Arabidopsis mutants impaired for T-DNA
integration [115].
Much can be learned about the T-DNA integration
mechanism by the inspection of borders, especially the
borders between adjacent T-DNA sequences in
multicopy insertions. The formation of heterodimers
during cotransformation argues in favor of T-DNA
concatemerization prior to integration. Although
inverted repeats around the right border are often
precise, those around the left border and those separat-
ing direct T-DNA repeats are often characterized by the
insertion of variable-sized regions of filler DNA, which
may be derived from the T-DNA sequence or from
plant genomic DNA [116]. This suggests either the
simultaneous integration of multiple T-DNAs at
a single locus, or a two-phase mechanism, in which
a primary T-DNA integration event stimulates further
secondary integrations in the same area, similar to
those proposed for particle bombardment (see
below). Zhu et al. [117] carried out a comprehensive
study of T-DNA border characteristics in a population
of transgenic rice plants including 156 T-DNA/geno-
mic DNA junctions, 69 T-DNA/T-DNA junctions, and
11 T-DNA/vector backbone junctions, which included
171 left borders and 134 right borders. Conserved
cleavage was observed in 6% of left and 43% of right
borders, microhomology was observed in 58% of
T-DNA/genomic DNA, 43% of T-DNA/T-DNA, and
82% of T-DNA/vector junctions, mostly at left borders,
and about one third of the T-DNA/genomic DNA and
T-DNA/T-DNA junctions showed evidence of filler
DNA (up to 344 bp). This was derived mainly from
the T-DNA region adjacent to the breakpoint and/or
from the rice genomic DNA flanking the T-DNA inte-
gration site, with T-DNA/T-DNA filler DNA showing
the greatest complexity. Interestingly, when two
T-DNAs were integrated in the inverted repeat config-
uration, significant truncation was always observed in
one of the two T-DNAs whereas with the direct repeat
configuration, large truncations were rare. These data
suggest that no single integration mechanism can
account for all observations but the presence of filler
DNA at many of the junctions argues that a template-
driven DNA synthesis mechanism must be involved.
The analysis of plasmid/plasmid and plasmid/geno-
mic junctions in transgenic plants generated by particle
bombardment reveals features characteristic of illegiti-
mate recombination similar to those seen for T-DNA
junctions, suggesting that the same overall integration
mechanisms may be involved [118]. For example,
such junctions are characterized by regions of
microhomology, filler DNA, trimming of the DNA
ends so sequences are lost, and AT-rich elements sur-
rounding the junction site, with similarity to topo-
isomerase I binding/cleavage sites (Fig. 3). In the
analysis of multiple plasmid/plasmid junctions in 12
transgenic rice lines, Kohli et al. [120] observed 10
plants with microhomology at the junctions and 2
plants where junctions appeared to be generated by
blunt ligation, with no overlap. A similar ratio of con-
served end-joining to microhomology-mediated
recombination was observed by Gorbunova and Levy
[121] and Salomon and Puchta [111]. Topoisomerase
I sites were also observed adjacent to 10 out of 12
junctions characterized in transgenic Arabidopsis plants
generated by particle bombardment [122] and in four of
the six junctions in the commercial SUNUP variety of
papaya [123]. Illegitimate recombination therefore
appears to be responsible both for the integration of
foreign DNA into the plant genome, and the linking of
multiple plasmid copies, which is similar to the mech-
anism proposed for T-DNA integration (see above).
When nuclei from the cells of transgenic cereal
plants generated by particle bombardment are analyzed
by fluorescence in situ hybridization (FISH) using
a transgene-specific probe, a curious phenomenon in
often observed in which a single fluorescent spot in the
interphase nucleus separates into multiple signals along
a metaphase chromosome [124]. Any model for

a
ACTGA
b G GACA
A c
ACTG
GACA
c G
ACTGT
d TGACA
Crop Plants Transformation Methods. Figure 3
Mechanism for transgene integration at regions of
microhomology [119]. A mixture of DNA fragments with
ragged ends (a) interacts with a double-stranded DNA
break with partially complementary ragged ends
(b). Repair synthesis across the gap (c) generates
a recombination junction (d) which may be completely
conserved if the homology is precise, or may involve either
the loss of terminal sequences or the insertion of filler DNA
if the homology is partial
transgene integration following particle bombardment
must take into account some form of three-tier orga-
nization, consisting of contiguous transgene arrays,
interspersed with short regions of genomic DNA to
generate local clusters, and the appearance of widely
dispersed signals at metaphase. Two-phase transgene
integration mechanisms have been proposed to explain
the first two levels of organization, and in such models
concatemerization is proposed to occur prior to inte-
gration, while interspersion occurs during the integra-
tion process [118, 120, 125] (Fig. 4). In each model,
penetration of the cell is proposed to elicit a wound
response, which would include the induction of DNA
Crop Plants Transformation Methods 577
RC RC
a RC
RC RC RC
b
RC RC
Genomic DNA Transgene DNA RC DNA repair complex
Crop Plants Transformation Methods. Figure 4
Explanation for the formation of transgene arrays and
transgene clusters interspersed with genomic DNA [119].
A mixture of DNA fragments interacts with a double-
stranded DNA break where a repair complex has already
assembled (a). The repair complex may stitch together
DNA fragments to form concatemers prior to integration,
or may integrate single copies. The first integration event
stimulates further repair complex activity nearby, resulting
in additional nicks and breaks in the genomic DNA that act
as further integration sites (b). This results in a cluster of
transgenes (single copies and concatemers) interspersed
with short regions of genomic DNA (c)
repair enzymes, such as nucleases and ligases. The
presence of these enzymes and an excess of foreign
DNA would result in the linking together of several
copies to form concatemers, which would be the sub-
strates for integration. This might be stimulated by
homology between individual copies of transforming
plasmids, and “backbone” homology might also result
in the concatemerization of plasmids carrying different
transgenes in cotransformation experiments. However,
as stated above, cotransformation and cointegration
also occur when two nonhomologous minimal cas-
settes are used for transformation, so homology
might not be as important as the presence of free
DNA ends [72]. Kohli et al. [120] suggested that
578 Crop Plants Transformation Methods

Nuclear matrix
Chromatin loops

Interphase
Metaphase
DNA stretched out

Cluster Cluster

Crop Plants Transformation Methods. Figure 5

Higher-order transgene locus organization in cereals transformed by particle bombardment [124]. Transformation occurs
during interphase, when the chromatin is distributed into specific nuclear zones and territories. If a metal particle causes
localized damage, DNA repair complexes will form at these sites and initiate transgene integration (a). During metaphase,
when FISH analysis is generally carried out, loci that are brought together in interphase may be separated, resulting in
multiple signals from the same transformation event (b). If the DNA were stretched out, this would reveal large (megabase)
interspersed sequences, which have also been observed in fiber-FISH experiments

transgene clusters arise in a second phase where
a primary integration event occurring by illegitimate
recombination at a chromosome break generates
a “hot-spot” for further integration events in the
same area. This might be due, for example, to the
presence of local repair complexes that can slide along
the DNA and introduce nicks which can be exploited by
more foreign DNA. Pawlowski and Somers [125]
suggested an alternative second phase where
a number of discrete transgene concatemers integrate
simultaneously at a site containing multiple replication
forks. Although there is no direct evidence for either
mechanism, it is interesting to note that DNA integra-
tion is stimulated in rapidly dividing cells, and is
blocked in Arabidopsis mutants lacking essential com-
ponents of the DNA recombination machinery.
The higher-order organization of transgenic loci
observed by FISH is thus far unique to particle bom-
bardment and demands a model which takes into
account the three-dimensional structure of the nucleus.
It is possible that the transformation event affects
a local region of the interphase nucleus, e.g., a metal
particle may cause damage to a particular area of chro-
matin arranged in loops attached to the nuclear matrix,
or to a localized transcription factory. If the particle
“skims” several loops or several transcription units,
there will be regions of DNA damage close together in
trans, but widely separated in the cis configuration were
the DNA to be stretched out (Fig. 5). Each of these sites
could act as a nucleation point where foreign DNA
diffusing from the metal particle is used to patch up
double-strand breaks, generating widely separated

arrays and/or clusters [124, 126]. In support of this
induced break and repair model, Svitashev et al. [127]
have shown that in 6 of 25 transgenic oat plants gener-
ated by particle bombardment, transgene integration
sites were associated with rearranged chromosomes.
This suggests that DNA breaks caused by incoming
particles are repaired with foreign DNA and may also
result in deletions, inversions, and translocations
involving genomic DNA.
Transgene Structure and Integrity
Transgene rearrangements following particle bombard-
ment have been widely reported in the literature and
many publications repeat the “lore” that direct
DNA transfer is more likely than T-DNA transfer to
generate complex rearranged loci. The number of
rearrangements that can be detected depends entirely
on the resolution of the method being used. Thus,
careful analysis of locus structure by Southern blot
hybridization, PCR, and DNA sequencing has shown
that rearrangements may be more widespread than first
envisaged in both transformation methods.
In the case of direct DNA transfer, the analysis of
transgenic oat loci by Somers et al. has shown that
transgene rearrangements can be extensive and
extremely complex, with multiple small insertions,
inversions, and deletions within any transgene, plus
the presence of filler DNA [127]. In corn, Mehlo et al.
[128] noted that every single plant among the popula-
tion they analyzed showed some form of
rearrangement, and they speculated that undetected
“minor” rearrangements could be responsible for
many instances of transgene silencing otherwise attrib-
uted to epigenetic processes. In particular, certain
transgene rearrangements were not detectable by
Southern blot hybridization because they were too
subtle, but they could be picked up by long-range
PCR and sequencing. Because Southern blot hybridi-
zation is normally the sole method used to determine
whether a given locus is intact or rearranged, this
suggests caution should be used in relying on such
results, since only “major” rearrangements can be
detected in this manner.
In the case of Agrobacterium-mediated transforma-
tion, Afolabi et al. [129] and Zhu et al. [117] found that
nonintact T-DNAs were present in >70% of transgenic
Crop Plants Transformation Methods 579
rice lines, in most cases reflecting loss of the mid to
right border portion of the T-DNA. Similarly, Rai et al.
[130] found that about 50% of rice plants transformed
with a T-DNA containing the phytoene synthase (psy)
and phytoene desaturase (crtI) genes showed evidence
of rearrangements, and in the majority of cases the
rearrangements occurred in the crtI expression cassette,
which was adjacent to the right T-DNA border.
Rearrangements involving the left border are often
characterized by the insertion of variable-size regions
of filler DNA, possibly derived from the T-DNA
sequence or from plant genomic DNA [116, 131].
Few researchers have characterized transgene
rearrangements in detail, but work by Kohli et al.
[132] has shown that rearrangements may involve pal-
indromic sequences in the transforming plasmid,
which tend to form secondary structures such as hair-
pins and cruciforms. These investigators characterized
12 transgenic rice lines created by particle bombard-
ment, which had been shown to contain rearranged
transgenes. Interestingly, they found that an imperfect
palindrome in the CaMV 35S promoter was involved in
one third of all rearrangements, i.e., the sequence of
this palindrome was adjacent to the rearrangement
junction. Similar phenomena have been noted in
T-DNA transformants containing the same promoter.
This sequence has the ability to adopt a cruciform
structure that may stimulate recombination events.
Many other promoters contain palindromic sequences
of variable length within 100 bp of the transcription
start site. The secondary structures formed at these sites
enable DNA–protein interactions for transcription
under normal circumstances, but may also participate
in aberrant recombination events. The fully sequenced
papaya genome [123] also revealed a number of previ-
ously unidentified transgene rearrangements, i.e.,
a 1,533-bp fragment comprising a truncated,
nonfunctional tetA gene and flanking vector backbone
sequence, and a 290-bp nonfunctional fragment of the
nptII gene, in addition to the intact, primary transgene
conferring virus resistance.
Transgene Silencing
A common issue raised in association with nuclear
transformation in plants is the phenomenon of trans-
gene silencing, where the phenotype corresponding to
580 Crop Plants Transformation Methods
the introduced transgene is not expressed. In the
absence of a genetic explanation (e.g., an undetected
mutation or rearrangement), silencing is an epigenetic
phenomenon that can occur at either the transcrip-
tional or posttranscriptional levels. Transcriptional
silencing involves the absence of transgene mRNA,
and often occurs due to the integration of the transgene
at a genomic position that is already repressed (posi-
tion-dependent silencing). However, transgenes
in active regions of the genome may also be silenced
if the promoter region is inactivated by
hypermethylation, which can occur in response to
unusual DNA structures and compositions that attract
de novo methylation, or DNA sequences that allow the
synthesis of short double-stranded RNA (dsRNA) mol-
ecules. In contrast, posttranscriptional silencing actu-
ally requires transcription to take place, but the mRNA
is rapidly degraded. This is confirmed by nuclear run-
on assays, which measure the amount of pre-mRNA in
the nucleus. Like transcriptional silencing, posttran-
scriptional silencing appears to have evolved as
a defense against invasive nucleic acids and is also
triggered by dsRNA, in this case matching the tran-
scribed region. There is considerable cross talk between
the transcriptional and posttranscriptional silencing
pathways and if a transgene is homologous to an
endogenous gene, the silencing effect can spread to
that gene resulting in a phenomenon known as
cosuppression.
Transcriptional silencing may be encountered in
plants where several copies of the same transgene or
part thereof are present in the transgenic locus, or
when the same promoter is used to control several
transgenes. However, the context is very important.
There have been plenty of reports describing transgenic
plants carrying multiple transgenes under the control
of the same promoter yet showing strong and stable
expression. For example, although Zhu et al. [133] used
five different endosperm-specific promoters in corn to
achieve the high-level expression of carotenogenic
genes, Naqvi et al. [134] achieved strong expression
of four genes in the same system using the barley
D-hordein promoter to control each transgene, with
no adverse effects. Transcriptional gene silencing
resulting from repetitive promoter regions is correlated
with increased promoter methylation [135] and
appears to be driven by the production of dsRNA
matching the promoter sequence [136]. This has been
demonstrated by deliberately expressing dsRNA
corresponding to the nos promoter in transgenic plants
carrying a second transgene driven by the nos promoter
[137] and by creating transgenic plants with
a transgene locus that triggers both transcriptional
and posttranscriptional silencing simultaneously, by
producing dsRNA corresponding to promoter and
transcribed sequences of different target genes [138].
In the absence of deliberately created promoter dsRNA,
the transcriptional silencing seen in some transgenic
plants carrying multiple copies of the same promoter
appears to arise from dsRNA produced either by unfor-
tunate transgene positioning or rearrangements that
create hairpin structures, or by transgenes with such
high levels of expression that the polyadenylation
machinery is saturated. Evidence from many transfor-
mation experiments indicates that there is no simple
correlation between transgene copy number and
expression level, with the exception of certain carefully
controlled experiments using boundary elements. In
some cases, higher copy numbers have suppressed
overall expression levels whereas in others higher copy
numbers have enhanced expression. Where suppres-
sion effects have occurred, it has been suggested that
“runaway expression” resulting in the generation of
aberrant RNAs lacking polyadenylate tails has triggered
potent silencing through the posttranscriptional silenc-
ing pathway [139].
The organization of a transgenic locus is difficult to
control, and it is therefore a common occurrence in
both Agrobacterium-mediated transformation and
direct DNA transfer that the juxtaposition of
transgenes or fragments thereof can result in the crea-
tion of hairpin promoter structures at the DNA level
that are transcribed into aberrant dsRNA species. Such
arrangements can be obvious and easy to detect, but
even where gross rearrangements are absent it is possi-
ble that undetected “micro-rearrangements” are pre-
sent in the transgenic locus, as observed by Mehlo et al.
[128] when investigating the structure of a transgenic
locus in corn generated by direct DNA transfer. The
siRNAs that trigger RNA-dependent DNA methylation
are just 24 bp in length, so it is conceivable that inverted
repeats of <50 bp could be sufficient for transgene
silencing, and such structures would be undetectable
using the coarse analysis methods typically employed

to study transgenic plants, such as Southern blot
hybridization. The likelihood of dsRNA production
depends not only on the presence of damaged or
rearranged transgenes, but also on the relative position
of intact transgenes, which is itself a reflection of the
mechanism of transgene integration. The organization
of integrated T-DNA sequences differs among
Agrobacterium strains, but a common feature of
nopaline-type derivatives such as C58 is the preferen-
tial integration of T-DNA as dimers with an inverted
repeat configuration, linked either at the left or right
borders [140]. Where cotransformation is carried out
with two T-DNAs containing different genes, the dif-
ferent T-DNAs often integrate as heterodimeric
inverted repeats, preferentially around the right border
[141]. If the same promoter is used for both genes, this
would favor the formation of hairpin structures that
could be transcribed from the opposite strand. The
structure of loci generated by direct DNA transfer is
more variable, but inverted repeat structures involving
promoter sequences are not uncommon, allowing the
same silencing mechanism to operate [126].
Plastid Transformation Methods
The introduction of DNA directly into the chloroplast
genome is considered beneficial for a number of rea-
sons including the high level of gene expression that
can be achieved, reflecting the presence of thousands of
chloroplasts in photosynthetic cells and the absence of
gene silencing. Chloroplast transformation also pro-
vides a natural containment method for transgenic
plants, since in most crops the transgene cannot be
transmitted through pollen (reviewed in [142]).
The first reports of chloroplast transformation were
serendipitous, and the integration events were found
to be unstable. For example, an early experiment in
which tobacco protoplasts were cocultivated with
Agrobacterium resulted in the recovery of one trans-
genic plant line in which the transgene was transmitted
maternally. Southern blot analysis of chloroplast DNA
showed directly that the foreign DNA had become
integrated into the chloroplast genome [143]. How-
ever, Agrobacterium is not an optimal system for chlo-
roplast transformation because the T-DNA complex is
targeted to the nucleus. Therefore, direct DNA transfer
has been explored as an alternative strategy and
Crop Plants Transformation Methods 581
efficient chloroplast transformation has been achieved
both through particle bombardment and PEG-
mediated transformation (reviewed in [144]).
Stable chloroplast transformation was first achieved
in the alga Chlamydomonas reinhardtii, which has
a single large chloroplast occupying most of the volume
of the cell [145]. Particle bombardment was used in this
experiment and the principles established using this
simple organism were extended to tobacco, allowing
the recovery of stable transplastomic tobacco plants
[146]. These principles included the use of vectors
containing chloroplast homology regions, allowing
targeted integration into the chloroplast genome, and
use of the selectable marker gene aadA (encoding
aminoglycoside adenyltransferase) which confers resis-
tance to streptomycin and spectinomycin [147]. Basic
vectors for plastid transformation include flanking
sequences and chloroplast-specific expression cassettes.
Species-specific chloroplast flanking sequences are gen-
erated by PCR using primers designed from the avail-
able chloroplast genomes. The chloroplast expression
cassette is composed of a promoter, the selectable
marker, and 50 /30 regulatory sequences to enhance the
efficiency of transcription and translation. The most
frequently used integration site is the transcriptionally
active intergenic region between the trnI and trnA
genes, within the rrn operon. The first-generation plas-
tid transformation vectors included the pPRV series and
plasmids pRB94/95, in which both the marker gene and
the primary transgene have their own 50 and 30 regula-
tory sequences (reviewed in [144]). More recent vectors
include modified restriction sites, loxP sequences for
posttransformation marker excision, and homology
regions targeting insertions to the rbcL-accD intergenic
region [148]. Thus far, chloroplast transformation
by particle bombardment has been achieved only in
crops that allow direct organogenesis, and this does
not include any monocots (reviewed in [142]).
Future Directions
The vast majority of the transgenic plants generated
thus far carry a single primary transgene plus
a selectable marker. The transgene integrates randomly
into the genome, which means it is subject to
unpredictable position effects that may result in silenc-
ing; the locus structure is also very difficult to control.
582 Crop Plants Transformation Methods
In the future, there will be a stronger emphasis on
strategies to increase the scope of gene transfer and
the predictability and preciseness of DNA integration,
and consequently the likelihood of stable and predict-
able transgene expression.
Transfer of Large DNA Molecules Using Modified
Conventional Vectors
A precise upper limit for T-DNA transfer has not been
established. It is greater than 50 kbp [2, 3], but using
standard vectors it is difficult to transfer inserts larger
than 30 kbp routinely due to instability in the bacterial
host. However, the analysis of very large genes or the
transfer of multiple genes linked in series can now be
achieved thanks to the development of high-capacity
binary vectors based on the artificial chromosome type
vectors used in E. coli.
The first to be described was BIBAC2 [149]. This
contains an F-plasmid origin of replication and is
modeled on the bacterial artificial chromosome. The
basic vector transforms tobacco with high efficiency,
but the efficiency of transformation drops substantially
when large inserts are used. This vector has been used
to introduce 150 kbp of human DNA flanked by
T-DNA borders into the tobacco genome, although
virulence helper plasmids supplying high levels of
VirG and VirE in trans were critical for successful
DNA transfer. An alternative vector carrying a P1 ori-
gin of replication and modeled on the P1 artificial
chromosome was constructed by Liu et al. [150]. This
transformation-competent bacterial artificial chromo-
some (TAC) vector was used to introduce up to 80 kbp
of genomic DNA into Arabidopsis, and while there was
some loss of efficiency with the larger inserts, it was still
possible to produce many transgenic plants. Both vec-
tors contain a kanamycin resistance marker for selec-
tion in bacteria and hpt for hygromycin selection in
transgenic plants. Both vectors also contain the Ri
origin for maintenance in Agrobacterium, and within
the T-DNA region, the sacB marker for negative selec-
tion, interrupted by a multiple cloning site for trans-
gene insertion. One of the most attractive uses of
high-capacity binary vectors is for the positional clon-
ing of genes identified by mutation. The ability to
introduce large segments of DNA into the plant
genome effectively bridges the gap between genetic
mapping and sequencing, allowing the position of
mutant genes to be narrowed down by complementa-
tion. Genomic libraries have been established for
several plant species in BIBAC2 and TAC vectors [151,
152] and cloning in high-capacity vectors has been
simplified by the inclusion of Cre/loxP and Gateway
site-specific recombination technology [153, 154].
Large (80–150 kbp) DNA molecules have also been
transferred to plants by direct DNA transfer [155], and
although this is not a routine procedure a novel trans-
formation method has been developed recently, based
on bombardment with DNA-coated “bioactive beads”
to deliver up to 150 kbp of DNA into rice protoplasts
[156].
Plant Minichromosomes
In bacteria, plasmid vectors are maintained as episomal
replicons to make cloning and isolating recombinant
DNA a simple procedure. When it comes to expressing
heterologous genes in eukaryotic cells, episomal vectors
are widely used to avoid position effects, hence the
development of yeast episomal vectors, yeast artificial
chromosomes, mammalian plasmid vectors carrying
virus origins of replication (e.g., SV40-based vectors,
herpes virus-based vectors), and plant expression vec-
tors based on plant viruses (all of which replicate epi-
somally). The yeast artificial chromosome (YAC)
system is the most relevant in this context because it
allows genes of any size to be introduced into the yeast
genome as an independent replicating unit that is
treated by the cell as an additional chromosome.
YACs comprise a yeast centromere and telomeres,
the origin of replication (autonomous replicating
sequence), and selectable markers.
More recently, analogous systems have been devel-
oped to maintain genes as episomal minichromosomes
in plants. These have many advantages for plant genetic
engineering including the ability to express large
transgenes or groups of transgenes, and the ability to
rapidly introduce new linkage groups into diverse
germplasm. Carlson et al. [157] created plant
minichromosomes by combining the DsRed and nptII
marker genes with 7–190 kb of corn genomic DNA
fragments containing satellites, retroelements, and
other repeat sequences commonly found in centro-
meres. The circular constructs were introduced into
embryogenic corn tissue by particle bombardment

and transformed cells were regenerated and propagated
for several generations without selection. The
minichromosomes were maintained as extrachromo-
somal replicons through mitosis and meiosis, and
showed roughly Mendelian segregation ratios (93%
transmission as a disome with 100% expected, 39%
transmission as a monosome crossed to wild type
with 50% expected, and 59% transmission in self
crosses with 75% expected). The DsRed reporter gene
was expressed over four generations, and DNA blot
analysis indicated the genes were intact.
Gene Targeting (Homologous Recombination)
Gene targeting is the directed modification of an
endogenous DNA sequence by homologous recombi-
nation, an efficient procedure in bacteria, yeast, certain
animal cells, and in the plastid genomes of plants, but
typically not in the nuclear genome. Only one plant
species has been shown to undergo efficient nuclear
homologous recombination and that is the moss
Physcomitrella patens [158]. Among higher plants,
low-level gene targeting has been achieved in certain
dicots with frequencies ranging from 103 to 106
[159]. However, targeting frequencies of up to 1%
have been achieved using a T-DNA-mediated gene
targeting strategy involving a long homology region
in combination with a strong counterselectable marker
in rice [160, 161].
There has also been interest in the use of zinc-finger
endonucleases to make targeted double-strand breaks
in the plant genome, so that homologous recombina-
tion is favored at such sites [162]. The modular nature
of zinc-finger transcription factors means that recom-
binant DNA technology can be used to “mix and
match” these DNA-binding domains to create recom-
binant proteins with unique sequence specificities.
Zinc-fingers are motifs approximately 30 amino acids
in length which coordinate a Zn2+ ion and bind to DNA
sequences 3-bp long. Combining different zinc fingers
in series allows proteins to be tailor made to bind
longer DNA sequences. When a nonspecific DNA
endonuclease is incorporated into such a protein, it
becomes a targeted DNA cutting tool [163, 164]. The
recent achievement of targeted transgene integration
and endogenous gene disruption in corn [165] and
tobacco [166] using zinc-finger endonucleases
Crop Plants Transformation Methods 583
provides a tantalizing glimpse of the future of plant
biotechnology in which precise changes can be made to
the genome of any plant genome that is amenable to
DNA transfer.
Site-Specific Recombination
Although site-specific recombination has already been
described as a cloning tool, particularly the Cre/loxP
and Gateway systems for rapid vector assembly, it can
also be used in transgenic plants to introduce DNA at
a specific, favorable locus, or remove DNA sequences in
vivo. The Cre/loxP system has been most widely used in
plants for the controlled excision of selectable marker
genes after transformation (e.g., [153]), but also for
controlled transgene insertion (e.g., [167]). Controlled
integration has been studied in transgenic plants
already engineered to contain recipient loxP sites
[168]. In this study, three different recipient wheat
lines were generated by bombarding plants with the
loxP sequence, and these were subsequently bombarded
with a gusA construct also containing flanking loxP
sequences, and a cre gene. Following transformation,
about 80% of lines contained gusA at the recipient site,
many with single-copy transgenes and others with
concatemers. Both types of locus were stably inherited.
There was much less variation in expression among the
single-copy lines [168]. Chawla et al. [169] generated
18 different transgenic rice lines containing a precise
single copy of gusA at a designated site. In seven of these
lines, additional copies of the transgene integrated at
random sites by illegitimate recombination while 11
showed “clean” integration by site-specific recombina-
tion only. The single-copy lines were stable over at least
four generations and showed consistent levels of
expression, which doubled in homozygous plants. In
contrast, the multicopy lines showed variable expres-
sion and some fell victim to transgene silencing. Inter-
estingly, where the site-specific and illegitimate
integration loci segregated in later generations, trans-
gene expression was reactivated in the plants carrying
the site-specific integration site alone, whereas close
linkage between the site-specific and random integra-
tion prevented segregation in other lines and the silenc-
ing persisted.
An exciting recent development is the GENE
DELETOR system, which is a hybrid of the Cre-loxP
584 Crop Plants Transformation Methods
and FLP-FRT systems. The GENE DELETOR is based
on a fusion recognition site (loxP-FRT), which is ineffi-
cient when both recombinases are expressed but highly
efficient when either one of the recombinases is expressed
alone, giving up to 100% efficiency in populations of up
to 25,000 T1 transgenic tobacco plants [170].
Another use for Cre-loxP is the simplification of
locus structure by resolving multicopy loci to a single
transgene copy [171]. A strategy was developed in
which the transformation vector contained a transgene
flanked by loxP sites in an inverted orientation. Regard-
less of the number of copies integrated between the
outermost transgenes, recombination between the out-
ermost sites resolved the integrated molecules into
a single copy. The principle was proven by resolving
four multicopy loci successfully into single-copy
transgenes.
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Crop Radiation Capture and Use
Efficiency
ERIK MURCHIE1, MATTHEW REYNOLDS2
1
Division of Plant and Crop Sciences, School of
Biosciences, University of Nottingham, Sutton
Bonington, UK
2
International Maize and Wheat Improvement Centre
(CIMMYT), Mexico DF, Mexico
Article Outline
Glossary
Definition of the Subject and Its Importance
Introduction and History
Determination of Radiation Use Efficiency
Radiation Capture by Crop Canopies
“Conversion” of Captured Radiation: Photosynthetic
Mechanisms
Metabolic and Regulatory Constraints to RUE
Source–Sink Processes and Partitioning of Assimilates
Theoretical Considerations Related to the Improve-
ment of RUE
Sources of Variation in Agricultural RUE
Future Directions
Bibliography
Glossary
C3 The C3 pathway of photosynthesis, found in most
plant species, for example, rice, potato, and wheat.
C4 The C4 pathway of photosynthesis, found in some
tropical species, for example, maize, sugarcane,
sorghum.
PAR Photosynthetically active radiation. Solar radia-
tion in the wavelength region 400–700 nm.
RUE Radiation use efficiency, the ratio of biomass
produced per unit radiation intercepted.
Definition of the Subject and Its Importance
The rate of growth (the rate of the accumulation of dry
matter) of all plants is entirely dependent on the inter-
ception of energy (electromagnetic radiation) from the
sun in the wavelength range 400–700 nm. This energy is
utilized by photosynthesis to synthesize carbohydrates
P. Christou et al. (eds.), Sustainable Food Production, DOI 10.1007/978-1-4614-5797-8,
# Springer Science+Business Media New York 2013
Originally published in
Robert A. Meyers (ed.) Encyclopedia of Sustainability Science and Technology,
Crop Radiation Capture and Use Efficiency 591
and other biological molecules needed for essential plant
processes.
The amount of energy intercepted or captured by the
whole plant and community system (the canopy) is
determined by the organization of leaves into an efficient
spatial structure with a large total surface area. The
amount of radiation captured will determine the rate of
photosynthesis possible and the rate of growth. However,
the final growth rate is then determined by losses in the
system that originate from a number of sources, includ-
ing the type of photosynthetic mechanism, metabolic
and hydraulic constraints, the relationship between
photosynthetic source and non-photosynthetic sink
organ, variability in environmental conditions, and
limitations imposed by management techniques.
The discovery that plant and crop growth is closely
linked to the amount of intercepted radiation led to the
establishment of methods for measuring radiation use
efficiency (RUE). RUE is measured as the amount of
dry matter produced per unit intercepted radiation
over a given time period and it is often separated into
key developmental stages within the life cycle of the
crop. It was quickly established that values of RUE tend
to be stable for a given species, growth stage, and
environment, but there are important differences
across crop species and plant types. In the absence of
other factors, RUE will set the theoretical limit to
biomass production and ultimately crop yield. It is
now accepted that RUE is a fundamental measurement
which underpins potential crop productivity and yield
and it has become embedded into modern methods of
crop growth analysis.
In many cases, plants absorb more photosyntheti-
cally active radiation (PAR) radiation than they utilize
for growth: given the current emphasis on global food
security, there is currently much interest in raising the
RUE of key crops in important agroecosystems.
Introduction and History
All green plants use sunlight as their sole energy source
for assimilation of carbon dioxide into carbohydrates.
At its most fundamental level, this process is the for-
mation of energy-rich bonds in a form that is easily
stored, transported, and utilized in most essential plant
functions. This process is of course photosynthesis and
# 2012, DOI 10.1007/978-1-4419-0851-3
592 Crop Radiation Capture and Use Efficiency
it is the sole supplier of energy for almost all human
nutrition and fuel requirements, via current and pre-
historic photosynthesis.
The continuing expansion of the human popula-
tion and the steady increase in consumption per capita
is placing pressure on land availability. A variety of
factors including urbanization and the erosion of
existing land certainly necessitate production of more
food and fuel per hectare with fewer inputs (water,
fertilizer) and all of this within the uncertainty of
changes in climate and increased pressure to use land
which may be currently unsuitable for cultivation. For
example, 700 million people depend on rice for calo-
rific intake. Each hectare can currently feed around
27 people; by 2050 this will have to rise to 43 people
per hectare [1]. This is compounded by the fact that
Asian rice yield potential appears to be stagnating [2].
Wheat is grown on over 200 million hectares of land
providing approximately one-fifth of the total calorific
input of the world’s population, and while there have
been steady increases in productivity since the green
revolution, global demand for wheat is predicted to
increase at a faster rate than the annual genetic gains
that are currently being realized [3]. Since the highest
yields of major crops are usually only achieved within
high input systems, it is not surprising that
many suggest a reappraisal of the basis of crop
productivity.
It seems clear that an increase in the rate of appli-
cation of fertilizer or water is neither desirable nor even
possible. Nitrogen fertilizer is heavily dependent on the
continued availability (physically, financially, and
politically) of fossil fuels. Water is increasingly scarce
not just in equatorial regions but in temperate regions.
One way to improve resource use efficiency is to
increase the rate of overall biomass production and
this is starting to be associated with yield progress of
major crops such as rice and wheat [3, 4]. As shall be
described, an increase in biomass production by crops,
whilst helping to improve upon current rates of breed-
ing progress in yield potential, would also (1) underpin
future genetic improvements in adaptive processes,
(2) improve the relative efficiency of resource use in
terms of biomass production per unit water, light, and
fertilizer, and therefore (3) increase the attractiveness of
using waste products of arable crops for secondary uses
such as fuel, and (4) if applied universally, reduce the
competition for land between biofuel crops and food
crops, and (5) increase the feasibility of using marginal
land for fuel and food production.
A History of Radiation Use Efficiency
Given that it underlies so much of human activity and
endeavor it is perhaps curious that the relationship
between radiation and plant growth did not become
explicitly defined and quantified until relatively recently.
It has been suggested that a tendency to retain methods of
classical crop growth analysis during the first half of the
twentieth century delayed a mechanistic analysis of the
processes of biomass accumulation [5]. In these early
studies, growth (crop mass) was described as a function
of time possibly because of the ease with which mass
could be measured through the growing season. This
approach was confounded by its inclusion in measure-
ments of relative growth rate where crop biomass increase
is closely related to existing biomass. The central role of
light interception in growth was recognized by Watson
[6] who suggested a measurement of net assimilation
rate which worked in some situations but did not
adequately account for the complex relationship
between leaf area and variation in assimilation rate
(photosynthesis) of individual leaves.
Another advancement in understanding came with
the consideration that efficiency of light use declined at
high levels (light saturation) [7, 8] and that light levels
were lower at the base of the canopy. In fact, as pointed
out by Hirose [9], the first mathematical model of can-
opy photosynthesis was first produced by Monsi and
Saeki in 1953 but this was not recognized for more than
a decade. This resulted in the first real considerations of
growth as a function of the amount of light intercepted by
the plant canopy. At this time, it was observed that the
amount of light intercepted by the canopy was closely
related to dry matter accumulation [5]. Despite this
there were many studies in the 1970s which used inci-
dent light rather than intercepted radiation to make key
studies in agronomy and ecology.
The sole use of incident radiation measurements
will lead to errors in the calculation of radiation use
efficiency especially before canopy closure when not all
radiation is absorbed and when crops possess differing
rates of canopy development. Over the whole life cycle
of the crop, the proportion of incident radiation that is

intercepted and available for assimilation can be quite
small. It was not until the mid 1970s that John
Monteith established the relationship between accu-
mulated intercepted radiation and accumulated bio-
mass within canopies [10]. This was accompanied by
experimental data demonstrating a level of conserva-
tion of radiation use efficiency of crop species when
grown under optimal growth conditions with high
resource availability and a consideration of the role of
leaf photosynthetic capacity in crop canopies. This has
since proved to be a robust approach and has remained
a central feature of crop growth analysis ever since.
Many studies attempted to provide values for RUE
across a wide range of species and growth conditions
across the world [11, 12]. It was quickly claimed
(1) that it was possible to attain consistent values for
a given species when growing conditions were good,
(2) that suboptimal (e.g., nitrogen or water deficient)
or stressful conditions caused RUE to decline, and
(3) that there were clear differences between crop spe-
cies: plants possessing the C4 photosynthetic mecha-
nism had the highest RUE followed by most C3 plant
species and finally legumes with the lowest RUE values.
Early data suggested that RUE was a conservative or
even constant value for a given species. However, data
published since has demonstrated significant variation
and it has been pointed out that close attention in each
case must be paid to the methods of analysis, growth
conditions, developmental phase, and genotype.
RUE is commonly used within the crop sciences
although it is applicable to growth of any autotrophic
organism and most ecosystems. Its calculation requires
knowledge of the amount of radiation absorbed in
a given time period and measurement of the resulting
biomass and energy content. These are easier to
measure in a uniform system like a monoculture.
The historical development of RUE was no doubt
dependent on the advancement in other areas of sci-
ence such as photosynthetic regulation and the techni-
cal development of instruments to accurately measure
key attributes, that is, radiation, photon flux density,
and the rate of photosynthetic carbon assimilation.
The understanding that RUE is fundamental to
crop productivity leads naturally to the question of
whether it can be improved. The observation that C4
plants possess a higher RUE leads to the notion that
such a change would give a higher biomass
Crop Radiation Capture and Use Efficiency 593
productivity which could be exploited to increase
yield per hectare. The green revolution provided
a step change in agricultural productivity that was
able to prevent the tragedy of mass starvation in some
regions and also to support population increase. As
outlined here, a step change of similar magnitude
now would probably require an improvement in RUE.
Determination of Radiation Use Efficiency
RUE is the ratio between accumulated plant biomass
and the accumulated radiation intercepted by the crop.
A note on terminology: some authors use the terms
“radiation conversion factor” [13] and “radiation con-
version efficiency.” Some authors have pointed out that
use of the word “conversion” is inappropriate because
energy is not being directly converted to matter but
rather converted from one form of energy to another
(i.e., solar radiation to higher energy state of chloro-
phyll molecules) [5]. The term radiation use efficiency
(RUE) shall be used.
To calculate RUE, it is necessary to know the
amount of radiation arriving at the top of the canopy,
the amount of radiation intercepted by the canopy, and
the biomass accumulated during a given period.
In many studies, the energy content of the dry
matter is also required. This section will summarize
how RUE is measured in a practical sense and tackle
some of the diversity in approaches that have been
taken.
Radiation arrives at the edge of the earth’s atmo-
sphere at a mean rate of 1.4 kJ m
2 s
1, the so-called
solar constant. By the time photons reach the earth’s
surface a number of geometrical and atmospheric fac-
tors have reduced this value significantly. Cloud cover
notwithstanding the greatest influx occurs in tropical
regions at low latitudes. The greatest measured flux at
the earth’s surface itself occurs in low latitude regions
where cloud cover is minimal (20–30 ). High latitudes
can have extremely low solar radiation levels.
Light is absorbed and scattered by molecules, aero-
sols, and particles in the atmosphere, reducing flux
even further. Gas molecules such as CO2, H2O, and
CH4 absorb energy at specific infrared wavelengths
whilst gas molecules O2 and O3 absorb at lower wave-
lengths. Photosynthesis is restricted to a range of wave-
lengths that approximate to those visible to the human
594 Crop Radiation Capture and Use Efficiency
eye: 400–700 nm. Higher wavelengths do not contain
sufficient energy to drive photosynthesis. At ground
level, this range of wavelengths makes up 49% of total
solar energy. In other words, the photosynthetic pro-
cess does not use over half of the energy available.
However, longer wavelengths have an important
heating effect which raises plant tissue temperature
and accelerates metabolic and developmental processes
such as leaf and canopy construction.
Variation from day to day means that it is necessary to
integrate measurements of radiation over long time
periods. This is often done by positioning devices above
the canopy and below the canopy. Most commonly these
have been fairly inexpensive tube solarimeters connected
to a device that continually stores data produced. Reflec-
tion can be measured by inverting the device. Instanta-
neous measurements (spot measurements) of fractional
interception (f ) are also used but these can be mislead-
ing because they are most reliably taken at midday
during the same, usually sunny, conditions and there-
fore do not account for cloud cover or low solar eleva-
tion and give lower values of f.
The proportion of PAR is higher in scattered light
than direct beam radiation and so it is useful to distin-
guish between the two and a few commercially available
devices are capable of doing so. This also means that the
proportion of PAR changes according to solar eleva-
tion, although it is around 0.45 at elevations greater
than 30 [5]. Growth in predominantly scattered radi-
ation commonly causes an increase in RUE.
Wavelengths used for growth (400–700) are prefer-
entially absorbed by chlorophyll within the canopy
resulting in transmitted light that is depleted in red
and blue and enriched in far-red. The formula for
fractional interception is presented in its simplest
form by Eq. 1.
ðI þ r Þ
f ¼1
ð1Þ
Io
where f = fractional interception, Io = incident radia-
tion, r = reflected radiation, I = intercepted radiation.
An alternative method for measuring f is spectral
reflectance. This compares reflectance from the canopy in
the red band and the near IR band to produce
a normalized difference vegetation index (NDVI). It has
been possible to show the relationship between NDVI
and f in many crops [14] and is a technique that is
possible to use remotely from satellites and aircraft.
Although it is clearly convenient and rapid, one must be
aware that it has the drawback of being a spot measure-
ment. Measurements made from satellites suffer from
problems of images that deviate from 90 to the earth’s
surface [15].
Photography is also used to estimate f using
a camera fitted with a fish-eye lens positioned below or
above the canopy. This has the advantage of
encompassing a large area, producing permanent image
and being taken rapidly in the field. This method has been
shown in many cases to correlate with f measured using
solarimeters; however, errors arise due to common bias
in positioning the camera. All indirect measurements
of f should consider the potential errors that may result.
RUE is usually calculated by measuring the difference
in biomass between many consecutive harvests (i.e., accu-
mulated intercepted growth) and plotting this against the
measured accumulated intercepted radiation. This is usu-
ally a linear relationship and RUE is calculated as the
slope of this relationship (Fig. 1). The points at which
measurements are made are critical because RUE varies
according to developmental state.
Care must be taken when comparing values from
different sources [5, 13]. Some of the common sources
of variation are as follows:
1. If root mass is not included then RUE values will be
lower. However, it is extremely difficult to measure
root mass directly and it is often ignored or assumed
to be a fixed percentage of total plant mass.
Nonuniform growth can therefore be a significant
source of error. Energy required to grow and maintain
roots is also a function of the biotic and abiotic
environment of the soil.
2. Solar radiation flux density is measured using
solarimeters, pyranometers, or radiometers which
usually cover the wavelengths 300–3,000 nm. The
photon flux density is commonly measured using
sensors that detect within the photosynthetically
active range of 400–700 nm. Therefore the conver-
sion between energy and quanta-based measure-
ments must take into account the energy of each
waveband. Data based on different regions of the
electromagnetic spectrum can cause inaccuracies,
for example, the transmission of shortwave radia-
tion through canopies differs from that of PAR [16].
 Crop Radiation Capture and Use Efficiency 595

Above ground dry weight, g m−2 Fractional of intercepted light

3000
1.0
2500
0.8
2000
0.6
1500
1000 0.4

500 0.2
0 0.0
0 100 200 300 400 500 600 700 0 20 40 60 80 100
a Accumulated intercepted PAR. MJ m−2 b Days after transplanting

Rice Maize Echinochloa glabrescens

Crop Radiation Capture and Use Efficiency. Figure 1

An example of the relationship between accumulated intercepted radiation and accumulation of dry matter by three
species : the C3 crop rice (circle), the C4 crop maize (square), and the C4 weed Echinochloa glabrescens (triangle). Note the
higher slope for maize in (a), that maize reached full interception before rice in (b), and that the weed is the first to form
a full canopy but does not achieve the same amount of biomass as the crop species (Redrawn from Sheehy et al. [1])

However, the solar spectrum is fairly constant,
and the reliable figure for this is 4.6 mmol quanta.
Inaccuracies can arise from badly positioned,
poorly leveled or uncalibrated instruments. It is
common practice to keep devices free of anything
that would attenuate radiation in a way that would
bias the results, for example, dead leaves are
removed and glass is cleaned.
3. It is important to distinguish absorbed from
intercepted radiation. The latter does not take into
account the reflection of PAR from the top surface
of the canopy. This is generally assumed to be about
5% of total PAR but variation is likely. Not all of the
plant tissues that absorbed energy are necessarily
alive or of equal photosynthetic potential.
4. When comparing different species of plant with
contrasting harvest organs it is important to consider
the energy content of the organs involved. Lipid has
a higher calorific content than protein, which in turn
has a higher calorific content than carbohydrates
[17]. The heats of combustion of carbohydrates,
proteins, and lipids are 17.3, 22.7, and 37.7 kJ g
1,
respectively. Since RUE is comparing the input of
energy to the output of dry matter it can vary
according to the energy content per unit dry matter
and it is essential to adjust RUE accordingly. For
example, the RUE of crops that produce particu-
larly oily seeds should decrease during the seed-
filling period. However, the nutrient content of
plant tissue does not greatly directly influence the
measurement of RUE.
5. The stage of growth can have a large influence on
the RUE measured (see section Source–Sink Pro-
cesses and Partitioning of Assimilates). It can be
common practice to calculate the RUE over the
entire growing season but this does not necessarily
represent the maximal RUE value. For example, in
many crops RUE appears to be steady during the
vegetative period but to decline following the onset
of the reproductive phase.
Radiation Capture by Crop Canopies
Canopy structure and therefore the efficiency of light
capture was a common feature in the domestication
and the later improvement of many crop species. For
example, the reduction of branching in maize and
sunflower allowed dense planting [18] and there is
evidence of further adaptation to higher planting den-
sity [19]. The reduction in height of cereals was a key
factor in the green revolution, permitting an increase in
harvest index and reduction in lodging. Crop canopies
596 Crop Radiation Capture and Use Efficiency
must intercept or capture as much radiation in the
400–700 nm bandwidth as possible. Most of the light
interception occurs by leaves; however, stems, petioles,
leaf sheaths, and reproductive structures can also
absorb significant amounts of radiation. To achieve
high interception, they must construct and present
a canopy with large leaf area and in many crops the
minimum leaf area index (L: the ratio of leaf area per
unit ground area) is around three. The L can be
expressed by the product of the number of plants per
unit ground area, the number of leaves per plant, and the
mean area of leaves per plant (plus green stem area). Light
can penetrate the canopy and strike the ground below as
direct beam radiation (so-called sunflecks), scattered
radiation, or radiation that has passed through leaves
and other plant organs, that is, transmitted. The amount
of penetrated radiation is dependent on the three-
dimensional arrangement of leaves in the canopy and
for a given L will be a function of a large number of
features such as leaf size, leaf density per cubic meter,
the angle of individual leaves, heterogeneity of leaves in
space (clumping), leaf thickness, and albedo. There is
clear variation between species and even between
crop varieties in these features. Additionally, it is pos-
sible to alter these features through management tech-
niques such as planting density and the application of
NPK fertilizer. Therefore there needs to be a way of
describing radiation distribution within canopies
mathematically and linking this to agronomic charac-
teristics such as leaf area index and nitrogen content.
The relationship between L and fractional intercep-
tion can be described by Monsi–Saeki equation (Eq. 2),
a modification of Beer’s law. This assumes that the
canopy is a homogeneous medium whose leaves are
randomly distributed in space, that is, there is no effect
of row structure or clumping and under these condi-
tions Beer’s law will apply [9]. The Monsi–Saeki equa-
tion can be applied if the canopy is considered to be
divided into horizontal layers with each layer
possessing a particular L and the irradiance within
each layer is measured. The irradiance at each layer
will depend on the three-dimensional characteristics
of the leaves both within that layer and the layers
above. It has been found that this provides an accurate
estimate in many crops in which this has been mea-
sured: ln(I/Io) against L provides a linear relationship,
the slope of which gives the value k which provides
a simple but useful mathematical description of the
architecture of the crop canopy in question.
I ¼ Io expð
kLÞ ð2Þ
I and Io as in Eq. 1, L = leaf area index, and k =
extinction coefficient for a given waveband
Crops have been found to vary for the value of k and
the major feature is the erectness of leaves. Canopies with
erect and narrow leaves such as cereals have a lower value
of K than those with flat, broad, and horizontal leaves.
This also applies to differences within species, for
example, rice varieties vary in erectness [20].
If the Monsi–Saeki equation holds and the k value
for a given canopy type is known then it becomes
possible to calculate f simply from a knowledge of L
(Eq. 3).
f ¼ 1
expð
kLÞ ð3Þ
Equation 3, letters as Eqs. 1 and 2
This analysis is useful when canopies are considered
to be a three-dimensional “box” of vegetation with
radiation penetrating only from above. It can fail
where plants are sparsely populated and do not achieve
full ground cover or are simply planted in rows with
space in between, which is the case for some crops that
are tended by hand. The calculation of intercepted
radiation in these cases can become quite complex
and impractical at high levels of heterogeneity although
estimations are often made on the basis of incident
radiation and leaf area per plant. Another source of
error is angle of solar elevation which can significantly
alter the proportion of reflected light and the propor-
tion of scattered light within the canopy. It also does
not account for the commonly seen variation in leaf
angle that occurs between the top and the bottom of
the canopy. Even at a point close to full canopy cover
there can be a “bimodal” type of variation in crop
canopies. For example, rice plants tend to form
“inverted cone” shapes and this causes a complex
three-dimensional variation in irradiance distribution
especially during canopy development.
The optimal “design” of plant canopies must con-
sider not just maximum interception but also the rela-
tionship with photosynthetic rate. In principle, a plant
could achieve close to 100% interception with a single,
planar chlorophyll-rich leaf and a L of 1.0. However,
 Crop Radiation Capture and Use Efficiency 597

Net single photosynthesis Net whole canopy photosynthesis

per unit leaf area per unit ground area
μmol CO2 m−2 s−1 mg CO2 dm−2 h−1
25

20
40
Amax
15 Θ 30
Φ
10 20
Φ = Quantum yield
5 Θ = ‘convexity’
b = light compensation point
10

0 0
b
−5

0 250 500 750 1000 1250 0 100 200 300 400

PAR (μmol m−2 s−1) Irradiance (W m−2)

Crop Radiation Capture and Use Efficiency. Figure 2

The relationship between photosynthesis and incident radiation levels for (a) single leaves and (b) a full canopy: (a) typical
features of the leaf response, the linear phase (maximum quantum efficiency), the light compensation point, convexity,
and the light saturated rate (Amax, and (b) schematic figure: note the linear response of whole canopies in comparison to
single leaves

this does not result in optimum productivity largely due Canopy Properties and Photosynthesis
to the fact that photosynthesis saturates below
The optimum design for biomass production in terms
full sunlight and this is especially marked in C3
of carbon gain is predicted to be one that permits
plants. This is a central point: the response of
a higher proportion of radiation to penetrate to lower
leaf photosynthesis to irradiance is shown in Fig. 2.
layers, whilst also reducing light saturation at the top
This is a useful measurement and easy to make with
(Fig. 3). Indeed, this seems to be the trend in species
today’s equipment. It provides not only a measurement
such as rice and wheat although the advantage here is
of the maximum rate of photosynthesis (Amax)
thought to be greatest at lower latitudes where light is
but also the potential quantum yield of photosynthesis.
overhead for a greater proportion of the year and
A greater canopy carbon gain is achieved by reduc-
therefore penetration into the canopy is greater. This
ing the proportion of leaves in the canopy that exist in
also has the advantage that it prevents the unwanted
the light-saturated state. The leaves lower in the canopy
senescence of leaves when light levels drop below or
are retained at light limitation while those at the top
close to the light compensation point [21].
will be prone to light saturation. If the irradiance
This raises the question of whether there is room for
increases (e.g., moving from cloud-cover to full sun-
improvement in canopy architecture to improve RUE.
light) then those lower in the canopy will be able to
Early work found that canopies with a low L benefitted
respond accordingly. This has lead to the widely
from horizontal posture while those with high L
observed phenomenon that when irradiance is plotted
benefitted from upright posture [5, 22]. This work
against carbon-gain (photosynthesis) one commonly
suggested that RUE would not be sensitive to extreme
observes a linear response for canopies but a saturation
upright posture of leaves. However, it is clear that
in leaves (normally described by a non-rectangular
upright leaves are associated with the highest yielding
hyperbola) (Fig. 3) [11]. This is important: if canopies
lines in wheat [23, 24] and it has been suggested that
demonstrated light saturation then this would severely
some cereal canopies may benefit from increasing
limit their ability to improve RUE.
598 Crop Radiation Capture and Use Efficiency

Canopy depth E, erect canopy e.g. rice F, flat, broadleaf canopy e.g. common bean
high L, low K Low L, high K

L=1.0

L=1.0

F
L=1.0

E
L=1.0

a PAR

50
Canopy photosynthesis

40 K=0.3
K=0.3
Biomass/area
(g m−2 d−1)

Point of canopy closure

30
K=0.6
20 K=1.0 K=0.9

10

0
0 2 4 6 8 10 c time
b Leaf Area Index, L

Crop Radiation Capture and Use Efficiency. Figure 3

Canopy structure and function according to leaf “architecture.” (a) The schematic figure on the left shows canopy depth
where the canopy has been divided into layers, each with a leaf area index (L) of 1.0. Attenuation of light in the canopy
follows the Monsi–Saeki equation. For a broadleaf canopy, the radiation reaches extinction at a higher point and a lower L
compared to an upright canopy. The canopy extinction coefficient value “K” is derived from Eq. 2 and provides
a mathematical description of the link between F and L. (b) The calculated relationship between canopy photosynthesis
and L, measured at irradiance saturating at the top of the canopy for species with two values of k. Note the higher
maximum value of the species with higher k (Redrawn from [9]). (c) A highly schematic figure redrawn and adapted from
Ong and Monteith [32] and shows the theoretical differences in canopy biomass accumulation over time in species with
different values of k. Maximum values of carbon gain are achieved after canopy closure which occurs earlier in broadleaf
species (high k). Species with a lower value of k have higher potential rates of total canopy photosynthesis

erectness further [3, 23, 25]. There are relatively few
genes controlling erectness and this should be straight-
forward to test, although work has shown that com-
pensatory effects such as leaf size may be hard to
account for.
Modern high-yielding rice lines also have highly
erect leaves and it is assumed that this also results in
higher potential productivity. In rice, this also permits
denser planting which accelerates canopy closure and
improves total radiation intercepted over a short trop-
ical growing season.
A further feature is that leaves have different char-
acteristics according to the light intensity to their can-
opy position. Most notably they have “sun leaf ” and

“shade leaf ” characteristics, the former having higher
light compensation points, higher photosynthetic
capacity, and higher nitrogen and enzyme content.
This is considered to be an efficient use of resources
with nitrogen being located only where it is required
for high photosynthetic activity. Additionally, there are
a number of acclimation mechanisms to improve light
harvesting in shade and diffuse light such as the syn-
thesis of light harvesting complexes enriched in chlo-
rophyll b [20, 26]. The efficiency of use of shade and
diffuse light within the canopy is a relatively
unexplored area in crops. This raises the question of
whether leaf ageing or leaf acclimation gives rise to the
nitrogen content of the leaf. There is evidence that both
processes are at play: in fact the distribution of nitrogen
in plant canopies should be closely related to photo-
synthesis and irradiance level [27, 28]. There is some
indication that this has been achieved although the role
of “storage” of assimilated nitrogen in leaves needs
further analysis [20, 21].
Canopy development occurs by the successive emer-
gence of leaves from primordia which are localized points
of tissue formed around the apical dome of the vegetative
shoot. The arrangements of these primordia will largely
determine the positions of the leaves in the final canopy.
Therefore there is great interest in the genetic manipula-
tion of developmental processes in crops [29, 30]. The
thermal time interval between the initiation of succes-
sive primordia is critical and determines the time taken
for production of successive leaves. The genetic and
molecular processes determining leaf development
and expansion is an exciting area of research [31].
For annual crops with a limited growing season, it is
critical that canopy development is synchronized with
periods of high radiation. In temperate regions, low
temperatures in Spring can increase the time taken for
canopy closure. Since maximum rates of production
are not attained until full canopy cover is achieved, this
can be deleterious and is referred to as “lost time” [32].
Faster rates of canopy closure can be achieved by dif-
ferent strategies such as the application of nitrogen
increasing planting density, irrigation, and the planting
of crops in autumn so that the time taken for establish-
ment is reduced. For example, in the UK, maximum
radiation receipts occur in June, and for Spring-sown
crops such as potato and sugar beet, productivity can
depend on the establishment of a high L before this
Crop Radiation Capture and Use Efficiency 599
period. In rice, the remarkable erectness of leaves in
some modern cultivars has allowed for extremely dense
planting and a shift toward a reduction in tillering. This
has the advantage that canopy closure can be achieved
quickly and the so-called lost time is reduced.
There is no doubt that canopy structure is a central
feature of crops with high RUE although the precise
three-dimensional characteristics are difficult to quan-
tify by direct measurement and this has meant that the
techniques to describe crop canopies mathematically
have remained relatively simple and continued to
assume that they exist as randomly distributed photo-
synthetic elements in space. As shall be described in the
next section, it will be essential to have a more sophis-
ticated approach which links leaf arrangement and light
distribution at higher resolutions and accounts for the
dynamics of both light distribution and localized pho-
tosynthetic responses. For this various photographic
and laser-based methods are available for “digitization”
of plant and crop canopies although the high density of
many crop canopies makes such measurements difficult
in a practical sense. Mathematical modeling of plant
canopy structure is progressing rapidly along with
increased computing power and more sophisticated
programming [33]. It would seem likely that methods
that can use empirical measurements to model canopy
architecture at high resolution and accurately predict
photosynthetic light responses are not far away.
“Conversion” of Captured Radiation:
Photosynthetic Mechanisms
The efficiency of radiation use by the whole canopy is the
sum of photosynthesis occurring by each leaf and each
leaf portion within the canopy. In turn, the photosyn-
thetic rate of each leaf is determined by the sum of
photosynthesis in the chlorophyll-containing organelles
(chloroplasts). Each leaf and organelle will exist in
a dynamic microenvironment and the photosynthetic
productivity will depend on the resources available to it
at a given moment in time (light, water, nutrients). Due
to limitations placed on the photosynthetic process such
as light saturation and CO2 diffusion, the rate of photo-
synthesis at the leaf level is a strong determinant of
canopy carbon gain. This section will describe the pho-
tosynthetic process, the different types of photosynthe-
sis, and limitations to leaf level photosynthesis.
600 Crop Radiation Capture and Use Efficiency
The vast majority of chloroplasts are located in
leaves and they are numerous, with each mesophyll
cell containing between 50 and 200 chloroplasts [34].
Chloroplasts are responsible for absorption of the
400–700 waveband in crop canopies and are the reason
that plants appear green. They are sac-like subcellular
organelles that contain membranous structures that
possess the chlorophyll used for light harvesting. Most
of the chlorophyll is contained within pigment protein
complexes called “light harvesting complexes” and
these are extremely efficient at absorbing visible light,
and through a series of resonance transfer mechanisms,
they pass the excitation energy to a reaction center
where a special pair of chlorophyll molecules use this
energy to generate a redox potential capable of oxidiz-
ing water via the oxygen evolving complex. The
resulting electrons are passed through an electron
transport chain and used to generate a proton gradient
that synthesizes ATP. Ultimately, the electrons produce
a reductant, NADPH. The NAPDPH and ATP are uti-
lized within the Calvin–Benson or photosynthetic car-
bon reduction cycle to reduce CO2 to triose phosphates
that are used in hexose and starch synthesis or exported
from the chloroplast for sucrose synthesis.
How Efficient Is Crop Photosynthesis?
It is useful to consider the amount of light energy
arriving at a canopy top or leaf surface and calculate
energy losses at each stage based on current knowledge
of the photosynthetic process in order to estimate the
potential system productivity [35, 36]. As mentioned,
only 49% of solar energy is available for photosynthe-
sis. Within the PAR range chlorophyll does not absorb
strongly in the green band and the reasons for this
adaptation have been well explored [37]. Green pho-
tons make up around 10% in the PAR range. Red and
blue photons drive photosynthesis with equal effi-
ciency despite the fact that blue light contains more
energy per photon than red light. Chlorophyll is excited
to higher energy states by blue photons but the extra
absorbed energy is not used to drive photochemistry.
This extra energy which is calculated to make up 6.6%
of incident solar energy is effectively lost [36].
C3 photosynthesis is the dominant form among
plant life which uses the enzyme ribulose bis phosphate
carboxylase (Rubisco) to fix CO2 where the initial
product of photosynthesis contains three carbon
atoms. Rubisco is part of the Calvin–Benson cycle.
Examples of C3 crop species are rice, wheat, potato,
soybean, cotton, and chickpea. In C3 photosynthesis,
three ATP molecules and two NADPH molecules are
used to assimilate one molecule of CO2 and regenerate
the acceptor, RuBP. Zhu et al. [36] provide a calculation
of the energy required to fix one carbon atom and
compare this to the energy contained within that one-
sixth of a mole of glucose. A calculation of the quantum
requirement of each ATP and NADPH molecule reveals
that 8 mol of photons are needed for the fixation of
CO2 molecule and this represents 1,388 kJ of energy
[36] whilst the energy content of this carbon atom
within the glucose molecule is 477 kJ.
C3 plants operate the process of photorespiration.
This is a consequence of the dual reaction of Rubisco:
in addition to the fixation of CO2 into carbohydrates,
Rubisco also reacts with O2 to produce glycolate. This
molecule must be metabolized through the
photorespiratory pathway, a process that consumes
ATP, reducing power and releases CO2. The product
of this is phosphoglycerate which reenters the Calvin–
Benson cycle. The losses caused by photorespiration are
significant and are greatly dependent on temperature:
the specificity of Rubisco for CO2 and the solubility of
CO2 relative to O2 in water declines with increasing
temperature. In tropical C3 plants the rate of
photorespiratory flux can be significant especially
when stomata limit the internal leaf CO2 concentra-
tion. In tropical rice in field temperatures of 35 C
under otherwise optimal conditions, saturating leaves
with CO2 resulted in an increase in leaf photosynthesis
of over 40% [38].
A subset of plants called C4 plants have evolved
a fascinating mechanism which minimizes the
oxygenase reaction of Rubisco to often insignificant
levels. It does this by restricting Rubisco to a bundle
sheath cell, which is oxygen free and non-leaky. Initial
fixation of CO2 takes place in mesophyll cells using an
alternative enzyme, phosphoenol pyruvate carboxylase
which generates C4 acids. These acids are pumped into
the bundle sheath cell where they are de-carboxylated,
and the CO2 released is concentrated around Rubisco
in the bundle sheath chloroplasts. This effectively
reduces the extent of the oxygenase reaction and the
photorespiratory pathway to insignificant levels. It is

thought to have evolved in a climate where high tem-
peratures, low CO2 levels, and low humidity could all
have been strong selective pressures. The C4 mecha-
nism is largely found in warm regions. Measurements
show that in many plants the advantage of the C4
mechanism over C3 starts to become particularly sig-
nificant at growth temperatures above 25 C. C4
requires additional ATP synthesis which increases the
quantum requirement of the photosynthetic process to
12 photons per CO2 molecule, in comparison with 8 for
C3 photosynthesis. However, this is more than over-
come by the elimination of photorespiration and low
activity of Rubisco resulting in a greatly enhanced
capacity for carbon assimilation at most light intensi-
ties in C4 plants. Other advantages become apparent,
for example, the higher efficiency of Rubisco means
that lower amounts of this protein are needed. Since
Rubisco can make up to 35% of total leaf protein in
some C3 plants [20], this increases the photosynthetic
nitrogen use efficiency of C4 plants. The CO2 concen-
trating mechanism of C4 plants means that stomatal
conductance does not need to be high and water loss
can be reduced.
A substantial “loss” of biomass is mitochondrial
respiration which has often been divided into growth
respiration and maintenance respiration in accor-
dance with the associated physiological process. Res-
piration is unsurprisingly difficult to measure and to
define in practice [39]. The relative rates can vary
substantially according to growth stage. For example,
maintenance respiration increases substantially as
a proportion of total carbon flux following ear emer-
gence in barley [35]. During vegetative growth, growth
respiration is substantially higher than maintenance
respiration. Respiration as a whole can vary from 30%
to 60% of total carbon exchange and therefore it has
a significant impact on RUE. Studies have shown that
it undergoes optimization according to the environ-
ment and developmental state: for example, it is well
known that the relationship between L and respiration
is not linear because this would result in a negative net
carbon balance in the lower, shaded regions of the
canopy. Attempts have been made to optimize respira-
tory rates to improve crop productivity but this
is a difficult task because of the problems with mea-
surement (especially with roots) on different scales
from tissue to whole canopy, and of the separation
Crop Radiation Capture and Use Efficiency 601
between maintenance and growth respiration. It is
often considered that there is greater scope for the
improvement of maintenance respiration because
this is more sensitive to prevailing conditions. Recent
work suggests that the possibilities should be revisited
[39, 40].
At any given moment in time photosynthesis can be
limited by light level (e.g., under cloud cover or self-
shading), stomatal conductance (this becomes severely
limiting under conditions of low soil water), and
Rubisco (under saturating irradiance and high stoma-
tal conductance Rubisco limits photosynthetic rate in
C3 plants). Interactions between growth condition and
genotype will determine the final rate of photosynthe-
sis. Growth under optimal conditions is considered: the
imposition of resource limitation on RUE will be
discussed in the final section.
Taking into account the minimum essential losses
that occur, the percentage of “biomass energy” pro-
duced per unit solar radiation (total solar spectrum)
for C3 and C4 plants is generally agreed to be 4.6%
and 6%, respectively (calculated on a kJ/kJ basis) [36].
The highest recorded values are 2.4% and 3.7% [36]
with common measured values being much lower than
this, for example, [41]. The large difference between
measured and attained photosynthetic efficiency of
crop canopies is the cause of some debate and some
mechanisms focusing on metabolic constraints
are discussed here. It is possible that canopy architec-
ture remains a limitation (discussed above). It is how-
ever noted that much of the observed reduction in
RUE and photosynthetic efficiency in agricultural
systems is caused by growth under suboptimal
conditions [36].
The above has considered photosynthetic effi-
ciency in the context of a leaf canopy. However there
is current interest in exploiting photosynthesis in non-
leaf organs such as the green ear in wheat [42, 43] and
the spikelets in rice. Despite the relatively small surface
area (compared to LAI) it seems clear that the high
exposure to radiation and close proximity to the grain
sink may mean that their photosynthetic contribution
has been underestimated [42]. In the case of oil seed
rape, the photosynthesis in seed pods is the dominant
supply due to a diminished leaf area [44]. This is
a source of photosynthate that deserves closer
investigation.
602 Crop Radiation Capture and Use Efficiency

Metabolic and Regulatory Constraints to RUE the Calvin–Benson cycle have been shown to have
promise for improvement such as sedoheptulose-1,7-
Although photosynthesis underpins growth and yield it
bisphosphatase [55].
has often been considered that it is a feature of primary
Elimination or reduction in photorespiration has
metabolism which has undergone optimization
been of interest for a long time even though the seem-
through natural selection and empirical breeding.
ingly wasteful process has been assigned metabolic and
This view has partly arisen from the conservative
photoprotective roles. The increased growth rates, bio-
nature of the basic mechanism and composition of
mass, and yield of plants grown under high CO2 where
the photosynthetic apparatus. However, firstly, it is
photorespiratory flux is reduced indicates that the pro-
clear that canopy photosynthesis has undergone
cess is largely wasteful. Methods for blocking the path-
improvement via total leaf area and nitrogen per unit
way have proved ineffective [56] probably due to the
leaf area (fertilizer) and, secondly, there is considerable
accumulation of intermediates. A recent and novel
genetic variation in leaf photosynthetic capacity and in
approach has avoided this problem by using bacterial
the response of leaf photosynthesis to environmental
enzymes to “shortcut” the pathway [57]. In Arabidopsis
factors and abiotic stress. Nevertheless, the role of any
thaliana plants, this has had the effect of improving
improvement in leaf photosynthesis in crop yield pro-
biomass production and shows great promise although
gress has been hard to quantify due to the difficulty of
the precise mechanism of improved growth has not
routinely measuring net leaf and canopy carbon gain
been described yet.
and of eliminating compensating processes, although
The greatest improvements in yield of crops native to
some success has been achieved [24, 45, 46]. and pho-
warmer climates would come from the introduction of
tosynthesis has been successfully linked to yield pro-
the C4 mechanism into C3 crop species. In the case of
gress in some cases, for example, [24]. There is some
rice, for example, it has been calculated that this is the
recent evidence that leaf photosynthesis can exert an
only way to bring about an increase in biomass produc-
effect when biomass production is the dominant lim-
tion sufficient to meet a 50% improvement in yield by
iting factor [47–49]. Nevertheless leaf photosynthesis is
2050 [1, 58]. Early attempts to introduce elements of the
considered to be the dominant factor determining RUE
C4 pathway into rice by transformation with C4 genes
[5] and its improvement is increasingly viewed as an
from maize or other C4 species [59] are considered
important target [49, 50].
ambiguous or partially successful at best and introduction
Many of the suggested routes to the leaf CO2 assim-
of the full “Kranz” anatomy seems to be the most likely
ilation rate of crops have focused on Rubisco. Increasing
way to achieve the required goal. Some natural C4 mech-
the amount of Rubisco in the leaf is problematic: it is
anisms exist in a single cell [60] but it is unclear whether
already at extremely high levels and to accumulate more
this would provide sufficient rates of assimilation.
would require an increase in nitrogen fertilizer applica-
The evolution of the C4 syndrome independently
tion. There are indications that Rubisco may be accumu-
on more than 60 occasions in angiosperms would sug-
lated to excess capacity in some leaves [51]. There may
gest there is no intrinsic reason why the C4 pathway
be opportunities to improve the properties of the
could not be introduced into a major crop such as rice
Rubisco enzyme [52] and there is some natural varia-
or wheat. A combination of advanced molecular tech-
tion among plants and algae in the properties of
niques, transformation of key genes and smart screen-
Rubisco. For example, forms of Rubisco present in
ing of germplasm may achieve this [61]. Indeed there is
the genus Limonium have a higher specificity factor
now a funded international consortium of scientists
than that in all crop species [53]. However, there is
formed to address this task in rice (www.irri.org).
a well-cited inverse relationship between specificity
Recent modeling work suggests that photosynthesis
for CO2 and maximum catalytic activity [54]. It has
may not be optimized in many plant species. For exam-
been suggested that different forms of Rubisco could
ple, Zhu et al. [62] used an evolutionary algorithm to
be assigned different roles within the plant according
partition nitrogen between enzymes associated with
to environmental condition, tailored, for example, to
different processes within a plant cell. The combination
high light or low light conditions. Other enzymes in
of enzyme amounts and activities that produced

a higher photosynthetic rate was allowed to proceed to
the next generation, and after 1,500 generations it was
found that photosynthesis was substantially increased.
It seems that an over investment in photorespiratory
metabolism and an underinvestment in enzymes of the
Calvin–Benson cycle may be critical. It is possible that
this represents a lack of adaptation to contemporary
higher CO2 levels.
Photosynthesis is a dynamic process and constantly
responds to changing environmental conditions. It is still
debated as to whether the responses observed in situ are
optimized. For example, when light is absorbed in excess
of that required for photosynthesis, a series of regulatory
mechanisms are activated which dissipate the excess exci-
tation energy within the thylakoid membrane [63, 64].
This is considered a photoprotective process which
reduces the likelihood of photooxidative stress. This pro-
cess often has no impact on the light-saturated rate of
CO2 assimilation but it does reduce the quantum yield at
low irradiance levels. Therefore, following a transfer to
low light (caused, e.g., by cloud cover or leaf and solar
movement), the slow relaxation of photoprotection
causes a potential reduction in the rate of CO2
assimilation. Given the large variation in irradiance in
canopies in space and time, this has long been consid-
ered a factor in canopy photosynthesis. Indeed the
manipulation of photoprotection has been shown to
influence fitness in A. thaliana [65]. Recently,
photoprotection was modeled in a tall three-
dimensional canopy using ray tracing algorithms
[66]. The reduction in canopy carbon gain was
predicted to be as large as 30% under low temperature
conditions. This would seem to indicate that there is
room for improvement in terms of optimization of
photoprotection.
Acclimation of photosynthetic capacity to environ-
mental conditions such as irradiance can occur over
longer timescales such as days and weeks. It has been
hypothesized that acclimation conferred an advantage
in terms of carbon gain [49]. Recent experiments using
A. thaliana indicate that over long growth periods under
naturally variable light levels this is indeed the case [67].
The question of whether plant and crop responses
are appropriate for any switch in environmental con-
ditions has been expanded to include those of growth
and storage of carbon. For example, there is evidence
that transfer of carbon to storage organs can occur at
Crop Radiation Capture and Use Efficiency 603
the expense of allocation to new organs [68]. Cross
et al. [69] observed that accessions that allocated less
carbon to storage at night had a higher growth rate.
This concept has yet to be tested in crop species. Genes
that are involved in the regulation are being identified,
for example, DELLA proteins are negative growth reg-
ulators of central importance which are thought to
integrate the effects of various growth-promoting hor-
mones such as gibberellins and have been shown to
have an effect on rates of tissue growth [70].
Source–Sink Processes and Partitioning of
Assimilates
This section discusses the different assimilate sinks that
have either direct or indirect effects on light intercep-
tion (LI) and RUE in crops. As discussed, radiation
capture is a highly dynamic process affected not only
by sun angle and fluxes in radiation intensity, but also
by gross morphological above ground structures that
evolve over a plant’s life cycle and include leaf area
development, stem dynamics, and the emergence
of floral structures. Within crop species, there is con-
siderable interest in genetic effects on morpho-
physiological traits that affect light interception and
distribution. Genetic effects include early vigor; stem
density (m
2) and dynamics; leaf anatomy and geom-
etry; the composition, distribution, and duration of
light harvesting and photosynthetic proteins in the
canopy; the architecture of floral structures; and the
continual interaction of these with crop development.
Crop management is an additional factor which will
affect radiation interception principally through row
spacing, N fertilization, and irrigation, and by control-
ling biotic stresses that may reduce LI.
Direct Effects of Sinks on RI and RUE
It is axiomatic that all photosynthetic structures begin
as carbon sinks while at some point becoming net
exporters (i.e., assimilate sources). The exact invest-
ment strategy in these photosynthetic structures will
determine the RUE of the “photosynthetic canopy” as
a whole. In this context, leaf area index (L) is a useful
parameter to consider. Typically L values above 3 are
considered optimal for maximizing RUE in annual
crops. However, theoretical considerations suggest
604 Crop Radiation Capture and Use Efficiency
that the relative distribution of leaf area and light
harvesting and photosynthetic proteins among differ-
ent layers of the canopy can also modulate RUE and are
currently targets for genetic improvement [25].
In the context of canopy photosynthesis, two other
important sinks with direct impact on RUE are the
stems and the reproductive structures. Stems, as well
as being covered with a green leaf sheath, provide the
skeleton for leaf display and, therefore, to a large extent,
determine the height and geometry of the leaf canopy.
Stems are highly dynamic and may appear and disap-
pear within the main part of the crop cycle. In small
grain cereals, full light interception is typically acceler-
ated through tiller development, a strategy which
results in the subsequent shedding of nonproductive
tillers after optimum L is achieved; the investment of
assimilates in tillers apparently being offset by the
increased L [71]. The strategy also has benefit under
favorable years when a larger proportion of tillers
achieve reproductive success. Reproduction requires
a further investment of assimilates in floral structures,
which, though often photosynthetic themselves, may
also intercept incident light and, therefore, shade other
photosynthetic tissue. Reducing panicle height for this
reason has proved beneficial in rice [72]. Genetic mod-
ification of tiller dynamics and spike photosynthesis
are both promising areas in crop breeding; neither has
been systematically addressed yet as outlined above,
and both traits interact with LI and potentially with
RUE. There is considerable genetic diversity for tiller-
ing capacity in small grain cereals and a tiller inhibitor
gene (Tin) has also been identified in wheat [73]. Con-
siderable morphological diversity is also apparent in
the reproductive structures of many crop species. How-
ever, those which are photosynthetic show a complex
physiology, the measurement of LI and RUE is
extremely challenging, and studies to establish genetic
diversity are scant [43]. Nonetheless, shading studies in
wheat have suggested genetic diversity for the contri-
bution of spike photosynthesis to grain filling under
drought, and given that reproductive structures inter-
cept a significant proportion of light in many crops, it
may be expedient to incorporate them into models of
canopy photosynthesis.
Finally, non-photosynthetic sinks such as roots and
structural components of the plant (stem wall, rachis,
etc.) may impact on LI and RUE by competing with
photosynthetic tissue for assimilates, however, these
effects are not well documented [74].
Indirect Effects of Sinks on RI and RUE
There are other non-photosynthetic sinks which may
also compete directly with photosynthetic tissue for
assimilates but whose indirect effects may be much
more significant in terms of overall RUE of the crop.
These are (1) the accumulation and remobilization of
carbohydrates (such as water-soluble carbohydrates in
wheat and starch in rice) to and from stems and (2) the
partitioning of assimilates to the reproductive spike.
While it seems clear that stem storage carbohydrates
accumulate to provide a buffer against post-anthesis
stress conditions when current photo-assimilates
may be insufficient for grain filling [75–77], it is
unclear what trade-offs may be involved in terms of
assimilate partitioning. Given that genetic variation for
storage carbohydrates is large and may constitute up to
50% of the stem dry weight shortly after anthesis, it
could affect not only competition for assimilates from
other sinks (e.g., roots, spike) but also RUE if, for
example, the demand for stem storage carbohydrates
is great enough to solicit feedback responses of the
photosynthetic apparatus. While the latter has not
been studied, feedback effects that influence RUE
have been shown in response to partitioning of assim-
ilates to the reproductive spikes.
A large body of evidence has shown that the num-
ber of reproductive sinks that are set in a crop is the
main factor determining yield potential [78, 79].
Indeed the post anthesis sink size in wheat has been
associated with RUE [80]. Sink strength associated
with grain number is the most likely explanation for
the relationships demonstrated between yield and pho-
tosynthetic rate, for example, in a historic series of
wheat cultivars [24]. Furthermore, in other studies in
spring wheat it was shown using both genetic and phys-
iological treatments that RUE responded to increased
spike fertility, resulting in increased yield and above-
ground biomass [3]. It has been suggested that one
way to enhance the sink capacity in wheat is to lengthen
the stem elongation phase. The stem elongation phase
encompasses the spike growth period and this would
therefore result in a heavier spike during this period [3].
Another strategy would be to alter the sinks that

compete with spike index, such as roots, stems, leaf
sheaths, and infertile shoots. It must be ensured that
any reduction in leaf lamina does not have an effect on
LI and RUE. Reducing the allocation of biomass to
roots may improve RUE by permitting increased
partitioning to spikes (discussed in Reynolds et al.
[3]). Some caution is urged since future yields may be
dependent on increasing the ability to access soil water
and nutrients. However, there is a possibility that the
efficiency of uptake of water and nutrients could be
improved with no change in root mass, for example,
partitioning root length density at greater soil depths
[81]. Structural stem carbohydrates (not the reserve
carbohydrates discussed above) could be reduced by
classical methods such as reducing plant height
although wheat plant heights of below 70–90 cm are
associated with lower biomass [3, 82]. Such approaches
must be offset against increased lodging susceptibility.
Other possible targets such as infertile tillers and awns
are discussed in Reynolds et al. [3].
In particular, the maintenance of fertility under
unpredictable environments is highlighted: in wheat,
kernel set can be very sensitive to a number of environ-
mental conditions such as moisture stress and irradi-
ance. This is part of a set of evidence suggesting that
plant signaling is involved in reducing grain number.
The signaling (local or long distance within the plant)
is a well-established phenomenon: the transport of
molecules regulates growth, partitioning, and metabo-
lism and is a fundamental feature of plant biology,
likened in some cases to neural networks that sense,
quantify, and memorize the environment around
them [49, 68, 83]. The most well-known example is
that drying soils induce the synthesis of abscisic acid in
roots, and transport of this hormone to shoots induces
a reduction in stomatal aperture to increase water use
efficiency of remaining soil water [84]. It is possible
that plant responses to environmental events are pre-
emptive certainly but may also be simply too “conser-
vative” predicting unfavorable conditions and setting
seed size accordingly, while a higher yield can be
attained by maintaining larger seed number under
well-managed conditions. Floral abortion in maize
in response to drought appears to be controlled by
the up- or downregulation of a few enzymes [85]. In
wheat, day length can alter sugar supply to fertile florets
leading to cell death [86], and ethylene is a high-
Crop Radiation Capture and Use Efficiency 605
temperature signal leading to kernel abortion. Cytoki-
nin appears to regulate the number of spikelets in rice
[87]. Therefore, better targeted regulation of grain
abortion before the onset of seed filling is a potential
target for improvement of RUE and yield potential
(Fig. 4).
Theoretical Considerations Related to the
Improvement of RUE
The concept of RUE has received some criticism as
being one that contains circular reasoning: one cannot
have growth without biomass accumulation and vice
versa. Additionally it is the product of almost every
process in plant canopy growth and development,
which makes a mechanistic description quite complex.
However, it is established as a unifying concept in crop
physiology, for example, [5, 10, 88]. Moreover the
understanding of the component parts of the system
and their integration is continually improving, and
increasingly these parts are not viewed as a “black
box,” giving more options in the future with regard to
RUE improvement.
For a given crop genotype, RUE is largely sensitive
to leaf photosynthesis (and therefore nitrogen content)
and the proportion of radiation that is diffuse or direct
[89]. The latter is a much under-studied area in plant
and crop physiology. The stability of RUE has been
used to question whether it is possible to improve
upon crops with existing C3 or C4 types.
Theoretical figures were outlined above for the
maximum efficiency with which photosynthesis can
operate and it was concluded that crops operate
below this. However, what is the realistic “spare capac-
ity” for RUE improvement in a major crop plant?
Reynolds et al. [23] estimate potential productivity
for the entire growth cycle of irrigated wheat in
a specific and well-characterized location, NW Mexico.
Radiation fluxes were measured throughout the season.
Due to the time it takes for canopy establishment, large
losses can occur before canopy closure, and this was
taken into account using a model that used measured
intercepted radiation and the time before canopy clo-
sure. A value of 1,748 MJ m
2 was obtained for radia-
tion absorbed by photosynthetically active tissue.
Estimates of field quantum requirement can vary
between 10 and 30 mol quanta mol
1 CO2, taking
606 Crop Radiation Capture and Use Efficiency
Phenology:
Time to maximum light interception
and changes in canopy architecture
Development and sink strength of tissues
Time to full canopy closure
Development and strength of sink tissue
Canopy architecture:
Full exploitation of erect habit
Optimization of leaf posture and N
distribution/duration
Partitioning:
Post anthesis sink strength, strength
of temporary vegetative sinks
Optimal partitioning between root,
shoot and other sink organs according
to environmental need
Crop Radiation Capture and Use Efficiency. Figure 4
Summary of the potential target areas which would result in an improvement of crop radiation use efficiency
into account photorespiration and photochemical
inefficiency. For a wheat crop, best estimates would
seem to be in the range 15–24 mol quanta mol
1 CO2
[90] which results in a range of RUE between 1.5 and
2.6 g carbohydrate MJ
1. The calculated value of bio-
mass ranges from 2,620 to 4,545 g m
2 whilst the
measured value for wheat in this environment is up
to 2,100 g m
2. This suggests that improvements in
field RUE are conceivable.
Sinclair and Horie [88] claimed that the observed
stability of RUE arose from consistently high light-
saturated leaf photosynthetic capacity (Amax). They
plotted the leaf photosynthetic capacity against RUE
for C3, C4, and leguminous crops types. The saturation
of this response indicated that further modest increase
in leaf photosynthetic capacity will only have a limited
impact on the RUE, hence the stability of the RUE
response [5]. However under conditions of restricted
nitrogen, water, or under stress much lower values of
photosynthetic capacity caused a larger reduction in
RUE. This has been used as an argument that seeking
Non-leaf photosynthesis:
Ear, panicle, stem, culm, leaf sheath
Metabolic efficiency:
Optimisation of maintenance
respiration, optimal partitioning
between component leaf processes
e.g. Calvin cycle, photorespiration,
CH2O synthesis, partitioning in time:
growth vs storage
Leaf photosynthesis:
Photorespiratory flux
Rubisco engineering
C4 engineering
Dynamic responses of
photosynthesis, photoprotection
Efficiency of light-limited tissue:
Efficiency of light harvesting
process, allocation of resources.
Efficiency of use of the ‘sunfleck’
resource
modest improvements in leaf photosynthetic capacity
may not be worthwhile. As pointed out here it is pos-
sible to trace improvements in biomass production that
are linked to Amax. Biomass, yield, and Amax were
associated in irrigated wheat cultivars in warm condi-
tions [23] and in temperate conditions [24]. In the
latter case, higher Amax may reflect a feedback
response caused by greater partitioning to reproductive
structures – the differences in Amax were greatest dur-
ing grain filling and not well associated with above-
ground biomass.
There has been a lot of attention paid to the use of
Amax in the selection of varieties with a higher photo-
synthetic potential, largely because it is convenient to
measure and shows genetic variability. However, it
should be clear from this entry that Amax will mostly
be expressed at the top of the canopy (most leaves will
not be at light saturation) and may not be a good proxy
for canopy photosynthetic rate especially when inte-
grated over long time periods. Care should be taken
when extrapolating from spot measurements to whole

canopy photosynthesis. It is not surprising then that
attempts to improve yield by simply selecting for Amax
have had largely unsuccessful results.
Perhaps more importantly, many of the improve-
ments that show great promise are not solely associated
with capacity at light saturation but rather to the dynamic
responses of photosynthesis over time and therefore to
total canopy carbon gain. These processes are much
harder to measure experimentally although advances in
techniques such as continual chlorophyll fluorescence
monitoring may provide breakthroughs. As the knowl-
edge of photosynthetic regulation improves, these should
be installed into agricultural photosynthesis. It would
seem necessary to tailor photosynthetic responses to
improvements in canopy architecture.
Sources of Variation in Agricultural RUE
In any comparative analysis of RUE, it is necessary to
ensure that the methods of analysis are directly com-
parable and provide an accurate estimate that do not
contain any of the potential errors in measurement
outlined above. Additionally, consideration must be
taken when comparing different environments because
varieties may not be well adapted to their locality and
therefore able to “express” their maximum RUE. Other
reviews, for example, Sinclair and Muchow [5] provide
a comprehensive survey of the literature for many key
crop species. A summary is provided here of the species
and of the environmental factors affecting RUE in
agriculture, and available information for a few key
species has been provided.
It is in fact not common to find studies that specif-
ically link RUE with yield progress; however, it has been
shown, for example, that yield progress in wheat asso-
ciates with both harvest index and total biomass pro-
duction [74]. Some evidence also suggests that older
wheat cultivars had a lower RUE and this was associ-
ated with a lower post-anthesis sink capacity [4, 80].
Species Variation in RUE
As pointed out by many workers, there is no “constant”
for RUE and there is considerable variation between
species. It is important to pay attention to the develop-
mental stage of each crop in question and maximum
attained RUE is referred to here. The highest recorded
Crop Radiation Capture and Use Efficiency 607
values for season-long RUE across C4 species are
3.4 g MJ
1 and those of C3 species 2.8 g MJ
1 [5, 35].
Mitchell et al. [13] reported that average values for RUE
in rice, wheat, maize, and soybean in the vegetative
stage were 2.2, 2.7, 3.3, and 1.9 g MJ
1, respectively.
Therefore the tendency for a distinction between C3
and C4 crops seems to be consistent. However, care
must be taken. For example, sorghum, a C4 species,
possesses a relatively low RUE and this has been attrib-
uted to low leaf N content. Conversely, sugarcane, also
a C4 species, seems to have an exceptionally high RUE.
Potato, a C3 species, has shown values for RUE higher
than all other C3 crops and even some C4 species
(sorghum). It must once again be emphasized that
the energy content of dry matter should be taken into
account. It has been suggested that the high values for
sugarcane and potato reported are due to the excep-
tionally low energy content of the products (sucrose
and starch). It is often claimed that sugarcane has the
highest radiation use efficiency among the plant king-
dom, although there are relatively few studies.
A few studies have indicated the relatively low RUE
of rice among C3 crops [13, 91], although there are
surprisingly few studies available for this species and
recent suggestions that RUE may not be currently
closely linked to high yield potential [92].
Leguminous species such as soya bean have lower
RUEs and a high percentage of PAR utilization. This is
due to the higher lipid and protein content (see earlier
section). However, even when the vegetative stage alone
is considered, soybean has shown lower RUE values
than wheat, rice, and maize [13, 93]. This has also
been observed in other grain legumes [5]. RUE of
legumes may be an important future target. For
detailed information on RUE values for each species,
the reader is referred to detailed reviews such as [5].
In recent years, there has been much interest in
“energy crops.” These are crops that are grown for the
sole purpose of fuel production or combustion for
energy generation. This has been stimulated by concerns
over emissions of greenhouse gasses and the growth of
crops represents a CO2-neutral strategy. Drawbacks
include the clear competition with food supply and
the possible threat to natural vegetation. Nevertheless
it has been shown that certain species have the potential
to be extremely productive in this regard, especially C4
grasses such as Miscanthus [94]. Miscanthus has the
608 Crop Radiation Capture and Use Efficiency
highest annual primary production of any crop species
producing 50% more biomass than corn due to a high
leaf area and longer duration, although the energy
conversion rate is about the same [95]. Other examples
of energy crops under study are willow, poplar, and oil
crops such as Jatropha, although radiation use effi-
ciency of this category of crop species is not well stud-
ied. These crops may find an application on land
unsuitable for food crops and low input environments.
Developmental Stage
The stage of crop development has a clear effect on the
RUE attained. Firstly the photosynthetic potential may
be dependent on growth stage. Secondly the appear-
ance and disappearance of vegetative and reproductive
sinks can influence photosynthesis via the presence of
feedback signaling mechanisms and carbohydrate
accumulation; this has been discussed in detail above.
In many species, a lower RUE was observed during
earlier crop establishment stages in comparison with
later stages and this was attributed to a lowered photo-
synthetic capacity [5]. The effect of senescence during
the post flowering has the effect of reducing photosyn-
thesis, and therefore RUE is usually observed to be
higher during the vegetative stages than post flowering.
Recent studies have indicated that post-anthesis RUE
in wheat is strongly linked to sink size: Older cultivars
with smaller sinks had significantly reduced RUE dur-
ing this phase [4].
There is an indication in some studies that in rice
this post-anthesis effect is not as pronounced [13]. In
a recent study, Takai et al. [96] suggested that
a maintenance of high growth rate and RUE in the
late reproductive phase of rice is a key to higher grain
yield potential in this species and may be linked to the
short tropical life cycle and requirement for rapid pho-
tosynthesis during grain filling.
Environmental Factors
There are many practical difficulties involved with
linking leaf-level photosynthesis with canopy-level
RUE, even though it is technically straightforward to
measure leaf photosynthesis. However, increasing the
photosynthetic rate per unit leaf area of leaves has
direct relationship with increasing RUE. It is frequently
observed that the response between light-saturated
photosynthetic capacity and RUE is curvilinear rather
than linear. However, as discussed above it is critical to
consider photosynthetic efficiency at all light levels. As
a generalization it is fair to say that any factor, biotic or
abiotic, that reduces the photosynthetic potential of
a canopy is also likely to reduce the RUE. This applies
to many of the common factors that reduce growing
conditions below optimum such as water availability,
nutrients, and extreme temperature.
It is not surprising that nitrogen has a significant
impact upon RUE due to the close and well-established
relationship with leaf photosynthesis. There is a strong
nitrogen-dependent effect on leaf area and consequently
light capture in plants, therefore, nitrogen content per
unit leaf mass or per unit leaf area is considered. This has
been the subject of a number of studies in several spe-
cies: Usually a curvilinear response with leaf nitrogen is
obtained, with RUE increasing up to a saturating value
beyond which response is limited such as sunflower
(average canopy leaf nitrogen [97]), soybean (specific
leaf nitrogen [98]), and maize and sorghum (leaf nitro-
gen per unit leaf area [99]). However, this relationship
is not always observed (see [5]). In examining species
differences, the plant matter energy content and the
nitrogen–photosynthesis relationship is important.
For example, C4 plants have a higher potential photo-
synthetic nitrogen use efficiency [88]. Naturally site-
variation in soil nitrogen is common and potentially
a large source of variation in RUE.
Soil water and atmospheric humidity have the
potential to reduce photosynthetic rate and therefore
RUE. In experiments that imposed soil water deficit,
reductions in RUE have been observed in some cases
[100] but not others [100]. It has been suggested
that the variability in response is a result of variation
in the extent of the soil water deficit: When this was
measured in a quantitative manner, it could be
established that RUE would decline when the level
of extractable soil water declined below a certain limit
(in this case 30%) [101].
An increase in vapor pressure deficit has been
associated with lower RUE in some studies. It is noted
that the impact in RUE was greater than that would
have been predicted from leaf photosynthesis alone
and it was suggested that environmental factors
associated with vapor pressure deficit are involved.

Other studies indicated only a small impact of vapor
pressure deficit on RUE [5, 102].
Temperature has been associated with RUE when it
has an influence on leaf CO2 assimilation rate and this
has also been shown to be related to the physiological
effects of nighttime temperature [5].
Future Directions
Due to the effects of climate change caused by anthro-
pogenic emissions, an altered environment for crop
growth is likely to be required [103]. Some of these
changes can be predicted with a high probability, for
example, CO2 is rising and is likely to continue at
a similar rate to the current one with a slowing of rate
according to internationally agreed emission cuts.
Since 1750, atmospheric CO2 has risen by around
100 ppm and continues to rise at the rate of around
2 ppm per year. By the end of the twenty-first century,
temperature is predicted to rise by between 0.5 C and
4 C in response to levels of the greenhouse gasses CO2,
methane (CH4), and nitrous oxide (N2O). Agriculture
itself is a major contributor to greenhouses gasses.
Studies have already indicated that climate change
is having an effect on productivity of crop systems
[103–105].
These changes must be considered according to
geographical region and in the context of deleterious
effects such as a likely reduction in water availability,
a rise in pollutants such as ozone, and the increasing
frequency of extreme damaging events such as
flooding, drought, and storms. They must also be
weighed against possible beneficial effects such as
higher photosynthetic rates induced by elevated CO2
and a longer growing season at higher latitudes leading
to higher biomass and higher yield. Accordingly,
a reduction in yield in the tropics and a rise in temper-
ate regions may be experienced. The effect of most of
these factors on RUE has been discussed above and
a detailed discussion of the wider impact of climate
change on agriculture is beyond the scope of this arti-
cle. Clearly suboptimal growing conditions will poten-
tially reduce RUE and yield. However, it is important to
ensure that the crops are adapted appropriately to
future climates.
Experiments using completely enclosed or open
topped chambers in the field in which CO2 levels are
Crop Radiation Capture and Use Efficiency 609
elevated above ambient have shown significant
increases in growth and yield. The reasons are clear
and related to the suppression of photorespiration
and consequently an increase in RUE. Additional ben-
eficial effects related to a lowered stomatal conductance
[25, 106]. These data have been used in models that
predict an increase in future crop production in many
northern regions. Experiments with more realistic field
conditions (Free Air CO2 Enrichment, FACE) have also
demonstrated an increase crop yields but these were
lower than those predicted by chamber experiments.
This is lowered further when additional effects such as
ozone and disease are accounted for and the net result
may be negative [107, 108]. This suggests that the
predicted beneficial effects of an increased atmospheric
CO2 on plant growth have been overestimated. How-
ever, as pointed out by Long et al. [107] there is still
a paucity of data for CO2 enrichment effects in many
agroecosystems so a level of uncertainty remains.
There is also a need for further work into the adap-
tation of crops to high CO2 levels: Theoretical work by
Zhu et al. [62] using evolutionary algorithms to predict
optimal levels of CO2 for photosynthesis have shown
that the relative levels of enzymes involved in primary
metabolism are not optimized for carbon assimilation.
It is possible that there has been insufficient adaptation
during crop improvement since the start of the indus-
trial revolution, raising the intriguing possibility that
plants should be bred or engineered for adaptation to
higher CO2 levels of the future in order to maximize
yield and RUE. Presumably this also applies to other
environmental changes.
Therefore it is clear that there is a demand for greater
crop yield per unit area of land with fewer resources
available. This step change in yield will arise from an
integrated set of targets, most of which have been
outlined in this article. Assuming that management of
the crop is optimal and improvement of partitioning and
sink capacity reach their limits, it will be increasingly
necessary to underpin any substantial yield improve-
ments with increased biomass production and this
means that RUE will need to increase.
RUE represents a complex system and as pointed
out by Reynolds et al. [23], it is a less frequent outcome
of the genetic recombination events required for breed-
ing, in comparison to manipulation of sink size. It will
be necessary to focus on methods for improving RUE
610 Crop Radiation Capture and Use Efficiency
of crops and to introduce this into breeding techniques.
In addition, there have been significant advances in the
plant molecular and genomic sciences in the past
2 decades and these should be exploited to raise RUE.
The discovery of new genes and combinations of genes
requires routine screening of large amounts of leaf
material for photosynthetic potential. It is difficult to
do this manually using traditional gas exchange tech-
niques; however, there are a number of techniques
developing rapidly that could be exploited to act as
surrogates or proxies of CO2 assimilation rate. For
example, chlorophyll fluorescence is a rapid method
that can be used to image leaf material for photosyn-
thetic efficiency extremely rapidly. It is routine to image
chlorophyll fluorescence; however, it is technically dif-
ficult to do this on a three-dimensional structure such
as a canopy. Another method is canopy temperature
depression which measures the difference between leaf
and air temperature caused by evaporation. It therefore
provides an indication of stomatal conductance and
has been correlated with plant performance [109].
Considerable recent interest has been placed on the
imaging plant growth leading to the emergence of the
field of “plant phenomics.” Advances in methods for
rapid and high-resolution screening of plant material
for photosynthetic performance can be anticipated.
Additionally, the complexity of RUE means that
a mathematical understanding of the integration and
functioning of component processes will be critical.
Given the difficulty of direct measurement, modeling
of canopy photosynthesis is already used routinely and
will be needed for the continued testing of new traits.
The Role in Wheat and Rice Research: Current
Research at CIMMYT and IRRI
The challenge of improving photosynthesis and there-
fore RUE in crops during both optimal and suboptimal
conditions has been recognized by the research organi-
zations that were central to the last great agricultural
revolution (the green revolution), the International
Rice Research Institute (IRRI) in the Philippines and
the International Maize and Wheat Improvement Cen-
tre (CIMMYT) in Mexico. IRRI has a number of
research programs that are centered toward the sus-
tainable improvement of rice yield in changing envi-
ronments. Perhaps the most relevant here is the C4 rice
consortium whose goal is a step change in rice photo-
synthesis through the introduction of the C4 pathway
of photosynthesis (http://beta.irri.org/projects15/en/
c4rice). The impact this would have on yield and
resource use efficiency of the poorer rice growing
regions of the world should not be underestimated.
Given the urgency of the current food crisis coupled
with the likely negative impact of climate change on
productivity, especially in less developed countries
[110], both IRRI and CIMMYT have strategic initia-
tives to raise the yield potential of rice and wheat,
respectively, by around 50%. The IRRI C4 rice initia-
tive is already underway as mentioned. CIMMYT is
currently facilitating a Wheat Yield Consortium
(WYC) among a group of leading scientists with the
view to raising the genetic yield threshold of wheat.
A central issue is that the fundamental bottleneck to
raising productivity, namely, photosynthetic capacity,
has hardly changed since wheat breeding started.
Nonetheless, basic research in photosynthesis suggests
that substantial improvements in yield are theoreti-
cally possible. While increasing photosynthetic poten-
tial will require considerable research focused at
cellular and subcellular processes (such as the genetic
modification of Rubisco and its regulation), interven-
tion at this level must go hand in hand with modifica-
tion of structural and reproductive aspects of growth,
since these will determine the net agronomic benefit of
increased RUE .For example, even at current levels of
yield potential, a significant portion of yield is lost to
lodging damage each year so improved structural integ-
rity will be prerequisite to realizing genetic gains in
RUE and biomass. The other major factor determining
yield potential is adaptation of the reproductive
processes which affect harvest index [3, 111] and
whose physiological and genetic basis is relatively
poorly understood [112]. The aim of the WYC is to
develop or identify sources and genes for the combina-
tion of traits necessary to realize improved expression
of wheat yield potential and combine them through
strategic crossing using the most expedient combina-
tion of conventional, physiological, and molecular
breeding approaches; the consortium approach is
expected to realize impacts in farmers fields
in a shorter time frame than if bottlenecks to yield
potential are identified successively and investigated
in relative isolation.

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 Crop Responses to Available Soil Water 615

Crop Responses to Available Soil Definition of the Subject

Water
MANUELA M. CHAVES1,2, O. ZARROUK1
1 tury scientific, social, and political communities, in
Plant Molecular Ecophysiology Laboratory (LEM),
Instituto de Tecnologia Quı́mica e Biológica,
Universidade Nova de Lisboa, Oeiras, Portugal
2 change could lead to a global crisis if efforts from
Instituto Superior de Agronomia, Universidade
Técnica de Lisboa, Lisbon, Portugal
Article Outline
Glossary
Definition of the Subject
Introduction
Revisiting Plant Strategies to Cope with Water Scarcity:
The Crop Perspective
Photoassimilates, Water Use, and Crop Yield
How Metabolic Processes Respond to Drought:
Photosynthesis, Respiration, Photorespiration
Yield and Quality Under Water Scarcity
The Breeding Achievements
Future Directions
Bibliography
Glossary
Dehydration avoidance Dehydration avoidance is the
strategy of the plants that are able to maintain tissue
water potential as long (and as high) as possible
under drought conditions.
Dehydration tolerance Dehydration tolerance is the
strategy of the plants that are able to cope with
severe tissue dehydration.
Harvest index Harvest index is the biomass of the
harvested product expressed as a percentage of the
total crop biomass.
Photoassimilates Photoassimilates is the energy-
storing carbohydrates produced by photosynthesis
in the green tissues of the plants.
Water-use efficiency (WUE) Water-use efficiency
(WUE) is the carbon gain (or biomass formed)
per unit of water transpired or the ratio between
photosynthesis (A) and stomatal conductance (gs),
termed as intrinsic WUE.
Sustainable intensification of global agriculture is
a major purpose (and challenge) for twenty-first cen-
order to guarantee food security, while preserving nat-
ural resources. Fast growing population and climate
different disciplines and countries are not congregated.
Among limiting factors is water scarcity, which may
dramatically decrease crop production worldwide.
Mitigation measures are therefore a major goal for
sustaining crop production and they are based either on
management practices that will enable water savings or
on breeding efforts for more adequate crops. Improved
physiological and molecular knowledge on plant’s
response to water deficits is essential to get improve-
ments in crop yield under adverse environments.
Because of the complexity of these responses, it is
imperative to integrate disciplines as functional geno-
mics, transcriptomics, proteomics, and metabolomics
with plant physiology to improve breeding strategies.
According to the present knowledge, the key factors
responsible for high yield under drought in annual
plants include an appropriate phenology that will
enable escaping drought and getting the timing of
flowering right, as well as high water-use efficiency
(WUE) and harvest index. On the other hand, the
basic knowledge for fruit tree crop breeding under
water scarcity is much more fragmented than for
annuals because of the highest complexity of fruit
trees. Therefore more efforts are needed in this area of
research.
Introduction
Scarcity of water resources is an increasingly important
issue since it will dictate global production of food and
feed for the next generations, as dramatically described
by The Economist (May 2010): “Water is the new oil:
a resource long squandered; now growing expensive
and soon to be overwhelmed by insatiable demand.
Aquifers are falling, glaciers vanishing, reservoirs dry-
ing up and rivers no longer flowing to the sea. Climate
change threatens to make the problems worse. Every-
one must use less water if famine, pestilence and mass
migration are not to sweep the globe.”

P. Christou et al. (eds.), Sustainable Food Production, DOI 10.1007/978-1-4614-5797-8,

# Springer Science+Business Media New York 2013

Originally published in
Robert A. Meyers (ed.) Encyclopedia of Sustainability Science and Technology, # 2012, DOI 10.1007/978-1-4419-0851-3
616 Crop Responses to Available Soil Water
The International Panel for Climate Change [1]
predicts that water scarcity, together with incidence of
high temperature, will increase in the near future in
many regions of the globe with dramatic effects on
agriculture. Of the world’s water resources, c. 80% is
currently consumed by irrigated agriculture. In this
context, the investment in research for improving
water use by the crops (“more crop per drop” as in
the CGR program Challenge Program on Food and
Water) [2], either through improved management
technologies [3, 4] or more efficient genotypes [5, 6],
is crucial. Increased water use by the crop can be
obtained by agronomic practices that decrease water
losses by soil evaporation, runoff, through-flow, deep
drainage, and competing weeds, thereby making more
water available for the crops, and in irrigated agricul-
ture by using deficit irrigation practices.
Crop resistance to drought has been the subject of
extensive research in the last decades, ranging from the
physiological traits underlying resistance to water def-
icits [7–12] to breeding efforts [13, 14] including clas-
sical and genetic engineering approaches [15–17]. The
world’s most important crops for human and animal
consumption in terms of total production – wheat,
maize, rice, soybean, barley, and sorghum – have
endured large yield increases during the twentieth cen-
tury [18–22] and recently became the target of investi-
gation aiming at improved performance under harsh
environments, namely water scarcity [5, 6, 15, 23]. To
articulate the knowledge obtained in different scientific
disciplines (agronomy, breeding, physiology, and
molecular biology) still remains a challenge, with the
ultimate goal of providing farmers with better
performing crops under water scarcity [7, 14]. Design-
ing robust field trials is also essential in order to test
improved crops under the multi-stress conditions they
generally face under natural conditions. Indeed, there
have been several reports of apparently promising bio-
technological manipulations that have failed to deliver
drought-tolerant crops in yield terms, when the novel
material is transferred from the growth room to the
field [4].
The key factors responsible for high yield under
drought in annual plants are an appropriate phenology
that will enable escaping drought and getting the
timing of flowering right, high water-use efficiency
(WUE) and a high harvest index [14, 24]. To get high
WUE would involve the improvement in crop transpi-
ration efficiency but the increased partitioning of bio-
mass into the harvested product will also lead by itself
to higher WUE. The relative importance of each of
these processes will depend on how water is available
during the crop cycle [13]. Indeed, it is noticeable that
under most dryland situations where seasonal rainfall
is unpredictable, maximizing soil moisture use is
a crucial component of drought resistance (avoidance),
which is generally expressed in lower WUE [25].
It is also important to recognize that many of
the traits that explain plant adaptation to drought
(e.g., phenology, the size and depth of the root sys-
tem, xylem properties or the storage of reserves) are
mostly constitutive [21, 26] and therefore can be
found within the species population when it exhibits
a large intraspecific variability. Moreover, there is
the general recognition that the efficacy of high
throughput screening of plant genetic material will
speed-up crop improvement via breeding programs
(see, e.g., [19]).
In fruit crop production, improvement under con-
ditions of water scarcity has been achieved mostly by
optimized management technologies such as deficit
irrigation [3, 27], with breeding efforts being much
more modest. Indeed the basic knowledge for tree
crop breeding under water scarcity is much more
fragmented than for annuals because of the highest
complexity of fruit trees. In tree crops economic return
is highly dependent on fruit quality, generally not
related with total plant biomass produced and there-
fore water availability [3]. Moreover, yield-determining
processes in fruit trees may not be sensitive to water
deprivation at some developmental stages [28] and
fruit quality may even beneficiate from a mild to mod-
erate water deficits, as is well documented in grapevines
[29]. High-density fruit orchards use composite
plants – the scion cultivar grafted into a rootstock.
The rootstock can alter the behavior of the scion,
including its vegetative growth, flower numbers and
flowering time, crop yield [30], and also drought
tolerance [31]. The mechanisms underlying the regu-
lation of scion growth and development by the root-
stock are not fully understood [32].
Research in the so-called new climate proof crops
such as Chenopodium quinoa Willd (quinoa) and
Amaranthus spp is also developing. Quinoa is an

Andean seed crop, very nutritious [33], showing high
tolerance to drought, frost, salinity, and biotic factors
[34–36]. Quinoa has been selected as one of the crops
to secure food production in the twenty-first century
[37]. The protein quantity and the quality of quinoa
seed is superior to those of cereals, because of the high
content of lysine, methionine, and threonine, in addi-
tion to a range of vitamins (B2) and minerals (iron,
calcium). Although amaranth is less tolerant to a range
of stresses as compared to quinoa, it tolerates higher
temperatures because it is a C4 plant [38]. The protein
content of amaranth grain ranges from 12% to 17%,
with a high level of lysine, similar to quinoa [39].
In this review the focus is on the key factors respon-
sible for sustained plant growth and production
under water scarcity, referring to both annual and
perennial (fruit) crops. During evolution, plants devel-
oped different strategies to successfully cope with water
stress; they comprised either acclimation to a slowly
developing water deficits or the response to a sudden
drought. These issues will be discussed in the context
of the agriculture needs and related to production
(fruit) quality, a major target of modern agriculture
nowadays.
Revisiting Plant Strategies to Cope with Water
Scarcity: The Crop Perspective
Plants respond and adapt to stress at whole plant, root,
reproductive structures, and leaves, by using mecha-
nisms that are being unraveled at the cellular and
molecular levels [7, 40].
Classically, plant resistance to drought has been
divided into escape, avoidance, and tolerance strategies
[7, 41, 42]. However, in practice, most plants combine
a range of these different strategies [43].
Plants that escape drought are able to adjust their
phenology to the environment, being able to complete
their life cycle before drought occurs. Escape strategies
rely on successful flowering and therefore reproduction
before the onset of severe stress. Plant ability to store
reserves in stems and roots and to remobilize them for
grain filling under water scarcity is extremely relevant
for grain crops under water scarcity [44, 45], as it has
been described in cereals such as wheat, maize, and
rice [46–48], and legumes [49]. When stem reserve
storage/utilization is insufficient to support fruit
Crop Responses to Available Soil Water 617
growth under stress, delayed-senescence genotypes
may be desirable in crops where yield is source-limited
[50]. High nitrogen availability can further increase the
reuse of stored carbohydrates in cereals under moder-
ate soil drying, leading to increased grain yield; under
non-limiting water conditions, however, abundant
nitrogen was shown to reduce grain yield in stay green
genotypes [47].
Dehydration avoidance, which is common to both
annuals and perennials, implies the maintenance of
tissue water potential as long (and as high) as possible
under drought conditions. This can be achieved either
by (1) minimizing water loss or by (2) maximizing
water uptake. Decreased water loss may derive from
stomata closure and/or reduction of absorbed light,
including leaf rolling [51], increased reflectance (with,
e.g., trichomes), or steep leaf angles [52]. Shedding of
older leaves is also relevant to reduce water loss, while
allowing the reallocation of nutrients to younger leaves,
when water deficit is relieved. Maximizing water uptake
may be accomplished by increased investment in the
roots at the expense of shoot growth (Fig. 1), which is
generally inhibited very early on in response to decreas-
ing water availability [8, 45]. Deep roots have been
reported as important drought resistance traits for
both annuals and perennials in semi-arid regions
[26]. However, in arid environments, where rainfall is
sporadic and of short duration, plants may take advan-
tage of shallow roots that proliferate quickly near the
soil surface allowing water uptake by the plant before it
evaporates [53]. For example, Zoysia japonica was
reported as having a relatively shallow root system,
moderate WUE, while exhibiting a high capacity for
osmotic adjustment [53]. High root plasticity is a key
factor in this strategy because it will determine how fast
roots grow in response to sporadic rain, following
a period of drought stress.
Under slowly developing water deficits some plants
show osmotic adjustment (OA), a cellular stress adap-
tive response that may improve tissue resistance to
desiccation, by maintaining cell turgor, likely
supporting crop yield under stress [25]. Because this
mechanism allows plant water uptake at lower soil
water potentials it is considered a dehydration avoid-
ance strategy. The alterations observed under OA com-
prise increases in soluble sugars (like fructans and
sucrose) [54], amino acids (e.g., proline, aspartic acid,
618 Crop Responses to Available Soil Water

Fruit trees Annual crops

SHOOT and BUD

-Reduced shoot growth
-Flower bud inititiation altered POLLEN
-Flower bud development reduced -Decrease pollen viability/pollen sterility
-Gibereline increase? -Early pollen maturity
-Gene response
FLOWER -Decrease acid invertase activity
-Ovary defects -Programmed cell death acceleration
-Aborted pistil
-Decreased germination potential
-Flower abscission SEED
-early maturity
-Reduced seed filling /small size
FRUIT -Seed abortion
-Early maturity + delay ripening -Reduced seed quality
-Increase fruit drop + reduced size
-Reduced quality OVARY
-Metabolic activity disturbed -Ovary abortion
-Decrease invertase activity
-Sucrose transport inhibition
-Starch depletion
LEAF -ABA increase?
-Root signal recognition -Gene response
-Stomatal closure -Senescence
-Reduced transpiration ABA signal
-Decrease C assimilation transport
-Gene response -Basal leaf abscission
-Reduce leaf expansion Reduced
-Metabolic activity disturbed carbohydrate
-Morphological changes reserve

-Increase absorption area

-Inhibition lateral roots -Increase root/shoot
-Increase root depth

Drought Stress
ROOTS
-ABA synthesis, ROS
-Root conductivity
-Osmotic adjustment

Crop Responses to Available Soil Water. Figure 1

Whole plant response to drought stress in fruit trees (left) and in annuals crops (right). Green boxes and letters correspond
to shared responses between the species

and glutamic acid), methylated quartenary ammonium
compounds (e.g., glycine betaine and alanine betaine),
and some proteins, such as dehydrins [55] and cyclitols
(e.g., D-pinitol, mannitol) ([56]; see also review [7]). In
addition to the role played via the decrease in osmotic
potential, these solutes may protect cell membrane and
metabolic machinery under dehydration. According to
Bohnert et al. [57] sequestration of H2O molecules,
reducing the solvent-protein interaction, may explain
stabilization of protein complexes and membranes.
The positive role of osmotic adjustment on yield
has been subject to much discussion since benefits were
often not observed (see review by [58, 59]). A possible
explanation is that turgor maintenance in cells is often
associated with slow growth. It is also likely that
osmoprotection mechanisms are not functional until
severe dehydration occurs, with the implication that
OA may be critical to survival rather than to promote
plant growth and crop yield under drought [7].
Mechanisms of protection against oxidative stress
are also fundamental to cope with drought and co-
occurring stresses under arid semi-arid environments,
as it will be discussed below.
Photoassimilates, Water Use, and Crop Yield
As proposed by Monteith [60], yield potential (Yp) of
a crop at a given location can be defined as: Yp=HI.Pn
with Pn=S(PAR ABSc ec) and PAR being the incident
solar radiation in the specific location, ABSc the frac-
tion of the radiation intercepted by the crop, ec the
efficiency of the conversion of intercepted radiation
into biomass and Pn the primary productivity (total
biomass produced over the growing season) and HI the
harvest index (see also [61]). Under water scarcity
a decrease in Yp may occur as a result of less intercepted
radiation (due to lower total leaf area and smaller leaf
angles) and a lower ec, as a result of a decrease in
photosynthesis.
On the other hand, as judiciously proposed by
Passioura ([62]; see also [14, 22, 63]), crop yield
under water-limited conditions can be estimated by
the product of transpiration efficiency (biomass/water
transpired)  water transpired (WU)  harvest index
(HI). Therefore, optimizing yield under such conditions
has to be performed by increasing either water-use
(amount and pattern), transpiration efficiency/WUE
Crop Responses to Available Soil Water 619
or partitioning of more biomass to grain. This can be
achieved through better water management, the adjust-
ment of crop phenology to the environment, or genetic
improvement. The latter implies getting varieties that
can give the so-called more crop per drop, either by
improved carbon fixation under water deficits or hav-
ing deeper roots to capture more water or converting
more of the biomass into grain (increasing HI).
Increasing WUE at the expenses o higher assimilated
carbon rather than decreased WU is a desirable way to
improve YP under water scarcity, but will generally
require biotechnological interventions [17].
In the last decades, increases in Yp under non-
limiting water conditions have been achieved with
genotypes of cotton, wheat, and rice that exhibit high
stomatal conductance and transpiration. This trait
allows a decrease in leaf temperature and a greater
CO2 fixation per unit of leaf and land area [25, 64].
Under water-limited environments crop production
has relied mostly in dehydration avoidance traits as
described above, which maximize soil moisture use
(when it becomes available for example, under sudden
rainfall), but is generally associated with lower WUE.
On the contrary, drought resistance traits that reduce
WU, and are therefore associated with high WUE, will
unavoidably reduce YP.
Few successful cases of breeding for water scarcity
environments have been reported, one being the dry-
land wheat grain yield improvement, with selection for
high WUE in Australia [65]. This success has been
explained by the fact that wheat is grown there mainly
on stored soil moisture. Furthermore, as suggested by
Blum [2], a major opportunity for yield improvement
under water scarcity is the control of WU during the
earlier part of the growing season in order to avoid lack
of soil moisture later on, during the reproduction
phase. An attempt to achieve this was done by Richards
and Passioura [66] by selecting for reduced root xylem
diameter.
How Metabolic Processes Respond to Drought:
Photosynthesis, Respiration, Photorespiration
Photosynthesis
Under water scarcity, restrictions in leaf carbon
uptake take place as a result of increased resistance to
CO2 diffusion induced both by decreased stomatal
620 Crop Responses to Available Soil Water

Physiological Drought

Drought
Low soil water content and high VPD

Stomatal Alterations Gene-Dependent

Closure in Metabolism Responses

pH Ci
VPD ABA Sugars
hydraulic
ROS Hormones
nitrate
PHOTOSYNTHESIS Redox state
+

gmes

Acclimation Protection
(e.g. reduced growth) (e.g. ROS detoxification)

Recovery Processes (e.g. Membrane fluidity)

Crop Responses to Available Soil Water. Figure 2

Direct effects of drought on stomata and mesophyll (gm) conductance as well as on gene expression, resulting in
alterations of photosynthetic metabolism and ultimately on plant acclimation (Adapted from [9])

aperture (Fig. 2) [7, 9] and decreased mesophyll con-
ductance [67]. At the whole plant level, total carbon
uptake is further reduced due to the concomitant or
even earlier inhibition of shoot growth [8]. Metabolic
inhibition of photosynthesis usually occurs under more
severe stress conditions or when various stresses co-
occur, such as high light and temperature [8, 10],
although claims for earlier alterations in photosyn-
thetic metabolism are reported by Lawlor and
coworkers ([12]; see a review by [68]). Interestingly,
alterations in gene expression are also observed very
early on, although these alterations are generally not
turned into differential expression of proteins or
enzymes in the short term (see revision [9]).
Stomata are able to detect a decrease in water avail-
ability either in the soil or in the atmosphere by feed-
back and feed-forward mechanisms. Feedback
mechanisms include the response to dehydration in
the leaf itself that is transmitted to the guard cells,
either by hydraulic or by chemical signals. Feed-
forward responses are generally mediated by hormonal
signals and may take place before any alteration in leaf
tissue water status occurred. They comprise responses
of guard cells to high vapor pressure deficit, whose
mechanisms area still not fully resolved [69–71] and
to dehydration taking place elsewhere in the plant,
namely in the roots [72]. Hormones, with particular
relevance to ABA, but also cytokinins and ethylene,
have been implicated in the root–shoot signaling,
either acting in isolation or concomitantly. This long
distance signaling by hormones may be mediated by
reactive oxygen species [73].
Primary events of photosynthesis including elec-
tron transport capacity are very resilient to moderate
drought [74], with the decline in PSII photochemistry
being explained by a decrease in substrate availability.
In fact, PSII activity often declines concomitantly with
carbon uptake under water deficits, suggesting that
photosynthetic electron chain is finely tuned by avail-
able CO2 [75]. However, under field conditions, when
high light co-occur with water deficits, CO2 depriva-
tion at the chloroplast (driven by stomatal closure) may

result in the production of excess reducing power and
therefore in the decline of the quantum yield of PSII.
These stresses also appear to inhibit the repair of PSII
through suppression of the synthesis of PSII proteins,
in particular, the production of D1 protein [76]. Pro-
tection mechanisms against reactive oxygen species
(ROS) that are formed under such conditions are an
essential component of plant survival [77]. Such pro-
tection may be achieved by the regulated thermal dis-
sipation occurring in the light-harvesting complexes,
involving the xanthophyll cycle [78, 79] and the lutein
cycle [80]. Photoprotective mechanisms compete with
photochemistry for the absorbed energy, thus leading
to the downregulation of photosynthesis. If the limita-
tion of the rate of CO2 assimilation is accompanied by
an increase in the activity of another sink for the
absorbed energy, for example, photorespiration [81]
or Mehler-peroxidase reaction [82], the decline in
non-cyclic electron transport will be proportionally
lower than the decrease observed in the rate of CO2
assimilation. It was estimated that, in the absence of the
repair mechanisms, photodamage would lower the
yield of photosynthesis to less than 5% of the yield
achieved now [83]. Although these responses have
mainly been documented in plants native to semi-arid
regions they may also occur in crop plants, even irri-
gated, when they are subjected to intense heat and
irradiance, during the summer period. This is likely
to increase its frequency in the near future.
As for Rubisco activity it was shown to be very
resistant to water deficits [84, 85], being generally
affected only after severe stress [86, 87]. The same
authors also found evidence that low CO2 concentra-
tion in the chloroplast (Cc) attained under severe
water deficits could induce deactivation of Rubisco
sites. It is further suggested that these effects are spe-
cies-dependent, with species adapted to low Cc being
able to maintain active Rubisco longer in response to
prolonged drought [87]. These findings are compatible
with earlier data suggesting that decreased sink capacity
(limited capacity to use photoassimilates), as induced,
e.g., by shoot growth inhibition under drought,
might feedback to decrease photosynthesis, namely by
downregulating the enzymes of the photosynthetic
carbon cycle [88].
When studying the effects of drought on photosyn-
thesis it is also important to recognize that crop
Crop Responses to Available Soil Water 621
productivity is dependent on photoassimilates pro-
duced at the whole plant level, which partly explains
why often there is no correlation between crop yield
and leaf photosynthetic rate [61]. Therefore, the impact
of drought on shoot and canopy growth may be as
important to crop yield as the effects produced at the
single leaf level. Indeed, water scarcity by inducing
a significant decline in total plant leaf area via the
inhibition of new leaf growth or the earlier senescence
of older leaves will decrease total carbon uptake by the
plant. Moreover, lower leaf angles induced by decreased
leaf turgor will reduce total intercepted irradiance, with
significant impact on crop yield (Pinheiro and Chaves,
unpublished results). However, it must be emphasized
that, at least in cereals (wheat, maize and soybean),
yield is generally more limited by the sink than by the
source [61, 89]. This explains why these crops have the
potential to cope with moderate drought and still fill
their grains. Of course, this is also possible due to the
capacity of remobilizing photoassimilates, previously
stored in the shoot, as was discussed above.
Respiration and Photorespiration
The effects of water deficits on dark respiration are still
unclear, with reports of either decreases, maintenance,
or increases in the rates of this process (see the review
[90]). Inhibition of respiration under drought has been
observed in mature leaves of crops and herbaceous
species as well as in roots, and is presumably related
with a decreased availability of substrate to mitochon-
dria under conditions of low photosynthesis and
growth (e.g., [86, 91, 92]). This may be explained by
the need of herbaceous species to quickly respond to
water scarcity, thereby lowering their respiration rates
in order to optimize their carbon gain over shorter
periods of time.
On the contrary, trees and shrubs seem to show
slower responses to drought than short-lived species.
As suggested by Flexas et al. [86] and Atkin and
Macherel [93], a higher demand for respiratory ATP
(higher respiration rates) may be required under severe
water stress to compensate for the lowered ATP pro-
duction in the chloroplasts. The increased maintenance
respiration will support repair mechanisms needed
under acclimation to drought that will ensure a better
performance under extended periods of water scarcity.
622 Crop Responses to Available Soil Water
In general, the changes observed in respiration in
response to drought are smaller than those observed in
photosynthesis, thereby implying that respiration
increases proportionally in relation to photosynthesis,
with likely impact on leaf intercellular CO2 and on
plant carbon balance [68].
The role of photorespiration during drought stress
has been scarcely studied [81, 94], partly due to diffi-
culties in quantifying the rate of photorespiration [95].
Since photorespiration and photosynthetic metabo-
lism are strictly linked and photorespiration depends
on the recycling of RuBP in the Benson–Calvin cycle, it
may be hypothesized that severe drought stress should
result in lower photorespiration. As suggested by
Osmond et al. [96], photorespiration and the Mehler-
peroxidase pathway could protect the photosynthetic
apparatus against photoinhibition in drought-stressed
leaves, by sustaining photon utilization in non-
assimilatory electron flow, when electron consumption
by CO2 assimilation is reduced due to low internal CO2
concentrations. However, other studies, such as the one
by Brestic et al. [97] concluded that photorespiration
was not important for photoprotection in drought-
stressed French bean.
In a recent work with glycine decarboxylase-
deficient plants, Igamberdiev et al. [98] showed that
photorespiration contributes to stomatal regulation.
The data obtained with these mutants revealed that
the photorespiratory mutants were able to decrease
the rate of photorespiration, but only at the expense
of increased water loss. Indeed, the necessity to
maintain a high CO2 concentration near the site of
carboxylation in the chloroplasts of plants deficient
in photorespiratory enzymes required an
increased opening of the stomata, with a corresponding
increase in water loss and decrease in water-use
efficiency.
Yield and Quality Under Water Scarcity
Agriculture depends to a large extent on the success of
plant reproduction [99]. Drought affects crop produc-
tivity as much as all other environmental factors com-
bined and its impact on crops differs according to the
attained developmental stage. The effects of water scar-
city are quite different in annuals and perennial species
[100]. Reproductive development of cereals, from
meiosis to seed set, is highly vulnerable to water deficit
[101] and the meiosis stage appears to be the most
stress sensitive period of reproduction in all studied
species [102]. However, anthesis, pollen fertility, polli-
nation, female fertility, and early zygote development
were also reported to be susceptible and finally their
failure altered the number and the final quality of grain
[102]. Later in the development, water stress tends to
reduce grain size [103]. In fruit trees, severe water stress
during flowering was reflected in the final fruit number
per tree, whereas water stress during the fruit-growth
and maturity phases was reflected mainly in fruit size
[104]. In addition, the timing and intensity of the
drought period dictate the extent of alterations occur-
ring in the final fruit quality [29].
Flower Initiation and Induction
Grain crops are very sensitive to drought during floral
initiation and floral pre-meiotic differentiation [105].
Water deficit at this stage causes pollen sterility, but
when stress is severe, it usually affects also female fer-
tility. In cereals, water stress during flower induction
and inflorescence development leads to a delay in
flowering or even to its complete inhibition [105].
The increase of abscisic acid (ABA) in response to
water stress has been suggested to play a role in this
delay [106].
In woody plants, flower bud initiation is an impor-
tant feature in fruit tree cultivation. However, the effect
of drought on floral meristems is among the least
understood aspects of crop reproductive development
under water deficit [107]. In species where flowering
takes place prior to leaf emergence as Prunus trees,
flower development occurs in the absence of new
photoassimilates and at the expense of pre-stored
reserves (starch), either in the flower itself or other
plant organs [108]. This explains why in Prunus water
stress effects are only detected in the subsequent repro-
duction event and not in the same year. This is the case
of peach, apricot, and sweet cherry trees, where
drought stress during flower bud initiation markedly
slowed the progression of floral differentiation
by delaying the differentiation of pistil primordia
[109, 110].
In opposition, water stress has been demonstrated
experimentally to induce flowering by a direct stimuli

on stem and bud in citrus [111], apple [112], and pear
[113]. In these species, water stress showed to stimulate
polyamine accumulation, which is linked to floral ini-
tiation, thus enhancing the production of floral
primordia and re-flowering. However, severe water
stress induces little flower production. Indeed, under
severe stress during flower bud induction periods, gib-
berellins level increased, inducing a low level of flower
production per shoot [114].
Flowering and Pollen Development
Loss of pollen fertility, spikelet death, and abortion of
newly formed seed are associated with a decline in the
water status of the reproductive structures and decline
of carbohydrate availability [105]. By reducing photo-
synthesis, drought directly interferes with inflorescence
and flower number, flowers life span, flowers opening,
and maintenance of floral organs in crops [115] as well
as maintenance of nectar production in floral organs
[116]. In addition, water stress reduces flower size and
sucrose content in nectar [117] leading to an extensive
loss in yield [118].
Drought also affects seed yield of plants through an
effect on availability and viability of pollen grains
[119]. The effect of drought on pollen fertility/avail-
ability also depends on the species and it is considered
a common symptom in angiosperms [120]. The
hypothesis that ABA is a primary controlling agent in
water stress induction of pollen viability still remains
unanswered [102]. Recently in chickpea [119], water
stress was shown to affect pollen growth in the pistil
rather than affecting pollen viability. In fact, pistils
from well watered plants pollinated with pollen of
stressed ones showed fewer germinated pollen grains
and fewer pollen tubes that reached the ovary, which
suggested that drought has an effect on pollen tube
growth, which is inhibited in the pistil [119]. Pollen
viability is highly dependent on sugar unloading and
pre-anthesis stem reserve accumulation is considered
a significant factor affecting flower development in
water stressed plants (see Fig. 1) [121]. The decrease
of acid invertase activity under low water potential
impedes pollen to metabolize incoming sucrose in hex-
ose in the developing pollen, which might lead to
pollen sterility [122, 123]. This hypothesis is confirmed
by the study of Koonjul et al. [124] who observed
Crop Responses to Available Soil Water 623
a downregulation of the soluble invertase gene Ivr1
and Irv5 in wheat microspores, which correlates with
accumulation of reducing (fructose and glucose) and
non reducing (sucrose) sugars in the ear [125]. Never-
theless, recent studies by Liu and Bennett [126] showed
that stress induced sterility in rice is not only caused by
disruption in sugar metabolism or by desiccation of
reproductive tissue, but also by disturbance of anther
pollen development and cell function. In wheat, micro-
spores lost contact with the tapetum at first pollen
grain mitosis and the filament degenerated in response
to water stress, which resulted in total sterility [127].
The expression of various proteins related to anther
wall degradation and cell wall modifications are mod-
ified in stressed rice anther [126]. Furthermore, pollen
from stressed plants might have a shorter life span
and reduced vigor, which explain that pollen tube
growth fails to reach the ovule [128]. The depletion of
the adenosine triphosphate pool, increased concentra-
tion of hydrogen peroxide, and downregulated tran-
scripts in anthers of drought-stressed rice lead
to a programmed cell death and may cause pollen
sterility [129].
In woody species, as observed in apricot trees, post-
harvest drought may induce an increase of aborted
pistils in the subsequent year, decrease germination
potential of pollen (see Fig. 1), and decrease the per-
centage of fruit set due to an increase of fruit drop
[130]. In almond, the number of fruiting positions
per tree is negatively associated with water stress
[131]. However, drought stress during flower initiation
does not influence the percentage of spurs that flow-
ered or set fruit during subsequent years. Although
water stress had no apparent effect on spur mortality
in the first year, more than a half of spurs died within
three subsequent years. In addition, water stress
reduces flower bud development in grapevine [132]
and peach [133] and increases flower abscission [134]
and young fruit drop [135].
Ovary Development
Consequences of inhibited photosynthesis under
drought and therefore insufficient assimilates are par-
ticularly striking around the time of pollination when
reproductive events occur rapidly, namely ovary
growth [136, 137]. In maize the pollen does not loose
624 Crop Responses to Available Soil Water
availability even at severe water stresses (water poten-
tial below 12.5 MPa) and low kernel number is
explained by a poor receptivity of silks and/or by an
ovary abortion. Maize ovaries are normally loaded with
glucose and starch on the day of pollination [138, 139].
Under drought, sucrose, the main translocated product
from the carbon fixed in photosynthesis, declines (see
Fig. 1). The enzymes that convert incoming sucrose to
glucose in ovary lose activity and starch starts to be
hydrolyzed [137, 138]. Cell wall acid invertase hydro-
lyses sucrose in the apoplast of the upper pedicel tissues
and its activity creates a steep gradient in glucose con-
centration between the upper pedicel and the nucellus
of young ovary. Ovary abortion under drought was
attributed to the decrease of glucose and invertase
activity in upper pedicel, inhibiting sucrose transport
[138]. Sucrose delivery decreased first in ovary and
probably triggers an early downregulation of genes
coding for sucrose processing enzymes (INCW2 and
IVR2). Glucose depletion occurs few days after and
triggers an up-regulation of putative senescence genes
as ribosome inactivation protein 2 [139], which suggests
the beginning of failed ribosome function and later
induces an up-regulation of phospholipase D1 gene
leading to loss of plasma membrane integrity, indicat-
ing the onset of senescence [137, 140]. This probably
causes the irreversible loss in viability found during
abortion [139]. Feeding sucrose during water stress
largely prevents these changes, which confirm that
senescence genes are “monitoring” the sugar status
of the ovary cells and when sugars content decreases
these genes turn on in sequence and orchestrate cell
death [137].
A transcriptional study of placenta and endosperm
of maize under drought showed that both tissues
responded differentially to water shortage [141].
While most of the responding genes in placenta
involved up-regulation, in endosperm these were
downregulated. Downregulated genes relate to cell
division and to endosperm growth, which may explain
arrested growth and thus decreased demand of photo-
synthates [141].
These described events are common across crop
species [137]. Although wheat requires water potentials
to be much lower than maize to lose the same amount
of photosynthetic activity [137], starch is also depleted
in floral structures after drought (see Fig. 1) [142],
inducing an impairment of ovule function [143]. Also
in grapevine, drought induces lower availability of
sugar due to decreased photosynthesis, which provokes
ovary abnormalities and flower abscission [144].
Setter and coauthors [145] suggest an interaction
between ABA and sugar that might induce a signal
cascade that leads to the abortion process to initiate.
The recent finding by Setter and Parra [146] supported
the model of the interacting influence of carbohydrates
and ABA at least at the pedicel-placenta tissues of basal
kernels. Several studies in maize reported that water
stress increases ABA in florets, suggesting it might
trigger the abortion process [145, 147]. Young and
Gallie [148] observed that regulation of programmed
cell death during maize endosperm development
involves the interaction of ABA and ethylene signaling
pathways. However, Boyer and Westgate [99] explained
that hormone effects are difficult to interpret because
their expression on dry matter or water content basis
can cause concentration to increase simply because the
ovary dry mass or water content decreased, as it was
shown in maize [149].
Yield
All described abnormalities of flowering, fertilization,
and zygote development that may occur under drought
stress induce yield losses, and the timing of the occur-
rence of drought stress determines the degree of dis-
turbance. Water stress during ear formation and milk
stages in maize was shown to induce early loss of lower
leaves and a decrease in plant dry matter and in grain
yield, as a result of reduced intercepted radiation [150].
Moreover, all the yield parameters were significantly
affected. When water stress occurred at pre-anthesis
a reduction in seed numbers was observed due to
pollen sterility and ovary abortion. Post-anthesis
water stress generally enhances whole-plant senescence
and lead to a faster remobilization of carbon from
vegetative tissues to seeds, thereby inducing earlier
maturity and small seeds [119, 151–155]. This is
a typical strategy of Mediterranean annuals, which
exhibit a phenological drought-avoidance producing
seed before water supplies are exhausted [45].
Remobilization of pre-stored carbon to the seed and
acceleration of seed filling rate are associated to an
alteration in the hormonal balance of grains, namely

the decrease in gibberellins and the increase in ABA
[48, 156]. The reduced duration of grain filling may
partly result from reduced number and size of endo-
sperm cells and therefore reduced capacity to accumu-
late starch following drought stress [157]. Additionally,
seed cell expansion is driven by water uptake [158] and
when cell expansion in the seed stops, the end of seed
growth and seed maturation are predetermined.
Indeed, Yang et al. [152] suggested the enhancing of
sink strength with water stress is promoted by alter-
ations in sucrose synthase and starch branching
enzyme activities taking place in grain during stress.
Severe drought stress during seed filling generally
resulted in seeds that are shriveled and deformed and
with a reduced weight [152, 159]. The shortening in the
duration of seed fill limits seed size because grain fill
process fails to finish in a short maturation period
[155, 160], consequently reducing seed yield [161].
Seed size reduction was highest in late maturing
wheat genotypes, suggesting that early maturing geno-
types partially escaped late-season water stress [162].
However if water stress occurs after cell division is
completed it does not affect yield since sink size per
kernel is already predetermined [152]. In sesame, most
drought-tolerant cultivars abort a higher proportion of
their seeds, and in that case more efficiently shunt
available nutrient resources to the healthy growth of
remaining seeds [163].
In woody plants and especially in fruit trees, it is
generally accepted that post-harvest water stress may
negatively influence fruit set and crop load in the next
season, as it is the case in peach [164–167], in almond
[168] and in sweet cherry [169]. This reduction in fruit
set is due to the limitation placed by reduction in the
accumulation of carbohydrate reserves. In fact, in cul-
tivated Prunus species, floral bud-break and fruit set
significantly precede net carbon export from leaves
[167]. In citrus, severe water stress taking place during
flowering led to decreased fruit number per tree,
whereas in water restrictions occurring during the
fruit-growth and maturity phases, the effects were
reflected mainly in fruit size [104]. Olive tree has
a reputation of being a drought resistant crop. How-
ever, drought incidence during winter, leading to a low
level of soil moisture, may reduce vegetative growth
[170] and have an important impact on flowering and
fruit set, resulting in a drastic decrease on olive fruit
Crop Responses to Available Soil Water 625
number and size [171, 172]. In fact, severe water stress
in phase I of fruit growth was shown to reduce meso-
carp cell division and number [170, 173] suggesting
a high sensitivity of olive mesocarp cell size to water
stress. Grapevines are also well adapted to semi-arid
climates, nonetheless severe water stress during the
summer largely limit grapevine yield and cluster weight
[27, 29]. This reduction is mainly observed in drought-
avoiding genotypes, which optimize survival at expense
of yield and reduced sugar allocation to reproductive
tissues and thereby reducing fertility [174].
The effect of drought in crop plants is enhanced by
high temperature and effects are usually difficult to
separate [118]. Differences among genotypes are
observed; for example barley is much less sensitive to
short periods of very high temperature than wheat, and
combined drought and high temperatures are more
likely than high temperature alone to explain the
reductions in grain weight observed in the field [175].
Fruit and Seed Quality
Drought influences end-use quality of major food
crops in the world, as it is the case of wheat [176],
barley, and maize [177]. Indeed, water stress in wheat
changes the patterns of proteins [178]; in barley, grain
b-glucan content and malt fine extract decreased with
drought stress [179]; in soybean drought during seed
development has a large effect on isoflavone concen-
tration in the seeds [180]; in maize, water stress
increases flavonoid content in seeds but reduces carot-
enoids and phenol content inducing a decrease in the
antioxidant capacity of kernel oil [181]. In lupin seeds,
raffinose quantity and accumulation pattern are
reduced by water stress, suggesting an increase in nutri-
tive values of seeds since raffinose is considered to
be a major cause of flatulence in animals and
humans [161].
Starch granule shape, volume and structure are
important factors contributing to starch quality in
wheat [182]. Post-anthesis water stress affects the
proportions of different types of starch granules
and increases the percentage of small granules per
seed [183].
Protein content has an important implication in the
processing qualities of cereals since it affects the func-
tional properties of processed wheat products [184].
626 Crop Responses to Available Soil Water
In wheat, grain protein content decreases linearly with
the severity of drought stress during grain development
[158]. Recently Zhao et al. [184] showed that the inten-
sity of drought dictates the intensity of the effect on
protein accumulation and reported a differential accu-
mulation of proteins (especially gliadins and glutenins)
in mature grains. The decrease in the accumulation of
oil due to water stress was reported in wheat [184],
soybean [185, 186], lupin [187] and chickpea [188].
In fruit trees, drought influences fruit development,
metabolism, and final composition, and its timing and
intensity dictate the extent of alterations occurring in
final quality [29]. With regard to the effect of water
stress on organoleptic properties, fruit quality is signif-
icantly affected by severe water stress during the fruit-
growth and maturity periods [104]. Under water
restriction, fruit maturity tends to be hastened,
supporting the hypothesis that increasing water restric-
tion might encourage early fruit maturity as observed
in peach [189, 190]. Post-harvest water deficit was
shown to increase fruit soluble solid concentration in
the following season in peach [191, 192]. The opposite
effect, reducing firmness and soluble solid concentra-
tion, was reported in sweet cherry [169].
Water stress was shown to decrease oil content and
yield in young olive trees and induce lower level of
phenolics in oil [172]. Olive fruits import assimilates
from the canopy, but also produce them in situ by
photosynthesis in the mesocarp; the fruit is capable of
retaining chlorophyll even when its color change [193]
and this makes a significant contribution to oil pro-
duction [194]. This “autonomy” of olive fruits may
explain the maintenance of oil quality in trees under
water deficits [172]. In fact, although irrigation led to
decreased contents of some undesirable sensory quali-
ties as phenols, some favorable intense green notes were
also reduced, which suggests complex effects of water
stress on olive quality [195]. In grapevine, severe water
stress influences final composition of the berries by
delaying their ripening but also through an indirect
effect on berry size [196, 197] that affects sugar and
flavonoids metabolism in the berry (review [29]).
However, moderate water deficit was shown to have
beneficial effects on grape berries, for example by
enhancing photoprotection mechanisms [198] and
likely having a positive effect on wine quality [29,
196]. Indeed, moderate water deficit promotes sugar
accumulation by inhibition of shoot growth with
a subsequent reallocation of carbohydrates to fruits
[199] and by the activation of ABA-mediated uptake
of hexoses [198]. Under such conditions, sugar accu-
mulation in berries accelerates anthocyanin accumula-
tion or/and biosynthesis [200, 201]. The enhancement
of carotenoids content and the up-regulation of genes
encoding enzymes involved in biosynthesis of berry
aroma were also observed after mild stress imposition
[198, 202]. Similar benefits of mild water deficit in fruit
quality were also observed in apricot [203], citrus [104]
and olive [204].
The Breeding Achievements
Conventional Breeding
To face drought, farm-management practices and plant
breeding are used for the improvement of crop yield
[18], being the second approach the most promising in
the long term [64]. Drought is the most recalcitrant to
breeders’ efforts due to the complexity of plant
drought-tolerance mechanisms [205] and breeders
have no reliable method of distinguishing sensitive
from tolerant germoplasm other than by measuring
yield [19]. Therefore, the main objective of a
drought-tolerance breeding program is to select the
variety presenting the better yield under stress condi-
tions [206]. However, yield is a trait which is charac-
terized by low heritability, polygenic control, and
a high genotype  environment interaction (G  E)
[21, 207].
During the last 50 years, most of the progress has
been derived from empirical (conventional) breeding
[208, 209] by selecting desired traits recognized at the
phenotypic level. Generally, there is a minimum of
4 years extensive yield testing to further selection at
multiple locations before a new variety is released to
farmers. Empirical selection for grain yield was effec-
tive during the last decades. However, when plants are
crossed some undesirable traits may be transferred
along with those of interest – including some with
negative effects on yield potential. In addition, breed-
ing can only be done between species inter-compatible,
which limits the new traits that can be added to those
that already exist in a particular species [206]. Further-
more, the variability of rainfall from year to year
increases G  E and reduces heritability for yield,

thereby limiting yield progress [19]. The indirect or
analytical approach based on an understanding of the
crop physiology, may help to target the key traits that
are limiting yield [64]. Such an approach may therefore
complement conventional breeding programs and has-
ten yield improvement [210–212].
A number of physiological studies have identified
some traits which presence/expression are associated
with plant adaptability to drought prone environment
[205] and have been already successfully exploited in
crop improvement [213]. Increasing water use by
increasing early shoot vigor to escape dryer periods is
a common strategy in Mediterranean region [214], but
in very dry years this strategy may result in reduced
yield. Early maturity by adjusting crop development
with seasonal rainfall pattern in environments where
they experiment terminal drought, or late flowering
with short grain filling period in environments with
early season drought, are examples of traits that are
being used in breeding. Leaf cuticle waxes appear to
play a key role in day and night transpiration and
selecting for waxy leaves may reduce water loss and
prevent leaf senescence during grain filling [215].
Enhancement of water extraction, by optimizing root
architecture that resulted in greater water capture, has
also the potential to significantly increase grain yield
[63]. In addition, enhancement of root osmolytes accu-
mulation was used in cereals, but a clear evidence of
their beneficial role in crop yield was not observed [58].
Carbon isotope discrimination (D) is an attractive
feature for C3 plants breeders since it may provide an
indirect sensing of transpiration efficiency (TE) [13].
In fact, genotypes showing higher TE (lower D) may be
more productive under certain environmental condi-
tions [216]. However, in the Mediterranean region the
opposite was found and positive correlation between D
and yield are reported [217], which is probably due to
the fact that genotypes with lower TE have maintained
a higher CO2 conductance due to a better water status
or by a faster plant development or better access to
water [216]. Aerial infrared (IR) methods on vege-
tative material offer the possibility to detect vari-
ation on TE and present the advantage to reduce the
cost of measuring carbon isotope discrimination
[19, 216].
Fruit tree species propagation is possible only by
vegetative methods. However, with the spreading of
Crop Responses to Available Soil Water 627
different species in new areas, the use of rootstock has
become the first alternative to adapt scion cultivars to
soils or climatic conditions that are otherwise not fully
suited to their cultivation. Climatic areas or soils
subjected to transient drought conditions require root-
stock capable of inducing efficient water use by the
scion cultivar. Unfortunately, there is only limited
understanding of how rootstock provides tolerance
to drought [218] and the majority of rootstock breed-
ing programs addresses fruit quality. Traditionally,
rootstocks have been produced using conventional
hybridization breeding techniques, which are time
and space consuming. Initial selection of main root-
stocks is made from open pollinated fruit tree germ-
plasm, generating thereby intra- and interspecific
hybrids. The extended use of interspecific hybrids
is due to the lower susceptibility of this type of root-
stocks to biotic and abiotic stresses and may also
overcome soil problems as drought [219], which
might derive from heterosis (hybrid vigor) or from
the elevation of ploidy levels of these rootstocks
[220]. However, the use of known drought-tolerant/
resistant rootstocks may not fulfill grower’s intents in
terms of fruit commercial traits, mainly because they
may provide excess vigor. In addition, the problem of
graft compatibility of hybrid rootstocks with commer-
cial scion complicated the use of a wide range of culti-
vars [221, 222].
New Breeding Approaches
Compared to conventional approaches, genomics offer
unprecedented opportunities for dissecting quantita-
tive traits into their single genetic determinants, the
quantitative trait loci (QTLs), thus paving the way to
marker-assisted selection (MAS) and, eventually, clon-
ing of QTLs and their direct manipulation via genetic
engineering. Several studies have reported QTLs for
roots architecture and investigated their effect on
yield under water stress in rice [223] and also in
maize, where QTL reported for leaf ABA concentration
showed an effect on root size and grain yield [224, 225].
In sorghum QTLs related to ‘stay green’ trait and yield
were identified [226].
Some QTLs of one character were shown to overlap
with QTLs of other characters. This is the case in maize,
where QTLs of leaf growth sensitivity to water deficit
628 Crop Responses to Available Soil Water
overlapped with QTLs for leaf responses to evaporative
demands, thus suggesting that hydraulic mechanisms
are involved in that response [227]. Similarly, overlap
between QTLs of leaf growth sensitivity with silk
growth was observed [228]. QTLs were also reported
for anthesis-silking interval in maize [15], for seed
weight and yield under different water conditions in
rice [229] and durum wheat [230]. It was also possible
to identify WUE QTLs in brassica [231], rice [232],
and wheat [233]. However, despite all the recent tech-
nological breakthroughs, the overall contribution of
genomics-assisted breeding to the release of drought-
tolerant cultivars has so far been marginal [234], in part
due to the fact that a given QTL can have positive, null
or negative additive effects depending on the drought
scenario [235]. In addition, cloning QTLs for yield
maintenance under drought conditions and drought
physiological traits is limited because the unavailable
information about the biochemistry of some of these
traits [236].
In recent years, proteomic and transcriptomic
studies have advanced the basic understanding of pro-
tein/gene regulatory networks that are active during the
exposure of plants to water stress [237, 238] and there
have been several reports of apparently promising bio-
technological manipulations emerging from these tools
[239]. Remarkable dehydration tolerance has been
obtained under laboratory conditions using bacterial
RNA chaperones overexpression [16], NFYB2 class
transcriptionals regulators [240], as well as modulating
the expression of dehydration response element bind-
ing (DBF/DREB1) transcription factor [241], using of
detoxification of reactive oxygen species that accumu-
late under stress [242, 243] and of hormone interven-
ing in drought signaling [244]. Introducing RNA
silencing in some crops to downregulate poly ADP
ribose polymerase and thus inducing tolerance to
wide range of stresses [245] and overexpression of
cyanobacterial flavodoxin [246] were also reported.
However, these reports and others have failed to deliver
drought tolerance in yield terms when the novel mate-
rial is transferred from the growth room to the field [4].
Fruit trees also have benefited from new technolo-
gies and it is now possible to reduce the breeding time by
MAS. Very recently fruit breeders have begun to exam-
ine the possibilities of using such techniques in the
production of new rootstocks. The aim is to improve
an already good rootstock for yield by modifying its
gene expression or introducing new genes to improve
resistance to abiotic factors. Unfortunately, the mecha-
nisms by which rootstocks bring about their beneficial
effects on the vigor and cropping of scions are still very
poorly understood. Until more research is conducted in
this field and genes that control such processes are
identified, progress in this area may be slow. Recent
studies showed that ectopic expression of the transcrip-
tion factor Osmyb4 gene in transgenic apple trees,
improved water stress tolerance and might result in
long term ameliorated productivity. Further experi-
ments are required to assess the agronomic value of
the plants produced and to verify to what extent the
expression of Osmyb4 may contribute to enhanced
drought tolerance under field conditions [247].
Summarizing, to date increased drought resistance
of the major crop plants (sustained yield under water
scarcity) has been dependent upon the screening of
a wide range of germoplasm in order to identify genetic
variation in major traits involved in stress resistance
[19, 248, 249]. In addition, the effective use by the crop
of a limiting water supply has been achieved by
adjusting crop phenology to its environment [2, 14]
or by using agronomic practices aiming at an improved
water use, such as deficit irrigation [4, 29]. A variety of
approaches have been successful, particularly in the
irrigation of top quality fruit and vineyards [3, 27, 29,
250, 251] but also in annual crops [252–255]. In addi-
tion to minimizing changes in shoot water status, def-
icit irrigation is able to control the balance between
fruit and vegetative growth, with likely positive impact
on fruit quality [8, 256].
Future Directions
Drought has a great impact on the vegetative and
reproductive development of annual and fruit tree
crops and thereby impacts on final yield and seed and
fruit quality. The key factors responsible for high yield
under drought in annual plants are an appropriate
phenology that will enable escaping drought and get-
ting the timing of flowering right, high water-use effi-
ciency and a high harvest index. The impact of drought
is highly complex and involves diverse processes as
photosynthesis, respiration, reserve accumulation, fer-
tilization, gamete and embryo development, and fruit

and seed development. Due to the complexity of crop
reproduction and the still incomplete knowledge of
mechanisms underlying the response of reproductive
events under water stress, it has been until now difficult
to construct a model system to guarantee successful
reproductive development and high yield under water
scarcity. How resource availability (namely carbohy-
drate reserve) influence the development of reproduc-
tive organs under water stress is among the least
understood aspects. On fruit trees, it is also urgent
to understand (1) how rootstock confers resistance
and/or tolerance to drought to the scion cultivar to
better adjust breeding strategies; (2) the key events
of fruit ripening. In addition, differences between
species imply different strategies to improve/maintain
yield and quality under such conditions. Spatial
strategies also differ: a particular trait may be associ-
ated with higher yield in one environment and may
have no/or negative effect in other environment [13].
Conventional breeding still appears to be the
most effective for a significant drought-tolerance
improvement of new cultivars. The application of mod-
ern molecular tools to understand responses of crops to
water scarcity has only recently begun. In addition, to
integrate them in breeding programs it is imperative
to incorporate an accurate measurement of the pheno-
type under water stress. However, the speed of molecular
technologies is not matched by the speed on
phenotyping, being the latter one of major limitations
to improve selection methods for water-limited environ-
ments [19]. Moreover, it is imperative to integrate dis-
ciplines, as functional genomics, transcriptomics,
proteomics and metabolomics with plant physiology
to improve breeding strategies. This would allow
researchers to address drought tolerance/resistance
through an integrated approach based on the knowledge
of expression of genes and their products at a particular
phenological and developmental stage of the crop.
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638 Crop Responses to Nitrogen

Crop Responses to Nitrogen Nitrogen absorption efficiency (NAE) Nitrogen

GILLES LEMAIRE
INRA, Lusignan, France
Article Outline
Glossary
Definition
Introduction
N in Plants
Crop N Demand
Regulation of Crop N Uptake
Diagnosis of Crop N Status: Nitrogen Nutrition Index
Crop Responses to N Deficiency
Nitrogen Use Efficiency in Crops
Conclusion
Future Directions
Bibliography
Glossary
Critical plant N concentration Critical plant
N concentration is defined as the minimum plant
nitrogen concentration of a crop corresponding to
its maximum crop mass.
Critical crop N uptake Critical crop N uptake is
defined as the minimum crop nitrogen uptake for
achieving maximum crop mass.
Harvest index (HI) Harvest index (HI) is the ratio
between harvested biomass Y (grains, tubers) and
aboveground crop mass W at crop maturity.
Intercepted photosynthetic active radiation
(IPAR) Intercepted Photosynthetic Active Radia-
tion (IPAR) is the proportion of the incident PAR
which is intercepted by the crop at a given time.
This proportion is related to the size of the canopy,
the Leaf Area Index, and depends also on canopy
structure: leaf angle and geometry.
Leaf area index (LAI) Leaf area index (LAI) is the total
canopy leaf area of a crop per unit of soil area. LAI
allows the estimation of the proportion of the inci-
dent light which is intercepted by the canopy, and
then which can be used for photosynthesis of the
whole crop.
absorption efficiency (NAE) is the increase in crop
nitrogen uptake per unit of supplemental N supply
rate.
Nitrogen conversion efficiency (NCE) Nitrogen con-
version efficiency (NCE) is the increase in crop dry
mass (dW) or in crop yield (dY) per unit of sup-
plemental crop N uptake corresponding to an
increase in nitrogen supply rate.
Nitrogen use efficiency (NUE) Nitrogen use efficiency
(NUE) is the increase in crop dry mass (dW) or in
crop yield (dY) per unit of supplemental N supply
rate. So NUE = NAE
NCE.
N dilution N dilution is the process corresponding
to more rapid accumulation of nitrogen-free
compounds than nitrogen compounds within
plant as plant grows, leading to decline in plant
nitrogen concentration with plant mass
accumulation.
Nitrogen nutrition index (NNI) Nitrogen nutrition
index (NNI) is an index which allows the estima-
tion of the crop nitrogen status. This index is cal-
culated at any moment as the ratio between the
actual plant nitrogen concentration of the crop
and the critical plant N concentration (see this
definition) corresponding to the actual crop mass.
Photosynthetic active radiation (PAR) Photosyn-
thetic active radiation (PAR) is the part of solar
radiation spectrum corresponding to wavelengths
that are active for photosynthesis.
Radiation use efficiency (RUE) Radiation use effi-
ciency (RUE) is the ratio between the quantity of
biomass accumulated within a crop and the quan-
tity of photosynthetic active radiation (PAR)
intercepted by this crop during the same period of
time.
RuBPc-o Ribulose bisphophate carboxylase/
oxygenase, the enzyme located within chloroplasts
which allows the carboxylation of CO2.
Definition
A prerequisite for the analysis of crop responses to
nitrogen (N) is the determination of the plant nitrogen
content and repartition. How much N is incorporated
within plants and crops? Within which plant tissue? For

P. Christou et al. (eds.), Sustainable Food Production, DOI 10.1007/978-1-4614-5797-8,

# Springer Science+Business Media New York 2013

Originally published in
Robert A. Meyers (ed.) Encyclopedia of Sustainability Science and Technology, # 2012, DOI 10.1007/978-1-4419-0851-3

which physiological function? Thus, according to the
answers to these questions, it is possible to determine
a critical plant nitrogen status as the minimum plant
N concentration that allows the maximum plant (or
crop) growth rate. It has been demonstrated that this
critical plant N concentration decreases as plant grows
as the result of an ontogenetic plant architecture devel-
opment leading to a dilution of N compounds within
increasing proportion of free-N compounds as plant
gets bigger. This N dilution process can be formulated
through a negative power relationship between plant
N concentration and crop mass. This critical N dilution
curve allows the discrimination of situations of
N deficiency (below the curve) and situations of
N luxury consumption (above the curve). So
a Nitrogen Nutrition Index (NNI) can be calculated
for quantifying the intensity of N deficiency or
N luxury of any crop at any stage of its life cycle. This
possibility for determination of crop N status and for
quantification of N deficiency either in terms of inten-
sity or timing allows the complete inversion of the
approach: instead of response of crop to N supply, the
problem is to study the response of crop to
N deficiency. By this way, the check treatment is the
non-limiting N conditions where crop growth poten-
tial is limited only by genetics and climate. Then the
effect of intensity and timing of N deficiency period on
the different plant growth processes and yield compo-
nent elaboration can be studied with a more generic
approach. This new method for analysis of the effect of
plant N nutrition on crop yield allows the identifica-
tion of physiological, agronomical, and genetical ways
for improving nitrogen use efficiency of crops.
Introduction
Nitrogen is considered to be the most important lim-
iting factor, after water deficit, for crop production
worldwide. Over the last 50 years, the worldwide use
of mineral fertilizers is one of the key elements for
producing sufficient food to meet the demand of
increasing human population [1, 2]. During this
period, the use of mineral N fertilizers in agriculture
systems increased sevenfold in parallel with the dou-
bling of agricultural food production. This huge
amount of mineral N is provided by industrial pro-
cesses of chemical reduction of atmospheric nitrogen.
Crop Responses to Nitrogen 639
So even if this resource can be considered as quantita-
tively non-limited, the energy cost of the Haber–Bosch
process necessary to obtain mineral N fertilizers and
the large greenhouse gas emission associated with
oblige to reconsider the sustainability of the use of
mineral N in agriculture. Moreover, the use of large
amounts of N in intensive agriculture production sys-
tems has led to important environmental problems
such as the eutrophication of freshwater [3] and marine
ecosystems [4], pollution of ground water, and gaseous
emissions of N oxides and ammonia in the atmosphere
[5, 6]. In consequence, problems associated with sus-
tainable development, global changes, environment
protection, and global food security are now
questioning the efficiency of use of N fertilizers in
agricultural systems [7].
During the last decades, the relatively high prod-
ucts/fertilizer price ratio, incited farmers to adopt an
insurance strategy in fertilizer management: applying
excess of N to avoid any restriction in crop N supply
and then any penalty in crop yield. These practices,
when they were generalized on large agricultural areas
led to a progressive increase in soil N surplus accumu-
lation and an elevated risk of N leaching with dramatic
consequences on ground water quality [8]. Adoption of
a more restricted strategy for supplying and timing of
N fertilizers is now a prerequisite for a more sustainable
agriculture development. But such a strategy is difficult
to be adopted in practice because of the non-
predictable variations in weather which determines
both soil N mineralization and crop growth potential.
In consequence, a reduction in N application rates to
avoid surplus of N in soils would increase the proba-
bility of temporary crop N nutrition deficiency, and
then, as a consequence, an increased risk for a lower
crop production and then a deterioration of the eco-
nomic outputs the farmers are expecting. Therefore,
optimizing crop production with the goal of reducing
environment hazards requires an improvement in the
understanding of the regulatory mechanisms by which
crops absorb nitrogen from the soil and use it effi-
ciently for yield component and quality elaboration.
But it requires also a better understanding of soil
microbial processes and the interaction between
C and N cycles in order to optimize crop N residue
cycling, soil organic matter dynamics, and to improve
the capacity to predict soil N supply.
640 Crop Responses to Nitrogen
Fertilization management has to be conceived as
a mean to match as closely as possible crop N supply
with crop N demand in order to limit accumulation of
mineral N within soils, and then emissions to hydro-
sphere and atmosphere. By this way, matching timing
of N supply with timing of crop N demand appears
very necessary. It is fundamental to underline here the
necessity for a high precision for adjustment of
N supply to N demand: for an intensive wheat crop
producing about 8 t ha1 of grain, the total N demand
is about 240 kgN ha1 and the loss of only 20 kgN ha1
through leaching (i.e., less than 10% of the total
N demand) can lead to pass over the admissible limit
of nitrate concentration (50 mg l1) in drainage water!
Moreover, the loss of only 2–3 kgN ha1 through emis-
sion of N2O to atmosphere (that corresponds to 1% of
the total N demand) is considered as highly detrimental
for greenhouse gas effect. So the problem to face now is
not to understand how the yield of the different crop
species can be improved by addition of N fertilizers, but
(i) to determine the dynamics of crop N demands all
along the crop development cycle; (ii) to determine the
timing of the soil N supply according to soil character-
istics, climate, and soil agronomic management; (iii) to
determine the crop responses to different intensity and
timing of N nutrition deficiency; and then (iv) to
manage timing of crop N fertilization using diagnostic
and decision tools for optimizing trade-off between
minimizing crop yield reduction and minimizing
environmental impacts.
N in Plants
For most of the crop species, plant life cycle can be
roughly divided into two main phases: (i) the vegetative
growth phase, when young developing roots and leaves
behave as sink organs that absorb and assimilate inor-
ganic nitrogen for amino acid and protein synthesis,
and (ii) the remobilization phase when senescing tis-
sues start to behave as source organs translocating
organic molecules to ensure formation of new devel-
oping and/or storage organs [9]. The first phase is
dominated by the dynamic of leaf area expansion as
a mean for light capture, and then the role of nitrogen
in both leaf growth and leaf photosynthesis is capital.
The second phase is dominated by the development
of reproductive organs such as seeds, fruits, tubers,
and bulbs, or by storage organs allowing survival for
perennial species such as trunks for trees or roots and
stubbles for herbaceous species like grasses. As
a consequence, the same N absorbed by the plant can
be used successively for different functions and then
analysis of crop response to N nutrition cannot be
simply reduced to an addition of different elementary
functions, but as a complex and integrated adaptive
system with strong interactions among different
processes.
In plants, N is required primarily for the synthesis
of proteins, both structural and enzymatic, as the more
important components of cells. There is large variation
in the composition of the different cell types within
a plant according to the different types of tissues. Cells
which store carbohydrates or are part of support and
transport tissues have relatively lower protein and
nucleic acid concentration than metabolically active
cells within meristems and photosynthetic parenchyma
[10]. The relative numbers of each type of cells deter-
mine the organ composition. So organs that have rela-
tively slow metabolism but are specialized in support
and transport, such as roots and stems, contain little
protein and thus have small N concentration in dry
matter. Organs which store starch or other carbohy-
drate, such as grains, fruits, and tubers, have also low
N concentration. On the contrary, leaves having a high
metabolic activity through photosynthesis have high
N concentrations. So, plants are very heterogeneous
systems in terms of N concentration at every level of
organization: cells, tissues, and organs. Therefore plant
N demand, i.e., the quantity of N required for the plant
to achieve its potential growth and development,
depends on the morphologic and histologic plant
composition.
Leaves are essential organs by which plants capture
light and assimilate carbon. Light capture, photosyn-
thesis, and the associated respiratory processes require
a large number of different enzymes, pigments, and pro-
teins [11]. About 50% of the soluble protein of the leaf
is in RuBPc-o enzyme alone, another 25% is in the light
harvesting and electron transport components [11].
Thus the chloroplast, as the location of RuBPc-o and
other photosynthetic enzymes, and according to its
high protein membrane component, contains the
major part of the total leaf N. Composition of leaves
and their N concentration varies with age, development

stage, and environment. Leaves of cereals have the
greatest concentration of N just after their full expan-
sion that corresponds to their maximum in photosyn-
thetic activity. After a period the N concentration in the
leaves decreases in parallel with metabolic activity
as senescence progresses [12]. Remobilization of
N-components occurs during the senescence process,
and then amino acids from proteolysis are transported
to younger developing leaves, or reproductive organs.
A consequence of this ageing and senescing processes is
that older leaves situated in the lower canopy where
light intensity is limiting for photosynthesis, provide
N for new leaf production at the top of the canopy. This
recycling process thus tends to optimize photosynthe-
sis in relation to N supply and light at the level of the
canopy [13].
To achieve large rates of photosynthesis in well-
illuminated conditions, leaves require a large concen-
tration of RuBPc-o and other N-components and,
hence, large N concentration [14]. As N supply rises,
the amount of N per unit area in leaves increases,
enhancing the rate of CO2 assimilation [12, 15]. Thus,
there is a good correlation between CO2 assimilation
rate and leaf N content. However, with very large
N supply, leaf N content may increase without any
increase of photosynthetic rate, unless larger CO2 con-
centrations are used [16]. Plants with C3 photosyn-
thetic pathway contain more N per unit leaf area than
those with C4 metabolism. So C4 crops use N more
efficiently than C3 ones [17]. Ample N increases the
number of chloroplast compared with deficient N. Also
the density of protein in the stroma is increased by large
N supply. All these processes contribute to accumulate
N within leaf tissues as plant N supply increases.
Although the primary role of RuBPc-o in plants is
to assimilate CO2, a storage function for N has been
mooted for this enzyme [18, 19]. Thus, N of RuBPc-o
have two successive functions within plants: CO2
assimilation and source of N supply for reproductive
and storage organs. Storage of N within the plants for
further reuse can be considered as an adaptive mecha-
nism for buffering plant N nutrition in highly variable
soil N supply conditions. In this way, plants can store
N reserves during periods of vegetative growth and
large soil N availability, and then to reuse these
N compounds at the end of their life cycle when min-
eral soil N is exhausted. Accumulation of RuBPc-o in
Crop Responses to Nitrogen 641
excess within leaf tissues is not the only way for plant to
store N. Accumulation of vegetative storage proteins
(VSP) within different plant tissues or organs is
a complementary way for storing N reserves, as dem-
onstrated in alfalfa [20] and in perennial grasses [21].
For perennial plants, the storage of N reserves within
perennial organs such as roots, rhizomes, tubers, or
trunks is a prerequisite for regrowth after defoliation
or after winter.
Due to the importance of light capture and photo-
synthesis for plant growth, much attention has been
paid to allocation of N to leaves within canopies. How-
ever, allocation of N to other vegetative tissues is also
quantitatively important. Green leaf N content repre-
sents only 53% of total shoot N in a lucerne crop [22],
and only 30% of shoot N in a wheat crop at the
beginning of the grain filling period [23]. Lemaire
et al. [24] showed that for a large number of crop
species there exists trade-off between accumulation of
N in leaves for optimizing crop photosynthesis and
accumulation of N within stem tissues for optimizing
the plant architecture development and the elaboration
of reproductive organs.
N compounds as amino acid and proteins are used
within meristematic tissues for initiation and expan-
sion of new organs: leaves, stem internodes, tillers, and
branches and roots, and also inflorescences, fruits, and
seeds. The use of N for leaf area expansion during
vegetative growth period is of first importance because
it determines the dynamics of LAI expansion of the
crop and then the capacity of the crop to intercept
light and to accumulate biomass. The rate at which
N is supplied to meristems greatly determines cell pro-
duction rate while the final cell size is only little affected
[25–27]. As cells enlarge in size, their N concentration
decreases through a dilution by accumulation of
greater quantities of N-free compounds until final size
is reached. So any limitation of N supply to meristem
tissues leads to a reduction in the cell production rate
and in the size of the final organ produced. For repro-
ductive meristems, shortage of N reduces more the
number of grains than the grain size [28–30].
When analyzed at the level of a plant population, it
has been clearly demonstrated that N is allocated to
individual plants according to their own contribution
to interception of light [31]. N resources are allocated
preferentially to dominant plants and then stressed
642 Crop Responses to Nitrogen
plants cannot respond to N supply because of the lack
of light. In consequence, competition for light among
individual plants within a dense canopy determines
greatly competition for N resources.
In conclusion, plants and canopies are very hetero-
geneous systems in terms of N content, concentration,
and repartition. This heterogeneity can be analyzed at
different levels of organization: organite, cell, tissue,
organ, whole plant, and plant population. N com-
pounds, mainly as proteins are involved in all meta-
bolic processes and then they are used successively for
different functions such as cell division, organ growth
and development, light capture, photosynthesis and
respiration, reserve formation and recycling. Once
absorbed by the plant, N is used several times through
internal recycling processes and reallocation to the
different plant parts. So analysis of plant N nutrition
is very complex because it involves several inter-
dependent metabolic functions and due to constant
feedback mechanisms, it can be studied only through
a dynamic approach.
Crop N Demand
Crop N demand (N expressed in kg ha1) at any time of
the crop cycle can be defined as the result of critical
crop mass (Wc) that is the maximum crop mass attain-
able in a given environment without any limitation of
nutrients and its critical plant N concentration %
Nc [32]:
N ¼ %Nc Wc ð1Þ
The critical plant N concentration (%Nc) has been
defined as the minimum plant N concentration
corresponding to maximum crop mass [33]. This con-
cept can be applied in dynamic terms, such that the
daily crop N demand (or critical N uptake rate
expressed in kg ha1 day1) is the quantity of N the
crop has to absorb each day to maintain its potential
growth rate over a given period of time.
Many studies conducted on a large range of crop
species (see [34] for a review) have shown that the
critical plant N concentration (%Nc) is regularly
decreasing as crop mass accumulates during the crop
growth period. Moreover, it has been shown that this
dynamic of decline in %Nc could be represented by
a unique and constant relationship whatever the
conditions and the genotypes for a same species [35]:
%Nc ¼ aðWcÞb ð2Þ
Coefficient a represents the critical plant
N concentration for Wc = 1t ha1. Coefficient b is
dimensionless, and it represents the dynamic of the
dilution of N within the dry matter.
Mixing Eqs. 1 and 2 allows the expression of the
dynamics of the critical crop N uptake, i.e., the crop
N demand, in relation with the dynamics of potential
crop biomass accumulation:
Nc ¼ a0 ðWcÞ1b ð3Þ
where coefficient a0 is the crop N demand (or the
critical N uptake) for a potential crop mass accumula-
tion of 1 t ha1. Value of a0 is equal to 10a, when a is
expressed in % and a0 is expressed in kg ha1.
Values of a (and a0 ) and b have been established for
the main cultivated species according to the method
developed on wheat by Justes et al. [36]. This method as
illustrated on Fig. 1 requires a family of response curves
of plant N concentration (%N) vs crop mass (W)
across increasing N supply rates. For each response
curve, the critical plant N concentration (%Nc) is esti-
mated as the intersection point between the oblique
line representing the response of both %N and W to
increased rate of N supply, and the vertical line
representing the increase in %N with further
N supply rates once the maximum crop mass (Wc) is
reached that correspond to luxury N accumulation. As
the determination of %Nc is done at different stages of
growth for a large range of value of W, then it is possible
to fit the series of %Nc–Wc data points for getting the
critical N dilution curve and to calculate the value of
coefficients a and b.
These values are presented in Table 1. For a given
species, coefficients a (and a0 ) and b remained constant
whatever the climatic conditions (years and locations).
Moreover, Lemaire et al. [37] showed that for wheat,
maize, canola, sorghum, and sunflower the same value
of coefficients holds either in temperate or in subtrop-
ical conditions. Comparison among species allows
a clear distinction between C3 and C4 groups for the
value of coefficient a (and a0 ) while coefficient b is
unaffected by the type of the metabolic pathway.
Within each of the metabolic groups, it is difficult to
 Crop Responses to Nitrogen 643

4.0

3.5

3.0
%N (g N (100 g DM)–1)

2.5

2.0

1.5

1.0

0.5

0.0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
W (t ha–1)

Crop Responses to Nitrogen. Figure 1

Determination of the critical plant nitrogen (N) concentration of maize crops as the intersection point between the oblique
line corresponding to the response of both plant nitrogen concentration (%N) and crop mass (W) to increasing
nitrogen supply rates, and the vertical line corresponding to the increases in plant N concentration without corresponding
increase in crop mass that corresponds to luxury nitrogen accumulation [46]

establish clear differences between species because of Crop Responses to Nitrogen. Table 1 Values of coeffi-
the high correlation between the coefficient a (or a0 ) cient a0 and b of Eq. 3, N = a0 (Wc)1b, for different crop
and b. Then the fitted curves corresponding to Eq. 2 for species
the different crop species are relatively close to each
a0 (kgN b
other [38]. Crop species ha1) (dimensionless) References
The derivative of Eq. 3 allows the expression of the
Temperate 48 0.32 [39, 40]
crop N demand in dynamic terms:
grasses (C3)
  Alfalfa (C3) 48 0.33 [41]
dN dN dW dW
¼ ¼ ac ð1  bÞW b ð4Þ
dt dW dt dt Pea (C3) 51 0.32 [42]
Wheat (C3) 53 0.44 [36]
Equation 4 shows that the daily crop N demand
Canola (C3) 45 0.25 [43]
follows the daily crop growth rate, but for a similar
daily crop growth rate, the daily crop N demand Rice (C3) 52 0.52 [44]
declines as the crop mass increases. Tomato (C3) 45 0.33 [45]
A theory has been developed for explaining Maize (C4) 34 0.37 [46]
the close relationship between crop N demand and
Sorghum (C4) 39 0.39 [47]
crop biomass accumulation rate [33, 35]. The hypoth-
esis is that plant mass W is composed of two compart- Tropical 36 0.34 [48]
grasses (C4)
ments: Wm, the metabolic tissues involved directly in
644 Crop Responses to Nitrogen

plant growth processes (photosynthesis and meristem crop growth. Just after seedling, and until a crop mass
activity) with a high N concentration %Nm; of approximately 1.5 t ha1 is reached the value of b
and Ws, the structural tissues involved in plant archi- and a is high, close to 0.90–0.95 while it decreases
tecture and transport with a low N concentration %Ns. rapidly to a value of 0.6–0.7 after this stage when
Then: competition for light between individual plants within
the canopy is established.
W ¼ Wm þ Ws ð5Þ Lemaire et al. [37] tried to determine among the
and: two parameters, crop mass or LAI, who is the funda-
mental driver of crop N demand. They concluded that
1 Eq. 3 was the more robust across genotypes and envi-
%N ¼ ð%NmWm þ %NsWsÞ ð6Þ
W ronments, but in fact the two Eqs. 3 and 11 each
Supposing that Wm increases allometrically with represent an incomplete expression of the feedback
W, then: regulation by shoot growth of N absorption capacity
of the roots and of N partitioning within the canopy as
Wm ¼ kW a ð7Þ
described within the above section. A more complex
and then: analysis of N allocation between leaf and stem and of
remobilization of N from old leaves would be necessary
%N ¼ k ð%Nm  %NsÞW a1 þ %Ns ð8Þ to better capture genotypic differences in N uptake
This equation is very close to the empirical Eq. 2. capacities. Nevertheless, the robustness of the relation-
The main difference is the asymptotic value of %Nc ship between critical crop N uptake (Nc) or critical
which is equal to %Ns and not to zero. But the crop N concentration (%Nc) with crop mass accumu-
difference is very little for the range of W from 1 to lation (W) across environments and genotypes allows
15 t ha1[35]. the use of these empirical relationships as a base for the
Following Hardwick [49] assumptions, it can be determination of crop N demand and for diagnosis of
postulated that because Wm is associated mainly with crop N status.
photosynthesis it scales for plant area, and then to crop
Leaf Area Index (LAI): Regulation of Crop N Uptake
Wm ¼ pðLAIÞ ð9Þ The theory developed above indicates that plant
and then: N uptake seems to be regulated by plant growth itself.
In steady state N supply conditions, plant N uptake is
k
LAI ¼ W a ð10Þ feedback regulated by shoot signals, with a positive
p signal from photosynthetic C supply and a negative
So Eqs 3 and 10 indicate that both N uptake and one from reduced N recirculation to roots [50–52].
LAI are allometrically related to crop mass. For a large So the relationship between N uptake and LAI as
range of crop species, cultivated either under temperate attested by Eq. 11 can be explained by the fact that
or sub-tropical climate, the hypothesis for a common LAI expansion allowing increased light interception
value of coefficients b and a cannot be rejected, but provides larger C supply to roots, and also increases
then a direct relation of proportionality could be N storage capacities within expanding leaves as
established between crop N uptake and crop LAI [37]: RuBPc-o that avoids the depletion of N uptake by
recirculating reduced N compounds in the phloem.
a0 p
N¼ LAI ð11Þ This leads to the proportionality between crop
k N uptake and LAI for most of the species, but the
So, at crop level, the dynamic of expansion of LAI is slope of the relationships, i.e., the N uptake per unit
driving the crop N demand. LAI, is variable across species according to their mor-
Lemaire et al. [37] showed that the two coefficients phology and, more particularly, their leaf/stem ratio. So
b and a evolves in parallel during the time course of the leaf area expansion is not the only way for plant to store

reduced N. Stem growth and leaf thickness increase are
also a possibility for sequestrating N to avoid the
repression of N uptake capacity of roots. In conse-
quence, a more general and stable relationship between
N uptake and crop mass can be obtained as attested by
Eq. 3. Nevertheless, this relationship is not linear
because N uptake per unit of crop mass decreases as
the LAI per unit of crop mass, i.e., the leaf area ratio
(LAR) of the crop, decreases, that determines the
N dilution effect.
In the field, in a variable N supply condition, plant
N uptake is co-regulated by both crop growth rate
potential and the soil N availability. Two groups of
transport systems, with low and high affinity for
nitrate, operate in plants [53]. Devienne-Barret et al.
[54] have proposed a model accounting for this co-
regulation of plant N uptake by soil nitrate concentra-
tion and plant growth:
 
dN b dW
¼ ac ð1  bÞ W
dt dt max
  ð12Þ
C C
VH þ VL
KH þ C KL þ C
where V and K are coefficients of the Michaelis–Menten
formula and subscripts describe the high (H) and the
low (L) affinity transport systems for nitrate; C is the
actual concentration of nitrate in soil solution, W is the
crop mass in t ha1 and b is the allometric coefficient of
Eq. 3.
Figure 2 represents the crop N uptake vs crop mass
trajectories for different steady-state N supply condi-
tions, i.e., C = constant. Among these curves, it is then
possible to identify the critical N uptake curve as
defined above. It is also possible to imagine that the
maximum N uptake curve corresponds to the higher
quantity of N a crop is able to accumulate at a given
crop mass. The difference between this curve and the
critical curve correspond to the quantity of luxury
N the crop is able to store.
So whatever the cause of the variation in crop mass
provided that N supply remains at steady state, any
increase in crop mass (DW) is accompanied by
a corresponding increase in crop N uptake (DN). As
soil N supply increases, plant N uptake increases as
a consequence of both (i) the increase in soil
N concentration (C) and (ii) the increase of plant
Crop Responses to Nitrogen 645
160
140
120
N uptake kg/ha
100
80
Maximum
60 Crit.
40 Fert.
Soil
20
0
0 5 10 15 20 25
W t/ha
Crop Responses to Nitrogen. Figure 2
Nitrogen (N) Uptake – Crop Mass (W) trajectories for
different steady states N supply: critical N uptake (Crit.),
maximum N uptake (Maximum), non-fertilized (Soil), and
suboptimal N application (Fert.). The two lines represent
either (i) two growth stages of the same crop, (ii) two crops
having different growth rate, or (iii) an environmental
effect. The lines represent the response curves to increased
N supply
growth rate itself. So, at any moment, crop growth
rate is the consequence of crop N uptake and vice versa.
Diagnosis of Crop N Status: Nitrogen Nutrition
Index
The main consequence of the theory developed above is
that neither the plant N concentration nor the crop
N uptake per se can indicate unequivocally the crop
N nutrition status. Eqs. 2 and 3 indicate that these
two variables have to be interpreted in relation with
crop mass.
As shown in Fig. 2, the critical N uptake curve
separate situations where N supply is limiting crop
mass accumulation for situations where N is accumu-
lated in excess without any supplemental increase in
crop mass. For a given situation and at any time course
of the growth period of the crop characterized by
an actual crop N uptake (Na) corresponding to an
actual crop mass Wa, it is possible to determine
a Nitrogen Nutrition Index (NNI) as the ratio between
Na and the critical N uptake, Nc, corresponding to the
same crop mass provided the critical N uptake of the
crop species has been determined. NNI can also be
646 Crop Responses to Nitrogen
Shoot N concentration
Critical shoot N
%Nc
Actual shoot N
%Na
Wa
Actual crop biomass
Crop Responses to Nitrogen. Figure 3
The use of the critical N dilution curve for the determination of the nitrogen nutrition index
determined directly from actual plant N concentration
and dilution curves as illustrated in Fig. 3:
%Na
NNI ¼ ð13Þ
%Nc
Values of NNI close to 1 indicate that at the date of
the determination of Na or %Na the crop were in
situation of non-limiting N supply. Values more than
1 indicate a luxury consumption of N. Values less than
1 indicate an N deficiency, the intensity of which can be
estimated by the value of the NNI: a value of 0.6
indicating that crop N availability was only 60% of
the critical level. Such an index of crop N status has
been used as a diagnostic tool for analyzing a posteriori
agronomical data from field experiments or farm
observations in order to explain variations in yield by
differences in crop N status [55].
Nevertheless, this approach does not take into
account that the minimum plant N concentration is
not 0 but is equal to %Ns (Eq. 8), which is the mini-
mum plant N concentration for the plant to stay alive
as postulated by Angus and Moncur [56]. So a more
complex nitrogen nutrition index can be calculated:
0%Na  %Ns
NNI ¼ ð14Þ
%Nc  %Ns
This new index, NNI0 , is therefore physiologically
more relevant than NNI, but it involves a greater degree
of uncertainty related to the value of %Ns which has
Supra-optimal N nutrition
Critical N “dilution” curve
%N = a (W )–b
Sub-optimal N nutrition
Crop biomass (W )
not been documented for many crop species. Lemaire
and Gastal, [35] by using Eq. 8 derived a value of 0.77%
and 0.82% for %Ns for wheat and maize, respectively.
So variation in %Ns across crop species could be not
very important. Therefore, the use of Eq. 2 for deter-
mining crop N status can be recommended owing to its
simplicity.
NNI estimates the instantaneous crop N status at
the period of time when actual plant N concentration
%Na and actual crop mass Wa are estimated. But under
changing N supply in the field, it is necessary to deter-
mine NNI several times during the growth cycle. An
integrated value of NNI can be obtained by the
weighted mean of NNI during the different growth
periods, each time interval representing the duration
in days or degree-days. Lemaire and Gastal [35] showed
that it was possible to establish a linear relationship
between the NNIint and the relative biomass accumu-
lation as expressed as the ratio between actual (Wa) and
potential (Wm) biomass:
Wa=Wm ¼ K ðNNIint  NNI0 Þ ð15Þ
where K is the response of the crop to increment in its
average N nutrition status and NNI0 is the minimum
crop N status to allow plant growth. This minimum
theoretically corresponds to %Ns.
Jeuffroy and Bouchard [57] characterizes the
N deficiency period of a wheat crop by both its length

NNIi
1 (LAR) as crop mass increases, and (ii) the preferential
ni
N NNI. Then, it would be possible to use the
Nb of days
Crop Responses to Nitrogen. Figure 4
Estimation of NNIint from a sequential determination of
NNIi : NNNint = 1/N SNNIini The intensity of N deficiency (ID)
is estimated by the lowest value of INNi, and the duration
of the deficiency (DD) is equal to the number of days
with NNIi < 1
(deficit N duration, (DD) and its intensity (ID) by
means of the 1-NNI minimum observed value, and
they calculated an integrated index of crop N status
by the mean of the product ID
DD = IDD that
represents about twice the area between the curve of
NNIi dynamic and the horizontal line NNIi = 1
(Fig. 4). They showed that IDD explained 96% of the
variation in grain number of wheat within a large
experimental sites x years data set.
So it is clear that NNI is a good basic tool for
analyzing actual plant status in crops, and then to
interpret agronomical data in field conditions in
order to detect if or not plant N deficiency periods
occurred, with which intensity and timing and to ana-
lyze the consequences on crop growth and the crop
yield components. But despite its high informative
value as diagnostic tool for crop N status, NNI is
difficult to use practically in field conditions and it
remains more a research than an agronomical manage-
ment tool. NNI determination is time consuming
because of the necessity to determine crop mass. Then
it is necessary to use noninvasive and cost-effective
methods for a rapid and operational determination of
plant N status, and then to use NNI as a reference for
calibration of these indirect methods.
The theory developed above showed that the
N dilution effect and then the dependency of plant
Crop Responses to Nitrogen 647
N concentration from the crop mass is the result of
two processes: (i) the decline in plant leaf area ratio
allocation of N to the well-illuminated upper layer of
leaves as canopy develops. Therefore, Lemaire et al.
[58] suggested that while plant N concentration
declines with crop mass accumulation, the N content
per unit of leaf area within the upper layer of leaves
was more stable and would correlate well with the crop
N concentration of the well-illuminated leaf layer as
an indicator of crop N status independently of the crop
mass. Farrugia et al. (2004), using this correlation, were
able to develop a method of diagnostics of grassland
N status [59] and of maize crop N status [60]. The leaf
N concentration can be measured directly from leaf
samplings, but it can also be estimated indirectly by
noninvasive methods. The leaf color chart (LCC) is an
easy-to-use and inexpensive diagnostic tool for moni-
toring the relative greenness of a rice leaf as an indicator
of the leaf and then of the plant N status [61]. The
estimation of chlorophyll content of leaves by portable
systems based on leaf transmittance or leaf reflectance
in specific wave bands is also well correlated with leaf
N content, and can be a method for crop N status
diagnosis [62, 63]. But these predictions are in general
dependant on cultivars and years [64]. Houlès et al.
[65] demonstrated that through remote sensing it was
possible to estimate both crop LAI and the quantity of
chlorophyll per unit of soil area, and then the quantity
of N within the canopy per unit of soil area. Therefore
it would be possible to recalculate the NNI of the crop.
Such an estimate can be intensively repeated in space
and time that allows very precise information on the
spatiotemporal dynamics of crop N status that is very
useful for precision agriculture.
Crop Responses to N Deficiency
The use of the concept of critical crop N concentration
and the possibility for diagnosis of the actual crop
N status totally reversed the approach of the response
of crop to N fertilizers. The response of crop to N was
studied by analyzing the response curve of yield to
increasing fertilizer rates.
The difficulty for such an approach is that (i) the
actual N supply for the crop is not known, because the
648 Crop Responses to Nitrogen
quantity of N provided directly by the soil, and then
the yield in absence of any fertilization is very variable,
and (ii) the potential yield, i.e., the yield achievable in
a given climatic condition when N supply can be con-
sidered as non-limiting is unknown. So, the crop
N demand and the total crop N supply are unknown
variables and it is possible to get only the regression
between increment in yield and increments in
N fertilization rates. This approach led to provide
huge families of crop response curves to N fertilizers
very variable to each other according to soils, climates,
years, species, and cultivars, and the only way was to
analyze this variability with statistic approaches with
any possibility to identify and to quantify processes in
order to develop prediction models.
The possibility for diagnosis crop N status allowed
the identification of situations where crop growth and
development were not limited by N deficiency, and
then to develop crop potential growth models as
resulting from climate conditions. Then using the
critical N uptake concept, it was possible to derive
from these models the dynamics of crop N demand
all along the crop cycle. So instead of having the check
treatment with no N fertilizer and to analyze response
of crop to increments in N supply, the check treatment
is now the crop with critical N status, and then the
response of the crop to the occurrence of periods of
N nutrition deficiency of different timing and inten-
sity all along the crop growth cycle and the conse-
quences on yield components and quality of the yield
can be analyzed.
N Deficiency Effects on Crop Mass Accumulation
According to Monteith [66], crop mass accumulation is
linearly related to the quantity of photosynthetic radi-
ation (PAR) intercepted by a crop during its life cycle.
The slope of this regression can be interpreted as the
radiation use efficiency (RUE).
As illustrated in Fig. 5, and as shown by Bélanger
et al. [67], N deficiency affects both the quantity of PAR
intercepted by the crop and the efficiency with which
the intercepted radiation is used for biomass elabora-
tion (RUE). These authors, using NNI, have shown that
in relative terms, RUE was more affected by moderate
N deficiency (NNI = 0.6–0.8) than the quantity of PAR
800
700
600
Biomass g m–2
500
400
300
200
N0
N60
100 N240
0
0 50 100 150 200 250 300 350
Intercepted PAR MJ m–2
Crop Responses to Nitrogen. Figure 5
Relationships between the quantity of photosynthetic
active radiation (PAR) intercepted by a tall fescue sward
and the accumulation of aboveground biomass for three
contrasted N supply rate: N0: no N application; N60: 60 kgN
ha1; N240: 240 kgN ha1. The slope of the regression
represents the radiation use efficiency [67]
intercepted while for more severe deficiency, the
response of the two variables converged. This type of
response has been confirmed on sunflower [68] and on
sorghum and maize [69]. LAI expansion seems a little
bit more responsive to N deficiency than RUE: for an
NNI of 0.6, RUE was reduced by 30% while LAI was
reduced by 40% (Fig. 6).
RUE is an integrated variable accounting for pho-
tosynthesis and respiration, but also the allocation of
assimilates to root [70]. These authors showed that, in
fact, canopy gross photosynthesis was less affected by
N deficiency than the accumulation of total biomass
reflecting higher respiration losses in N-deficient crops,
and the accumulation of shoot biomass was more
affected than the accumulation of total biomass
reflecting then an important increase of allocation of
biomass to roots in N deficient situations. The lower
shoot/root ratio in N deficient crops is widely
documented [71, 72]. In fact this increased allocation
of assimilate to roots is the consequence of the lower
activity of shoot meristems (leaf and stem extension)
that allows a greater quantity of carbon to be used for
root growth. So the more sensible plant growth process
to N deficiency appears to be the leaf area expansion
rate, with two major consequences: (i) a reduction

1,2
1 That demonstrates the high responsiveness of leaf
Relative responses
0,8
0,6
0,4
0,2
0 reduction in crop mass (i.e., simply a crop size effect)
0 0,2 0,4 0,6 0,8
Nitrogen nutrition index
Crop Responses to Nitrogen. Figure 6
Effects of crop N status determined by the nitrogen
nutrition index (NNI) of tall fescue swards receiving
different N application rates and (i) the relative quantity of
intercepted PAR, PARact/PARmax (●), (ii) the relative
Radiation Use Efficiency, RUEact/RUEmax (■), and the
relative LAI, LAIact/LAImax (▲). (Redrawn from [67])
in the dynamic of light interception and then in
the canopy photosynthesis and crop C supply and
(ii) a preferential allocation of C for root growth that
contributes to increase root foraging activity for a fur-
ther increase in N uptake capacity.
The response of leaf area of plants and canopies to
N deficiency is brought about by a decline in the
expansion rate and size of individual leaves combined
with reduction of branching or tillering. The accumu-
lation of nonstructural carbohydrates in N-deficient
leaves indicates that C supply is not the cause of the
leaf area expansion under N deficiency [38].
N deficiency decrease the rates of cell division and
cell expansion with little effect on final cell length [26],
so the reduction of leaf size in N-deficient plants is
mainly due to a reduction in cell number. Gastal et al.
[73] proposed a quantitative relationship between the
leaf elongation rate (LER) of tall fescue and the NNI of
the sward:
LERactual
¼ 1:39  1:9e 1:49NNI
LERcritical
Subscript “actual” corresponds to any suboptimal
N condition, and subscript “critical” refers to a non-
limiting N nutrition. For a severe limitation in
N nutrition, NNI = 0.4, LER is then reduced to about
Crop Responses to Nitrogen 649
30% of its maximum value in non-limiting conditions.
expansion to N deficiency.
Lemaire et al. [24] used the relationship between
LAI and crop mass (W) of Eq. 10 for studying the
different types of response of crop species to
N deficiency. This approach allows the separation of
the reduction of LAI being directly associated with the
1 1,2 1,4 from any specific reduction of LAI at similar crop mass
(i.e., a modification of plant architecture through
a reduction of leaf area ratio. They demonstrated that
under a similar intensity of N deficiency as estimated
through NNI, the reduction of LAI of maize is totally
allometrically related to its reduction in crop mass
(crop size effect), while for wheat N deficiency pro-
vokes in supplement a reduction of LAI at same crop
mass, i.e., a reduction in LAR. These two types of
responses represented two extremes and tall fescue
behaves like maize while canola behaves like wheat;
sorghum and sunflower having intermediate responses
[24]. Hence classification of crops in either metabolic
(C3 vs C4) or botanical (monocots vs dicots) groups
does not correspond to any particular response type.
In response to N deficiency, some species such as maize
or tall fescue tend to maximize light interception by
minimizing the reduction in LAI, at the expanse of
N concentration per unit leaf area, and then leaf pho-
tosynthesis, while other species such as wheat and
canola tend to develop an inverse response. Do these
two opposite strategies mean that there exists a trade-
off between photosynthesis per unit of LAI and leaf
area expansion as already proposed by Sinclair and
Horie [74]?
N Deficiency Effects on Leaf and Canopy
Photosynthesis
The response of leaf photosynthesis to irradiance
largely depends on the leaf N content. Leaf photosyn-
ð16Þ thesis at saturating light intensity (Amax) increases
asymptotically with leaf N content [75]. This relation-
ship shows a positive intercept on the leaf N content
axis, indicating that when leaf photosynthesis rate
becomes zero, leaves would still contain significant
amount of N, corresponding the structural leaf
650 Crop Responses to Nitrogen
N (Ns) of Eq. 8. The variation in the Amax/SLN (leaf
N content per unit leaf area) relationship seems rela-
tively limited among cultivated species of the same
metabolic group [10, 76], despite Sinclair and Horie
[74] showed a lower Amax at similar SLN for soybean.
This variation among species could be due to (i) dif-
ferences in nitrogen costs of PEP-carboxylase and
RuBPc-o and the relative amounts of these two
enzymes in leaves, and/or (ii) the possible accumula-
tion of vegetative storage proteins within leaves of
legume species such as soybean.
There is a large discussion whether Amax has to be
related to either leaf N content per unit mass or per unit
leaf area [75]. Lemaire and Gastal [32] indicated that
none of these two relationships are completely right.
The two components of photosynthesis, light
harvesting by chlorophyll and CO2 reduction by
RuBPc-o, are affected by leaf N status, and these two
processes have to be expressed on a leaf tissue volume
basis, and not only on an area basis. Then leaf thickness
is an important parameter to take into account. Spe-
cific leaf area (SLA) is the parameter allowing corre-
spondence between leaf N content per unit area and per
unit dry matter basis. But relationship between leaf
thickness and SLA is weak because of the variations of
nonstructural carbohydrate content within leaves.
The quantum efficiency that is the response of leaf
photosynthesis to light at low irradiance is only little
affected by N deficiency [77, 78]. Moreover, the dark
respiration of leaves seems to increase with increasing
leaf N [78]. Hence, as the leaves are progressively
shaded within the canopy the effect of N deficiency
on leaf net photosynthesis becomes lower and lower
and then negligible.
Gastal and Bélanger [77] showed that canopy gross
photosynthesis of a tall fescue sward at high irradiance
(CGPmax) only responds smoothly to N deficiency:
a reduction in NNI from 1 to 0.4 reduced the relative
CGPmax from 1 to 0.6 only. This low responsiveness of
canopy photosynthesis to N deficiency is due to the fact
that as canopy develops, a greater number of leaves are
shaded and then their photosynthesis does not respond
to N shortage. When irradiance becomes more limited,
the responsiveness of canopy photosynthesis to
N deficiency becomes more limited. So when canopy
photosynthesis is integrated over day and for a long
period where crop LAI is high, the response of canopy
photosynthesis to N deficiency appears relatively
limited, which explains the limited influence of
N deficiency on RUE.
N deficiency Effects on Harvest Index and
Components of Grain Yield
For grain crops, yield is closely related to grain number
per unit area of soil. The elaboration of grains depends
on flows of C and N compounds to reproductive
meristems during a narrow window period around
flowering, anthesis, and very initial grain development,
and also on environmental conditions (temperature,
radiation, and water stress) during this period. This
critical period coincides with the maximum rate of
crop N uptake [79]. So any limitation of crop growth
rate at this period by N deficiency decreases grain
number and then grain yield on cereals such as maize
[28], wheat [29, 80]. Jeuffroy and Bouchard [57]
established for wheat a relationship between grain
number and the severity and duration of the
N deficiency before anthesis as calculated by NNI
method. The effect of N deficiency on grain
number is the consequence of two simultaneous effects:
(i) a lower crop growth rate at anthesis restricting
C supply to spikelets and then a spikelet abortion
[81]; and (ii) a decrease in the N content in the
spike stems [29] corresponding to a direct effect of
N deficiency on floret fertility.
The other grain yield component, grain weight, is
generally less affected by N deficiency at anthesis than
the grain number [47]. However, it is necessary to take
into account the negative correlation between grain
weight and grain number: a reduced grain number
resulting from pre-anthesis N deficiency can lead to
a more favorable source:sink ration during grain filling
period and then to an unaffected grain weight. Grain
filling in both carbohydrates and proteins depends on
(i) the recycling of C and N compounds previously
accumulated within vegetative organs during pre-
anthesis growth period, and (ii) post-anthesis photo-
synthesis and N absorption. The relative importance of
these two components depends on plant species and
their capacities to store C and N compounds in their
vegetative organs. So some species such as wheat or rice
are able to develop large LAI [7–9] and then to store
large quantities of N, then they are able to feed 80–90%

of the N demand for grain development by recycling
N stored within vegetative biomass, while other species
such as maize, because they develop less LAI [4, 5]
with lower N content because they have C4 metabolism,
are obliged to feed 40–60% of their grain N demand
through post-anthesis N absorption. As a consequence,
crop species like maize are more susceptible to terminal
soil N shortage than crop species like wheat or rice.
Delaying leaf senescence for species like maize or
sorghum should allow these species to continue to
maintain high N absorption rate after anthesis. The
supplement of carbohydrates allocated to roots permit-
ted by this delayed leaf senescence allows the mainte-
nance of root absorption capacity [82]. So using
stay-green genotypes in low N supply conditions
seems to be beneficial for both sorghum [83] and
maize [84].
Nitrogen Use Efficiency in Crops
From an agronomic point of view, nitrogen use effi-
ciency (NUE) of a crop represents its capacity to pro-
duce a supplement of yield (dY) for each added unit of
N fertilizer (dNf) that corresponds to the derivative of
the crop yield response curve to the rate of N supply:
Y = f(Nf). As this response curve is asymptotic, NUE
generally declines with the higher rate of N supply,
indicating that the first N unit applied is more effi-
ciently converted into yield than later applications.
According to the crop species types, Y can represent
either the aboveground biomass, as for forage crops, or
the grain part as for cereals, grain legumes, or oil seed
crops.
Such a global approach does not allow a clear mech-
anistic analysis of the physiological traits controlling
NUE between different species and cultivars because
crops respond to total N supply (Nt) that include
fertilizer supply (Nf) and soil N mineralization (Ns).
So according to variations in Ns due both to soil and
climatic conditions and to previous crop management,
different value of Nt can be obtained with the same Nf,
leading to large differences in NUE. For this reason,
Moll et al. [85] propose to define NUE as the yield
produced per unit of N available in the soil, considering
two components:
1. The Nitrogen Absorption Efficiency (NAE) which
measures the ability of a crop to uptake N from soil
Crop Responses to Nitrogen 651
2. The nitrogen conversion efficiency (NCE) which
measures the ability of a crop to use absorbed
N for dry matter and grain production
NUE ¼ðNAEÞðNCEÞ ð17Þ
Nitrogen Absorption Efficiency
Genotypic differences in N uptake at different levels of
N supply have been shown in rice [86], wheat [87], and
maize [88]. Modern cultivars have a higher N uptake
capacity because they have a higher biomass produc-
tion. This effect is accounted by Eq. (x) as it shows that
the crop N uptake rate increases with crop growth rate.
So any factor enhancing the potential crop growth rate,
genotype or environment, increase de facto the
N uptake capacity of the crop. More interesting from
a plant breeding point of view would be to increase the
N uptake capacity of crop at similar crop biomass
production. Lemaire and Gastal [32] reported data
comparing N uptake of tall fescue and cocksfoot swards
at similar biomass. These data show that cocksfoot
had a higher N uptake capacity than tall fescue
under a sub-limiting N supply condition, while the
two species had similar N uptake capacity under non-
limiting N supply. Similarly, Lemaire et al. [89] showed
that grain sorghum had a higher N uptake capacity
than maize under limiting N supply, while the two
species had similar N uptake under non-limiting
N supply.
The fact that N uptake capacity and then nitrogen
absorption efficiency (NAE) does not vary too much
among crop species is due to the fact that the critical
N uptake curve, as described by Eq. 2 is not very
different among species of the same metabolic group
(C3 vs C4). Then differences between crop species in
NAE under non-limiting N supply conditions would
reflect their differences in their potential biomass
accumulation according to their respective metabolic
group. Under limiting N supply conditions, clear dif-
ferences emerge among species. As for cocksfoot vs tall
fescue, sorghum appears to have a greater root length
density than maize. This would confer these species
a better capacity of interception of soil mineral N.
Soil N recovery that determines NAE is the result of
the nitrogen balance between crop N uptake rate,
immobilization by soil microbial communities and
losses through leaching, denitrification, and
652 Crop Responses to Nitrogen
volatilization [90]. Root architecture and biochemical
composition, and perhaps root exudates could play
an important role in this balance, and then on NAE.
So because of a permanent turnover of N in soil,
plants having a dense root system are able to compete
more efficiently against microbes for capturing min-
eral N when the principal source of N is the minerali-
zation of organic matter, i.e., in limiting N supply
conditions. In non-limiting N supply conditions,
there is ample mineral N in soil, the immobilization
capacity of microbial community is saturated and
then the differences among species tend to disappear.
Only the difference in N uptake capacity linked to
difference in crop growth capacity can then be
observed.
Some coincidences between quantitative trait loci
(QTL) for root architecture and NAE have been
detected on wheat [91]. A large genotypic variability
has been identified across maize lines for the density
and length of lateral roots [92], suggesting that it would
be possible to breed this species for improving its NAE
under low N supply conditions. Similarly, genetic stud-
ies showed that NAE was the most important compo-
nent of the Nitrogen use Efficiency in rice [93] and
wheat [87].
Nitrogen Conversion Efficiency
The efficiency of conversion of absorbed N by crop into
yield has to be analyzed at two levels:
– The efficiency for crop biomass production (W),
i.e., the supplement of biomass (dW) associated to
the supplement of N uptake (dN) when N supply
increases
– The harvest index HI, i.e., the proportion of crop
mass allocated to grain
Figure 7 allows a more detailed analysis of the NCE
for crop mass production. This figure represents two
N uptake vs crop mass (W) trajectories for crops at two
level of N supply: a limiting N supply rate and a non-
limiting one corresponding to critical N status. At each
date the slope of the line joining data points
corresponding to the two N supply rates is equal to
DNupt/DW which is the reciprocal of NCE. The sup-
plement of nitrogen taken up by the crop, DNupt, due to
the increase in N supply rate, can be defined as the sum
of two components (i) (DNupt)1, the quantity of N the
limiting N supply treatment should have been absorb
in supplement for reaching the corresponding critical
level, and (ii) (DNupt)2, the supplemental increase in
N uptake associated to the accelerated growth rate
resulting to the higher N supply level. The NCE of
(DNupt)1 is 0 as the increment in N uptake is made at
a constant crop mass. The NCE of (DNupt)2 is not
constant and can be approached only for a given crop
mass by the derivative of Eq. 3:
dNupt
¼ abðW Þb1 ð18Þ
dW
This equation indicates that the efficiency of con-
version of N absorbed into crop mass dW/dNupt
increases as crop mass increases. Such an analysis
allows the identification of two different sources of
variation for NCE: (i) when W is increased only by
other factors than N supply, climate and/or genotype,
then NCE increases with crop mass, and (ii) when W is
increased by N supply, then NCE is lowered by the cost
in N uptake for restoring the plant N status. This
approach allows the study of NCE as a dynamic process
where the time has to be explicitly taken into account
through crop growth rate: the higher crop growth rate,
the higher NCE of the crop. Then it allows the identi-
fication of the trivial effect of plant mass per se: a small
crop, either because genetically small or because of
unfavorable environmental conditions will have
a lower NCE as compared to a bigger crop. This reason
explains why a good correlation is observed between
genotypes growing either in high or low N supply con-
ditions [88]. In consequence, breeding for a high NCE
must lead to the selection of genotypes to higher
growth potential. There is no clear indication until
now if NCE variation among genotypes would persist
when comparisons are made at same crop mass. Since
coefficients a and b of Eq. 2 differ little or not at all
between species within C3 or C4 groups, the chances to
find intraspecific differences in NCE when comparing
plants having a similar growth potential remains very
limited.
When considering yield (Y) and not only crop
biomass (W), NCE can be highly variable among crop
species as resulting to large genotypic variations in

N uptake
Δ (Nupt) 2
Δ (Nupt) 1
W0
Crop Responses to Nitrogen. Figure 7
Schematic representation of the components of the nitrogen conversion efficiency. The first component D(Nupt)1
corresponds to the quantity of N necessary for crop to reach its critical N status, and its NCE is 0. The second component
D(Nupt)2 corresponds to the quantity of N necessary for the synthesis the supplement of biomass DW and whose the
NCE is dW/dN = 1/ab(W)1b that increases with crop mass
harvest index (HI). For grain crops as cereals, grain
legumes, and oil seed species, HI is an important source
of variation in grain yield per unit of N uptake. The
repartition of N between the harvested and non-
harvested plant parts that is the nitrogen harvested
index (NHI) is then an important aspect of the grain
nutritional quality [95]. Grain produced per unit of
N uptake and grain N concentration are in general
inversely related [96], and a variable proportion of
the variation in yield per unit N uptake is accounted
for by grain protein concentration or by NHI,
according to crop type.
Grain protein N concentration is the result of two
concomitant processes: (i) the rate of accumulation of
proteins during grain filling and (ii) the rate of accu-
mulation of free-N compounds (carbohydrates) within
grains that lead to a dilution of N as grains develop. So
a low grain N concentration can result in both a N
deficiency during grain filling period and a large accu-
mulation rate of starch. Thus, late application of
N fertilizer to avoid any N shortage during grain filling
can lead to an increase of the grain protein concentra-
tion. But as the capacity of roots for N absorption at
this stage of the crop is largely impaired by the begin-
ning of leaf senescence and the shortage of C allocated
Crop Responses to Nitrogen 653
Critical N uptake
Nc = a c(Wc)b
ΔNupt /ΔW = 1/NCE
Limiting N uptake
N0 = a0(W0)b
Wc
Biomass
to roots, the recovery of this N application is low, and
then, large mineral N residues in soil increase the risk
for subsequent N leaching.
Conclusion
Since soil N availability, N uptake and distribution
within plant and crop and finally crop growth are per-
manently interrelated during crop development and
growth, the traditional view where crop N uptake was
totally regulated by soil N supply must be reconsidered
and replaced by a more dynamic approach where plant
N uptake rate at any moment is co-regulated by both soil
N supply and plant growth capacity itself. This co-
regulation lead to the concept of critical plant
N concentration and N dilution that link plant and
crop N status to plant and crop mass. Such an approach
allows the determination of the dynamics of plant
N demand and plant N status all along their life-cycle.
Critical N-curves are now available for most of the crop
species growing either in temperate or tropical condi-
tions. This allows the determination of the nitrogen
nutrition index of any crop in any condition for diagnosis
of its actual N status and estimating the necessity for
applying N fertilizers.By this way, it would be possible
654 Crop Responses to Nitrogen
now to adjust more precisely the quantity and the
timing of N fertilizer supply for matching the crop
N demand according to target yields. As
a consequence, a reduction of the risk for N losses to
atmosphere and hydrosphere should be obtained while
the crop productivity would be maintained.
One other perspective is to improve the efficiency of
use of N within agro-systems. It appears that improv-
ing the ability of crops to absorb and accumulate
N efficiently from soil (nitrogen absorption efficiency)
is the first objective for high nitrogen use efficiency in
cropping systems. This ability of plants and crops to
take up mineral N from soil has to be investigated both
at high or low N supply conditions. As demonstrated
above, in both conditions, N uptake capacity of a crop
is directly dependent upon its growth capacity as deter-
mined by (i) its own genetic potential, (ii) environ-
mental conditions such as soil and climate, (iii)
cropping management techniques, and (iv) interactions
between these variables. So any improvement in crop
growth capacity by both breeding cultivars and crop
management (irrigation, P, K, S fertilization, planting
density) will increase NAE of the crop and then will
reduce the risk of accumulation of soil N mineral sur-
plus with environmental hazards. But more important
would be to improve NAE of crop species at similar crop
growth potential. This would allow achieving a given
target yield with less N fertilizer. Such an objective
requires a more efficient root system to increase the
competitive ability of the plant for using soil mineral
N against microbial communities. This objective could
be reached through a combination of plant breeding
strategy and crop management techniques such as soil
tillage and soil structure conservation.
Future Directions
The improvement of the ability of plants and crops to
absorb and accumulate N efficiently from soil, that is
measured by NAE, appears to be the first objective for
a plant breeding strategy. As demonstrated above, the
N uptake capacity of a crop primarily depends on its
growth potential under given climatic conditions. So
adaptation of genotypes for fast growth potential is the
first way for improving its N uptake capacity. However,
it should be also possible to increase the plant N uptake
capacity at similar growth potential. By this way, it
could be possible to increase the quantity of N a crop
is able to uptake from soil in low N supply conditions
and then to reduce the quantity of N fertilizers neces-
sary to obtain a given target yield. Under low N supply
conditions, root development and architecture as well
as the interactions with rhizosphere through root exu-
dates may be of major importance for the determina-
tion of the N uptake capacity of crops. Under high
N supply conditions, it is the down regulation of root
absorption capacities by shoot signals which determine
the crop N uptake capacity. So for breeding genotypes
with higher N uptake capacities both in low and in high
N supply conditions, it is necessary to analyze more
deeply all the regulation processes of N absorption at
physiological and molecular levels. This kind of
approach should be one of the major tasks in the next
decade. The development of large-scale genomic, pro-
teomic, and metabolomic studies is necessary for such
an objective. However, the difficulty will be to integrate
the huge amount of data generated by these studies. All
elementary processes studied at cellular or molecular
level have to be scaled up to the level of whole plant and
crop where they are agronomically relevant. The nitro-
gen nutrition of crops and its efficiency for yield pro-
duction is controlled by elementary physiological
processes such as nitrate or ammonium transport
through cell membranes in roots, nitrate reduction,
ammonium assimilation, and protein synthesis. Each
of these metabolic processes is regulated at molecular
level, and then is susceptible to have more or less
genetic variability. But when all processes are scaled
up to whole plant or crop level, a large part of this
elementary variability is buffered because of existing
trade-off among elementary processes and feedback
mechanisms. Quantitative genetics using both mutants
and genetic engineering appears to be one of the most
promising tools to allow the identification of key reg-
ulatory genes that are likely to control a variety of
physiological and developmental processes involved
in the determination of crop nitrogen absorption effi-
ciency. But this approach must be always accompanied
by a modeling approach in order to analyze all the
feedback and trade-off at whole plant and crop level.
Breeding plants to increase nitrogen conversion
efficiency for aboveground dry matter production
appears to be more difficult because, apart from the
difference between C4 and C3 groups, no clear

interspecific differences are observed among cultivated
crops. So it seems that intraspecific variability should
be very low. However, if the N efficiency for grain yield
is considered, it should be possible to detect inter- and
intraspecific differences. Detailed analysis of grain
development and grain-filling processes should pro-
vide information about the genetic variability. The
development of models describing both N partitioning
and translocation within plant, and dynamics of
grain development, taking into account the coupling
between N and C fluxes between plant organs are nec-
essary for detecting the key regulation processes for
optimizing grain yield and quality. This approach will
require a cooperative and integrated effort between
plant molecular physiologists, geneticists, crop physi-
ologists, and agronomists and the intensive use of
bioinformatics.
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Books and Reviews
Gastal F, Lemaire G (2002) N uptake and distribution in crops: an
agronomical and ecophysiological perspective. J Exp Bot
53:789–799
Grindlay DJC (1997) Towards an explanation of crop nitrogen
demand based on leaf nitrogen per unit leaf area. J Sci Food
Agric 63:116–123
Hirel B, Lemaire G (2005) From agronomy and ecophysiology to
molecular genetics for improving nitrogen use efficiency in
crops. J Crop Imp 15:213–257
Justes E, Mary B, Meynard JM, Machet JM, Thellier-Huché L (1994)
Determination of a critical nitrogen dilution curve for winter
wheat crops. Ann Bot 74:397–407
Lemaire G, Gastal F (1997) N uptake and distribution in plant
canopies. In: Lemaire G (ed) Diagnosis on the nitrogen status
in crops. Springer, Heidelberg, pp 3–43
Lemaire G, Gastal F (2009) Quantifying crop responses to
nitrogen deficiency and avenues to improve nitrogen use
efficiency
Lemaire G, Jeuffroy MH, Gastal F (2008) Diagnostis tool for plant
and crop N status in vegetative stage. Theory and practices for
crop N management. Eur J Agron 28:614–624
Lemaire G, Recous S, Mary B (2004) Managing residues and
nitrogen in intensive cropping systems. New understandings
for efficient recovery by crops. In: Proceedings of the
4th international crop science congress, Brisbane, Australia,
2004
Lemaire G, van Oosterom E, Jeuffroz MH, Gastal F, Massignan A
(2008) Crop species present different qualitative types of
response to N deficiency during their vegetative growth.
Field Crop Res 105:253–265
Lemaire G, van Oosterom E, Sheehy J, Jeuffroy MH, Massignan A,
Rossato L (2007) Is crop demand closely related to dry matter
accumulation of leaf area expansion during vegetative
growth? Field Crop Res 100:91–106
Sinclair TR, Horie T (1989) Leaf nitrogen, photosynthesis, and crop
radiation use efficiency: a review. Crop Sci 29:90–98
Sinclair TR (1998) Historical changes in harvest index crop N
accumulation. Crop Sci 38:638–643
Van Keulen H, Goudrian J, Seligman NG (1989) Modelling the
effects of nitrogen on canopy development and crop growth.
In: Rusell G, Marshall LB, Jarvis PG (eds) Plant canopies: their
growth, form and function. Cambridge University Press, Cam-
bridge, pp 83–104

Crop Science and Technology,
Introduction
PAUL CHRISTOU1,2, ROXANA SAVIN3
1
Department de Produccio Vegetal i Ciencia Forestal,
Universitat de Lleida/ICREA, Lleida, Spain
2
Institució Catalana de Recerca i Estudis Avançats,
(ICREA), Barcelona, Spain
3
Department of Crop and Forest Sciences, University
of Lleida, Lleida, Spain
Article Outline
Transgenic Crops and Molecular Breeding
Crop Physiology and Agronomy
Population growth in the coming decades will put
severe pressure on human food, animal feed, and
fiber production. Bioenergy applications are already
exerting increasing pressure on agricultural commod-
ities and land use with severe economic consequences,
particularly in the developing world. Any crop pro-
ductivity increases must come necessarily from
enhancing crop performance as further land expan-
sion for agriculture is unlikely to take place. Environ-
mental sustainability and social justice issues are
becoming increasingly key elements in debates on
how to assure adequate food for the ever-increasing
global population. It is likely that the efficiency
of increasing productivity would benefit by
complementing the conventional empirical
approaches with opportunities presented by the devel-
opment of new knowledge and technologies in the
field of Crop Science. Therefore, in this section a
number of seminal articles by world experts in the
field are featured. Through this collection of articles
key advances in the field are highlighted and the
reader is pointed to future directions in terms of
opportunities and constraints for a more productive
and sustainable agriculture. This is not meant to be an
exhaustive list of topics; rather the aim is to highlight
particular technologies and topics that have a real
potential to make a substantial contribution to
a more productive and environmentally friendlier
agriculture in the short to medium term. This section
P. Christou et al. (eds.), Sustainable Food Production, DOI 10.1007/978-1-4614-5797-8,
# Springer Science+Business Media New York 2013
Originally published in
Robert A. Meyers (ed.) Encyclopedia of Sustainability Science and Technology,
Crop Science and Technology, Introduction 659
comprises 45 articles that can be divided into two
parts: (a) molecular approaches and (b) crop physiol-
ogy and agronomy.
Transgenic Crops and Molecular Breeding
World food and feed security are increasingly depen-
dent on continuous crop improvement and in partic-
ular the development of crops with increased resistance
to abiotic stresses (▶ Abiotic Stress Tolerant Crops:
Genes, Pathways and Bottlenecks). Plants unlike ani-
mals are not mobile, consequently they need to develop
strategies to combat natural enemies such as herbivores
and environmental stresses. Drought, salinity, submer-
gence, and temperature stresses amongst others are all
important abiotic constraints which limit crop yields.
Significant advances in our understanding of molecular
mechanisms underpinning a plant’s ability to combat
abiotic stresses has resulted in the creation of transgenic
plants with improved resistance to such stresses.
Sustainable, renewable resources are those derived
from biological sources, primarily plant biomass which
can be regenerated with minimal inputs using energy
from the sun (▶ Biomass Crops for Biofuels and Bio-
based Products). Biomass for biofuels includes many
sources of material derived from agricultural harvests
including grains, agricultural residues such as stalks
and leaves, perennial crops such as hay and trees, ani-
mal manures, building waste wood, municipal solid
waste such as paper, and various food industry wastes.
Humans currently consume at least 25% more raw
materials every year than are replaced through biolog-
ical growth. In order to sustain quality of life and
maintain adequate environmental resources, those
resources must be balanced and renewable. Pressure
on those resources has never been greater with the
world population currently at seven billion people,
and estimated to plateau at 10.5 billion by 2050.
Once a gene is introduced into a plant its expression
may be controlled by a number of different factors
(▶ Transgene Expression in Plants, Control of).
Among these, promoters are most important as they
regulate expression temporally and spatially. Transcrip-
tional fusions of genes, with the Cauliflower Mosaic
Virus 35S promoter when integrated into the plant
genome (mostly dicotyledonous plants), result in
# 2012, DOI 10.1007/978-1-4419-0851-3
660 Crop Science and Technology, Introduction
transgenic plants with high and constitutive expression
of the transgene. However, 35S-driven transgene
expression is very variable with ca: 20% of the
transformants exhibiting high expression levels, while
the majority (ca: 80%) display an intermediate or low
and unstable transgene expression. The possible causes
of variation in transgene expression in a population of
transgenic plants need to be identified and addressed
in order to generate useful plants with stable and pre-
dictable levels of expression of introduced transgenes.
Plant transformation is a fundamental component
of both basic and applied plant biology (▶ Crop Plants
Transformation Methods). For basic research, transfor-
mation allows scientists to study how genes function
and allows the study of both endogenous genes and
transgenes. This has increased our understanding of
how plants grow, develop, and defend themselves
against pests, diseases, and harsh environments; how
photosynthesis is controlled; and the basis of primary
and secondary metabolism. For applied research, trans-
formation can be used to improve the agronomic per-
formance of crops, making them hardier, more
nutritious, more productive, or converting them from
conventional crops into green factories producing
chemical precursors, novel oils, industrial enzymes,
and pharmaceuticals. Plants have been cultivated for
food and animal feed, fibers and structural materials,
and small molecules that can be used as dyes, scents,
and medicines since the dawn of history, and for the
same length of time people have sought to improve
plants by breeding them and selecting the better-
performing and most useful varieties. The limitations
of this approach, that is, the fact that breeders are
restricted to the existing gene pool in each group of
sexually compatible species, and that breeding takes
a long time to achieve its goals, can be overcome by
plant transformation, thus accelerating the develop-
ment of plants with novel, beneficial traits. Plant trans-
formation includes both the uptake of naked DNA
(direct DNA transfer) and the transfer of DNA by the
conjugation-like method adopted by Agrobacterium
tumefaciens and A. rhizogenes (Agrobacterium-
mediated transformation).
Genetics, molecular biology, genomics, and other
disciplines have now provided us with an insight to the
genes that encode important crop traits on which
humans now depend. Knowledge of the molecular
genetic basis of valuable crop traits will help provide
solutions to our requirements for sustainable exis-
tence, faced with a growing global population and
diminishing natural resources (▶ Crop Traits: Gene
Isolation). Isolated genes encoding useful traits and
their use in plant transformation experiments allow
the development of an in-depth understanding of the
mechanisms that control such important crop traits.
Thus, through such investigations that utilize isolated
genes, one is able to understand and harness traits
involved in plant domestication such as plant architec-
ture, to those offering solutions for high crop produc-
tion purposes such as growth under unfavorable
environments or disease outbreaks. Cloning or
identification of the genes involved in crop traits pro-
vide an insight into factors which determine gene–trait
relationships. Understanding the molecular basis of
crop traits provides a route to their controlled modu-
lation ultimately leading to the development and
implementation of novel genetic engineering solutions
to create plants with superior and sustainable
characteristics.
Conventional plant breeding practices alone will
not be able to achieve sustainability in today’s agricul-
ture. Advances in plant genomics research are opening
up a new era in plant breeding where the linkage of
genes to specific traits will lead to more efficient and
predictable breeding programs (▶ Crop Breeding for
Sustainable Agriculture, Genomics Interventions in).
Plant genomics is a rapidly developing field, which is
radically improving our understanding of plant biol-
ogy by making available novel tools for the improve-
ment of plant properties relevant to sustainable
agricultural production. Recent advances in high
throughput genomics technologies including next gen-
eration sequencing and high-throughput genotyping
have helped immensely in understanding the functions
and regulation of genes in crop plants. The ever-
increasing availability of genome sequences in crop
plants has facilitated greatly the development of geno-
mic resources that will allow us to address biological
functions and a number of basic processes relevant to
crop production leading to sustainable agriculture. It is
therefore expected that genomics will be an integral
part of the agricultural/plant breeding practices of the
future for improving crop productivity to achieve food
security and sustainable production.

Plants have been used as sources of small molecular
weight compounds (secondary metabolites) with
applications as medicines, flavors, and fragrances for
millennia. However, many plants containing such high-
value secondary metabolites are difficult to cultivate or
are becoming endangered because of overharvesting. In
addition, the chemical synthesis of plant-derived com-
pounds is often uneconomical due to their highly com-
plex structures. The biotechnological production of
valuable secondary metabolites is an attractive alternative
to the extraction of whole plant material (▶ Medicinal
Plants, Engineering of Secondary Metabolites in Cell
Cultures). Functional genomics may open entirely new
avenues to screen unexplored medicinal plant species
for their pharmacological value.
Molecular breeding (MB) allows the stacking of
favorable alleles, or genomic regions, for target traits
in a desired genetic background, thanks to the use of
polymorphic molecular markers (MM) that monitor
differences in genomic composition among cultivars,
or genotypes, at specific genomic regions, or genes,
involved in the expression of those target traits
(▶ Molecular Breeding Platforms in World Agricul-
ture). The use of MM generally increases the genetic
gain per crop cycle compared to selection based on
plant phenotyping only, and therefore reduces the
number of needed selection cycles, hastening the deliv-
ery of improved crop varieties to the farmer. In contrast
to the private sector, MB adoption is still limited in the
public sector, and is hardly used at all in developing
countries. The situation is critical in developing coun-
tries due to resource-limited breeding programs. As
a result, the developing world has yet to benefit from
the MB revolution, and most countries indeed lack the
fundamental prerequisites for a move to informatics
powered breeding. A sustainable web-based Molecular
Breeding Platform (MBP) as a one-stop-shop for infor-
mation, analytical tools and related services to help
design and conduct marker-assisted breeding experi-
ments in the most efficient way will alleviate many of
these bottlenecks in the developing world. Such
a platform will enable breeding programs in the public
and private sectors in developing countries to acceler-
ate variety development using marker technologies for
different breeding objectives.
With the advent of molecular techniques, plants
have the potential to serve as production vehicles for
Crop Science and Technology, Introduction 661
natural or engineered products that were previously
limited to other hosts. Plant molecular pharming of
industrial proteins refers to recombinant proteins used
in industrial processes and produced in plants (▶ Plant
Molecular Pharming, Industrial Enzymes). Enormous
quantities of a variety of enzymes go into making
products such as paper, leather, detergents, pharma-
ceuticals, food, beverages, chemicals, and fabric, to
name a few, and economical production of these indus-
trially important enzymes is crucial to commerce. This
production must be balanced with the need for sus-
tainability and environmental stewardship. Sustainable
production of industrial enzymes requires that
resources are not overexploited and wastes are not
polluting. The use of plants as “green” factories can
meet both criteria. Combined with modern farming
and containment methods, transgenic plants have the
potential to produce large quantities of target material
safely and sustainably.
The demand for recombinant medical proteins has
increased in recent years and modern biotechnological
methods have, until recently, ensured the production of
safe and effective biopharmaceuticals to meet this
demand. Various production platforms are currently
used in the pharmaceutical industry, most based on the
fermentation of engineered pro- and eukaryotic micro-
organisms, insect cells, or mammalian cells (▶ Plant
Molecular Pharming, Pharmaceuticals for Human
Health). The growth of the market for biopharma-
ceuticals is predicted to outpace production capacity
using these platforms in the next decade, so alternatives
are necessary. The production of pharmaceutical
proteins in plants began with a monoclonal antibody
expressed in transgenic tobacco plants more than
20 years ago. Since then many different plant species
have been genetically engineered to produce valuable
pharmaceutical proteins. Major progress has been
achieved in transformation and expression technology,
downstream processing of transgenic plant material,
and the adaptation of regulatory procedures to encom-
pass the new production platforms, allowing the first
plant-made pharmaceuticals to begin clinical trials.
Since the successful expression of complete anti-
bodies in transgenic plants and the first report of
plant-based vaccine production in 1992 a large number
of different vaccines, antibodies, as well as antibody
fragments have been produced in plants for medical
662 Crop Science and Technology, Introduction
or veterinary purposes (▶ Plant Molecular Pharming,
Veterinary Applications). Novel processing methods
have been developed over the past several years to
facilitate the development of recombinant proteins
for veterinary applications.
Humankind has had an ever-increasing impact on the
environment. With the increasing intensification of agri-
culture, particularly during the twentieth century, this
impact has become even more pronounced, often with
undesirable or unacceptable consequences, including
water pollution, soil erosion, and loss of habitat, often
accompanied by a loss of biodiversity (▶ Transgenic
Crops, Environmental Impact). Pests, particularly
weedy plants, demonstrate a remarkable ability to
adapt to agricultural production systems. The practice
of growing monocultures, typically used in intensive
agriculture, increases the number of pests; these are
currently predominantly controlled through use of pes-
ticides. However, with increasing exposure to pesticides
many pest populations are evolving resistance to these
compounds. An additional problem encountered with
many pesticides, and particularly insecticides, are their
nontarget effects on beneficial insects. Transgenic crops
expressing genes conferring resistance to insect pests
and/or herbicides are becoming increasingly more
widely grown and have the potential to eliminate many
of the problems in conventional agriculture thus reduc-
ing agriculture’s negative impact on the environment.
Annual losses worldwide due to plant diseases are
estimated to be 14% of total losses and about $220
billion. In addition, the need for measures to control
diseases limits the acreage of land available for cultiva-
tion; restricts the crops that can be grown in fields already
contaminated with certain pathogens; and necessitates
the use of agrochemicals for treating seeds, fumigating
soils, spraying plants, and applying postharvest treat-
ments. Such control measures add to the cost of food
production and toxic chemicals can be harmful to
human health and the environment (▶ Transgenic
Crops Resistant to Fungal, Bacterial and Viral Patho-
gens). Resistance to pathogens can be achieved by
application of disease-suppressing cultural practices,
use of plant defense-promoting substances, deploy-
ment of biological agents antagonistic to the pathogens
that cause disease, agrochemicals, conventional breed-
ing strategies, and genetic engineering. The need for
controlling plant diseases effectively is not only a major
challenge, but also a necessity to reduce food losses
while improving food quality and safeguarding the
environment.
Genetic Engineering can increase the nutritional
quality of crops by increasing the availability of
essential nutrients, which are often limited in human
diets and lead to specific deficiency diseases
(▶ Biotechnology and Nutritional Improvement of
Crops). Food insecurity is one of the most important
social issues faced today, with nearly one billion peo-
ple enduring chronic hunger and an additional two
billion people suffering from nutrient deficiencies,
most in the developing world. Strategies to address
food insecurity must ultimately address underlying
problems such as poverty and poor governance/infra-
structure, but the improvement of agricultural pro-
ductivity in the developing world is an important
goal, and biotechnology is one of a raft of measures
being considered to achieve it.
The economic impact of transgenic crops has been
immense (▶ Global Economic Impact of Transgenic/
Biotech Crops (1996–2008)). This has been a major
driver in their rates of adoption amongst farmers in
the USA and other industrialized countries but perhaps
more importantly by small holders in the developing
world. Analysis on farm income effects through exten-
sive analysis of existing farm level impact data for
biotech crops confirms this to be a major reason for
their broad adoption worldwide.
Biotechnology offers efficient and cost-effective
means to produce a diverse array of novel, value-
added products and tools. The first generation of
commercialized biotechnology products were crops
focusing largely on input agronomic traits whose
value was often opaque to consumers. The coming
generations of crop plants can be grouped into broad
areas each presenting what, on the surface, may appear
as unique challenges and opportunities (▶ Transgenic
Crops, Next Generation). The present and future focus
is on continuing improvement of agronomic traits such
as yield and abiotic stress resistance in addition to the
biotic stress tolerance of the present generation,
crop plants as biomass feedstocks for biofuels and
“bio-synthetics,” value-added output traits such as
improved nutrition and food functionality, and
plants as production factories for therapeutics and
industrial products. From a consumer perspective,

the focus on value added traits, especially improved
nutrition, is undoubtedly one of the areas of greatest
interest.
The rapid development and deployment of modern
biotechnology in the last decades has made biosafety
a major issue. Although modern biotechnology can
benefit agricultural productivity in developing coun-
tries transgenic crops remain an issue with regard to the
conservation and sustainable use of biodiversity, as well
as to human health. The perceived risks, which relate to
the release of transgenic crops into the environment as
well as the placement of genetically engineered crops
onto the market, have as much to do with social values
as scientific concerns. Regulatory frameworks for
transgenic crops and the underpinning legislative
frameworks have been or are being developed world-
wide (▶ Commercialisation of GM Crops: Compari-
son of Regulatory Frameworks). These are viewed as
essential components for the prudent deployment of
transgenic crops worldwide.
The safety measures associated with transgenic crop
deployment are embedded in process- or product-
based regulatory approaches. The EU regulatory
approach is process-based, precautionary, and includes
mandatory labeling and traceability requirements for
transgenic crops and their derived food and feed prod-
ucts (▶ Transgenic Crops, Risk Assessment and Regu-
latory Framework in the European Union). During its
development, the EU regulatory system has become
increasingly more stringent and unduly onerous. In
the EU, the risk analysis consists of three components:
risk assessment, risk management, and risk communi-
cation. When analyzing potential risks, it is important
to bear in mind that the real choice is not between
transgenic crops that are inherently risky and tradition-
ally bred ones that are completely safe. The cultivation
of existing crops and those with novel traits (including
transgenic crops) will have both positive and negative
consequences. To fully acknowledge the overall out-
come of adopting specific crops, and to assess and
manage more effectively the environmental footprint
of agriculture as a whole, broader and more balanced
legislative oversight is needed in the EU.
A framework for a better communication about
science and regulation and production of GM crops is
described in ▶ GM Crop Risk Debate, Science and
Socioeconomics.
Crop Science and Technology, Introduction 663
While transgenic herbicide resistant crops have
been a boon to agriculture, reducing both production
costs and ecological impacts of farming, weeds have
rapidly evolved resistance to the major herbicide used
in transgenic crops (glyphosate), rapidly rendering the
technology less sustainable than had been thought
(▶ Sustainable Herbicide-Resistant Crops). While no
practice in agriculture has been sustainable forever, the
period of sustainability can be extended. Methods to
extend both the usefulness to crops where needed as
well as the sustainability of transgenic herbicide tech-
nologies such as rotations of crops and herbicides,
increasing the targets of herbicide action, suppressing
herbicide targets in rotation, are needed.
Crop Physiology and Agronomy
Seed dormancy is a means of restricting germination to
the season when environmental conditions are suitable
for plant establishment. From an agricultural perspec-
tive, dormancy is a problem (▶ Seed Dormancy and
Agriculture, Physiology). Many important challenges
face agriculture in relation to dormancy and these
apply to cultivated crops as well as noxious weeds.
The physiological mechanisms responsible for the
expression of the character are now better understood
and molecular information underpinning the process
is gradually being generated and incorporated into
strategies to solve dormancy-related problems.
One of the first decisions a farmer needs to make
is to choose the particular genotype to be grown in
the fields based on anticipated or projected economic
returns. This is a critical choice that determines the
sustainability of the agricultural system (▶ Genotype
by Environment Interaction and Adaptation). Identi-
fying breeding implications on specific adaptive traits
and the different statistical approaches for genotype by
environment interaction characterization is important.
Attaining global food security by means of increased
crop productivity will require an increase in gains from
selection achieved through conventional breeding. The
identification of molecular markers associated with loci
controlling traits of agronomic interest coupled with the
exploitation of marker-assisted breeding (MAB)
approaches provides the opportunity to accelerate
gain from selection (▶ Marker-Assisted Breeding in
Crops). Genomic selection is already having a positive
664 Crop Science and Technology, Introduction
impact on the improvement of crop yield, mainly in the
private sector where high-throughput infrastructures
allow breeders to handle the large number of molecular
data points that are needed for deploying genomic
selection effectively. Ultimately, an effective exploita-
tion of MAB to enhance crop performance will rely on
a closer integration between molecular approaches and
conventional breeding.
The next generation of highly productive crops in
an increasingly variable and changing climate, will rely
on genetic interventions based on process understand-
ing, selection of target traits in managed environments,
and high-throughput phenotyping and genotyping
(▶ Plant Breeding Under a Changing Climate). There-
fore, it is crucial to understand the recent advances in
plant breeding for high yield potential environments
and also those where abiotic stress is a major limitation
to productivity.
Agronomic systems are defined as site-specific
management of soils and crops on the basis of eco-
regional and physiographic characteristics, and in the
context of socioeconomic and policy environments.
These systems are strong determinants of agricultural
production, sustainable use of resources, and their
environmental impact. Agricultural soils and ecosys-
tems can also be used for sequestration of atmospheric
CO2 by enhancing photosynthesis, increasing net
primary productivity (▶ Agronomic Interactions with
CO2 Sequestration).
Crop management comprises a set of agronomic
practices such as tillage systems, methods of fertiliza-
tion, and crop rotations (▶ Cropping Systems: Shaping
Nature). Cropping system may vary among farms
depending on availability of resources and particular
constraints. The different cropping systems may deter-
mine water and nutrient availabilities, carbon cycle,
erosion, and the pathogen inoculum in the soil.
Understanding plant development or the progres-
sion of plants through their life cycle is important
because of the need to know and predict when harvest-
able products are at their optimum (▶ Crop Develop-
ment Related to Temperature and Photoperiod).
Current knowledge on how temperature (including
vernalization) and photoperiod regulate crop develop-
ment is of major interest and determines how crops
adapt in a wide range of environments. Thus, it is
critical to understand the physiology and genetic basis
of crop development, and to predict as accurately as
possible the timing of key developmental events.
Reduced crop productivity from the theoretical
potential maximum commonly occurs because of
high temperatures. In addition, air temperatures are
predicted to increase during the twenty-first century.
Crop physiological and developmental processes are
sensitive to temperature so that high temperatures do
frequently affect negatively crop productivity
(▶ Sustainable Productivity, Heat Tolerance for). The
effects of high temperature, elevated CO2 and their
interaction on crops, are therefore important to under-
stand and subsequently to mitigate potential negative
effects of genes that confer heat tolerance.
Since plants are immobile, their distribution greatly
influences the ability of a crop to capture and use
environmental resources (radiation, water, and nutri-
ents), which are necessary for growth and yield. The
spatial arrangement of plants and the temporal devel-
opment of their structures (mainly leaves and roots)
define the ▶ Spatial Crop Structure in agricultural sys-
tems. Density and spatial arrangement of crops may
affect intraspecific competition and resource use effi-
ciency, allowing full or partial use of available
resources.
The rate of accumulation of dry plant matter is
entirely dependent on the interception of energy from
the sun in the wavelength range 400–700 nm. This
energy is utilized by photosynthesis to synthesize car-
bohydrates and other biological molecules needed for
essential plant processes (▶ Crop Radiation Capture
and Use Efficiency). Given the current emphasis on
global food security, there is currently much interest
in raising the radiation use efficiency of key crops in
important agro-ecosystems.
Scarcity of water resources is an increasingly impor-
tant issue since it will dictate global production of food
and feed for the next generations (▶ Crop Responses to
Available Soil Water). Key factors responsible for
sustained plant growth and production under water
scarcity, for annual as well as perennial (fruit) crops
are of paramount importance.
Irrigated agriculture is currently responsible for
over 40% of total production on 17% of all cultivated
land area. It is therefore imperative that irrigated agri-
culture not only sustains its current rates of productiv-
ity but also increases (▶ Irrigation Management for

Efficient Crop Production). Irrigation expansion is
currently under pressure from other sectors to reduce
its share of the fresh water resources. Efficient crop
production under irrigation in the future would be
essential to produce more food with less water. This is
an immense challenge, not easy to achieve without
novel and innovative approaches in irrigation manage-
ment and crop productivity.
Sustainability of fertilizer use is very important
(▶ Fertilizer Science and Technology), as fertilizers
are indispensable because nutrient supplies from the
soil are normally inadequate for high-yielding crops
and compensate for nutrient removals by previously
cultivated crops. In addition, fertilizer may also
improve the quality of human food and animal feed.
Nitrogen is the most important limiting factor, after
water deficit, for crop production worldwide (▶ Crop
Responses to Nitrogen). Therefore, understanding how
the yield of different crops can be improved by addition
of nitrogen (N) fertilizers is critical. There are
several important issues regarding the dynamics of
crop N demands during the crop development cycle,
the timing of the soil N supply according to soil char-
acteristics, climate and soil agronomic management,
the crop responses to different intensity and timing of
N nutrition deficiency, and the time management of
crop N fertilization using diagnostic and decision-
making tools to optimize trade-offs between
minimizing crop yield reduction and minimizing envi-
ronmental impacts.
A substantial increase in the effectiveness with
which available water and nutrients are used is required
to ensure food security and environmental protection.
An essential component of crop improvement is
breeding for deeper or denser root systems. These char-
acteristics promote soil moisture and nutrient capture
and high dry matter production in cultivars subjected
to water and/or nutrient stresses (▶ Roots and Uptake
of Water and Nutrients). The current understanding of
the structure and functions of crop root systems and
the avenues for the optimization of root anatomy and
morphology traits that could be applied to the genetic
and agronomic improvement of crop root systems for
more effective below-ground resource capture are thus
very important.
Lodging is the process by which the shoots of small
grained cereals are permanently displaced from their
Crop Science and Technology, Introduction 665
vertical stance (▶ Lodging Resistance in Cereals).
The reduced lodging risk of shorter varieties enabled
them to respond to greater amounts of fertilizers and
this was a significant reason for the steady improvement
in global cereal grain yields starting in the late 1960s.
However, lodging is still a major problem in many coun-
tries and there is an urgent need to improve lodging
resistance to further increase the yield of cereal species.
Plant growth and yield are severely affected by
saline soils. High concentrations of salt in the soil
make it difficult for plants to take up water, whilst the
accumulated salts in cells, particularly sodium and
chloride ions, are toxic to plant metabolism
(▶ Increasing Salinity Tolerance of Crops). These two
factors result in a reduction in plant growth, an
increase in the rate of leaf senescence, and a loss in
crop yield. Crop salinity tolerance can be improved,
but a more in-depth understanding of osmotic and
ionic stresses is needed.
Agroecology provides the basic ecological princi-
ples needed for studying, designing, and managing
agroecosystems that are productive, sustainable, and
economically viable (▶ Agroecological Basis for
Managing Biotic Constraints). Rather than focusing
on one particular component of the agroecosystem,
agroecology emphasizes the interrelatedness of all of
its components and the complex dynamics of ecologi-
cal processes including all environmental and human
elements. From a management perspective, the agro-
ecological objective is to provide balanced environ-
ments, sustained yields, biologically mediated soil
fertility, and natural pest regulation through the design
of diversified agroecosystems and the use of low-input
technologies.
Plant diseases cause substantial crop losses every
year (▶ Crop Diseases, Management and Control of).
Controlling plant diseases is therefore vital to
maintaining crop productivity. Crop diseases can be
controlled using a variety of methods; however, plant
pathogens are genetically adaptable and can overcome
plant resistance, and the toxic effects of pesticides.
Ensuring that crops are adequately protected from
diseases depends therefore on being a step ahead of
the pathogens by improving existing control measures
and developing new approaches.
Increase of crop yields may be achieved by maxi-
mizing the proportion of sunlight energy that is fixed
666 Crop Science and Technology, Introduction
by the crop or by reducing the amount of energy that is
lost by insect pests, diseases, and weeds. More than 50%
of the potential yield of agricultural crops is lost by the
three constraints. ▶ Integrated Pest Management
(IPM) aims to diminish losses caused by insect pests
in agriculture, in an economically, ecologically, and
sociologically acceptable manner. A major challenge
for ecology is the development of a scientific approach
to better understand processes in agroecosystems in
order to implement more rapidly sustainable IPM
systems.
Yield increases will continue to play a dominant
role in world food security (▶ Crop Yields Around
the World: Closing the Gap and Raising the Potential).
Understanding the gaps between potential and actual
yield is of paramount importance in order to increase
actual yields. These include several aspects with respect
to the natural resource base of the plot (climate, soil
type, topography) and long-term management
investments.
Grain quality of field crops is related to seed
structure and composition. Grain composition is the
major reason why only a limited number of plant
species are used for food and fiber (▶ Grain Quality
in Oil and Cereal Crops). It is impossible to put
forward a unique grain quality definition for any
species because this depends on the specific product
end use. Therefore, understanding the physiological
bases of seed composition and structure is essential
to produce grains with a particular quality
specification.
Agricultural production takes place under erratic
and unpredictable conditions, particularly the avail-
ability and timing of radiation and rainfall patterns
which are extremely difficult to predict. Their effects
are compensated to some extend by the qualities of the
land and the interventions of the farmer. Any method-
ology that would improve the predictability of the
availability of resources and their impact on the per-
formance of the production system could in principle
improve performance and reduce the level of uncer-
tainty (▶ Simulation Models as Tools for Crop Man-
agement). Crop growth simulation models are viewed
as excellent tools for the reduction of this uncertainty.
Advantages and limitations of such models need to be
understood in the context of past experiences and the
current state of the art in order to ascertain their best
possible uses.

Crop Traits: Gene Isolation
KURNIAWAN RUDI TRIJATMIKO1, ANDY PEREIRA2
1
International Rice Research Institute, Manila,
Philippines
2
University of Arkansas, Fayetteville, AR, USA
Article Outline
Glossary
Definition of the Subject and Its Importance
Introduction
Crop Traits: Gene Isolation
Future Directions
Bibliography
Glossary
Backcross To cross the progeny of a hybrid to one of
its parents, which when repeated for many genera-
tions would yield advanced backcross progeny.
cDNA Complementary DNA (cDNA) refers to the
DNA copy of RNA transcripts, which can be cloned
into vectors to generate a library (collection) of
different cDNAs.
Chromosomal recombination Breakage and rejoining
of parental homologous chromosomes during
meiosis, resulting in the exchange of chromosomal
segments.
Cloning vector DNA molecule into which another
DNA fragment can be integrated and replicated to
produce large quantities of the cloned DNA. Exam-
ples are plasmids, lambda phage, cosmids, yeast
artificial chromosomes (YACs), and bacterial arti-
ficial chromosomes (BACs).
Complementation A method of validating a gene clon-
ing by using wild-type allele to rescue the function of
mutant allele through genetic transformation.
Crop trait Any morphological, physiological, or other
biological feature measurable at the plant level,
present in different forms in different individuals
that enable genetic analysis.
Domestication An artificial selection process
conducted by humans to produce plants that have
more desirable traits than wild plants.
EST Expressed sequence tag is a short subsequence
from a transcribed cDNA sequence.
P. Christou et al. (eds.), Sustainable Food Production, DOI 10.1007/978-1-4614-5797-8,
# Springer Science+Business Media New York 2013
Originally published in
Robert A. Meyers (ed.) Encyclopedia of Sustainability Science and Technology,
Crop Traits: Gene Isolation 667
Forward genetics A strategy to identify or clone genes
that are responsible for a phenotype of interest.
Gene A gene is an ordered sequence of nucleotides that
encodes a specific functional product (i.e., a protein
or RNA molecule).
Marker Molecular or genetic marker is a DNA
sequence at a known location on a chromosome.
Mutation Changes in a genomic sequence, occurring
naturally or artificially, that can either have
no effect, alter the product of a gene, or prevent
the gene from functioning properly or completely.
NIL Near-isogenic lines (NILs) refers to genotypes or
lines that are genetically almost identical except for
a small chromosome fragment or DNA sequence by
which they differ.
ORF Open reading frame (ORF) refers to a DNA
sequence that does not encode a stopcodon and
predicted to encode a protein.
PCR Polymerase chain reaction, a method in which
DNA is amplified or increased in number of copies,
using oligonucleotide primers flanking the DNA
sequence and an enzyme that carries out the reaction.
Polyploid Organism that has more than two paired
sets of chromosomes that is present in a diploid.
Positional cloning A method of cloning genes based
on their position in the genome, using molecular
markers in a genetic map located close to the gene
and then identifying cloned DNA fragments
containing the gene of interest.
Quantitative trait locus A region on the genome asso-
ciated with a particular phenotype showing contin-
uous and measurable variation such as height or
weight.
Reverse genetics A strategy to identify the function of
genes revealed by DNA sequencing, by analyzing
the phenotypic effects of the gene sequences by
mutations or changes in expression.
RFLP Restriction fragment length polymorphism
(RFLP) is a molecular marker revealed by differ-
ences in restriction fragments between homologous
(similar) fragments of DNA.
STS marker A sequence-tagged site (STS) marker
is a short DNA sequence at known location
in the genome, with nucleotide differences/
polymorphisms that make it a useful marker for
mapping.
# 2012, DOI 10.1007/978-1-4419-0851-3
668 Crop Traits: Gene Isolation
Transposon A piece of DNA that can move within the
genome of an organism, and when inserted in genes
can cause a mutation.
Definition of the Subject and Its Importance
Crop plants have been selected from their wild ances-
tors by humans for food, fiber, health, recreation,
industry, and other special purposes. Crop breeding
has then added on desired traits for convenience in
crop production. Genetics, molecular biology, geno-
mics, and other disciplines have now provided us an
insight to the genes that encode these very important
crop traits on which the human race now depends.
Knowledge of the molecular genetic basis of valuable
crop traits will help provide solutions to our require-
ments for sustainable existence, faced with the threats
of growing population needs, climate change, and
dwindling natural resources. This entry describes the
historical and conceptual discoveries underlying the
basis of important crop traits, leading from plant
domestication such as in plant architecture to those
offering solutions for high crop production purposes
such as growth under unfavorable environments or
disease outbreaks. Detailed descriptions are provided
on the cloning or identification of the plant genes
involved in crop traits that give an insight on the
evidence required for establishing the relationship. In
addition, the molecular basis of the crop trait provides
an understanding of the biological processes involved,
the interaction to other traits, and a framework that
can be built on to engineer novel solutions to the future
needs from crop plants.
Introduction
The domestication of the major crop plants from around
10,000 to 4,000 years ago resulted in the selection of
crops on which humans are now dependent [1]. The
crop traits involved in domestication, transformed the
wild species to a crop species catered to the needs of this
paradigm shift of human civilization to an agricultural
lifestyle. These early crop traits that made agriculture
possible were selections from wild species that allowed
humans to collect grain from the sown crop involving
non-shattering, larger grain and fruit, determinate
growth with more synchronous harvest, easier accessi-
bility to the grain from protective hard outer coat, and
an overall increase in edibility along with a plethora
of many specialized traits. The genetic analysis
and molecular isolation of key domestication traits is
now uncovering the gene, the regulatory processes,
and the selective sweeps from the nearest
wild ancestors that accompanied the domestication
process [2].
Gene cloning and DNA sequencing from the simple
bacterial organisms to that of the complex polyploids
such as wheat, has enabled researchers to examine gene
sequences one at a time and make conclusions on
similarity between close and distantly related species,
propose functions based on molecular and biological
properties. In fact the generation of gene sequences of
a multitude of organisms is the fastest growing dataset,
which promises to reveal the identity and function of
all living forms. Genome sequencing started with
model and standard genomes. The model plant
Arabidopsis thaliana, a common weed, was selected
for genome sequencing and molecular genetics analysis
[3], with the DNA sequence of a specific ecotype
Columbia [4]. Now, the 1001 genomes project intends
to finish off high-quality resequencing of Arabidopsis
[5] that will provide sequences of genes from the eco-
types adapted to diverse environments, which might
reveal the effect of natural selection and adaptation of
this very effective weed species. This also opens up
a new era of going beyond a “reference” genome
sequence of an organism toward sequence-based
maps of closely related genotypes, which can provide
a more accurate landscape of differences between genes
and genomes and the relationships to expressed traits.
The power of this technology was demonstrated
by revealing the low level of mutations that occur
between plants following a few generations of self-
fertilization [6].
Gene isolation and cloning in higher plants has now
come a long way since the early days of struggling with
genome size and complexity. Isolation of mRNA and
characterization of translated proteins was one of the
primary methods to demonstrate the function of spe-
cific nucleic acids as shown for soybean leghemoglobin
mRNA [7], which was then proven to be transcribed
from the soybean genome [8]. One of the first
approaches in plants to characterize the different com-
ponents of a plant cell in terms of the complexity of
nuclear and polysomal RNA, was using tobacco an

early amenable model for biological studies [9], and
then analysis of the proportion of the genome and the
genes that were transcribed [10]. One of the first plant
genes cloned from genomic DNA was a soybean ribo-
somal DNA (rDNA) gene from a lambda genomic
DNA library [11]. Soybean continued to be a species
of interest, resulting in the generation of a complemen-
tary DNA (cDNA) library for the analysis of auxin
response [12]. In maize, differential expression of the
gene for the chloroplast encoded large subunit of
ribulose bisphosphate carboxylase was shown in bun-
dle sheath cells and absent in mesophyll cells [13],
describing the regulation of enzymes distinguishing
C4 plant cell types. These early studies showed the
potential to address different plant traits by the analysis
of cloned or isolated characterized genes, and the novel
insights they provided.
In the course of improvement in techniques for the
isolation of plant genes, methods were developed to
make a correlation or causative association between
specific functions and gene sequences. These functions
could be defined on the basis of specific proteins with
a biological function or genes determining a phenotype
as revealed by isolated mutants or naturally occurring
variants.
One of the important technologies that helped
bridge the gap between genes and functions was the
development of transformation technologies that
allowed the expression of isolated genes in plants and
the monitoring of the resultant phenotype. Following
early demonstrations of the transfer and expression of
the Agrobacterium tumefaciens Ti-plasmid into plant
cells [14], these Ti-plasmids were engineered to transfer
DNA (T-DNA) and express genes in transformed plant
cells, demonstrated by a number of research groups
around the same time in 1983 [15–19]. In addition to
Agrobacterium-mediated transformation systems,
other methods were developed such as direct DNA
transfer to plant protoplasts [20], electroporation
[21], and particle bombardment [22].
Cloning of plant genes can presently be accom-
plished by a variety of high-throughput ways. The
cloning of expressed genes in the form of cDNA librar-
ies has been done in plasmids, lambda vectors, and
other expression systems that allow the selection of
specific genes based on hybridization, DNA sequenc-
ing, or functional expression assays. The plant genome
Crop Traits: Gene Isolation 669
can be fractionated into representative fragments and
cloned in a variety of sizes in appropriate cloning
vectors. Genome fragments, in progressively increasing
sizes from a few kilobases (Kb) to megabases (Mb), can
be cloned in vectors such as plasmids, lambda phage,
cosmids, bacterial artificial chromosomes (BACs),
yeast artificial chromosomes (YACs), and modifica-
tions to these. While the smaller clones are useful for
characterizing single genes or fragments thereof, the
larger clones are used in positional cloning methods
to identify genes using molecular markers located close
to the gene of interest.
To enable the isolation of plant genes that are asso-
ciated with plant biological functions/processes or crop
traits, a causal relationship has to be made between
DNA sequence and observed phenotype. Genetic anal-
ysis of mutants or other genetic variants that are local-
ized on the chromosome can be used to clone the
corresponding genes by methods such as map-based
cloning (Fig. 1) and transposon tagging (Fig. 2), which
are the two most commonly used methods for cloning
genes by a forward genetics strategy centered around
the phenotype of interest. In positional or map-
based cloning, with a general strategy described in
Fig. 1, the mapped position of a gene on the chromo-
some/genome, which specifies a specific trait or phe-
notype is the starting point to identify molecular/
genetic markers close to the gene and then using inte-
grated physical and genetic maps “walk” or “land”
closer to the gene. The proof of isolating the gene is
by recombination analysis, complementation by trans-
formation, analysis of genetic variants by sequencing,
or mutational analysis of the candidate genes. In trans-
poson tagging, described in a scheme in Fig. 2, an
insertion sequence from a native or heterologous
transposon is used to generate mutants of a specific
phenotype or genetic locus, and analysis of the trans-
poson/insertion-tagged mutant by co-segregation
analysis to a transposon probe, transposon revertant
analysis or complementation of the mutant by trans-
formation, and among the many ways evidence can be
obtained to prove the identity of the tagged gene. With
the availability of genome sequences of many plant
species, these quite anonymous sequences need to be
assigned a function, generally through a reverse genet-
ics strategy. In reverse genetics strategies many tools
are available, in most models and important crops, to
670 Crop Traits: Gene Isolation

90.0

LOD
Locus
3.0

SSR601
SSR602
SSR603

SSR604
SSR605

SSR606
SSR607
SSR608
a
4Mb

Locus
SSR7004
SSR7005

SSR7008
SSR7001
SSR7002
SNP14
SSR7003
SSR604

SSR605
SSR7006

SSR7007
b SNP15
SSR20001

SSR20002

SSR20003

SSR20004
100Kb
SSR7004

SSR7005
SNP101

SNP102

SNP103

SNP104
BAC

Gene1 Gene2 Gene3

Recombinant Phenotype
R1 High
R2 High
R3 Low
c R4 Low

Crop Traits: Gene Isolation. Figure 1

Example of map-based cloning of a gene. (a) Locus mapped to a chromosome region between markers SSR604 and
SSR605 via rough-scale mapping, with LOD (log of odds) score indicating linkage. (b) The gene further delimited to a
100-kb genomic region between markers SSR7004 and SSR7005 via fine mapping and physical mapping. (c) Precision
mapping identified break points flanking a region containing a single gene. Black bars represent DNA from parent 1
(phenotype: high), white bars represent DNA from parent 2 (phenotype: low), and patterned bars represent DNA of
heterozygote. The vertical lines show where recombination break points are positioned along the chromosome

generate or select mutations in specific genes and thus Crop Traits: Gene Isolation
attribute functions by the analysis of the mutant
Dwarfing Genes in Cereals
phenotypes.
This entry describes the specific methods that have The biggest increase in agricultural production in the
been used in the isolation and characterization of genes modern era, around the middle of the last century, is
that determine important crop traits that agriculture is attributed to the “Green Revolution” where grain yields
based on. The crop traits identified in many plants have of the major world cereals, wheat and rice were
homologues in the model plant Arabidopsis that can improved along with complementary crop production
provide clues to the function. The examples described practices [23, 24]. The spectacular increases in wheat
are described in some detail and others summarized in and rice yields during the Green Revolution
a table (Table 1) for the reader to refer to. were enabled by the introduction of dwarfing traits
 Crop Traits: Gene Isolation 671

chromosome
transposon specific primer
mobile non-autonomous

transposition
arbitrary degenerate primer

RB
RB

segregation
TAIL-PCR
LB immobile autonomous

LB

Use flanking DNA sequence as probe to screen Sequence PCR product to get information on
genomic or cDNA library of wild type plant DNA Sequence flanking transposon tag

Sequence selected genomic or cDNA clones to get Use the wild type genomic clone for functional
information on gene disrupted by transposon tag complementation in the transposon mutant

Crop Traits: Gene Isolation. Figure 2

Example of gene tagging using heterologous transposons. An “in cis two-component transposon construct”
integrated into plant chromosome via Agrobacterium-mediated transformation. The construct contains
a mobile nonautonomous transposon and an immobile transposase source. The transposase mediates transposition of
the mobile nonautonomous transposon into new chromosomal positions. Segregation in the next generation creates
stable mutants containing the transposon inserted in a gene with no transposase source. These stable mutants can be
used in forward or reverse genetics. Identification of integration position is typically done by sequencing of DNA flanking
the transposon isolated using techniques, for example, TAIL-PCR, Inverse PCR, or plasmid rescue. The flanking sequence is
used as a probe to screen genomic and cDNA clones. Functional complementation is done by transforming wild-type
genomic clone containing the tagged gene into the insertional mutant line

into the crop varieties. The founder of this Green Rev- the DELLA proteins and a repressor of GA responses
olution, Norman Borlaug received the Nobel Peace [28]. The wheat Rht-B1b and Rht-D1b dwarfing
prize in 1970 in recognition for his efforts in bringing mutants had similar characteristics to the Arabidopsis
food security to major parts of the developing gai mutant, including reduced plant height, reduced
world [25]. Identification of the genes responsible for responses to gibberellin, and increased in planta gib-
these traits showed that they interfere with the berellin levels. Compared to the wild-type Rht-1 allele,
action or production of the gibberellin (GA) plant the Rht-B1b and Rht-D1b dwarfing alleles each
hormones [26]. contained a nucleotide substitution that created
The dwarfing gene of wheat, Reduced Height-1 a stop codon near the N-terminus of the protein [27].
(Rht-1), was isolated based on sequence similarity to It was proposed that translational reinitiation after these
the previously isolated Arabidopsis GIBBERELLIN introduced stop codons might result in an N-terminally
INSENSITIVE (GAI) gene [27]. GAI is a member of truncated product that confers the mutant phenotype.
672 Crop Traits: Gene Isolation

Crop Traits: Gene Isolation. Table 1 Some isolated genes in crops

Cloning Validation Causative
Gene (s) Crop Molecular and phenotypic function methoda methodb changec Reference (s)
1. Dwarfing gene
Rht-1 Wheat Transcriptional regulator (SH2); plant height SS Mut, HE EStp [27]
sd1 Rice GA20 oxidase; plant height SS, MBC Var Coding [30–32]
d Tomato Cytochrome P450 enzyme; plant height TT FC [141]
2. Flowering/heading date
Hd6 Rice Protein kinase; flowering time MBC FC EStp [33]
Hd1 Rice Transcriptional regulator (zinc finger); MBC FC Coding [34]
flowering time
Hd3a Rice Phosphatidylethanolamine-binding protein; MBC OE, Sil - [37]
flowering time
Ehd1 Rice B-type response regulator; flowering time MBC FC AC [142]
Vgt1 Maize Transcriptional regulator (AP2); flowering time MBC, AM OE, Sil Reg [143]
E3 Soybean Phytochrome A; flowering time MBC Mut AC? [144]
Vrn1 Wheat Transcriptional regulator (MADS); MBC Var Reg [145]
vernalization
Vrn2 Wheat Transcriptional regulator (ZCCT); vernalization MBC Sil AC [146]
3. Fruit ripening, shape, and weight
Rin Tomato Transcriptional regulator (MADS-box); fruit MBC FC, Sil Coding [147]
ripening
Ovate Tomato Regulatory protein (OVATE); fruit shape MBC FC EStp [148]
fw2.2 Tomato Cell signaling; fruit weight MBC FC Reg [40]
4. Grain yield
Gn1 Rice Cytokinin oxidase/dehydrogenase; grain MBC Sil Reg/EStp [42]
number
GS3 Rice VWFC module-containing protein; grain MBC Var EStp [44]
weight
GW2 Rice RING-type protein with E3 ubiquitin ligase MBC OE, Sil EStp [47]
activity; grain width
5. Disease resistance
Hm1 Maize HC toxin reductase; resistance to the fungus TT Co Reg [49]
Cochliobolus carbonum
Cf-9 Tomato Leucine-rich repeat family of proteins; TT Co Wild [54]
resistance to leaf mold fungus Cladosporium
fulvum
RB/Rpi- Potato CC–NBS–LRR-class R-gene analog; resistance MBC FC Wild [58, 59]
blb1 to oomycete pathogen Phytophthora infestans
Rpi-blb2 Potato NBS–LRR protein; resistance to oomycete MBC FC Wild [60]
pathogen Phytophthora infestans
 Crop Traits: Gene Isolation 673

Crop Traits: Gene Isolation. Table 1 (Continued)

Cloning Validation Causative

Gene (s) Crop Molecular and phenotypic function methoda methodb changec Reference (s)
mlo Barley Membrane-anchored protein; resistance MBC Mut, RA AC/EStp [62]
against the fungal pathogen Blumeria
graminis f. sp. hordei (Bgh)
Mi Tomato NBS–LRR protein; resistance to root-knot MBC FC Wild [72]
nematodes (Meloidogyne spp.)
Tm-22 Tomato CC–NBS–LRR class of R proteins; resistance to TT FC Wild [76]
tomato mosaic virus (ToMV)
Pto Tomato Protein kinase; resistance to Pseudomonas MBC FC Wild [149]
syringae pv. tomato
Xa21 Rice Protein kinase; resistance to Xanthomonas MBC FC Wild [150]
oryzae pv. oryzae
Cf-2 Tomato LRR protein; resistance to leaf mold fungus MBC FC Wild [151]
Cladosporium fulvum
N Tobacco LRR protein; resistance to tobacco mosaic TT FC Wild [152]
virus (TMV)
Hs1pro-1 Sugar LRR protein; resistance to beet cyst nematode MBC FC Wild [153]
beet
I2C Tomato NBS–LRR protein; resistance to Fusarium MBC Sil, OE Wild [154]
oxysporum f sp 1ycopersici
Ve Tomato Cell surface-like receptors; resistance to MBC FC - [155]
Verticillium dahliae
R1 Potato Leucine-zipper/NBS/LRR protein; resistance to MBC FC Wild [156]
Phytophthora infestans
Rpg1 Barley Receptor kinase; resistance to Puccinia MBC Var EStp, AC, [157]
graminis f. sp. tritici FS
Hero Tomato NBS–LRR protein; resistance to potato cyst MBC FC Wild [158]
nematodes Globodera rostochiensis
Lr10 Wheat CC–NBS–LRR protein; resistance to Puccinia MBC, HS OE CD [159]
triticina
Lr21 Wheat NBS–LRR protein; resistance to Puccinia MBC FC Wild [160]
triticina
Pm3b Wheat CC–NBS–LRR protein; resistance to Blumeria MBC STA - [161]
graminis f. sp. tritici
Pi9 Rice NBS–LRR protein; resistance to Magnaporthe MBC FC Wild [162]
grisea
Rpg5 Barley Protein kinase; resistance to Puccinia graminis MBC Sil FS [163]
f. sp. secalis
Yr36 Wheat Kinase-START protein; resistance to Puccinia MBC FC Wild [164]
(WKS1) striiformis f. sp. tritici
Rdg2a Barley CC–NB–LRR protein; resistance to MBC FC Reg [165]
Pyrenophora graminea
674 Crop Traits: Gene Isolation

Crop Traits: Gene Isolation. Table 1 (Continued)

Cloning Validation Causative

Gene (s) Crop Molecular and phenotypic function methoda methodb changec Reference (s)
6. Plant and inflorescence architecture
tb1 Maize Transcriptional regulator (TCP); plant and TT Co Reg [80]
inflorescence structure
tga1 Maize Transcriptional regulator (SBP); seed casing MBC Mut AC [85]
Q Wheat Transcriptional regulator (AP2); inflorescence MBC Mut Reg/AC [92]
structure
vrs1 Barley Transcriptional regulator (HD-ZIP); spikelet MBC Mut AC/FS [98]
morphology
nud Barley Transcriptional regulator (ERF); seed casing MBC Var, Mut CD [103]
MOC1 Rice Transcriptional regulator (GRAS); tillering MBC FC TE [107]
PROG1 Rice Transcriptional regulator (ZF); growth habit MBC FC Reg/AC [109]
7. Seed quality and color
opaque-2 Maize Transcriptional regulator (bZIP); endosperm TT Co TE [111]
characteristic
Wx Maize Starch synthase; sticky grains TT Rev TE [113]
Wx Rice Starch synthase; sticky grains SS Var Splice [115, 116]
Sh2 Maize Pyrophosphorylase; supersweet sweet corn TT Rev TE [118]
su1 Maize Isoamylase; sweet corn gene TT Co AC [119]
Rc Rice Transcriptional regulator (bHLH); seed color MBC Mut Coding [121]
c1 Maize Transcriptional regulator (MYB); kernel color TT Co Reg [166]
y1 Maize Phytoene synthase; carotenoid content TT Rev Reg [167]
R Pea Starch branching enzyme; seed sugar content IS Co TE [168]
Brix9-2-5 Tomato Invertase; fruit-soluble solid content MBC IR Reg [169]
8. Seed shattering
sh4 Rice Transcriptional regulator (Myb3); abscission MBC FC Reg/AC [128]
layer formation, shattering
qSH1 Rice Transcriptional regulator (homeodomain); MBC FC Reg [129]
abscission layer formation, shattering
sh-h Rice CTD phosphatase; abscission layer MBC Mut, Sil Splice [131)
differentiation, shattering
Jointless Tomato Transcriptional regulator (MADS); abscission MBC FC, Sil Coding [170]
zone development, shedding
9. Tolerance to abiotic stresses
SKC1 Rice HKT-type transporters; salt tolerance MBC FC AC [134]
Sub1 Rice Transcriptional regulator (ERF); submergence MBC FC CD [137]
tolerance
 Crop Traits: Gene Isolation 675

Crop Traits: Gene Isolation. Table 1 (Continued)

Cloning Validation Causative

Gene (s) Crop Molecular and phenotypic function methoda methodb changec Reference (s)
Alt1 Wheat Aluminum-activated malate transporter; SH Co, Var, HE Reg/AC [140]
tolerance to aluminum toxicity
Snorkel1, Rice Transcriptional regulator (ERF); deepwater MBC Var, OE CD [171]
Snorkel2 tolerance
a
MBC map-based cloning, TT transposon tagging, AM association mapping, HS haplotype study, SS sequence similarity, SH subtractive
hybridization, IS immunological screening
b
FC functional complementation, Rev transposon revertant analysis, Co co-segregation analysis, RA recombination analysis, Var analysis of
genetic variants by sequencing, Mut mutational analysis of the candidate genes, OE overexpression, Sil silencing, IR intragenic recombi-
nation, HE heterologous expression, STA single-cell transient assay
c
AC amino acid change, Coding disrupted coding sequence, FS frame shift, EStp early stop codon, Reg regulatory change, Splice intron
splicing defect, CD complete deletion, Wild introgression from wild relative,TE transposon insertion (Following Doebley et al. [1] with
modification)

The rice sd-1 dwarfing allele, with origin from cul-
tivar Dee-Geo-Woo-Gen and used in the Green Revo-
lution dwarf variety IR8, was mapped to the long arm
of chromosome 1 [29]. Characterization and isolation
of this gene was reported in 2002 by three different
groups [30–32]. Biochemical analysis of the sd-1
mutant showed that the activity of GA20 oxidase
(GA20ox), a key enzyme in the biosynthesis of gibber-
ellin, did not function effectively in the mutant. A wild-
type GA20ox gene (GA20ox-2) was amplified by PCR
using primers based on the conserved domain of
GA20ox genes. Linkage mapping showed that this
gene mapped to the long arm of chromosome 1, tightly
linked to the sd1 locus. Compared to the wild-type Sd-1
allele, the sd-1 dwarfing allele (in Dee-Geo-Woo-Gen
and IR8) contained a 383-base-pair deletion which
produces a frame shift that creates a stop codon [30].
Another study showed RFLP markers flanking the sd-1
locus were positioned on a physical segment of chro-
mosome 1 covered by contiguous BAC clones by using
the physical map of the reference genome available in
the database. A candidate gene search identified the
GA20ox-2 gene in this region. This gene was amplified
in the wild type and mutant, and sequence comparison
revealed a 280-bp deletion in the coding region of this
gene in the mutant allele [31]. A third independent
analysis of 3,477 segregants using several PCR-based
markers localized the sd-1 locus in a 6-kb candidate
interval on chromosome 1, containing only one
predictable ORF, that of GA20ox-2. The 3,477 segre-
gants were derived from selfing of a backcross inbred
line (BIL) having close chromosomal similarity to
Sasanishiki (normal-type parent) over the whole
genome length with the only heterozygous sequences
located on sd-1 locus. Sequence comparison showed
that Habataki (semidwarf parent) has a 383-bp dele-
tion from the middle of exon 1 to upstream of exon 2,
including a 105-bp intron, resulting in a frame shift
that produces a termination codon in exon 3 [32].
These evidence clearly defined the identity of the Sd-1
dwarfing gene and the mutant sd-1 alleles.
Flowering or Heading Date
Heading date or flowering time is an important trait for
the adaptation of crops to different cultivation areas. In
rice, heading date is determined mainly by two factors:
duration of the basic vegetative growth and photope-
riod sensitivity (PS) [33]. Genetic analysis of heading
date in rice cultivars Kasalath and Nipponbare showed
that a number of quantitative trait loci, termed Hd
(heading date) determine the genetic variation for
flowering time [34]. Day-length treatment sensitivity
tests with an NIL of Hd6 [NIL(Hd6)] revealed the Hd6
PS phenotype, with the Kasalath allele increasing days-
to-heading under natural and long-day conditions but
not under short-day conditions. Linkage analysis of an
advanced backcross progeny mapped Hd6 on the long
676 Crop Traits: Gene Isolation
arm of chromosome 3 as a single Mendelian factor
[35]. High-resolution mapping using 2,807 segregating
plants derived from an advanced backcross progeny, in
which the region around Hd6 was heterozygous and
almost all other regions were homozygous for
Nipponbare, delimited Hd6 to a 26.4-kb genomic
region. The sequence analysis of this region identified
one gene with deduced amino acid sequence having
high homology (>90%) to the a subunit of protein
kinase CK2 (CK2a) in maize and Arabidopsis.
Sequence comparison showed a single-nucleotide sub-
stitution within the coding region, which changed the
lysine codon (AAG) in Kasalath to a premature stop
codon (TAG) in Nipponbare. Functional complemen-
tation was performed by introducing the Kasalath
genomic fragment carrying the CK2a gene into
Nipponbare and the transgenic plants scored under
natural day-length condition showed late heading,
indicating that the Kasalath allele of CK2a increases
days-to-heading [33].
Another heading date locus Hd1 was characterized
by high-resolution mapping using 1,505 early heading
BC3F3 segregants derived from a cross between
Nipponbare and Kasalath, which resolved the locus to
a genomic region of 12 kb. Sequence analysis of the
region identified one putative gene with considerable
similarity to the CONSTANS (CO) gene, known to be
involved in photoperiod response in Arabidopsis.
Sequence comparison showed a 2-bp deletion in the
second putative exon of the Kasalath allele resulting in
a premature stop codon and a predicted shorter protein
than the wild-type Nipponbare protein. Functional
complementation was performed by transferring the
candidate Hd1 region from Nipponbare into a NIL of
Nipponbare carrying an introgression of Kasalath
nonfunctional Hd1 allele. Transgenic plants showed
earlier heading under short-day conditions than the
NIL control and null-segregants. These results provide
clear evidence that the Hd1 sequence in the candidate
genomic region retains the function of photoperiod
response [34].
The Hd3a locus was roughly mapped on chromo-
some 6. NIL(Hd3a), an NIL homozygous for the
Kasalath allele at the Hd3a locus in the genetic
background of Nipponbare, headed earlier than
Nipponbare under SD conditions and headed at almost
the same date as Nipponbare under LD and natural
field conditions [36]. High-resolution mapping using
2,207 segregants delimited Hd3a to a 20-kb genomic
region, whose sequence revealed one putative gene with
high similarity to the FLOWERING LOCUS T (FT)
gene, which promotes flowering in Arabidopsis. In
this study, the nucleotide polymorphism that caused
the allelic difference between Kasalath and Nipponbare
Hd3a could not be clarified [37].
Fruit Weight and Grain Yield
The domestication and improvement of crops has been
accompanied by increase in size and yield of harvest-
able products. Cultivated tomatoes (Solanum
lycopersicum) have hence undergone more than a
100-fold increase in mass over their wild relatives.
A major quantitative trail locus (QTL) for fruit weight,
fw2.2, was mapped to the same position on
chromosome 2 in an introgression line F2 derived
from S. lycopersicum x Lycopersicon pennellii and
a backcross 1 (BC1) population derived from
S. lycopersicum x. Lycopersicon pimpinellifolium. The
fw2.2 locus accounts for 30% and 47% of the total
phenotypic variance in the L. pimpinellifolium and
L. pennellii populations, respectively, indicating that
this is a major QTL controlling fruit weight in both
species. The small-fruit L. pennellii allele for fw2.2 is
semidominant to the large-fruit S. lycopersicum allele
[38]. High-resolution mapping using 3,472 F2 plants
derived from a cross between S. lycopersicum and a NIL
containing a small introgression from L. pennellii
narrowed down fw2.2 to a less than 150-kb region
[39]. A yeast artificial chromosome (YAC) containing
fw2.2 was used to screen a cDNA library constructed
from the small-fruit L. pennellii. Four unique tran-
scripts were identified and used to screen a L. pennellii
cosmid library, identifying four positive,
nonoverlapping cosmids (cos50, cos62, cos69, and
cos84), one corresponding to each unique transcript.
These four cosmid clones were transformed into two
tomato cultivars carrying the partially recessive large-
fruit allele of fw2.2. R1 progeny of primary
transformants carrying cos50, but not other cosmids,
showed a statistically significant reduction in fruit
weight compared to null-segregants, indicated that
fw2.2 is contained within cos50. Sequence analysis of
cos50 revealed two open reading frames (ORFs): one

corresponding to cDNA44, which was used to isolate
cos50, and another 663-nucleotide (nt) gene, ORFX,
for which no corresponding transcript was detected in
the initial cDNA library screen. Analysis on a single
recombination event used in the previous mapping
showed that ORFX is the likely cause of the fw2.2
QTL phenotype. Semiquantitative reverse transcrip-
tase-polymerase chain reaction (RT-PCR) analysis
showed that the relative level of the ORFX transcript
in the carpels of the small-fruited NIL was significantly
higher than in the large-fruited NIL. Analysis of the
predicted amino acid sequence of ORFX indicated that
it is a soluble protein with alpha/beta-type secondary
structure, and has a structural similarity to the human
oncogene c-H-ras p21. Sequence comparison of
L. pennellii and S. lycopersicum ORFX regions indicated
that the fw2.2 phenotype is probably not caused by
differences within the coding region of ORFX, but by
one or more changes upstream in the promoter region
of ORFX [40].
Grain yield is a complex crop trait determined
mainly by the three component traits, number of pan-
icles, number of grains per panicle, and grain weight; all
of which are typical quantitative traits. In rice, the
developments in genome mapping, sequencing, and
functional genomic research have provided the neces-
sary tools for dissecting the genetic and molecular bases
of these quantitative traits [41].
The rice grain number gene Gn1 was first roughly
mapped on the short arm of chromosome 1 using 96 F2
individuals derived from heterozygote (Gn1/gn1)
plants of NIL-Gn1 carrying the Gn1 region from culti-
var Habataki in the Koshihikari background. Habataki
plants have been known to produce more grains in
their main panicle than Koshihikari plants. It was
found that Gn1 consisted of two loci, QTL-Gn1a and
QTL-Gn1b. High-resolution mapping using 13,000 F2
plants derived from heterozygotes (Gn1a/gn1a) of NIL-
Gn1a narrowed down the Gn1a region into a 6.3-kb
interval containing one reading frame with high simi-
larity to cytokinin oxidase/dehydrogenase (CKX),
named OsCKX2. Sequence comparison between
Habataki and Koshihikari revealed several nucleotide
changes, including a 16-bp deletion in the 50 -
untranslated region, a 6-bp deletion in the first exon,
and three nucleotide changes resulting in amino acid
variation in the first and fourth exons of the Habataki
Crop Traits: Gene Isolation 677
allele. However, protein expression study in Saccharo-
myces cerevisiae showed both Habataki and Koshihikari
alleles of OsCKX2 encode functional enzymes.
Sequence analysis of 5150, a rice variety from China
producing higher grain number, detected 11-bp dele-
tion in the coding region of this gene creating
a premature stop codon. The coincidence of the
OsCKX2 null allele and a higher grain number
suggested that a reduction or loss of function of
OsCKX2 enhanced grain production. Confirmation
was done by introducing antisense strands of OsCKX2
into an easily transformable cultivar Taichung 65
(TC65), which possesses the Koshihikari allele of
OsCKX2. Transgenic plants with reduced levels of
expression developed higher grain numbers. Reverse
Transcription (RT)-Southern blotting showed that
the highest levels of OsCKX2 expression in inflores-
cence meristems were found in Koshihikari, but were
less abundant in Habataki and NIL-Gn1a and
extremely low in 5150. These results suggested that
the phenotypic differences observed might have been
caused by differential transcription of OsCKX2 [42].
The rice GS3 grain weight gene was mapped to the
pericentromeric region of chromosome 3 [43]. Near-
isogenic lines (NILs) of GS3 were developed by succes-
sive crossing and backcrossing Minghui 63 (large
grain) with Chuan 7 (small grain), using Minghui 63
as the recurrent parent. High-resolution mapping
using 1,384 BC3F2 individuals with the recessive phe-
notype (large grain) derived from one BC3F1 plant
heterozygous for the GS3 region and containing the
least genetic background from Chuan 7 delimited the
GS3 region to 7.9 kb. A full-length cDNA was identified
that matched well with the region, and sequence anal-
ysis of the predicted GS3 protein revealed the presence
of a combination of multiple domains. Comparative
sequencing analysis using six cultivars including three
with long grains and three with short to medium grains
indicated that all the large-grain varieties tested share
the same nonsense mutation in the second exon of the
GS3 gene that causes a 178-aa truncation in the
C-terminus of the predicted protein. These findings
suggest that GS3 may function as a negative regulator
to prevent the growth and size of the grain [44].
Two QTLs for grain width were mapped to the short
arm of chromosome 2 [45, 46). Subsequently, a major
QTL for grain width GW2, was mapped to the same
678 Crop Traits: Gene Isolation
region using an F2 population derived from a cross
between WY3 (1,000-grain weight, 41.9  1.3 g) and
Fengaizhan-1 (FAZ1) (1,000-grain weight, 17.9 
0.7 g). High-resolution mapping using 6,013 BC3F2
plants narrowed the GW2 locus to an 8.2-kb region.
Only one predicted ORF was considered a viable can-
didate for GW2 in this region [47]. Homology search in
databases showed that GW2 encodes a previously
unknown RING-type protein with E3 ubiquitin ligase
activity, which is known to function in the degradation
by the ubiquitin–proteasome pathway. Comparison of
the nucleotide sequences of the FAZ1 and WY3 alleles
of GW2 uncovered a 1-bp deletion resulting in
a premature stop codon in exon 4 of the WY3 allele.
The premature stop codon led to truncation of 310
amino acid residues; the remaining portion of the
protein consisted of a 115-residue polypeptide of
13 kDa. Sequence analysis of GW2 from Oochikara,
another rice variety that has a wider grain width, sim-
ilar to WY3, showed a nucleotide sequence identical to
the WY3 allele. These data indicate that reduction or
loss of function of GW2 results in increased grain
width. Rice transformation was used to produce trans-
genic plants expressing different levels of GW2. The
transgenic plants with antisense strands of GW2 and
with reduced levels of endogenous expression had
a significantly wider grain width than plants containing
the vector control. Transgenic plants overexpressing
GW2 cDNA under the control of the 35S promoter,
which produced high levels of expression, showed
reduced grain width [47].
Disease Resistance
Disease resistance loci (R genes) conferring resistance to
specific races of the pathogen, carrying corresponding
avirulence (Avr) genes, have been studied in light of the
gene-for-gene hypothesis proposed by Flor [48]. Plants
that contain such race-specific R-genes with resistance
to specific pathogen races, offer an interesting system to
study plant pathogen interactions, although the genes
are very rarely useful in the crop as they are quickly
overcome by the pathogen gaining virulence.
In addition, there are non-race-specific disease
resistance loci which provide plant resistance to
a wider range or races of the pathogens. The first
plant resistance gene cloned was Hm1 from maize,
which belonged to this class of resistance genes, was
isolated by transposon tagging [49]. Two RFLP probes
were mapped to 5 centimorgan (cM) proximal and
distal to the Hm1 locus, respectively, using progeny
from the cross K61/Pr1  K61 [50]. These two probes
were used to classify segregating progeny of transposon
insertion hm1 mutants to determine which HM1 alleles
they had inherited. A transposable element probe was
then used to identify a restriction fragment that
cosegregated with hm1 mutant allele. This fragment
was isolated and sequenced, and the DNA flanking
the transposon insertion was used as probe in northern
blot analysis. A 1.3-kb RNA band was detected in
polyadenylated [poly(A)+] RNA from the resistant
inbred strain Pr1 (Hm1-Pr1), while the susceptible
strain, K61 (hm1-2), and the mutants either had no
detectable hybridizing mRNA, or an mRNA of aberrant
size. Difference at the transcriptional level between
resistant and susceptible genotypes makes it unlikely
that susceptible genotypes possess an alternative form
of HM1 with specificity for a substrate other than HC
toxin. A 1.6-kb cDNA clone was isolated by homology
with the probe and sequenced. DNA sequence data-
bases revealed homology between the HM1 cDNA and
the NADPH-dependent dihydroflavonol-4-reductase
(DFR) genes of maize, petunia, and snapdragon. This
homology supports the prediction that HM1 encodes
HC toxin reductase (HCTR) [49].
Among the race-specific R-genes, the tomato-leaf
mold interaction has been well studied and yielded the
first examples of cloned R-genes. The avirulence gene
Avr9 in the leaf mold fungus Cladosporium fulvum was
shown to specify a 28-amino acid secreted peptide that
elicits a necrotic response when injected into tomato
plants carrying the Cf-9 resistance gene [51]. The Cf-9
gene was introgressed into cultivated tomato from wild
species L. pimpinellifolium accession PI126915 [52].
The Cf-9 locus was mapped to the short arm of chro-
mosome 1 [53], and a targeted transposon tagging
strategy was employed to isolate the Cf-9 gene [54].
A tomato line homozygous for Cf-9 was crossed to
a transgenic tomato line carrying a Ds element located
3 cM from the Cf-9 locus, and additionally to a trans-
genic tomato plant containing a stabilized Ac (sAc)
element. The F1 plants were crossed and the progenies
selected to produce tagging parents heterozygous for
Ds and sAc and homozygous for Cf-9. To tag Cf-9 the

tagging lines were crossed as female parents to a tomato
line homozygous for the Avr9 transgene and lacking
Cf-9. The progeny of this cross, which were heterozy-
gous for Cf-9 and Avr9, became necrotic and died
shortly after seed germination, but the mutants for
Cf-9 survived. Approximately 160,000 progeny were
germinated and 118 survivors were recovered. A total
of at least 37 independent Ds insertions into Cf-9 were
identified. Specific Cf-9 primers were used in conjunc-
tion with Ds primers to map the Ds insertions on the
basis of PCR product size. Twenty-eight Ds insertions
were mapped to the same 3-kb region of the tomato
genome. All stable mutants tested were susceptible to
race 5 of Cladosporium fulvum, which indicates con-
cordance between the loss of response to the Avr9
transgene and loss of resistance to a race of the fungus
carrying Avr9. Isolation via plasmid rescue and
sequencing of the flanking genomic sequence showed
that Cf-9 encodes a putative membrane-anchored
extracytoplasmic glycoprotein with homology to the
leucine-rich repeat family of proteins [54].
Late blight caused by the oomycete pathogen
Phytophthora infestans, and responsible for the Irish
Potato Famine of the mid-nineteenth century that
induced vast migration, is still one of the most devas-
tating of plant diseases causing more than $3 billion
loss annually [55]. The P. infestans pathogen easily
overcomes the R-genes crossed in from various wild
relatives of potato. Diploid Solanum bulbocastanum
from Mexico, which was not easily crossable to potato
(Solanum tuberosum) cultivars, had been characterized
to have high resistance to P. infestans and was used to
characterize the resistance gene loci. The RB (for
“Resistance from S. bulbocastanum”) locus was
mapped to chromosome 8 of S. bulbocastanum [56].
BAC walking by the reiterative screening of a BAC
library using probes derived from the ends of previ-
ously identified BAC clones, was initiated using linked
RFLP markers and the contig subsequently used to
develop PCR-based markers to enhance map resolution
in the RB region. High-resolution mapping using 542
BC2, 1,060 BC3, and 206 BC4 genotypes delimited the
RB region to approximately 55 kb [57]. This region
contained one truncated and four complete CC–
NBS–LRR (coiled coil–nucleotide binding site–
Leu-rich repeat)-class R-gene analogs (RGAs). Each of
the four complete RGAs was amplified from
Crop Traits: Gene Isolation 679
S. bulbocastanum using Long Range-PCR and cloned
into a binary vector for complementation studies.
Katahdin, a late blight susceptible potato variety was
transformed with the four complete R-gene analogs.
Only the transgenic plants containing RGA2-PCR con-
struct displayed resistance to all six isolates of
P. infestans, including 126C18, a “super race” that over-
comes all 11 major R genes identified in Solanum
demissum, the complementation demonstrating that
RGA2 represents the functional RB gene [58].
In a simultaneous effort to the analysis of the RB
locus, the Rpi-blb1 resistance locus was characterized in
intraspecific crosses between S. bulbocastanum acces-
sions segregating for resistance, and mapped closely to
marker CT88 on chromosome 8 [59]. A BAC contig
was isolated across the Rpi-blb1 locus, and sequence
analysis revealed 4 RGAs of the CC–NBS–LRR class.
One of the genes termed Rpi-blb1 complemented
potato and tomato cultivars to confer resistance
to complex races of the P. infestans pathogen. In
subsequent analysis of resistant complex inter-
specific hybrids designated ABPT derived from
S. bulbocastanum, the Rpi-blb2 locus was identified and
mapped to a position on chromosome 6 at a similar
position as the tomato Mi locus (see below) conferring
resistance to nematode, aphids, and white flies. The
Rpi-blb2 locus harbored 15 Mi-1 gene homologues, one
of which conferred P. infestans resistance in tomato and
potato. The Rpi-blb2 protein shares 82% sequence
identity to the tomato Mi-1 protein and exemplifies the
evolution of a resistance gene cluster to confer diverse
resistance specificity to a wide range of organisms such as
nematodes, insects, and oomycetes [60].
The barley recessive mlo locus conferring resistance
against the fungal pathogen Blumeria graminis f. sp.
hordei (Bgh) has been mapped on chromosome 4 [61].
High-resolution mapping using 2,022 F2 segregants
identified a DNA marker cosegregating with mlo and
two flanking markers at a distance of 0.24 and 0.4 cM,
respectively. Screening of a large insert yeast artificial
chromosome (YAC) library identified three YAC clones
containing the cosegregating marker and two flanking
markers. Subcloning experiments of one YAC clone
into bacterial artificial chromosome (BAC) vector and
further mapping and sequencing delimited the mlo
region to 30 kb. Sequence analysis identified one
sequence contig of 5.8 kb, including the cosegregating
680 Crop Traits: Gene Isolation
marker, revealing an extensive region of high coding
probability. Reverse transcriptase-PCR using a series of
primers deduced from this region and sequencing
revealed a single extensive open reading frame (ORF)
of 1,599 bp. The deduced 60 kDa protein was predicted
to be membrane-anchored by at least six membrane-
spanning helices. Genomic PCR-based sequencing of
11 mutagen-induced mlo resistance alleles and their
corresponding wild-type DNAs identified nucleotide
alterations (point mutations or deletions) in all tested
mutant alleles that at the amino acid level result either
in single amino acid substitutions or truncated versions
of the predicted wild-type protein. A comparison
among the wild-type gene sequences of seven tested
barley cultivars indicated not a single amino acid dif-
ference. Inter-mutant crosses were performed using
lines containing different mutant alleles. F2 seedlings
were screened for rare disease–susceptible individuals
after inoculation with an isolate of powdery mildew
which is virulent on each of the parental Mlo wild-type
cultivars. Homozygous susceptible F3 progeny were
isolated and used for molecular analysis using RFLP
markers tightly linked (<4 cM) on each side of the Mlo
locus. Seven susceptible individuals exhibited flanking
molecular marker exchange, indicating reciprocal
crossover (CO) events. Genomic PCR-based sequenc-
ing demonstrated that all seven CO type recombinants
restored wild-type sequences [62].
Root-knot nematodes (Meloidogyne spp.) are endo-
parasites of thousands of crop species and are impor-
tant pests of tomato worldwide [63]. The root-knot
nematode resistance gene (Mi) was introduced into
cultivated tomato, S. lycopersicum, from its wild relative
Lycopersicon peruvianum in the early 1940s [64], and
today it provides the only form of genetic resistance
against this pathogen. The Mi locus was localized to the
short arm of chromosome 6 [65] and multiple markers
linked to Mi were identified [66–69]. In these studies, it
was found that the nematode-resistant tomato line
Motelle, and related lines, retained only a small
introgressed region (650 kb) from L. peruvianum. Efforts
to localize the Mi gene were hampered for many years
because of the severe repression of recombination near
this gene in Lycopersicon esculentum lines carrying
the introgressed L. peruvianum DNA [67, 68, 70].
This handicap was circumvented by screening
large populations of tomato for recombinants and by
identifying recombinants within L. peruvianum
populations [71]. After data from S. lycopersicum and
L. peruvianum recombinant analyses were combined,
Mi could be localized to a genomic region of <65 kb.
Subcloning experiments of one YAC clone containing
the Mi locus identified four overlapping BACs hybrid-
izing to the Mi-flanking DNA probes. Large-scale
sequencing of two BACs identified six open reading
frames; three of these are homologous to each other
and to previously identified R genes of the nucleotide
binding–LRR class. Two of them, Mi-1.1 and Mi-1.2,
appear to be intact genes; the third is a pseudogene
predicted to not encode a functional product. Comple-
mentation studies were performed by transforming
a nematode-susceptible tomato line using constructs
containing the Mi-1.1 and Mi-1.2 genes. Eighty-seven
percent of transformants carrying Mi-1.2 were resistant
to the root-knot nematode, whereas all transformants
carrying Mi-1.1 were completely susceptible,
confirming Mi-1.2 as the functional Mi-1 gene [72].
In cultivated tomato, tomato mosaic virus (ToMV)
infections are controlled by the introgressed Tm-1, Tm-2,
and Tm-22 resistance (R) genes [73, 74]. Among these
resistances the Tm-22 resistance was shown to be
remarkably durable and, therefore, of ongoing practical
importance. The Tm-22 locus has been localized to
tomato chromosome 9, but map-based cloning of this
gene has been shown to be difficult, especially due to
the lack of recombination in the centromeric region
[75]. The two-component Ac/Ds transposon system
was utilized to isolate the tomato Tm-22 gene.
A tomato line (homozygous Tm-22) was crossed with
another line (homozygous for sAc). One F2 plant from
this cross (homozygous for both sAc and Tm-22) was
subsequently used in a cross with a tomato line having
Ds transposon on chromosome 9 with distance 2 cM
from the resistance gene. About 100 independent
plants with the genotype Ds/–; sAc/–; Tm-22/Tm-22
were selected and used as males and females in a cross
with a transgenic tomato line which is homozygous for
the ToMV MP (Movement Protein) gene for a large-
scale tagging experiment. Previous observation had
shown a lethal combination of the Tm-22 gene and
the ToMV MP transgene in the seedling stage of tomato
plants. From about 30,000 seeds obtained from these
crosses, five putative mutants were obtained. All
contained the Ds element and the MP gene and only

one mutant plant still contained sAc. To test whether
these five putative mutants were really mutants in the
Tm-22 gene, cuttings of these plants were inoculated
with ToMV. All the five putative mutants were suscep-
tible to ToMV infection. Plasmid rescue was used to
obtain transposon-flanking plant DNA from the
mutant plants. Sequencing of 9.8 kb of rescued plant
DNA identified an open reading frame (ORF) of
2,586 bp, which corresponds to a polypeptide of 861
amino acids. In silico analysis of the ORF did not
predict the presence of introns. The polypeptide
encoded by the 2,586-bp ORF shows the characteristics
of the CC–NBS–LRR class of R proteins, and the 50 -
and 30 -RACE experiments confirmed the prediction of
the gene structure. Functional complementation was
performed by introducing either Tm-22 gene under the
control of its own promoter and polyadenylation signal
or Tm-22 gene under the control of the CaMV 35S
promoter and the NOS polyadenylation signal into
a susceptible tomato line. The transgenic plants of the
two constructs were resistant to ToMV [76].
Plant and Inflorescence Architecture
Maize Domestication Traits Most crop plants differ
considerably from their wild ancestors in major mor-
phological phenotypes that have probably resulted
from visual selection of desired plant type. This is
most dramatic in maize, where the major difference
between maize and its probable wild ancestor teosinte
is that teosinte typically has long branches with tassels
at their tips whereas maize possesses short branches
tipped by ears [77]. Most of the variation for the dra-
matic differences in inflorescence morphology between
maize and teosinte is explained by five quantitative trait
loci (QTLs) [78]. Complementation tests indicated
that one of these QTLs, which is on chromosome arm
1L, is the locus for the maize mutant teosinte branched l
(tbl) [79]. To isolate the tb1 gene, homozygous tb1-ref
(a spontaneous mutant in a maize population) plants
were crossed to an active Mu transposon line, and
26,000 F1 plants were grown. Among these, three new
tb1 mutants (tb1-mum1, tb1-mum2, tb1-mum3) were
observed. Each new mutant was crossed to maize
inbred A532, and 14 progeny from each of these crosses
were used for Southern blot analysis with marker
loci that closely flank tb1 to discriminate progeny that
Crop Traits: Gene Isolation 681
possessed the tb1-ref versus the new Mu alleles.
The progeny were also screened by Southern blot
analysis with Mu element probes, and a Mu3 element
that cosegregated with tb1-mum1 was identified.
Overlapping genomic restriction fragments carrying
the cosegregating Mu3 element were cloned into l
vectors. Subclones of these were used for Southern
blot analyses of the 20 progeny of each of the three
mutant plants, showing that each of these mutants has
an insertion into this region of the genome that did not
exist in their sibs and the progenitor stocks. These
observations provided the crucial evidence that the
tb1 has been newly tagged. Portions of the l clones
were sequenced and oligonucleotide primers were
designed to be used in conjunction with a Mu-specific
primer to amplify the insertion fragments for each of
the three Mu alleles. DNA sequence analysis of these
fragments identified the Mu insertion point of each
allele. A 0.8-kb restriction fragment flanking the Mu
element was used to screen a cDNA library derived
from immature ear. A single clone with a 1,360-bp
insert, excluding the poly(A)tail was obtained. The
cDNA sequence is fully collinear with the genomic
sequence of maize inbred A619, without any evidence
for introns. BLAST analysis with the sequence showed
that tb1 shares two short regions of homology with the
snapdragon cycloidea gene. The pattern of tb1 expres-
sion and the morphology of tb1 mutant plants suggest
that tb1 acts both to repress the growth of axillary
organs and to enable the formation of female inflores-
cence. The maize allele of tb1 is expressed at twice the
level of the teosinte allele, suggesting that gene regula-
tory changes underlie the evolutionary divergence of
maize from teosinte [80]. Analysis of nucleotide poly-
morphism in tb1 gene of maize and teosinte
populations showed that during maize domestication
the effects of selection were limited to the gene’s regu-
latory region and could not be detected in the protein-
coding region [81]. Fine mapping showed that the
intergenic sequences approximately 58–69 kb 50 to the
tb1 cDNA confer pleiotropic effects on Zea mays mor-
phology. Moreover, an allele-specific expression assay
showed that sequences >41 kb upstream of tb1 act in
cis to alter tb1 transcription [82].
The most critical step in maize (Zea mays ssp. mays)
domestication was the liberation of the kernel from the
hardened, protective casing that envelops the kernel in
682 Crop Traits: Gene Isolation
the maize progenitor, teosinte [83]. This evolutionary
step exposed the kernel on the surface of the ear, such
that it could readily be used by humans as a food
source. A large effect QTL for glume induration
(husk hardening) was mapped to chromosome 4
using two F2 populations derived from crosses between
maize race Reventador (Nay 15) and Balsas teosinte, Z.
mays ssp. parviglumis or between maize race Chapalote
and Z. mays ssp. mexicana [84]. This large-effect QTL
segregates as a single Mendelian locus in an isogenic
background, and has been designated tga1 [83]. Maize
Mo17 bacterial artificial chromosome (BAC) libraries
were screened using a marker on maize chromosome 4,
which is tightly linked to tga1 and a BAC contig near
tga1 was identified. The BAC end and other sequences
from this contig were used to BLAST search the rice
genome sequence, and a region on rice chromosome
8 that is collinear with the region near tga1 on maize
chromosome 4 was identified. Subsequent BLAST
searches using the collinear rice sequence identified
a second maize Mo17 contig near tga1. Marker analysis
showed that these 2 contigs flank tga1. Maize B73
contigs that correspond to the two Mo17 contigs were
subsequently identified. DNA sequence analysis
revealed that the two B73 contigs overlap, and could
be merged to a single supercontig of 1.5 megabases.
Fine mapping using 3,106 F2 plants delimited tga1 to
a 6-kb region. Further BLAST searches using the 6-kb
region at tga1 revealed that it has homology to SBP
(squamosa-promoter binding protein) transcriptional
regulators. DNA sequence analysis of the SPB gene for
the tga1-ems1 stock, another tga1 allele generated by
ethyl methanesulfonate mutagenesis of maize line W22
that matched the phenotype of the teosinte allele in
homozygous state, revealed that it differs from its
parental (W22) allele by a nonconservative amino
acid substitution of a phenylalanine for a leucine at
position 5. This mutation in the tga1-ems1 allele con-
firmed the conclusion from positional cloning that tga1
is the SBP gene, and demonstrated that a single amino
acid substitution was sufficient to confer the difference
between the maize and teosinte phenotypes. Northern
blots, real-time PCR, and in situ hybridization did not
show any quantitative or qualitative differences in tga1
expression between the isogenic lines (W22 and W22:
tga1) or between the maize inbred W22 and teosinte
itself, suggesting that differences between the maize and
teosinte proteins may be critical to phenotype. Further
mapping using seven additional recombination events
within the 6-kb region narrowed the location of the
causative site for the functional difference between
maize and teosinte phenotypes to a 1,042-bp segment.
DNA sequence analysis of this 1,042-bp segment using
16 diverse maize and 12 teosinte individuals identified
seven fixed differences between maize and teosinte. Six
of these seven are single base-pair polymorphisms that
lie just 50 of the coding sequence and potentially affect
tga1 expression. The seventh difference encodes an
amino acid substitution of lysine (K) in teosinte to
asparagine (N) in maize at position 6. Western blot
analysis showed that tga1 protein encoded by the teo-
sinte allele is more abundant than the one encoded by
the maize allele over a range of developmental stages
that might underlie the phenotypic differences.
The K ! N substitution might alter protein stability,
or it might affect translation efficiency or protein
function [85].
Wheat Inflorescence In wheat, the Q allele confers
the square-headed phenotype and free-threshing char-
acter, and is possessed by most of the cultivated wheats,
but most wild wheats have the q allele and, therefore,
speltoid spikes that are not free threshing [86]. Early
experiments involving the cytogenetic analysis of aneu-
ploid (abnormal chromosome number) plants located
the Q gene on the long arm of chromosome 5A [87, 88].
Using chromosome deletion lines, Endo and Gill
(1996) physically mapped the Q gene to
a submicroscopic deletion interval on the long arm of
chromosome 5A [89]. Comparative mapping of anon-
ymous RFLP clones, AFLP (amplified fragment length
polymorphism) markers, and mRNA differential dis-
play analysis of lines which have deletion break points
that flank the Q locus identified 18 markers within the
Q gene deletion interval. These markers were used to
construct a genetic linkage map of the region in F2
populations derived from chromosome 5A disomic
(homologous chromosome pair) substitution lines.
The genetic map corresponding to the deletion
segment was 20-cM long, and markers as close as
0.7 cM to the Q gene were identified [90]. The closest
marker to the Q gene was used to screen four high-
density Triticum monococcum BAC filters resulting in

the identification of one BAC clone. Chromosome
walking involved the reiterative screening of the BAC
library using probes derived from the ends of previ-
ously identified BAC clones. A BAC contig was then
constructed that spans a physical distance of 300 kb
corresponding to a genetic distance of 0.9 cM. The
physical map of T. monococcum had perfect colinearity
with the genetic map of wheat chromosome arm 5AL.
Analysis of fast neutron q mutants using markers
derived from the BAC contig at the Q locus confirmed
that the T. monococcum BAC contig spanned the Q
locus and narrowed the region for prospective Q gene
candidates to a 100-kb segment, which contains an
APETALA2 (AP2)-like gene that cosegregates with Q
[91]. Sequence analysis showed that this AP2 gene
consists of ten exons and nine introns. The M2 gener-
ation of a population of Triticum aestivum cv.Chinese
Spring (CS) EMS mutants was screened for the speltoid
phenotype to identify putative knockouts. Three
speltoid mutants harboring point mutations within
the AP2 gene were identified. One mutant had
a single base substitution in an AP2 DNA binding
domain (exon 5) that resulted in the change of
a cysteine to a tyrosine. Two other mutants had point
mutations in the donor site of intron 2 and the acceptor
site of intron 7, respectively, resulting in alternate splic-
ing in these mutants. The sequence analysis of the
mutants validated that the AP2 gene is the Q locus.
Comparison of the genomic sequences of the AP2 gene
revealed six conserved differences between Q- and
q-containing genotypes. Four of these differences,
including a variable microsatellite (DNA sequence
repeat), were present in introns and one was in the
30 UTR. One conserved nucleotide difference changed
a predicted amino acid where, at position 329,
all Q-containing genotypes possessed an isoleucine
while all q-containing genotypes possessed a valine.
Yeast two-hybrid analysis showed that the full-length
Q protein has the ability to form a homodimer,
whereas the point mutation of q greatly reduced
homodimer formation of the full-length q protein.
Rachis fragility, glume shape, and glume tenacity mim-
icked the q phenotype in transgenic plants exhibiting
posttranscriptional silencing of the transgene and the
endogenous Q gene. Variation in spike compactness
and plant height were associated with the level of trans-
gene transcription due to the dosage effects of Q [92].
Crop Traits: Gene Isolation 683
Barley Inflorescence Barley spikes have a unique
structure consisting of three one-flowered spikelets at
each spike node. Cultivated barley, Hordeum vulgare
subsp. vulgare, can be divided into two forms according
to the morphology of the spikelets: two-rowed and six-
rowed. The two-rowed condition is believed to be
primitive, because wild barley, Hordeum vulgare
subsp. spontaneum, is two-rowed. Row type is con-
trolled by multiple alleles at the vrs1 locus (formerly v
for vulgare) on chromosome 2H [93, 94]. A recessive
mutation from Vrs1 to vrs1 changes two-rowed barley
to six-rowed barley. High-resolution mapping using
373 BC7F1 plants and 278 BC6F2 plants identified
four RFLP-derived STS markers closely linked to the
vrs1 locus [95]. The orders of four marker loci and vrs1
locus were the same in six different mapping
populations developed from nine different barley cul-
tivars (H. vulgare subsp. vulgare) or mutant and wild
barley (H. vulgare subsp. spontaneum) [96]. Five AFLP
markers within the 0.9-cM region associated with the
vrs1 locus were subsequently developed using well-
characterized near-isogenic lines as plant materials
[97]. Recombinants within the 0.9-cM region were
analyzed further using STS markers generated from
ESTs (expressed sequence tags) and BAC DNA
sequences. PCR screening of BAC clones of cv. Morex
using an STS marker located 0.01 cM proximal to the
vrs1 locus isolated one BAC clone. Chromosome walk-
ing identified six bacterial artificial chromosome
(BAC) clones covering completely the candidate geno-
mic region. The contig containing vrs1 is composed of
518,343 bp. Annotation showed three predicted genes:
HvHox1 and HvEP2 appeared to be intact genes,
whereas HvEP1 is highly degenerated and interrupted
by several insertions of transposable elements. HvHox1
is the only gene that lies between two markers that
define the break points and is thus a likely candidate
for Vrs1. The ORF of two-rowed barley encoded
a polypeptide composed of 222 amino acid residues,
including a homeodomain-leucine zipper (HD-ZIP)
motif. Expression of Vrs1 was strictly localized in the
lateral-spikelet primordia of immature spikes,
suggesting that the VRS1 protein suppresses develop-
ment of the lateral rows. Loss of function of Vrs1
resulted in complete conversion of the rudimentary
lateral spikelets in two-rowed barley into fully devel-
oped fertile spikelets in the six-rowed phenotype [98].
684 Crop Traits: Gene Isolation
The wild progenitor of barley, H. vulgare subsp.
spontaneum, has covered (hulled) caryopses in which
the hull (outer lemma and inner palea) is firmly adher-
ent to the pericarp epidermis at maturity. In cultivated
barley, the hulled or naked caryopsis is one of the most
important agronomic traits because of the direct link to
its use. Most cultivars have the hulled caryopsis and are
mainly used for animal feed and brewing malts. In
contrast, naked barley has a caryopsis with easily sep-
arable husks upon threshing and is suitable for edible
purposes. The naked caryopsis is considered a key
domestication character in barley because extensive
pearling to remove the hull is unnecessary [99]. The
covered/naked caryopsis in barley is controlled by
a single locus (nud, for nudum) located on chromo-
some arm 7HL [100]; the covered caryopsis allele
(Nud) is dominant over the naked one (nud). Bulked
segregant analysis on an F2 population derived from
a cross between Kobinkatagi (naked type) and Triumph
(hulled type) was performed using 1,894 primer com-
binations, and 12 AFLP markers were selected. Among
them, five closely linked and two cosegregating AFLP
markers were mapped around the nud locus using 151
F2 individuals [101]. High-resolution mapping using
2,380 segregants derived from five cross-combinations
identified AFLP-derived markers flanking the nud
locus at the 0.6-cM proximal and the 0.06-cM distal
side, respectively [102]. Further mapping using 2,828
progeny segregating for the trait from two cross-
combinations delimited the nud locus to a 0.64 cM
interval. Integration of the flanking markers into
a high-density barley expressed sequence tag (EST)
map selected two barley ESTs flanking the nud locus.
BLASTN analysis identified their respective homolo-
gous rice ESTs 370 kb apart on rice chromosome arm
6L. Two rice genes within the collinear region were used
as vehicles to develop closer barley markers. A BAC
library of the covered barley cultivar Haruna Nijo was
screened using the closest marker to the nud locus.
Seven rounds of chromosome walks selected
20 BAC clones and a 500 kb-contig spanning the nud
locus was constructed. In the physical map, the
nud locus was covered completely with four
overlapping BAC clones. An ethylene response factor
(ERF) family transcription factor was the only gene
that lies in the region delimited by the genetic and
physical mapping and, therefore, is considered as
a Nud candidate gene [103]. Sequencing of the nud
region obtained from two naked lines [Kobinkatagi
(a Japanese landrace) and nud-Bowman (an isogenic
line carrying the nud allele in the genetic background of
the covered cultivar Bowman)] revealed a deletion of
16,680 bp relative to the corresponding region of the
Haruna Nijo BAC contig sequence. The 16,680-bp
deletion included the entire ERF gene. Thus, the gene
structure analysis of naked cultivars supports the can-
didacy of the ERF gene. Sequence analysis of the can-
didate gene in two X-ray-induced naked mutants
showed that each of the two mutants carried
a different single base mutation in the putative func-
tional motif of the ERF gene, but their wild-type vari-
eties (Haisa’s and Ackermann’s Donaria, respectively)
had a nucleotide sequence that is identical to that of
Haruna Nijo. RNA in situ hybridization using the anti-
sense probe revealed that, in Bowman, Nud was
expressed strictly in the testa where adherence occurs,
while no signal above background was detected in
nud-Bowman. The Nud gene has homology to the
Arabidopsis WIN1/SHN1 transcription factor gene,
whose deduced function is control of cuticular wax and
cutin-related lipid biosynthesis pathways [104–106].
Staining with a lipophilic dye (Sudan black B) detected
a lipid layer on the pericarp epidermis only in covered
barley [103].
Rice Plant Architecture Tillering in rice (Oryza
sativa L.) is an important agronomic trait for grain
production. Screening of a collection derived from
spontaneous mutations identified monoculm 1 (moc1)
mutant. The moc1 plants nearly completely lose their
tillering ability, producing only one main culm, in
contrast to the multiple tillers in wild-type plants.
Genetic analysis with reciprocal crosses between moc1
and wild-type plants revealed that moc1 is a single locus
mutation. The MOC1 locus was mapped to the long
arm of chromosome 6 of O. sativa using 280 F2 mutant
plants generated from the crosses between moc1 and
Minghui 63. Fine mapping using 2,010 F2 mutant
plants and newly developed molecular markers
delimited the MOC1 locus to a 20-kb region. Annota-
tion of the 20-kb sequence identified an ORF that
encodes a protein highly homologous (44% identity)
to the tomato LATERAL SUPPRESSOR (LS). The
corresponding ORF from moc1 and wild-type plants

were amplified by PCR and sequenced. DNA sequence
comparison revealed a 1.9-kb retrotransposon inserted
in this ORF in the moc1 mutant. Transformation of
a binary plasmid carrying a 3.2-kb wild-type genomic
fragment containing the entire ORF plus a 1.5-kb
upstream sequence and the 316-bp downstream
sequence, but not the one carrying a 30 truncated
MOC1 gene, was able to rescue the monoculm pheno-
type of the moc1 mutant [107].
Typical common wild rice (Oryza rufipogon) tends
to have a prostrate growth habit during the vegetative
phase and develop erect panicle-bearing stalks during
the reproductive phase. Cultivated rice has an erect-
growth habit throughout the entire growth phase,
which may increase plant density, enhance photosyn-
thesis efficiency, and improve grain yield. A set of
introgression lines was constructed using an accession
of Yuanjiang common wild rice (YJCWR, Oryza
rufipogon) with prostrate growth habit as a donor,
and an elite indica cultivar Teqing (O. sativa) with
erect-growth habit as a recipient [108]. One introgres-
sion line (YIL18) displaying prostrate growth was
obtained, which harbored two YJCWR chromosomal
segments on the long arm of chromosome 3 and the
short arm of chromosome 7. The tiller angle of YIL18
was larger than that of Teqing. The grain number on
the main panicle (GNP) in YIL18 was only 57.6% of
that in the recipient Teqing, a result of the lesser num-
ber of primary branches and secondary branches on the
main panicle. Genetic linkage analysis within 246 F2
individuals derived from the cross between YIL18 and
Teqing showed that prostrate growth was completely
associated with a marked decrease of GNP and con-
trolled by a single semidominant gene, PROG1
(PROSTATE GROWTH 1), located on short arm of
chromosome 7. High-resolution mapping using 3,600
recessive homozygote plants with erect growth from
the F2 population delimited prog1 within an 8.8-kb
region. Only one ORF was identified within this region
that encodes a putative single Cys2-His2 zinc-finger
protein. Transformation of a binary plasmid carrying
the entire O. rufipogon PROG1 with 596-bp or 2,914-bp
50 -flanking regions, but not the one carrying only the
2,914-bp 50 -flanking region, showed complementation
of the prostrate growth phenotype. Comparison of the
coding sequences of PROG1 in YJCWR and prog1 in
Teqing showed 15 SNPs and 6 insertion/deletions
Crop Traits: Gene Isolation 685
(indels) that encoded 23 amino acid changes between
PROG1 and prog1. Sequencing of the prog1 coding
regions of 182 erect-growth varieties of cultivated rice,
including 87 indica and 95 japonica cultivars from
17 countries showed that all the cultivars contained
identical mutations as prog1 in Teqing, including 15
SNPs and 6 indels [109].
Cereal Seed Quality and Color
Cereal grain is the staple food of most of the world,
representing the major carbohydrate energy source in
the diet. One of the most important crop traits is grain
quality, which determines the price and variety of food
that can be prepared from the cereal crop. Thus the
major cereal crops such as rice, wheat, maize, barley,
and oats have undergone selection from the onset of
the crop domestication process. In comparison to
other crop traits, seed quality and color are primarily
biochemical traits and have been studied at the protein,
gene, and metabolite or grain component level.
Maize Lysine Content The protein nutritional qual-
ity of maize is improved by increase in the essential
amino acid lysine in the opaque-2 (o2) mutant locus,
which was localized on the short arm of chromosome 7
[110]. To isolate the gene by transposon tagging, nor-
mal maize strains (O2/O2) carrying a mutable allele of
C1 containing an autonomous Spm (c1-m5) or
a mutable allele of Wx containing a nonautonomous,
defective derivative of Spm (dSpm) (wx-m8) were used
as pollen donors for opaque plants (o2/o2). Three
opaque-mutable o2/o2-m kernels were selected from
approximately 530,000 F1 seeds. These kernels were
grown to maturity and self-pollinated. DNAs prepared
from leaf samples of F2 individual plants were used for
Southern blot analysis using a Spm-specific probe. The
opaque-mutable plants contained a novel 8.4-kb band
absent in both parents and missing from at least some
of the individuals that had been classified as opaque.
Those kernels that had been classified as opaque but
possessed the fragment were evidently not mutable
because of failure of the Spm to transpose, or else
transposition occurred so late in endosperm develop-
ment that revertant sectors were undetectable, giving
the kernels an opaque rather than an obvious opaque-
mutable phenotype. The fragment was cloned and
686 Crop Traits: Gene Isolation
restriction enzyme mapping of the clone revealed the
presence of a full-length, autonomous 8.3 kb Spm and
an adjacent sequence of about 150 bp. This non-Spm
region was sequenced and used as a probe on Southern
blot using a BC1 population derived from a cross
between opaque (o2/o2) and normal (O2/O2) plants.
All the plants derived from opaque (o2/o2) seeds
showed a single 6.5-kb fragment, whereas plants from
seeds with a normal phenotype (o2/O2) had a 10-kb
fragment in addition to the 6.5-kb fragment. This
demonstrates that the 150-bp Spm-fanking sequence
derived from the opaque-mutable plant represents
a single-copy sequence that cosegregates with alleles
of the o2 locus. This fragment was subsequently used
as a probe to clone a 17-kb HindIII fragment of the
wild-type O2 allele from the c1-m5 parent [111].
A cDNA library prepared from wild-type endosperms
was screened with probes derived from the O2 genomic
clone. Analysis of O2 cDNA sequence showed that the
deduced 02 protein sequence contains a “leucine-zip-
per” domain characteristic of some mammalian and
fungal transcription activation factors. DNA binding
assays demonstrated that the O2 protein or only
a fragment specifying the leucine-zipper domain
bound to two specific regions on the 50 side of the
coding sequence in a zein genomic clone [112].
Cereal Amylose Content The maize Waxy locus
determines starch quality by the level of amylose and
amylopectin. Isolation of the gene was carried out by
a combination of identification of the protein and the
gene associated with the kernel mutant phenotype. The
mutant wx-m6 and wx-m9 alleles have Ds transposon
inserts at the Wx locus, while the wx-m8 allele is attrib-
utable to insertion of a defective transposon of the Spm
transposon family. Starch granules from immature
endosperm tissue of kernels carrying the wx-m6, wx-
m9, and wx-m8 alleles were isolated and the starch
granule-bound proteins were analyzed on SDS-
polyacrylamide gels [113]. When the kernels did not
contain an autonomous controlling element (either Ac
or Spm) and showed no somatic reversion of the
wx-m6, wx-m9, or wx-m8 mutations, the 58 kd protein
was either not detectable or present at a very low level.
When the Spm element was present with the wx-m8
allele, sectors of endosperm tissue showed the Wx phe-
notype and a 58 kd protein was present in isolated
starch granules. By contrast, when Ac was present
together with the wx-m6 allele, endosperm tissue
showed sectors that were intermediate between the
Wx and the wx phenotypes and a novel 60 kd protein
was detectable on starch granules. Hence, reversion of
the wx-m6 mutation gives a protein that is structurally
abnormal and appears to have an altered enzymatic
activity. Only the 58 kd protein is present in starch
granules from kernels that are heterozygous for the
wx-m6 and wx-m8 alleles, but contain only the Spm
element. Therefore, the structurally altered protein
detected upon reversion of the wx-m6 allele in the
presence of Ac is not detected in the presence of Spm.
Were the Wx locus a regulatory locus, both the 58 kd
and the 60 kd proteins would be present in a kernel that
is heterozygous for the wx-m6 and wx-m8 mutations
and contains the Spm element. It was concluded that
the Wx locus is the structural locus for the 58 kd
protein [113].
Poly(A)+ RNA purified from Wx and wx endo-
sperms was translated in a rabbit reticulocyte in vitro
translation system, and an antigenically related poly-
peptide was identified using antiserum raised against
the Wx protein. No major 58 kd polypeptide was evi-
dent among the translation products, but a major 65 kd
polypeptide was translated from Wx, and not from wx
poly(A)+ RNA. The 65 kd in vitro translation product
may be a precursor that is synthesized, but not
processed in the rabbit reticulocyte extract. Poly(A)+
mRNA fractions substantially enriched for Wx mRNA
were used to construct cDNA clones. Screening of
cDNA clones was performed by selective hybridization
to the mRNA sequence encoding the immunopre-
cipitable 65 kd Wx polypeptide. Further testing showed
that the Wx cDNA hybridized to an abundant 2.3-kb
poly(A) + RNA present in immature endosperm of
plants homozygous for the Wx allele, but absent from
immature endosperm tissue of a plant homozygous for
a stable recessive wx allele. Finally, the Wx cDNA was
shown to hybridize to a unique sequence in maize
genomic DNA. That the unique sequence corresponds
to the Wx locus was deduced from the results of hybrid-
ization analyses of DNA isolated from maize strains
with and without a Ds insertion mutation at the Wx
locus. The presence of the insertion at the Wx locus was
correlated with the presence of a 2.4-kb insertion in the
sequence homologous to the cloned Wx cDNA,

unequivocally establishing the homology of the cDNA
to the Wx locus [113]. Sequence analysis of genomic
and cDNA clones of the wild-type Wx gene showed that
the coding region comprises 3,718 bp and is composed
of 14 exons and 13 small introns. N-terminal sequenc-
ing of the mature Wx protein led to the identification
of a maize amyloplast-specific transit peptide of 72
amino acid residues [114].
The Wx gene homologues were isolated from
a number of species by isolating homologous clones
or identification of DNA/protein sequences homolo-
gous to the maize Wx gene/protein. A rice genomic
library was constructed in lEMBL-3 vector and
screened using the maize Waxy gene DNA as probe.
Two overlapping genomic clones containing the Wx
gene sequence were identified. Sequencing and align-
ment with Wx gene of maize and barley revealed that
the Wx gene in rice contains 13 introns and 14 exons.
The full-length of rice waxy preprotein is 609 amino
acid residues [115]. An analysis of Wx transcripts, Wx
protein, and amylose content of 31 rice cultivars
revealed that endosperm amylose and Wx protein con-
tents are correlated with the ability of the cultivar to
excise intron I from the leader sequence of the Wx
transcript [116]. Sticky or glutinous rice quality is
related to the low amount of amylose due to the mutant
wx allele.
Sweet Corn The maize sh2 mutant used in
“supersweet” corn has negligible starch. The Sh2 gene
was predicted to be involved in carbohydrate metabo-
lism, since the mutant sh2 endosperm contains reduced
starch. Candidate Sh2 clones were identified from
a cDNA library prepared from endosperm by differen-
tial screening with labeled cDNA of near-isogenic W64
Sh2 and W64 sh2 mRNA. The 1.3-kb cDNA clone
pES6-66 hybridized to wild-type cDNA and not sh2
cDNA. Clone pES6-66 was subsequently shown to
hybridize to a restriction fragment length polymor-
phism (RFLP) associated with normal Sh2 function in
F2 segregants of Sh2  sh2 crosses in three backgrounds
[117]. To determine whether this clone was indeed Sh2
or simply a closely linked gene, a series of maize stocks
that differed only at the Sh2 locus were used, including
a wild-type Sh2 allele (progenitor), sh2-m1 (mutable)
allele containing Ds at the sh2 locus, and Sh2 revertants
[118]. Southern blot analysis using 500-bp fragment
Crop Traits: Gene Isolation 687
derived from pES6-66 as probe showed that sh2-m1
yielded a fragment that is approximately 1,600 bp
larger than that seen in the progenitor or revertant.
The data are compatible with sh2-m1 containing
a 1,600-bp insertion. RNA gel blot analysis indicated
that the size of the wild-type transcript is approxi-
mately 2.0–2.2 kb. A cDNA library constructed from
poly A+ RNA of shrunken-1, bronze-mutable4 (sh1 bz-
m4) was screened using probes derived from pES6-66.
Eight of the 14 clones isolated had the 1.95-kb insert
and were almost full length. The 900-bp and 1,050-bp
EcoRI fragments from one of these clones were
subcloned and sequenced. Examination of the 1.95-kb
cDNA sequence identified only one open reading frame
(ORF) coding for 542 amino acids and making up
a polypeptide of 59,845 Da. Homology search
identified the Escherichia coli glucose-l-phosphate
adenyltransferase (ADP-glucose pyrophosphorylase or
ADP-glucose synthetase, EC 2.7.7.27) was the only
protein having significant similarity to the amino acid
sequence of the Sh2 cDNA [118].
The maize sugary1 (su1) mutant accumulates twice
as much sugar as sweet corn, having reduced starch.
Mutations at the su1 locus were generated by crossing
active Mutator (Mu) plants with standard lines. Mutant
alleles were identified following the self-pollinations of
the F1 plants. Normal and sugary sibling kernels from
a population segregating 1:1 for the nonmutant allele
Su1 and su1-R4582::Mul were germinated, and geno-
mic DNA used for Southern blot analysis using the
Mu1 fragment as probe. A 4.0-kb EcoRI fragment
containing sequences homologous to the transposon
Mu1 that cosegregated with su1-R4582::Mu1 was iden-
tified. This 4.0-kb EcoRI fragment was subsequently
cloned by screening a BAC library, based on hybridiza-
tion to a probe internal to Mu1, and the genomic DNA
insert was subcloned as part of plasmid pMJ60. The
nucleotide sequence of both termini of the transposon
were determined and found to match the known
sequence of Mul. A 1.0-kb BamHI-EcoRI genomic frag-
ment flanking Mu1 in the cloned DNA was purified and
used as a hybridization probe (termed BE1000). This
genomic probe detected a 4.0-kb EcoRI fragment that
was present in all su1-R4582::Mu1/su1-Ref plants but
was missing from all Su1/su1-Ref plants. Thus, the
cloned Mu transposon is the same element that is
within or tightly linked to the su1 gene locus. Probe
688 Crop Traits: Gene Isolation
BE1000 identified a second EcoRI fragment of 2.6 kb
that was present in all plants examined, and represen-
tative of the nonmutant progenitor allele. Presumably,
insertion of the 1.4-kb transposon Mul within this
2.6-kb region resulted in the 4.0-kb EcoRI fragment
that cosegregated with su1-R4582::Mu1. Mutations of
the su1 gene locus other than su1-R4582::Mu1 were
analyzed to determine whether they also cosegregated
with physical alterations in the cloned region of the
genome. Populations segregating for the nonmutant
allele Su1 and either su1-R2412, su1-R7110, or su1-
R3162 was digested with EcoRI and probed in DNA
gel blot analysis with genomic fragment BE1000. In the
su1-R7110 and su1-R3162 families, the 4.0-kb EcoRI
fragment was present in all the seedlings grown from
sugary kernels but was not observed in any seedlings
grown from nonmutant sibling kernels. A different
EcoRI fragment, 4.6 kb in length, was found to
cosegregate with su1-R2412. The mutation su1-R2412
is likely to have occurred via insertion of a 2.0-kb
element into this same region. Approximately 200,000
lambda clones from a maize endosperm cDNA library
were screened with genomic probe BE1000. DNA from
eight different hybridizing clones was digested with
EcoRI; the largest cDNA insert was 2.4 kb in length.
Rapid amplification of cDNA ends (RACE) protocol
was used to obtain the 50 end of the sul cDNA. Sequenc-
ing of su1 cDNA and BLAST search of the deducted
amino acid sequence showed that su1 cDNA specified a
polypeptide of at least 742 amino acids, which is highly
similar in amino acid sequence to bacterial enzymes that
hydrolyze a-(1 ! 6) glucosyl linkages of starch [119].
Rice Seed Color Red pericarp is ubiquitous among
the wild ancestors of cultivated rice (Oryza rufipogon),
in which it is closely associated with seed shattering and
dormancy. On the other hand, most rice (Oryza sativa)
that is grown and consumed throughout the world has
white pericarp, showing that white pericarp is associ-
ated with domestication and remains under strong
selection in most rice breeding programs today.
A QTL associated with red pericarp, rg7.1, was mapped
on chromosome 7 using two independent BC2
populations derived from crosses between an accession
of O. rufipogon (IRGC-105,491) from Malaysia and, in
one case, a US tropical japonica cultivar, Jefferson, and
in the other case, a widely planted tropical indica
cultivar, IR64. The log of the odds scores associated
with the rg7.1 QTL peaks in these two populations were
99 and 33, respectively [120, 121]. The peak of both
QTLs corresponded to the previously mapped position
of the mutant locus, brown pericarp, Rc [122]. Fine
mapping using 1,410 BC2F3 plants and high-
resolution mapping using 4,000 BC2F6 plants
narrowed the rg7.1 QTL to an 18.5-kb region. Two
genes encoding CACTA type, En/Spm subclass, trans-
poson proteins, and one gene encoding bHLH protein
were detected within the 18.5-kb target region.
Sequence comparison of bHLH locus between cv Jef-
ferson and H75, an Rc mutant stock belonging to the
japonica subspecies, carrying a functional allele, but
much more closely related to cv Jefferson than to O.
rufipogon, showed that the coding sequence of the
bHLH allele in H75 was identical to the cv Jefferson
sequence except for a 14-bp indel in exon 6. This 14-bp
sequence was present in the H75 stock as well as in O.
rufipogon, but was deleted in cv Jefferson and cv
Nipponbare. The deletion induces a frame shift in the
sequence, resulting in two premature stop codons
before the end of exon 6. The stop codons truncate
the protein before the bHLH domain. Sequence analy-
sis of bHLH of Surjamkuhi, an indica line that carries
a third allele, Rc-s, conditioning light red seed pigmen-
tation, showed that the sequence of this line differed
from the O. rufipogon allele at only four sites (positions
96, 660, 1353, and 1833–1844). The first two changes
proved to be synonymous substitutions. The change at
position 1353 consisted of a C-to-A change in exon 6.
This single-nucleotide polymorphism (SNP) was inde-
pendent of any change seen in previous comparisons
and represented a premature stop codon before the
bHLH domain, truncating the protein and rendering
the effect of the remaining indel immaterial. Conven-
tional reverse transcriptase-PCR showed no expression
of Rc in leaf tissue; however, expression was seen in
panicles before fertilization, pericarp from grains in
the milk or dough stage of filling, and pericarp from
mature seeds. Similar expression levels of Rc were
detected in cv Jefferson and O. rufipogon [121].
Seed Shattering
Wild plant species shatter their seed from the mature
infloresence and thus disperse seed for the next

generation to be seeded, while during domestication
humans have selected for non-shattering plant types
from which the seed can be harvested or gathered from
the crop. Study of shattering has been very fruitful in
Arabidopsis, setting up the genetic models for
shattering phenotypes [123] that have been also
revealed in other crops like the cereals.
In rice, a major QTL for seed shattering, sh4, has
been mapped on chromosome 4 using segregating
populations derived from crosses between O. sativa
ssp. indica and the wild perennial species O. rufipogon
[124, 125], between O. sativa ssp. japonica and
O. rufipogon and two other closely related wild species
O. glumaepetula and O. meridionalis [126, 127], and
between O. sativa ssp. indica and the wild annual spe-
cies Oryza nivara [128]. High-resolution mapping
using 12,000 F2 (O. sativa ssp. indica x O. nivara)
delimited sh4 to a 1.7-kb region of a gene with
a previously unknown function. The comparison of
the 1.7-kb sequences between the mapping parents
revealed seven mutations located in the intron, exon,
or 50 upstream of the start codon. Sequencing of this
1.7-kb region from an additional 14 rice cultivars of
O. sativa, 21 accessions of O. nivara, 6 accessions of
O. rufipogon, and 1 accession of each of the four
remaining wild A-genome species and their association
with shattering phenotypes showed that a nucleotide
substitution of G for Tor an amino acid substitution of
asparagine for lysine in the first exon was selected for
the development of non-shattering cultivars during
rice domestication. Annotation of the sh4 protein
identified a Myb3 DNA binding domain and a
nuclear localization signal, suggesting that sh4 is
a transcription factor. Transformation of a binary plas-
mid carrying sh4-GFP fusion under the control of Ubi
promoter into rice cv. Taipei 309 determined the nuclear
localization of GFP-tagged sh4. Transformation into rice
cv. Taipei 309 of a binary plasmid carrying O. nivara
sequence from the 30 nontranslated region to the inclu-
sion of the G-T mutation site and O. sativa sequence for
the rest sequence until the 50 regulatory region, but
not the one carrying a shorter O. nivara sequence
excluding the G-T mutation site, showed significantly
reduced strength of grain attachment to pedicel [128].
A major QTL for seed shattering, qSH1, was first
mapped on chromosome 1 using 182 F2 plants of
a cross between Kasalath (a shattering-type indica
Crop Traits: Gene Isolation 689
cultivar) and Nipponbare (a non-shattering-type
japonica cultivar). A near-isogenic line (NIL) was
developed that contained a short chromosomal seg-
ment from Kasalath at the qSH1 region in
a Nipponbare genetic background. The NIL had
a stronger seed-shattering phenotype than either
Kasalath or Nipponbare. High-resolution mapping
using 10,388 plants from the BC4F2 and BC3F2
populations segregating at the qSH1 region succeeded
in mapping the functional natural variation in 612 bp
and only one single-nucleotide polymorphism (SNP)
was found within this region. No distinct open reading
frame (ORF) was identified in the SNP region based on
gene prediction for the qSH1 region in both
Nipponbare and Kasalath genome sequences. However,
one ORF for a rice ortholog of the Arabidopsis
REPLUMLESS (RPL) gene was found 12 kb away
from the SNP. The RPL gene encodes a BEL1-type
homeobox and is involved in the formation of
a dehiscence zone (or abscission layer) alongside the
valve in the Arabidopsis fruit (silique). Transformation
of 26-kb Kasalath genomic fragments scanning the
predicted ORF and the SNP regions into the non-
shattering Nipponbare cultivar showed complementa-
tion of the complete seed-shattering phenotype. The
other fragments were not able to complement the phe-
notype, even if they contained the entire ORF region or
the SNP region. These results indicated that both the
ORF and the SNP regions were required for full
shattering function. In situ hybridization analysis
revealed that in the NIL the ORF was expressed at the
inflorescence meristem in the stage of rachis meristem
establishment [inflorescence stage 1 (In1)]. It was also
expressed at both the anther region and the provisional
abscission layer at the base of the spikelet in the stage of
floral organ differentiation (In7) and in the stage of
rapid elongation of the rachis and branches (In8). On
the other hand, in Nipponbare, the ORF was expressed
in the same way as in the NIL, except that it was not
expressed at the provisional abscission layer in either
In7 or In8. These results, together with the comple-
mentation results, indicated that this RPL ortholog was
the qSH1 gene and that the identified SNP affected only
the spatial mRNA expression pattern of qSH1 at the
abscission layer [129].
A shattering mutant line of rice, Hsh, was derived
from a non-shattering japonica variety, Hwacheong, by
690 Crop Traits: Gene Isolation
N-methyl-N-nitrosourea (MNU) treatment. Optical
microscopy revealed that Hsh had a well-developed
abscission layer similar to the wild rice Oryza nivara,
while Hwacheong did not produce an abscission layer.
Genetic analysis showed that the easy shattering of Hsh
was controlled by the single recessive gene sh-h. Using
an F2 population consisting of 240 individuals derived
from the Hsh/Blue&Gundil cross the sh-h was mapped
on chromosome 7 [130]. Fine mapping using six newly
developed SSR markers delimited the sh-h locus to
a 150-kb region. Further mapping using three newly
developed SNP markers on five F3 recombinant lines
derived from F2 heterozygous plants narrowed the sh-h
locus to a 34-kb region. Analysis of genomic sequences
from Hwacheong, Hsh, and Blue&Gundil lines identi-
fied eight genes in this region. Among these, a gene
which encodes a protein containing a conserved
carboxy-terminal domain (CTD) phosphatase domain
was considered to be a strong candidate for the sh-h
locus because of the presence of a point mutation at the
30 end splice site of its seventh intron. Sequencing of the
RT-PCR products of Hwacheong and Hsh revealed
that, compared with Hwacheong, a 15-bp deletion
was induced by altered splicing in the mRNA isolated
from Hsh. Because the consensus sequence for splicing
at this “AG” site was changed to “TG,” the next “AG”
sequence, 15-bp downstream, was used as a new splice
site. The 15-bp deletion in the Hsh mRNA resulted in
the deletion of five amino acids in the C-terminal
region of the CTD-like phosphatase domain (CPDc),
which are situated within the phosphatase active site.
Two transferred DNA (T-DNA) insertion mutants and
one point mutant exhibited the enhanced shattering
phenotype, confirming that this CTD phosphatase-like
gene is indeed the sh-h gene. RNA interference (RNAi)
transgenic lines with suppressed expression of this gene
exhibited greater shattering [131].
Tolerance to Abiotic Stresses
Wild species are adapted to the environment where they
are found; survive over long periods of time by creating
diversity where environmental selection pressures allow
the fit to reproduce and survive. The environmental
stresses include abiotic stresses such as heat, cold,
drought, salt, light, radiation, heavy metals, soil pH, and
others. Plant response to these stresses has been studied
and shows common and unique responses [132].
Salinity Stress Tolerance Salinity tolerance has been
amenable to study for phenotypic variation and the
genetic dissection into quantitative traits. In rice,
a major QTL for shoot K + content under salt stress,
SKC1, was mapped to chromosome 1 using 133 lines of
an F2 and an equivalent F3 population derived from
a cross between a salt-tolerant indica variety Nona
Bokra, and a susceptible elite japonica variety
Koshihikari [133]. Fine mapping was performed
using 192 BC2F2 plants, and a high-resolution map
was subsequently generated with 2,973 BC3F2 plants
using markers newly developed on the basis of the PAC
clone sequences. Progeny testing of fixed recombinant
plants (BC3F4) delimited the SKC1 locus to a 7.4-kb
region, and only one predicted open reading frame
(ORF) was identified in the region. The SKC1 promoter
region (2,554-bp upstream of the initial codon) and the
coding region of the cDNA clone (1,665 bp) were
ligated and subcloned into a plant binary vector. This
construct was transferred into the japonica variety
Zhonghua 11, which contained the same SKC1 allele
as Koshihikari and is salt susceptible. Shoot K + con-
centrations were substantially higher in all six T1 prog-
eny containing the Nona Bokra SKC1 transgene
compared with plants containing the vector control
under salt stress but not under normal condition.
Database searches showed substantial similarity
between SKC1 and the HKT-type transporters found
in plants, bacteria, and fungi. Comparison of nucleo-
tide sequences between Nona Bokra and Koshihikari
alleles showed six nucleotide substitutions in the cod-
ing region that lead to four amino acid changes, which
may be responsible for the functional difference
between the two alleles. RT-PCR analysis in both
Koshihikari and NIL (SKC1) showed that SKC1 tran-
script levels were upregulated by salt stress in the root
but not in the shoot. Under normal condition, K+ and
Na+ contents in NIL (SKC1) shoots were not substan-
tially different from those in Koshihikari. But under salt
stress, NIL (SKC1) shoots had a higher K+ content and
lower Na+ content than Koshihikari shoots. No sub-
stantial differences in K+ and Na+ contents were
observed in the roots under either normal or stress
condition. Consistent with the results of shoot analysis,
K+ and Na+ contents in xylem sap were not different
between NIL (SKC1) and Koshihikari; under salt stress,
however, the xylem sap of NIL (SKC1) contained more

K+ and less Na+ than that of Koshihikari. These results
indicated that SKC1 is involved in regulating K+/
Na+ homeostasis in the shoots [134].
Submergence Tolerance A major QTL for submer-
gence tolerance, Sub1, was mapped to chromosome 9
using 169 F2 plants and the resulting F3 families of
a cross between a tolerant indica rice line, IR40931-26
(a descendant of FR13A), and a susceptible japonica
line, PI543851 [135]. High-resolution mapping using
2,950 F2 individuals and the resulting F3 families of
a cross between DX18-121 (a tolerant F3 plant from the
first population) and M-202 (a submergence-
susceptible japonica cultivar that is widely used in
California) identified 2 AFLP markers localized within
0.2 cM of Sub1 [136]. The Sub1 locus was delimited to
an interval of 0.06 centimorgan using 4,022 F2 individ-
uals from the cross between DX18-121 and M-202.
Physical mapping with five overlapping bacterial arti-
ficial chromosome (BAC) clones derived from submer-
gence-intolerant indica rice varieties and a nearly
complete contig of 13 binary clones from IR40931-26
showed that Sub1 region physically spans over 182
kilobases (kb). This interval encodes three genes
containing ethylene response factor (ERF) domains
and designated Sub1A, Sub1B, and Sub1C, ten non-ERF
genes including four transcribed and six hypothetical
protein-coding genes, and >50% retrotransposon-
related sequences. The corresponding region of the
japonica genome represented by the sequenced variety
Nipponbare spans 142 kb and is considerably
rearranged. Notably, Sub1A is absent from the rice
reference Nipponbare genome. Accumulation of
Sub1A and Sub1C messenger RNAs was strongly but
transiently promoted by submergence in seedling
leaves of tolerant FR13A, while Sub1B transcripts
increased only slightly during submergence. The ten
non-ERF genes in the indica Sub1 region showed no
evidence of expression in seedling leaves before or
during submergence in IR40931-26 or the intolerant
variety M-202. A survey of the Sub1 locus haplotypes in
17 indica and 4 japonica varieties identified 2 Sub1A, 9
Sub1B, and 7Sub1C alleles on the basis of variation in
amino acid sequence. The Sub1A-1 and Sub1C-1 alleles
are limited to all six submergence-tolerant accessions.
There was no Sub1B allele identified as being specific to
submergence tolerance. In the tolerant Sub1A-1 allele,
Crop Traits: Gene Isolation 691
a single-nucleotide polymorphism at position 556 is
responsible for a Pro 186 (intolerant) to Ser 186 (tol-
erant) substitution in a MAPK site. Conversely, the
Sub1C-1 allele of tolerant lines lacks a MAPK phos-
phorylation site present in the alleles of the intolerant
accessions. A Sub1A-1 full-length cDNA under the
control of the maize Ubiquitin1 promoter was
transformed into an intolerant japonica variety
Liaogeng. A screen of seedlings after 11 days of sub-
mergence identified four T1 families, derived from
independent T0 Ubi:Sub1A + lines, with submer-
gence-tolerant transgenic individuals, and progeny
from two families were examined in detail. T1 families
one and three showed a correlation between high
expression of the Sub1A-1 transgene and submergence
tolerance. As observed in the FR13A descendant
IR40931-26, tolerant Sub1A-1+ plants showed a signif-
icant impairment of shoot elongation under submer-
gence compared with the intolerant parent Liaogeng
and non-transgenic siblings [137].
Aluminum Tolerance In wheat (Triticum aestivum L.)
mechanisms that minimize the harmful effects of Al
ions have been investigated using near-isogenic lines
[ET8 (Al-tolerant) and ES8 (Al-sensitive)] that differ in
Al tolerance at a single dominant locus designated as
Alt1. The Alt1 locus cosegregates with an Al-activated
malate efflux from root apices [138, 139]. One cDNA
clone more highly expressed in the root apices of ET8
compared to that of ES8 was isolated by subtractive
hybridization. The full-length cDNA was obtained by
rapid amplification of cDNA ends (RACE)-PCR. The
predicted protein is hydrophobic having six to eight
putative transmembrane regions. Heterologous expres-
sion of this gene in Xenopus oocytes indicated that this
gene encodes an Al-activated transporter that facilitates
the efflux of malate but not citrate. Based on this result,
the gene was named ALMT1 (aluminum-activated
malate transporter). Sequencing the ALMT1 cDNAs
derived from ET8 and ES8 showed that the sequences
differed at six nucleotides that resulted in the deduced
proteins differing at two amino acid residues. Sequence
of the ALMT1 coding region in Atlas66 (Al-tolerant
cultivar) was identical to that of the ET8 allele, while
the sequence in Scout66 (Al-sensitive cultivar) was
identical to that of ES8. As found for the ET8 and ES8
lines, the expression of ALMT1 in the root apices was
692 Crop Traits: Gene Isolation
greater in Atlas66 compared to Scout66. Expression
analysis of ALMT1 in the 57 F2 individuals derived
from a cross between ET7 and ES5, the near-isogenic
progenitor lines of ET8 and ES8, showed that all the
Al-sensitive seedlings expressed the ES8 allele only,
whereas Al-tolerant seedlings expressed either ET8
allele only or both ET8 and ES8 alleles. Thus, the ET8
allele was expressed only in the Al-tolerant seedlings,
indicating that, in this population, the Al-tolerant phe-
notype was correlated with the higher expression of this
allele. Evaluation of Al tolerance and genotype analyses
using an RFLP marker and a PCR-based assay on 204
F3 families derived from a cross between ET8 and ES8
showed that the ET8 allele of ALMT1 completely
cosegregated with the Al-tolerant phenotypes and the
Al-tolerance locus, Alt1. Heterologous expression of
ALMT1 in cultured tobacco cells increased the toler-
ance of tobacco cells to Al treatment [140].
Future Directions
The present era of plant sciences is distinguished by
an integration of multiple disciplines toward a
transdisciplinary/interdisciplinary approach of doing
science, utilizing the technology and knowledge from
computer science and bioinformatics, genomics, dedi-
cated instrumentation for high-throughput automa-
tion of laboratory and field tasks, which can all
together provide integrated models and technologies
for the improvement of crop traits. The paradigm shift
in genetics, due to the reduced cost in genome sequenc-
ing, is to be able to generate the genome sequence of
any plant genotype of interest that will totally revolu-
tionize the approach to use this information. With
a few genetic crosses followed by whole genome
sequencing of all genotypes, it would be possible to
identify genes and useful alleles that can be incorpo-
rated into plant improvement programs. This would
make the traditional ways of isolating or identifying
gene sequences for crop traits obsolete. The lag will be
in phenotyping individual genotypes, which will
remain the challenge for the biologist. The association
of genes to a trait will still require that traits be charac-
terized, dissected into components and parameters
that can be measured, and the candidate gene(s)
validated by some reverse genetics strategies using
methods such as mutant analysis, transformation, and
complementation, at least till the function of every
gene/allele is known and how these genes and their
alleles interact together to determine the phenotype
of a crop trait.
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 Crop Yields Around the World: Closing the Gap and Raising the Potential 699

Crop Yields Around the World: Definition of Subject

Closing the Gap and Raising the The entry assumes that yield increase will continue to
Potential play a dominant role in world food security, as it has
over the last 60 years. It is restricted to annual grain
R. A. (TONY) FISCHER crops, since these dominate the world’s arable land-
CSIRO Plant Industry, Canberra City, Australia scape (>70%) and humankind’s food supply
(>70%), including grain used as livestock feed. Crop
Article Outline yield is the weight of grain, at some agreed standard
moisture content, harvested per unit of land area per
Glossary crop (note, there can be two or even three annual crops
Definition of Subject per year in some favored environments, meaning
Introduction a cropping intensity of 200% or 300%, respectively).
Estimates of Yield Gaps The starting point for yield is usually the field, district,
Closing Yield Gaps regional, or national average yield in kg or t per hectare,
Lifting Potential Yield as reported in surveys or local or national statistics.
Future Directions and Synthesis Here this is referred to as farm yield (FY, t/ha). This and
Bibliography many related cropping statistics are collated annually
for all countries by FAO (http://faostat.fao.org/site/
Glossary
567/default.aspx#ancor). FY is usually expressed rela-
Allocative efficiency Efficiency with which given var- tive to harvested land area (note: this can fall well below
iable input(s) is used. planted area in some situations). Although FY is
Attainable yield Yield reached by farmers with eco- quoted and used widely, it may not be as accurate as
nomically optimum management and reasonable it appears due to uncertain grain admixtures and/or
risk aversion. poor collection of the statistics. With survey data sam-
Ecotilling Seeking related alleles across germplasm pling error can also arise.
collections based on DNA homology. At the highest end of the yield scale it is useful to
Farm yield Average grain yield across farms in a region. define potential yield (PY), which is the yield to be
Harvest index Ratio of grain dry weight to total expected with the best adapted cultivar, the best man-
above-ground dry weight at maturity. agement of agronomic inputs, and in the absence of
Molecular markers Short sequences of DNA that are manageable abiotic and biotic stresses [1]. Many com-
homologous with sequences close to or within plications are hidden within this apparently simple
important genes. definition. PY is usually determined in plots, with of
Potential yield Grain yield in the absence of manage- course sampling error. In order to be relevant to the
able abiotic and biotic stresses. surrounding district, the natural resource base of the
Potential yield water limited Grain yield in the plot (climate, soil type, topography) needs to be com-
absence of manageable abiotic and biotic stresses parable, including any long-term management invest-
apart from that imposed by water supply through ments to improve this aspect of the site (e.g., liming,
inadequate rainfall. tile drainage). Water supply must be adequate for PY
Radiation use efficiency Above ground biomass pro- determination as defined, and this can come from well-
duced per unit of total solar radiation intercepted distributed rainfall close or equal to crop potential
by green crop tissue. evapotranspiration (crop water use from sowing to
Technical efficiency Practices, timing, and technical harvest without water limitation) or from full or sup-
skills adopted by farmer in using inputs. plemental irrigation; in addition, pests, weeds, and
Yield gap Difference between farm yield and potential diseases must be held at negligible levels through the
yield. use of biocides if necessary. Finally, crops experiencing

P. Christou et al. (eds.), Sustainable Food Production, DOI 10.1007/978-1-4614-5797-8,

# Springer Science+Business Media New York 2013

Originally published in
Robert A. Meyers (ed.) Encyclopedia of Sustainability Science and Technology, # 2012, DOI 10.1007/978-1-4419-0851-3
700 Crop Yields Around the World: Closing the Gap and Raising the Potential
relatively rare weather damage such as crop lodging or
unseasonal frosting are excluded from PY measure-
ment. Taken overall, it would seem PY might be impos-
sibly difficult to measure, but it is reported often in the
crop science literature, although not always with
proper attention to the above caveats.
Since PY is usually measured in plots, edge effects
arising from extra solar radiation or soil moisture or
nutrients reaching outside plants must be avoided by
discarding the plot edges. Two types of PY plots are
commonest. Often they are well-managed yield trials to
compare new varieties or advanced lines against older
ones, or even historic ones, to give measures of breed-
ing progress by plotting variety yield against year of
release. The most useful such trials, for example those
for wheat conducted by the UK Home Grown Cereals
Authority (HGCA http://www.hgca.com/content.tem-
plate/0/0/Home/Home/Home.mspx), measure yields
with and without fungicide protection. Only the for-
mer yields are a measure of true PY, but fungicide
protection is still not very common in such yield trials
around the world, although visible disease levels are
usually reported and can be negligible. The second
source of PY data comes from experiments conducted
by crop physiologists to calibrate and/or validate crop
simulation models: the models, driven largely by var-
ious aspects of solar radiation and temperature, can
then be used to predict PY in other environments (e.g.,
sowing dates, years, locations). For such purposes
modeling accuracy has steadily improved, but models
need to be updated with the latest varieties every few
years, since breeders are steadily altering varieties
(e.g., phasic development, improving PY). Some-
times, crop contest yields or crop record yields are
considered to be synonymous with PY. But they need
to be treated with caution, because they usually exceed
PY measured as defined here, probably because they
refer to very favorable circumstances (e.g., soils,
weather, management), relative to the district average;
nevertheless, we can learn from such yields if all site
and management variables are quantified. For exam-
ple, properly verified world record yields, invariably
higher than PY values, not only extend our simulation
models but also reveal that there is no anatomical
limitation to very high yields, the main limitation
evident with such yields being the stem strength
needed to support them.
Since much of the world’s grain crops are grown in
rainfed situations where water supply from precipita-
tion plus starting soil storage falls well below the poten-
tial evapotranspiration, it is also useful to define
a water-limited potential yield (PYw, t/ha): it is the
yield obtained with no other manageable limitation
to the crop apart from the water supply. Obviously, it
will depend on the amount of water, so PYw is usually
plotted relative to water supply (or use), the slope being
the crop water use efficiency or water productivity,
commonly reported in kilograms per hectare per
millimeter. Complications can arise due to variation
in rainfall distribution with respect to crop develop-
ment stages, but PYw, defined as a linear function of
the water supply, is a very useful simple benchmark, as
argued in a recent in-depth review by Passioura and
Angus [2], while simulation modeling has been espe-
cially useful in dealing with expected deviations due to
variation in the distribution of water supply.
In any given region, between FY and PY (or PYw),
there is another useful yield notion, namely attainable
yield (AY, t/ha), which is defined here as the yield
attained by a farmer with average natural resources
adopting economically optimal practices and levels of
inputs. Since risk of financial loss is almost always part
of a farmer’s decision to invest in increased inputs, the
AY definition must temper “optimum level” with “pru-
dent attention to risk”; as an example this could mean
input investments must be expected to return at least
50% in net profit at the margin. Of course, AY will
reflect the economic circumstances of the crop and
region, in particular grain prices relative to input
ones, all measured at the farm gate. Thus, it is not
easy to know AY, but general experience suggests that
it lies around 20–30% below PY in situations where
world prices and reasonable transport costs operate. FY
for wheat in the UK probably meets these conditions
(Fig. 1). Where this does not occur, for example, in
much of sub-Saharan Africa where infrastructure and
institutions are weak, the AY as defined above may be
much lower; alternatively where inputs and grain prices
are heavily subsidized, AY could approach closer to PY.
Because of these uncertainties, it is easier to talk of the
yield gap in terms of the FY to PY gap, bearing in mind
that even in the most advanced cropping situations in
developed economies, operating at close to world
prices, FY will remain significantly below PY. Since it
 Crop Yields Around the World: Closing the Gap and Raising the Potential 701

12
Potential yield
11 y = 0.061x - 111
R2 = 0.532
Grain yield (t/ha) 10

9
y = 0.053x - 98
8 R2 = 0.457

7
Farm yield
6

4
1985 1990 1995 2000 2005 2010
Year

Crop Yields Around the World: Closing the Gap and Raising the Potential. Figure 1
Change in farm yield (FY) and potential yield (PY) with time for wheat in the UK. Source: FAOStat and HGCA. Note that
in this and other figures (except Fig. 4), PY is plotted against year of variety release, for varieties are compared side by side
in recent yield trials under modern management

is more appropriate to express this gap as a percentage
of FY, it can be assumed from the literature that this
minimal gap is reached when the PY–FY gap is about
30% (of FY). Any larger gap that this, which is usually
the case, is often defined as an “economically exploit-
able yield gap,” but bearing in mind that the expected
exploitation can be as much a task for national and
local government and agribusiness, as for the farmers.
Introduction
Crop yield has been the subject of attention since the
days of Malthus and earlier. More recently, many have
pointed out that the improved supply of food calories
per capita globally, and reduced real costs of grain, over
the last 60 years has been due almost entirely to global
crop yields growing faster than the burgeoning world
population (e.g., [4]), for there have been only small
increases in cropped area (Fig. 2). The world popula-
tion will grow to over nine billion by 2050, and grain
demand is projected to increase around 60–70% (com-
pared to 2000) [5], with others projecting even greater
increases, and certainly more rapid increase coming
early in this period. Seventy percent over 50 years
amounts to an exponential increase of 1.1%; output
growth must match this or real prices will increase (see
also later). There is some potential new arable land of
reasonable quality in South America and sub-Saharan
Africa (and in northern latitudes of Russia), but
increase in arable area demands heavy capital invest-
ment and brings negative environmental consequences;
indeed, maintenance of arable area could be a challenge
in densely populated fast-urbanizing regions like South
and East Asia, and where there is continuing arable
land degradation. Crop area can also increase through
intensification of annual cropping but that usually
depends on expansion in irrigation, which has in fact
slowed markedly, especially in Asia where water avail-
ability for agriculture is now constrained. As
a consequence, most argue that the projected increase
in grain demand must continue to be met by yield
increase.
Many grain crops have exceeded 1% exponential
yield growth in the early decades of the modernization
of agriculture last century, including the Green Revo-
lution period for rice and wheat in Asia, but global
yield growth trends for most crops over the last 20
years have slowed and have become linear, with current
702 Crop Yields Around the World: Closing the Gap and Raising the Potential
7.00
6.00
5.00
Population (billion)
4.00
3.00
2.00
1.00
0.00
1880 1900
Crop Yields Around the World: Closing the Gap and Raising the Potential. Figure 2
Change in world population, global arable area, and cereal yield. Adapted with permission for Evans (1998)
growth rates at or below 1%; maize at 1.6% and soy-
beans at 1.1% are the only major crops exceptions to
this (Fig. 3; note growth rate is expressed here by
calculating the linear slope as a percentage of current
predicted yield for each crop). Increasing the propor-
tion of a crop which is irrigated can increase yield
growth, as happened with wheat in Asia in the 1960s,
but further increases in irrigation area are unlikely, due
to cost and/or lack of water, except possibly in sub-
Saharan Africa. This leaves the maintenance, or prefer-
ably boosting, of rates of yield increase in existing
cropped situations as the major route for meeting
growing demand for grain, and the major challenge
facing agricultural science. And despite some media
sentiment to the contrary, agricultural scientists are
fully aware that this goal has to be met while sustaining,
or better still, improving the natural resource base of
cropping, and while confronting the uncertain and
predominantly negative projected impacts of climate
change.
From the definition of the subject, it can be seen
that there are two sources of yield increase: raising PY
and closing the PY to FY gap (see also Figs. 1, 4 and 5).
3.5
Population
Arable area
3
Cereal yield
2.5
Arable area (billion/ha),
Cereal yield (t/ha)
2
1.5
1
0.5
0
1920 1940 1960 1980 2000 2020
Year
In most situations, both processes contribute simulta-
neously to FY increase, with farmers adopting new
technologies (varieties, management techniques,
input levels) that have been developed years or decades
earlier by researchers at which time the associated rise
in PY occurred. Recently, two trends in this process are
becoming evident. Firstly, management or agronomic
innovations for increasing PY, including their positive
interactions with new varieties (e.g., the universal
nitrogen by variety interaction in wheat and rice, or
the plant density by hybrid variety interaction in
maize), are becoming fewer, leaving a greater propor-
tion of PY progress to breeding advances. This is
a somewhat controversial observation but is clearly
supported by analysis of the winter wheat yield changes
in the UK [6]. Secondly, in some special situations, as
mentioned earlier, the yield gap is approaching the
minimal 30% or so dictated by current economics;
examples include winter wheat in the UK (Fig. 1) and
probably all of Western Europe), rice in Egypt (Fig. 4),
and maize in the US corn belt (e.g., Iowa, see Fig. 6).
While it is not always easy to separate PY advance
from gap closing, since this volume has given more
 Crop Yields Around the World: Closing the Gap and Raising the Potential 703

y = 0.0818x - 159
5
Maize R2 = 0.90

y = 0.0394x - 75
4
R2 = 0.96
Grain yield (t/ha)

Rice

3 y = 0.0268x - 51
Wheat R2 = 0.85
y = 0.0290x - 55
R2 = 0.83
2
Soybean

0
1985 1990 1995 2000 2005 2010
Year of production

Crop Yields Around the World: Closing the Gap and Raising the Potential. Figure 3
World average yield versus time for wheat, rice, maize, and soybeans over last 20 years. Source: FAOStat

attention to the former, in particular the possibilities 14

Potential yield y = 0.075x - 139
arising with new molecular biological techniques 12 R2 = 0.712
Grain yield (t/ha)

becoming available to plant breeders, this entry will 10 y = 0.185x - 361

give more attention to closing the yield gap than to 8 R2 = 0.959
raising PY. Both issues have been more fully discussed 6
in Fischer et al. [5]. Another reason for concentrating Farm Yield
4
here on closing the yield gap is that, notwithstanding 2
the evidence of small yield gaps in some situations, it is 0
likely that yield gap closing offers better prospects 1975 1985 1995 2005
globally for quickly lifting FY progress, as is needed, Year
than does boosting PY progress. The entry continues by Crop Yields Around the World: Closing the Gap and
looking at the size of the yield gap in various key Raising the Potential. Figure 4
situations around the world. Passing then to gap clo- Change in PY and in FY for rice in Egypt. Source: Badawi [3];
sure, traditionally this is considered to arise from A. E. Draz, 2009, personal communication
farmer adoption of yield-enhancing technologies, the
area of agricultural extension or technology transfer.
While there is a lively body of socioeconomic research
on this process, which will be considered briefly, the impact should be on PY as defined, actually increasing
way the yield gap is defined here, it is hard to see the the gap, followed by their gradual adoption which, if
new products of breeding and agronomic research complete, might be expected to reduce the gap to the
reducing the yield gap. This is because their initial same relative value as before, other things being equal.
704 Crop Yields Around the World: Closing the Gap and Raising the Potential

10.0
Potential yield y = 0.024x - 38
9.0 R2 = 0.237

8.0

7.0
Grain yield (t/ha)

6.0 y = 0.049x - 92
R2 = 0.522
5.0

4.0
Farm yield
3.0

2.0

1.0

0.0
1950 1960 1970 1980 1990 2000 2010
Year

Crop Yields Around the World: Closing the Gap and Raising the Potential. Figure 5
Wheat PY and FY changes with time, Yaqui Valley, Mexico. Source: [5]

16
y = 0.261x - 510
R2 = 0.490
14
Potential yield
y = 0.116x - 221
12 R2 = 0.732
Grain Yield (t/ha)

10
Farm yield y = 0.205x - 401
R2 = 0.639
8

0
1980 1985 1990 1995 2000 2005 2010 2015
Year

Crop Yields Around the World: Closing the Gap and Raising the Potential. Figure 6
Change in maize FY in Iowa compared to that in PY as estimated by Pioneer (Hammer et al. ([7], red squares) and de Kalb
(Edgerton [8], green triangles) hybrid yields plotted against year of hybrid release
 Crop Yields Around the World: Closing the Gap and Raising the Potential
Needless to say the real situation is more complex and
examples will be given of how such research, including
progress in molecular biology, can help directly reduce
the gap (e.g., more adoptable technologies, less costly
ones, and better biotic stress resistance). The entry
finishes by discussing briefly the current situation
with progress in PY and PYw themselves. What will
not be discussed further is the likelihood that atmo-
spheric CO2 increase will continue to improve all grain
yields, other things remaining equal, but the rate of
increase is currently around only 0.2% in a C3 crop like
wheat [9], and less for C4 crops.
Estimates of Yield Gaps
Fischer et al. [5] attempted to estimate yield gaps and
their rate of change for a number of key representative
cereal-producing regions in the world. The most reliable
estimates are summarized in Table 1, supplemented
with a few new crops and numbers.
The yield gap estimates for wheat are remarkably
consistent at around 50% except for the UK, where as
mentioned before the 25% gap probably implies that
FY is approaching AY, an AY largely determined by
ruling world prices in the UK. Figure 5 illustrates the
Yaqui Valley data, where there was substantial yield gap
closing as the modern semidwarf varieties took over in
the 1960s; lately, however, the yield gap has remained
fairly steady at 50%. Lobell et al. [12] recently
presented 12 published estimations of the yield gap
for wheat in developing countries, the gap ranged
from 5% to 150% but the average was close to that in
Table 1, namely 55%.
For rice, Table 1 shows gap values around 100% or
more for most developing country situations except for
Central Luzon in the wet season (65%), and
a remarkably low 15% for Egypt (see also Fig. 4). The
value of 55% for Japan reflects the heavy emphasis on
producing high-quality grain, acting as brake on FY
progress, because food-quality rice varieties and agron-
omy deliver lower yields. From the 41 rice estimates
from developing countries in [12], the average gap was
65%, with modeling evidence that the gap was 120% in
northwest India (cf. 110% in the Indian Punjab in
Table 1).
Table 1 has only two cases for maize: Iowa at 25%
contrasts with Kenya at 200+%; sub-Saharan Africa as
705
a whole is similar to Kenya. Tittonell et al. [13] in
a detailed survey of farm yields in three districts in
the favored Kenyan Highlands confirms the figure
shown in Table 1, recording a mean FY of 1.5 t/ha
when PY was 6–7 t/ha. Lobell et al. [12] give nine
examples of rainfed maize in tropical and subtropical
developing countries (average gap 200%) and examples
of irrigated and rainfed maize in Nebraska based on
simulation of PY and PYw which suggest gaps of 35%
and 55%, respectively, both larger than that for rainfed
maize in the adjacent wetter state of Iowa (Table 1).
While there is no doubt about the huge yield gap with
maize in Africa, some uncertainty exists in these critical
estimates from the USA, where maize is probably the
most intensively researched and promoted crop in the
world. No doubt the gap is quite small in Iowa and
Nebraska, where it has been closing lately, and FY may
be close to AY (Fig. 6), although more PY progress data
is needed for a clearer conclusion.
The soybean examples in Table 1 also show a large
difference in the yield gap between a developed and
developing country, but do not show developing coun-
tries Argentina and Brazil, where FY across more than
35 M ha with favorable rainfall is as high as the USA,
and PY is unlikely to be any higher. Finally, millet,
grown in India under very harsh conditions, shows
a gap no worse that of soybeans in adjacent more
favorable areas of India.
Closing Yield Gaps
Yield gaps are reduced by farmers adopting new tech-
nologies or practices (new at least for them) or
adopting higher rates of inputs (e.g., many farmers in
western Kenya use added nutrients, either fertilizer or
organic ones, but the rate of nutrients applied may be
very low relative to the need/optimum (e.g., [13])).
Some of this is usefully illustrated in Fig. 7 derived
from Byerlee [14].
FY under traditional practices with low inputs is
shown by point A on curve 1. Adopting new technol-
ogies such as an improved new variety and practices
(e.g., line sowing and fertilizer) lifts technical efficiency
and brings curve 2 into play. The farmer may move
from point A to point B, as in the first years of the
Green Revolution. Allocative efficiency could then rise
further as fertilizer levels increase, following curve 2 to
706 Crop Yields Around the World: Closing the Gap and Raising the Potential

Crop Yields Around the World: Closing the Gap and Raising the Potential. Table 1 Estimates of current yield gaps for
key crops and regions (compiled from [5] and elsewhere as noted)

Yield (t/ha)a and gap(%), 2007 or 2008

Region Environment Crop area (M ha) FY PY Gap
Wheat
Yaqui V, Mexico Irrigated, low latitude 0.16 6 9 50
Punjab, India Irrigated, low latitude 3.9 4.3 6.25 45
b
Western Australia Subhumid, low latitude 4.5 1.8 2.6 45
b
N. Dakota Subhumid, high latitude 3.4 2.5 3.7 50
UK High rain, winter wheat 1.8 8.2 10.4 25
Eastern China Irrigated, winter wheat 16 4.7 7 50
b
Kansas Subhumid, winter wheat 3.6 2.6 3.9 45
Rice
Central Luzon Wet season + irrigation 0.8 3.8 6 60
Punjab, India Wet season + irrigation 2.4 3.8 8 110
Japan Wet season + irrigation 3 6.5 10 55
South Asia Wet season, rainfed 28.5 1.8 3.6 100
Central Luzon Dry season, irrigated 0.4 4.5 9 100
Egypt Dry season, irrigated 0.7 10.1 11.6 15
Maize
Iowa Temperate, high rainfall 5.3 10.8 15.5c 45
b
Kenya All altitudes, moderate rain 1.75 1.8 6 200+
Soybeans
USAd Temperate, high rainfall 30 2.8 3.6 30
e b
India Subtropical, tropical, moderate 9 1 2.2 120
rainfall
Millet
Indiaf Subtropical, rainfed 11 0.9 1.8b 100
a
Predicted from linear trends
b
Actually PYw
c
Reestimated in Fischer and Edmeades [9]
d
Estimate by R. A. Fischer, 2010 unpublished
e
Bhatia et al. [10]
f
Murty et al. [11]

point C, approaching some economic optimum. An
even newer variety and the best available practices
(e.g., herbicides, conservation tillage) which together
define current PY (point F) could lift the technical
frontier further (the uppermost curve 3). Farmers
might choose to further increase technical efficiency
by moving to the uppermost curve, maximizing
allocative efficiency at around point D, or sacrificing
allocative efficiency, but not yield, by reducing inputs
(point E). Of course, curve 3 is not static: for any given

4
E
Grain yield (t/ha)
3 C
B
2
1 A
0
0 2
Crop Yields Around the World: Closing the Gap and Raising the Potential. Figure 7
Illustration of pathways from low FY to close the gap with PY; modified from Byerlee [14]
environment it may be moved further upward with
new technologies, and it may also shift downward due
to problems of resource degradation. This figure also
serves to illustrate that the yield gap is not the same as
an efficiency gap, gaps between curves are technical
efficiency ones, gaps along curves are allocative ones,
both combine to make up the FY to AY gap.
Adoption of New Technology
There is a rich agricultural economic literature on the
adoption of new technologies by farmers. Examples for
the adoption of two new technologies in the rainfed
cropping in Australia are shown in Fig. 8a (a new crop,
lupins, and a new practice, no till). The new crop was
adopted faster than the new agronomic technique.
Developing countries have proved equally fast in
adopting new varieties (Fig. 8b) when the conditions
are favorable. The well-recognized patterns of lag, rate
of adoption, and ceiling adoption level clearly differ
between technologies and farmers (regions); in addi-
tion, the infrastructural and institutional context is
important. For the technology itself, key elements are
its perceived relative advantage (return over cost, risk of
loss, convenience, etc.) over that which it replaces, and
its trialability, meaning the ease with which a new
Crop Yields Around the World: Closing the Gap and Raising the Potential 707
D F
3.Modern practices
2.Intermediate practices
1.Traditional practices
4 6 8 10 12
Input level
technology can be tested on a small scale. Farmer char-
acteristics influencing adoption are very diverse and
can also interact with the nature of the innovation:
education, age, health, exposure to extension and
related media and demonstrations, group pressure,
and support can be important. But there is little
doubt that farmers around the world, large and espe-
cially the small, women and men, aspire to increase
monetary return and given the means to do so, will
adopt more profitable practices.
It is in the area of rural infrastructure and institu-
tions that developed and developing country farmer
circumstances differ most. One obvious example is the
effect of poor roads on increasing input costs and
reducing prices for surplus farm output. For example,
the price ratio of N fertilizer to grain is on average
double that in other regions of the world and higher
still in inland landlocked regions of sub-Saharan Africa
[18]. The high cost of credit, uncertainties surrounding
contracts (including land tenure), the risk of poor-
quality inputs, theft or unrest, and the prevalence of
unfavorable and uncertain tax and pricing policies, all
make adoption less attractive in many developing situ-
ations. Sometimes, price subsidy on inputs and outputs
can compensate for these brakes on adoption, such as in
the recent situation where a well-crafted fertilizer supply
708 Crop Yields Around the World: Closing the Gap and Raising the Potential
100
90
Lupins
80
Percent adoption by farmers
70
No-till
60
50
40
30
20
10
0 0
1970 1980 1990 2000
a Year
Crop Yields Around the World: Closing the Gap and Raising the Potential. Figure 8
Adoption of new technologies by (a) farmers in regions of Australia (lupins in Wongan Hills district [15] and no till in
Loddon district [16]), and (b) semidwarf wheat varieties in Mexico and in Bangladesh [17]
and subsidy policy permitted Malawi, a nation of small
holders, to produce an unprecedented surplus of maize
and to do so without a maize price collapse. But in
general and especially in sub-Saharan Africa the lack of
public investment in infrastructure, institutions and
agricultural extension, and the lack of sound policy are
the major contributors to the yield gap.
Looking at farm-level technical constraints that
contribute to the yield gap, in any situation there are
usually multiple constraints, and the challenge is to
determine which constrains should take priority,
while recognizing that interventions often interact pos-
itively and are thus more effective when adopted
together [19, 20]. This can only be answered by on-
farm survey and experimentation. Such work started
many years ago with farming system research, farm
management clubs, and rapid rural appraisal. It con-
tinues in many guises in the developed world, especially
influenced by the privatization of agricultural exten-
sion, the use of remote sensing and ICT advances, and
the entry of input suppliers, in particular seed compa-
nies, into agricultural extension.
In the developing world the more traditional
approaches remain, although with growing emphasis
on farmer participation [21]. Lobell et al. [12] recount
how IRRI conducted on-farm rice experiments in Asia
100
90
Mexico Bangladesh
80
Percent farmer adoption
70
60
50
40
30
20
10
2010 1955 1960 1965 1970 1975 1980 1985 1990
b Year
in the 1970s to test high inputs, learning that farmer
yields varied greatly between fields, as did responses to
inputs especially fertilizer and insecticide, which were
often uneconomic. This pointed to the importance of
field-to-field variability, and the need to adjust inputs
accordingly and as the season unfolds, whether by site-
specific nutrient management, which reached maturity
some 20 years later [22], or via field-level pest moni-
toring as part of IPM packages. Titonell et al. [13]
recount a similar picture of substantial variability in
soil fertility, resource use intensity, and yields among
small farmer maize fields in western Kenya. Another
lesson is surely that this is scientist-intensive expensive
research, usually taken over by the farmer and his
advisers in the industrial world, and explaining why
large yield gaps often persist in the developing one,
where circumstances demand innovative approaches
in order to reach the billion small farmers (e.g., [21]).
Very recently IRRI again looked at rice yield gaps,
this time using expert knowledge to assess constraints
and possibilities for irrigated rice in South Asia
[23]. For this crop, FY is currently 5.1 t/ha over 34.3
M ha; it was estimated that on average yield was
constrained 1.9 t/ha (37%) by yield-limiting factors,
which included individual constraints from nutrient
insufficiency (10%), diseases (7%), weeds (7%),
 Crop Yields Around the World: Closing the Gap and Raising the Potential
water shortage (5%), and rats (4%). The exercise was
repeated for the 28.5 M ha of rainfed lowland and
upland rice in South Asia with a current FY of 1.8 t/ha:
yield-limiting factors amounted to 68% of FY, including
nutrients (23%), disease (15%), and weeds (12%). The
IRRI paper predicted that with a substantial research,
development and extension effort, the adoption of
existing technology and ongoing breeding over the
next 15 years could reduce these losses by one third
(irrigated rice) or one quarter (rainfed rice) adding
about 1% to FY growth rate.
With wheat in the Yaqui Valley we have a recent
concerted effort to understand the yield gap, PY–FY
(currently 50%, Fig. 5, Table 1), this time using the
latest high-resolution satellite imagery to estimate
field-level yields for all fields in the Valley [24] and
supplement a long history of farm surveys. Despite
the moderate size and wealth of farms relative to
India or Kenya, again field-to-field variability in yield
was substantial. It was estimated from images over
several years that wheat yields were constrained by
late planting, delays in the first post-plant irrigation,
and summer fallow weeds [25]. Improved institutions
and farm management decisions could largely elimi-
nate these constraints, which averaged over years
totaled about 10–15% of FY, and would bridge about
half of the gap to estimated AY in the Valley. These
authors [26] used inter alia classification and regression
trees to relate yield to constraints in their complex data
sets. The same technique was used by Tittonell et al.
[13] to explore management and soil constraints to 150
field-level maize yields in highland Kenya: the rate of
added nutrients was the strongest explanatory variable,
followed by date of planting and plant density, as yield
ranged from 1.2 (low nutrients, late plant) to 4.2 t/ha
(high nutrients, normal plant, high density).
Two recent studies illustrate for rainfed cropping
the power of simulation modeling and water produc-
tivity boundary functions in understanding yield gaps
in surveyed farmer fields and in particular removing
that part of the apparent gap which is actually due to
non-manageable weather (mostly rainfall distribu-
tion). Grassini et al. [27] found these distribution
effects quite important for sunflower yield variation
among fields in the western pampas of central Argen-
tina. Hochman et al. [28] looking at farm wheat yields
across Australia, found less influence of rainfall
709
distribution, and more scope for farmers to lift water
productivity via improved management (e.g., earlier
planting, higher seeding density, higher N input).
A weakness of both these studies is that they refer to
better farmers rather than to a random sample of fields,
for which the opportunities of gap-closing interven-
tions are likely to be greater.
The persistence of large yield gaps in the developing
world especially draws attention to situations where
these gaps have been closed. Rice in Egyptian is an
obvious example where strong R, D, and E engaged
a small geographically focused industry of small
holders under a sound price policy (Fig. 4). A second
example of dramatic technology adoption, albeit with
lesser immediate implications for FY than for sustain-
ability of the whole cropping system, relates to the
uptake of conservation tillage for wheat, maize, and
soybeans in southern South America (Argentina,
Brazil, and Paraguay), rising from nothing in 1970 to
24 M ha in 2000. This was very much driven by farmer
groups and the farmers themselves faced with the threat
of serious soil degradation and by the opportunity
provided by knock-down herbicides and knowledge
spillover from the North (e.g., [29]). This revolution
has yet to reach other developing continents (but is
beginning in northwest South Asia). A third success
story among small poor farmers has recently appeared
with the introduction and expansion of winter maize in
northeastern India and Bangladesh.
Despite individual success stories like rice in Egypt,
yield gaps in general appear to be quite persistent and
close only slowly; this happens even when gaps are well
above that to be expected from economics and risk
aversion and even when PY progress has slowed such
that catch-up through eliminating excessive lags in
varietal adoption is not a big issue. The problem is
that gap closing on the large scale needed requires
massive investments in rural infrastructure and insti-
tutions as well as technology transfer, and these are not
forthcoming, as maize in sub-Saharan Africa exem-
plifies. Elsewhere public sector agencies, in particular
reaching the billion small farmers in Asia [21], aided by
the private sector, in particular in Latin America,
have made some inroads on the yield gap; they should
continue to do so largely in proportion to the invest-
ments made, but there is also scope for innovation,
for example, based on modern ICT technologies.
710 Crop Yields Around the World: Closing the Gap and Raising the Potential
The employment of agronomists by private seed com-
panies is a pattern that is bound to be followed in the
developing world as its seed industry grows in strength
and competitiveness. With gap closing, there are no
spill-ins as there are in the case of PY advance through
R and D, innovations need to be adapted locally, but it
can be argued that the Internet and mobile phones are
relevant spill-in technologies for delivering informa-
tion to farmers small and large, a role which could
greatly expand.
Innovative Adoption-Friendly Crop Management
and New Varieties
Research can facilitate the adoption of technologies to
lessen those constraints which contribute to the FY to
PY gaps. Table 2 lists common constraints, separating
research targeting agronomic solutions from that
involving breeding, while institutional and infrastruc-
tural solutions already mentioned are shown in the last
column. Some agronomic technologies are still under
development (e.g., improved seasonal weather fore-
casts), but much of the necessary agronomic research
is adaptive, generally fitting technologies from more
advanced farming systems. For example, the widespread
adoption of direct seeding of wheat after rice in north-
west India, which by saving on land preparation time
has meant that sowing dates are less likely to be late and
yield expectations are hence improved, required
research and development on appropriate small-scale
drills for this direct seeding operation. Sometimes, the
agronomic technology comes from a “less-advanced”
field cropping situations or from farmers themselves:
for example, the use of plastic film mulches, quite com-
mon in northern China with wheat, maize, and oilseeds.
Such mulches give substantial yield benefit through
retaining soil moisture and aiding soil warming in the
spring, and challenge agronomic researchers to adapt
them in a sustainable fashion elsewhere, including
developed countries.
It is with breeding that there are greater gap-closing
opportunities. Considering that actual losses from dis-
eases and insects globally exceed 20% of yield with
wheat, rice, and maize [30], any improvement in
genetic resistance has an immediate benefit for FY
when the improved varieties are adopted. Conventional
host plant resistance breeding continues to make
progress on this front, while genetic engineering has
brought exciting new opportunities. Although
engineered resistance does not lift PY, it lifts FY wher-
ever farmers cannot control pests and diseases with
traditional means. The experience with Bt cotton in
India shows how important this can be: small holder
yields are at least 30% better simply because before the
advent of Bt cotton they were unable to eliminate
damage no matter how much insecticide was used
[31]. It is likely that the advent of GM corn resistant
to root worm is boosting farm yields in the USA
because root worm damage went unnoticed before-
hand (and is very difficult to treat with chemicals).
Fungal diseases have yet to succumb to genetically
engineered host plant resistance, but it is reasonable
to expect varietal releases in this area in the next
decade; engineered viral resistance has already been
deployed in some crops [31]. Actual yield losses
due to weeds are estimated globally at about 10% by
Oerke [30], to which should be added the costs of
current control measures. More competitive cultivars
can help, but again genetic engineering has brought
revolutionary advances in ease, cost, and effectiveness
of weed control. Of course, with any chemical-based
susceptibility of biotic stress agents, there will be the
risk of resistance evolving in the target organism, but
integrated management, albeit requiring greater farmer
skills, can prevent that. Given proper R, D, and exten-
sion, the potential impact of herbicide-tolerant culti-
vars on FY in labor-limited African cropping is likely
substantial. Of course, all breeding solutions to aid gap
closing presuppose an effective seed production and
distribution system. Systems are gradually improving
in developing countries and most commercial farmers
grow improved varieties although the rate of turnover
of varieties is often too slow. There is little doubt that
wherever plant breeding is privatized, competition
drives quicker variety turnover; in advanced systems,
it also drives significant agronomic extension by the
breeding firms keen to maximize variety by manage-
ment interactions and to retain clients.
Lifting Potential Yield
Although separated by a yield gap and a time lag, many
situations show a close relationship between progress
in PY and that in FY, as new technologies eventually
 Crop Yields Around the World: Closing the Gap and Raising the Potential 711

Crop Yields Around the World: Closing the Gap and Raising the Potential. Table 2 Constraining factors contributing
to the farm yield–potential yield gap and their alleviation so that farm yield can approach the attainable yield
corresponding to the current potential yield with realistic economics
Resolution
Research
Constraint Agronomic Breeding Institutional/infrastructural
General farmer constraints
Lack of farmer On-farm demonstration On-farm testing and selection Education, media campaigns,
awareness or conviction extension
or skill
Risk aversion by farmer Forecasts, tactical decision Tolerance of extreme weather Insurance schemes, favorable
making (e.g., for N top events, like drought, flooding, credit terms, price stability
dress) hail, frost, wind
Inadequate labor Mechanization, reduced Select for uniform maturity to Facilitate labor migration; credit
supply tillage, herbicides favor mechanical harvesting for mechanization
Technical constraints
Lacking major long- Drainage, land leveling, Waterlogging, acidity, Long-term credit for major soil
term soil amelioration liming, deep tillage, and salt tolerance amelioration, secure tenure
gypsum
Excess tillage and loss Conservation tillage Suitable varieties: disease and Credit for new machinery
of moisture, soil options and suitable herbicide tolerance
compaction machinery, controlled
traffic
Manageable topsoil soil Ameliorate (e.g., lime for Acidity, aluminum tolerance Input suppliers, credit for lime
toxicities acidity)
Suboptimal nutrient Diagnostics, application of Some scope for improved N, P, Infrastructure, input suppliers,
supply nutrients, slow release and Zn uptake and utilization credit, quality control
forms, tactics to match efficiency
supply with need
Soil variation within and Diagnostics to aid Greater tolerance of soil
between adjacent fields adjustment of application stresses
rates
Growing old varieties, Better on-farm seed F1 hybrids, licensed traits, Strong seed industry and proper
or use of poor seed management and storage royalties to encourage strong regulation, credit
seed industry
Incorrect time of sowing Mechanize, reduced tillage Make available varieties with Policy to favor mechanization,
to speed sowing; range of maturities; herbicide contract seeding
treatments to warm soil for and cold tolerant varieties
spring sowing
Poor plant population Better drilling procedures More robust varieties (e.g., Seed testing and regulations
and machines, quality seed long coleoptile in wheat, more
storage tillering)
Diseases and pests, Biocides, sanitation, crop Host plant resistance Input suppliers, pesticide
above and below rotation, IPM quality control
ground
712 Crop Yields Around the World: Closing the Gap and Raising the Potential
Crop Yields Around the World: Closing the Gap and Raising the Potential. Table 2 (Continued)
Resolution
Research
Constraint Agronomic
Weeds Herbicides, cultivation,
sanitation, crop rotation
Poor water Improve water application Greater tolerance water
management in techniques and skills, land shortage and excess
irrigated systems levelling
Long-term soil Crop rotation, fertilizer,
degradation green manuring, farm yard
manure, conservation
tillage, amelioration
find their way to farmers. Often the process is facili-
tated by new varieties linked to agronomic advances,
for new varieties are fairly quickly adopted in most
commercial farming. Sometimes, the PY advances owe
more to innovative farmers than to researchers, as for
example, when farmers moved to earlier planting per-
mitted by direct seeding in southern Australia. In all
cases, increase in PY is an important component driv-
ing increased FY, inevitably so where the yield gap is
approaching the economic minimum (e.g., wheat in
the UK), but less so where it is large (e.g., paddy or
rainfed rice in India), and even less when the gap is
huge (e.g., maize in sub-Saharan Africa).
Current rates of PY progress were summarized for
the key cereals in [5]. These numbers come from
breeders’ and researchers’ trials containing varieties of
different vintage grown under modern agronomic
management with little or no biotic stress; thus, the
rates capture as well the positive interactions between
new varieties and modern management. Linear regres-
sion is used to calculate the absolute rate of progress
which is then expressed as a percentage of most recent
predicted PY in the series, hopefully a recently released
variety (>2006) but such data is not always available.
For wheat, progress ranged from 0.3% to 1% p.a. with
an average of 0.6% (n = 6) and with little difference
between water-unlimited and water-limited PY pro-
gress (see also Figs. 1 and 5). For rice, the range was
0.2–0.7% (average of 0.4%, n = 4). Progress in PYat the
International Rice Research Institute (IRRI) is
Breeding Institutional/infrastructural
Enhance crop plant Herbicide quality control,
competitiveness, herbicide release regulation
tolerance
Efficient supply systems to farm
Varieties adapted to biotic and Tenure regulations ensuring
abiotic stresses of high plant land ownership by farmer
residue levels, and with good
residue production
disappointing, at close to zero; however, there is
a one-off yield jump of 10–15% through the recent
exploitation of F1 hybrids feeding into the tropical
rice regions, and following upon on a similar gain in
China over the last 20 years as hybrids moved to >50%
of their acreage. For maize, there is only one estimate in
[5], coming from Iowa and showing 1%. Data of Luque
et al. [32] in a somewhat similar environment in Argen-
tina give a rather similar number for hybrids released
up to 1997 (1.3%), but since then PY progress may have
slowed (M. Otegui, 2009, personal communication).
A subsequent report from Iowa with hybrids of a rival
company showed progress at 1.7% but the breeding
period sampled was only 2001–2006 [8]. In an irrigated
favorable Mediterranean environment, data presented
by Campos et al. [33] indicate PY progress in the Iowa
Pioneer hybrids of 1% (but interestingly progress in
PYw of 1.5% under artificial severe mid-season water
stress). PY progress in cereals is discussed in more
detail in [9]. Potential yield for soybeans in the USA
appears to be progressing at 0.7% p.a. (R. A. Fischer,
2010, unpublished).
From the above, it is apparent that even in advanced
situations with substantial breeding investment, poten-
tial yield progress from this source is currently at
or well below 1%, with maize showing more progress
than other major crops. Such PY progress is unlikely
alone to drive FY progress at the rates needed.
Prospects of lifting this rate of PY progress were now
discussed briefly.
 Crop Yields Around the World: Closing the Gap and Raising the Potential
New Yield-Positive Agronomic Techniques
Improved agronomy has played a large if not dominant
role in past yield increase (e.g., increased fertilizer use,
better weed control, better seeding techniques for ear-
lier seeding and more reliable crop stands, and water
conserving reduced tillage), and most improvements
have interacted positively with variety improvement
(e.g., [20]). While many authors have consistently
failed to anticipate agronomic advances in the past
[34], currently it is hard to see any new ones raising
PY or PYw (as distinct from advances specifically
targeting improved input efficiency). One example
might be improved seasonal forecasts which would
permit the farmer to better tailor management and
variety to expected weather, something currently lim-
ited by the low skill of forecasts. Another, which would
clearly lift PYw, would be the reduction of soil evapo-
ration with inexpensive plastic films (e.g., spray-
applied nanofilms), copying a common practice seen
in northwest China field crops, but designed so as not
to contaminate the environment. Also it is possible
advances can still come from better management of
soil–root–microbe interactions, a complex and
neglected area of research: poorly explained observa-
tions of “yield decline” and “break crop effects” point
in this direction, as do reports of positive effects of
increased soil organic matter (independent of nutri-
tion), controlled traffic and reduced soil compaction.
Greater Investment in Current Conventional
Breeding Efforts for Yield
Yield improvement through plant breeding is generally
regarded as delivering benefits far in excess of the costs
[35, 36]. Nevertheless, it has been suggested that
returns from the investment in breeding personnel are
diminishing, for example in maize, and that yield gains
are getting harder to achieve [37]. This is occurring
despite greater efficiency through computerization,
advanced biometrics, mechanization, robotics, and
techniques for rapid generation advance, all part of
any conventional breeding today. What is quite unclear
however is the marginal return on investment in con-
ventional breeding; for example, what would happen if
the number of breeders, and of crosses, selections, and
yield trials, were increased say 50% in any currently
substantial program? In crops where breeders are
713
devoting many resources to maintaining disease resis-
tance (e.g., wheat) or eating quality (e.g., wheat, rice),
and thus unable to give full attention to yield, PY
progress is likely to increase, although probably less
proportionally that the proportional investment
increase. A related question is whether any extra
funds would be better spent on new breeding tools.
New Tools to Increase Progress in Conventional
Breeding
Genetic variation is the basis of yield progress. It is
unlikely that all or even the major part of the existing
genetic variation has been utilized in any crop. Sam-
pling germplasm collections is a formidable task, but
new tools are becoming available for seeking out new
and useful variation, such as the Focused Identification
of Germplasm Strategy (FIGS, [39]) and Ecotilling
[38]. Because of the low chance of success, this work
tends to be in the area of publically funded
prebreeding, with only medium to long-term impacts
on breeding, but it is worth noting that the steady
reduction in genome sequencing costs is also opening
up new ways of allele searching [40].
Both parental and especially early generation
selection can benefit from the use of low cost perfor-
mance-related markers. For the last 50 years or so,
physiologists have attempted to identify such markers
or traits, physiological or morphological, or even whole
ideotypes, which would lead to greater yield. There
have been some successes, for example, the first high-
yielding semidwarf tropical rice variety, IR8, resulted
from the pursuit of a specific ideotype [41], and breed-
ing for resistance to some toxicities is usually based on
early generation physiological screening (e.g., alumi-
num tolerance). However, indirect selection for both
PYw and especially for PY has proved challenging, and
much physiology has been restricted to the retrospec-
tive identification and understanding of traits which
have changed as direct selection has increased yield.
A good example are the many traits that have been
found to change as maize yields increased in North
America under direct selection for yield, lodging resis-
tance, dry down, and disease resistance alone [37, 42].
As understanding of yield determination improves,
including the recognition that seemingly useful traits
can carry trade-offs, and as measurements of many
714 Crop Yields Around the World: Closing the Gap and Raising the Potential
traits become cheaper, especially through the use of
remote sensing in the field, the possibilities for physi-
ological traits have improved. At CIMMYT, maize
breeding for drought (PYw) has definitely benefitted
from the inclusion of physiological traits such as anthe-
sis to silking interval in managed drought environ-
ments [43]. More recently, with wheat there has been
testing of canopy temperature as an early generation
selection criterion, a surrogate for stomatal conduc-
tance and possibly photosynthetic rate under irriga-
tion, or possibly for rooting depth under drought; it
shows promise under both conditions [44], as does
multispectral canopy reflectance under irrigation
[45]. In CSIRO, Australia, ideotrait selection for greater
PYw has been under way for several decades, with focus
on traits such as water use efficiency via carbon isotope
discrimination, coleoptiles length, low tillering, and
high stem carbohydrate content at flowering, with
moderate success [46]. Very recent research points to
genetic differences in wheat for grain set under drought
and ways for screening this trait which is likely to be
a significant bottleneck in PYw determination [47].
While physiology may be beginning to look more
useful in early generation yield selection, it is now being
challenged by the rapidly growing use of molecular
markers for traits, initially qualitative ones but lately
also quantitative traits, including yield itself. The huge
decline in cost of detecting molecular markers, and
their increasing density across crop genomes is driving
this. Heffner et al. [48] review the possibilities of such
marker-based genome selection in crops. Earthington
et al. [49] describe in some detail the extent to which
molecular markers are being incorporated into the
Monsanto crop breeding programs: rates of progress
in multiple trait indices (e.g., including yield, grain mois-
ture, standability, and test weight for maize) can be dou-
bled; indeed, for these authors marker-aided selection is
the “new” conventional breeding. These approaches pre-
sume accurate phenotyping of genotypes for yield and its
interactions with target environments, a challenge in all
breeding. However, it is interesting that physiology hardly
features in this new scenario, although other groups are
busily mapping putatively useful physiological traits at
the molecular level (e.g., [50, 51]). It remains to be seen
whether physiological traits, either observed directly
or identified by accurate markers, will have a place
in marker-aided yield breeding, but it is possible some
combination of markers and early generation trait
phenotyping using smart remote sensing will prove an
even more efficient strategy for PY advance. In addition
physiology, just like plant pathology, will continue to
identify individual traits whose incorporation into lead-
ing varieties may lift PY or PYw, a process which could
benefit from the use of molecular trait markers, just as is
becoming the case with new hard-to-screen host plant
disease resistance genes.
Multilocation multiple-year yield testing remains
the essential final step in all plant breeding for yield;
testing under different management regimes (plant
density, planting date, soil stored water) is also recog-
nized as increasingly important. Crop simulation
models are now good enough to help breeders under-
stand the environmental and sometimes the genetic
bases of the interactions encountered, and thus deploy
the testing more efficiently, and more efficiently
indentify the genotypes best suited to major environ-
ment  management combinations. An excellent
example is the analyses of multiple environment
rainfed trials of sorghum in Queeensland [52].
Genetic Engineering for Yield
There are many claims of engineered traits that may
increase crop yield, but very few of these have been
backed up by yield measurements in the field. Crop
physiologists have regularly pointed out how different
is the performance of a genotype growing in a pot in the
glass house from that of one at normal plant density in
the field environment [53, 54]. For this reason, they
remain skeptical of the value of these claims and await
with much interest the results of field tests, for, to date,
it appears that only a handful of papers describe per-
formance in proper field tests. In an excellent example,
wheat transformed with a more active ADP glucose
pyrophosphorylase in the grain to increase grain sink
strength and grain size, was thoroughly field tested, but
there was no yield benefit [55]. More recently,
Monsanto has reported increased PYw in field plots
with maize transformed with two separate events [56,
57]; results in the latter case point to modest PYw
increases and no change in PY, but the nature of the
drought and of the crop physiological responses are not
well described. More experimental data is needed
before much confidence can be placed in this event,
 Crop Yields Around the World: Closing the Gap and Raising the Potential
or in engineering for yield in general, for both genetic
and physiological understanding of yield determina-
tion indicates that many processes are involved, and
that the impact of any single transformation is likely to
be small even if key regulatory genes or bottleneck
processes are targeted. Indeed, it is now being recog-
nized that if functional genomics is to have a significant
impact on yield through engineering yield processes, it
must pay more attention to the relevant plant and crop
physiology connecting gene action to field performance
[58]; even the current fashion for automated
phenotyping in controlled facilities must be balanced
by substantial investment in phenotyping in field plots
in managed environments.
Some Tentative Conclusions on Future Potential
Yield Progress
As PY increases, relative rates of PY progress are
decreasing; this is probably more than the consequence
of a linear rate of progress relative to a rising denom-
inator, and may, in situations where the breeding
investment is large (e.g., maize in North America,
wheat in the UK and the rest of Western Europe), also
reflect diminishing returns as biological limits are
approached. Theoretical limits to harvest index are
already being reached, but those to radiation use effi-
ciency appear well above current measured values [59].
There have been a couple of attempts to project PY into
the future. For example, Sylvester-Bradley et al. [60]
propose that a longer period of light capture and
a higher radiation use efficiency could lift PY of winter
wheat in the UK to 17.4 t/ha by 2050, a linear rate of
progress of 167 kg/ha/year, or two and half times the
current rate of progress seen in Fig. 1. Taking a different
tack, Monsanto has predicted a doubling of US maize
yield between 2000 and 2030, based on equal contribu-
tions from conventional breeding, marker-aided selec-
tion, and genetic engineering, and amounting to
a linear annual rate of progress which is 3.3% of today’s
yields. Physiological theory and commercial optimism
both, however, have their limitations. Looking more
cautiously at the ways of impacting PY progress
outlined above, it is concluded that breeding progress
will dominate in the future, that conventional breed-
ing, including the exploitation of heterosis, will con-
tinue to deliver small but declining gains, while new
715
approaches may boost gains enough to maintain cur-
rent rates of linear PY progress at between 0.5% and
1%, other things such as climate remaining equal (note
that given the way PY is defined here, this progress is
independent of any direct effect of atmospheric CO2
increase). Observers expect the investment in the rele-
vant R and D to remain large, as the private sector
involvement increases to balance a declining public
sector, and many argue that it should grow further,
although the marginal rate of return is not well known.
Future Directions and Synthesis
The key issue for future food security is the real grain
price needed to balance burgeoning demand against
increasing supply. World food production demand
and supply to 2050 have recently been projected by
Hubert et al. [61] with the IFPRI Impact model, and
by Tweetin and Thompson [62]. Thus, the baseline case
of the former paper projects a 56% increase in cereal
production over 2000, accompanied by substantial real
price increases over the 2000 base, with negative effects
on malnutrition. Demand growth is greater for maize
and least for rice, such that the projected real price
increases over 2000 are maize (97%), wheat (90%),
and rice (60%). Clearly, a 56% increase in production
is not enough. For Tweetin and Thompson [62], cereal
supply rises somewhat more, by around 75% in 2050,
but real prices still increase (on average 44%). The
IFPRI projections allow for biofuel demand (peaking
in the late 2020s at 16% of total maize consumption,
after which second-generation biomass biofuel takes
over), rising energy costs and likely restrictions on
irrigation water supply. They also point to a much
greater increase in grain demand in the first 25 years
(1.4% pa) than in the second 25 years (0.4%), clearly
placing more pressure on short- to medium-term yield
progress. The IFPRI model contains crop area as
a variable but, given the cost of developing new lands
and its scarcity, this only increases significantly if prices
more than double under a very low yield progress
scenario.
In the IFPRI baseline projection, yield grows to
meet demand at around 1% exponential projected
over the whole period. In [62], it is expected to grow
linearly at a rate equivalent to 1.4% of current yields.
These rates compare to the current rates of world yield
716 Crop Yields Around the World: Closing the Gap and Raising the Potential
growth, which are wheat and rice (0.9%), maize (1.6%),
and soybeans (1.1%), but it must be recalled that the
actual rates all appear to be linear, not exponential, and
that the projected rates in neither of the models are
enough to hold real prices steady at 2000 levels. In
addition, the elasticity of demand is such that a supply
shortfall due to below baseline yield growth would be
disastrous (as we saw 2 years ago although some other
factors added to the sharp doubling of prices then): the
projections in [61] suggest a 40% fall in yield growth
below baseline (to 0.6% exponential) would lift 2050
real cereal prices to more than 200% above 2000 real
prices!! Alternatively, 1.4% yield growth would mean
continuing declines in real grain prices.
These numbers highlight the importance of yield
growth that has been the subject of this entry and reveal
that current FY growth rates are inadequate to hold real
prices down. Since PY growth can contribute to FY
growth everywhere, and is the only source of growth
in advanced agricultural systems where FY is
approaching PY, it assumes great underlying impor-
tance, even as current rates of growth, except possibly
for maize, seem to be well below 1% and gradually
falling as a percentage of yield. Unfortunately, the
brief examination here suggests that the prospects of
boosting PY growth, largely now relying on plant
breeding, are small, or at least rather uncertain, espe-
cially in the short to medium term when yield increases
are most critical. Much will depend on whether the
molecular marker-aided yield selection can boost
rates, for example, double them, as claimed by some;
genetic engineering seems unlikely to lift PY in the
short to medium term, although the prospects to lift
PYw may be somewhat better if water stress-related
yield bottlenecks can be relieved. And none of these
projections have factored in the possible negative
effects of climate change.
As a consequence of the above, yield gap closing
assumes great importance for future world food secu-
rity, especially as there still appear to be large exploit-
able gaps (>50%) in many situations, and as the way
forward is clearer. There are few uncertainties regarding
what needs to change at the farm level, but less opti-
mism regarding how to achieve this quickly enough
and from where the necessary resources will come. The
biggest gaps are associated with small farmers in the
developing world, and there is little doubt that
strategies to help these farmers will have the greatest
benefits for alleviating poverty and food insecurity.
And note that the association between large yield gaps
and small farmers is not an inevitable association, as
can be seen among small holders in Egypt or parts of
eastern China. This entry has emphasized that
improvements in rural infrastructure and institutions
are critical, but that breeding and agronomic research
also has a role to play in gap closing, as is illustrated by
the impact of simple direct seeding machinery and
earlier sowing on wheat yields in the Indo-Gangetic
Plains or of Bt cotton with Indian small holders. It is
a great pity, however, that the types of on-farm adaptive
research and development needed for gap closing in
such situations are often thwarted by policy inertia
toward agriculture in general, and poor rewards by
science funders in particular, although the situation
may be gradually improving. In developed countries,
new paradigms seem to be emerging in which private
sector companies, especially breeding and seed compa-
nies, invest not only in developing better varieties but
also in applied agronomic research linked to variety
development and promotion, and this may account
for some of the gap closing in the USA and Western
Europe. In addition, and especially in Australia and the
southern cone of South America, privately hired agron-
omists are assuming a critical role in yield gap closing
and are rewarded accordingly. Taken overall such
changes auger well for ongoing yield gap closing, even
in the short to medium term, in the developed world,
but their impact at the farm level in developing coun-
tries, and that of more traditional approaches, will
remain limited by infrastructure and institutional con-
straints in the near to medium-term future. The cau-
tious optimism expressed a decade ago by leading
agricultural scientist Evans [4] regarding world food
security may well be overly optimistic.
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Working paper AEDE-WP 0044-08, Ohio State University

Cropping Systems: Shaping Nature
RAFAEL J. LÓPEZ-BELLIDO, LUIS LÓPEZ-BELLIDO
Departamento de Ciencias y Recursos Agrı́colas y
Forestales, University of Córdoba, Córdoba, Spain
Article Outline
Glossary
Definition of the Subject
Introduction
Components and Design
Anthropological View
Agricultural Practices’ Footprint
Praise of Agricultural Science
Eradication of Fallow, Tillage, and Monoculture
Biological Synergism and Technical Synchronization
Dynamic Cropping Systems
Future Challenges
Bibliography
Glossary
Agriculture Agriculture and farming are often consid-
ered to be the same concept. However, both con-
cepts can vary in their territorial scope of
application. Agriculture is the production of food
and goods through farming and forestry. Agricul-
ture encompasses a wide variety of specialties and
techniques. For this reason, its definition has devel-
oped to become: The science, art, and business of
cultivating soil, producing crops, and raising live-
stock. The major agricultural products can be
broadly grouped into foods, fibers, and raw mate-
rials. As of late, agriculture also uses plants to pro-
duce biofuels, biopharmaceuticals, and bioplastics.
Agricultural practices Agricultural practices are a set
of techniques applied to on-farm production and
postproduction processes, resulting in food and
nonfood agricultural products.
Agronomic crops Agronomic crops typically involve
a crop that is grown for grain, feed, or for
processing into oil, starch, protein, or flour. Major
agronomic crops include corn (grown for feed,
ethanol, or processing), soybeans, wheat, hay
(alfalfa and legume and grass mixtures), rice, pea-
nuts, and cotton. Hay is also considered forage [1].
P. Christou et al. (eds.), Sustainable Food Production, DOI 10.1007/978-1-4614-5797-8,
# Springer Science+Business Media New York 2013
Originally published in
Robert A. Meyers (ed.) Encyclopedia of Sustainability Science and Technology,
Cropping Systems: Shaping Nature 719
Agroecosystem An agricultural system or agricultural
ecosystem is the basic unit of study for an
agroecologist. This term is somewhat arbitrarily
defined as a spatially and functionally coherent
unit of agricultural activity, and includes the living
and nonliving components involved in the unit as
well as their interactions. An agroecosystem can be
viewed as a subset of a conventional ecosystem. As
the name implies, at the core of an agroecosystem
lies the human activity of agriculture. However, an
agroecosystem is not restricted to the immediate
site of agricultural activity (e.g., the farm), but
rather includes the region that is impacted by this
activity, usually by changes to the complexity of
species assemblages and energy flows, as well as to
the net nutrient balance.
Agronomy The science which establishes the theory
and practice of crop production and soil manage-
ment [1].
Ecosystem A functioning community of nature that
includes fauna and flora together with the chemical
and physical environment with which they interact.
Ecosystems vary greatly in size and characteristics,
and can be a mud puddle, a field, or orchard, or
a forest. An ecosystem provides a unit of biological
study and can be a unit of management [2].
Environment The totality of the surrounding external
conditions (biological, chemical, and physical)
within which an organism, community, or object
exists. The environment can be defined at any scale.
The term is not exclusive in that organisms can be
and usually are part of another organism’s environ-
ment. Thus, one can speak of the environment as
that within which humankind lives (i.e., separate
and external) or, of humankind as a component of
the environment [2].
Definition of the Subject
Cropping system (CS) is a general term that describes
how a producer or farmer might grow a crop [2].
Pragmatically, CSs effectively address the what to
grow, when to grow it, and how to grow it considerations
of crop production in the context of optimizing mul-
tiple goals [3]. A CS must bring together the biological,
technical, economic, and sociological aspects of
# 2012, DOI 10.1007/978-1-4419-0851-3
720 Cropping Systems: Shaping Nature
the land area farmed [4]. Therefore, a CS is a set
of agronomic or agricultural practices used in
a crop under specific conditions. The CS generates an
agroecosystem, agri-environment, or agricultural sys-
tem. Common synonyms of CS include crop manage-
ment system, crop production system, farming system,
crop production practices, etc. For example, a farming
system is defined as a population of individual farm
systems that have broadly similar resource bases, enter-
prise patterns, household livelihoods, and constraints,
and for which similar development strategies and inter-
ventions would be appropriate [5].
The classification of CSs has been based on
the following criteria: available natural resource base,
including water, land, grazing areas, and forest; climate,
of which altitude is one important determinant;
landscape, including slope; farm size, tenure and orga-
nization; dominant pattern of farm activities and
household livelihoods, including field crops, livestock,
trees, aquaculture, hunting and gathering, processing
and off-farm activities; and taking into account the
main technologies used, which determine the intensity
of production and integration of crops, livestock,
and other activities [5]. Multiple cropping, in which
several crops are grown sequentially in 1 year, and
intercropping, when several crops are grown at the
same time, are other kinds of annual CSs known as
polycultures [6].
In general, each and every agronomic practice
used in a CS affects different aspects of the system,
i.e., the tillage system prepares the seedbed but affects
the availability of water and nutrients, carbon cycle,
erosion, diseases, etc. The use of the term CS is
often applied to a rotation (wheat-fallow CS), tillage
system (no-tillage CS), method of fertilization (organic
CS), or agroecosystem (semiarid CSs) to simplify
the designation of the CSs. However, it is important
to note that a CS involves much more than a rotation,
tillage system, fertilization method, or climatic
conditions.
Introduction
Since the dawn of settled agriculture about 10 millennia
ago, human beings have integrated different agronomic
practices for application to selected plants (crops), and
as a result generated CSs that transformed the natural
ecosystem into agroecosystems. Agricultural practices
such as genotype selection, crop rotation, natural fer-
tilizers, irrigation, etc., were developed long ago, but
have made great strides in the past century. The history
of agriculture has played a major role in human history
as agricultural progress has been a crucial factor in
worldwide socioeconomic change. When human
beings began to produce food beyond their needs,
others activities began to take place. It is this agricul-
tural surplus that made the development of civilization
possible. A crucial time in the history of agricultural
began with the discovery of America. A global exchange
of previously local crops and livestock breeds occurred.
Key crops involved in this exchange from the New
World to the Old included the tomato, maize, potato,
manioc, cocoa bean, and tobacco, with several varieties
of wheat, spices, coffee, and sugar cane going from the
Old World to the New. With the rapid rise of mecha-
nization in the early twentieth century, particularly in
the form of the tractor, farming tasks could be accom-
plished with a speed and on a scale previously impos-
sible. Also during this period, the Haber–Bosch
method for synthesizing ammonium nitrate
represented a major breakthrough. Synthetic nitrogen,
along with mined rock phosphate, pesticides, and
mechanization, allowed crop yields to overcome previ-
ous constraints. Furthermore, global yield increases
were experienced later in the twentieth century when
high yield varieties of wheat, corn, and rice were intro-
duced as a part of the Green Revolution. The Green
Revolution exported technologies (including pesticides
and synthetic nitrogen) from the developed world to
the developing world. However, concerns have been
raised over the sustainability of intensive agriculture.
Intensive agriculture has become associated with
decreased soil quality, and there has been increased
concern over the effects of fertilizers and pesticides on
the environment, particularly as the population
increases and food demand expands.
As a result, in the past few decades, a move toward
sustainability in agriculture has also developed, inte-
grating ideas of socioeconomic justice and conserva-
tion of resources and the environment within an
agricultural system [7, 8]. Sustainable agriculture is
defined as practices that meet current and future soci-
etal needs for food and fiber, for ecosystem services,
and for healthy lives, and that do so by maximizing the

net benefit to society when all costs and benefits of the
practices are considered. If society is to maximize the
net benefits of agriculture, there must be a fuller
accounting of both the costs and the benefits of alter-
native agricultural practices, and such an accounting
must become the basis of policy, ethics, and action.
Additionally, the development of sustainable agricul-
ture must accompany advances in the sustainability of
energy use, manufacturing, transportation, and other
economic sectors that also have significant environ-
mental impacts [9]. Sustainable agriculture has
renewed research in alternative technologies such as
no-tillage, integrated pest management, etc. Recent
mainstream technological developments include
genetically modified (GM) crops. Also, agriculture
has needed – and still needs – subsidies from many
governments to ensure an adequate food supply and its
competitivity in global markets. However, at times,
these subsidies, especially when instituted by developed
countries, have been noted as protectionist, inefficient,
and environmentally damaging [10].
This chapter is not an overview of a subject as
complex and broad as worldwide CSs, where oversights
are inevitable. It must first be pointed out that about
85% of the Earth’s cultivated land is planted with
annual crops [11], and thus the principle CSs are used
in herbaceous plants. Of these, wheat, rice, and maize
provide more than 60% of human dietary calories,
either as cereals for direct human consumption or
embodied in livestock products produced from ani-
mals fed with feed grains and their by-products
[9, 12]. Moreover, it is likely that these same cereal
crops will continue to account for the bulk of future
human food supply because they produce greater yields
of human-edible food, are easily grown, stored, and
transported, and require less fuel and labor for
processing and cooking than other food crops [12].
Given their importance, this chapter focuses primarily
on these crops, but also provides a general view that can
be applied to any CS. The chapter starts off with a brief
description of CS components and factors that must
be taken into consideration during their design
(Components and Design). To provide a new focus on
defining CSs, and as a result agroecosystems, an
anthropological view of a CS is then provided
(Anthropological View). Following, the most important
effects of these “human ecosystems” on the
Cropping Systems: Shaping Nature 721
environment are reviewed (Agricultural Practices’ Foot-
print). At this point, the chapter goes on to analyze the
achievements of agricultural research and their impor-
tance to man (Praise of Agricultural Science). From
here, agricultural practices that were unsuccessful in
CSs and which must be urgently eliminated are studied
(Eradication of Fallow, Tillage, and Monoculture). After-
ward, the basic principles on which current and future
CSs should be inspired are discussed. In fact, the appli-
cation of these principles to a certain degree involves
returning to techniques from the past, based on mon-
itoring technologies of the future (Biological Synergism
and Technical Synchronization). The application of
these principles logically leads to what are called
Dynamic Cropping Systems, which shift away from the
application of a series of rigid agricultural practices to
the agroecosystem at each growth stage. Finally, the
chapter tackles the Future Challenges for CSs in the
twenty-first century.
Components and Design
All agroecosystems generated by a CS break away from
“natural” ecosystems, and the main effects are the loss
of biodiversity and the export of resources. This causes
environmental imbalance which means more or less of
a struggle, depending on the intensity of the CS, to
stabilize it. The more distant it is from a “natural”
stable ecosystem, the more inputs are required by the
CS as the result of a greater imbalance. In fact, one of
the greatest concerns caused by agriculture, which has
been a problem from the beginning due to the selection
of few species, is the lack of biodiversity in many CSs.
Advocates of diversification argue that it provides
greater income stability [2].
Many CSs throughout the world are characterized
by variable climate and soils, resulting in a high-risk
condition for agricultural producers due to extreme
variability in precipitation and seasonal temperatures
[13]. In other words, crop production occurs in an
environment that is always changing. With every grow-
ing season, producers must attend to numerous factors
that influence their management decisions. Cropping
systems vary among farms depending on the available
resources and constraints; geography and climate of the
farm; government policy; economic, social, and polit-
ical pressures; and the philosophy and culture of the
722 Cropping Systems: Shaping Nature
farmer [5, 6]. This is a daunting challenge, especially
when one considers that producers’ decisions are car-
ried out in a financial environment of diminishing
economic returns, where one wrong decision could
mean financial hardship and potentially the end to
a way of life [14].
A CS involves numerous interactive factors that
may limit or facilitate crop production. The factors
can be divided into the following groups [14]: (1) bio-
physical environment: weather/climate and soil;
(2) socioeconomic externalities: market conditions
and government programs; and (3) available technol-
ogy. These three factors affect or guide the agricultural
practices of the CS (crop election, rotation, tillage sys-
tem, planting date, fertilization, crop protection, etc.),
and are conceived on the basis of productive and eco-
nomic objectives, as well as the social and environmen-
tal objectives that must be imposed by sociopolitical
externalities.
Most developed countries’ agriculture operates in
a market-driven economy, although government poli-
cies can have an influence on what farmers produce and
how they produce it. As with other businesses, agricul-
tural producers respond to economic incentives and
disincentives, and make decisions to maximize their
welfare; usually measured as net income. Specifically,
the farmer must consider the influence of technological
advancements, income supports embodied in farm leg-
islation, and changes in market structure and consumer
demand. Many of the technological advancements have
required large-scale production units to justify the
investment. The influence of commodity support pro-
grams has been ambiguous. As farm legislation has
evolved to decouple production decisions from pro-
gram benefits, the incentives to specialize in program
crops (crops that receive price and/or income benefits
under governmental legislation) have diminished.
However, wealth and risk effects, albeit small, may
have promoted or inhibited the adoption of a more
sustainable system. Changes in market structure, chan-
nels, and consumer demand in the past five decades
have been dramatic with consolidation and specializa-
tion in both production and marketing sectors. How-
ever, the diversity of consumer demand has also created
opportunities for more integrated farm operations.
There is an increasing number of consumers who
have become concerned about how and where their
food has been produced (food security). While price
and income supports may have been biased toward
specialization (as these programs were targeted to spe-
cific commodities), the reduction in risk associated
with the programs has enabled producers to expand
the number and diversity of their production enter-
prises [15].
The rapid change in the agricultural industry driven
by continuously arising challenges (climate change,
market globalization, environmental concerns)
requires the development of new methods of produc-
tion in order to guarantee sustainable agriculture [16].
The design of a CS undergoes a study of the biophysical
environment and socioeconomic externalities, and an
inventory of available technologies. The first step in
developing a CS is the definition of goals and con-
straints for the new CS. Constraints may result from
soil and climate but also from environmental or eco-
nomic concerns [17]. Afterward, a crop portfolio must
be established, usually based on climate, and
containing a diverse array of adaptable crop species,
economic potential, crop production practices, and soil
and water management considerations. The crop port-
folio is used to screen adaptable crops for a region and
includes the best management practices for the pro-
duction of each adaptable crop [14]. In others words, it
is the selection of crops compatible with the set of
constraints. Next is the comparison and choice of the
most satisfying crop and management options [16].
Designing new CSs is a long process and occurs in
a rapidly changing environment. This is exemplified
by climate but also by the economy (prices and poli-
cies) and the changing demands and functions that
society assigns to agricultural systems. The design pro-
cess must therefore integrate objectives of the resilience
and flexibility of CSs in an unpredictable environment
[18]. Obviously, the use of models for the design of CSs
can be an important tool for the farmer, although there
are some drawbacks [16]. Geographic information sys-
tems, qualitative decision-matrix analyses, a simple
rule-based model using multi-criteria evaluations,
and a machine learning-based land-transformation
model can be used harmoniously to study complex
socio-ecological systems. Models evaluate how each
technique performs in the study of complex socio-
ecological systems using a multi-tier framework, detail-
ing how each method analyzes the resource system,

resource units, governance system, users and interac-
tions, and outcomes in the system. Model use enhances
our understanding of the land-use decision-making
process [19].
Anthropological View
Beginning around 8,500 BC, the transition from the
hunter–gatherer lifestyle to food production enabled
people to settle down next to their permanent agricul-
tural lands, instead of migrating to follow seasonal
shifts in wild food supplies. Food production was
accompanied by a human population explosion that
has continued unabated to this day, resulting from two
separate factors. First, the sedentary lifestyle permitted
shorter birth intervals. Nomadic hunter–gatherers had
previously spaced out birth intervals at 4 years or more
because a mother shifting camp can carry only one
infant or slow toddler. Second, plant and animal spe-
cies that are edible to humans can be cultivated in
much higher density in our agricultural land than in
wild habitats [20].
Agriculture also led to an explosion of technology
because sedentary living permitted the accumulation
Proportion of Landscape (%)
100
Natural Clearing
Ecosystems Forests
75
50
25
0
Pre-settlement Hunter–gatherer
agriculture
Cropping Systems: Shaping Nature. Figure 1
Stages in “domestication” of nature by human being in relation with the area occupied (Adapted from [23])
Cropping Systems: Shaping Nature 723
of heavy technology such as forges that nomadic
hunter–gatherers could not carry and because the stor-
able food surpluses resulting from agriculture could be
used to feed full-time craftspeople and inventors. By
also feeding full-time kings, bureaucrats, nobles, and
soldiers, those food surpluses led to social stratifica-
tion, political centralization, and standing armies. All
of these overwhelming advantages are what enabled
farmers to eventually displace hunter–gatherers [21].
Like all species, humans have exercised their
impulse to perpetuate and propagate themselves. In
doing so, humankind has “domesticated” ecosystems
in ways that enhance its food supplies, reduce exposure
to predators and natural dangers, and promote com-
merce. On average, the net benefits to humankind of
“domesticated” nature have been positive. We have, of
course, made mistakes, causing unforeseen changes in
ecosystem attributes, while leaving few, if any, truly
wild places on Earth [22]. Domestication of plants
and animals may be the single most important feature
of the human domination of our planet (Fig. 1).
Domestication involves the selection of traits that fun-
damentally alter wild species to become more useful to
us. Conservation has often been framed as the science
Protected/
recreational lands
Subsistence Urban areas
agriculture
Agriculture
Primitive Modern era Current
agriculture agriculture
Stages in land use
724 Cropping Systems: Shaping Nature
aimed at protecting nature, and especially protecting
nature from people. However, there really is no such
thing as nature untainted by people [24]. Facing this
reality should change the scientific focus of environ-
mental science. Instead of recounting doom-and-
gloom statistics, it would be more fruitful to consider
the domestication of nature as the selection of certain
desirable ecosystem attributes, such as increased food
production, with consequent alteration to other eco-
system attributes that may not be desirable. Under this
paradigm, our challenge is to understand and thought-
fully manage the trade-offs among ecosystem services
that result from the inescapable domestication of
nature [22].
It is clear that cities are the main consumers of most
ecosystem services. This is important because the desire
and value for these services determines the traits that
humans select for preservation or elimination. For
example, if humans want to maximize food produc-
tion, landscapes will be domesticated to accommodate
a few high productivity species, plus the human-
associated species able to survive in these modified
landscapes. If people want more wildlife for recrea-
tional hunting, populations of predators of game spe-
cies will be reduced, and the edge habitat that a few
game species prefer will be increased. The choices and
actions of urban dwellers influence nature far removed
from cities, yet urban dwellers are increasingly unaware
of these impacts [22].
Agricultural Practices’ Footprint
The huge magnitude of human impacts in the environ-
ment is recent, but the presence of impacts such as
purposeful wildfires goes back thousands of years.
The reality of the human footprint renders discussions
about what areas of the world to set aside as wild and
protected areas as somewhat irrelevant; more germane
is a discussion of what trade-offs we are willing to
accept as a result of the domestication of nature [22].
The main environmental impacts attributed to
agriculture come from the conversion of “natural” eco-
systems to agroecosystems by CSs [9]. Clearing land for
agriculture, humans target wild species for harvest or
elimination. Humans have so tamed nature that few
locations in the world remain without human influence
[22] (Fig. 1). The whole notion of a “virgin rainforest”
may be erroneous, with extensive prehistoric human
activity evident in what were once thought to be
untouched forests in the Amazon and Congo [25].
Global maps of human impact indicate that, as of
1995, only 17% of the world’s land area had escaped
direct influence by humans [22]. Between 1700 and
1980, the total area of cultivated land worldwide
increased 466%, and yields increased dramatically, par-
ticularly because of selectively bred, high-yielding vari-
eties, fertilizers, pesticides, irrigation, and machinery.
For example, irrigation increased corn yields in eastern
Colorado by 400–500% from 1940 to 1997 [9].
Roughly 50% of the world’s surface area has been
converted to grazed land or cultivated crops [26].
Cropping systems (areas where at least 30% of the
landscape is in croplands, confined livestock produc-
tion, or freshwater aquaculture) now cover a quarter of
the Earth’s surface, partly by conversion of temperate
grasslands, Mediterranean climate forests, and many
tropical ecosystem types. More than half of the world’s
forests have been lost in this land conversion [26].
Forests have essentially disappeared from 25 countries,
with 9.4 million hectares lost annually from the Earth’s
surface [27]. Species and populations of species are
being lost at unprecedented rates, while at the same
time, the global biota is becoming homogenized, owing
to the introductions of alien species to new regions.
These examples represent major losses of pieces of the
biosphere machinery, which have a serious impact on
the delivery of ecosystem-regulating services – impacts
such as greater prevalence of infectious diseases in
disrupted ecosystems, adverse effects on local climates
by ecosystem modification, and the loss of flood
protection [27].
Environmental damages have come from agricul-
tural nutrients that pollute aquatic and terrestrial hab-
itats and groundwater, and from pesticides, especially
bioaccumulating or persistent organic agricultural pol-
lutants. Agricultural nutrients enter other ecosystems
through leaching, volatilization, and the waste streams
of livestock and humans. Pesticides can also harm
human health, as can pathogens, including antibiotic-
resistant pathogens associated with certain animal
production practices [9]. Many CSs have degraded
soil quality and necessitated the expense of increased
fertilization, irrigation, and energy to maintain pro-
ductivity [28]. Today, only 30–50% of applied nitrogen

fertilizer [29] and 45% of phosphorus fertilizer [30]
is taken up by crops. A significant amount of the
applied nitrogen and a smaller portion of the applied
phosphorus are lost from agricultural fields. Nitrogen
fertilization can increase the emission of gases that
have critical roles in tropospheric and stratospheric
chemistry and air pollution. Nitrogen oxides (NOx)
emitted from agricultural soils and through combus-
tion increase tropospheric ozone, a component of
smog that impacts human health, agricultural crops,
and natural ecosystems. NOx from agroecosystems can
be transported atmospherically over long distances and
deposited in terrestrial and aquatic ecosystems. Finally,
nitrogen inputs to agricultural systems contribute
to emissions of the greenhouse-gas nitrous oxide.
Rice paddy agriculture and livestock production
are the most important anthropogenic sources of the
greenhouse-gas methane [31]. Much of agriculture
in the developed world had for a time embraced
potentially non-sustainable systems for economic
reasons, over-utilizing monocropping, specialization
and mechanization, which was damaging to soils and
the environment [32]. Modern agroecosystems are also
depleted in biodiversity and habitat heterogeneity,
often with a reduction in resilience as a result of their
biological monotony [22].
Cities are a good place to start when considering the
broader implications of agroecosystems. The cumula-
tive resource demands of cities are often expressed
as the total land area required to supply those
resources, called the “ecological footprint” [33]. Every
city imports resources and exports waste into a region
that is spatially much larger than the city’s area. How-
ever, there is substantial variation in per capita ecolog-
ical footprints between rich and poor regions, with the
average resident of the United States using 6 times the
area of the average sub-Saharan African [34].
Praise of Agricultural Science
To a conservationist interested mainly in biodiversity,
we have degraded nature, but to an agronomist, we
have shaped the nature to make it better serve humans
[22]. It is paradoxical that current urban dwellers look
down upon and lack an appreciation of agriculture
when it is the key to our existence and to obtaining
the energy needed for their daily activities. There is no
Cropping Systems: Shaping Nature 725
question that humans have been successful in their
efforts to produce food, thereby enhancing their well-
being [22]. Contrary to Malthus’s predictions, food
production has kept up with, and even outpaced,
human population growth [35]. Malthus did not take
into account that “there is no gene for the human
spirit” [36]. The massive increase in food supply has
been achieved by focusing efforts on planting and con-
suming a small variety of plants [22]. As of 1999, barley,
maize, rice, and wheat occupied almost 40% of global
cropland [37].
Nowadays, few scientists think of agriculture as the
chief, or model science. Many, indeed, do not consider
it a science at all. Yet it was the first science – the mother
of all sciences; it remains the science that makes human
life possible; and it may well be that, before the century
is over, the success or failure of Science as a whole will
be judged by the success or failure of agriculture [38].
For too many years, the agricultural sciences have been
disparaged in the science and education communities,
perhaps because agronomy, soil science, plant pathol-
ogy, and animal science use a problem-solving
approach rather than simply seeking knowledge [39].
But as we move into a new era of shared accountability
and responsibility, let’s keep in mind that agricultural
sciences affect us all, and when agricultural science is
thriving, our communities likely are thriving too [39].
Agriculture is not seen as a source of solutions to many
of the world’s most pressing challenges. When science
research funds are handed out, agriculture often gets
left off the list. It is possible to suspect this because
policy-makers and some scientists see “agriculture” as
synonymous with “agribusiness” rather than as
a purely scientific discipline, and they assume private
funding will take care of agriculture-related research
needs [39].
The benefits of agriculture have been immense.
Before the dawn of agriculture, the hunter–gatherer
lifestyle supported about four million people globally
[40]. Modern agriculture now feeds 6,000 million peo-
ple. Global cereal production has doubled in the past
40 years, mainly from the increased yields resulting
from greater inputs of fertilizer, water and pesticides,
new crop strains, and other technologies of the
“Green Revolution” [41] (Fig. 2). This has increased
the global per capita food supply, reducing hunger,
improving nutrition (and thus the ability of people to
726 Cropping Systems: Shaping Nature

Wheat grain yield (Mg ha−1)

10
Broadbalk experiment
established in 1843 at Fungicides
Rothamsted, Harpenden, UK

8 [3 year wheat + 1 year fallow + 1 year potatoes]

Rotation + Nitrogen

Semidwarf
6 Varieties

Herbicides

4 Periodic
Fallowing
Monocrop + Nitrogen

2
Monocrop-no Nitrogen

0
1850 1880 1910 1940 1970 2000
Year

Cropping Systems: Shaping Nature. Figure 2

Changes with time in winter wheat yields from 1850 to 2000 with explanations for the trends due to new
agricultural practice technologies introduced. Treatments show how important is nitrogen fertilization and crop
rotation (Adapted from [42])

better reach their mental and physical potential), and
sparing natural ecosystems from conversion to agricul-
ture [43].
Agriculture is the science that has had and con-
tinues to have the greatest impact on humanity. The
advancements are the result of countless researchers
unknown to the general public and who deserve proper
tribute and acknowledgement. We should be eternally
grateful to people, many of whom are consigned to
oblivion, such as Justus von Liebig, (1803–1873), who
is known as the “father of the fertilizer industry,” for his
discovery of nitrogen as an essential plant nutrient
and his formulation of the Law of the Minimum
which described the effect of individual nutrients on
crops; Sir John Bennet Lawes, (1814–1900) who
experimented with crops and manures at his farm at
Harpenden, nowadays known as Rothamsted, and
established the first long-term field experiment in the
world, the Broadbalk (1843); Daniel Albone, who
invented, in 1902, the world’s first successful light
farm tractor; Fritz Haber and Carl Bosch, whose work
led to the synthesis of the first N fertilizer in 1913,
seeing that without the use of synthetic fertilizers,
world food production could not have increased at
the rate it did and more natural ecosystems would
have been converted to agriculture [9]; Sir Ronald
Aylmer Fisher, who, working in the long-term experi-
ments at Rothamsted in 1925, established the basis of
the modern statistics that so greatly benefited agricul-
tural research and all other sciences; Erling Johnson,
who in 1927 developed an industrial method for pro-
ducing nitrophosphate; Paul Hermann Müller, who
discovered that DDT was a very effective insecticide
in 1939, in spite of the fact that it was later discovered
to be hazardous for many living beings, the good it
accomplished for humanity is immeasurable as it con-
trolled mosquitoes spreading malaria and lice trans-
mitting typhus; Judah Hirsch Quastel, who working
at Rothamsted, developed the first herbicide (2,4-D)
in 1946; Norman Ernest Borlaug, father of the “green

revolution,” led the introduction of high-yielding vari-
eties, doubling wheat grain yield in many countries
during the 1960s (Fig. 2); John E. Franz, who discovered
Glyphosate in 1970, a broad-spectrum systemic herbi-
cide; Marc Van Montagu and Jeff Schell discovered the
gene transfer mechanism, developing the first transgenic
plant in the 1980s; in addition to a long list of anony-
mous researchers who contributed their part to provid-
ing humanity with the food needed through techniques
which have become increasingly more respectful of the
environment and safer for consumption.
Eradication of Fallow, Tillage, and Monoculture
There are three agricultural practices that must tend to
disappear from present and future CSs because it has
been proven, in general, that they are more harmful
than beneficial. Two of these, tillage and fallow, have
been used since the beginning of agriculture, while the
third, monoculture, emerged during the last century as
a consequence of the incorporation of other techniques
that made it apparently viable in the context of a great
demand for food. These practices must be considered
errors of the past that the present has allowed us to
discover.
Soil is not naturally intended to be in a state of bare
fallow. CSs must be inspired by or consider nature in
their design. Soil is meant to be covered by vegetation
as can be observed in most terrestrial ecosystems; there-
fore, when man included bare fallow in its group of
agricultural practices, he committed a big mistake.
Even in the conditions employed to allow low-fertility
soils to rest, the most ideal option would be a change in
crop, or if necessary, another option would be to not
remove the crop. Although it was already known at the
end of the last century that bare fallow was not useful,
there have been policies, as is the case of the EU, which
encouraged this practice as a result of market forces.
Efforts to stabilize production of cereal crops led to
the adoption of wheat–fallow CSs. This system, while
popular with producers because it required limited
equipment and managerial skills, has proven to be
agronomically inefficient and environmentally
unsustainable as shown through poor precipitation-
use efficiency and decreased soil quality [44, 45]. In
fact, at least 60% of the precipitation received during
fallow is lost to evaporation [46]. Recognition of the
Cropping Systems: Shaping Nature 727
drawbacks of wheat–fallow as well as advances in weed
and residue management technology led to a reduction
in the frequency of fallow [13]. Fallow time must be
limited to those periods in which no water is available.
Leaving a soil without residue shifts away from natural
processes since in naturally dry ecosystems, the dry
pasture leaves its residue protecting the soil against
aggressive climatic events. All of the suggestions for
intensifying cropping under dry-land conditions are
contingent on maximum water capture and minimum
losses to weeds, runoff, and evaporation. Ideally, crops
should be synchronized so that there is always one crop
in the field available to intercept possible rainfall [44].
Increased emphasis on crop diversity within annual
CSs has allowed producers to take advantage of positive
agronomic benefits derived from crop rotations [14].
Realizing these benefits requires knowledge of soil-
water depletion and recharge characteristics for indi-
vidual crops. Such knowledge is especially critical for
areas where soil-water status can vary greatly between
growing seasons [47].
Tillage, as is, is also not observed in nature. It was
man, with the purpose of domesticating nature, who
introduced it. No-tillage was used since ancient times
by indigenous cultures. This was because tillage to any
depth required more energy and power than was gen-
erally possible with hand labor. The ancient Egyptians
and the Incas in the Andes of South America used
a stick to make a hole in the ground and put seeds by
hand into unprepared soil [48]. For thousands of years,
agriculture and tillage were considered synonymous.
It was simply not thought possible to grow crops without
first tilling the soil before planting and for weed control
[49]. Intensive tillage and use of heavy machinery have
accelerated soil erosion, soil-C loss and nutrient deple-
tion, soil compaction, acidification, pollution, and sali-
nization [50]. The advent of modern herbicides
permitted no-tillage to be developed and practiced on
actual working family farms. No-tillage is generally
defined as planting crops in unprepared soil with at
least 30% mulch cover [49]. Adoption of no-tillage
after its successful demonstration in the 1950s was
slow. Today, approximately 23% of the total cropland
in the United States is planted using no-tillage [49].
No-tillage has revolutionized agricultural systems
because it allows individual producers to manage
greater amounts of land with reduced energy, labor,
728 Cropping Systems: Shaping Nature
and machinery inputs. Maintenance of surface residue
cover is essential to optimize CS performance as resi-
due coverage minimizes erosion, enhances retention
of limited precipitation, and improves soil quality
[51]. Moreover, no-tillage substantially improves soil
carbon, an area that is currently of great importance
[45]. Crop residues, including the presence of crop
stubble, can alter the soil environment in a number of
ways by acting as a physical barrier, exudate of chemical
suppressants (allelochemicals) from the residue, or
enhancer of biological activity, and by providing
a habitat for weed seed predators. These factors, as
well as the buffering effect of crop residues on soil
moisture and soil temperature and impacts on light
availability, have a significant impact on weed-seed
germination and emergence [52]. Lastly, the adoption
of no-tillage has an economic benefit for farmers,
reducing crop costs [53].
Monoculture, the lack of biodiversity, was
a contributing factor to several agricultural disasters
in history. Monoculture causes a loss of biodiversity
in the “ecosystem” soil as a result of the monotony,
leading to a yield loss with respect to a crop in rotation
[54]. Practices that change species’ composition or
reduce biodiversity in agricultural systems may also
diminish goods and services because the ability of eco-
systems to provide some services depends both on the
number and type of species in an ecosystem [9]. The
supply of agricultural products and ecosystem services
are both essential to human existence and quality of
life. However, recent agricultural practices that have
greatly increased global food supply have had inadver-
tent, detrimental impacts on the environment and on
ecosystem services, highlighting the need for more
sustainable agricultural methods [9]. Wheat, rice, and
maize crops have become the three most abundant
plants on Earth. A central conclusion of epidemiology
is that both the number of diseases and disease inci-
dence should increase proportionally to host abun-
dance, and this disconcerting possibility illustrates the
potential instability of a global strategy of food pro-
duction in which just three crops account for so high
a proportion of production [9]. The relative scarcity of
outbreaks of diseases on these crops is a testament to
plant breeding and cultivation practices. For all three
cereals, breeders have been successful at improving
resistances to abiotic stresses, pathogens, and diseases,
and at deploying these defenses in space and time so as
to maintain yield stability despite low crop diversity in
continuous cereal systems [9]. However, it is unclear if
such conventional breeding approaches can work
indefinitely. Both integrated pest management and bio-
technology that identifies durable resistance through
multiple gene sources should play increasingly impor-
tant roles [55]. Nonetheless, the evolutionary interac-
tions among crops and their pathogens mean that any
improvement in crop resistance to a pathogen is likely
to be transitory [9]. Although the “Green Revolution”
significantly increased rice yields in Asia, yield increases
have not occurred in the past 15–20 years. The genetic
“yield potential” has increased for wheat, but the yield
potential for rice has not increased since 1966, and the
yield potential for maize has “barely increased in 35
years.” It takes a decade or two for herbicide-resistant
weeds to emerge, and insects become resistant to insec-
ticides within about a decade. Within about one or two
decades of the introduction of each of seven major
herbicides, herbicide-resistant weeds were observed.
Crop rotation helps to prevent resistances [9].
Biological Synergism and Technical
Synchronization
Agriculture should fulfill the economic, environmental,
and social objectives of sustainable development. It is
thus necessary to tailor current CSs to meet these needs
as they are often too intensive and dependent on exter-
nal inputs [56]. It is clear that the more we imitate
nature or encourage natural processes, the greater the
benefits for the CSs and fewer inputs that will be
required. Two basic concepts should illustrate the
design and management of CSs of the future: biological
synergism and technical synchronization.
Biological Synergism
Biological synergic CSs should synchronize
edaphoclimatic resources with the natural rhythms of
crops, this being a question of common sense that has
not always been applied, and clearly speaks of the
diversity which has been lost with intensive systems.
Therefore, the increase in biodiversity in CSs is a clear
consequence of the search for biological synergism in
addition to the promotion of natural processes. Biodi-
versity is essential to ecosystem processes in ways that

are not yet fully understood [57], and it is considered
worth protecting in its own right. Crop diversification
by itself, however, is of limited use without knowledge
of how individual crops affect each other in a sequence.
Consequently, a thorough understanding of crop
sequencing effects – both positive and negative – on
agronomic parameters is necessary to optimize CS
performance [13, 46]. One problem associated with
synergic CSs is how to choose and sequence crops to
develop the inherent internal resources of the system
while taking advantage of external resources such as
weather, markets, government programs, and new
technology [14]. Present CSs rely on extensive use of
fertilizer and pesticides and the low cost of fossil fuel
energy [46]. Future challenges for CSs will exploit
synergism through crop sequencing to improve crop
yields without additional inputs and to reduce deteri-
oration of the environment [58].
Diversification of farming systems cannot be
managed on a farm or field scale alone but also requires
management on a regional scale. To improve biodiver-
sity, the interaction with other stakeholders in
the region is necessary as part of regional planning
processes. This has consequences for agronomic
research which thus far has had only limited attention
for the regional scale. Spatial planning aimed at
multifunctional agriculture can be seen as a negotiation
process on environmental, social, and economic aspects
of land use. Complexity arises due to the high number
of stakeholders and due to limited knowledge, which is
often organized along disciplinary divides [59].
Crop rotation is defined as the growing of different
crops (spatial and temporal), in recurring succession,
on the same land in contrast to monoculture cropping.
Rotation usually is done to replenish soil fertility, create
a diverse soil organism, and reduce pest populations in
order to increase the potential for high levels of pro-
duction in future years [2, 46]. Each crop or a closely
related crop species should not be grown more than
every 4 years because of increased pest problems
[60, 61]. One of the most effective and inexpensive
methods to control plant diseases in CSs is through
crop rotation. Through the use of this practice, the
need to breed for new disease resistance and to discover
new pesticides can be reduced. Recently, an important
and costly pathogen of rice was controlled in a large
region of China by planting alternating rows of two rice
Cropping Systems: Shaping Nature 729
varieties [62]. This tactic increased profitability and
reduced the use of a potent pesticide. Specific crop-
rotation effects on plant diseases, however, are poorly
understood and contradictory results have been
reported [13].
Changes in the weed flora of agroecosystems can
occur as long-term changes or temporary fluctuations
in species composition [52]. Agricultural weeds are
a unique group of plant species because of their ability
to infest and thrive in intensively disturbed habitats
despite extensive efforts to eliminate them. Weed floras
differ between fields, farms, regions, climatic zones,
and CSs. Weeds are successful because they are gener-
ally plastic plants that adapt to and survive changes in
the environment. In continuous CSs, changes in the
environment may be as a result of differences in crop
species, tillage, fertilizers, herbicides, and other weed-
control tactics. Long-term population shifts are usually
observed after repeated use of a control measure, which
exerts a high selection pressure on a population, for
example, the increased incidence of some annual grass
weed species due to the intensification of cereal pro-
duction and selective herbicides [52]. Studies have
shown that the more diverse the CS, the more diverse
the weed community, with less dominant and trouble-
some species as would occur in a simple rotation.
With diversified continuous cropping, there is
greater opportunity to utilize crops that vary in
N requirements [63]. Rotations with legumes are of
great interest for strengthening the synergism of a CS.
Legume crops are often planted to enhance nutrient
cycling and availability to other crops in the rotation.
Crops with high N demands are usually grown after
legume crops. Grain legumes conserve soil N by fixing
atmospheric N, thereby leaving residual N in place for
the next crop. Wheat yields are often higher following
legumes than following other cereals [54] and the N use
efficiency of a wheat crop is greater following legumes
than others crops [64]. The inclusion of a legume crop
decreases its reliance on external fertilizer, supplying
a significant proportion of the N for the next crop and
may moderate nitrate levels in the soil to avoid the
potential for nitrate leaching [65] (Fig. 3).
Alternatives such as plurispecific CSs are of consid-
erable interest for biological synergism because plant
associations can provide environmental benefits in
terms of better use of resources and provision of
730 Cropping Systems: Shaping Nature
Relative other crops yield
3.0
Benefit only for others crops
2.5
Chickpea
2.0 Sunflower
Maize
1.5
Proso millet
Buckwheat
1.0
Wheat
Detrimental for both crops
0.5
0.8 0.9
Cropping Systems: Shaping Nature. Figure 3
Effect of 14 previous crops on relative wheat yield and the effect of wheat, as a previous crop, on those crops in the Great
Plains (Adapted from [46, 66])
ecological services [67]. Yet they should also be able to
maintain a level of productivity and production stabil-
ity that fits with farmers’ objectives. Intercrops (grass
cover in the inter-rows) are now being introduced to an
increasing extent due to the potential positive impacts
on perennial crop and their environment: increased
infiltration rate and decreased runoff due to modified
soil surface characteristics [68], mitigation of soil ero-
sion [69], limitation of herbicide use and weed control,
better water-resource utilization by roots [70], and
limitation of risks of diseases by reducing vegetative
development [71]. However, intercropping also
induces competition for soil resources, and vegetative
development and yield can consequently be limited
[56]. These different functions should be promoted
through simultaneous adapted management of the
two crops of the system.
Finally, an important link of biological synergism
is the return provided by livestock to the CSs. There
was always a crop–livestock pairing in ancient agricul-
ture, but specialization caused this very important
Benefit for both crops
FLAX
Safflower
Lentil
Dry pea
Barley
Canola
Crambe
Soybean Dry bean
Benefit only for wheat
1.0 1.1 1.2
Relative wheat yield
aspect of biodiversity and synergism to disappear.
Research is needed to better understand interactions
between crops and livestock with the intention of
identifying management practices that maximize
agroecosystem productivity and operational efficiency.
This would save having to remove residues, part of
which would be used directly by the livestock, partially
allowing the recycling of nutrients. Inclusion of live-
stock in a CS complements both crops and livestock by
adding value to grain, improving nutrient-use effi-
ciency, and providing alternative uses for forages and
crop residues [72]. It may create synergisms between
the two enterprises, resulting in far greater productivity
than either enterprise could attain alone [13, 73]. The
inclusion of livestock in CSs would also help alleviate
the problems generated by livestock: high-density ani-
mal production operations can increase livestock dis-
ease incidence, the emergence of new, often antibiotic-
resistant diseases, and air, groundwater, and surface
water pollution associated with animal wastes. Current
livestock operations are vulnerable to catastrophic loss

of animals to disease. The handling and disposal of
animal wastes are significant problems of high-density
animal confinement facilities. Manure lagoons can
release high levels of hydrogen sulfide and other toxic
gases, volatilize ammonium that greatly increases
regional nitrogen deposition, and contaminate surface
and ground waters with nutrients, toxins, and patho-
gens. These animal wastes pose health and environ-
mental risks similar to those of human wastes and
should be treated accordingly. For example, animal
wastes could be treated by composting to create
a crop fertilizer that no longer harbors pathogens,
and that is applied at appropriate rates and times
and with methods that minimize nutrient leaching.
This closing of the nutrient cycle decreases dependence
on synthetic fertilizer production, and is more
efficient when animal and crop production are com-
bined locally [9].
Technical Synchronization
Technical synchronization must be defined as the con-
cept on which precision agriculture is based, i.e., the
temporal and spatial synchronization of agricultural
techniques. All agricultural practices must be synchro-
nized in time with the rhythms of the agroecosystem
(time-specific management). This synchronization
must take into account the growth stage of the crop,
different soil characteristics (nutrients and water),
weather forecasts, etc. On the other hand, there is the
spatial synchronization that divides the land into
homogeneous units for management (site-specific
management) [63]. A basic principal of technical syn-
chronization is the energetic efficiency when making
decisions in relation to the agricultural practices to be
carried out.
The key to the application of technical synchroni-
zation in a CS is the acquisition of data in order to
make management decisions in time and space. There
are an increasing number of data acquisition devices
available to assist with decision making: global posi-
tioning (GPS), satellites or aerial images, information
management tools (GIS), weather stations, soil sensors
(water content, temperature, etc.), optical sensors (e.g.,
chlorophyll meters), and remote sensors (visible and
near infrared spectral), etc. These tools allow the farmer
to have a better understanding of the interaction of
Cropping Systems: Shaping Nature 731
climate and management that causes tremendous
year-to-year variation in on-farm yields and crop
requirements.
Nonetheless, technical synchronization poses a
problem when applied by the farmer. There is a large
body of published research on technologies for increas-
ing the technical synchronization of agricultural prac-
tices, but relatively few have been adopted by farmers
because they are not cost-effective or practical. Adop-
tion of improved technologies typically requires addi-
tional skills and labor or investments in new
equipment. Information on expected costs and eco-
nomic returns from such investments is required to
convince farmers of the benefits from adoption [12].
A possible solution is cooperativism for the acquisition
and sharing of these new technologies.
Dynamic Cropping Systems
It could be said that, until now, CSs have been static,
i.e., year after year the same group of agricultural prac-
tices have been repeated, causing soil monotony in the
agroecosystem. A dynamic CS must be flexible and go
against repeated management practices, and the deci-
sions must be made based on measured parameters and
their determining factors. A dynamic CS is only the
consequence of the application of the two previously
stated principles: biological synergism and technical
synchronization. Cropping systems need to be inher-
ently flexible to take advantage of economic opportu-
nities and/or adapt to environmental realities [13, 18].
The implementation of a management strategy
requires knowing, understanding, planning, measur-
ing, monitoring, and record keeping at each step of
the production process. Dynamic CSs require the
development and implementation of agricultural pro-
duction systems that are highly productive, energy
efficient, and environmentally non-degrading [12].
Dynamic CSs are a form of agricultural production
that relies on an annual or pluri-annual strategy to
optimize the outcome of (1) production, (2) economic,
and (3) resource-conservation goals using ecologically
based management principles [74]. Implicit to this
strategy is the need for producers to possess informa-
tion necessary to respond to continual change. The key
factors associated with dynamic CSs are diversity,
adaptability, reduced input cost, multiple enterprise
732 Cropping Systems: Shaping Nature
systems, and awareness of environment and informa-
tion [14]. Greater crop diversity and sequencing flexi-
bility within dynamic CSs may result in reduced weed,
pest and disease infestations, greater nutrient- and
precipitation-use efficiency, decreased requirements of
exogenous inputs, and lower production risk [74].
To remain competitive in agricultural markets,
increased diversity of systems will be required to min-
imize inputs and improve economic margins.
To increase responsiveness to externalities, oppor-
tunity/flex CS [75] or dynamic CS [14] concepts have
been developed. These systems allow producers to
adjust CS intensity and/or diversity based on external-
ities as well as management goals, such as soil-water
status at planting, soil residual nitrogen, market
demands, etc. This approach to crop sequencing pos-
sesses an inherent flexibility to adapt to high-risk con-
ditions, and therefore may be more economically and
environmentally sustainable than other approaches to
crop sequencing [13]. However, the authors who
established the principles of dynamic CSs have focused
solely on rotation selection, which is erroneous, since
a CS is more than its rotation selection, although the
crop selection is without a doubt the most important
decision made in a CS. Perhaps the concepts should be
called dynamic or flexible crop election system. How-
ever, a dynamic CS makes more sense if each year all of
the crop techniques used are reconsidered, i.e., crop
selection, tillage, residue management, nitrogenous fer-
tilization, etc. in such a way that there are dynamics in
the selection of each and every one of the agricultural
practices that depend on externalities.
Future Challenges
Agricultural science is ripe for a renaissance [39] as
a consequence of the future challenges it must con-
front. The widespread adoption of CSs that are sustain-
able and environmentally benign is essential for the
long-term survival of civilization [32]. Furthermore,
through the use of strategic alliances, cooperation
among producers on a regional basis may eventually
lead to greater integration and diversification than
could be achieved for the individual farm operation
[15]. The primary challenges facing CSs in this century
are: covering the demands of an increasing world pop-
ulation; water shortage; the inclusion and management
of GM crops; harmonization of the production of
biofuels and food production; and climate change,
and the focus it brings to agricultural research. Obvi-
ously, there are many other challenges which have not
been presented here, but which are intimately related to
those mentioned such as: the biodiversity of CSs – as
already previously mentioned – which should redound
to a reduction of inputs and, therefore, a reduction
of the negative effects on the environment, strategies
to manage anthropogenic carbon emissions from
terrestrial systems as well as fossil fuel and industrial
sources, etc.
Nevertheless, together with these future challenges
are a series of forces that may contribute or hinder their
fulfillment: research policies, transference, and financ-
ing. In view of these challenges, governmental policies
play an important role that may help or hurt the
achieving of these objectives. For this reason,
policymakers have a great responsibility, and their deci-
sions should be guided by agricultural science profes-
sionals. Political intervention has an economic facet
that arises as the result of the following question:
How can society accomplish the dual objectives of
improving food production and stability and of pre-
serving the quality and quantity of ecosystem services
provided by the Earth’s land and water resources?
Clearly, appropriate incentives are needed [9]. What
incentives and policies could lead to the adoption of
sustainable agricultural practices? Several policy initia-
tives have tried to level the playing field between agri-
cultural production and the production of ecosystem
services.
Another problem in the attempt to fulfill these
objectives is the transference and application of new
technologies in CSs. The earlier paradigm of science
being developed at the international or perhaps
national level and then disseminated to farmers should
be replaced by an active exchange of information
among scientists and farmers. Scientists in developing
countries who understand the ecosystems, human cul-
ture, and demands on local agricultural systems must
be actively trained, promoted, and brought into the
international scientific community [9].
The economic investment in agricultural research is
the third problem. Organized public and private invest-
ment in agricultural R&D was a primary driver of the
comparatively rapid growth in agricultural

productivity experienced in the latter half of the twen-
tieth century [76]. Substantially greater public and
private investments in technology and human
resources are needed internationally, especially in low-
income nations, to make agricultural systems more
sustainable. Global research expenditures are less than
2% of agricultural gross domestic product worldwide
[77], being roughly 5.5% of agricultural GDP in devel-
oped countries, but less than 1% in developing coun-
tries (where most of the increased food demand will
occur during the next 50 years). At present, there are
few incentives for the private sector to increase invest-
ments in lower-income developing countries [78].
Moreover, funds have been redirected away from farm
productivity toward other concerns, such as the envi-
ronmental effects of agriculture; food safety and other
aspects of food quality; and the medical, energy, and
industrial uses of agricultural commodities. Without
adequate investments, yield gains and environmental
protection may be insufficient for a transition to sus-
tainable agriculture [9].
Feeding the World: Priority Aim
In a recent update of earlier estimates, the Food and
Agriculture Organization [79] of the United Nations
reported that more than one billion people now suffer
malnutrition [80]. By 2050, global population is
projected to be 50% larger than at present and global
grain demand is projected to double [28, 82], especially
the economic growth of the fast-growing economies of
Asia [81]. This doubling will result from a projected
2.4-fold increase in per capita real income and from
dietary shifts toward a higher proportion of meat
(much of it grain fed) associated with higher income.
Further increases in agricultural output are essential for
global political and social stability and equity. Dou-
bling food production again and sustaining food pro-
duction at this level are major challenges [82]. Doing so
in ways that do not compromise environmental integ-
rity and public health is a greater challenge still [9].
Agricultural productivity growth will be a pivotal
determinant of long-term growth in the supply, avail-
ability, and price of food over the coming decades [81].
However, a slowdown in growth of agricultural
productivity and grain yields has been documented.
If this slowdown in productivity persists, it could
Cropping Systems: Shaping Nature 733
have profound implications for food-price trends in
the future [81].
A key trade-off in cultivated systems is between
increasing the amount of cropland needed to meet
growing food needs versus increasing the productivity
of each hectare of cropland. The “land-sparing” impact
of modern farming practices has mainly been achieved
by yield increases from the use of crop monocultures
with improved crop varieties, fertilizer inputs, and
irrigation. For example, if yields of the six major crop
groups that are cultivated on 80% of the total cultivated
land area had remained at 1961 yield levels, it would
require an additional 1.4 billion hectares of land in
2004 – more than double the amount currently used.
This represents 34% of the total land area suitable for
crop cultivation and would have required conversion of
large areas of uncultivated land that support rain for-
ests, grassland savannahs, and wetlands. In Asia alone,
it would require an additional 600 million hectares,
which represent 25% more land area than is suitable
for cultivation on this continent. Asia would now be
heavily dependent on food imports if crop yields had
remained at 1961 yield levels. Although this increase in
productivity has saved some land from conversion,
it has resulted in greater impact on other services
through water withdrawals, excessive nutrient loads
and pesticide use. The key ecological question is, there-
fore, whether environmental services – other than food
production at regional and global scales – would be
enhanced by focusing food production on less land
under intensive management with high yields, versus
expanding cultivated area in lower-yielding systems
using farming practices that preserve environmental
services at the field and local levels. Few studies have
addressed this issue using sound, ecological, analytical
methods [27].
Water: Colorless Gold
Water is the principal factor limiting crop yield and is
considered a luxury in many agroecosystems. In many
developing countries, water is a major factor
constraining agricultural output, and income of the
world’s rural poor [83]. Forty percent of crop produc-
tion comes from the 16% of agricultural land that is
irrigated [84]. Irrigated lands account for a substantial
portion of increased yields obtained during the Green
734 Cropping Systems: Shaping Nature
Revolution [9]. Unless water-use efficiency is increased,
greater agricultural production will require increased
irrigation. However, the global rate of increase in irri-
gated area is declining, per capita irrigated area has
declined by 5% since 1978, and new dam construction
may allow only a 10% increase in water for irrigation
over the next 30 years [84]. Irrigation return-flows
typically carry more salt, nutrients, minerals, and pes-
ticides into surface and ground waters than in source
water, impacting downstream agricultural, natural sys-
tems, and drinking water. Technologies such as drip
and pivot irrigation can improve water-use efficiency
and decrease salinization while maintaining or increas-
ing yields. They have been used in industrialized
nations on high-value horticultural crops, but their
expanded use currently is not economically viable for
staple food crops. In developing countries, 15 million
hectares have experienced reduced yields owing to salt
accumulation and waterlogging [85]. The water-
holding capacity of soil can be increased by adding
manure or reducing tillage and by other approaches
that maintain or increase soil organic matter. Cultiva-
tion of crops with high water-use efficiency and the
development – through the use of biotechnology or
conventional breeding – of crops with greater drought
tolerance can also contribute to yield increases
in water-limited production environments. Investment
in such water-efficient technologies, however, is
best facilitated when water is valued and priced
appropriately [9].
However, most of the world’s agricultural area cor-
responds to dry-land areas and therefore the challenge
is to increase the efficiency of water use in these areas.
Efficient use of limited water supplies in dry-land CSs is
critical to system success. In the future, increases to
semiarid dry-land system water-use efficiency and pre-
cipitation-use efficiency may come from continued
improvement in managing residues (especially no-
tillage), cropping sequences that minimize fallow
periods, herbicides, and crop choice. However, contin-
uous cropping under dry-land conditions remains
risky due to the limited precipitation (erratic in distri-
bution and frequency) and high-potential evaporation.
In the future, the following are potentially fruitful areas
of research that may improve system water-use effi-
ciency [86]: (1) Increasing the amount and persistence
of crop residues. (2) Implementation of flexible rota-
tions (i.e., opportunity cropping). The occurrence of
precipitation and, hence, the availability of adequate
stored soil water for a crop is highly variable, especially
in semiarid regions. Sometimes stored soil water at
normal planting times for a crop in a given CS is
limited; at other times, adequate water for a crop is
available when the planting of a crop had not been
planned. By practicing opportunity cropping, some
crop generally could be planted when water becomes
available. The goal should be to grow a crop whenever
conditions are or become favorable and not according
to some predetermined schedule [87]. (3) Matching of
crop cultivar selection to prevailing weather condi-
tions. Genetic yield potential is linked positively with
maturity, so cultivar evaluation trials conducted under
conditions of adequate soil water often favor longer-
maturity cultivars and influence farmer choice.
(4) Improvement of the timeliness of cultural opera-
tions, including early seeding of crops and optimum
timing of weed control, and time operations to coin-
cide with favorable conditions as predicted by short-
term (48–72 h) weather forecasts.
Genetic Modified Crops: Essential
Genetically modified crops are a very important part of
the “second Green Revolution,” which may help to
break the tendency toward the stabilization of work
agricultural production. Genetically modified crops
may offer the best hope for producing crops that can
withstand drought, impoverished soils, and disease.
Future GM products are likely to carry traits that will
improve nutrition and health, help guard against
drought, heat and cold, and allow plants to access and
more efficiently utilize plant nutrients. All of these
technologies have more benefits to offer society, and
especially, poor farmers and consumers even more than
rich ones [88].
Therefore, GM crops are an essential tool in feeding
the growing world population and reducing the nega-
tive effects on the environment from the use of pesti-
cides. Nevertheless, this technology must be used
properly by growers. Growers who adopt herbicide or
pest tolerant crops in all cycles of the crop rotation will
be at greater risk than those who choose to rotate crops

and/or use other management strategies [89].
However, farmers must be proactive in protecting the
longevity of the technology by reducing the likelihood
of resistance occurring and providing alternative selec-
tion pressures on weeds and pests [52]. We must not
forget that life always finds a way through barriers [90].
Biofuel Crops: A Controversial Birth
Recent analyses of the energy and greenhouse-gas per-
formance of alternative biofuels have ignited
a controversy that may be best resolved by applying
two simple principles. In a world seeking solutions to
its energy, environmental, and food challenges, society
cannot afford to miss out on the global greenhouse-gas
emission reductions and the local environmental and
societal benefits when biofuels are done right. However,
society also cannot accept the undesirable impacts of
biofuels done wrong [91]. Biofuels done right can be
produced in substantial quantities with little or no
competition with food production.
The biofuel industry could have many positive
social and environmental attributes, but it could also
suffer from many of the sustainability issues if not
implemented the right way. Putting biofuel crops on
marginal lands, rather than on our most productive
croplands, could mean preventing competition with
food production and concomitant effects on commod-
ity prices, as well as minimizing or even avoiding the
carbon debt associated with land clearing. However,
marginal lands can also be rich in biodiversity,
may require sizable inputs of nutrients and water to
make production economically viable, and may carry
the opportunity cost of forgone future carbon
sequestration [92].
Globally, the production of an important amount
of energy with biofuels will require a large amount of
land – perhaps as much as is in row crop agriculture
today. This will change the landscape of Earth in
a significant way. The identification of unintended
consequences early in the development of alternative
fuel strategies will help to avoid costly mistakes and
regrets about the effects on the environment. Policies
that support long-term sustainability of both our land-
scapes and our atmosphere are essential if we are to
chart a low-carbon economy that is substantially better
Cropping Systems: Shaping Nature 735
than business as usual. Sustainable biofuel production
systems could play a highly positive role in mitigating
climate change, enhancing environmental quality, and
strengthening the global economy. But it will take
sound, science-based policy and additional research
effort to make this so [93].
Until now, the most convincing option for the
production of biofuel has been the use of perennial
plants as they require fewer inputs and can be grown
on degraded lands abandoned from agricultural use
[91]. Use of such lands minimizes competition with
food crops. This also minimizes the potential for direct
and indirect land-clearing associated with biofuel
expansion, as well as the resultant creation of long-
term carbon debt and biodiversity loss. Some initial
analyses on the global potential of degraded lands sug-
gest that they could meet meaningful amounts of cur-
rent global demand for liquid transportation fuels
[92, 94, 95]. The option of using crop residues from
food crops, such as corn stover and straw from rice and
wheat is a more controversial matter. Recent research
suggests that it is to the benefit of farmers to leave
substantial quantities of crop residues on the land
[96], but that, nonetheless, even conservative removal
rates can provide a sustainable biomass resource about
as large as that from dedicated perennial crops grown
on degraded lands.
Good public policy will ensure that biofuel produc-
tion optimizes a bundle of benefits, including real
energy gains, greenhouse-gas reductions, preservation
of biodiversity, and maintenance of food security.
Performance-based policies that provide incentives
proportional to the benefits delivered are needed.
This is a complex question that cannot be addressed
with simplistic solutions and sound bites. It needs
a new collaboration between environmentalists, econ-
omists, technologists, the agricultural community,
engaged citizens, and governments around the
world [91].
Climate Change: Technical Plasticity
The scenario produced, if indeed climate change
becomes a greater reality, is unclear. There is
a “climate of suspicion” as published in the journal
Nature at the beginning of January 2010. Climate
736 Cropping Systems: Shaping Nature

researchers must emphasize that – although many Agricultural Research: Return to the Field
holes remain to be filled – there is little uncertainty
In recent years, an important movement in agricultural
about the overall conclusions: greenhouse-gas emis-
research has emerged on an economic as well as human
sions are rising sharply, they are very likely to be the
resource level, toward molecular aspects of plant
cause of recent global warming and precipitation
growth and development. Progress in understanding
changes, and the world is on a trajectory that will
plant molecular biology has been impressive, and use-
shoot far past 2 C of warming unless emissions are
ful applications are evident [101]. However, agronomy
cut substantially [97]. It is evident that CSs must have
has been marginalized, with its field laboratories which
the ability to adapt to a more unpredictable amount
require much more time to produce results. This
of rainfall and high temperatures. According to the
migration could have something to do with the scien-
IPCC definition, the extent to which CSs are vulnerable
tific productivity stimulated by the present scientific
to climate change depends on the actual exposure
system since many agricultural research areas that do
to climate change, their sensitivity, and adaptive
not conduct field work require less than a year from the
capacity [98]. The adaptive capacity refers to the ability
start of experimental design until the results are
to cope with climate change, including
published. On the contrary, field research requires at
climate variability and extremes, in order to (1) mod-
least 2 years before the results can be accepted by the
erate potential damages, (2) take advantage of emerg-
scientific community. Most records used to assess eco-
ing opportunities, and/or (3) cope with its
system changes are based on short-term data or satellite
consequences [99].
imagery spanning only a few decades. In many
Nonetheless, in spite of the uncertainty regarding
instances, it is impossible to disentangle natural vari-
climate change, primarily as a consequence of the
ability from other, potentially significant trends in
increase in atmospheric CO2 levels, there are some
these records partly because of their short time scale
positive effects. When you step into a commercial
[102]. It is evident that the improvement of CSs
greenhouse, the chances are you are stepping into the
requires research in CSs, and specially long-term exper-
future. To plants, CO2 is food, and greenhouse opera-
iments. Unless a return is made to the roots of research
tors, knowing this, use it to fatten them up. While
under field conditions, many of the challenges faced
today’s atmosphere contains about 380 parts per mil-
will not be overcome. The return to field research will
lion of CO2, commercial greenhouses often contain
contribute to the global public good by restoring and
CO2 concentrations of twice that or more – the sort
sustaining productivity growth over the long run,
of concentration that we might expect in the open air at
which in turn will mitigate hunger and poverty and,
the end of the century [100]. Many crop scientists
at the same time, reduce pressure on the natural
believe that this carbon dioxide fertilization effect will
resource base [81].
go at least some way toward offsetting the losses in yield
Nevertheless, in recent years, some agronomists
to be expected as a result of the higher temperatures,
have begun to once again stress the importance of this
flooding, drought, and rising sea levels that the CO2
marginalized dimension of agricultural research, which
greenhouse effect will bring. But some are not so sure.
is time, in relation to the consequences that CSs
These researchers point to the known negative effects
have on productivity and the environment. Within
that increased CO2 concentrations have on the protein
this framework, long-term experiments acquire a
content of crops. When Bruce Kimball started out in
noteworthy relevance. The aim is to compare the bio-
the 1970s, available technology for high-CO2 research
logical and economic productivity of different CSs. The
was limited. Based on current knowledge, he says, the
high annual fluctuations in rainfall, and resulting crop
net effect of increasing CO2 is a good one: “As far as
yields, may make several crop cycles necessary in order
crops go, I think higher CO2 is a definite benefit. Yes,
to detect significant differences. Likewise, the differ-
a little less nutrition than before, but we get more food”
ences between soils, water–soil relationship, and vari-
[100]. But while Kimball thinks that, in general, the
ability of pathogens and plagues could provide
gains in yield are the most important thing, he is not
information that explains the differences in yield
blind to the drop in protein levels.

among CSs and suggest strategies in which crop
sequences and management practices can be selected
to increase efficiency in the use of water and nutrients,
and control the populations of weed, plagues, and
diseases. In this respect, long-term studies act as “lab-
oratories” in which specific problems or mechanisms
can be studied in continuous field conditions, where
the crop and “input/output” history is well known, and
also where tendencies of the quality of resources over
time and crop yields can be examined [103]. The most
convincing evidence regarding the sustainability of an
agroecosystem comes from long-term experiments
with positive results.
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