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Plant biotechnology for crop improvement

Article  in  Biotechnology Advances · February 1995

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 Biotechnology Advances, Vol. 13, No. 4, pp. 673-693, 1995
Copyright © 1995 Elsevier Science Inc.
Pergamon Printed in Great Britain. All fights reserved
0734-9750/95 $29.00 + .00
0734-9750(95)02010-1

PLANT BIOTECHNOLOGY FOR CROP IMPROVEMENT

K.P. PAULS

Department of Crop Science, Universityof Guelph,

Guelph, Ontario, Canada, N1G 2JV1

ABSTRACT

The typical crop improvement cycle takes 10-15 years to complete and includes germplasm
manipulations, genotype selection and stabilization, variety testing, variety increase, proprietary
protection and crop production stages. Plant tissue culture and genetic engineering procedures
that form the basis of plant biotechnology can contribute to most of these crop improvement
stages. This review provides an overview of the opportunities presented by the integration of
plant biotechnology into plant improvement efforts and raises some of the societal issues that
need to be considered in their application.

Key Words: Plant bioteehnology, crop improvement, genetic engineering, plant tissue culture.

INTRODUCTION

Humans began to modify the characteristics of plants used for food and fibre
approximately ten to twenty thousand years ago. Even primitive seeding, cultivating, harvesting
and storing practices would have exerted selection pressures on those plant species which
became domesticated that were different from the pressures their progenitors encountered in the
wild. Over time, but particularly in the last 150 years, plant breeding has developed into a
complex discipline that now incorporates information from many branches of science and
mathematics. The most recent development is the utilization of biotechnology for plant
improvement (Ratner, 1989).
Plant biotechnology can be defined as the application of tissue culture and molecular
genetics to develop or produce a commodity from plants. Tissue culture refers to the
673
674 K.P. PAULS

maintenance and propagation of plant parts (as small as a single cell) in biologically pure
(axenic) and controlled environments (Fig. 1; Evans et al., 1983; Vasil, 1984). Molecular

~..~ .~ ~ plantexplant
matureplant hUl~ujedewiplthant

a,,us

suspension
culture
"germination"~ Psr~ll~P~~ ~ "

embryos ~ embryogenicel
c ls
collectedonmesh ~ ~) largecellclumps
hormone t ~ ~ L~v( [
treatmentand~ somatic / ~'~ ~ / /~] / siremoved evi
n g
by
desiccation embryos ¢

embryogenic
callus

Figure 1. Plant tissue culture. Plant cells can be induced to proliferate in a variety of forms
including nondifferentiated callus and suspension cultures in vitro. Single ceils like protoplasts
express totipotency, i.e. the ability of a single cell to regenerate into a whole plant.
Regeneration may occur via various processes including somatic embryogenesis. In this process
structures that resemble zygotic embryos are formed in the tissue culture.
 PLANTBIOTECHNOLOGYFOR CROP IMPROVEMENT 675

genetics includes techniques for isolating, characterizing, recombining and multiplying and
transferring discrete fragments of DNA that contain genes coding for specific traits (Fig. 2;

foreign gene and

, right hand

~actefium
~aciens

callus forming on
co-cultivated leaf disks from
cells that have taken up the
gene that confers resistance
to the selective agent

shoots developing
transgemc ~lant containing from resistant
the selectable marker callus
gene and the foreign gene
of interest

Figure 2. Plant molecular genetics. A foreign gene, coupled to a selectable marker gene, is
cloned into a disarmed Ti plasmid of Agrobacterium tumefaciens. The foreign gend and the
selectable marker gene axe transferred to the plant by cocultivating the bacteria with the explant
(leaf disk) and regenerating plants from the cells that express resistance to the selective agent
in tissue culture. The foreign gene becomes stably integrated into the DNA of the plant and
is inherited by future generations of plants derived from the original transgenic plant.
676 K, P. P A U L S

Maniatis et al., 1982; Gelvin et al., 1988; Watson et al., 1987). The fact that a whole plant
can be regenerated from a single cell makes tissue culture a valuable procedure for proliferating
genetically identical material and selecting interesting variants. Totipotency also allows a
genetic change, made at the cellular level, to become an established trait of a whole plant. The
newly introduced or selected trait can, subsequently, be passed on to future generations of the
species by conventional crossing methods.
To be effective, plant biotechnology must be well integrated into established plant
breeding and crop production practices. For many field crop species the average amount of
time that is required to develop, test and release a new variety is 10-15 years. The procedure
has many stages including: germplasm manipulation, parent selection, genotype selection,
genetic stabilization, testing, variety increase, proprietary protection, crop production and crop
quality control. Fig. 3 illustrates that biotechnology can contribute to most stages of crop
development and production.

GERMPLASM MANIPULATIONS

Germplasm Preservation

The variety of forms that occur in a plant species reflect its genetic diversity. Since
this germplasm represents the raw material with which plant breeders work there is considerable
interest in preserving representatives of old varieties, land races and related wild species
(Plucknet et al., 1987). These lines may be a valuable source of agronomic genes in the future.
Biotechnology makes diverse germplasm sources more valuable than ever before because
genetic material from different species, genera and even kingdoms can now be stably
incorporated and expressed in plants.
Techniques such as minimal growth in tissue culture and cryopreservation (in liquid
N2) have been used to store plant materials from a wide variety of species (Kartha, 1985;
Withers, 1987). These techniques are particularly important for plant species that are
vegetatively propagated since they reduce the labour associated with maintaining a line as well
as the risk of losing the material to disease. International exchange of germplasm is also
facilitated because of the disease-free nature of the plant materials in culture.
Molecular techniques can be used to make libraries of the genetic material of plants
in bacteria or viruses (Sambrook et al., 1989). Germplasm can be stored in these forms for an
indefinite period in low temperature freezers (-70°C) and the libraries take up little space.
 PLANT BIOTECHNOLOGY FOR CROP IMPROVEMENT 677

Minimal growth
Invitm
Genetic distance test
Cryo~on
' ~ GERMPLASM
Gene libraries
Relatedwildspecies T
Protoplastfusion
somadon~ , Unimproved
germplasm PARENT I F
1 F
2
variation i Land races SELECTION
RECOMBINATION /F
iN
In vitro selection p 9r GeneticallYmodified
t: 3
o°°
°°o"
Agrobacfe#um genotypes
transformation
Direct D ~
uptake
Registered varieties

,,ng,= ng SEED F "".

Pollinationcontrol INCREASE larker
Agrobacterium assisted
V transformation selection
Pestmonitoring • CROP DirectDNAUptake
To,in =says r PRODUCTION

Figure 3. Inputs from biotechnology into the plant improvement cycle. In a typical plant
breeding program parents for new varieties are selected from a germplasm pool that contains
currently registered varieties, land races, unimproved germplasm collected from the wild and
related plant species. Today genetically-modified plants created by biotechnology are also used
for plant breeding. Parents for crosses on which new varieties are based are selected on the
basis of their proven agronomic characteristics (like high yield) or because they have useful
traits like disease resistance. The best characteristics of the parents are combined into one
population by making crosses among the parents. The progeny are evaluated on the basis of
a variety of agronomic criteria and the best few percent of the plants are used for further rounds
of crossing and testing. The genetic makeup of the plants in the breeding population is
stabilized (fixed) by the crossing program and can be accelerated by crossing the plants to
themselves or by crossing closely related plants. The filial (F 1 to Fs) generations that result are
increasingly uniform. This process ensures that the variety that is produced breeds true and
gives uniform crop stands in the field. Today haploidy can be used to rapidly produce
homozygous lines of breeding material. High performing plant lines are introduced into
registration trials to test their performance against current standard varieties. After
demonstrating superior performance in these trials a new line may be licensed for sale as a
variety in Canada. Several years of seed increase are required to obtain sufficient seed to
establish a new variety. Bioteclmological procedures can enhance this crop improvement cycle
in the various ways described in the text.
678 K.P. P A U L S

Thus, gene libraries and individual genes (Newman et al., 1994) are rapidly becoming important
components of germplasm exchange among plant breeders.

Germ~lasm D/versification

A common first step in plant breeding is to create a genetically diverse population by

crossing unrelated parents or by inducing mutations in the breeding germplasm. Several
biotechnological procedures have greatly expanded the possibilities for increasing the genetic
diversity of crop species including: molecular assisted selection of potential parents, tissue
culture-induced variability, protoplast fusion and the production of transgenic plants.
The selection of parents is one of the most critical decisions a plant breeder makes
because the amount of genetic diversity that is incorporated into the first cross determines the
range of characteristics that can be expressed in the varieties that develop from it as well as the
time it takes to achieve the goals of the breeding program. DNA analyses results can be used
to maximize genetic distances among parents in a conventional cross, thus assuring that a broad
base of genetic diversity exists in the population from which selections are made. Genetic
distances, based on DNA analyses, have also been shown to be good predictors of hybrid vigour
(heterosis) in single cross hybrid combinations of corn inbreds (Smith et al., 1990).
Although the purpose of tissue culture can be to preserve the genetic fidelity of the
stocks, long-term tissue culture can also be used to increase useful genetic variation. Genetic
variability in tissue culture-derived material, called somaclonal variation (Larkin & Scowcroft,
1981) is especially prevalent if the material is kept in a rapidly dividing, non-differentiated
state, (callus or cell suspension) for an extended period. In fact, the frequency of somaclonal
variation can be 10,000 times higher than spontaneous mutation rates in whole plants (Larkin
& Scowcroft, 1981). Variants such as lettuce with improved seedling vigour, a scented
geranium, rice with improved protein quality, flax tolerant to salt, and disease-resistant tobacco
have been isolated from mutant cell lines that arose spontaneously in tissue culture and some
of these variants have been used to develop plant varieties (Evans, 1989).
Protoplast fusion is a method for making large changes in the genetic composition of
plants. Protoplasts are released from plant tissues after incubation in cell wall degrading
enzymes (Cocking, 1960). Several procedures, including incubation in PEG (Kao &
Michayluk, 1974) or treatment with electrical pulses (Zimmerman, 1982), can induce protoplasts
from different plants to fuse. Somatic hybrid plants can be regenerated from cultures of these
fusion products. Protoplast fusion has been used to:
 PLANTBIOTECHNOLOGYFOR CROPIMPROVEMENT 679

I. make very wide combinations between distantly related species that are not possible
by conventional crossing procedures [like tomato and potato (Melchers et al., 1978)
or mustard (B. juncea) and rapeseed (B. napus) (Sjodin & Glimelius, 1989)],
2. bring about limited gene transfer, [for example, disease resistance from a wild relative
of potato (Solanum brevidens) to potato (S. tuberosura) (Gibson et al., 1988)], and
3. create new nuclear cytoplasmic combinations [for example, to obtain new cytoplasmic
male sterility systems for hybrid variety production oferops like canola and rice
(Pelletier et al., 1983; Pelletier and Chupeau, 1984; Kyozaka and Shimamoto, 1989;
Schell & Vasil, 1989; Pauls, 1991)].
Recombinant DNA procedures can be used to make discrete changes in the genetic
makeup of plants. The most successful systems to date harness the natural ability of a
tumorigenic plant pathogen called Agrobacterium tumefaciens to transfer genes into plants
(Schell & Vasil, 1989). Agrobacteria are usually altered by recombinant DNA technology to
remove tumour-inducing genes and introduce a foreign gene(s) before they are used for plant
transformation. The modified Agrobacteria no longer induce the disease state but retain the
ability to transfer foreign genes into plants. Applications of the Agrobacterium-mediated gene
transfer techniques have been limited by a host range that is restricted primarily to dicots but
successes with direct DNA uptake methods, like protoplast electroporation (Potrykus, 1990) and
biolistic introduction (Klein et al., 1987), indicate that plant transformation will be achievable
with virtually all crop species. The most significant development in the past few years in plant
transformation has been the demonstration that transgenic monocots like rice (Datta et al.,
1990), corn (Gordon-Kamm et al., 1990) and wheat (Vasil et al., 1992) can be created.
Transgenic plants of over 45 species that contain genes from other plant species,
bacteria, viruses and animals, are currently available (Oxtoby & Hughes, 1990; Fraley, 1992).
The foreign genes expressed in transgenic plants confer on them a variety of important
agronomic traits including:
1) herbicide resistance (Oxtoby, 1990; Quinn, 1990; Schulz et al., 1990),
2) insect resistance (Htfte & Whiteley, 1989; Ryan, 1990; Koziel et al., 1993),
3) viral resistance (Baulcombe, 1989; Beachy et al., 1990; Hull & Davies, 1992),
4) microbial resistance (Lamb et al., 1992; Staskawicz, 1995),
5) altered macromolecular composition (Hiatt et al., 1989; Altenbach et al., 1990;
Krebbers & Vandekerckhove, 1990; Knutzon et al., 1992; Voelker et al., 1992; Visser
& Jacobsen, 1993; Ttpfer et al., 1995),
6) modified reproductive capacity (Mariani et al., 1990; Mariani et al., 1992; Lee et al.,
680 K.P. PAULS

1994) and
7) delayed senescence (Sheehy et al., 1988; Smith et al., 1988 Oeller et al., 1991; Gray
& Grierson, 1993).
Completely new roles for crop plant as factories for recombinant DNA products such as
antibodies have been suggested (Conrad and Fielder, 1994; Ma et al., 1995). Proponents of this
approach cite high quality products, easy scale-up and cost effective development and
production economics as some of the attributes of using plants to produce speciality protein
products.
The transgenic plants created by Agrobacterium transformation or direct DNA uptake
procedures have also been very useful for isolating genes when used in conjunction with
transposon tagging (Springer et al., 1995) or T-DNA tagging procedures (Gierl & Saedler,
1992). Furthermore, transgenic plants have been used for testing the functions of genes and
their promoters (Schell, 1987). These studies have resulted in a more profound understanding
of plant biology and have led to the evolution of strategies for tissue developmental- and
environmental-specific expression of inserted genes (Katagiri, 1992; Quail, 1995).

GENOTYPE SELECTION AND GENETIC STABILIZATION

In most plant breeding programs line selection and genetic stabilization occurs
simultaneously and consists of a procedure whereby the breeder selects a few of the best lines
to advance to the next round of selfing or backcrossing. The process serves to fashion the raw
germplasm into varieties that are superior to those that already exist and to stabilize their
genetic makeup by making them homozygous. Biotechnological techniques that can contribute
to this stage of variety development are: in vitro selection, haploidy, and marker-assisted
selection.

Selection

An efficient method for obtaining plants with desired characteristics is to add a

selective agent that will kill the majority of the cells (except the resistant ones) to a tissue
culture. This procedure is called in vitro selection (Chaleff, 1983). Since the in vitro unit of
selection can be a single cell the selection pressure can be uniformly and reproducibly applied.
Also, in vitro selection is potentially more efficient than whole plant selection. For example,
a single flask of cells can have the same number of selection units as 30,000 plants in a three
 PLANTBIOTECHNOLOGYFOR CROP IMPROVEMENT 681

hectare nursery. This method has been particularlyeffective for selecting herbicide- and
disease-resistantplants (Shaner & Anderson, 1985; Swanson et aI., 1989; Dix, 1990; Frame et
al., 1991; lleret aI., 1993).
The difficulties that are associated with selecting plants that express recessive alleles,
genes that have only minor effects on plant phenotypes or genes whose expression is strongly
modified by the growth environment are major limiting factors in plant improvement. The
utilization of molecular marker-assisted selection may allow plant breeders to overcome some
of these limitations. Differences (polymorphisms) in isoenzyme, DNA restriction fragment and
polymerase chain fxagment patterns are useful as molecular markers and have been shown to
be genetically linked to both simple and complex traits such as disease resistance and fruit
composition in crop plants (Bernatzlcy & Tanksley, 1986; Lander et al, 1987; Landry et al.,
1987; Tanskley et al., 1989; Landry et al., 1992).
In a breeding program which uses the molecular markers, progeny from each mating
cycle are selected on the basis of the presence of a particular electrophoretic band that has been
shown to be tightly linked to the desired characteristic (Ralfalski & Tingey, 1993). This
approach circumvents masking effects by dominant alleles, eliminates variability due to
environmental effects and can greatly simplify the patterns of inheritance for complex traits.
Marker-assisted selection also allows early stage selection to be carried out since the genetic
pattern does not change during plant development. Furthermore, after a linkage map of
molecular markers has been constructed a whole genome selection procedure can be utilized,
thus, decreasing the time required to fix a gene in an agronomically useful background
(Paterson et al., 1988; Lander & Botstein, 1989).

Genetic stabilization

Homozygosity is required for many crop varieties to ensure their uniformity in the
field as well as their stability from year to year. Generally, three to four years are required to
achieve homozygosity in a field crop by conventional techniques. However, haploidy can
replace several generations of inbreeding (usually 7 or 8) normally required to achieve
homozygosity. Haploid plants are obtained by culturing gamete cells and they are,
subsequently, doubled by a treatment with colchicine to produce homozygous lines (Kasha,
1974; Hu & Yang, 1986). The use of doubled haploid populations also makes breeding for
recessive traits much simpler because the homozygous recessive individuals occur at a greater
frequency in these populations than in conventional breeding populations (Siebel & Pauls, 1989;
682 K.P. PAULS

Henderson & Pauls, 1992). Haploids have been obtained from over 200 species, and varieties
based on double haploid lines have been produced in a variety of field crops including: barley,
wheat, tobacco, rice and rapeseed (Morrison & Evans, 1988).

VARIETY TESTING

After a variety is developed it is tested in several locales over several years. In some
countries the results of these trials are used to decide whether the variety can be licensed for
sale. The movement of genetically altered material from the laboratory or greenhouse to the
field is highly regulated. In many countries the regulations state that field tests of transgenic
material can only be done under a permit that is granted on a case by case basis (Anonymous,
1988; Anonymous, 1995 a,b).
Some of the concerns that have been raised about the production and use of transgenic
plants include:
1) escape of genes from the transgenics to related wild species to produce super weeds
(Wrubel et al., 1992; Dale, 1992),
2) intergeneric or interkingdom transfer of genes like antibiotic resistance genes
(Am/Lbile-Cuevas, 1993; Dixon, 1993),
3) questions of the safety of foods derived from plants possessing crop protection genes
like the insecticidal Bacillus thuringiensis (Erickson, 1992; Beck & Ulrich, 1993) and
4) issues related to ownership of genetic resources (Witt, 1985; Berland & Lewontin,
1986; Stone, 1995).
The results from initial studies indicate that the use of biotechnology does not impose
extraordinary risks on society (Flavell & Fraley, 1992; Huttner et al., 1992). However,
considerable knowledge gaps exist in these areas of concern and a cautious approach to the
introduction of transgenic plants into the environment or the utilization of transgenic foods has
been advocated (Bryant & Leather, 1992)

VARIETY INCREASE

The plant propagation step cain be a major cost in a variety development program. For
many field crops this is simply done by producing seed from pure stands of one variety.
However, the interest in using pollination control systems such as cytoplasmic male sterility,
self incompatibility and artificial male sterility to produce hybrid seed in a wide variety of
 PLANT BIOTECHNOLOGYFOR CROP IMPROVEMENT 683

crops has grown as the techniques to manipulate these traits have developed. This interest can
be attributed to the facts that hybrid varieties generally have higher yields than conventional
varieties and that the investment associated with their development is protected because they
do not breed true.
Molecular biology has been used to identify some of the genes (Cornish et al., 1987;
Dzelzkalns et al., 1993) that determine self incompatibility and there is some hope that this
information can be used to develop hybrid seed production systems for crops that are now sold
as inbreds or homozygous varieties. Protoplast fusion has been used to transfer organelles (in
particular mitochondria) from one species to another. This approach has been used to introduce
cytoplasmic male sterility into crop plants for use in pollination control and hybrid seed
production (Pelletier & Chupeau, 1984; Kyozuka et al., 1989; Scbell & Vasil, 1989; Pauls,
1991).
An artificial genetic male sterile system has been created by transforming plants with
a RNAse gene from Bacillus amyloliquefaciens under the control of a promoter that is specific
for cells (called tapetal cells) that surround the pollen sacs (Mariani et al., 1990). In these
transgenic plants the tapetom is destroyed and they do not produce pollen. They represent the
female parent in a hybrid seed production system. The male parents are transgenic for a RNAse
inhibitor gene which is also under the control of the tapetum promoter (Mariani et al., 1992).
Both the male parents and F I hybrids obtained by crossing the female and male transgenics
produce pollen.
Plant cloning is a method that has been used with success by horticulturaiists and
ornamentalists for many years to rapidly propagate desired genotypes. The multiplication of
fruit varieties by bud grafting is an example of vegetative propagation. Vegetative propagation
can be greatly accelerated by utilizing tissue culture procedures because a small piece of tissue
can be used to produce hundreds of plants. This procedure has been called micropropagation
and has been applied to a wide variety of plant species (Klausner, 1986). In addition, the
material that is produced in vitro is disease free which facilitates international plant exchange
Giles & Morgan, 1987).
For many field crops vegetative propagation has not been used because the cost of
production greatly exceeds the value of an individual plant (Sluis & Walker, 1985). However,
from recent work on somatic embryogenesis (Gray & Purohit, 1991) and automated plant tissue
culture (Levin et aL, 1988; Vasil, 1991) it appears that it may be possible, in some instances,
to produce large numbers of cheap synthetic seed that are vegetative clones of one plant. Such
techniques may be particularly important for cross pollinating species like alfalfa where it is
684 K.P. PAULS

difficult to produce seed from self pollinations and, therefore, difficult to maintain and increase
superior plants (McKersie & Bowie)', 1993).

PROPRIETARY PROTECTION

Because of the long time it takes to develop a variety there is a need to prevent its
unauthorized multiplication and sale. The ability to distinguish one variety from another is
important in establishing ownership and protecting investments related to variety development.
Isoenzyme, restriction enzyme and PCR fragment polymorphisms have been used to describe
the uniqueness of plant varieties. In particular, the latter two techniques, can be used to obtain
fingerprints of a plant that are based on the sequences in the DNA itself (SoUer & Beckman,
1983; Beckman & Soller, 1986). A DNA branding procedure has been described where a
unique sequence of DNA is inserted into nontranslated regions of the DNA used for
transformation (Beckman & Bar-Joseph, 1986). This positively identifies plant material derived
from a molecular transformation experiment.
Over 100 US patents have been issued since the 1980's for transgenic plants or genetic
engineering approaches to altering plants (Stone, 1995). However, some very broad patents
issued in the United States and Europe for rights to all genetically altered forms of a particular
crop or all crops that utilize a particular molecular engineering procedure have raised concerns
that this practice will severely restrict the utilization of plant biotechnology.

CROP PRODUCTION AND QUALITY CONTROL

Once the crop is standing, biotechnology can play a role in its management and in the
quality control of products derived from it. For example, some DNA hybridization-based
disease assays, and antibody-based toxin assays can be used to monitor disease levels in crops
and products derived from them (Klauaner, 1986). Early detection of diseases in the field is
valuable where economical control measures exist and they can be applied before major
outbreaks occur (Miller et al., 1988). Also, rapid immunoassays for toxins produced by plant
pathogens such as Fusarium toxins in corn or agrochemical residuals such as herbicides and
insecticides (Vanderlaan et al., 1988), can be used to test crops before they are used for animal
or human consumption.
 PLANTBIOTECHNOLOGYFOR CROPIMPROVEMENT 685

SUMMARY

Biotechnology is applicable to many aspects of plant improvement and crop

production. Seed, agrochemical and food processing companies have made substantial financial
commitments to implement these technologies in plant improvement programs and research
carried out at universities and in government research institutes have been important in
developing and evaluating these techniques (Moses et al., 1988; Kalton et al., 1989; Hodgson,
1992). A measure of the level of interest in plant biotechnology can be obtained by examining
the number of field trials of transgenic plants conducted throughout the world in the last five
years (Beck and Ulrich, 1993). For example, in Canada this value has grown from less than 10
in 1985 to almost 500 in 1993 (Tomlin, 1993). Plant varieties based on transgenic plants are
just beginning to be introduced into the market. However, it is obvious from the level of
activity in this area that the pipeline is full with a large variety of potential products to follow
the recent introduction of the Flaw Saw tomato by Calgene.
Given the range of expertise and level of integration that is required in a modem plant
breeding program it is important that mutually beneficial partnerships among industry,
government and university institutions are developed in the application of plant biotechnology
to plant improvement. Not only is this partnership approach likely to be necessary to collect
a sufficient critical mass around a particular topic, but it is probably the only way in which the
societal issues related to the application of biotechnology will be properly addressed (Wrubel
et al., 1992; Buttel, 1986).

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