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Eucalyptus

Chapter · April 2009

DOI: 10.1002/9781405181099.k0902

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JWBK245-Kole k0902 May 10, 2008 14:1

Eucalyptus
Carlos Alberto Labate1 , Teotônio Francisco de Assis2 , Shinitiro Oda3 , Eduardo José
de Mello3 , Edson Seizo Mori4 , Mario Luiz Teixeira de Moraes5 , Luis Pedro Barrueto
Cid6 , Esteban Roberto González3 , Acelino Couto Alfenas7 , Edival A. Valverde
Zauza7 , Celso Foelkel8 , David H. Moon9 , Mayra Costa da Cruz Gallo de Carvalho9 ,
Danielle Gregorio Gomes Caldas9 , Raphael Tozelli Carneiro9 , Alexander de Andrade9
and Guilhermo Rafael Salvatierra9
1
Departamento de Genética, Universidade de São Paulo, Sao Paulo, Brazil, 2 Assistech Ltda,
Guaiba, RS, Brazil, 3 Suzano Papel e Celulose, São Paulo, Brazil, 4 Departamento de Agricultura e
Melhoramento Vegetal, Universidade Estadual Paulista-UNESP, Sao Paulo, Brazil,
5
Departamento de Fitotecnia, Tecnologia de Alimentos e Sócio-Economia, Sao Paulo, Brazil,
6
Embrapa Recursos Genéticos e Biotecnologia, Brası́lia, Brazil, 7 Departamento de Fitopatologia,

o
Universidade Federal de Viçosa, Viçosa, Brazil, 8 Graus Celsius Ltda, Guaiba, RS, Brazil,
o f
r
9

P
Departamento de Genética, Universidade de São Paulo, Sao Paulo, Brazil

1. INTRODUCTION
Fi
In the last century, the genus Eucalypts became
an important multipurpose source of timber in
many industrial applications for production of
pulp and paper, charcoal, energy, furniture, and
housing. The genus Eucalyptus is a member of
the Myrtaceae family, mainly originated from
Australia, comprising of more than 700 species
(Brooker, 2000). The trees form tall open forests,
woodlands, and occur in environments ranging
from areas of high rainfall to semi-arid regions,
and from sea level to subalpine altitudes. A few
species have been described as occurring outside
the Australian territory (Eldridge et al., 1994);
Eucalyptus deglupta is endemic to the Indonesian
islands of Sulawesi and Ceram, Mindanao in
the southern Philippines, northern New Guinea,
and New Britain; Eucalyptus urophylla, Eucalyptus
P a ge
rst orophila, and Eucalyptus wetarensis occur in Timor
and adjoining islands of the Lesser Sunda group
(Pryor et al., 1995). Several factors contributed
to the success of Eucalypts; the fast growth
rate and large biomass production, the ability to
grow in a wide range of environments and soils,
good wood quality for solid wood products and
short cellulose fiber, suitable for pulp production,
particularly for paper and tissue. A considerable
number of Eucalyptus plantations were established
in the early 1900s in Brazil and South Africa
along the railway tracks providing charcoal
for the locomotives. Today, several countries
in Asia, South America, southern Europe, and
Africa, have an estimated area of planted forest
around 16–19 million hectares (FAO, 2000). The
predominance species in commercial plantations
(around 80%) are Eucalyptus grandis, Eucalyptus
globulus, Eucalyptus camaldulensis and their

A Compendium of Transgenic Crop Plants: Forest Tree Species. Edited by Chittaranjan Kole and Timothy C. Hall


C 2008 Blackwell Publishing Ltd. ISBN 978-1-405-16923-3
JWBK245-Kole k0902 May 10, 2008 14:1
9-2 FOREST TREE SPECIES
hybrids (Potts, 2004). E. grandis, E. urophylla and
their hybrids are mainly planted in tropical and
subtropical regions, while E. globulus is preferred
in temperate climates (Potts, 2004).
One important factor determining the expan-
sion of large areas of commercial Eucalyptus plan-
tations was the development of cloning techniques,
initiated in 1975 in the Popular Republic of Congo
(Delwaulle et al., 1983). Outstanding genotypes,
usually hybrids, having large and straight trunk
with good wood properties were selected to make
rooted cuttings and planted in large extensions of
land. Vegetative propagation has been intensively
used by the pulp and paper industry, producing
highly uniform timber and allowing further gains
in productivity in the pulping process. In the mid
1970s the development of tissue culture techniques
and in vitro propagation of Eucalyptus spp.
provided new opportunities for mass propagation,
on a commercial scale, of selected genotypes.
At the same time, the growing importance of
Eucalyptus for the pulp and paper industry,
particularly in South America and Africa, led to
the establishment of breeding programs selecting
ge
better hybrids for cloning and to improve the basic
populations introduced from Australia.
P a
First
In the last 10 years, the advances in tissue
culture techniques, plant regeneration, and genetic
transformation, using mainly the Agrobacterium
system, allowed the development of the first
transgenic Eucalyptus trees. Also, advances in
the use of molecular markers have played an
important role in helping breeders to select
Eucalyptus trees with better wood quality, disease
resistance, and stress tolerance. More recently,
the development of genomic approaches, such as
major expressed sequence tag (EST) sequencing
programs, have increased the interest in the
application of biotechnological tools in order to
produce better Eucalyptus trees.
Our chapter provides an overview of the
main topics involved in Eucalyptus biotechnology,
from cloning and hybrids production, breeding,
application of molecular markers, tissue culture
and plant regeneration, genetic transformation,
main diseases affecting the Eucalyptus plantations,
wood quality for the pulp and paper industry,
energy production and biomaterials, and also
the identification of genes involved in disease
resistance and wood formation through functional
genomics.
1.1 Hybridization, Cloning, and Breeding
1.1.1 Hybridization and cloning of
eucalyptus spp.: evolution and its
importance for the forest sector
in Brazil
The development of forestry plantations for
industrial purposes must be oriented to increasing
industrial competitiveness in the distinct market
segments. In such a scenario, forestry-based
companies must take into account the influence
that forestry raw material can have on their
competitive capacity. The modern concept of
competitiveness includes generating products to
meet the customer’s requirements at low costs,
in a sustainable manner and with minimum
environmental impact. Therefore, the development
of tree breeding programs to obtain quick
gains, and a well-established cloning system
have become important. Vegetative propagation
methods should rapidly transform the genetic
o o f
gains, obtained through breeding, into benefits
P r
for the industry. One of the most efficient tools
to acquire these objectives is the combination
of interspecific hybridization and establishment
of clonal forestry derived from superior hybrid
individuals (Assis, 2000).
In this context, hybridization can have a great
impact on tree breeding programs, combining
superior wood characteristics with tolerance to
biotic and abiotic stresses, thus representing
a significant source of superior individuals,
capable of introducing genetic gains into forest
productivity and wood properties. Crossing species
with different characteristics allows the production
of complementary wood properties in trees to meet
special industrial requirements.
The effective and quick integration of genetic
gains obtained with hybrids into the industrial
process depends basically on the existence of
functional large-scale cloning systems. Mass
vegetative propagation perfectly complements
hybridization for producing clonal forestry and
has some advantages over the sexual methods of
mass reproduction of selected families, besides
being the best way to commercially exploit the
heterosis found in several Eucalyptus hybrid
crosses. By capturing the total genetic variance
(Zobel, 1992), vegetative propagation allows for
maximum benefits from wood properties and
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
productivity, besides allowing for the production
of more uniform raw material, which from an
industrial point of view is highly beneficial to the
industrial process and product quality. Therefore,
tree breeding programs that focus on these
aspects of the forest industry will have a great
impact on the three important components of the
competitive process: productivity, product quality,
and production costs (Assis, 2001).
Recognizing the importance of hybridization
and cloning in the context of industrial production
has led to a rapid evolution of the techniques
and processes that constitute these activities. As
a result, methods to produce hybrid seeds and
commercial cloning are now well understood and
highly effective from the technical and operational
perspective in Eucalyptus species. Developing the
concept of indoor breeding orchards and the
creation of artificially induced protogyny (AIP)
has made controlled crosses operationally feasible.
Hybrid production by controlled pollination is
thus a simple and functional process. Indoor
breeding orchards do not require flower isolation,
saving a lot of time. On the other hand, AIP enables
ge
pollination without the need to emasculate the
P
flowers. These two technologies allow large-scale
a importance of hybrids for industry was recognized,
Fi rst
controlled crossing, which is technically difficult to
carry out and also economically unfeasible using
traditional methods.
In the cloning systems for commercial-scale
propagation, the development of microcutting and
mini-cutting to replace rooting stem cuttings led
to the development of super-intensive systems to
produce vegetative propagules, which can now
be done in much more controlled environments,
allowing greater physiological and phytosanitary
control. The use of more appropriate propagation
facilities, with better environmental control,
has contributed to improve rooting rates. The
development of these technologies for Eucalyptus
marked the beginning of a new cycle in the
commercial cloning of vegetatively propagated
plants, especially for woody species (Assis, 2001).
In the last two decades, cloning Eucalyptus spp.
has produced relevant progress for the forestry
companies, especially solving problems associated
with diseases such as canker (Cryphonectria cuben-
sis) and productivity improvement (Campinhos
and Ikemori, 1983). Currently, the focus on cloning
has shifted to industrial requirements, rather
than remaining limited to disease resistance and
9-3
increased volume. Wood properties that positively
influence industrial processes and product quality
are considered, especially where cloning has an
important role to play.
1.1.1.1 Historical and technical evolution
of Eucalyptus hybridization and
cloning in Brazil
Hybridization Considering the length of forest
production cycles, the commercial use of Eu-
calyptus hybrids is relatively recent in Brazil.
Spontaneous hybrids randomly formed at the tree
farm in Rio Claro, São Paulo, at the Companhia
Paulista de Estradas de Ferro, provided the base
for the first plantations for industrial purposes.
These hybrids highlighted aspects of resistance to
Eucalypt canker (Ferreira, 1997; Alfenas et al.,
2004) and to the presence of heterosis at the
individual level during the 1970s. Therefore, these
hybrids were also, the starting point for the
o o f
introduction of the concept of clonal forestry
P r
in Brazil, during the same decade (Campinhos
and Ikemori, 1980). In the 1980s, when the
hybridization became an essential tool, helping to
create a strong and competitive forestry industry
(Assis and Mafia, 2007).
The first synthetic Eucalyptus hybrids were
produced in the 1970s by open-pollination between
compatible species (Assis, 1985; Gomes, 1987;
Martins and Ikemori, 1987). Since this technique
was limited by crosses between species that
flowered at the same time, in the 1980s a controlled
crossing method was developed in South Africa
(Hodgson, 1967; Van Wick, 1977). Although it
was difficult to perform, it enabled the expansion
of hybrids produced artificially in Brazil (Brigatti
et al., 1983; Assis, 1985; Martins and Ikemori,
1987). This method is based on the exploitation
of protandry; where pollen reaches physiological
maturity in anthesis, but the stigma is not
receptive for another few days. To make controlled
crosses using this technique, the flowers must be
emasculated, isolated, and pollinated after 5–7
days. Therefore, the method has a low operational
yield, mainly because of the number of visits
needed to make the crosses (Harbard et al., 2000).
Moreover, relatively high losses are caused by
injury to the flower buds during emasculation and
JWBK245-Kole k0902 May 10, 2008 14:1
9-4 FOREST TREE SPECIES
isolation, contributing to reduce the efficiency of
the crossings.
In 1996, a new technique for controlled crossing
was developed, called AIP. This new technique
was initially developed at Riocell S.A. (Assis
and Jardim, unpublished results) and its great
advantage is higher operational yield and better
use of the flower buds. This technique was
conceived based on two discoveries made in Chile
and in Portugal. OSP (one stop pollination) was
developed in Chile (Harbard et al., 2000), allowing
receptiveness induced by cutting lengthwise or
cutting off the upper third of the pistil of
emasculated flowers. In Portugal, it was found
that this receptiveness could be achieved before
anthesis (Trindade et al., 2001). AIP was developed
by combining these two discoveries. This tech-
nique consists of the artificial transformation of
protandry into protogyny, by cutting the top of the
floral bud operculum. At the same time, the upper
third of the pistil is cut during the preanthesis stage,
i.e., when the flower is still closed, and pollen is
applied immediately after induction. Thus, there
is no need to emasculate the flowers to make
ge
crosses.
P
Later, this new technique was tested simulta-
a as aspects of rejuvenation in Eucalyptus and
Fi rst
neously in Brazil, at Aracruz Guaı́ba, and at
Commonwealth Scientific and Industrial Research
Organisation (CSIRO) in Australia (Assis et al.,
2005) with the same success rate observed at
both sites. Besides reducing the number of visits,
AIP does away with the need for emasculation
and made it possible to obtain more seeds
in Eucalyptus-controlled crosses. Operational
productivity rose from 35 to 400 flowers per
man/hour using this technique.
At the same time as AIP was created, the concept
of indoor breeding orchard was developed, in
screened houses, doing away with the need to
isolate individual flowers, umbels or branches,
enabling the isolation of whole plants in a collective
way (Assis et al., 2005). When some type of
contamination is acceptable, this technique can be
implemented in open areas that are not isolated.
Currently it is the most widely used method for
Eucalyptus-controlled crosses. The development
of indoor breeding orchards was enabled by
the development of technologies to induce early
flowering in potted plants. Flower production in
small plants is necessary to cultivate them within
greenhouses and also to allow easier access to the
floral buds. In order to obtain flowered plants in
pots, grafting of physiologically adult branches is
used combined with application of the flowering
inductor paclobutrazol.
The combined use of these technologies (AIP
and indoor breeding orchards) allows large-scale
controlled crosses and enables the multiplication
of highly superior full-sib families, a task
formerly considered technically and economically
impossible. This is very important to improve
difficult-to-root species, since the superiority of
pure or hybrid full-sib families can be captured and
multiplied on a large scale. This technique is widely
disseminated in forestry companies and now
accounts for most controlled crosses performed
(Assis and Mafia, 2007).
Cloning techniques The first rooted eucalyptus
cutting was obtained in Australia in the 1940s
(Eldridge et al., 1993). During the next decade,
studies carried out by French researchers in
the Congo, Tunisia, and Morocco played an
o o f
important role in understanding physiological
P r
phenomena involved in the rooting of Eucalyptus,
such as juvenility and maturation, as well
their importance in cloning adult trees. This
knowledge was important to establish the concept
of Eucalyptus clonal forests, which occurred in
the 1970s in Tunisia (Chaperon, 1987) and in
Aracruz in Brazil (Campinhos and Ikemori, 1980).
However, the first large-scale cloning system was
established in Brazil, by Aracruz, and it served as a
model for the development of clonal forests, whose
use was extended to other Brazilian companies and
to several parts of the world. After the success
achieved by Aracruz, it was quickly disseminated,
and now it is the main system for the reproduction
of Eucalyptus species and clones.
Until the 1990s, the development of cloning
techniques for Eucalyptus species evolved relatively
slowly. For over a decade, the commercial
production of vegetative propagules continued to
be done by the original system, in clonal banks,
before being replaced by the clonal hedges system.
On the other hand, the techniques initially used
for the stem cutting rooting of Eucalyptus, as
well as the rooting facilities, remained practically
the same until the early 1990s. In 1992 the first
great change in the rooting technique occurred
when microcutting (Assis et al., 1992; Xavier and
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
Comério, 1996) and later mini-cutting were created
(Assis, 1996; Higashi et al., 2000).
Microcutting is a rooting method where the
propagules are obtained from shoot apices
originating from micropropagated plants and in
mini-cutting originating from auxiliary sprouts of
plants cloned by stem cuttings. After rooting of
the first shoot, the two techniques are identical,
varying only in the origin of the initial propagule
source. In some clones using mini-cutting,
some propagation cycles (serial propagation)
are required to reactivate before acquiring full
rooting capacity potential. In microcuttings such
propagation cycles come naturally by monthly
in vitro subculturing of explants. Micropropa-
gation is unnecessary for easy-to-root species
because high levels of juvenility can be obtained
easily by inducing basal shoots, therefore in such
cases, mini-cutting is technically and economically
feasible.
When plants are established in the mini-clonal
hedges to form the mini-stumps by whatever
means, multiplication is performed using the
mini-cutting technique (Assis and Mafia, 2007).
ge
Microcutting and mini-cutting are the most
P
modern concepts for cloning Eucalyptus species
a rooted stem cuttings has worked well for these
Fi rst
in large scale and they are currently the most
widely used techniques in Brazil. The creation
of these technologies was the watershed in the
evolution of Eucalyptus cloning systems and they
resulted in profound changes in the main technical
segments that constitute this activity. It marked
the beginning of a new cycle in the propagation of
woody species.
A major consequence of creating microcutting
and mini-cutting was the great change in the
methods to produce vegetative propagules in large
scale. The clonal hedges that had replaced the
original clonal banks system at the beginning of
the 1980s were replaced by the mini-clonal hedges.
Propagule production began to be performed in a
super-intensive manner, supported by hydroponic
systems in controlled environments (indoor mini-
clonal hedges). Currently, two hydroponic systems
are used: sand bed with drip irrigation (Higashi
et al., 2000) and intermittent flooding (Campinhos
et al., 2000).
The development of these technologies also
influenced changes in the conception of rooting
facilities that were important for their evolution
and improvement. When Eucalyptus cloning
9-5
attained industrial proportions, most of the
physical structures used to root stem cuttings were
extremely simple, consisting of partial shading
and a misting system. When the mini-cutting
technology began to be used, greenhouses had to
be used to achieve greater control over the rooting
environment. Their importance in rooting became
clear and led to their widespread use. Therefore, at
the same time as rooting methods were developed,
a major evolution occurred in the propagation
structures. On the other hand, the development
of super-intensive vegetative propagule production
systems in hydroponic mini-clonal hedges, with
better nutritional and phytosanitary control of
the mother plants, is technically and economically
advantageous in the commercial production of
Eucalyptus plantlets. Currently, mini-cutting is the
cloning technique most widely used by the large
Brazilian forestry companies.
In easy-to-root hybrid clones, micropropagation
is not commonly used as a rejuvenation method
or simply as a method to rapidly broaden the
o o f
clonal base of recently selected clones. Except in
P r
the systems that use microcutting, where it is a
mandatory phase, mini-cutting from sprouts of
species and the tendency is that micropropagation
will no longer be used for this purpose, especially
due to its high costs. Nevertheless, there are
technical advantages in using micropropagated
plants to form mini-clonal hedges, even in easy-
to-root clones. In clones that are difficult to
root, micropropagation is still a technique that
can potentially increase predisposition to rooting.
This class includes E. globulus hybrid clones
that are increasingly used in plantations for pulp
production.
1.1.1.2 Importance of hybridization
and cloning in Brazil
In recent years, the growing integration between
the different segments of industrial production
based on planting forests has been a major
factor in the development of technologies dealing
with forest production for industrial purposes.
The greater proximity between forestry, industry,
and commerce has enabled an integrated view
of the segments: forest × industrial process ×
product quality, which is the base for acquiring
JWBK245-Kole k0902 May 10, 2008 14:1
9-6 FOREST TREE SPECIES
competitive differentials in industries. In this
way, new prospects arose in the development
of technologies that could promote a positive
impact on the industrial production chain and,
consequently, on industry performance. On the
other hand, the perception of the great economic
gains by industry as a result of the increased
quality of raw materials has significantly enhanced
the genetic improvement programs, above all due
to their capacity to promote quantitative and
qualitative changes in the raw material.
In this context, increasing forest productivity
and improvement of wood properties for industrial
use are important demands on Eucalyptus
breeding programs in Brazil. The main objectives
of these demands are the reduction of operational
costs, improving raw material performance in
industrial processing, and rendering it appropriate
for the manufacture of high quality products
for different market segments (Assis, 2000). The
main contribution of genetic improvement for
the forestry-based industry is the generation of
superior individuals that can lead to gains in
forest productivity, industrial process, and product
ge
quality. The greatest challenges for tree breeders
P
have been to use effective strategies to obtain
a with 65% of the planted area. Fifteen percent of
Fi rst
individuals that are superior in both growth and
wood quality. The main obstacles to overcome
these challenges are that the species traditionally
planted in Brazil present certain limitations
concerning the wood quality, compared with other
Eucalyptus species. In this context, interspecific
hybridization is a very useful tool, especially since
it allows transfer of genes from species with
high wood quality but slow growth, to species
that have an inferior wood quality but are well
adapted to the planting sites and with excellent
growth. The main advantage of hybridization,
besides the capacity to allow the combination of
differentiated characteristics in distinct species, is
the possibility of producing trees with superior
growth as a result of heterosis or hybrid vigor,
a common phenomenon in Eucalyptus (Martin,
1989; Denison and Kietzka, 1992; Nikles, 1992).
The differences between the various species
of Eucalyptus adapted to this country, with
respect to wood properties, tolerance to biotic and
abiotic stresses, as well as to the manifestation
of heterosis has been the main factor to produce
individuals with superior growth, adaptation, and
wood quality, through hybridization. Thus, the
shortest path between industrial demands and
their fulfillment by genetic improvement programs
is to seek complementarity among species, by
increasing the number of desirable characteristics
of the wood, combined with the commercial
utilization of heterosis. Enhancement of the forest
quality by the commercial use of heterosis, through
the vegetative propagation of Eucalyptus hybrids
in Brazil is clear and there is evidence that it is
a very useful phenomenon for a faster gain in
forest productivity. In this sense, the contribution
of Eucalyptus hybridization to forest development
in Brazil is very significant. Most benefits from
hybrid use, especially in forest productivity, are
accredited to the manifestation of heterosis for
growth and to the complementarity of certain
species concerning characteristics that combine
to produce more appropriate genotypes for the
different planting environments (Assis, 2000).
Currently, the vast majority of eucalyptus
improvement programs in Brazil are based on
hybrids. In 2005, 84.5% of the clonal plantings
o o f
of Eucalyptus in Brazil were hybrids, where 66.5%
P r
of which were hybrids from controlled crosses. The
hybrid E. grandis × E. urophylla is most often used,
the remainder is still natural and spontaneous E.
urophylla hybrids, 3% Rio Claro hybrids, 0.5% E.
urophylla × E. camaldulensis, 0.4% E. urophylla ×
E. globulus and 0.6% E. camaldulensis × E. grandis
(Assis, 2004).
Wood properties are very important for industry
because they have an impact on processing
costs, production gains, and the technological
qualification of the products and are therefore
essential in production processes of raw material
for industrial use. In this sense, hybridization
tends to play an increasingly important role
in the Eucalyptus planting programs in Brazil.
Because of the success achieved in southern Brazil
concerning the incorporation of E. globulus genes
into site-adapted species and clones, mainly due to
the significant enhancement of the wood quality to
make pulp, there is a tendency to seek the source of
wood quality in this crossing, by pulp companies.
A major breakthrough is expected in wood quality
to make short-fiber pulp using E. globulus hybrids.
From the perspective of industrial processes, E.
globulus is the most appreciated Eucalyptus species
by the pulp mills, because its wood presents tech-
nological properties that are especially important
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
for the pulping process. Either because it provides
greater production gains, or due to the lower
manufacturing costs, its wood tends to be a major
competitive differential point for the companies.
Evaluations performed on E. globulus hybrids
show that it is possible to maintain the same
forest productivity levels obtained in E. grandis,
Eucalyptus saligna, Eucalyptus dunnii, E. urophylla
and several of their hybrids, but with positive
changes in wood and pulp quality. Compared
to the traditional genetic materials, these hybrids
allow increasing wood density; reduction of 4%
in lignin content; a 3% increase in pulp yield; 3%
increase in hemicellulose content; 1 m3 reduction
of wood per ton of pulp in specific consumption
and a 5 kg reduction of the chlorine dioxide per
ton of cellulose in bleaching, among others.
The forests that are being planted using the
new genetic materials from this new type of
crossing will provide major gains for the industries,
significantly improving their competitive capacity
on the short-fiber pulp market. Together with
the gains in production and cost reduction, the
variability found in the selected individuals also
ge
allows the obtention of fibers with anatomical
P a
characteristics that are appropriate for different
Fi rst
market segments, allowing the qualification of
pulp for each specific product.
The efficiency of hybridization to produce
genetic gains is fully acknowledged, being used
by most forestry companies in Brazil. However,
since the hybrid offspring are heterogeneous,
transforming these gains quickly and effectively
into benefits for the industry depends on
functional, large-scale cloning methods. Thus, if
on the one hand, interspecific hybridization in
Eucalyptus has been the fastest way to achieve
genetic gains, cloning on the other hand is the
most efficient way of incorporate these gains into
the industrial production processes. Cloning as
a tool for the implementation of clonal forests
derived from high-quality hybrids, is still the ideal
technical complement to maximize the benefits of
hybridization in the context of forest production
for industrial purposes. Currently its maximum
potential has been achieved in establishing clonal
forests derived from controlled interspecific hy-
brids, which produce better quality wood, greater
volume, growth, and higher resistance to biotic
and abiotic factors. In this way, cloning plays a
major role and for this reason it has received much
9-7
attention in forestry research programs in Brazil.
The consolidation of cloning as a method for the
commercial reproduction of superior Eucalyptus
trees had a positive effect on productivity, costs of
industrial processes, and product quality. Cloning
Eucalyptus in large scale is currently one of the
most important factors in promoting increased
productivity, improvement, and homogenization
of wood technological properties as an industrial
raw material (Assis et al., 2004).
Almost 30 years after the introduction of the
concept of clonal forests in Brazil, cloning is
definitely a part of the raw material production
processes used in various sectors of industrial
activity, above all pulp and paper and charcoal,
which account for 78% and 21% of the area
planted with clones, respectively. In 2004, the area
planted with clones surpassed 1 000 000 ha. The
current annual rate of new clonal planting is over
250 000 ha, with a tendency for further growth.
Brazilian clonal forestry was initially developed
from spontaneous hybrids and natural hybrids
o o f
presenting heterosis for growth, besides resistance
P r
to diseases, above all Eucalyptus canker. Although
these hybrids were produced empirically, they were
responsible for the great breakthrough in forest
productivity that occurred in recent years. Forest
productivity was increased threefold by cloning
them. Currently the clonal forests are derived
from individuals generated in genetic improvement
programs, whose main strategy is based on the
production of controlled interspecific hybrids.
The cellulose increment (AMI cell) was around
6 t ha−1 year−1 in the 1970s. This productivity
is now around 12 t ha−1 year−1 for the leading
companies in this industry. Using E. globulus in
the composition of hybrids, it is expected that
trees planted from 2010 onward will achieve
productivities of 16 t ha−1 year−1 .
1.1.1.3 Future perspectives
Due to the current importance of hybridization
and cloning for industrial forests, their use has been
consolidated and they are increasingly becoming
part of the forest-based industrial production
processes. The progress achieved in the different
techniques involving these activities will ensure
new levels of quality and constitute new platforms
for the development of Brazilian forestry.
JWBK245-Kole k0902 May 10, 2008 14:1
9-8 FOREST TREE SPECIES
The advances that have occurred in controlled
pollination techniques, for instance, will allow the
intense use of hybrids in breeding programs of the
industries that use Eucalyptus as raw material.
On the other hand, hybrid production will be
increasingly oriented to crosses with E. globulus
and its subspecies, mainly because of the quality of
this wood for pulp production. The use of adapted
species and clones, combined with this species, has
allowed a significant improvement in the wood
quality, in environments where the pure species
is not well adapted. This will allow the use of E.
globulus hybrids both in subtropical and tropical
areas, with significant gains in wood quality.
Although there has been significant progress in
techniques and Eucalyptus rooting methods cul-
minating in a robust, functional, well-established
system, several studies on cloning are being
performed to develop new systems that could be
technically and economically more appropriate
than the current ones. Propagule production
methods evolved from an extensive system, with
low propagule production per square meter,
passing through an intensive system in clonal gar-
ge
dens until they reached the present super-intensive
P
systems, with high propagule productivity per
a constitution of other loci).
Fi rst
square meter in the mini-clonal hedges supported
by hydroponics. The search for new advances in
this field is directly related to establishing produc-
tion systems for mini-propagules in bioreactors,
using minimum space and with a significantly
higher propagule production per square meter. A
further advantage of these technologies is that they
use highly rejuvenated tissues, which may provide
better technical results both for plant production
and for the quality of the clonal forests.
1.2 Factors Affecting Expected Genetic
Gains in Forest Breeding Programs
Using Seeds
Genetic improvement has the main objective
of obtaining genotypes, through selection, that
present adaptations to environmental conditions,
resistance to disease and insects, characteristics
desirable for productivity, and the multiple uses
of wood. Many species of the genera Eucalyptus
and Pinus have been intensely used in reforestation
programs in Brazil and many other countries.
The importance of Eucalyptus seeds of the
best quality, from the best origins and the
establishment of test species, seeds and progeny
allowed the selection of genetic material with the
potential for adaptation and productivity under
the edaphoclimatic conditions found in different
regions of Brazil.
The beginning of any breeding program is
the selection of the species and populations
to be used, by species tests and the origin
of the desired characteristics (Resende et al.,
2005). The strategies for genetic improvement
of exotic forest species has been based on the
selection of species/origins and the selection of
individuals from the base population, exploring
the natural genetic variability existing amongst
populations and individuals (Ferreira, 1992).
Vencovsky and Barriga (1992) concluded that
the knowledge of the type of genetic action
that predominates in the genetic basis of a
character is an important ingredient in an efficient
breeding program. Gene effects are classified into
two basic types, allelic interactions (dominance
o o f
interactions between alleles at an interlocus level)
P r
and nonallelic (epistatic actions that arise when
alleles or genotypes are influenced by the genetic
According to Resende et al. (2005), based on
experimental evaluation, the selection should be
based on means and variance, recommending that
the selected material should have an elevated mean
and ample genetic variability, allowing continuous
gains with selection through various generations.
A way to estimate the progress of the selection
process is very important when using quantitative
genetics in plant improvement (Vencovsky and
Barriga, 1992). By the general expression of gain
(
G) “
G = ds × h2 ”, it can be observed that
the progress is a direct function of the magnitude
of the differential selection (ds = difference
between the mean of the selected group and
the mean of the original population) and of the
heritability coefficient (h2 ) (Wright, 1976; Zobel
and Talbert, 1984). The expected progress varies
in accordance with the selection scheme and is
detailed by Shelbourne (1969), Vencovsky (1978),
Vencovsky and Barriga (1992), and Cruz and
Regazzi (2001).
Ferreira (1985) stated that the gains obtained
depend on the standard used for comparison
(selected material or not), the effect of age on
the evaluation, and environmental variation at the
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
sites where the progeny were tested. Sometimes
the expected genetic gain at a determined phase
of the improvement program cannot be achieved.
Factors related to the management of the improved
populations, factors related to the production
of seeds and saplings, genotype × environment
interaction and others, can contribute negatively
and these factors are discussed individually below.
1.2.1 Selection of genetic material
The selection based on indices has been used
in forest improvement, offering larger gains
when compared to other selection methods
(Cotterill, 1986). The utilization of selection
indices in Eucalyptus spp. improvement requires
the estimation of a series of parameters, indices,
and progress with selection, to permit the breeder
to choose an index adequate for each situation
(Resende et al., 1990). Resende et al. (2005) states
that selection methods applicable to the perennial
species can be classified in accordance with the
selection units and the procedure used to predict
ge
the genetic values. According to the author, the
a
P
procedure currently used to estimate the variation
rst
Fi
components and predict the genetic values is
AM-BLUP (animal model, evaluation of genetic
variation) associated with DFREML (derivative
free maximum likelihood).
Oda et al. (1989) described the problems in
classic genetic improvement with Eucalyptus due
to high selection intensity, affirming that the
risks of intense individual selection in genetic
improvement can be reduced if the species
used are pure and adapted, the matrices were
selected under normal environmental condition
(without stress), the size of the effective population
is maintained high, and used for short-term
programs. In asexual improvement, the authors
suggest that better results could be obtained if
hybrid species are used, the matrices were selected
under stress, heterosis could eventually appear,
and high intensity selection was used. The authors
present results from experiments with E. grandis
obtained under high selection intensities. A total of
51 selected trees, as well as the half-sibling progeny,
were evaluated with respect to cloning. The
selection of the trees was made using populations
originally form Coff’s Harbour (NSW, Austrália),
under the intensity of 1:5000 and populations from
9-9
Zimbabwe, at 1:2000. Based on the evaluations
of the materials from the different experiments,
the following relationships between the characters
were established:
(a) Relationship between the flowering period,
the typical qualities of the trees, and the
behavior of the respective progenies (material
from Coff’s Harbour). It was observed that
the same trees that originated from inferior
progenies presented early flowering and
atypical fruits, demonstrating the occurrence
of hybrids in the selected material and a loss
in productivity due to the heterogeneity in the
trees.
(b) Correlation of the clonal behavior with the
behavior of the progenies using diameter at-
breast-height (DBH) and height data and
comparisons with the inheritance estimates
of the progenies (material from Zimbabwe).
A clone-progeny correlation for DBH (r =
0.020) was very weak (Figure 1) and low for
f
height (r = 0.33) (Figure 2), this shows that
r o o
clones with good performance do not always
P
present good progenies. Gene dominance
could favor the selection of heterozygotic
trees and heterogenic descendents with low
productivity.
This study shows that intense selection can
reduce the effective size of the population, leading
to the presence of different degrees of endogamy,
demonstrating the loss of vigor in the saplings orig-
inating for endogamous crosses. In relation to the
concept of the effective population size, Vencovsky
(1987) discussed the genetic representativity con-
tained within a sample in relation to immediately
previous generation. Thus, the effective size is
related to the genetic size of the population and not
the number of individuals within the population
(Resende and Vencovsky, 1990).
1.2.2 Correlations and correlated responses
between characteristics
The study of the correlations between charac-
teristics is important in genetic improvement,
because the general preoccupation is to improve
the material, not only for isolated characteristics
but simultaneously for a group of characteristics
(Vencovsky, 1978). According to the author,
JWBK245-Kole k0902 May 10, 2008 14:1

9-10 FOREST TREE SPECIES

8,33

7,83
Progênies—DAP (cm)

7,33

6,83

6,33
6,00 7,00 8,00 9,00 10,00 11,00 12,00 13,00 14,00 15,00 16,00
Clones - DAP (cm)

Figure 1 Correlation between the DBH values (cm) for progenies (2 years) × clones (3.5 years) [Source: Oda et al., 1989]

the expected alteration in the mean of a

o f
difficulties, such as low heritability or in the

o
determined characteristic Y, when the selection is
r
measurement or identification (Cruz and Regazzi,
P
ge
for characteristic X is a response correlated with 2001). According to the authors, in genetic studies
the system of selection considered. The correlation
P a it is important to differentiate and quantify the

rst
visualized directly in an experiment is phenotypic degree of genetic and environmental association

Fi
in nature, provoked by two factors: genetic and between the characteristics, such that the genetic
environmental (Vencovsky and Barriga, 1992). correlations are of a heritable nature and as such
In genetic improvement it is important to know usable in breeding programs.
the association between characteristics, especially Cruz and Regazzi (2001) discussing simulta-
when the selection of one of them presents neous selection of characteristics stated that the

10,50

10,00
Progênies—altura (m)

9,50

9,00

8,50

8,00

7,50
5,00 6,00 7,00 8,00 9,00 10,00 11,00 12,00 13,00 14,00 15,00
Clones - altura (m)

Figure 2 Correlation between the height (m) for progenies (2 years) × clones (3.5 years) [Source: Oda et al., 1989]
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
use of the index theory of selection is an efficient
alternative, permitting the combination of multiple
information contained within the experimental
unit and making possible selection based on a
number of variables that unite various attributes
of economic importance. The authors reviewed
various proposals to obtain selection indices
(classic index proposed by Smith, 1936 and Hazel,
1943; index based on desired gains; base index;
index based on the sum the ranks; “weight free”
and “parameter free” index).
In Pinus radiata, Dean et al. (1983) studied
30 families of half-siblings and detected negative
correlations between volume and basic wood
density. The gain estimates for these character-
istics, combined into one index, revealed that
it is not possible to improve wood density and
volume simultaneously. Moraes (1987) detected a
low negative genetic correlation between growth
characteristics and basic wood density in open-
pollinated E. grandis progeny. However, this
correlation was not sufficient to seriously weaken
the possibility of gains in basic wood density.
Reviewing other genetic and phenotypic corre-
ge
lations between basic wood density and growth
characteristics, Moraes (1987) observed the
P a
Fi rst
existence of high correlations between height,
DBH, and volume, however a correlation between
these characteristics and basic wood density was of
different magnitudes, both positive and negative.
For Sturion (1993) these contrasting results
demonstrate that generalization could provoke
losses in a selection process, depending on the
adopted strategy.
1.2.3 Endogamy
According to Wright (1976), endogamy signifies
the crossing of related individuals or descendents
of the same individual. The most extreme case of
endogamy is self-fertilization; however, there are
various degrees of endogamy before this, such as
crossing between siblings and members of small
isolated populations. The authors further state that
endogamy does not always cause a reduction in
vigor or depression by endogamy, which is caused
by the accumulation of recessive deleterious genes
and not solely because of self-fertilization.
Eldridge (1978), working with the proportion
of self-fertilization and open-crossing in Euca-
9-11
lypts, confirmed predominance for open-crosses,
although this proportion differed between species.
Reddy and Rockwood (1989) reported that the
quantity of self-fertilization in Eucalypts is higher
than 7%. Estimates for the degree of natural self-
fertilization vary among the species of Eucalyptus:
24% in E. obliqua (Brown et al., 1975), 18% in E.
stoatei (Hopper and Moran, 1981) and 10–38% in
E. grandis (Hodgson, 1976a).
Hodgson (1976b) studied some aspects of
flowering and the reproductive system in E. grandis
and observed that progenies from self-fertilization
were subject to various forms of endogamic
depression, including deformation of the leaves
and stem, reduction in vigor in comparison
with progenies from open-crosses. The author
concluded that the product of a seed producing
plantation suffers a degree of reduction because of
the quantity of self-fertilization, estimated at 30%
(Van Wyk, 1981).
Various studies were carried out in order to
verify the effects of different degrees of endogamy
o o f
in Eucalypts, such as Van Wyk (1981), Eldridge
P r
and Griffin (1983), Maêda (1987), Griffin and
Cotterill (1988), and Hardner and Potts (1995).
Eldridge (1976) stated that endogamic depression
in Eucalyptus is expressed as low growth of the
saplings in the field and low viability of their
seeds. Eldridge and Griffin (1983) working with
Eucalyptus regnans, observed that the number of
seeds varied from 42 per 100 in self-fertilized to
71 seeds in open-pollinated and 90 seeds in cross-
pollinated flowers.
Irregular flowering and a deficiency in pollina-
tion are factors that can provoke endogamy. The
presence of endogamy causes various prejudicial
effects in Eucalypts, including deformed leaves
and stems, reduction in vigor of the saplings,
reduced height, reduced survival of saplings in the
field, and reduction in the production of viable
seeds.
1.2.4 Progeny tests
Individuals selected using an estimation of genetic
parameters are then analyzed using progeny
tests to ascertain their reproductive value as
seed producers and later used to form seed
producing plantations (Kageyama, 1980). Shimizu
et al. (1982) presented the procedures and
JWBK245-Kole k0902 May 10, 2008 14:1
9-12 FOREST TREE SPECIES
recommendations for the installation of progeny
tests.
The best way to evaluate the genotypic
value of the selected trees is through their
progenies, permitting the estimation of their
value as progenitors (Weir, 1977). This procedure
permits the separation of trees with phenotypic
superiority caused by the good planting conditions
from those that present genotypic superiority
(Allard, 1971; Weir, 1977). Hodge et al. (1996)
compared the genetic parameters of progenies
by open-pollination and controlled pollination,
using E. globulus and Eucalyptus Nitens, and
evaluated using two types of populations resulting
from open-pollination (native population and a
seed producing plantation) and two types of
populations resulting from controlled pollination
(using families of siblings from self-pollination
and pollination between individuals). For E. nitens
two populations were evaluated, families from the
seed producing plantation using open-pollination
and self-pollinated sibling families. The progeny
tests were established at various sites and after
2 years height, DBH, and cylindrical volume
ge
were measured. The authors observed that early
P a
estimates of heritability for the open-pollinated
First
progenies derived from native populations could
be exaggerated, probably due to the high degree
of endogamous depression. It was concluded
that genetic gain estimates of open-pollinated
populations can be overestimated because of (1)
inflated heritability estimates, and (2) flaws in the
evaluation genotype × environment interaction.
One point to be discussed during the installation
of the progeny test is sapling preparation. Gener-
ally the selection of saplings is made in order to
make the forest more productive and homogenous.
The appearance of abnormal saplings could be
linked to endogamous effects; however Nogueira
(2005) observed that a controlled cross carried
out using two unrelated E. grandis trees, resulted
in progeny with a 3:1 proportion of normal
and abnormal individuals, respectively. The study
identified two random amplified polymorphic
DNA (RAPD) markers linked to the principal
gene that causes the abnormal phenotype.
The preparation of saplings for the installation
of progeny tests for E. grandis was studied by Mello
et al. (1993). The authors studied the influence
of two systems for the utilization of seeds on the
behavior of 14 progenies and three controls, using
two methods of sapling preparation: (a) seeded
directly into tubes, thinned and selected; and (b)
seeded into a germinator, planted and utilize all the
seedlings. At 2.7 years of age, the results indicated
that significant differences exist between the two
systems. The progenies presented superior DBH,
basal area, and cylindrical volume when submitted
to sapling selection in the nursery. It was also
observed that there was a significant interaction
between the progenies and the sapling production
system.
1.2.5 Seed production
When the seed production plantation is estab-
lished, it is expected that the seeds produced
represent the desired crosses, and it is important
that the pollination is effective between the
genetic material present in the plantation. Mora
et al. (1981) reviewed the aspects of seed
production in forest species, citing various authors
o o f
who emphasize that abundant flowering and
P r
the production of good quality seeds depend
on various factors related to the processes
of pollination, pollen germination, pollen tube
growth, fertilization, and embryo development.
Thus, the success of the plantation depends,
among other things, on the quality of the pollen
produced and germination efficiency. Contrasting
differences in the germination efficiency of the
pollen between the selected genetic materials in
the plantation, would suggest that not all the
materials will participate equally as the masculine
progenitor. Thus, it is important to evaluate pollen
viability in the trees selected as part of the breeding
matrices and to detect male sterility. It is also
important to study tree management techniques
that influence pollen quality.
Weir and Zobel (1975) discussed seed produc-
tion plantations for selected material. According
to the authors, the strategy to develop and test
material adequate for future generations should be
elaborated early, during the first generation of the
improvement program. If the program is of long
duration, the consequences of a restricted genetic
base and associated risk of endogamy should be
avoided by the continuous inclusion of new genetic
material within the population. Thus, the ideal
way to select the maximum number of unrelated
individuals for advanced generations is diallelic
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
or partial diallelic, both give good estimates of
the parental value of the clones, and general and
specific combining abilities of the parent trees.
Kellison (1969) stated that due to the fact that
seed productivity is dependent on environmental
variables, the geographic location, plantation area,
and clone dispersal should be considered before
a seed producing plantation is installed. For the
installation of plantations for the production of
hybrid seeds by open-pollination, the following
points should be considered: (a) that the species
have coincident flowering; (b) the ratio of pollen
receptors to pollen donors should be adequate
to favor hybridization; (c) the pollen receptor
species should have a shorter period of flowering
in relation to the pollen donor species; (d) the
pollen receptor species should be relatively or
completely self-incompatible; (e) the selection of
hybrid saplings in the nursery is essential and
should be preceded by studies on the architecture
and inheritance of the characteristics of interest
to assess its operational viability. The hybrid
seeds produced by controlled pollination and the
progeny tests are the base of this study.
ge
Basic studies such as the phenology of the
P a
selected clones, determination of the general and
Fi rst
specific combining abilities and self-compatibility
studies are necessary to conduct a prudent
improvement program in order to achieve the pre-
established objectives.
1.2.6 Pollen viability
Studies on Eucalyptus pollen, in general, aim
at developing techniques adequate for pollen
management, including genetic conservation,
controlled production of hybrids, and the deter-
mination of pollen quality/viability. Sousa and
Pinto Jr. (1993) reported the existence of intra-
and interspecific differences in pollen germination;
these could be genetic or physiological in origin,
suggesting the need to develop specific culture
media that imitate natural conditions. Studies on
the in vitro germination of Eucalyptus pollen were
carried out by Boden (1958), Gabrielli et al. (1965),
Borges et al. (1973), Griffin et al. (1982), Cangiani
(1988), Sousa (1988), Menck et al. (1990), and
Sousa and Pinto Jr. (1993).
Sousa (1988) stated that the nutritional state
of the plant during pollen development could
9-13
affect longevity. Due to the general opinion that
boron is necessary and germination efficiency is
poor in media with only sucrose, Sousa (1988)
studied the effect of boron and calcium on
E. urophylla and Eucalyptus tereticornis pollen
germination. The authors concluded that boron
was necessary although they were unable to define
the exact concentration. Therefore, the nutritional
status of the seed producing trees, with particular
respect to boron, is very important. On the other
hand, calcium doses of 220 ppm improved the
germination efficiency of the two species tested.
It would be interesting to observe the effect of
boron supplements in the seed production plan-
tations with respect to pollen viability and seed
quality.
Suzano Company evaluated 55 E. grandis
matrices of a second-generation clonal seed
production plantation, from different origins,
that are part of Suzano’s breeding program.
Branches containing flowers were collected, the
open flowers and the green buds were removed,
o o f
and the branch was placed in a recipient with
P r
water and left in the greenhouse. The buds in
anthesis and the open flowers were cut from the
branches and the stamens were removed for pollen
extraction. A large variation in the efficiency of
pollen germination was observed between the trees
analyzed, the amplitude of the variation was from
0.15 to 93.96%, with a mean of 44.77%. This
variation in the efficiency of pollen germination
was also observed by Boden (1958). As these
trees are part of the Suzano’s clonal seed orchard,
second generation, this data is important to
evaluate the pollination among the selected genetic
material. Thus, variations in pollen germination
efficiency will influence the pollination within the
plantation and as such influence the genetic quality
of the seed. To investigate pollen germination
efficiency between locations, the variation in
five different locations was determined: Location
1 = 2.56–92.45%, Location 2 = 10.78–69.92%,
Location 3 = 25.03–79.17%, Location 4 =
0.15–82.74%, and Location 5 = 1.22–93.96%.
From these data, it can be observed that there
is an ample variation in germination efficiency
within the progenies used, with the range of
efficiencies varying with location probably due
to physiological/environmental factors. Thus, any
variation detected at the same location should be
due to genetic effect.
JWBK245-Kole k0902 May 10, 2008 14:1
9-14 FOREST TREE SPECIES
According to Eldridge (1976) there are only a
few examples of male sterility in Eucalypts. The
detection of these individuals is important since
these trees will not participate as pollen donors.
1.2.7 Pollination
In natural populations, Eucalypts exhibit a mixed
crossing system, but predominantly allogamy
(Griffin, 1989). The pollination of the genus
Eucalyptus is mainly entomophilic with the
principal pollinator being the bee, Apis mellifera L.
(Pacheco, 1982). Novelli et al. (1982) investigated
the influence of bee pollination on seed production
in a clonal plantation of Eucalyptus citriodora
and demonstrated an increase in the number of
viable seeds per kilogram and the number of
capsules per panicle. Pacheco (1982) studied the
effect of bee hives (A. mellifera L.) in a seed
producing plantation of E. saligna on production
and quality of the seeds. The pollen from the
trees in the center of the plantation were labeled
using P32 and it was shown that the number of
ge
flowers containing labeled pollen decreased with
P a
rst
the increase in distance from the source up to
Fi
a distance of 300 m. Other results demonstrated
a gradual reduction in the number of seeds per
fruit with an increase in distance from the hive.
At 0–50 m, the number of seeds was 40% higher
than that from 300 to 350 m from the hive, with
the authors demonstrating pollination activity of
the bees up to a distance of 350 m from the hive.
Maêda (1987) estimated for an E. grandis seed
producing plantation, the fertilization rate for
the characteristic that produces albino plantlets.
The observed rate was 5.57, which represented
a coefficient of endogamy of 0.03. The author
considered this rate to be low and the variability
of the plantation was maintained by efficient insect
pollination. Although there appeared to be a large
number of insects present on the plants, only a few
effectively participated in the movement of pollen
between flowers. When the quantity of pollen
transported, visiting frequency and movement
among the flowers was observed, A. mellifera was
confirmed as the most important insect in E.
grandis pollination.
According to Griffin (1989), pollen is frequently
transported to larger distances than those inferred
by direct observation of the insect flight behavior.
The pollen collected from one flower is not
necessarily deposited in the next to be visited.
The biological aspects of the Eucalyptus flower
suggests that (a) feeding behavior is not strongly
conditioned by flower structure, therefore sticky
pollen could be distributed over the whole insect,
not solely the part that enters into contact with the
stigmas; and (b) pollen can maintain its viability
for approximately 8 days on the insect’s body,
indicating that the transported pollen remains
functional during this time.
Barbour et al. (2006) evaluated the phenology
of flowering in species of the subgenus Sym-
phyomyrtus native to Tasmania and E. nitens,
introduced species. The difference in the flowering
period of the native and an introduced species was
highlighted as the principal barrier to gene flow by
pollen. The authors state that the results show the
importance of knowing the factors that affect gene
flow, with the objective of identifying species and
populations, native and exotic, with the highest
risk of gene flow.
Griffin and Hand (1979) stated that knowledge
o o f
about the receptiveness of the stigma, besides
P r
the processes of floral development and pollen
release, is fundamental to the study of reproductive
biology. Sousa and Pinto Jr. (1993) studied stigma
receptiveness in E. dunnii in order to suggest ways
to improve the efficiency of controlled pollination.
The authors observed that for maximum efficiency,
consequently better seed production, pollination
should be carried out on the 6th day after
anthesis. The receptive period for Eucalyptus
stigma can vary within the subgenus (Pryor,
1951), between species of the same subgenus
(Griffin and Hand, 1979) and within the same
species. Griffin and Hand (1979) highlighted that
there could be differences in floral receptiveness
within the same tree. The similarity in floral
development rates of different species within the
same subgenus Symphyomyrtus, but growing in
different hemispheres, suggests to the authors that
this process is under strong genetic control.
According to Eldridge (1976), the flowers of
Eucalyptus are hermaphrodite (male and female
in the one flower), because protandry has been
observed in some species, with examples of
self-incompatibility and male sterility; however,
allogamy can be considered as predominant in
the genus. Although protandry occurs, this does
not eliminate the possibility of self-fertilization,
since flowering in the canopy lasts longer than the
receptive period of a flower.
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
Kageyama (1979) studied the isolation of seed
production areas, trying to minimize undesirable
pollination problems because of the facility in
intercrossing between large numbers of Eucalyptus
species. The fact that pollination is predominantly
entomophilic within the genus highlights the need
to take care in the minimal distance between
plantations to prevent possible contamination.
A minimum distance of 200 m between seed
production areas is recommended. The authors
also suggested the use of barriers consisting of
other genera or unimportant Eucalyptus species
to effectively isolate the seed production areas.
1.2.8 Floral development, phenology of
flowering, and fruit formation in
eucalypts
The period and intensity of flowering varies within
and between species, especially where they grow as
exotic plants (Griffin, 1989). This could cause two
sorts of problems: firstly, when the populations
selected for improvement contain material from
ge
different origins they could function as a series
P a
rst
of subpopulations partially isolated because of the
Fi
different flowering periods, decreasing the effective
size of the population and increasing the risk of
endogamy; secondly, variations in the interaction
between the species and the insect vector could
affect the pollination efficiency between members
of the population.
Studies on floral development and flowering/
fruiting intensities in Eucalyptus populations
have been carried out by Moggi (1959) with E.
camaldulensis, Eucalyptus resinifera, Eucalyptus
rudis, E. tereticornis, Eucalyptus Botryoides, and
Eucalyptus gomphocephala; Hodgson (1976a, b)
with E. grandis; Ashton (1975) with E. regnans;
Ferreira (1977) with E. grandis; Mora and Ferreira
(1978) with E. urophylla; Aguiar and Kageyama
(1987) with E. grandis; Graça (1987) with E.
dunnii; and Souza (1996) with E. camaldulensis.
Mora and Ferreira (1978) studied the flowering
of E. urophylla clones in Piracicaba (SP, Brazil).
For each clone a branch was chosen to represent
flowering in order to follow floral development
in accordance with the following stages: (a)
inflorescence formation, without individualization
of the floral buds until the first operculum falls; (b)
from the fall of the first operculum to the fall of the
second; (c) flowering (opening of the floral bud);
9-15
Table 1 Comparison of the number of viable seeds per
kilogram in populations of E. grandis with different degrees
of genetic improvement
Number of viable seeds
Degree of genetic improvement (kg−1 )
SCA (seed collection 400 000 to 800 000
area)—Austrália
SCA—Brasil 800 000 to 1 000 000
SSO (seed orchard by seeds) 80 000 to 400 000
CSo (clonal seed orchard) 500 000 to 1 000 000
(d) fruit development; and (e) natural seed release.
Proposing a vision more quantitative, percentages
were calculated related to the production and
development of the flower buds, flowers, and fruits.
The authors observed that, in general, from the
initial development of the buds to flowering takes
4–5 months and fruit formation to natural seed
release 7–8 months. The natural loss of floral buds
f
during flowering, fertilization, and fruit formation
o o
was 54.85%. The flowering period varied among
r
P
the clones and could be put into the following
categories: clones that did not flower during the
experimental period; clones with an extremely
short flowering period; clones that flowered almost
the whole year; clones with a flowering peak from
November to February; clones that flower from
April to August; and clones with incompatible
flowering characteristics.
Mori et al. (1988b) suggested that to obtain
maximum production, it is necessary to know
the factors that affect productivity and use man-
agement techniques to alleviate these problems.
Thus, basic knowledge about the biological process
involved in seed production is important to
predict and study the factors that could reduce
productivity (Mora et al., 1981).
The variations in the number of viable seeds
per kilogram could be related to the pollination
efficiency, frequently weakened by the high
variability in flowering or restricted genetic bases
are shown in Table 1.
1.2.9 Seed vigor and size, germination and
initial development of the seedlings
The level of physiological quality of the seeds
should be evaluated through their capacity to
JWBK245-Kole k0902 May 10, 2008 14:1
9-16 FOREST TREE SPECIES
germinate and seedling vigor (Popinigis, 1977;
Aguiar, 1984). Popinigis (1977) suggested an index
for the speed of emergence (SEI) in the determina-
tion of relative vigor between lots of seeds and an
index for the speed of germination (SGI).
Valeri et al. (1984) commented that previously
published papers demonstrate that the size of the
seed is one of the factors that affect germination,
with larger seeds producing more vigorous
plantlets that develop faster. Thus, a classification
by size would reduce losses associated with the
less vigorous plantlets produced by small seeds
that are quickly dominated by the plantlets form
larger seeds (Aguiar et al., 1979). The authors
studied the influence of E. saligna seed size
on germination and initial plantlet development.
The results demonstrated that plantlets from
larger seeds reached the size for thinning and
transplanting quicker than those from smaller
seeds. Similar results were obtained by Pereira
and Garrido (1975) with E. grandis, observing
that larger seeds presented a higher SEI than the
smaller seeds.
The occurrence of self-pollination contributes to
ge
the loss of seed quality, provoking the appearance
of albino and abnormal plantlets (Kageyama,
P a
Fi rst
1981; Maêda, 1987). Oda et al. (1991) studied
the influence of the number of viable seeds
per kilogram and the size of the seeds on the
percentage of albino and abnormal plantlets. This
study used E. grandis seeds collected at different
times of the year and the seeds were classified into
three groups (<0.50, 0.59–0.71, >0.71 mm). The
results demonstrated that larger seeds were less
likely to produce albino or abnormal plantlets and
suggested that the production of small seeds could
be linked to self-pollination.
Suzano (1994a) evaluated the behavior of
different progenies of E. grandis with respect to
seed vigor and initial plantlet growth, looking
for possible links between these variables and
the quality of the sapling and the future forest.
The seeds of six progenies from the clonal seed
orchard (second generation) were divided into
two groups, >0.59 mm but smaller than 0.71 mm
and >0.71 mm. The germination efficiency, SEI
and the height of the plantlets at 32 and 40
days after seeding were evaluated. Differences
between the progenies were demonstrated, thus
during thinning the plantlets that have higher
SEIs and faster initial growth will be preferentially
represented in the final product from the nursery.
In this way, when breeding populations and
commercial plantations are formed, not all of the
progenies are equally represented, thus resulting
in lower genetic variability, due to the reduction
in the effective population number, and failure to
achieve the expected results.
1.2.10 Quality of the seedling for planting
The selection of seedlings in the nursery by height
is one of the parameters used to assess quality.
The advantage of this selection is to form more
uniform lots with a better use of the seedlings in
the field (Coelho, 1984). According to the author,
the practice of sapling selection in the nursery is a
controversial question. Some authors state that a
certain time after planting, the forest presented
a more homogenous development even though
the seedlings from the nursery were not selected.
However, other studies report that the selection
o o f
of taller seedlings give rise to taller trees. The
P r
removal of defective seedlings or seedlings with low
vigor is, however, necessary. For example, of the
total number of seedlings produced in the nursery
by Companhia Suzano de Papel e Celulose, 20%
were considered as abnormal, those presenting
defects such as bifurcations and loss of vigor.
Part of these abnormalities could be explained
by different degrees of endogamy in the seed
production plantation.
Coelho (1984) evaluated the silvicultural behav-
ior of E. grandis seedlings up to 1 year old, based
on seed, plantlet, and sapling size. The author
observed that selection by size of the seedling had
highly significant effects on height and diameter,
suggesting that the selection for this species by
seed or seedling should be encouraged in order to
obtain trees with better growth qualities. Various
studies demonstrate that the selection of seedlings
can enable an initial gain in height, but with time
this difference is reduced or disappears (Donald,
1976; Balloni et al., 1978; Morais and Brune, 1983;
Suzano, 1994b).
In this way, the selection of seedlings has an
effect on the formation of commercial plantations
where all the progenies do not participate in equal
quantities, thus the genetic variability could be
reduced due to the reduction in the size of the
effective population.
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
1.2.11 Interaction genotype × environment
(G × E)
Zobel and Talbert (1984) stated that when the
tests are carried out at the same location, any
effect is limited only by the genotype. This fact
has two consequences: (a) the heritability could
be overestimated, resulting in overestimation of
the expected gains when the material is planted
in an untested environment; (b) the genotypes
selected in one environment may not be the best
in other environments resulting in a reduction
in the genetic gain. The authors also suggested
that the G × E interaction affects the strategy
in forestry breeding programs as seen by tree
mortality and/or reductions in growth.
Various studies have reported that stability
is genetically controlled (Barriga, 1980; and
Torres, 1988). Kageyama (1980) evaluated the
genetic variation in E. grandis (origin Coff’s
Harbour, Australia) generated by open-pollination
in five locations: Agudos, Anhembi, Brotas,
Lençois Paulista, and Resende (SP, Brazil). The
evaluation characteristics were as follows: form
ge
of the tree trunk, DBH, and height at 2-
P a
year-old. The effect of the location, significant
First
on all of the characteristics, reflected in the
reduction in heritability (at the level of the
location, individual plants, mean of the families,
and within the families) and, therefore, in the
selection for the locations as a group. The same
tendency was observed by Pinto Jr. (1984) in
progenies of E. urophylla S.T. Blake from Island
of Flowers (Indonésia), using conjugated origin
testes and locations in four Brazilian states,
Aracruz (ES), Anhembi (SP), Bom Despacho
(MG), and Planaltina (DF).
Mori et al. (1986) estimated the genetic
parameter for E. saligna from three locations
(Itatinga (ACS); Areia Branca (PSC); and Salto
(PS)) and planted at three test sites: General
Câmara (RS), Brotas (SP), and Bom Despacho
(MG). The characteristics evaluated were DBH,
height, and cylindrical volume at 3 years old. The
coefficient of heritability when analyzed together
was inferior to the majority of the coefficients
obtained by test location, demonstrating losses
in heritability because of the progeny × location
interaction. The losses in the selection due to
this interaction were estimates at up to 88.3% for
cylindrical volume.
9-17
Mori et al. (1988a) studied the progeny ×
location interaction in 30 7-year-old progenies
of E. urophylla generated by open-pollination
from the Island of Flowers (Indonesia) at four
locations: Aracruz (ES), Belo Oriente (MG),
Bom Despacho (MG), and Grão Mogol (MG).
The values of the mean heritability (heritability
coefficients at the plant level, progeny mean, and
within the progenies) for the four locations were
superior to the values obtained when the data
was analyzed together, demonstrating the great
effect of progeny × location interaction. The
losses in genetic gain due to the progenies ×
location interaction for the cylindrical volume,
DBH, and height, were 26.73%, 15.74%, and
8.14%, respectively. The authors compared the
genetic gains (%), using the progeny mean of the
cylindrical tree volume and three improvement
strategies: multiple populations with selection
of progeny for each location; the selection of
individuals with a greater capacity to adapt to
the study location, stable selection; traditional
o o f
selection not considering environmental difference
P r
and the progenies × location interaction. For
this comparison, a selection proportion of 1:6
between progenies was used, which made possible
the selection of progenies predictably more
productive (selection of 5 progenies from 30).
The genetic gains in percentage are shown in
Table 2.
Carvalho (1989) evaluated the G × E interaction
in 50 clones of E. grandis, in the region of
São Mateus (ES) in three traditionally reforested
areas, using the following genetic parameters:
heritability, standard deviation from the estimate
of heritability, coefficient of genetic variation, and
the quotient between genotypic variation and the
coefficient of experimental variation, calculated
using the measurable parameters DBH, height,
cylindrical volume, and basic wood density. It was
observed that the estimated heritability values were
relatively high for all the measured characteristics
when considered at each location. When the data
was analyzed together, there was a general decrease
for all the characteristics involved in growth (DBH,
height, and volume), and less pronounced for basic
density. Van Wyk et al. (1989) tested the stability
of 31 hybrid Eucalyptus clones at 10 locations in
South Africa, observing that many clones were
stable for tree height. According to the authors,
the results indicated that the selection of clones
JWBK245-Kole k0902 May 10, 2008 14:1
9-18
Table 2 The genetic gains expressed in percentage (%) for the three strategies and the four locations
Strategy Aracruz (ES) Belo oriente (MG)
Multipopulation 18.81 10.18
Stable 5.09 0.79
Traditional 1.15 1.93
should be cautious since the behavior of each clone
is location specific.
Cruz and Regazzi (1994, 2001) discussed the
effect of the G × E interaction on the prediction
of selection gains obtained using a particular
improvement strategy. The authors presented
situations in which it had been desirable to make
gain predictions. These studies demonstrated that
the effects of the G × E interaction should
be considered in breeding programs, since it is
possible that losses in the expected gains could
occur in the selection of genotypes. According to
Mori et al. (1988a) the G × E interaction, when
badly administered, could result in a reduction
of expected gains making it difficult for the
breeding program to achieve its goals. The authors
suggested, in order to minimize losses, a more
P a
rst
adequate multipopulation strategy is necessary
Fi
that considers the specificity of the genetic material
for particular environmental conditions, through
the selection of individuals more adapted and
productive for each ecological region.
A genetic improvement program should have
well-defined strategies, planned for short- and the
long-term objectives, in order to obtain genetic
material improved for the desirable characteristics.
This material should guarantee the sustainability
of commercial plantations and the continuity of
the improvement program through the selection of
advanced generations of trees and superior clones.
The breeder should be acquainted with the factors
that could reduce the expected genetic gains and
how to intervene in the improvement strategies to
minimize these losses.
1.3 Application of Molecular Markers
in Eucalypt Breeding
The success of a breeding program depends on the
genetic resources, which are being explored, the
selection procedures, and the strategy used. There
are numerous examples of successful Eucalypt
FOREST TREE SPECIES
Bom despacho (MG) Grão mogol (MG) Mean
14.18 26.10 17.32
10.72 13.25 7.46
1.09 1.94 1.53
breeding programs throughout the world. The
average Eucalypt productivity has been improved
in three decades from 12 m3 ha−1 year−1 in the
1960s, to a current value of 60 m3 ha−1 year−1 . The
current performance has been reached because of
applied silvicultural research and especially the
breeding programs using high performance clones
with fast growth, excellent wood quality, rusticity
on poor soil, and resistance to diseases and pests.
The success of the breeding programs depends
on the ability to distinguish heritable and envi-
ronmental factors. Genetic markers are heritable
when associated with the characters of interest
and can increase the efficiency of selection. The
o o f
segregation analysis of molecular markers permits
P r
the construction of genomic or linkage maps,
ge
to estimate gene frequency, especially useful to
characterize the population gene diversity and
germplasm in breeding programs for the genetic
conservation of Eucalypts (Mori, 1993). Paternity
tests and the study of mating system of different
Eucalypt species have contributed to obtain
superior clones in breeding programs (Camargo,
2001).
A genetic marker is a character capable
of detecting differences between two or more
individuals or organisms, be able to distinguish
progenitors and progenies, and should present
a series of attributes such as high level of
polymorphism, stability in different environments,
detection of high numbers of unlinked loci and of
simple inheritance. In the following will be present
the main types of molecular markers as well as their
main applications in Eucalypt breeding programs.
1.3.1 Application of molecular markers
in breeding programs
The selection of Eucalypt phenotypic characters
for commercial purposes is time consuming
because of the time taken to make field evaluations.
One way to shorten this time is the use of molecular
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
markers. Various types are appropriate for use in
Eucalypt breeding programs and some examples
are given below.
1.3.1.1 Characterization of genetic diversity
The characterization of gene diversity in breeding
programs is very important for researchers.
Molecular markers are very useful to study the
structure of populations, families, clones, cultivars,
and also to discriminate gene pools from different
populations.
Polymorphism based upon isoenzyme loci was
studied in populations of E. urophylla, E. grandis,
and E. saligna to develop tests for discrimination
of interspecific hybrids from contaminants. Mori
et al. (1996) concluded that the Est-1, Est-2, and
Idh-1 loci were useful for the purpose because some
alleles were present in a population and absent
in others and vice versa. Clones and progenies
of five E. grandis subpopulations were analyzed
by isoenzyme loci observing that the average
inbreeding (f) was 0.08 and outcrossing rate (t)
ge
was 0.88. Much of the genetic diversity was within
the subpopulations and there was practically no
P a
rst
diversity between subpopulations (F ST = 0.011)
Fi
(Mori and Kageyama, 2001).
Sixty-nine progenies were analyzed representing
one open-pollinated family of E. urophylla trees.
RAPD markers allowed the identification of 72
loci that were analyzed using Jaccard’s coefficient,
generating matrices of genetic distances. The
genetic distances between individuals were 0.40
through 12 groups. The progenies also showed
different bark patterns, allowing the establishment
of distinct groups. However, the groups based
on genetic distances using DNA analysis did not
correspond of those based on bark pattern (Pigato
and Lopes, 2001).
A total of 44 natural hybrids of Eucalyptus,
cultivated in central Brazil were analyzed. The
RAPD markers presented efficient discriminating
power, determining a mean genetic distance of
54% among them and genetic divergence from
24% to 73%. This demonstrated that there is a
wide genetic base among individuals, which is
desirable in breeding programs. Clustering analysis
established by UPGMA (Unweighted Pair Group
Method with Arithmetic mean) method, using
80% as a cut off criterion for the total genetic
9-19
distance, established nine distinct groups with
average genetic divergence above 60% (Caixeta
et al., 2003).
The study of genetic diversity among trees
is an important stage of any breeding program
that targets the exploration of heterozygosis. The
prediction of heterozygosis has been extensively
used in Eucalyptus to obtain better hybrid
combinations. In a study using 40 E. grandis and E.
urophylla trees (two 10 × 10 circulate partial diallel
crosses) were analyzed for their genetic diversity.
The crosses were produced in a previous study
based on the tree diversity using RAPD markers.
Seventeen microsatellites amplified 75 allelic forms
and gave a heterozygosis value of 26.1%. The
genetic distance varied from 10% to 100%, whereas
the mean genetic distance was 64.6%. The cluster
analysis for the 40 trees showed high diversity
and forming two groups (one for each species),
although some genotypes were outside their
species-specific group. In comparison with groups
formed by the RAPD markers, microsatellite
o o f
markers were more efficient in discriminating
P r
species. However, the correlation values among
RAPD and microsatellite markers were low and
negative. Microsatellite markers were efficient to
discriminate trees of E. grandis and E. urophylla
(Muro-Abad et al., 2005).
1.3.1.2 Fingerprinting
The characterization and identification of in-
dividuals are important procedures in plant
breeding. Rocha et al. (2002) used RAPD and
simple sequence repeat (SSR) markers to obtain
an exclusive fingerprint for 15 genotypes of
Eucalypt hybrids with high potential for vegetative
propagation used in breeding programs. Those two
molecular techniques produced a set of markers
that allowed an accurate identification of all
genotypes. The RAPD procedures were also used
by Pimenta et al. (2001) to obtain information on
origin, genetic distance, and relationship among
clones.
Hybridization between three species of Euca-
lyptus in the Series Curviptera, Eucalyptus macro-
carpa, Eucalyptus pyriformis, and Eucalyptus
youngiana was investigated using RAPD markers.
The dendrogram based on genetic similarities
showed the relative proximity and distance among
JWBK245-Kole k0902 May 10, 2008 14:1
9-20 FOREST TREE SPECIES
the individuals. Two clusters were identified by the
UPGMA dendrogram. One of them included all
of the E. macrocarpa genotypes and also one of
E. macrocarpa hybrid. The other included all of
the E. youngiana and E. pyriformis genotypes and
their hybrids (Neaylon et al., 2001).
1.3.1.3 QTL mapping
Most Eucalyptus breeding programs have used
interspecific hybridization, making it possible to
capture nonadditive genetic variance. Propagating
genotypes via vegetative propagation greatly en-
hanced the possibility to use genetic linkage maps
for accelerating breeding programs by marker-
assisted selection (MAS) and recombination
(Grattapaglia and Sederoff, 1994).
Linkage maps were constructed mainly by
RAPD dominant markers (Verhaegen et al., 1997),
amplified fragment length polymorphism (AFLP)
dominant markers (Gaiotto and Grattapaglia,
1997), and SSR co-dominant markers (Brondani
et al., 1998). The maps could identify genomic
e
Pag Some studies were carried out using isoenzyme
regions in eucalypts with significant effects on
expression of economically important characters.
t
Firs
Various characters were studied by quantitative
trait loci (QTL) analysis, such as, characters
associated to eucalypt rooting (Marques et al.,
2002), insect resistance (Shepherd et al., 1995),
plant growth (Squilassi and Grattapaglia, 1998),
and Puccinia psidii rust resistance in E. grandis
(Junghans et al., 2001). There are also studies for
different eucalypt species and cultures in which
few loci with major effects controlling relatively
large proportion (from 10% to 40%) of total
phenotypic variation for quantitative characters
of silvicultural importance, such as, wood volume
and wood density were detected.
Squilassi and Grattapaglia (1998) mapped
QTLs in eucalypts for wood volume by RAPD
markers, using linkage disequilibrium on selected
progenies. The study showed that the MAS was
efficient to select through the family mean level;
however, it was not efficient at the individual level.
Gion et al. (2001) studied 201 full-sib
families from interspecific hybrids between E.
urophylla and E. grandis for eight genes in-
volved in the lignin biosynthesis (PAL (pheny-
lalanine ammonia-lyase), COMT1 (caffeate/5-
hydroxyconiferaldehyde O-methyltransferase 1),
COMT2, 4CL (4-coumarate:coenzyme A ligase),
C4H (cinnamate 4-hydroxylase), CCoAOMT (caf-
feoyl coenzyme A O-methyltransferase), CAD2
(cinnamyl alcohol dehydrogenase 2), and CCR
(cinnamoyl-coenzime A reductase)) and three
Myb transcription factors. The genes were mapped
using the single strand conformation polymor-
phism (SSCP) technique. These genes were located
on the two parental genetic maps constructed with
polymerase chain reaction (PCR)-based markers.
The lignin monomeric compositions (S/G ratio)
were obtained using the thioaciolysis method and
the QTL analysis for S/G ratio was performed
using the interval mapping procedure. Several
regions controlling part of the variation were
identified, showing that multipoint estimates of the
total variation explained through the QTL were
38.0% and 18.5% for E. urophylla and E. grandis,
respectively. The study shows that it should be
possible to follow the manipulation of lignin
quality in breeding programs using molecular
markers.
r o of
P
1.3.1.4 Marker-assisted selection (MAS)
markers to allow earlier selection of superior
genotypes. Menck (1996) studying the acid
phosphatase isoenzyme in clones of E. grandis
cultivated in vitro found a correlation between
superior genotypes for growth characters and
those for efficiency by the use of phosphorous.
The author also observed correlations between
superior genotypes on the field and higher
activities of acid phosphatase enzyme.
The transfer of disease resistance alleles in plants
can be expedited by the use of DNA markers. If
the markers are tightly linked to the resistance
alleles they can be used for MAS. One effective
use of MAS is found in the process of pyramiding
resistance alleles. The use of MAS is based upon
the principle that a gene or a block of genes
is associated with a molecular marker of easier
identification, making the selection for that marker
more efficient than the selection for the phenotypic
character. MAS in eucalypt has focused mainly on
wood growth, which is an important commercial
character, easily measured but of low heritability.
Experimental full-sib families of E. grandis and
E. urophylla were used to evaluate the comparative
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
efficiency of selection based on molecular markers
associated with a favorable QTL and the regular
phenotypic selection for diameter at breast height
(Squilassi and Grattapaglia, 1998). The authors
did not study all possible crosses, evaluating only
the maternal QTL allelic contribution, because an
effective assessment of MAS in eucalypt depends
on experiments where the phenotypic data are
gathered at adequate ages and QTL information is
preferentially available for all progenitors involved
in the pedigree (Squilassi and Grattapaglia, 1998).
Using the BSA (bulked segregant analysis)
procedure, Bortoloto (2006) developed a SCAR
(sequenced characterized amplified region) marker
to detect the flowering time region on E. grandis
map. The marker presented linkage to the
EMBRA 7 (Brondani et al., 1998) microsatellite
marker on a paternal map. The author also
detected linkage of the EgLFY flowering gene
to the EMBRA 6 (Brondani et al., 1998)
microsatellite marker indicating the region for
flowering control.
ge
1.3.1.5 Recombination of genotypes
and seed orchard
P a
F i
Eucalyptus species are widely used for r st
planting
through tropical and subtropical regions of the
world and they are predominantly outcrossing,
highly heterogeneous, and genetically diverse
(Moran and Bell, 1983). Using microsatellites
and AFLP markers, Zelener et al. (2005) studied
six provenances and 37 selected half-sib families
of E. dunnii to make a selection to establish a
seed orchard. The estimated genetic differentiation
showed low values among provenances (θ P =
0.026) and high values among families (θ S =
0.174). A high proportion of the total variation
observed within families suggested that the
orchard design should be based upon individual or
family selection rather than provenance selection.
A E. dunnii breeding population of 46
provenances from Australia and selected for fitness
through subtropical and cold environments was
screened by AFLP and microsatellite markers
to estimate the genetic diversity. The markers
presented no significant differences between the
original breeding population and the selected
genotypes of seed orchard confirming that the seed
orchard can be established with a limited number
9-21
of individuals without problems of inbreeding
(Poltri et al., 2005).
Microsatellite markers were used to genotype
potential pollen donor and maternal progenies
from a E. globulus seed orchard in Chile and an
E. grandis seed orchard in Uruguay. The resulting
data were used to infer the most likely pollen
parents for the seed from each seed orchard. The
estimated distances from maternal tree to the most
likely male parent served as the data set basis to
model the pattern of gene flow in both orchards.
The results indicate that the pollen dispersal may
reach distances as far as 300 m or more (Russell
et al., 2001).
1.3.1.6 Mating system
Eucalypts present hermaphrodite flowers making
possible selfing and outcrossing rates at different
levels. Different authors have observed that
eucalypts present a model of mixed mating, pre-
o o f
dominantly by outcrossing, however inbreeding
P r
can occur by selfing and crossing amongst relatives.
Moran and Brown (1980) studying a population
of Eucalyptus delegatensis by isoenzymes, observed
an average outcrossing rate of 0.77, with
differences occurring between young (r = 0.66)
and adult (r = 0.85) plants. Through time, the
populations change and usually there is a tendency
for endogamous individuals to die, increasing the
level of heterozygosity in the population. Moran
et al. (1989) studying a seed orchard of E. regnans
observed an outcrossing rate (r = 0.91) higher than
that of the natural population (r = 0.74). The lower
rate of outcrossing in natural populations could
be due to inbreeding between physically close
relatives. In a seed orchard there are no relatives
and the inbreeding occurs via selfing. Mori (1993)
investigating a seed orchard of E. grandis, also
using isoenzymes found an outcrossing rate of
0.88.
Patterson et al. (2001) determined the out-
crossing rates at two positions on the tree:
the top and the bottom of the canopy in E.
globulus, in a remnant of a native stand in
southern Tasmania using isoenzyme markers. In
trees previously determined to have high levels of
self-incompatibility, outcrossing rates were high at
both, the top and bottom of the canopy (from
0.87 to 0.99). In contrast, the outcrossing rates
JWBK245-Kole k0902 May 10, 2008 14:1
9-22 FOREST TREE SPECIES
in self-compatible trees were significantly different
in both positions of the canopy: lower at the
bottom (from 0.27 to 0.66) than the top (from
0.74 to 0.90). These results were of particular
significance to collection of open-pollinated seed
for breeding or deployment, where unfortunately,
the most accessible seed may also be the most
inbred.
Mori et al. (2004) studying E. grandis progenies
by microsatellite markers observed an outcrossing
rate of 0.67 indicating that the species shows
crossing among relatives and selfing.
1.3.1.7 Diagnosis of disease resistance
Eucalypt rust, caused by P. psidii, is one of the
most serious diseases in Brazil and considered to
be the most serious threat to eucalypt plantations
worldwide. Junghans et al. (2001) analyzed the
number of sorus and pustule size to characterize
rust severity on E. grandis seedlings which were
further screened with RAPD markers. One of
the markers (AT/917) showed complete co-
gePigato and Lopes (2001) studied the diversity
segregation with Ppr-1 (P. psidii resistance, gene 1).
All the resistant plants showed the marker,
P a
Fi rst
while no susceptible plants had this marker. The
AT9/917 sequence was used to obtain specific and the genetic distances in four generations
primers (SCAR), which will be used for screening
clones in an E. grandis genomic library.
Using microsatellite markers, Mori et al. (2004)
also studied rust resistance character in progenies
of E. grandis. Nei’s genetic distance varied up to
0.400 and the index of gene diversity varied from
0.383 to 0.713, having an average of 0.587.
1.3.1.8 Analysis of paternity
It is very common to use molecular markers to
check the paternity of a superior elite tree obtained
by open-pollination. The analysis of paternity is
especially important to determine the other parent
contributing to the genotype with superior gene
combination.
Estimates of the level of multiple paternities
correlated with outcrossing within and between
fruits in a pre outbred population of the bird-
pollinated mallee, Eucalyptus rameliana, were
made by Sampson (1998) using six isoenzyme loci.
The correlation of outcrossing paternity (rp ) was
positive and significant within fruits (0.26) and
the effective number of mates for a single fruit
was estimated to be 3.85. The specialization of
floral structure and phenology in E. rameliana
for bird pollination has probably contributed to
correlation of paternity within fruits because there
are fewer male parents available at any one time
when compared to the mass-flowering species.
Paternity analysis using microsatellite markers
was conducted on a E. nitens seed orchard
in Victoria, Australia. An average of 40 seeds
per clone were germinated and screened for
paternity using the four loci. Paternal contribution
varied among clones, suggesting that panmitic
pollination was not occurring, probably due to
differences in flowering time and flower numbers
between the clones such that each clone was
subjected to a different pollen pool. Distance
between clones was another important factor
influencing paternal contribution (Grosser et al.,
2001).
r o of of breeding generations
P of Eucalyptus
1.3.1.9
Comparison
of E. urophylla, which provided data to help
guiding the breeding program. The initial base
population was introduced by seeds collected
in Indonesia (P0 generation). In the subsequent
segregating generations originated by open-
pollination, recombinations were designated as
P1 , P2 , and P3 . One hundred and seventy four
individual trees representing the four generations
were analyzed. The RAPD technique allowed
the identification of 86 positions analyzed using
the Jaccard Coefficient. The genetic distance
from P0 generation was 0.33, 0.34 from P1 , 0.40
from P2 , and 0.38 from the P3 generation. The
genetic distances between individuals increased
in relation to the base population, being 0.15%
from P1 generation, 18.93% from P2 , and 13.31%
from P3 , showing an increase in genetic diversity
in the advanced generations, despite selective
processes. Genetic diversity of 14 populations
of E. grandis were studied by Mezzena (2003)
utilizing microsatellite markers. The populations
were at different levels and breeding generations.
The inbreeding coefficients within the populations
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
were very similar (f = 0.16). The author observed
a little loss of heterozygosity from F1 (Ho = 0.64)
to F2 (Ho = 0.60) breeding generations.
2. DEVELOPMENT OF TRANSGENIC
EUCALYPTS
2.1 In Vitro Culture in Eucalypts as a
Prelude to Genetic Transformation
From the agribusiness point of view, eucalypts is
important in many aspects. Essential oils can be
extracted from the leaves; tannins from the bark;
and wood, methanol, charcoal, and cellulose for
paper production can be extracted from the trunk.
Hence, it is not estrange that millions of hectares
are planted in the world. Currently, Brazil has
the biggest collections of Eucalyptus spp., even
bigger than Australia and Indonesia (Ferreira,
1992). In addition, this genus has species with
various interesting attributes such as fast growth,
adaptation to poor soils, fertile hybrid formation,
and capacity to produce roots from stump sprouts
ge
(coppiced).
Commercially, Eucalyptus propagation has
P a
Fi rst
been traditionally done by seed, but, the cellulose
and paper industry has stimulated research
to attempt clonal multiplication (Xavier and
Comério, 1996; Rosse et al., 1996), to optimize
field production, reduce costs, and to achieve
better genetic gain (Thorpe and Harry, 1990).
Ikemori et al. (1994) showed that pulp and
cellulose production was significantly higher in
cutting cloned forests (10.9 t ha–1 year–1 ) than
in unimproved seedling forest (5.9 t ha–1 year–1 ).
However, the propagation via rooted cuttings
offers difficulties such as loss of rooting capacity
in the adult trees (Brune, 1982; Burger and Lee,
1987) and plagiotropic, a bushlike growth pattern
(Durand-Cresswell et al., 1985; Flynn et al., 1990).
Eucalyptus spp., despite the fact that many species
can propagate by cutting due to the presence of
an epicormic bud at the base of adult trees others,
such as E. regnans, E. nitens, Eucalyptus fraxinoides
and E. deglupta, are recalcitrant (Hartney, 1980).
In cloning, tissue culture has been used for
several years as a tool to propagate uniform and
selected material with major commercial value
(Karnosky, 1981; Haissig et al., 1987; Hartmann
et al., 1990; Kozai and Kubota, 2001). On
9-23
the other hand, the development of breeding
programs with woody plants for additive or
nonadditive genetic variation has been too slow.
Therefore, tissue culture has been effective in
these programs, aiming to diminish the time to
obtain intra-and interspecific hybrids. Recently, in
vitro culture has contributed efficaciously to the
research on biolistic or Agrobacterium tumefaciens
transformation with the objective to reach new
genetic commercial arrangement for forestry at
short, medium, and long terms (Rochange et al.,
1995; Machado et al., 1997; González et al., 2002).
There are many reviews reporting in vitro
techniques showing morphogenesis either via
organogenesis or embryogenesis to obtain whole
plants in Eucalyptus spp. (Hartney, 1982; Durand-
Cresswell et al., 1985; LeRoux and Staden,
1991a; Sita, 1993). In this context, problems with
genotype, contamination, and phenol oxidation
have been reported as serious challenges to be
overcome. For the time being, problems with
somaclonal variation and genetic stability (Tibok
o o f
et al., 1995; Azmi et al., 1997; Rani and Raina,
P r
1998) or vitrification (Bunn, 2005; LeRoux and
Staden, 1991b) have not been cited as important
drawbacks.
Tissue culture is a strong biotechnological tool
for micropropagation with potential large-scale
propagation of Eucalyptus. Having elite material
with determined degrees of genetic gain so as
to replace seeds by cloned propagation, with
low cost and high quality products for wood
and cellulose and competitiveness is the basic
premise of any company. Here we attempt to
review the use of in vitro tissue and organ cultures
to produce Eucalyptus clonal forestry, showing
different experimental procedures according to the
literature available by the author.
2.1.1 The explants
Table 1 shows that different explants have been
utilized to initiate Eucalyptus tissue culture and
that the use of nodal segments from elite adult
plants is more frequent. Although contamination
has been recorded (Fossard et al., 1977; Durand-
Cresswell et al., 1985), this has not been described
as a problem in Eucalyptus, even if explants
from field or simply surface-sterilized by HgCl2
or NaOCl was used (Figure 3) (Rao and
JWBK245-Kole k0902 May 10, 2008 14:1
9-24
Figure 3 Seedlings of E. grandis × E. urophylla approximately 14-day-old in SP medium, prior seeds surface disinfected with NaOCl
(9%) for 10 min, showing hypocotyls, cotyledons, roots, and contamination free. Growth conditions: 24 ± 2 ◦ C, 16 h photoperiod,
and photosynthetic photon flux density at 50 µm m−2 s−1
Venkateswara, 1985; Gupta and Mascarenhas,
1987; Bennett et al., 1994). However, it seems that
this cannot be generalized since 70% of in vitro-
P a
Fi rst
inoculated nodal explants from forest trees show
contamination, while greenhouse nodal fragments
remained virtually contamination-free (Fossard
et al., 1977). On the other hand, with respect
to surface sterilization of explants, most reports
show use of NaOCl or HgCl2 with varying periods
from 10–20 min for NaOCl, and from 5–15 min for
HgCl2 . Concentrations varied from 0.12% to 9.0%
of NaOCl and from 0.02% to 0.1 % for HgCl2 .
Prior to the treatments, fungicide has sometimes
been used (LeRoux and Staden, 1991a, b). Also,
the material sometimes was thoroughly rinsed with
tap water and detergent (Gupta and Mascarenhas,
1987; Das and Mitra, 1990), before sterilizing with
HgCl2 or commercial bleach.
Ca(OCl)2 is an energetic oxidant, with low
phytotoxicity, similar to NaOCl. However, due to
its powder formulation and the need to be
filtered, few studies have used it as a surface
sterilizer for the inoculation material. In these
cases, concentrations varied from 5% to 7% for
5–20 min (Cresswell and Nitsch, 1975; Fossard
et al., 1977; Trindade and Pais, 1997).
Other chemical substances against bacteria and
fungi have rarely been mentioned in Eucalyptus
FOREST TREE SPECIES
o o f
P r
studies, besides ethanol and H2 O2 . Alkydimethyl-
ge
benzalkonium chloride, ADBAC for example,
was a product rarely cited (Bunn, 2005), and
biocide-type isothiazolones such as PPM (Niedz
and Bausher, 2002) were not used in Eucalyptus
micropropagation. The later is heat stable, with
a broad spectrum against plant contaminants
(Compton and Koch, 2001). Physical methods
such as the use of hot water and UV light
have practically not been mentioned in explant
sterilization procedures for Eucalyptus.
In general, phenolic oxidation is mostly
inconvenient in in vitro cultures (Prieto et al., 2005)
often demanding the use of various alternative
substances to overcome it (Ziv and Halevy,
1983). Changes in peroxidase activity and level of
phenolic compounds were showed by Arezki et al.
(2001) in E. camaldulensis.
In Eucalyptus, browning has hindered the
establishment of some protocols such as those
related to protoplasts (Teullères and Boudet,
1991), cell suspension (Teulières et al., 1989;
Barrueto et al., 1997), nodal explants from
adult trees (Rao, 1988; Das and Mitra, 1990),
or floral origin (Warrag et al., 1991), being
necessary the use of antioxidant substances such
as polyvinylpyrolidone and ascorbic acid in several
cases, or adsorbents of toxic substances such as
JWBK245-Kole k0902 May 10, 2008 14:1

EUCALYPTUS 9-25

Table 3 Summary of some protocols for Eucalyptus micropropagation

Basal Rooting
Species Explant medium Growth regulators (µM) Organogenesis (via) (µM) References

Eucalyptus Nodal explants from MS BAP: 2.2 + Shoot induction NAA: 11 Gupta et al.,
torelliana tree KIN: 0.93 1983.
E. sideroxylon Nodal explants from MS BAP: 2–4 + Shoot induction IBA: 10 Burger, 1987
coppice regrowth NAA: 0.5–1
of tree
E. citriodora Mature seed B5; NAA: 16.0 Directly somatic Muralidharan
B5M NAA: 27 embryogenesis et al., 1989
B5R —

E. macarthurii; Nodal explants from MS BAP: 0.88 + Shoot induction IBA: 10 LeRoux and
E. smithii 1-year-old plant NAA: 0.05 Staden, 1991a
Hybrid MG25
E. saligna
E. globulus Nodal explants from MS BAP: 2.5 + Shoot induction IBA: 10 Bennett et al.,
coppice stumps of NAA: 1.25 1994
tree
E. grandis Leaf explants from KG, BAP: 0.2; Callus IBA: 1.25 Lainé and David,
in vitro plants G22, BAP: 2.0 + NAA: 2.5; 1994
R5, Zeatin: 8.0 +
GBA NAA: 0.5;
BAP: 5.0 +

f
NAA: 0.5;

roo
E. urophylla Hypocotyl explants MS BAP: 0.9 +NAA: 1.1; Callus NAA: 5.4 Tibok et al., 1995

P
from 14-day-old Zeatin: 4.6 BAP: 0.04
E. dunnii Seedlings—3 days B5 NAA: 16.5

ge Callus—somatic Termignoni

Pa
old embryogenesis et al., 1996

t
E. grandis × E. Hypocotyls,

Firs
SP TDZ: 2; Callus IBA: 2.5 Barrueto et al.,
urophyla cotyledonary- BA/NAA: 2.5/0.5, 1999
node, from 5/0.5, 10/0.5;
14-day-old Zeatin/NAA: 2.5/0.5,
seedlings 5/0.5, 10/0.5
Primary leaves with
different ages
from in vitro
plants
E. impensa Shoot segments MS BAP: 0.25 + Shoot induction IBA: 5 + Bunn, 2005
from trees Kin: 2.5 NAA: 0.5

activated charcoal. Other methods reported had
been periodic subcultures, initial dark period, or
using explants from established shoot cultures
(LeRoux and Staden, 1991a, b).
2.1.2 Shoot multiplication and
hormone treatment
Once the explants have been established in a basal
medium, multiplication follows via organogenesis
or embryogenesis. Usually the basal medium used
in this phase is an extension of the medium used
previously, frequently a Murashige and Skoog
(MS) medium (Table 3). Salt and vitamins from
the MS nutrient medium have been used in their
original form (Rao, 1988; MacRae and van Staden,
1990; Bennett et al., 1994; Yang et al., 1995) or
with modifications (Hartney, 1982; Barrueto et al.,
1999) depending on the purpose (induction of
organogenesis or somatic embryogenesis) and the
genotype used.
Auxin and cytokinin have been useful to
obtain in vitro plants from basal medium at
different levels. Calli have been obtained with
2 µM thidiazuron (TDZ) using juvenile material
from a E. grandis × E. urograndis hybrid (using
cotyledon, hypocotyls, and cotyledonary node;
JWBK245-Kole k0902 May 10, 2008 14:1
9-26 FOREST TREE SPECIES
Figure 1), and plants were obtained when these
calli were submitted to different concentrations of
6-benzylaminopurine/α-naphthalene acetic acid
(BAP/NAA) or Zeatin/NAA. The best results
were obtained with Zeatin: 5.0 µM and NAA
0.5 µM, respectively, using cotyledonary node
explant (Barrueto et al., 1999).
Calli were also obtained with hypocotyl juvenile
material from E. grandis using a combination
of 21.5 µM NAA and 4.6 µM kinetin (Kin) and
organogenesis was observed when the material was
transferred to a hormone-free medium (Warrag
et al., 1991). Callus induction was also observed
in E. citriodora, from cotyledons and hypocotyls
submitted to different auxins: IAA (indole-3-acetic
acid), NAA, 2,4-D (2,4-dichlorophenoxyacetic
acid) (range: 0.1–5.0 mg l−1 ) plus coconut milk
(15%); however, organogenesis occurred when
calli were placed in 2.7 µM Zeatin and 1.1 µM
IAA (Sita, 1979). According to the same author,
calli from leaves originated from mature plants
induced with 27 µM NAA and 28 µM IAA, but
no organogenesis was observed. Organogenesis
was observed in E. urophylla, from hypocotyl
ge
explants when MS was supplemented with several
P a
NAA/BAP concentrations, showing better results
rst
Fi
with NAA at 1.1 µM and BAP at 0.88 µM (Tibok
et al., 1995); however, shoot production of E.
grandis × urophyla was also effective, at 13.7 per
explant with NAA/BAP at 0.54 µM and 0.44 µM
on apical shoots and auxiliary bud, respectively
(Yang et al., 1995).
At low concentrations (NAA 0.54 µM and BAP
0.9 µM), shoot multiplication from nodal explants
of juvenile Eucalyptus of many species (Eucalyptus
smithii, E. saligna, etc.) showed good results
(LeRoux and Staden, 1991a, b). Using auxiliary
meristems, shoots were also obtained with
different genotypes of E. tereticornis, inoculated
onto MS basal medium. Among the growth
regulators tested, NAA/BAP/IAA at 5.3, 4.4,
1.1 (µM), respectively, was more effective giving
32–45 shoots per explant. Repeated subculture
in MS plus IAA 0.1 µM and BAP 0.44 µM,
produced around 200 shoots per explant (Rao,
1988). With nodal segments from an 8–10-year-
old E. tereticornis, 18–22 shoots per explant were
achieved with modified MS supplemented with
NAA 0.54 µM and BAP 4.44 µM (Das and Mitra,
1990). Low concentrations of growth regulators
were also used with Eucalyptus impensa to obtain
shoot multiplication in half-strength MS medium
plus BAP 2.5 µM and Kin 2.5 µM from shoot
segments as explants (Bunn, 2005).
Regeneration competency from auxiliary and
apical buds was observed in 20-year-old E.
citriodora. This was achieved by incubating the
explants at 15 ◦ C for 72 h, and then incubating
them in a liquid MS-2 media (120 rev min−1 ).
Five to eight shoots per bud developed. After
this, individual shoots were transferred to solid
MS-2. In this new condition, 10–15 shoots were
obtained per explant (Gupta et al., 1981). The MS-
2 medium was supplemented with Kin 0.93 µM,
BAP 1.33 µM, calcium pantothenate, and biotin
(0.1 mg l−1 of each). According to the authors, it
could be possible to get 100 000 viable plants per
year from a single bud using this protocol.
Somatic embryogenesis was obtained with
E. citriodora using NAA 16.13 µM and zy-
gotic embryos as a starting point. The basal
medium (B5) was supplemented with sucrose 50 g
(Muralidharan et al., 1989). In a peculiar protocol,
o o f
somatic embryogenesis was also described for E.
P r
grandis. This was obtained with leaves from in
vitro seedlings using 2,4-D (2.3 µM) for callus
induction. Callus proliferation was reached in
MS medium with 4 g l−1 of activated charcoal
containing (µM): 0.05 NAA, 0.44 BAP, and
0.27 gibberellin. However, additional study was
mentioned for implanting large-scale production
(Watt et al., 1991). In conclusion, low concen-
trations of BAP and NAA seem to be more
effective for Eucalyptus shoot multiplication under
a wide range of experimental conditions (Table 3);
however, these wide experimental conditions,
basically, included Eucalyptus solid cultures and
not liquid cultures, probably because liquid
medium can cause vitrification—hyperhydricity
(Monsalud et al., 1995).
2.1.3 Shoot elongation
Usually, shoot elongation has been obtained using
a half-strength medium. In E. camaldulensis,
regenerated shoots were transferred to quarter
strength B5 basal medium plus NAA 0.05 µM
(Kawazu et al., 1996). In E. grandis × E.
urophylla shoot elongation was more efficiently
obtained with MS medium containing half-
strength potassium nitrate and sucrose (Yang et al.,
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
1995). On the other hand, the elongation of shoot
clusters may be mediated with gelling agents. In E.
grandis, the MS medium containing Gelrite plus a
combination of NAA/BAP/GA (µM): 0.05, 0.44,
and 0.27, respectively, improved shoot-cluster
elongation (MacRae and van Staden, 1990). In
contrast, optimum shoot elongation of Eucalyptus
torelliana and E. camaldulensis was achieved with
solid and liquid MS-2 media, respectively, using
Kin/BAP: 0.23 µM and 0.44 µM, respectively
(Gupta et al., 1983). In addition, E. grandis shoot
elongation was promoted in MS plus gibberellic
acid 2.7 µM prior to rooting (Sita and Rani, 1985).
2.1.4 Rooting
The formation of roots from excised shoots in one-
fourth strength MS containing IAA 10 µM with
no cytokinin was reported by Hartney (1982). In
addition, shoots of Eucalyptus sideroxylon from
modified MS (half-strength salts, full-strength
organic constituents) rooted with 10 µM IBA
(indole-3-butyric acid). IBA was more effective
than NAA in adult or coppice tissues (Burger,
a e Temperature and light
g2.1.5
P
1987). Differences in rooting between juvenile
Fi rst
and old tree material were also noted in E.
In general, the culture conditions for in vitro
citriodora (Gupta et al., 1981). In general, this is a
physiological characteristic of woody species that
in vitro regeneration and rooting ability decrease
with age. For E. tereticornis, the presence of IBA
in the basal medium was essential for rooting, as
well as the use of a dark period before the transfer
of shoots to light (Das and Mitra, 1990). A period
of 72 h in darkness, prior to a 16 h photoperiod
along with hormonal treatment, was used to root
four Eucalyptus genotypes, giving different rooting
ability, inclusive, deficient rooting was observed in
Eucalyptus macarthurii and E. smithii. In addition,
these species also showed poor rooting from
coppice cuttings (LeRoux and Staden, 1991a).
Eucalyptus globules showed better results when
low concentrations of IBA (1.0–2.5 µM) were used
and when NH4 NO3 was removed from the root-
induction medium (Bennett et al., 2003).
Rooting of different clones of E. globulus was
verified after different periods of rejuvenation
using subculturing routines (Trindade and Pais,
1997). Root induction improved when riboflavin
and choline chloride were included in the medium
and boron was removed from the rooting medium.
9-27
The authors also reported that IBA dipping before
transfer to the rooting medium improved the
appearance of adventitious roots. Experiments
with E. grandis × E. urophylla using modified
SP supplemented with IBA 2.5 µM for 5 days
and transfer of shoots to a similar medium
with activated charcoal (1 g l−1 ) greatly improved
rooting but resulted in little callus formation at the
base of some shoots (Barrueto et al., 1999).
Despite auxin being an important factor in
root induction, care must be taken to avoid callus
induction at the base of the shoots (Mehra-Palta,
1982; Rao, 1988; Cheng et al., 1992). On the
other hand, cytokinin is a critical component of
the multiplication media, but inhibitory for the
rooting media. However, subsequent adventitious
root production in rooting medium was better
when Kin was used during the last multiplication
(Bennett et al., 1994). As seen above, rooting is a
highly clone-dependent characteristic, hence, one
must be careful when testing or improving rooting
conditions, especially with shoots from a mature
o o f
tree, older the tree, smaller the rooting ability.
P r
Eucalyptus plant establishment were a constant
temperature of 25 ± 2 ◦ C and a 16 h photoperiod,
involving different ranges of photosynthetic pho-
ton flux density (PPFD) from cool fluorescent light
bulbs (Rao and Venkateswara, 1985; Subbaiah and
Minoch, 1990; Termignoni et al., 1996, Sartoretto
et al., 2002; Bennett et al., 2003).
2.1.6 Hardening plantlets
The transfer from tube to soil under greenhouse
condition is an important step in the microprop-
agation process. Usually, intermittent mist or a
plastic cover is required as well as plastic pots
with sterile vermiculite and adequate natural or
artificial light and temperature. When established,
plantlets are transferred to polyethylene bags with
a mixture of soil and sand. Survival is variable,
but, usually high. Generally, a hardening protocol
contains steps similar to those previously described
with variation in the composition of the rooting
substrate (inclusion of fertilizer, fungicide, etc).
JWBK245-Kole k0902 May 10, 2008 14:1
9-28 FOREST TREE SPECIES
Finally, the plants are taken to the field and
their performance is evaluated in terms of survival,
growth rates, and biomass production. On the
whole, micropropagated Eucalyptus plants have
the same appearance as their sources (Mehra-
Palta, 1982; Gupta and Mascarenhas, 1987;
Warrag et al., 1989; LeRoux and Staden, 1991a).
Consequently, tissue culture techniques have been
observed with great interest in agronomy, forestry,
and ornamental plant production, due to the
economic benefits of clonal propagation on a
commercial level; however, the productivity of
commercial clones can be reduced by viral
infections, but culture of meristem tips is a good
way to combat this (Rutledge and Douglas, 1988).
2.2 Genetic Transformation of Eucalypts
To maintain and sustain forest vegetation,
conventional approaches have been exploited for
propagation and improvement, but tree breeding
efforts are restricted to the most valuable and fast
growing species. However, such methods are lim-
ge
ited with several inherent bottlenecks because trees
P a
are generally slow growing, long-lived, sexually
First
self-incompatible, and highly heterozygous plants.
Due to the prevalence of high heterozygosity in
these species, a number of recessive deleterious
alleles are retained within populations, resulting
in high genetic load and inbreeding depression.
This limits the use of traditional breeding methods
such as selfing and backcrossing, and makes it
difficult to fix desirable alleles in a particular
genetic background (Williams and Savolainen,
1996). Thus, conventional breeding is rather
slow and less productive and cannot be used
efficiently for the genetic improvement of trees. To
circumvent these impediments clonal or vegetative
propagation has been deployed for recovering
dominant, additive, and epistatic genetic effects
to select superior genotypes. Plant tissue culture
and genetic transformation methods offer an
important option for effective multiplication and
improvement of trees within a limited time
frame. Biotechnological approaches for in vitro
regeneration, mass micropropagation techniques,
and gene transfer studies in tree species have been
encouraging, particularly in the last decade. With
these techniques, genetic engineering assumes
additional significance, allowing introduction of
desired gene(s) in a simple step, for precision
breeding of forest trees (Giri et al., 2004).
Until recently, trees were considered to be
recalcitrant for genetic transformation studies
involving molecular techniques. The main obstacle
for genetic transformation of trees is the regen-
eration of transformed plantlets. Agrobacterium-
based genetic transformation is normally the
main method used for developing transgenic trees.
Further regeneration of plants from single cells
is a requisite for Agrobacterium-mediated gene
transfer to achieve homogenetically transformed
plants (Giri et al., 2004).
Choice of explants having competence for
transformation and regeneration is a crucial
factor. At this point in time efficient tissue
culture techniques become the foundation for
genetic transformation studies. In addition to
the regeneration through organogenesis, somatic
embryogenesis definitely offers the advantage of
single cell regeneration and currently appears to
be the most promising approach to introduce new
o o f
genes into woody tree species (Giri et al., 2004).
P r
Despite the progress in recent years, challenges
remain for the transformation of hardwood trees.
Although elite individual of hardwoods such
as poplar, eucalypts, and sweet gum can be
maintained by vegetative propagation methods,
genotypic variation in regeneration prohibits the
inclusion of many genotypes in tree improvement
programs. Even for the elite genotypes that are
propagated for clonal deployment, transformation
can be limited by Agrobacterium susceptibility or
in vitro regeneration. At the moment hardwood
transformation is largely limited to either the
clones that are easy to transform and regenerate
or juvenile materials that have higher regeneration
and transformation potential. Maturation is
another common problem. While seedling or
juvenile tree explants are relatively easy to
regenerate and transform, explants of mature trees
tend to lose regeneration potential. Unfortunately,
elite clones are selected from older trees with years
of field performance data (Nehra et al., 2005).
A limitation to the use of this technique lies
in the general recalcitrance of eucalypts to trans-
formation and poor regeneration capacity (Poke
et al., 2005). At this time, the stable transformation
has only been successful in a small number of
species including E. camaldulensis, E. globulus,
E. urophylla, E. grandis, and E. urophylla ×
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
E. grandis hybrids (Mullins et al., 1997; Ho et al.,
1998; Moralejo et al., 1998; González et al., 2002;
Tournier et al., 2003; Valerio et al., 2003). These
are predominantly laboratory studies. Field studies
of transgenic eucalypts have been reported in the
United Kingdom and Spain in 1995, in Portugal
and South Africa in 1997, and Uruguay and
Chile in 1997/1998 (Potts et al., 2001) including
field testing of glyphosate-resistant transgenic E.
grandis (Llewellyn, 2000).
Despite the clear potential of genetic engineer-
ing for improving woody plants during the last
10 years, progress has been slow on eucalyptus,
which is still considered recalcitrant. In particular,
regeneration and tissue culture is often very poor,
probably due to the high concentration of phenolic
compounds in the cells (Tournier et al., 2003) or
to low endogenous cytokinin content as shown
by Azmi (1999). Regeneration capacity is even
lower on a selective antibiotic-containing medium
making it often impossible to recover transgenic
shoots even when stable transformation is achieved
(Serrano et al., 1996; Sartoretto et al., 2002).
ge
a
P
2.2.1 Gene transfer
i r t
stransfer
F
From a scientific point of view, the
of specific genes with a known function is
very similar to classical breeding, but due to a
restricted approach, the efficiency is increased. The
prerequisites for gene transfer aiming at cultivar
improvement are as follows:
r Availability of the trait to be transferred as
cloned-DNA;
r Availability of a powerful transfer system; and
r Availability of a reliable regeneration system
predominantly from a single transformed cell.
The last two points are solved in princi-
ple, although regeneration is still a problem.
Regeneration is more an art than a science,
particularly in transformation of Eucalyptus
clones. However, recipes are available and, with
sufficient trials and the use of a range of different
genotypes, success should be achievable. Transfer
studies are predominantly via Agrobacterium,
as such transformations are more stable than
microbombardment techniques during subsequent
meiosis. Microbombardment has, however, the
advantage that it can transform meristematic cells,
9-29
therefore the regeneration of plants is relatively
easy to achieve. The most critical problem
is still gene isolation. Most of these isolated
genes are used in transformation experiments for
basic research to elucidate biochemical pathways
and add knowledge particularly in the field
of metabolomics (Wenzel, 2006). Most of the
patented genes available in the market, which are
expected to give a determinant trait, have not
been sufficiently tested in Eucalyptus. However,
patented genes for specific traits in engineered
plants have not generated improved commercial
Eucalyptus clones, yet.
2.2.2 The use of biolistics in the genetic
transformation of Eucalyptus
Microprojectile bombardment, which bypasses
the problems associated with host specificity of
Agrobacterium and regeneration systems, offers
an alternative approach for the delivery of DNA
o f
into cells that are also competent for regeneration.
o
r
Because the particle gun concept involves physical
P
introduction of DNA into the cell, it potentially
allows the transformation of any living cell,
independent of its type or localization in the tissue
(Sartoretto et al., 2002).
Serrano et al. (1996) obtained stable trans-
formation of E. globulus cells for the first time,
using biolistic DNA delivery in zygotic embryos
as the target material; however, no transgenic
plants were regenerated. Sartoretto et al. (2002)
reported a procedure for genetic transformation
of calli from a E. grandis × E. urophylla hybrid
using particle bombardment of calli derived from
cotyledon and hypocotyl. However, the calli were
unable to regenerate transgenic shoots, suggesting
that the conditions suitable for regeneration are
unsuitable for transformation and vice versa.
In spite of some interesting advances using the
particle gun delivery system, no transgenic plants
of any Eucalyptus species have been reported
using the biolistic strategy.
2.2.3 Eucalyptus transformation
using A. tumefaciens
Mullins et al. (1997) obtained stable transforma-
tion of one clone of E. camaldulensis using leaf
explants inoculated with A. tumefaciens. In this
JWBK245-Kole k0902 May 10, 2008 14:1
9-30 FOREST TREE SPECIES
work, the reporter gene β-glucuronidase and the
NPTII gene were used for selecting transgenic
tissues. Ho et al. (1998) reported an efficient system
for transformation of seedling material from E.
camaldulensis by the inoculation of hypocotyls
segments with A. tumefaciens. The work used the
Ti-plasmid vector harboring chimeric neomycin
phosphotransferase and β-glucuronidase genes.
Moralejo et al. (1998) regenerated transgenic E.
globulus plants using hypocotyls and cotyledons
from young seedlings. The explants were inocu-
lated with A. tumefaciens and the genes introduced
were GUS and NPTII. Due to confidentiality
problems, no precise transformation protocol
has been published for most of the eucalypt
species. Maunders et al. (1997), even though
the protocol is not described, reported a reliable
transformation system for several species of
Eucalyptus including commercially important E.
grandis and E. globulus, starting from either mature
elite clones or improved seed material.
Transformation of forest tree species remains
difficult, particularly within the genus Eucalyptus.
Harcourt et al. (2000) produced transgenic
ge
plants of E. camaldulensis containing both the
P
insecticidal cryA3 gene and the bar gene by A.
a a decrease in transcription. The wood chemical
Fi rst
tumefaciens-mediated transformation of seedling
explants. The transgenic lines exhibited tolerance
to the broad-spectrum herbicide and resistance
to the first instars of chrysomelid beetles. These
plants are likely to provide better insect and weed
control options in plantations, particularly during
the vulnerable establishment phase, mainly in elite
genetic background.
Chen et al. (2001) regenerated transgenic E.
camaldulensis plants of elite clones. The genes
introduced were cinnamate 4-hydroxylase (C4H)
and NPTII. The main objective of this work
was to alter the lignin content of the wood. The
transgenic plants were obtained but the wood
modification was not evaluated. For Eucalyptus
species, transformation and plantlet regeneration
are generally more efficient with juvenile materials,
such as hypocotyls, cotyledons, and leaf disks from
in vitro-germinated seedlings, than with clonally
derived material from field-grown trees that have
poor regenerability in tissue culture (MacRae
and van Staden, 1999). For forest tree species,
the true value of genetic engineering lies in its
integration into conventional breeding programs
to improve economically important traits that
cannot be modified by conventional means within
a reasonable time frame. González et al. (2002)
obtained transgenic E. grandis × E. urophylla
plants using seedling material and A. tumefaciens.
The chimeric construct contained the nptII
gene and the Lhcb1*2 gene, coding the 28 kDa
chlorophyll a/b binding pea protein from LHCII
antenna. The transformation system used the
inoculation of seeds with A. tumefaciens followed
by sonication for a few seconds. The objective
of the work was to develop a transformation
protocol and evaluate the alteration in the process
of photosynthesis.
Tournier et al. (2003) regenerated E. grandis × E.
urophylla juvenile clones with the antisense CAD
gene and NPTII like selective gene. The transgene
expression was demonstrated with high inhibition
of endogenous CAD gene, but physiological
and biochemical wood modifications were not
evaluated.
Valerio et al. (2003) developed a procedure for
A. tumefaciens-mediated genetic transformation of
o o f
a juvenile E. camaldulensis clone using antisense
P r
CAD gene. Some transgenic lines exhibited a
strong inhibition of CAD activity, associated with
analysis showed no differences in lignin quantity,
composition or pulp yield, compared to control
trees. These results underline the problems of
extrapolating genetic engineering results from a
model to a genetically distant target plant species.
Other techniques such as RNA interference-type
procedures may be used for more complete gene
suppression.
Recent developments in transgenic trees can
have multidirectional benefits. The benefits range
from manipulating generation time, plant protec-
tion, wood quality, production of compounds of
pharmaceutical value, and recovery of polluted
soils.
These successes have opened up avenues to
include agronomically more useful genes for
transfer into tree species as has been demonstrated
in crop plants (Giri and Vijaya, 2000). Following
the global trend in forestry biotechnology there
are now 24 genetically modified tree species that
have been approved for field trials. Recently, it has
been emphasized that genetically modified trees
can be excellent tools for physiological research
(Herschbach and Kopriva, 2002). The research
completed so far demonstrates the potential of
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
these techniques in the improvement of forest tree
species. One of the most important aspects of
transgenic trees is integration of the introduced
gene into the genome and its expression. For long-
lived tree species, new questions arise regarding
the stability of the integration and expression of
foreign genes. Biosafety considerations, including
the impact of transgene dispersion through pollen
and unexpected effects on nontargeted organisms,
are now receiving attention. With recent research
developments, molecular genetics provide tools
that may allow genetic improvement to make
up lost ground. If current progress in tissue
culture and genetic transformation combined with
biotechnological applications continues, the future
may witness super tree species tailored for special
agronomic and economic characteristics (Giri
et al., 2004).
Private companies claim to possess routine
transformation protocols for different Eucalyptus
species and hybrids; however, because of the
confidentiality issues, these methods are not widely
available (MacRae and van Staden, 1999).
ge
2.3
P
Eucalyptus Diseases, Control Strategies,
a intraspecific genetic variability for resistance to
Fi rst
and Genomic Approaches to Identify
Genetic Resistance
Eucalyptus cultivation in Brazil was limited only
to a few thousand hectares until the 1970s,
when eucalypts were considered to be practically
immune to diseases. The increasing demand for
forest products and the need to conserve the
native forests pushed the expansion of Eucalyptus
plantations to about 3.4 million hectares. About
1 million hectares of this are planted with
362 different clones, of both pure species and
hybrids, distributed in areas varying from 10 to
34 000 ha/clone. It is expected that within this
year an additional 250 000 ha of eucalypt forests
comprising 55 clones will be planted, occupying
areas varying from 10 to 9000 ha/clone/company.
The expansion of plantations to warmer and
more humid regions conducive to infection by
plant pathogens, the regional peculiarities of
climate and soil, combined with the possible
introduction of diseases from other countries and
adaptation of some local pathogens from native
Myrtaceae to Eucalyptus species have resulted
in the incidence of various fungal and bacterial
9-31
diseases that cause significant losses in some highly
susceptible clones. In spite of the risks, cloning has
proved to be an excellent tool for disease control.
The knowledge of Eucalyptus species and their
interactions with different potential diseases is
important to establish strategies to ensure stability
of wood production. This review summarizes the
main eucalyptus field diseases and their control.
2.3.1 Rust
The symptoms are the appearance of yellow-
colored powdery urediniosporic sporulation over
the affected organs is the typical feature for rust
diagnosis (Figures 4a–c). In highly susceptible
materials, the infection causes deformation,
necrosis, hypertrophy, mini-cankers, and death
of the apical meristems (Figure 4d). Although
the uredinial phase is more common and is the
main form of pathogen dispersal, less frequently,
teliospores can be produced, during the warmer
o o f
periods, on fully expanded leaves.
P r
Causal agent: Puccinia psidii Winter.
Control: The existence of high inter- and
rust allows for disease control by planting resistant
clones, progenies, or species. Corymbia citriodora,
C. torelliana, E. camaldulensis E. microcorys, E.
pellita, E. pilularis, E. propinqua, E. resinifera,
E. robusta, E. saligna, E. tereticornis, and E.
urophylla are important sources of resistance. In
regions, favorable to rust infection, planting of
E. grandis seeds (provenances: South Africa and
Coff’s Harbour 9583), E. phaeotricha, Eucalyptus
cloeziana, E. globulus, and E. nitens should be
avoided. There is, however, ample intraspecific
variability, which permits selection and cloning
of resistant genotypes for planting. In E. grandis,
resistance might be dominant and controlled by a
major gene (Ppr-1) (Junghans et al., 2003). Thus,
rust can be controlled through use of resistant
progenies, whose seeds are harvested from resistant
homozygous mother plants, as practiced by some
Brazilian forest industries in São Paulo and
southern Bahia. It is also possible to select trees
with rapid growth characteristics, which rapidly
escape the disease by virtue of the microclimate in
the upper canopy being unfavorable for disease
development. Similarly, selection can be made
from clones of E. globulus and related species
JWBK245-Kole k0902 May 10, 2008 14:1

9-32 FOREST TREE SPECIES

(a) (b)

o o f
P r
P a ge
Fi rst
(c) (d)

Figure 4 Eucalyptus rust (Puccinia psidii). (a) Infected apical shoots of Eucalyptus urophylla; (b) Pathogen esporulation on E.
globulus leaf; (c) Rust incidence on stem and leaves of Syzygium jambos; and (d) Necrosis and death of apical shoots of E. grandis

that rapidly pass through the susceptible juvenile
leaf stage to produce resistant adult leaves. In the
case of highly susceptible genetic materials of high
commercial value, the disease can be controlled
by systemic fungicide spray such as triadimenol
(Bayfidan 25 PM ou 25 CE) (0.5 g i.a. l−1 ) and
azoxystrobin (Amistar 500 WG) (0.1 g i.a. l−1 )
at 2 weeks intervals. In nurseries, especially in
clonal hedges and mini hedges, the disease is
controlled by fortnightly spraying of triadimenol
or azoxystrobin, using the above concentrations.
2.3.2 Cylindrocladium leaf blight
The lesions can develop at the base, at the apex, or
at the leaf margins, and can cover a large portion of
the leaf area, inducing severe premature defoliation
of the lower, middle, and apical thirds of the tree
canopy during the 1st and 2nd years after planting
(Figure 5a). It is believed that, when only the basal
or middle third of the canopy is affected, the trees
tend to recover. However, under disease favorable
conditions, the apical portion is also defoliated,
especially in highly susceptible materials, leading
to reduced plant growth. Defoliation also allows
for high light intensity penetration into the stand
leading to growth of competing weeds (Figure 5a).
Disease symptoms can vary depending upon the
species of Cylindrocladium and Eucalyptus. The
leaf spots of C. pteridis are smaller, circular, or
elongated and light gray progressing to light brown
in color (Ferreira et al., 1995) (Figure 5b), while
those of C. candelabrum, C. floridanum, C. ilicicola,
JWBK245-Kole k0902 May 10, 2008 14:1

EUCALYPTUS 9-33

o o f
(a)
P r
P a ge
Fi rst

(b) (c)

Figure 5 Cylindrocladium leaf blight. (a) Defoliation in commercial plantation of eucalyptus hybrid (E. grandis × E. urophylla),
the arrows indicate growth of weeds due to light penetration increase; (b) Smaller leaf spot, typical of C. pteridis; and (c) Larger leaf
spot, typical of Cylindroclaium spp

C. parasiticum, and C. scoparium are larger, light to
dark brown with a gray green halo (Alfenas et al.,
2004) (Figure 5c). In most Eucalyptus species, the
lesions are light or pale brown, but in E. cloeziana
they are dark brown.
Causal agents: C. candelabrum, C. floridanum,
C. ilicicola, C. ovatum, C. parasiticum, C. pteridis,
and C. scoparium.
Control: Considering the natural recovery
of infected plants during subsequent periods,
climatically unfavorable to the disease, no control
measure is currently used. However, because of
high reduction of the photosynthetic area, a
significant loss in volume is expected, which
may justify adoption of control measures to
reduce potential losses. In this case, planting
JWBK245-Kole k0902 May 10, 2008 14:1

9-34 FOREST TREE SPECIES

blight-resistant progenies, provenances, species, 2.3.4 Bacterial leaf blight

and clones is the best control strategy. The
determination of inheritance model and genetic
base is essential for a breeding program to obtain
resistant materials.
2.3.3 Rhizoctonia leaf blight
In field plantations and clonal hedges, the infection
starts on the leaves of the lower branches and pro-
gresses to the plant apex, causing intense leaf blight
and defoliation (Silveira et al., 2000) (Figure 6a).
The disease is characterized by large and irregular
leaf spots (Figures 6b and c). Depending on
the disease intensity, whitish-mycelium-covered
branches and leaves can be observed (Figure 6b),
with the possible presence of whitish or light to
dark-brown sclerotia (Figure 6d). Initially, the
affected leaves show irregular light-gray to light-
brown lesions of different sizes leading to blight
of almost all leaves that become pale in color.
At the initial phases of infection, the disease
is characterized by water-soaked translucent leaf
spots (Figure 7a), resulting from water leakage
into the intercellular spaces, and is followed by
intense defoliation, girdling, and mortality of
the apical portion of highly susceptible materials
(Figure 7b). As the disease progresses, the lesions
become necrotic and dry with perforations and
deformation of the leaf blade (Figure 7c). Its
precise diagnosis requires laboratory examination,
using exudation tests in which bacterial slime
emanates from newly formed lesions.
Causal agents: Species of Xanthomonas ax-
onopodis, X. campestris, Pseudomonas cichorii, and
others (Gonçalves, 2003).
Control: Selection and planting of resistant
genotypes.
2.3.5

o o f
Phaeophleospora leaf blight
Initially, infected leaves remain attached to the
P r
ge
Angular purple brown spots, distributed on both
plant, but tend to fall with time. Other marked
a
sides of mature leaves (Figures 8a–c), result from
P
characteristics of the disease are hanging leaves

rst
exudation of conidial masses (cirri) (Figure 8d),

Fi
attached by the fungal hyphae, adhering to one
black sporulation resembling black mold. The
another, and connected by hyphae resembling
disease is sometimes confused with the leaf
a web (web-blight) (Figure 6c). The pathogen
spots caused by Cylindrocladium, plant bacterial
survives in soil, from which it disseminates by
infection or phosphorus deficiency, especially if
water splash to the surface of lower leaves or by
observed on the upper leaf surface. However,
growing epiphytically up the trunk reaching the
typical sporulation of the pathogen is the major
higher portions of the canopy. In general, the
characteristic for diagnosing the disease. It
fungus does not sporulate, and the most important
generally occurs on old leaves of plants in the field
features are sclerotia formation along the infected
or in the nursery.
organs, right angle branching of hyphae, and
Causal agent: Phaeophleospora epicoccoides
presence of a constriction at the first septum in
(Phaeoseptoria eucalypti = Kirramyces epicoc-
the branched hyphae when observed under the
coides), teleomorph Mycosphaerella suttoniae
microscope. The sexual phase of some isolates of
(Crous, 1998).
Rhizoctonia solani (Thanatephorus cucumeris) can
Control: No specific control measure has been
be produced under controlled conditions, but is
used, but selection and planting of resistant
rarely found in natural infections (Silveira et al.,
genotypes may be effective.
2000).
Causal agents: Rhizoctonia solani (AG1-1B)
and binucleate species of Rhizoctonia not yet
identified. 2.3.6 Pilidiella leaf spot
Control: Although there are no studies about the
genetic variability for resistance, it is unlikely that Large, light brown to pale leaf spots, with
resistant genotypes will be found within eucalypt dark concentric halos (Figure 9) formed by the
species. However, artificial inoculations should be exudation of spore mass. Typical conidiophores
conducted to examine this hypothesis. and conidia of the pathogen can be observed by
JWBK245-Kole k0902 May 10, 2008 14:1

EUCALYPTUS 9-35

o o f
P r
(a)
P a ge (b)

t
Firs

(c) (d)

Figure 6 Rhizoctonia leaf blight. (a) High defoliation; (b) Leaves with lesions and pathogen mycelium (arrows); (c) Leaves with
blight symptoms stuck each other, with the fungal mycelia holding the dead leaf; and (d)Dark-brown sclerotia over infected leaves
JWBK245-Kole k0902 May 10, 2008 14:1

9-36 FOREST TREE SPECIES

o o f
P r
(a)
P a ge (b)

Fi rst

(c)

Figure 7 Bacterial leaf blight. (a) Water-soaked translucent leaf spot on Eucalyptus cloeziana plants; (b) Death of apical portion of
E. urophylla; and (c) Differences in the symptoms

microscopic examination of histological sections reduced in case of high disease severity, but
through the pycnidium. This fungus penetrates defoliation is not usually observed.
host tissues through wounds (leaf friction by the Causal agent: Pilidiella eucaliptorum
strong winds), insect (thrips, larval, and aphids), (= Coniella fragariae).
or mite injuries and also through the lesions Control: No specific control measures are
caused by other leaf pathogens as P. psidii and employed, but planting of resistant clones is the
Cylindrocladium spp. The photosynthetic area is best strategy of control.
JWBK245-Kole k0902 May 10, 2008 14:1

EUCALYPTUS 9-37

(a) (b)

(c) (d)

o o f
P r
ge
Figure 8 Phaeophleospora leaf spot. (a) Angular purple brown spot with fungi sporulation; (b) Healthy leaf and leaves with

P a
symptoms; (c) Detail of angular spots; and (d) Exudation of conidial masses (cirri)

2.3.7 Aulographina leaf spot Fi rst
Circular or elongated dark-brown corky spots
occur over the main vein, petiole, and twigs
(Ferreira, 1989) (Figures 10a and b). Superficial
dark-brown to black, elongated, curved, or
branched fruiting bodies with a longitudinal
slit (hysterothecia) are formed over the lesions
(Ferreira, 1989) (Figure 10c). Asci and the
ascospores of the fungus can be observed by
microscopic examination of histological sections
of the ascomata.
Causal agent: Aulographina eucalypti.
Control: The disease does not cause important
damage, therefore, no specific control measures are
being used.
circular to irregular circular shape and are light
to pale brown, being darker on the lower leaf
surface (Figures 11a–d). Dark ascomata, asci, and
ascospores are formed on the lesions.
Causal agents: The disease is caused by various
species of Mycosphaerella. Although it is not well
known, it is believed that M. parkii (anamorph =
Stenella parkii), M. suberosa (anamorph = not
determined), and M. suttoniae (anamorph =
Phaeophleospora epicoccoides) are the most com-
mon species on Eucalyptus in Brazil.
Control: No control measures have been
adopted in Brazil, but for species such as E.
globulus, E. maidenii, E. dunnii, and E. nitens it
is possible to select faster growing clones, which
rapidly pass to the less susceptible adult stage and,
thus escape the disease.

2.3.8 Mycosphaerella leaf spot

The fungus infects young leaves of E. globulus, E.
nitens, and E. dunnii and mature leaves of many
other species, including E. grandis, E. saligna, E.
urophylla, and their hybrids. The spots vary from
2.3.9 Cryptosporiopsis leaf spot
The pathogen infects leaves on the basal third
branches without causing significant defoliation.
The lesions are light to grayish-brown of varying
JWBK245-Kole k0902 May 10, 2008 14:1

9-38 FOREST TREE SPECIES

o o f
P r
P a ge
Fi rst

Figure 9 Pilidiella leaf spot. Leaf spot showing pathogen penetration by wounds or other pathogens lesions

sizes, semicircular or circular in shape (Figure 12a).
They are encircled by a dark halo and the center
has a dark rust colored spot of up to 6 mm
diameter (Ferreira et al., 1998) (Figure 12b).
Typical conidiophores and conidia can be ob-
served microscopically in the histological sections
of conidiomata. Like Coniella fragariae and
Hainesia lythri, the fungus penetrates the host
through wounds.
Causal agent: Cryptosporiopsis eucalypti.
Control: Since the disease does not cause
significant economical losses, no specific control
measures have been adopted. However, it is
believed that planting of resistant genotypes may
be the best strategy.
2.3.10 Ralstonia wilt
The first symptoms appear on 4- to 8-month-old
plants. Initially, the leaves show wilting, becoming
reddish, yellowish (Figure 13a), and latter pale to
dark-brown in recently dead plants (Figure 13b).
Stem section of wilted plants, exudates bacterial
pus as cream-colored drops (Figures 13c and d).
JWBK245-Kole k0902 May 10, 2008 14:1

EUCALYPTUS 9-39

(a)

o o f
P r
P a ge
Fi rst

(b) (c)

Figure 10 Aulographina leaf spot. (a) Typical symptoms; (b) Detail of corky spots over the main vein; and (c) Typical hysterothecia
JWBK245-Kole k0902 May 10, 2008 14:1

9-40 FOREST TREE SPECIES

(a) (b)

o o f
P r
P a ge
Fi rst

(c)

(d)

Figure 11 Mycosphaerella leaf spot. (a) Typical spot of Mycosphaerella suberosa; (b) Typical spot of M. parkii; (c) Lesions caused
by M. marksii; and (d) Typical spot of M. juvenis
JWBK245-Kole k0902 May 10, 2008 14:1

EUCALYPTUS 9-41

(a)

o o f
P r
P a ge
Fi rst
(b)

Figure 12 Cryptosporiopsis leaf spot. (a) Lesions on the lower (left) and upper side (right) of leaf; and (b) Detail of spot lesions

Causal agent: Ralstonia solanacearum 2.3.12 Eucalyptus canker

(= Pseudomonas solanacearum).
Control: Production of plantlets exempt of
contamination with the pathogen.
2.3.11 Ceratocystis wilt, dieback,
and canker
Initially are observed epicormic shoots along the
trunk, and depression in the bark, then dieback,
canker, wood discoloration, and wilt, leading to
plant death (Figures 14a–d, Figures 15e–g).
Causal agents: Ceratocystis fimbriata.
Control: Planting of resistant genotypes.
Infection may be observed on 6-month-old plants
(Figure 16a). When it occurs on young or adult
plants of small stem diameter or on mini-stumps
in clonal hedges, usually causes death by stem
girdling. The canker can occur at any height of
the stem, but usually occurs at the tree base (basal
canker), causing superficial or deep lesions on
the bark surrounded by callus (Figures 16b–d).
A typical canker is formed if the lesion is deep and
localized at a specific point of the trunk, while in
superficial lesions, not reaching the cambium, the
plant responds by producing new tissues resulting
in the trunk swelling and bark cracking at the
JWBK245-Kole k0902 May 10, 2008 14:1

9-42 FOREST TREE SPECIES

(a) (b)

o o f
P r
P a ge
First

(c) (d)

Figure 13 Ralstonia wilt. (a)—Plant with wilt symptoms and basal defoliation; (b) Dead plants; (c) Symptoms in the stem section
immediately after cut; and (d) Exudates bacterial pus (arrows)

infection point (Figure 16e). The weakened trunk
at this point can break (Figure 16f), especially in
regions of strong winds. Dark pycnidia and or
perithecia produced on dead bark are the signs
of the disease, essential for unequivocal diagnosis.
Causal agent: Chrysophorte cubensis (= Cry-
phonectria cubensis = Diaporthe cubensis =
Endothia eugeniae).
Control: Planting of resistant species, prove-
nances, families, or clones. C. citriodora,
JWBK245-Kole k0902 May 10, 2008 14:1

EUCALYPTUS 9-43

(a) (b)

o o f
P r
P a ge
Fi rst

(c) (d)

Figure 14 Dieback, canker, and wilt caused by Ceratocystis fimbriata. (a) Development of basal shoots due to veins colonization
by the fungus; (b) Natural infection showing dieback; (c) Dead plant; and (d) Canker
JWBK245-Kole k0902 May 10, 2008 14:1

9-44 FOREST TREE SPECIES

(e) (f)

o o f
P r
P a ge
Fi rst

(g)

Figure 15 Continue from Figure 14. Transversal (e) and radial (f) xylem discoloration symptoms; and (g) inoculated plant with wilt
symptoms (left), and detail of stem discoloration (right)

C. torelliana, E. cloeziana, E. pilularis, E.
paniculata, E. pellita, E. urophylla, E. robusta,
E. resinifera, and E. microcorys are the more
resistant species, while provenances of E. grandis
and E. saligna are the most susceptible. However,
there is a high intraspecific variability as to
resistance, which allows for selection and cloning
of resistant genotypes for planting. In clonal
hedges, canker can be controlled by selective
shoot harvesting that reduces the stress on
the mini-stumps, avoiding predisposition to the
disease.
JWBK245-Kole k0902 May 10, 2008 14:1

EUCALYPTUS 9-45

(a)

o o f
P r
P a ge
(b) (c)
Fi rst (d) (e) (f)

Figure 16 Eucalyptus canker caused by Chrysophorte cubensis. (a) Scattered death of Eucalyptus saligna plants in the field; (b)
Typical canker without bark removal; (c) Typical canker (deep lesion flanked by callus); (d) Basal canker; (e) Trunk swelling, and
bark cracking on the infection point; and (f) Plant with canker, broken by wind at the infection point

2.3.13 Pink disease or rubelosis Control: Selection and cloning of resistant

Lesions and girdling are observed on the stem and
branches of 1- to 3-year-old plants. A pink colored
mycelium grows on young lesions (Figures 17a
and b). Epicormic shoots emerge from below the
girdled portion of the stem (Figure 17a). Later,
the lesions dry out and lose the typical color
(Figure 17c), leaving behind cankers (Figure 17d)
on the thicker nongirdled stem and branches.
The stem may break and lose apical dominance
(Figures 17e and f). Pink to salmon colored
mycelium, containing basidia and basidospores
may be observed on the lesions.
Causal agent: Erythricium salmonicolor (=
Corticium salmonicolor).
genotypes.
2.3.14 Coniothyrium canker
Small discrete necrotic lesions with a strong
depression in the bark are formed along the trunk
(Figures 18a–c). The infection occurs through
the younger tissues of the green stem. In highly
susceptible genotypes, the lesions coalesce and
cause extensive necrosis, generally followed by
kinopocket formation (gummosis) (Figure 18d)
and reduced plant growth, dieback, and emission
of epicormic shoots along the trunk, due to partial
cambium death and stem girdling. The fungus
JWBK245-Kole k0902 May 10, 2008 14:1

9-46 FOREST TREE SPECIES

(a) (b) (c)

o o f
P r
P a ge
First
(d) (e) (f)

Figure 17 Pink disease or Eucalyptus rubelosis, caused by Erythricium salmonicolor. (a) Pink mycelium and epicormic shoots
below infected area; (b) and (c) Stem lesion showing mycelium growth and bark cracking; (d) Typical canker; (e) Trunk breaks on
the infection point; and (f) Trunk break and lose apical dominance

produces globose and substomatal pycnidia,
containing conidiospores and conidia.
Causal agent: The disease, first described in
South Africa, was attributed to Coniothyrium
zuluense (Wingfield et al., 1997). In Brazil, the
disease was found on E. grandis and attributed
to Coniothyrium sp. (Ferreira, 1997).
Control: In Brazil, it has not caused serious
damage to the affected plants, thus no control
measures have been adopted.
Generally, the lesions are superficial and confined
to the bark region, showing fructification of
the pathogen. Pseudothecia containing asci and
ascospores are formed on the lesions.
Causal agent: Botryosphaeria ribis.
Control: No specific control measures have been
adopted.
2.4 Sources of Disease Resistance and
Breeding Strategies

2.3.15 Botryosphaeria canker Considering that a relatively small number of

The infection occurs in young tissues, resulting
in breakage with the forking of the main stem
at the infection point, gum exudation, darkening,
depression, and bark cracking (Figures 19a–f).
clones, possibly with narrow genetic base, are
planted in some regions of Brazil, it is imperative
to establish an interspecies breeding program to
obtain new resistant genotypes. Predicting the
eventual dry periods and predominance of high
JWBK245-Kole k0902 May 10, 2008 14:1

EUCALYPTUS 9-47

o f
(a) (b)

P r o
P a ge
Fi rst

(c) (d)

Figure 18 Coniothyrium canker. (a), (b), and (c) Necrotic lesions with a depression in the bark; and (d) Details of the xylem infection
with kinopocket formation
JWBK245-Kole k0902 May 10, 2008 14:1

9-48 FOREST TREE SPECIES

(a) (b) (c)

o o f
P r
P a ge
Fi rst

(d) (e) (f)

Figure 19 Botryosphaeria canker. (a) Canker; (b) Depression and bark cracking; (c) Gum exudation; (d)—Break of apical trunk;
(e) Forking of main stem in the infection point; and (f) Details of superficial infection in the bark region

temperatures, drought resistant species, such as E.
camaldulensis, should be considered for crossings.
Other species, such as E. pellita (Papua New
Guinea provenance) and E. urophylla, are excellent
source of resistance to canker. The former is
also an important source of resistance to leaf
blights. On the other hand, E. globulus can be
used as a gene source for reducing the lignin
and extractive contents, increasing wood density,
improving pulp yield, and fiber quality, while E.
grandis (Atherton or Coff’s Harbour) has high
adaptability and is suitable for high cellulose yield.
Introgression of genes of E. globulus should be
carried out using pollen obtained from elite mother
plants from the southern part of Brazil, Uruguay,
Spain, Chile, Portugal, or Australia, where it
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
is possible to cultivate these plants as a pure
species. In the final composition, hybrids should
contain 50–75% genes of E. grandis, obtained
by a series of crossing and back crossings, and
selfings if necessary. However, E. deglupta, E.
resinifera, and E. robusta can also be tested,
although little is known about their resistance
to diseases, fiber quality, and adaptability. Since
E. globulus and E. pellita have contrasting
characteristics, for example, disease resistance, site
adaptability and extractives, and lignin content,
they should contribute a maximum of 12.5%
of genes in constituting interspecific hybrids.
Since wood lignin and extractives have essential
functions in defense mechanisms, reduction of
their concentrations can potentially result in
higher susceptibility. Thus, in each generation
of crosses, the resistant genotypes should be
selected through artificial inoculation with specific
pathogens of interest for each region. It is also
essential to determine the resistance of commercial
clones and its genetic basis to different diseases
such as stem canker, rust, leaf blights (fungal
and bacterial), ceratocystis wilt, and bacterial
ge
wilt through artificial inoculations of controlled
P
crossing progenies. When resistance is dominant,
a contains approximately 15 000 Eucalyptus ESTs
Fi rst
resistant homozygous mother plants should be
used for seed harvesting, since irrespective of the
pollen origin, the progeny should be resistant.
2.4.1 Genomic approaches to identify
genes for rust resistance
Currently, one of the biggest threats to Eucalyptus
plantations is neotropical rust caused by the
biotrophic fungus P. psidii. This disease attacks
young trees, normally younger than 2 years old,
thus it principally affects nurseries and recently
planted areas, and depending on the severity
of the infestation can result in a significant
to almost total loss of production. In order
to investigate the genes differentially expressed
during P. psidii infection, Moon et al. (2007)
constructed two SAGE (serial analysis of gene
expression, Velculescu et al., 1995) libraries
representing susceptible and resistant material.
Susceptible and resistant individuals were selected
from a segregating population of half-siblings of
E. grandis (Suzano Papel e Celulose), after being
naturally infected with P. psidii Winter under field
9-49
Table 4 The percentage of transcripts for the identified
genes within each functional category (Rison et al., 2000)
Susceptible Resistant
Category library (%) library (%)
Metabolism and energy 4.4 20.1
Processes 32.3 31.4
Transport 1.1 2.3
Structural and organization 4.8 16.5
of structure
Information pathways 19.3 1.7
Unknown function 38.1 28.0
conditions. Bulks representing each phenotype
were formed using ten susceptible and ten resistant
individuals selected from an E. grandis segregating
population of half-siblings (Suzano Papel e
Celulose). 31 645 and 39 964 tags, representing a
cumulative gene count of 4095 and 5213, were
generated from the susceptible and resistant bulks,
respectively. The Z-test indicated that 239 were
preferentially expressed in the susceptible library
o o f
and 232 in the resistant. Using the National Center
P r
for Biotechnology Information (NCBI) public
database (http://www.ncbi.nlm.nih.gov), which
and cDNAs, the authors were able to associate
nucleotide sequences to 40 and 72 tags, susceptible
and resistant, respectively.
Table 4 shows the differences in the number of
transcripts within each functional category for the
differentially expressed genes for the susceptible
and resistant libraries. It can be seen that in the
susceptible library the expression of genes involved
in metabolism and the production of energy is
considerably reduced showing about 25% of the
level observed in the resistant material, probably
reflecting the generally debilitated state of the
infected susceptible plants. Genes preferentially
expressed in the resistant library in this category
were associated with the metabolism of sugar
nucleotides and carbohydrates, indicating a higher
biosynthetic activity directed toward the produc-
tion of structural components.
Another interesting category was structural
and organization of structure, which represents
the genes involved in cell wall synthesis and
cytoskeletal organization. This category was
reduced in the susceptible material, approximately
33% of the level observed in the resistant library.
Genes involved in the synthesis of the cell wall were
JWBK245-Kole k0902 May 10, 2008 14:1
9-50 FOREST TREE SPECIES
preferentially expressed in the resistant library,
including those associated with cellulose and lignin
biosynthesis. We also observed genes involved in
the formation of the cytoskeleton, the tubulins,
preferentially expressed in the resistant material.
Although the number of transcripts produced
for the processes category was almost the
same in the two libraries, the genes that they
represent show differing pictures in the presence
of the pathogen. In the susceptible library, the
preferentially expressed genes demonstrated a
response to oxidative stress and general stress
response proteins. However, the resistant material
showed the induction of genes more specifically
for defense responses, including a catalase and a
kinase.
The category representing the information
pathways was almost ten times more expressed in
the susceptible than the resistant material. These
genes were associated with ubiquitin regulated
protein turnover and proteases were mainly
expressed in the susceptible library indicating a
very active protein degradation system.
From these preliminary results Moon et al.
ge end-uses
(2007) suggest that two completely different
P a
processes are occurring in the susceptible and
First
resistant plants. Firstly, from the generalized
lesions observed in the susceptible material, itEucalyptus wood has gained a respectable position
is obvious that plant is fighting a loosing battle
against the pathogen and is trying to limit the
oxidative damage within the infected leaves and
active proteolysis is occurring within the areas
surrounding the necrotic lesions. Secondly, in
the resistant plants various distinct processes
are occurring simultaneously contributing to the
expression of the resistant phenotype. Primary
metabolism shows an increase in the expression
of genes involved in the production of the
raw materials for cell wall formation, sugar
nucleotides, and carbohydrates. Coupled to this is
the preferential expression of the genes associated
with cellulose biosynthesis and the principal genes
involved in lignin biosynthesis and polymerization,
most probably re-enforcing the cell wall structure
making fungal penetration more difficult. The
preferential expression of genes involved in the
formation of the cytoskeleton and vesicular trans-
port factors would also indicate that the process
of cellular polarization is occurring (reviewed
by Schmelzer, 2002). Cellular polarization is
defined as the process of cytoskeletal organization
that allows the translocation of the cell nucleus
and transport of vesicles containing cell wall
matrix material or specific defense related proteins
directly to the site of infection. This process is
documented as being an important process during
the host cell response to pathogens. The authors
also observed catalase and a kinase, preferentially
expressed in the resistant material, probably
involved in signal transduction during the initial
infection and later in the process of systemic
acquired resistance. Unfortunately, they were
unable to identify any gene specifically involved
in the destruction of the fungal pathogen, such as
chitinases, thaumatins, or R genes, probably due
to the lack of publicly available Eucalyptus ESTs
that represent these genes. The authors suggest that
sustainable resistance to P. psidii in E. grandis is the
result of more than one predominant gene.
2.5 Wood Quality and the Transcriptome
Involved in Eucalyptus Wood Formation
o f requirements for pulp
oquality
2.5.1
r
P and paper, charcoal biomass fuel
Wood
as raw material for several utilizations. Thanks
to an excellent forestry technology, which was
developed in countries like Brazil, Portugal, South
Africa, Spain, Chile, Uruguay, and Australia,
Eucalyptus has reached the status of “super-tree”.
In a historical first moment, the efforts coming
from tree breeding and silvicultural techniques
were directed to the production of volume and/or
weight in the trees. The acquired productivity
would provide the desired competitiveness to
the wood-based businesses. Wood-specific unit
costs and forest operation costs were dramatically
reduced by these technologies. Thanks to the high
productivity of the planted forests and to the low
production costs, the wood of Eucalyptus soon
gained importance with several industries, such as
pulp and paper, charcoal for steel manufacturing,
lumber, and furniture. This raw material could
easily supply fibers and biomass fuel, in unbeatable
conditions. In a short period of time, the world
quickly became surprised and enchanted with
Eucalyptus for its fast growth rates and wood
quality. The quality targets are becoming more
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
and more sophisticated for each of the wood
utilizations. In the beginning, the selection of the
raw materials was based on productivity (volume
or dry weight), yields in the manufacturing
conversion processes, and low wood costs. Today,
the needs for product differentiation and for
adding value in the production chain have oriented
wood and trees to new requirements. Besides
the traditional needs for productivity, yields, and
costs, there are two new and important wood
requirements: tree and wood uniformities. The
control of forest variability may be seen as a
simple task, but it is tremendously difficult. The
anticipation using the cloning techniques was
very high with the aim of controlling variability.
However, the dream of having very similar trees has
not been realized. Although a lot more uniform,
the cloned trees show a considerable level of
variability. This happens in the trees and in the
wood quality (anatomical, physical, and chemical
properties).
Currently, the foresters have placed attention on
the production of trees having good shape, straight
trunks, higher shape factors, and low percentage
ge
of bark. Additionally, the foresters also consider
P a
the resistance of the trees to pests and diseases,
Fi rst
and tolerance to detrimental weather conditions
(frost, winds, water deficits in the soil, flooding,
etc). All these issues are demanding a lot of
attention and research. The target for high quality
Eucalyptus planted forests is not that simple.
Even being healthy, with good shape and fast
growth, the trees may not be suitable to some end-
uses. For example, E. grandis trees, with fantastic
growth rates, and tree shapes, may not be well
suitable to the production of charcoal. The wood
density may be too low for this purpose, the logs
become very cracked when dried and the charcoal
would be poor in density, in calorific value per
volume, very bulky, and generate more fines during
manufacturing and handling. On the other hand,
trees of Eucalyptus robusta may give low pulp yield
in the conversion to kraft pulp due to the excess of
extractives and/or lignin.
In short, now it is just the right time not only
to have forests and trees, but also with end-use
oriented qualities. A very good forest oriented
for the production of charcoal will not be so
desirable for kraft pulping, since both utilizations
have opposite requirements in terms of wood lignin
content. Whereas for charcoal, the best wood
9-51
quality is the one with very high lignin content
and wood density but for kraft pulping, lower the
lignin content the better. The wood density for
pulping cannot be very high due to the problems
with liquor impregnation.
Although the knowledge for the required
wood characteristics is well understood by most
researchers, the truth is that we are still finding
many mistakes in the way breeding programs being
performed. The major mistake is the precarious
sampling procedure being used in the majority of
the tree and wood quality improvement programs.
As far as the variability remains high for many
qualitative parameters, even for cloned forests,
the size of sampling should be larger than is
generally used. Additionally, the sampling should
be more representative, collecting samples from
all segments of the population according to their
frequency. Currently, there is a trend to take few
trees with the average volume. Average sized trees
are not synonymous of trees with average basic
density, or average lignin content, etc. The first
o o f
recommendation has to be made related to trees
P r
and wood sampling. Definitively, the size and
the representativeness of these samples need to
be improved. The second recommendation is to
increase the number of analyses, the number of
replications for each required quality parameters
being evaluated. In general, the analyses are
made with a single measurement and the mean
value is used in the tree and wood improvement
program. Depending on the statistical error that
the forest breeder is willing to allow, the numbers of
replications have to be considerably higher. There
is a chance that the breeder is accepting what is
accepted as a good genome but is definitively is
not good for a given property of the tree or wood.
This is something that needs to be immediately
reevaluated.
2.5.2 Eucalyptus wood quality requirements
for the production of kraft pulp
Independent of the pulp mill, the pulp sector has
fundamental issues including high productivity,
high operational efficiency (no losses, no problems,
no breaks, and no stops), low production costs,
and uniform quality of the process and products.
For achieving these targets, the raw material must
be as uniform as possible so as not to cause
JWBK245-Kole k0902 May 10, 2008 14:1
9-52 FOREST TREE SPECIES
strong impacts on the pulping process and pulp
qualities. To reduce this variability, the pulp mill
engineers blend woods. Blending very different
wood is acceptable if the result has a reasonable
average quality. This is not an ideal situation, but
it is the most usual.
When the pulp maker asks for uniform wood
he is not only referring to basic wood density,
but also to a series of wood quality parameters
that are very important in the conversion process:
proportion of bark in the wood chips, wood
chip dimensions, decay level of the wood, wood
moisture, wood chip bulk density, etc. The
objective is to have a cooking and bleaching
operation with the minimum variability, without
undesirable surprises. The final quality must be
uniform and within the specification limits and
the process losses should be minimal. When the
mill manager standardizes the wood intake, the
first thing is to guarantee an appropriate quantity
of dry wood being fed to the digesters. It is
important to keep chip bulk density as uniform
as possible for continuous addition of the same
dry weight of wood to the digesters. The mill
ge
manager is carrying out “management of the wood
P a
dry quantity intake to the mill.” When a uniform
First
flow of wood dry weight is guaranteed, the liquor,
steam, and chemical flows do not sharply change
and the process runs smoothly, and the process and
product quality is more easily achieved.
The second type of management is “manage-
ment of wood variability” that tries to guarantee
a low variability of wood parameter such as
wood basic density, lignin content, extractives
content, and active alkali consumption during
kraft cooking, bleaching chemical consumption,
yields in the conversion of the wood to bleached
pulp, etc. There are other associated goals, such as
to minimize the overload of dry solids to aid boiler
turnover; reduce the specific wood consumption in
cubic meters of wood by air-drying metric tons of
pulp; guarantee stable quality and to reduce the
production costs.
Management of dry quantities and management
of wood variability are the basic requirements
in any pulp mill. Having fulfilled these needs,
the next type of management is “management
for product differentiation” or “tailor making
orientation in the manufacture of products.”
This type of management requires substantial
changes and offers important challenges to the
mill personnel. The changes may happen in wood
quality (for example, low and high basic density
woods), process conditions (for example, ECF or
ECF-Light bleaching sequences), or even other
recently added qualities (certified or noncertified
wood). Differentiation of products is more easily
achieved in mills with more than one fiberline. This
means that the mill may run each fiberline with a
differentiated product, without experiencing the
usual troubles in making the transition from
one product to another using a single fiberline.
Anyhow, the tailor-making concept only will be
winner when the pulp maker has guaranteed the
two first mentioned types of management: dry
quantity and variability. It is very simple to say,
but very difficult to understand and to implement.
Conflicts and misunderstandings are frequent
between commercial, production, and product
innovation areas in a pulp mill. Each of these areas
has their own needs, product uniformity, product
uniqueness, and product differentiation. As a
result, few Eucalyptus pulp mills have products
o o f
that may be said to be completely differentiated
P r
in their products portfolio. Most of the pulp
manufacturers aim to have a single product, as
uniform as possible, with the minimum cost,
and maximum in productivity and in operational
efficiency.
It is relatively difficult to say what is the
single most important wood characteristic for
a given pulp mill. The reason is that there is
not a universal wood property to be managed.
Depending on the pulp mill bottleneck, wood
quality is defined to guarantee the maximum
performance of a particular mill. The most
common bottlenecks are the digester capacity,
recovery boiler, drying machine, lime kiln and
alkalinization, pulp washing, pulp bleaching
chemicals. As a conclusion, it may be said that
the type of mill bottlenecks will define the most
desirable wood quality. This is the case for
existing mills. For new greenfield mills, the quality
may be previously built at the forest. However,
soon the mill starts up, and the bottlenecks will
appear to define the new wood quality standards.
This is the reality, no doubt about it. This is
also the cause of domestic conflicts within the
company.
A list of important properties of Eucalyptus
wood that influence the kraft pulp production
process is presented.
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
r Wood cleanliness. This is an item not clearly
understood by many forest managers, who
try to mechanize their operations to reduce
costs and do not realize the impact on wood
cleanliness, which in turn affects pulp mill
efficiency. The wood be as clean as possible
in terms of bark, soil, leaves, stones, decayed
wood, etc.
r Wood basic density. To the pulp maker, the
uniformity of wood density is very important.
The more uniform is the wood, the better and
simpler is the management of the quantity and
variability. In many mills the design capacity
allows the use of light woods, which are
easier to be impregnated with cooking liquor
and demand a lower alkali charge. However,
when the digester is the bottleneck, denser
wood is used to raise mill production in the
digester. Again, the kind of bottleneck is the
source of specifications for the wood quality.
When the mill solves the bottleneck using
wood quality, normally it is the forest breeders
who need to produce wood with the desired
specification, sometimes simply by changing
ge
clones or searching for new clones that fit
P
the specifications. Pulp mills are short-term
a and pulp yield should be significant. As a
Fi rst
oriented and forest breeders are long term. The
conclusions and behaviors are very different
in both cases. Depending on the bottleneck
experienced, some mills prefer to use low-
density wood (0.40–0.45 g cm–3 ), other medium
(0.50–0.55 g cm–3 ), and others high density
(0.55–0.60 g cm–3 ).
r Active alkali consumption and pulp yield.
These variables are a consequence of the wood
characteristics such as lignin, extractives, ash,
density, Eucalyptus species, tree age, etc. The
pulp maker wants to manufacture more and
better, without overloading the production
process. For this reason, the wood quality
improvement programs need to be oriented by
processing qualities in pulp manufacturing.
r Specific wood consumption (m3 /adt = cubic
meters of wood by air-dry ton of pulp). This
consumption is the result of many intercon-
nected wood and kraft process variables: wood
basic density, pulp yield, decay of wood, process
losses of fibers, wood chipping operations,
etc. The specific consumption of the wood
is responsible for important fraction of the
pulp cost. Wood is the main component in
9-53
the pulp production cost, even in low-cost
wood countries. For this reason, it is one of
the most vital indicators to the mill manager.
All qualitative wood characteristics that may
impact the specific wood consumption should
be optimized.
r Lignin content and lignin type. Lignin is
abundant in Eucalyptus wood, especially those
planted in Brazil. The total lignin content in
Brazilian Eucalyptus wood varies from 24%
to 32%, relatively high for hardwoods. This
affects the pulp yield in the conversion to
kraft pulp, as well as the consumption of
active alkali and dry solid generation in the
recovery boiler. There are Eucalyptus species
with lower lignin contents and as such are more
appropriate for pulping operations. E. globulus
and E. dunnii when compared to E. urograndis,
E. urophylla, E. grandis, and E. saligna, offer
woods of 2–8% less total lignin (based on
dry wood). For this reason, tree breeders are
trying to combine in hybrids for cloning these
o o f
wood characteristics in association with the fast
P r
growth rates from other species. Thus, gains
in wood-specific consumption, alkali charges,
rule, for 1.2–1.5% reduction in lignin, the
kraft pulp yield increases 1% based on dry
wood. The active alkali consumption reduces
around 0.2–0.3% for the same lignin reduction.
Therefore, the production of low lignin clones is
a new challenge for hybridization and cloning,
without the need for genetic modification to
insert genes for low lignin in the wood. At the
same time, in Portugal, the need to maintain
the purity of E. globulus and to improve the
forest productivity should be possible through
genetics and silviculture. With this in mind,
the Portuguese foresters are willing to improve
the productivity of E. globulus in equivalent
tons of pulp per hectare per year. In Brazil,
Eucalyptus commercial forests produce from
9 to 15 adt ha–1 year–1 , while in Portugal the
E. globulus forests produce 6–8 adt ha–1 year–1 .
Even with lower growth rates, the E. globulus
trees are offering a competitive raw material
thanks to the better basic wood density,
lignin content, and pulp yield. However, not
only the quantity of lignin in the wood is
important, but also its quality. Lignin with high
syringyl/guaiacyl ratio offers easy cooking and
JWBK245-Kole k0902 May 10, 2008 14:1
9-54 FOREST TREE SPECIES
pulp bleaching because guaiacyl type lignin
is more difficult to be removed during the
chemical reactions. This ratio in Brazilian
Eucalyptus varies from 2 to 3, but in Portuguese
E. globulus it may vary from 2.5 to 6.
r Extractives content. Extractives are undesir-
able in the pulping process because they have
an impact on pulp yield and contaminate the
process and the final product with pitch. The
total extractives content in the Eucalyptus
wood varies as a function of species, age,
silvicultural stresses, pests, and diseases, etc.
Values are variable from 1.5% to 6%. Extractive
can be removed in several ways, normally
extracting them with a solvent (water, caustic
soda, ethanol, toluene, dichloromethane, etc.).
In all cases, the lower the extractives, the better
is the pulping, bleaching, and cleanliness of the
final product.
r Ash content. Wood is rich in mineral elements
and these are absorbed by the trees as
nutrients. When the wood is harvested, the
minerals are exported from the soil through
the trees to the mills. Minerals are measured
ge
in the wood as ash content, after burning the
P
sawdust of the sampled wood. Ash content in
a costs.
Fi rst
Eucalyptus wood varies from 0.3% to 1.0%,
the most important minerals being calcium,
potassium, and magnesium. With the trend
to use closed water cycles in the pulp mills,
these minerals are able to build up in these
systems, bringing enormous problems with
incrustations, pitch formation, formation of
“stones” in the recovery boiler, etc. Ash content
is very variable among the Eucalyptus species
and for this reason it is an important parameter
for tree breeding. When low ash woods are
selected, the exportation of nutrients from the
soil is minimized. The trees are more efficient
in wood formation using or immobilizing fewer
minerals in their tissues. These minerals, also
known as nonprocess elements, leave the pulp
mills as pollution (solid residues, air pollution
particulate or dust, and dissolved ions in
liquid effluents). This means that a fantastic
natural resource is transformed into pollution,
in mills not oriented to prevent or to develop
clean production techniques for controlling
and recycling these minerals. For this reason,
the emphasis today is to prevent the intake of
minerals into the mills, both those in the wood
composition, or as contaminants (soil, stones,
etc.). Bark is also very rich in ash (about 5–10
times richer in ash content than wood), thus
the contamination of logs and chips with bark
brings additional amounts of minerals into the
milling process affecting pulp making.
2.6 Eucalyptus Wood Quality Requirements
for the Production of Paper
All these wood characteristics and related
consequences mentioned till now favor the pulp
mills in their targets for productivity, costs, and
efficiency. However, they are only part of the wood
features to be evaluated. Eucalyptus pulp is a raw
material for the manufacture of several grades
of papers. For each paper grade and for each
paper mill design, different are the wood and pulp
quality requirements. It is important to mention
that, independent of the type of paper to be
manufactured, all the paper makers have what are
o o f
known as basic physiological needs. These needs
P r
are similar to those for the pulp manufacturer:
productivity, operational efficiency, quality, and
Productivity requires a high-speed paper
machine, fast drainage at the wet end, high
consistency after the wet presses, and minimum
number of paper sheet breaks along the machine.
Quality implies in maximum percentage of paper
in the specification range and minimum generation
of broke. Machine operation efficiency is the
dream of any paper manufacturer. He wants
his machine working smoothly, at the maximum
speed as possible, no breaks, and achieving the
required quality in the manufactured products.
The consequence of all this is that the specific
unit cost is also here optimized. No doubt that
a good pulp is the one able to provide good paper
machine runnability and appropriate quality in the
end product.
Some of the desired pulp properties are closely
related to these performances and directly related
to wood quality, others depend on the conversion
of wood to pulp (cooking, bleaching), and many
are a combination of these two factors influencing
the pulp quality. For example, some properties
that are related to pulping and bleaching are
viscosity and degradation of cellulose chains,
fiber deformations and individual fiber strengths,
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
surface charges in fibers, etc. One very important
pulp property that is related to wood quality and
pulp conversion is the hemicellulose content in
the pulp. This parameter depends on the wood
content and the ability of the pulping process to
preserve it in the fibers.
There are many pulp properties that are
dependent both on wood quality and on
pulping/bleaching processes. There are also many
cases where the exigencies are placed a lot on the
wood quality, when the wood is not the only factor
to determine the pulp quality for paper. Wood
quality affects properties such as WRV—water
retention value, WWS—wet web strength, fiber
bonding, and individual fiber strength. However,
other pulp properties are directly related to
pulp manipulation processes, for example, the
fine content of the pulp are generated in
operations such as wood chipping, pulp pumping,
pulp dynamic mixers, pulp dewatering presses,
etc.
As a rule, there are some physiological
properties that any pulp has to fulfil to be
acceptable to papermakers. They are related to the
ge
following:
r
P a
rst
Drainage at the wet end section in the paper
Fi
machine. This behavior is very much affected
by the fiber population (number of fibers per
gram of pulp), by the initial or refined pulp
freeness (drainability of the pulp measured
as Canadian Standard Freeness or Schopper
Riegler degree), by the Water Retention Value
(hydration and swelling ability of the pulp
furnish), and by the fine content in the pulp
furnish.
r Paper sheet strength along the paper machine,
mainly at the wet end and press section.
Sheet behavior is very much dependent on
the individual fiber strength, fiber bonding,
furnish contaminants (shives, sand, solid
debris, etc.) and consolidation of the paper
web. Individual fiber strength is related to fiber
wall thickness, fibril angle, fiber deformations,
and micro-fractures and the Eucalyptus species
used.
As long as the basic physiological needs are
achieved it is possible to differentiate the product
to be supplied to different markets or customers.
One of the most important ways to reach
differentiated products is through the production
9-55
of wood with different qualities providing very
different pulp fibers allowing the manufacture of
different products. This is what is known as to
tailor making the wood to the end product. The
following wood and pulp quality parameters are
important drivers in the differentiation of paper
products:
r Fiber population or the number of fiber per
gram of pulp. The fiber population is related
to the weight of each individual fiber, to
fiber coarseness and to the percentage of
fiber wall in the fiber volume. There are a
number of fiber properties associated to fiber
population and fiber coarseness: fiber wall
fraction (ratio between cell wall thickness and
fiber ray), Runkel index, fiber flexibility index
(ratio between the lumen diameter and fiber
diameter), index of fiber collapsibility, ratio
fiber wall thickness and fiber perimeter, wood
basic density, and fiber length. Pulps with lower
fiber population show better drainage in the
o o f
wet end and the paper sheets are more porous,
P r
bulkier, more permeable, and absorbent. They
are very much appreciated by paper makers
because they allow faster machine speeds, if
they furnish enough strength to the wet paper
sheet.
r Individual fiber strength. This fiber characteris-
tic is very difficult to be measured in short fibers
such as those from Eucalyptus. There are tests
correlated to this parameter such as the zero
spans, very useful for predicting pulp quality
and behavior in the pulp machines.
r Fiber bonding ability. This test is measured
by the wet/dry zero span technique or by
other equipment for bonding tests, such as
the Scott bond tester. It is also related to
the hemicellulose content of the fibers, fiber
population, fiber drainability (CSF or SR),
fiber fines content, and fiber collapsibility.
r Fiber swelling. This fiber property is affected
by the pulping and bleaching operations
during pulp manufacture and by the pulp
hemicellulose content. Several properties are
associated to the swelling of fibers: water
retention value, fiber charges, fiber wall
microporosity, and fiber wall microfractures.
r Fiber deformations. The deformations in the
fibers are measured as curl index, fiber kinks,
fiber latency, and fiber microfractures in the
JWBK245-Kole k0902 May 10, 2008 14:1
9-56 FOREST TREE SPECIES
cell wall. They affect fiber strength, but they
provide substantial improvements in the paper
sheet porosity, bulk, smoothness, and water
absorption.
Eucalyptus pulps are special products in the
manufacture of bulky and/or opaque papers.
Today, Eucalyptus pulps are preferred raw
materials in the manufacture of tissue, printing
and writing, carton boards, industrial filter,
impregnation based, cigarette, and many other
papers. Eucalyptus fibers may be the sole fiber in
the pulp furnish or to be part of a blend with other
short and/or long fibers.
Tissue papers demand softness, smoothness,
absorption, bulk, and the exact strength to provide
machine runnability and very fast drainage in
the wet end. The fibers cannot collapse because
this will flatten the paper surface, the paper
becomes stronger in tensile, but all the tactile
properties are lost. Pulp fines are also undesirable
for two reasons: fiber bonding and building
up in the paper machine white water system,
reflecting in drainability losses. The most indicated
ge
Eucalyptus fibers for tissue manufacture are those
showing low fiber population and consequently
P a
Fi rst
high coarseness, low fine content, low bonding
ability, low hemicellulose content, high bulk,
and water absorption in the manufactured paper
sheets. Fiber deformations are also important,
since these deformations improve the bulk,
porosity and absorption of the paper. It is
important to remember that fiber deformations
may be artificially created in the pulp mills.
The manufacture of industrial filter papers and
impregnation-based papers demand the same
properties, but at different levels. This means, to go
to these specialty paper markets, the differentiation
must be even more pronounced. Thus the simplest
way to move between very specialty markets
is to work toward very high coarseness (low
fiber population, high wood basic density), low
hemicellulose content, and to intensify fiber
deformations (by high consistency presses, fiber
shredding, or pulp flash drying).
For printing and writing papers, the desirable
paper properties are formation, paper strength,
porosity, dimensional stability, and opacity. A
higher fiber population provides improved opacity
associated with lower fiber coarseness. Fiber
bonding and hemicellulose and pulp fine content is
important to improve strength. However, there are
limits depending on each paper machine system
and operation. A very high fiber population may
improve opacity and formation, but drainage at
the wet end and consistency after wet presses may
deteriorate and machine speed is reduced. Fiber
deformation may not be so important, but may
help to balance the pulp properties, since it may
be created by machines. A higher hemicellulose
content favors refining, bonding, consolidation of
the paper web and strength properties (tensile,
burst, tear, folding). An ideal pulp should have
high strength at the low levels of refining. However
pulp refining raises energy costs, reduces the
life of refiner discs, reduces machine drainage
and machine speed, increases steam consumption
and a very important paper property that is
dimensional stability. Definitively, the best pulps
are those showing good strength at low levels
of refining. Paper maker is very sensitive to this.
Besides these properties, there is another wood
anatomical characteristic that is very important to
o o f
printing grade paper: vessel element content and
P r
vessel dimensions (especially the diameter). Large,
wide, and numerous vessels are undesirable for
P&W (printing and writing) papers giving a defect
known as vessel picking. The paper maker needs
to have special conditions to combat the vessel-
picking tendency in the paper. Thus, wood with
smaller and less numerous vessels is preferred.
There are many other grades of papers man-
ufactured with Eucalyptus pulps, but generally
Eucalyptus fibers are used to improve paper
formation, opacity, smoothness, dimensional
stability, bulk, and porosity. The Eucalyptus fiber
population in the pulps is rigid and difficult to
collapse an important property for paper making.
There is another key driver to paper makers for
using fibers: the market pulp prices of this fiber.
Thanks to the low production costs, high pulping
yield, and lower chemical and wood consumption,
Eucalyptus pulps are generally less expensive than
softwood pulps. No doubt that the production
costs are also key issues for paper makers. The
same are to the entire Eucalyptus pulp and paper
production chain.
Eucalyptus pulps have today gained the status
of the most admired fiber supply. They are growing
in an unbeatable rate in the paper world business.
Eucalyptus pulps are versatile and may be used
as the single fiber or blended with others, such
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
as hardwoods, softwoods or recycled fibers. Wood
and fiber quality improvements due to genetic
and silvicultural operations and of the conversion
process may contribute to further worldwide
appreciation of Eucalyptus.
2.7 Eucalyptus Wood Quality Requirements
for Wood Charcoal Manufacturing and
Biomass Fuel
Brazil has had enormous success in the utilization
of Eucalyptus biomass as firewood and for
charcoal production. The first biomass fuel-
oriented forest plantations were based on high
wood density Eucalyptus species, such as E.
paniculata, E. camaldulensis, E. tereticornis, C.
citriodora and C. maculata. Basic wood density is a
fundamental wood characteristic because it leads
to better quality charcoal and a higher calorific
value per volume of wood or charcoal. However,
tree growth rate is also important in order to
reach the maximum possible production of dry
biomass per hectare (trunk, branches and bark).
gr e
Thus, if an Eucalyptus species leads to high-density
P
wood, but its growth rate is poor, the production
a low as possible.
Fi rst
of total biomass may not economically attractive.
For this reason, the wood biomass segment has
directed its efforts to the diversification of species,
and hybridization. Currently, more species are
being explored in the forest breeding: E. cloeziana,
E. pellita, E. urophylla, and hybrids, such as E.
urograndis. These species are adapted to grow in
tropical regions of Brazil, where the attacks of
pests and diseases are more frequent, due to higher
temperature and humidity. In case that charcoal
production would migrate to temperate regions,
there are other species very suitable such as E.
dunnii, E. viminalis, E. benthamii, E. saligna, E.
globulus, and hybrids.
It is important to mention that wood density
is a key property, but there are other wood
characteristics that are important when wood
is destined for fuel purposes. The trees should
be as straight as possible to favor the feeding
of the charcoal ovens or biomass furnaces or
chippers. The bark content is required to be as
low as possible, since the wood for energy is
not debarked and Eucalyptus bark has a higher
percentage of minerals, lower carbon content,
basic density and calorific value. Furthermore,
9-57
the high phosphorous content in the bark may
make difficult the utilization of this material in
the production of some grades of charcoal.
The following wood properties should be
included in a forest breeding program when wood
is for biomass fuel and charcoal manufacture.
r Basic Wood density and forest growth rate. It is
essential to optimize these two key parameters
at once. Faster growth and higher wood density
are desirable with the goal of maximizing dry
biomass production per hectare.
r Lignin content, lignin composition, and carbon
content. The lignin calorific value is higher
than that obtained from the cellulose and
hemicelluloses carbohydrates. The carbon
content in lignin is somewhat higher than in
the wood carbohydrates. For this reason, the
higher the lignin content, the higher the carbon
content, and the better the wood is for fuel.
The ratio syringyl/guaiacyl is another point to
be considered since guaiacyl lignin is richer in
o o f
carbon content than syringyl. This means that,
P r
contrary to the wood for pulping, the wood for
energy the syringyl/guaiacyl ratio should be as
Ash content. Wood minerals do not generate
heat, but they consume heat during combustion
and they also reduce the carbon content of the
wood and bark based on dry weight content.
Both wood and bark should have minimal ash
content.
r Volatile extractives. Charcoal production im-
plies substantial dry weight losses and the
usual yields when manufacturing charcoal
with Eucalyptus wood varies from 30% to
40%. Many types of wood extractives are
very volatile and they are lost in the exhaust
flue gases during controlled combustion.
Depending on the manufacturing process, the
extractive content of wood and bark may affect
the charcoal yield.
r Wood moisture. High-density woods have
lower moisture content due to the fact that
they have less open spaces or porosity to hold
water. For this reason, they are easily dried for
consumption as fuel raw material. High speed
drying is a good quality parameter for wood
selection, thus the lower the wood and bark
moisture, the better is the raw material is for
biomass fuel.
JWBK245-Kole k0902 May 10, 2008 14:1
9-58 FOREST TREE SPECIES
r Anatomical composition of the wood. It
has been proven that wood that is rich in
vessel elements and parenchyma cells is less
recommended for charcoal manufacturing. In
general, the lower the density and the quantity
of these anatomical elements, the more friable
and poorer quality charcoal is produced.
r Fissured and cracked logs. It is very important
that the logs have good dimensional stability,
with minimal cracks and fissures. These wood
defects give a higher fine content in the charcoal
and a lower yield as consequence, since fines
have to be screened out from the charcoal to
improve its quality.
Wood may be engineered and improved by
selection, genetics and silvicultural operations,
therefore for maximization of results, it is impor-
tant to know and to understand the parameters
that need to be improved. They need to show good
heritability and to add value to the conversion
processes and to the end-use products. Sound
planning, representative sampling, high quality
evaluation, and data interpretation are vital to
ge
the success of any wood quality improvement
program. Many of the current successes can be
P a
Fi rst
attributed to the excellent opportunities offered
by hybridization and cloning techniques. Basic
wood density has been the most important quality
parameter to predict wood quality but it is not
the unique wood characteristic. Another point is
that basic wood density is a very good parameter
to compare wood from the same species, or
with similar behavior for a given utilization. For
example, even with the same wood density, two
very different species of Eucalyptus may show very
different behavior in pulping and papermaking.
E. saligna wood with 0.5 g cm–3 usually shows a
completely different performance than E. robusta
wood with exactly the same wood basic density.
Definitively, there are many roads to walk in the
direction to the future. Thus, future improvement
programs need to be efficient and efficacious if
they are to maintain continuous competitiveness
of Eucalyptus as source of industrial raw material.
These programs must not only face economic and
quality issues, but also environmental and social
performances. These new roads are demanding
new additional challenges, and because of this,
science, technology, knowledge, and goodwill are
to be soundly matched.
2.8 Characterization of the Transcriptome
Involved in the Process of Wood
Formation in Eucalyptus
Wood is formed from the vascular cambium,
which is the secondary meristem, responsible
for the diametric growth of the stem. When
the cambial initials divide, one cell remains at
meristematic status while the other is destined
to become a xylem or a phloem mother cell.
During differentiation, the xylem mother cells
undergo an ordered series of developmental steps
that include cell division, cellular expansion, and
deposition of secondary cell wall, giving rise to
supporting, storage, and conducting cells (Larson,
1994). The xylem transport function is carried out
by a specific cell type, the vessel elements, which
undergo a differentiation process characterized by
successive deposition of secondary cell wall layers,
culminating in PCD (programmed cell death) and
autolysis.
The genetic factors controlling wood formation
o o f
in Eucalyptus are still not fully understood.
P r
In recent years, many genes involved in wood
formation have been identified by large-scale
genomic approaches in Poplars, Pines, and
Eucalyptus (Hertzberg et al., 2001; Lorenz and
Dean, 2002; Paux et al., 2004). Hertzberg et al.
(2001) established a hierarchical pattern of gene
expression through different zones of developing
xylem in Populus by isolating cells at different
stages of xylogenesis. By microarray analysis, the
authors showed that genes encoding enzymes
involved in cellulose and lignin biosynthesis, as
well as a large number of transcription factors
and potential xylogenesis regulators are under
strict control at each xylem differentiation stage.
Paux et al. (2004) developed a targeted approach
of functional genomics by the construction of
a xylem-leaves subtractive library to identify
genes involved in the control of Eucalyptus
wood formation. The two main classes of ESTs
preferentially expressed in xylem were related to
auxin signaling through ubiquitin proteolysis, cell
wall biosynthesis, and remodeling. More recently,
the induction of tension in wood has been used as
a model to study wood formation due to its higher
cellulose content and poor lignification (Paux
et al., 2005; Andersson-Gunnerås et al., 2006).
In the attempt to investigate the genes expressed
during juvenile wood formation in E. grandis,
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
(a) (b)
Figure 20 Biological material sampling. (a) Removal of the tree bark. (b) Sampling the cambial region by scraping the tissue. (c)
Transversal section of Eucalyptus wood before removal of the bark. (d) Transversal section of the Eucalyptus wood after removal of
the bark. Bars = 100 µm
when growth is maximized, Carvalho et al. (2008)
produced two SAGE libraries (Velculescu et al.,
1995) from the cambial region of 3- and 6-year-
old trees representing juvenile wood. Overall, the
authors identified 444 genes involved in cellulose
biosynthesis, nucleotide sugar metabolism, and
lignin biosynthesis, as well other physiological
ge
processes related to wood formation. The tissue
samples were collected from a half-sibling
P a
Fi rst
population of E. grandis originated from a single
mother tree of a second generation of clonal
seeds orchard, introduced from Coff’s Harbour,
Australia. A total of 40 3-year-old and 40 6-year-
old trees were sampled from stands located in
Itapetininga, State of São Paulo, Brazil (23◦ 35 20
S and 48◦ 03 11 W) at an altitude of 656 m.
The 3- and 6-year-old trees were spaced at
3 × 1.5 m and had an average height of 18 and
25 m, respectively. The cambial region of the trees
were harvested in the same morning during the
summer of 2003, by opening a window (20 ×
15 cm) in the bark at breast height, scraping the
stem exposed tissue and the inner surface of the
bark, and immediately freezing the sample in liquid
nitrogen (Figures 20a and b). The stem tissue
was scraped until the fibrous material below the
differentiating cells was reached. The inner side of
the bark was also scraped to guarantee that all the
meristematic material would be represented in the
SAGE libraries. This procedure was firstly used by
Foucart et al. (2006) who showed that the cambial
cells get adhered to the removed bark. Figures
20(c) and (d) also shows two transversal sections
of Eucalyptus wood (xylem, cambial region, and
9-59
(c) (d)
phloem) where it is possible to observe that
the meristematic cells tend to follow the phloem
cells when the bark is removed. Two bulks, one
representing the 3-year-old trees and the other the
6-year-old trees, were made by grinding and mixing
o o f
all the samples material.
P r
The 3- and 6-year-old SAGE libraries were
produced from the sequencing of 737 and
703 clones generating 22 660 and 22 024 tags,
respectively. As the number of tags produced by
the two libraries was very similar, the authors
combined the sequencing reads from both and
analyzed the data as one library representing
the cambial region of juvenile E. grandis trees.
The SAGE 2000 software extracted 43 304 tags
from the sequencing data file and produced 26 958
tags with three or more copies representing 3066
unique tags or genes. From this total the authors
have assigned ESTs to 657 unique tags (data not
shown), 444 and produced 26 958 tags with three
or more copies representing 3066 unique tags or
genes. From this total the authors have as signed
ESTs to 657 unique tags (data not shown), 444
with a defined function, and 213 with unknown
functions. For most of the unique tags (79%) it
was not possible to associate an EST or a complete
cDNA due to the small number of Eucalyptus
sequences currently available in public databases
(Figure 21a).
The 444 tags that presented a tag–gene associ-
ation were annotated and assigned to functional
categories as defined by Rison et al. (2000). The
most expressed categories were metabolism and
energy and structure and organization of structure,
JWBK245-Kole k0902 May 10, 2008 14:1

9-60 FOREST TREE SPECIES

14%
21%
27%

7%

24%

79% 20%

8%
Tags mismatchs
Expressed sequences Metabolism and energy
Identified genes Cellular processes

Transport

Structure and organization of structure

(a) (b) Information pathways

o o f
r
Figure 21 Distribution of the 3066 genes analyzed. (a) Tags mismatches: tags with no homology to the Eucalyptus public EST

ge P
sequences, Expressed sequences: tags associated to an EST with unknown function in the GenBank, Identified genes: tags associated
to an EST with a probable function defined in the GenBank. (b) Functional classification of the 444 identified genes. Functional

P a
Categories: Metabolism and energy, Cellular processes, Transport, Structure and organization of structure, and Information pathways

Fi
followed by information pathways and processes
rst
(Figure 21b).
It is important to note that among the
tags in the subcategory autotrophic metabolism,
there are genes associated with glycolysis, TCA
cycle, alcoholic fermentation, and ATP synthesis.
In nonphotosynthetic organs, carbohydrates are
consumed through respiration to produce energy
and carbon skeletons for cellular metabolism and
biosynthesis of structural molecules, including
cell wall polymers. However, the quantity of
free O2 in the cambial region might be limited
by the physical barrier imposed by the bark
and, also due to the O2 consumption during
respiration. Thus, a proportion of the necessary
energy to secondary growth could be provided by
alcoholic fermentation (Kimmerer and Stringer,
1988). The possible role of alcoholic fermentation
as an alternative or principal supply of energy
in juvenile E. grandis trees is also suggested
by the presence of alcohol dehydrogenase and
pyruvate decarboxylase transcripts. Other recent
studies have reported the presence of transcripts
or proteins associated with anaerobic respiration
during xylem formation and secondary growth
(Gion et al., 2005; Juan et al., 2006; Ranik et al.,
2006).
Another interesting observation in the work
of Carvalho et al. (2008) was the existence
of tags representing light induced proteins and
components of the photosystems I and II in
wood forming tissues (Carvalho et al., 2008).
The presence of functional chloroplasts with
active photosystems in the outer peridermal layers
(chlorenchyma) and also in deeper stem tissues
such as ray cells and pith, have been reported
in many woody species (Pfanz et al., 2002), what
could explain the presence of transcripts related
to photosynthesis in Carvalho’s data. Supporting
this result Celedon et al. (2007) also found
rubisco proteins using 2D-LC-MS/MS in the same
samples used in the previous study. The outer
bark of the stems have a rather low permeability
to gaseous diffusion, leading to accumulation
of CO2 in the intercellular air spaces, around
500–800 times higher ambient air (Pfanz et al.,
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
2002). Thus, stem photosynthesis may be an
important mechanism to partially reduce the
internal anaerobiosis, avoiding a further restriction
to dark respiration and ATP production (Pfanz
et al., 2002).
2.8.1 Sugar-nucleotide metabolism
and cell wall biosynthesis
Sugar-nucleotide metabolism provides the pre-
cursors for biosynthesis of hemicelluloses and
pectins during wood formation. These precursors
represent 65% and 26–36% of the primary and
secondary cell walls, respectively (Mellerowicz
et al., 2001). The UDP-glucose dehydrogenase
(UGDH) and UDP-glucuronate decarboxylase
(UXS), responsible for UDP-glucuronate and
UDP-xylose production, respectively, were rep-
resented by highly frequent tags (Figure 22a).
However, only one UGDH tag with 22 copies
was observed, while the total expression of the
three UXS tags accounted for 47 copies. This
expression pattern is consistent with previously
ge
reported findings pointing to a low activity of
UGDH when compared to other enzymes in
P a
Fi rst
subsequent reaction steps in the same pathway,
suggesting its function as rate limiting in the
synthesis of matrix polysaccharides (Dalessandro
and Northcote, 1977).
Among the UXS tags, one (TACTCGGTTG)
with 27 copies is associated with the Arabidopsis
AtUXS3 soluble form. The other two, occurred at
a frequency of 20 copies and are associated with
the Arabidopsis AtUX4 Golgi membrane form
(Harper and Bar-Peled, 2002). A more detailed
analysis of the ESTs associated with the AtUXS4
tags reveled that the tag (TCATTATCAA) was
present in both ESTs sequences distant 27
and 146 bp from the poly-A, suggesting that
probably two alternative transcripts of AtUXS4
are expressed in E. grandis wood forming
tissues.
Besides UDP-xylose production, another im-
portant UDP-glucuronate-derived branch leads
to pectin metabolism. The pectic polymers are
mainly important in the primary cell wall
where they comprise approximately 47% of the
polysaccharides. Despite its importance in cell wall
structure, only a few genes responsible for pectin
biosynthesis have been identified to date.
9-61
In dicotyledonous plants, the primary cell
wall consists basically of a cellulose microfibrils
framework embedded in a polysaccharide matrix
of pectin and cross-linked glycans (Carpita and
Gibeaut, 1993). During cell extension occur
modifications in the structure and composition
of the cross-linked pectin-xyloglucans (Bourquin
et al., 2002). For example, xyloglucan endotrans-
glycosylases (XETs) are responsible for cell wall
remodeling during primary cell wall biosynthesis
by cutting and rejoining the xyloglucan chains.
XETs probably act during secondary cell wall
deposition, as well as, by creating and reinforcing
the connections between primary and secondary
wall layers (Bourquin et al., 2002). Despite its
well-known importance in cell wall remodeling,
only one XET tag with 19 copies was observed,
although this result could be due to the lack
of Eucalyptus XET ESTs publicly available.
Moreover, an increased number of transcripts
for pectinesterases and pectate lyases were
observed during increased secondary growth in
o o f
poplar tension wood (Andersson-Gunnerås et al.,
P r
2006). However, under normal growth conditions
experienced in our study, two pectinesterases tags
at a frequency of 35 and 11 copies were observed
in the cambial region library, and only one low
expressed tag for a polygalacturonase (Figure 22a)
in contrast to the 11 highly expressed genes
identified in active poplar cambium (Geisler-Lee
et al., 2006).
2.8.2 Cellulose biosynthesis
Current models of cellulose biosynthesis involve
not only CESA proteins but also membrane-
associated proteins like KORRIGAN (endo-1,4-
β-glucanase) and SUSY (sucrose synthase) (Joshi
et al., 2004). Five tags corresponding to Eucalyptus
grandis cellulose synthase (EgCesA) genes were
identified, one showing high similarity to the
primary cell wall related cellulose synthase gene
EgCesA4, and the other four showing high
similarity to the secondary cell wall related
genes EgCesA1, EgCesA2, and EgCesA3. Two
tags (AATTGATATG and GAATCAAAAT)
represented the EgCesA1 gene, the first occurred
in both ESTs sequences at a distance of 151 and
30 bp from the poly-A tail, indicating a possible
alternative transcript (Figure 22a).
JWBK245-Kole k0902 May 10, 2008 14:1

9-62 FOREST TREE SPECIES

o o f
P r
P a ge
Fi rst
Figure 22 Transcript profiling of genes related to the main biosynthetic routes of cell wall components. For each enzyme the
corresponding tag(s), as well as its expression level are presented. * Tags identified as potential alternative transcripts. Pathways related
to (a) Sugar-nucleotide interconversions, pectin, hemicellulose, and cellulose biosynthesis. (b) Lignin biosynthesis. GT8D, glycosyl
transferase family 8D; UXS, UDP-xylose synthase; UGDH, UDP-glucose dehydrogenase; PAL, phenylalanine ammonia-lyase; C4H,
cinnamate 4-hydroxylase; 4CL, 4-coumarate: coenzyme A ligase; CCR, cinnamoyl-coenzyme A reductase; CAD, cinnamoyl alcohol
dehydrogenase; C3H, coumarate 3-hydroxylase; HCT, hydroxycinnamoyltransferase; COMT, caffeate/5-hydroxyconiferaldehyde O-
methyltransferase; CCoAOMT, caffeoyl-coenzyme A O methyltransferase; F5H, ferulic acid/coniferaldehyde 5-hydroxylase; SAMS,
S-adenosyl-methionine-synthetase; GS1, cytosolic glutamine synthetase

According to Ranik and Myburg (2006), some
genes implicated in Eucalyptus secondary wall
formation (EgCesA1, EgCesA2, and EgCesA3)
have higher expression levels than those involved in
primary cell wall formation (EgCesA4, EgCesA5,
and EgCesA6) in xylem. Carvalho et al. (2008)
observed the same transcriptional profiles for
primary cell wall EgCesA genes. The EgCesA4
tag shows low expression (Figure 22a) and the
EgCesA5 and EgCesA6 tags are represented only
as single-copy transcripts (Carvalho et al., 2008).
The higher expression of the secondary cell wall
associated CesA genes was expected since the
sample contained more cells from the xylem
side of the cambial region. Ranik and Myburg
(2006) reported the EgCesA3 gene as the highest-
expressed CesA gene in secondary xylem. In
contrast, the results of Carvalho et al. (2008)
pointed out the EgCesA1 as the most expressed
(71 copies) cellulose synthase gene in the E. grandis
juvenile wood forming tissue (Figure 22a).
The pool of UDP-glucose destined to cellulose
synthesis can be produced either by UDP-
glucose pyrophosphorylase or sucrose synthase.
In Carvalho’s study, the higher frequency of
SUSY transcripts compared to UDP-glucose
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
P a ge
First
Figure 22 (Continued)
pyrophosphorylase transcripts strongly suggests
that SUSY activity is probably the main source
of UDP-glucose for cellulose synthesis in E.
grandis differentiating xylem (Figure 22a). SUSY
transcripts were the most abundant carbohydrate-
active enzymes (CAZyme) transcripts in poplar
and have been shown to be highly expressed during
secondary cell wall biosynthesis in tension wood
formation (Andersson-Gunnerås et al., 2006). The
membrane-associated SUS (SUSY) was detected
in developing cotton fibers giving rise to a
functional model for cellulose biosynthesis where
this enzyme directly channels UDP-glucose to
the membrane-bound cellulose synthesis complex
avoiding competition from the cellular metabolic
pool of UDP-glucose, and providing a more
efficient cellulose synthesis (Amor et al., 1995),
especially important during active secondary
growth.
9-63
o o f
P r
2.8.3 Lignin biosynthesis
The phenylpropanoid pathway starts with the
deamination of phenylalanine to cinnamic acid by
PAL. Cinnamic acid is then converted to coumaric
acid by C4H and diverted to monolignol synthesis
(Figure 22b). Both enzymes from the early steps
of phenylpropanoid biosynthesis were represented
by highly frequent tags (Figure 22b). Both PAL
genes (AtPAL1 and AtPAL2) are believed to be the
most important players in lignin synthesis during
vascular lignification among the four Arabidopsis
PAL genes (Raes et al., 2003). The C4H tag
was observed at a frequency of 36 copies and is
associated with the poplar PtreC4H2 gene, which
is more xylem specific than the PtreC4H1 and
more weakly expressed in phloem (Lu et al., 2006).
Only one tag representing the E. camaldulensis
4CL1 gene (Figure 22b) was observed. The enzyme
JWBK245-Kole k0902 May 10, 2008 14:1
9-64 FOREST TREE SPECIES
4CL produces CoA thioesters of hydroxycinnamic
acids, which are the precursors for H (p-coumaryl
alcohol), G (coniferyl alcohol), and S (sinapyl
alcohol) lignin subunits. One poplar 4CL gene was
reported to be up-regulated in the cambial zone
undergoing lignification (Hertzberg et al., 2001)
confirming its important role in the initial steps of
lignin synthesis. Lignin polymers in angiosperm
wood are composed by great amounts of G and
S units while only small amounts of H units are
added. This is because H units are predominantly
deposited into the middle lamella and cell corners
followed by G units, which are mainly laid down in
the secondary wall, and S units that are deposited
at the late stages of lignification (Lewis, 1999).
Thus, it is expected that the key enzymes in
the biosynthesis of the monolignols G and S
show higher expression levels during secondary
growth. The tags for COMT, CCoAOMT, and
S-adenosylmethionine synthase (SAMS) showed
a general higher expression level than those for
4CL and CCR (Figure 22b). These results agree
with Paux et al. (2005), who suggested that the
expression of 4CL, CCR, and CAD is under
ge
a common transcriptional control while COMT
and CCoAOMT form another co-regulated
P a
First
transcriptional cluster. Similar expression profiles
for these two enzymes was also observed by
Hertzberg et al. (2001) supporting this hypothesis.
The determining step for the diversion of p-
coumaryl-CoA for G and S monolignols synthesis
is its conversion into p-coumaroyl shikimic
acid/quinic acid by hydroxycinnamoyltransferase
(HCT), since p-coumarate 3-hydroxylase (C3H)
is not able to use p-coumaryl-CoA as substrate
(Schoch et al., 2001). It has been shown that p-
coumaroyl shikimate and p-coumaroyl quinate are
important intermediates in the phenylpropanoid
pathway with HCT acting both upstream and
downstream of C3H, producing caffeoyl CoA
(Hoffmann et al., 2004). Interestingly, the C3H
tag, similar to the Arabidopsis CYP98A3 class of
P450 gene whose expression is more evident in
lignifying vascular cells (Nair et al., 2002), was
almost four times more expressed than the HCT
tag (Figure 22b). Alternatively, C3H can also act
on p-coumaric acid precursors producing caffeic
acid, which in turns can be diverted to ferulate, by
COMT, or to caffeoyl CoA, by 4CL (Figure 22b).
Although COMT was first believed to convert
caffeic acid into ferulate (Dixon, 2001), it was
subsequently shown that COMT preferentially
catalyzes the conversion of 5-hydroxyferulate, 5-
hydroxyconiferaldehyde, and 5-hydroxyconiferyl
alcohol into sinapic acid, sinapaldehyde, and
sinapyl alcohol, respectively, and thus acts
preferentially on ferulic acid/coniferaldehyde 5-
hydroxylase (F5H) derived products (Parvathi
et al., 2001). A differential regulation for F5H
and COMT genes is supported by recent findings
obtained through a proteomic approach where
F5H proteins were not found among the expressed
proteins during poplar cambial regeneration, while
COMT isoforms were detected at all stages
(Juan et al., 2006). Consistent with this previous
result, our data showed that COMT transcripts
expression was almost twice the F5H expression
level (Figure 22b).
According to Carvalho’s data, the CCoAOMT
transcripts had the highest expression level of
all lignin biosynthetic enzymes observed in E.
grandis wood forming tissue (Figure 22b). This
is in agreement with the results of Paux et al.
o o f
(2004) that showed a preferential expression of
P r
this gene during Eucalyptus wood formation.
CCoAOMT adds a methyl radical to caffeoyl
CoA, producing feruloyl CoA in an alternative
route for monolignol production (Zhong et al.,
1998). The importance of this alternative route
was demonstrated by the down-regulation of
CCoAOMT in transgenic tobacco and poplar
plants leading to an altered S/G ratio and
significantly decreased lignin content (Zhong et al.,
1998, 2000). As the number of CCoAOMT
transcripts is much higher than the number for
COMT, we suggest that the synthesis of S and
G units is being preferentially carried out directly
from caffeoyl CoA in E. grandis.
It is interesting to note that CCoAOMT and
SAMS tags have similar high expression levels
(Figure 22b), even though the presence of a
common transcriptional regulatory system for
both genes is still unknown. Although SAMS is
a housekeeping enzyme involved in methionine
metabolism, its activity occurs in xylem tissue
undergoing secondary growth (Juan et al., 2006).
According to Cantón et al. (2005), SAMS might
be functioning in a local providing of methyl
groups consumed by CCoAOMT and COMT thus
ensuring high rates of lignification.
The lignification processes also requires a
large nitrogen input to support the first step
JWBK245-Kole k0902 May 10, 2008 14:1
EUCALYPTUS
in the phenylpropanoid biosynthesis catalyzed
by PAL. Extensive amounts of ammonium are
liberated through phenylalanine deamination and
an efficient system to recycle nitrogen is needed
to prevent a severe N deficiency in plants during
active lignification (Cantón et al., 2005). Therefore,
the authors proposed a mechanism in which
the liberated nitrogen is locally recycled and re-
incorporated into glutamine by GS1 (glutamine
synthetase). Carvalho’s results also suggest a
possible role for GS1 during lignification as the
GS1 tag showed high expression (101 copies),
occupying the 17◦ position among the 50 most
expressed tags in E. grandis juvenile wood forming
library (Table 4; Figure 22b).
The final step in lignin biosynthesis is the
polymerization of monolignols that is conducted
by peroxidases and laccases (Baucher et al.,
2003). Although the role of laccases in lignin
polymerization is still a matter of debate, their
importance during wood formation has been
reported (Paux et al., 2004). The results observed
by Carvalho et al. (2008) corroborate a greater
role for laccases in lignin polymerization during
ge
Eucalyptus wood formation due to the lack of tags
a
P
representing peroxidases (Figure 22b). Supporting
rst
Fi
this idea, no significant decrease in peroxidase
expression was observed in poplar tension wood
(characterized by decreased lignin content), while
the laccase gene lac3 is co-regulated with the lignin
biosynthesis genes (Andersson-Gunnerås et al.,
2006). However, Hertzberg et al. (2001) identified
two peroxidase genes up-regulated in poplar
lignifying tissues, as well as, two laccases with an
overlapping expression pattern, indicating specific
but different roles in the polymerization process.
The authors also demonstrated the induction of a
dirigentlike protein coincident with lignification.
Dirigent proteins can mediate the monolignols
coupling mainly in the S (1) sublayer and middle
lamella of lignifying secondary xylem cell walls
(Burlat et al., 2001). Carvalho et al. (2008) found
one low expressed tag similar to the Forsythia ×
intermedia dirigent protein and a highly expressed
tag similar to the Picea glauca dirigent protein
(Figure 22b). The authors suggested that probably
there are similar roles for laccases and dirigent
proteins in monolignol polymerization in E.
grandis.
The data obtained by Carvalho et al. (2008)
offers an insight into the expression of functionally
9-65
related genes involved in cell wall biosynthesis
during the first years of development of Eucalyp-
tus, and this information is just beginning to be
combined to produce an overview of the important
genes for wood formation. In the near future,
the information generated by functional genomics,
molecular markers, transgenesis, will provide the
breeders with important information to decide in
the selection of the best genotypes to improve
productivity, resistant to biotic and abiotic stresses,
and better wood quality.
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JWBK245-Kole k0902 May 10, 2008 14:1

EUCALYPTUS 9-75

Abstract: The genus Eucalyptus is currently an

important source of timber, cellulose pulp, and
energy in a world that is becoming increasingly
more conscious of the need to use renewable
resources. This chapter initially explains the im-
portance of hybridization and cloning techniques
in intensive breeding programs that occurred in
Brazil during the last few decades. The strength
of these programs is based on the selection of the
genetic material and the evaluation of the factors
that can influence the expected genetic gains in
forestry breeding programs using seeds. Factors
such as endogamy, floral development, pollen
viability, and pollination are discussed together
with seed vigor and the influence of environmental
factors on correct evaluation of candidate
material. The recent application of molecu-
lar markers in eucalypt breeding programs is
also mentioned with particular emphasis on
genetic diversity, genotyping, and their association
with disease resistance. An introduction into
Eucalyptus tissue culture is also given as a prelude
to the current status of genetic transformation
o of
using biolistics and Agrobacterium tumefaciens in
P r
ge
this genus. The final sections describe the current

P a
challenges, disease resistance, and wood quality for

Fi rst
large-scale cultivation and utilization of eucalyptus
wood as prime material.

Keywords: Eucalyptus, hybridization, cloning,

breeding, molecular markers, tissue culture,
genetic transformation, genetic resistance, wood
quality
JWBK245-Kole k0902 May 10, 2008 14:1

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P a ge
First

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