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In Vitro Cell. Dev. Biol.

Plant 41:91101, March April 2005


q 2005 Society for In Vitro Biology
1054-5476/05 $18.00+0.00

DOI: 10.1079/IVP2004587

TRANSGENIC TREES FOR A NEW ERA


M. JOSEFINA POUPIN

AND

PATRICIO ARCE-JOHNSON*

Laboratorio de Bioqumica, Departamento de Genetica Molecular y Microbiologa, Facultad de Ciencias Biologicas,


Pontificia Universidad Catolica de Chile, Santiago, Chile
(Received 7 June 2004; accepted 22 July 2004; editor E. C. Pua)

Summary
Traditional breeding has been widely used in forestry. However, this technique is inefficient because trees have a long
and complex life cycle that is not amenable to strict control by man. Fortunately, the development of genetic engineering is
offering new ways of breeding and allowing the incorporation of new traits in plant species through the introduction of
foreign genes (transgenes). The introduction of selected traits can be used to increase the productivity and commercial
value of trees and other plants. For example, some species have been endowed with resistance to herbicide and pathogens
such as insects and fungi. Also, it has been possible to introduce genes that modify development and wood quality, and
induce sexual sterility. The development of transgenic trees has required the implementation of in vitro regeneration
techniques such as organogenesis and somatic embryogenesis. Release of transgenic species into the agricultural market
requires a standardized biosafety regulatory frame and effective communication between the scientific community and
society to dissipate the suspicions associated with transgenic products.
Key words: Agrobacterium; biolistics; reporter genes; resistance; selection markers.

Introduction

IN VITRO Propagation and Tree Genetic Transformation

Conventional tree breeding is being complemented with genetic


engineering (GE) techniques that allow a controlled modification of
the species phenotype through the introduction of foreign genes.
The resulting transgenic plants are able to express new traits that
can confer on them an enhanced potential and commercial value.
These new traits are particularly important for the worldwide timber
industry because trees have complex reproductive cycles, long
juvenile periods, self-incompatibility and high heterozygosity
levels, all of them features that increase the variability and cost
of this industry. In this regard, new traits such as herbicide
resistance, pest and disease resistance, and modifications of wood
properties are being introduced through GE, with wide potential
applications. Tree improvement programs based upon GE also
require improved in vitro regeneration techniques such as macroand micropropagation, and a more thorough understanding of the
key metabolic pathways of trees.
This review focuses on the research approaches used to obtain
transgenic trees within a commercial framework, and how these
studies have helped the scientific community to better understand
tree physiology. In general, it covers most of the tree species
transformed by GE, techniques, tissues used, and results for each
example chosen. In addition, some of the biosafety issues involved
in the release of transgenic plants are also discussed.

In vitro propagation techniques were developed in order to


multiply desired genotypes as clones, and through time these
techniques have been further developed to provide plant materials
susceptible to being genetically transformed (Merkle and Dean,
2000). The more current systems used for the regeneration of tree
species are somatic organogenesis and somatic embryogenesis.
Somatic organogenesis can be defined as the production of plants
through de novo organ formation from a wide range of explants or
cellular masses. In somatic embryogenesis instead, embryo
formation is induced from cells other than gametes (Pena and
Seguin, 2001). Somatic embryogenesis has become the most
preferred technique for tree regeneration since it allows the
development of a complete plant from individual cells, thus
maintaining clonal identity and avoiding undesired phenomena like
chimeras. Due to its advantages, somatic embryogenesis has been
used to genetically transform different tree species such as walnuts
(Junglans regia) (McGranahan et al., 1998), avocado (Persea
americana) (Cruz-Hernandez et al., 1998), American chestnut
(Aesculus glabra) (Trick and Finer, 1999), and casuarina
(Allocasuarina verticillata) (Franche et al., 1997). Unfortunately,
all these examples of embryogenic cultures were developed only
from isolated seed zygotic embryos. This approach has the
disadvantage of not knowing the exact phenotype (Franche et al.,
1997). In addition, introducing transgenes in seed cells or tissues is
less attractive than using developed embryogenic tissues derived
from clonal material, since in the former, sexual stages may involve

*Author to whom correspondence should be addressed: Email parce@


bio.puc.cl

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POUPIN AND ARCE-JOHNSON

genomic rearrangements and a dramatic reduction in genetic gains.


Even though in many tree species embryogenic tissues are useful
starting materials for embryogenic cultures, many other species,
especially angiosperm tree species, have been genetically
transformed using somatic organogenesis. In these studies, explants
have been prepared from tissues as diverse as foliar disks,
cotyledons, hypocotyl sections, and nodal stem sections. Somatic
organogenesis has been used to produce transgenic trees of poplar
(Fillati et al., 1987), Eucalyptus spp., birch, Liquidambar spp.,
black locust (Igasaki et al., 2000), and Ulmus spp. (Gartland et al.,
2000). In addition, fruit species like apple, pear, several Citrus
species, plums, almonds, and apricots have been genetically
transformed using similar techniques (Singh and Sansavini, 1998).
Genetic transformation can be achieved by biological or physical
means. In the former, trees are transformed with genes using bacteria
such as Agrobacterium tumefaciens. In the physical transformation,
called particle acceleration or biolistics, selected tissues are
bombarded with DNA-coated tungsten or gold microparticles. This
technique is particularly useful for species that are not amenable to
genetic transformation with Agrobacterium. This is the case for most
monocotyledonous and forest species. Most coniferous tree species
are recalcitrant and require complex regeneration systems (Wenk
et al., 1999). In coniferous tree species, biological transformation
techniques have been less successful, but promising results have
been achieved using biolistics (Campbell et al., 1992). Using this
technique it has been possible to obtain a transient expression of a
number of transgenes in Picea mariana (Duchesne and Charest,
1991), Pinus taeda (Stomp et al., 1991), Pinus banksiana, Pinus
contorta, Pinus sylvestris, and Pinus radiata (Campbell et al., 1992).
However, stable expression of transgenes and subsequent plant
regeneration has only been achieved in a limited number of species,
such as Picea glauca (Ellis et al., 1993), Picea mariana (Charest
et al., 1996), Larix laricina (Klimaszewska et al., 1997), and Pinus
radiata (Walter et al., 1994, 1998a, b).
Unfortunately, transformation using biolistic techniques is
generally associated with undesirable side-effects such as complex
DNA integration and production of large numbers of transgene copies
(Walter et al., 1998b). These phenomena generally result in transgene
silencing in different stages of tree development with the consequent
loss of the traits introduced through GE (Meyer and Seadler, 1996).
In contrast, transformation with A. tumefaciens normally produces
stable integration patterns with one or a few transgene copies (Medina
et al., 1998). These features make this technique the currently
preferred system for genetically transforming tree species.
A stable and reproducible genetic transformation of Pseudotsuga
spp. (Dandekar et al., 1987), Picea abies, Picea glauca and Picea
mariana (Klimaszewska et al., 2001), Pinus taeda (Wenk et al.,
1999), Pinus strobus (Levee et al., 1999), and Pinus radiata (Stange
et al., 1999; Cerda et al., 2002) (Fig. 1) has been achieved using
embryogenic tissues and Agrobacterium tumefaciens-mediated
transformation. These results are strong proof of the successful
production of genetically transformed plants.
Reporter genes and selection markers. Reporter genes and
selection markers are required in order to identify effectively
transformed cell or cellular groups. They are generally used in the
preliminary stages of genetic engineering experiments before
attempting stable introduction through genetic transformation of a
given transgene into a plant recipient. An important example of
selection markers are genes encoding resistance to antibiotics

FIG . 1. GUS expression in embryogenic tissue from Pinus radiata. a, b,


Masses of embryogenic tissues. c, d, Light microphotograph of proembryo
and suspensor cells (200 and 400 , respectively). e, f, Early somatic
embryos (100 ). a, c, e, Non-transformed tissues. b, d, f, GUS expression in
transformed tissues.

and/or herbicides. For instance, the selection marker genes npt II


(neomycin phosphotransferase II) and aph IV, encoding resistance
to kanamycin and hygromycin, respectively, were used to
genetically transform Pinus radiata and Picea abies (Wagner et al.,
1997). Nevertheless, selection systems based upon antibiotic
selection allow a rather frequent escape of non-transformed cells
(Pena and Seguin, 2001); thus reporter genes like uidA encoding
the enzyme b-glucuronidase (GUS) have been used in addition.
Since a significant drawback of the reporter gene uidA is the
destruction of assessed tissues, non-destructive reporter genes like
the green fluorescent protein gene (GFP) have been developed in
order to identify in vivo-transformed explants from tree species
(Tian et al., 1999). Reporter genes like the above-mentioned are
generally driven by transcriptional promoters such as cauliflower
mosaic virus (CaMV) 35S or even the artificial promoter Emu
(Merkle and Dean, 2000). This approach has allowed confirming
genetic transformation experiments in softwood species like conifers
Pinus taeda (Wenk et al., 1999), Pinus strobus (Levee et al., 1999)
and Pinus radiata (Cerda et al., 2002).
In this regard, recently developed methodologies like markeraided selection, genetic fingerprinting, and functional genomics will
assist the early and successful detection of introduced and expressed
transgenes in tree species (Wilcox et al., 1996).

Genetic Transformation in Tree Species


There are many traits being introduced and modified in tree
species, most of them having commercial value. Table 1 displays

Pinus pinea

Pinus contorta
Pinus halapensis

Picea mariana

Picea glauca

Picea abies, Pinus radiata

Ohio buckeye (Aesculus


glabra)
Pear (Pyrus communis)
Pear

Norway spruce
Norway spruce

Norway spruce

Norway spruce (Picea abies)

Larch (Larix decidua)


Larch (Larix laricina)

English elm (Ulmus


procera), European chestnut
Hybrid aspen

Black spruce, white spruce,


white pine
Cherry rootstock colt
(Prunus avium P. pseudocerasus)
Citrus

Embryogenic
tissues
Embryogenic
callus
Embryogenic
culture
Roots
Embryogenic
cultures
Cotyledons
Biolistics

A. tumefaciens

uidA

uidA
uidA

uidA

Biolistics

A. rhizogenes
A. rhizogenes

bar gene: resistance to herbicide


phosphinothricin (Buster, Basta)
uidA, Bt (CryIA endotoxin)

Humara et al., 1999

Duchesne and
Charest, 1991
Lindroth et al., 1999
Tzifira et al., 1996

Charest et al., 1993

Ellis et al., 1993

Bell et al., 1999


Bishop-Hurley et al., 2001

Growth regulation: gene rolC


uidA

A. rhizogenes
A. tumefaciens
Biolistics

Trick and Finer, 1999

Martinussen et al., 1994


Wenk et al., 1999

Robertson et al., 1992

Walter et al., 1999

Shin et al., 1994


Klimaszewska et al., 1997

Eriksson et al., 2000

Gartland et al., 2000

Hypersensitivity to Agrobacterium infections (high necrosis


levels)

Six different promoters were evaluated


Stable or transient transgene expression in different tissues

Transient transgene expression

Stable transgene expression


Tobacco promoters responsive to Erwinia amylovora infection
were tested. They differentially responded to infection or
abiotic stress
Herbicide resistance even in plants with very low bar
expression levels (as assessed through Northern blots)
Stable transgene expression

Stable transgene expression

Transient transgene expression


Transient transgene expression

Stable and transient transgene expression in different lines

Stable transgene expression

Increased growth and diameter, and slow root growth in


juvenile plants. Longer and more abundant xylematic fibers
Stable transgene expression
Stable transgene expression

Heritable changes (i.e.) shortened juvenility, early flowering,


normal flowers and seeds. LFY expression created alterations,
but API expression developed a normal phenotype
Stable transgene expression

Pena et al., 2001

Hygromycin resistance and uidA

Luciferase
uidA

uidA

uidA

Overexpression of GA-20 oxidase


from Arabidopsis
Bt, glyphosate tolerance (aroA)
uidA

uidA

Increased in rooting ability and number of seminal roots

Successful transformation in all tissues

Stable transgene expression

Resistance to fungal species Venturia inaequalis;


slow plant growth
Stable transgene expression. High performance liquid
chromatography analysis revealed the accumulation of a
resveratrol-derivate, a glycoside, in transgenic plants.

Reduction in plant growth

Main effects

Gutierrez-Pesce
et al., 1998

Cruz-Hernandez
et al., 1998
Tian et al., 1997

Szankowski et al., 2003

Bolar et al., 2000

Zhu et al., 2001

Authors

A. tumefaciens

Biolistics
A. tumefaciens

Biolistics

Biolistics

A. rhizogenes
Biolistics

ND

ND

Sprouts
Somatic
embryos
Embryogenic
tissue
Somatic
embryos
Pollen
Embryogenic
suspensions
Somatic
embryos
Sprouts
Leaves

A. tumefaciens

A. tumefaciens

Sprouts

ND

Ri T-DNA

A. rhizogenes
LFY, API

gfp

A. tumefaciens
Biolistics

Avocado

A. tumefaciens

Antifungal endochitinase from fungal species Trichoderma harzianum


Stilbene synthase gene from grapevine, polygalacturonase-inhibiting
protein
(PGIP) from kiwi
uidA

Growth regulation: rolA, rol B

Genes

Somatic
embryos
Pollen, somatic
embryos
Roots

ND

A. tumefaciens

Leaf segments

Apple (cvs. Elstar and


Holsteiner)

A. tumefaciens

Leaf segments

Apple (Malus
domestica Borkh)
Apple

Technique

Tissues

Species

EXAMPLES OF TECHNIQUES, TISSUES USED, AND EFFECTS OBTAINED IN THE PRODUCTION OF TRANSGENIC TREE SPECIES

TABLE 1

TRANSGENIC TREES

93

Biolistics
A. tumefaciens

ND

ND

ND

ND

Protoplasts,
calluses

Stem and leaf


segments

Poplar

Poplar

Poplar (Populus tremula


P. tremuloides)

Poplar

Poplar (P. alba P. grandidentata), (P. nigra P. trichocarpa)


Robinia pseudoacacia

Cambium
callus
Internodal
segments
Proembryogenic
masses

Sour orange (Citrus


aurantium)
Trifoliate orange (Poncirus
trifolliata)
Yellow poplar (Liriodendron
tulipifera)

ND, not described in the reference.

A. tumefaciens

Anther callus

Biolistics

A. rhizogenes

A. tumefaciens

A. tumefaciens

Anther callus

Rubber tree (Hevea


brasiliensis)
Rubber tree

A. rhizogenes

A. tumefaciens

A. tumefaciens

Phytoremediation: mercury
reductase gene

Anti-oxidative stress: constitutive


expression of superoxide dismutase
gene
Citrus tristeza virus protein coat
gene
Growth regulation: rolC

uidA

uidA

Growth regulation: auxins and cytokinins biosynthesis genes from


A. tumefaciens
Growth regulation: rolA, rol B, rolC
from A. rhizogenes
Modified Bt gene

Flowering: LEAFY and PTLF genes

uidA
uidA
Cystein proteinase inhibitor from
Arabidopsis thaliana
Oxalate oxidase from wheat

Biolistics
Biolistics
ND
A. tumefaciens

uidA

A. tumefaciens

uidA

Pinus taeda
Pinus taeda
Poplar (Populus sp.)

Pinus strobus

Pinus radiata

A. tumefaciens

uidA

Biolistics

Pinus radiata

uidA
uidA and luciferase
uidA

Biolistics
Electroporation
Biolistics

Cotyledons
Protoplasts
Embryogenic
suspensions
Embryogenic
cultures,
sprouts
Somatic
embryos
Embryogenic
tissues
Cotyledons
Apical stem
ND

Pinus radiata
Pinus radiata
Pinus radiata

Genes

Technique

Tissues

Species

TABLE 1 continued

Stable transgene expression

Levee et al., 1999

Transgenic plants were able to withstand high concentrations


of mercury

Transgenic plants showed a depressed size

Kaneyoshi and Kobayashi,


1999
Rugh et al., 1998

Ghorbel et al., 2000

A transformation frequency of 4% was achieved.


The morphology of the transgenic plants was similar to that of
untransformed plants
The aim was to avoid the juvenile stage

The transformation frequency from stem segments was


approximately 24%, and the morphology of regenerated
plants resembled that of the original parental strain
Stable transgene expression

Dwarf transgenic plants. Apical dominance was repressed and


growth rates reduced

Reduced growth rates, plant size, leaf size and stem diameter

Jayashree et al., 2003

Arokiaraj et al., 1998

Igasaki et al., 2000

McCown et al., 1991

Nilsson et al., 1996

Tuominen et al., 1995

Rottmann et al., 2000

Liang et al., 2001

Transient transgene expression


Transient transgene expression
Overexpression of protease inhibitors associated with smaller
feeding rates in insects
Transgenic plants showed a reduced infection
by Septoria musiva
Early flowering and meristematic changes in two out of 19
poplar lines expressing PTLF from Populus trichocarpa
Transgenes were constitutively expressed in some plants only.
Abnormal growth patterns

Stable transgene expression

Cerda et al., 2002

Stomp et al., 1991


Loopstra et al., 1992
Delledone et al., 2001

Stable transgene expression

Transient transgene expression


Transient transgene expression
Stable transgene expression

Main effects

Walter et al., 1994

Rey et al., 1996


Campbell et al., 1992
Walter et al., 1998b

Authors

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TRANSGENIC TREES

the most frequently used procedures and genes introduced using


transgenic approaches into tree species.
Herbicide resistance. Most tree species are susceptible to the
herbicides used for weed control. These compounds interfere with
key metabolic pathways of trees, hampering their normal growth and
development with great impact on their commercial value in
forestry. Thus, introduction of the herbicide resistance trait in tree
species has great economic potential. It will allow, for instance, the
establishment of tree plantations in weed-infested sites that would
otherwise be economically unsuitable to plant (Walter et al., 2002).
The strategies used to confer herbicide resistance to trees are based
either on the induction of the herbicide metabolism by the plant or
in the introduction of resistant isoforms of the herbicides target
enzymes or proteins.
Glyphosate, a herbicide used worldwide, has low toxicity and a
broad action spectrum. This compound interferes with the plants
synthesis of aromatic amino acids by interfering with 5-enolpyruvylshikimate-3-phosphate aroA synthase (EPSP) activity, coded by the
aroA gene (Fillati et al., 1987). A lower sensibility to glyphosate has
been reported in an aroA transgenic Populus hybrid (Fillati et al.,
1987). Glyphosate tolerance mediated by aroA was also reported in
European larch and results in the survival, albeit with reduced
growth, of transgenic plants following a spray with glyphosate (Shin
et al., 1994). It has been reported that a much higher resistance to
glyphosate can be achieved if EPSP is expressed in the chloroplast
rather than in its original cytosolic location (Donahue et al., 1994).
This was achieved by attaching the appropriate signal peptide to the
aroA gene sequence. In hybrid poplars, glyphosate resistance was
obtained by an approach that combines herbicide detoxification with
target enzyme reductions (Strauss et al., 1997).
In other species like Picea abies and Pinus radiata, resistance to
the commercial herbicide Buster (phosphinothricin) was attained
through the use of the bar gene. This gene encodes a
phosphinothricin acetyltransferase from Streptomyces hygroscopicus that renders Buster inactive. In this study, transgenic plants
survived and continued to grow with minor or no damage to their
needles, even with a low rate of transgene expression, whereas nontransgenic plants regenerated from the same cell lines died within
8 wk of spraying. These results show that herbicide resistance is a
feasible concept in conifer forestry and that a low level of gene
expression may be sufficient to give useful levels of resistance to
Buster (Bishop-Hurley et al., 2001).
Resistance to oxidative stress. Reactive oxygen species (ROS)
such as hydrogen peroxide (H2O2) and hydroxyl radicals (OH2) are
formed in plants in response to several biotic and abiotic
environmental stresses including cold, ozone, herbicides, and
pathogens (Polle and Rennenberg, 1993; Noctor and Foyer, 1998).
These reactive species exert an oxidative pressure and thus pose a
continuous threat to the plants survival. The cellular defense
mechanisms of plants against ROS depend largely on the efficiency
of the plants detoxifying pathways; one of the mechanisms used by
cells depends in turn on the availability of reduced glutathione
(Herschbach and Kopriva, 2002).
Thus transgenic poplars with modified glutathione pathways or
capable of over-expressing the enzymes involved in ROS detoxification showed improved stress resistance (Nicolescu et al., 1996; Arisi
et al., 1998). Likewise, transgenic poplar plants that produce abovenormal levels of reduced glutathione are less susceptible to
photoinhibition stress than the wild-type species (Foyer et al.,

95

1995). However, these approaches have been insufficient to increase


tolerance to ozone stress in these plants (Will et al., 1997). Likewise,
the increases in the glutathione pools achieved by manipulation of its
biosynthetic enzyme g-glutamylcysteine synthase (gGCS) failed to
increase tolerance of transgenic poplar to heavy metals like cadmium
(Rennenberg and Will, 2000). It is noteworthy, however, that these
transgenic trees had a much increased root accumulation of cadmium,
a characteristic that may make them useful for phytoremediation
purposes (Gullner et al., 2001).
Abiotic stresses, such as drought, salinity, extreme temperatures,
chemical toxicity, and oxidative stress are serious threats to
agriculture and the natural status of the environment. Molecular
control mechanisms for abiotic stress tolerance are based on the
activation and regulation of specific stress-related genes. These
genes are involved in the whole sequence of stress responses, such
as signaling, transcriptional control, protection of membranes and
proteins, and free-radical and toxic-compound scavenging (Wang
et al., 2003). Recently, research into the molecular mechanisms of
stress responses has started to bear fruit and, in parallel, genetic
modification of stress tolerance has also shown promising results
that may ultimately apply to agriculturally and ecologically
important plants.
Insect resistance. Genetic engineering currently allows the
production of plants resistant to an ample range of insects through
the introduction of transgenes derived from plants, microorganisms,
and mammals. These transgenes code for an extensive range of biomolecules (e.g., toxins, proteases, inhibitory proteins, amylases, and
lectins) that attack the digestive systems of insects by different
mechanisms (Schuler et al., 1998).
Of particular interest are the transgenes derived from the bacterium
Bacillus thuringiensis (Bt) that code for proteins active against diverse
Lepidoptera species. Genes isolated from different Bt strains have
been expressed in tree species as diverse as apple, spruce, larch, and
poplar (Schuler et al., 1998; Tzifira et al., 1998). These insecticidal
proteins, effective against several pests, are environmentally
innocuous (Schuler et al., 1998). However, the use of these proteins
themselves is limited by the fact that they are sensitive to UV radiation
and, under normal environmental conditions, are active only for
approximately 24 h. Recently, Kleiner and coworkers tested the
hypothesis that ontogenetic variation in leaf chemistry could affect the
efficacy of genetically expressed Bt cry1A(a) d-endotoxin in
transgenic poplar, and could delay the resistance of folivores. The
results indicated that the presence of foliar allelochemicals of poplar
could enhance the effectiveness of at least some aspect of genetically
expressed Bt d-endotoxin against gypsy moth larvae. The benefits of
combining novel resistance mechanisms with natural ones will
depend upon the specific folivores adaptation to natural resistance
mechanisms, such as allelochemicals (Kleiner et al., 2003).
Moreover, some of the greatest benefits from transgenic resistance
may arise from the need to protect trees from multiple pests, some of
which may not be deterred by, or may even prefer, allelochemicals that
confer protection from a few species. The enormous advantage of
transforming plants with the Bt transgenes is that they become able to
synthesize their own insecticide and, consequently, acquire immunity
to insect attack. In the near future, the use of this new technology could
reduce the use of insecticides and its impact on field workers and the
environment in general (Schuler et al., 1998).
Fungal resistance. Trees are susceptible to infection by many
pathogens, including fungi. For instance, Pinus radiata can be

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POUPIN AND ARCE-JOHNSON

attacked by fungi such as Spharopsis sapinea, Phytophthora spp.,


and Cilindrocarpon destructans (Everett et al., 1997). The
development of trees resistant to fungal attack can be approached
through the incorporation of genes encoding products with
antifungal activity. One of the problems inherent to this approach
is the fact that resistance to fungi is a trait determined by multiple
genes. Thus, a large number of genes need to be transferred and
coordinately expressed in order to confer the tree protection against
fungi (Dixon, 2001). A second problem is that many fungal species
quickly develop detoxification systems against plant compounds
with antifungal activity (Maloney and Vanetten, 1994).
In spite of these difficulties, it has been possible to express
hydrolytic enzymes such as chitinases and b-1,3-glucanases in
tobacco, using GE techniques. These enzymes degrade the
polysacharides present in the cell wall of many fungal species.
The resulting transgenic tobacco plants acquired resistance to the
fungus Botrytis cinerea (Carstens et al., 2003). Resistance to fungal
attacks has also been reported in transgenic plants expressing
synthetic antimicrobial peptide (Cary et al., 2000). The successful
production of transgenic poplar plants with an enhanced resistance
to the fungus Septotria musiva has also been achieved (Dixon,
2001). These plants express the enzyme oxalate oxidase from wheat.
Even though full resistance was not achieved, the symptoms
observed during fungal infection were reduced. Szankowski and
collaborators are working to introduce genes with antifungal
potential in commercially important apple cultivars, in order to
establish resistance against fungal diseases. They introduced, in
apple cultivars, the gene encoding stilbene synthase (Vst1) from
Vitis vinifera L., responsible for the synthesis of the phytoalexin
resveratrol in grapevine, and the gene for a polygalacturonaseinhibiting protein (PGIP) from kiwi (Actinidia deliciosa) via
Agrobacterium tumefaciens-mediated transformation. However, the
efficiency of these genes in antifungal activity has not been reported
(Szankowski et al., 2003).
The effect of two stilbene compounds, pinosylvin and resveratrol,
on the growth of several fungi was evaluated in plate tests. Then a
pinosylvin synthase-encoding gene from Pinus sylvestris was
transferred into aspen (Populus tremula) and two hybrid aspen
clones (Populus tremulax tremuloides) by A. tumefaciens-mediated
transformation. Transgenic plants accumulated pinosylvin synthasespecific mRNA and showed stilbene synthase enzyme activity in
vitro. One transgenic aspen line showed increased resistance to
Phellinus tremulae, while two hybrid aspen transformants decayed
faster than the control trees. However, it was not possible to detect
the accumulation of stilbenes in the transgenic plantlets (Seppanen
et al., 2004).
Alteration of developmental patterns, tree architecture, and wood
quality. Plant development and growth depends on the concerted
action of diverse phytohormones like auxins (indole-3-acetic acid,
IAA), gibberellins (GAs), cytokinins, or abscisic acid (ABA) (Kende
and Zeevaart, 1997). A better understanding of the regulation of these
hormones may help us devise new tools to modify the development,
form, and quality of tree species. So far, some bacterial genes have
been used to alter the developmental patterns and form of plant
species. For instance, the introduction of IaaM (trp-2-monooxygenase) and Iaa H (indole-3-acetamide hydrolase) from
A. tumefaciens, two genes involved in auxin biosynthesis, significantly
altered the wood properties of aspen (Tuominen et al., 1995).
Interestingly, the resulting transgenic trees were smaller and had

lower growth rates, leaf size, and stem diameter than untransformed
controls. These features are apparently unsuitable for commercial
applications. However, other traits like reduced lateral branching and
modifications in the structure and properties of xylem may have new
important applications (Tuominen et al., 1995).
The genes rolA, rol B, and rolC that code for growth hormones in
A. rhizogenes have been introduced in transgenic poplar and aspen.
The overexpression of genes rolA, rol B, and rolC in these tree
species resulted in a modified plant growth and development and a
change in root characteristics. In particular, the overexpression of
the gene rolC in hybrid aspen produced dwarf individuals with
diminished apical dominance, growth rates, and internode intervals
(Nilsson et al., 1996). This rolC-related phenotype is also associated
with low auxin levels, modified GA biosynthesis, and increased
cytokinin levels (Tzifira et al., 1998). In contrast, the expression of
the native fragment of rolABC derived from A. rhizogenes in
transgenic aspen improved in vitro growth rates and root properties
(Tzifira et al., 1998). This apparent inconsistency may relate to
differences in the gene expression levels determined by the specific
promoters used in these experiments. In the latter, gene expression
was driven by the native bacterial promoter. The use of native
promoters is associated with lower levels of expression that may
determine a positive effect on a plants growth. Even though the
expression of the rolABC fragment resulted in some apparently
undesirable properties, such as reduction in apical dominance, this
effect could be extremely useful for developing dwarfing rootstocks
in fruit tree species and to produce cellulose in forest operations
(Tzifira et al., 1998).
The group of GA hormones participates in processes like sprout
extension, leaf form and expansion, flowering, seed germination,
and differentiation of xylem fibers (Kende and Zeevaart, 1997). GA20-oxidase is the key gene in the control of GA biosynthesis. When
this gene is overexpressed in transgenic aspen, the resulting trees
show enhanced growth and an increased biomass (Eriksson et al.,
2000). Besides the obvious economic applications of this result, it
helps us understand the molecular mechanisms involved in the
regulation of growth and developmental processes by GAs
(Herschbach and Kopriva, 2002). Another gene affecting the
metabolism of plant growth regulators is the homeobox gene OHS1.
Its overexpression in transgenic poplar induces morphological
aberrations that include dwarfing, loss of apical dominance, and
thinning of leaves (Mohr et al., 1999).
In sum, the ability to modify the metabolism of plant hormones
through GE is a powerful mechanism for both the production of
more efficient trees and the advancement of our knowledge of the
growth and development of tree species.
Flowering and sexual sterility. The flowering process in
angiosperm species is regulated by a complex system controlled by
a family of transcription factors encoded by MAD-box genes. These
genes code for proteins that have a highly conserved domain of 59
amino acids that binds to specific DNA sequences (Decroocq et al.,
1997). The specific formation of each of the diverse floral organs
depends upon the expression of a reduced number of homeotic genes
carrying the MAD-box domain. These genes can be classified into
three classes: A, B, and C, each acting independently or associated
with others to create different floral whorls (Mouradov et al., 1999).
Even though MAD-box genes have always been associated with
flowering plants, recent phylogenetic investigations, including
morphological and evolutionary studies, demonstrated the presence

TRANSGENIC TREES

of genes orthologous to the MAD-box genes in diverse species of


angiosperms and gymnosperms, for example, in conifer species like
Norway spruce (Picea abies) (Sundstrom et al., 1999), black spruce
(Picea mariana) (Rutledge et al., 1998), and Pinus radiata
(Mouradov et al., 1999).
A better understanding of the flowering process in tree species
may serve two different purposes. On the one hand, it may help to
speed up flowering and thus reduce the normally extensive juvenile
period of trees and increase productivity. On the other hand, a
better understanding of the flowering process may help us to modify
tree species through GE and create sterile trees lacking flowers or
parts of them. In order to speed up flowering and therefore reduce
the length of generation, heterologous homeotic genes like MADbox, LEAFY (LFY) and APETALA (AP1) from Arabidopsis have been
introduced into Citrus species. Constitutive expression of either
LFY or AP1 Arabidopsis genes in Citrus seedlings shortened the
juvenile phase and promoted precocious flowering. These traits
were transmitted to the progeny, resulting in trees with a generation
time of 1 yr from seed to seed. Whereas LFY lines showed
alterations in growth and development, AP1 plants were adult and
fully normal (Pena et al., 2001).
In transgenic poplar, the influence of flowering genes is quite
complex. For example, the gene PTLF, a gene homologous to LFY,
is expressed even in vegetative tissues (Rottmann et al., 2000). As a
result of this, the overexpression of the Arabidopsis LFY gene affects
the stem meristems and accelerates flower development (Weigel and
Nilsson, 1995). In addition, the overexpression of PTLF derived
from Populus trichocarpa in several Populus species induced early
flowering in only two out of 19 transgenic lines (Rottmann et al.,
2000). These Populus transgenic lines produced anthers instead of
carpels, further confirming the role of LEAFY in the floral induction
of male structures (Frohlich and Parker, 2000). Poplar transgenic
lines with high expression levels of the PTLF gene showed an
abnormal vegetative growth that precluded their analysis throughout
their complete life cycles. The study of reproductive changes in
trees is a complex process, due to their long reproductive cycles.
In a tree like poplar, 5 6 yr are required to achieve reproductive
maturity or to notice changes in flowering behavior (Herschbach
and Kopriva, 2002).
With regard to the production of sterile plants, this can be highly
attractive from two standpoints. On one hand, the use of sterile
organisms makes the release of transgenic organisms a safer process
(Strauss et al., 1995). The deployment of sterile individuals allows
control of the transgene flow and prevents the unexpected transfer of
transgenes into natural populations. On the other hand, the use of
sterile transgenic plants may help to redirect into vegetative growth
the energy invested in reproductive processes.
Two approaches have been developed to create sterile plants
through genetic engineering: (1) use of promoters that regulate the
expression of cytotoxic genes and (2) inhibition of expression of
genes important for the flowering process at the RNA or protein
levels (TGERC, 1999). In this respect, the discovery of floweringrelated genes, such as MAD-box genes, in tree species and its
overexpression or inhibition may provide us with key knowledge on
the flowering process and could help us to design new tools to
develop sterile trees.
One of the main difficulties in the study of flowering processes is
the dependence of tree development upon environmental
conditions. For instance, LEAFY and other flowering-related

97

genes may interact with genes controlling the photoperiodic


response (Haughn et al., 1995). Even though the exact role of
different photoreceptors is still largely unknown, phytochrome A is
known to control flowering in Arabidopsis. Overexpression of the
phytochrome A gene from oat in transgenic poplar resulted in
transgenic individuals whose auxin and gibberellin levels were not
changed by short-day conditions and whose growth was not halted
even in conditions of a 6 h photoperiod. This suggests that
phytochrome A affects hormone metabolism and could be
responsible for the response to diverse photoperiodic conditions
(Olsen et al., 1997). GAs are involved in the regulation of flower
development in Arabidopsis. The GA-deficient ga1-3 mutant shows
retarded growth of all floral organs, especially abortive stamen
development that results in complete male sterility. Until now, it
has not been clear how GA regulates the late-stage development of
floral organs after the establishment of their identities within floral
meristems. Various combinations of null mutations of DELLA
proteins can gradually rescue floral defects in ga1-3. In particular,
the synergistic effect of rga-t2 and rgl2-1 can substantially restore
flower development in ga1-3. These results indicate that GA
promotes the expression of floral homeotic genes by antagonizing
the effects of DELLA proteins, thereby allowing continued flower
development (Yu et al., 2004).
One of the most recent discoveries in flowering is the role of small
RNAs (microRNAs) in flowering control. Elevated microRNA172
accumulation results in floral organ identity defects similar to those
in loss-of-function APETALA2 (AP2) mutants, showing that
miRNA172, which can base-pair with the messenger RNA of
AP2, regulates AP2 expression primarily through translational
inhibition. Therefore, miRNA172 likely acts in cell-fate specification as a translational repressor of AP2 in Arabidopsis flower
development (Chen, 2004).
All these experimental approaches could help us to get a better
understanding of the flowering process, and to use the knowledge for
the development of new biotechnology tools in trees.
Wood quality. By means of genetic engineering it is possible to
modify wood properties. Our knowledge of key enzymes involved in
polysaccharide biosynthesis makes this area of research a promising
one which is expected to have a large impact upon forest
biotechnology. Up to now, all the data concern the genetic
modification of cellulose and starch content in tobacco. In this
species it has been possible to modify the expression of genes
encoding the enzymes involved in cellulose biosynthesis. Plant
mutants of the enzyme UDP-glucose pyrophosphorylase show
cellulose deficiency, suggesting target biosynthetic pathways could
be modified in order to increase cellulose biosynthesis (Delmer and
Amor, 1995).
Lignin is another key polymer, accounting for between 15 and
35% of dry weight in tree species (Pena and Seguin, 2001). Lignin
is a hard tree component and consists of monomers derived from
three aromatic alcohols: para-coumaryl alcohol, coniferyl alcohol
and sinapyl alcohol, possessing none, one and two methoxyl groups,
respectively. The relative composition of each residue defines the
extent of condensation of the lignin molecule. A larger number of
methoxyl residues in lignin makes it more compact and difficult to
remove. Conifer species naturally contain mostly coniferyl alcoholderived residues, and to a lesser extent residues derived from paracoumaryl alcohol, whereas angiosperm species possess roughly
equivalent amounts of coniferyl- and sinapyl-derived residues

98

POUPIN AND ARCE-JOHNSON

(Whetten and Sederoff, 1995). Lignin extraction during the pulp and
paper production processes is not only highly expensive but it also
creates large amounts of chemical waste. Therefore, it has been
suggested that GE may serve to modify the metabolic pathways
involved in lignin biosynthesis in order to develop transgenic trees
with improved pulp properties. The resulting transgenic trees would
have a huge value for forestry (Franke et al., 2000).
In spite of the complexity of the biosynthetic pathways involved
in lignin production, transgenic trees with reduced levels of lignin
or modified lignin properties have been produced already. The
modification of lignin patterns was achieved by gene-silencing
processes induced in poplar hybrids through antisense technology,
targeting key enzymes such as O-methyltransferase (OMT) (Jouanin
et al., 2000). In aspen (Populus tremuloides Michx), the antisense
regulation of the gene encoding the enzyme para-4-coumarate-CoA
ligase (4CL) allowed a reduction in lignin of over 45%,
compensated by a 15% increase in cellulose content. Furthermore,
growth rates were improved and the structure of transgenic trees
was maintained (Hu et al., 1999). Many studies have targeted the
gene encoding the enzyme ferulate-5-hydroxylase (F5H). This
enzyme hydroxylates ferulate to 5-hydroxyferulate, thus creating a
substrate able to generate sinapyl alcohol, from which syringyl
momomers are derived. When this gene was expressed in poplar, an
increased proportion of syringyl monomers was found in the lignin
synthesized (Franke et al., 2000). Modifying F5H can be of great
importance for conifers such as Pinus and Picea species, since it
would allow modifications in the amount of lignin accumulated and
in the relative proportion of monomers.

such efforts is the development by Rugh et al. (1998) of transgenic


yellow poplar that can grow under high mercury concentrations,
thanks to the introduction of a gene encoding the enzyme mercury
reductase.
Conclusions and Perspectives
The worldwide importance of forestry, summed to the lengthy
generation cycles of tree species, makes unavoidable the
development of new technologies that complement conventional
tree breeding programs in order to obtain improved genotypes. In
this regard, the development of transgenic trees carrying traits, for
example, herbicide resistance and enhanced growth rates, becomes
an alternative not only to reduce production costs and increase
gains to the industry, but also to provide ecological advantages as in
phytoremediation or in the establishment of artificial plantings in
weed-infested sites. The use of more efficient trees may reduce the
soil areas required to produce the goods and services derived from
tree plantations, thus reducing the use of natural ecosystems for
agricultural purposes.
The genetic engineering of tree species is a novel technology that
requires better understanding of the physiology and genetics of the
species it will be applied to. Also needed are improvements in
current in vitro propagation techniques and development of new
molecular markers in order to identify specific genes in any given
species. These genes will be then be introduced through genetic
transformation and the transgenic trees cloned and propagated
through tissue culture, improving considerably the genetic gains
achievable in tree species.

Release of Transgenic Trees


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Despite their lengthy reproductive cycles, sterile transgenic trees
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reassured that the transgenes will remain in the transformed
organisms. Also, effective communication between scientists and
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