Trees (2009) 23:1125–1135
DOI 10.1007/s00468-009-0362-8
REVIEW
Transgene stability and dispersal in forest trees
Mulkh Raj Ahuja
Received: 31 March 2009 / Revised: 9 June 2009 / Accepted: 12 June 2009 / Published online: 1 July 2009
Ó Springer-Verlag 2009
Abstract Transgenics from several forest tree species,
carrying a number of commercially important recombinant
genes, have been produced, and are undergoing confined
field trials in a number of countries. However, there are
questions and issues regarding stability of transgene
expression and transgene dispersal that need to be
addressed in long-lived forest trees. Variation in transgene
expression is not uncommon in the primary transformants
in plants, and is undesirable as it requires screening a large
number of transformants in order to select transgenic lines
with acceptable levels of transgene expression. Therefore,
the current focus of plant transformation is toward fine
tuning of transgene expression and stability in the trans-
genic forest trees. Although a number of studies have
reported a relatively stable transgene expression for several
target traits, including herbicide resistance, insect resis-
tance, and lignin modification, there was also some unin-
tended transgene instability in the genetically modified
(GM) forest trees. Transgene dispersal from GM trees to
feral forest populations and their containment remain
important biological and regulatory issues facing com-
mercial release of GM trees. Containment of transgenes
must be in place to effectively prevent escape of transgenic
pollen, seed, and vegetative propagules in economically
important GM forest trees before their commercialization.
Therefore, it is important to devise innovative technologies
in genetic engineering that lead to genetically stable
transgenic trees not only for qualitative traits (herbicide
resistance, insect resistance), but also for quantitative traits
Communicated by F. Cánovas.
M. R. Ahuja (&)
60 Shivertown Road, New Paltz, NY 12561, USA
e-mail: mrahuja@hotmail.com
(accelerated growth, increased height, increased wood
density), and also prevent escape of transgenes in the forest
trees.
Keywords Forest trees
Recombinant genes

Transgenic trees
Transgene stability
Gene flow

Transgene dispersal
Containment
Introduction
Genetic improvement of forest trees has mainly advanced
by using time-honored selection and breeding approaches
(Zobel and Talbert 1984; White et al. 2007). However,
improvement of forest trees by traditional approaches is a
slow process, because of long life cycles, with extended
vegetative phases ranging from one to many decades, in
forest trees. Genetic engineering (GE), on the other hand,
offers prospects of transferring desired traits into selected
genotypes at a comparatively faster rate by bypassing the
reproductive process. Thus, transfer of a desirable trait by
traditional approaches, involving breeding and recurrent
selection that would take decades to centuries in forest
trees, can be accomplished by GE in a single generation.
Further, GE overrides the incompatibility barriers and
allows gene transfer not only in unrelated tree species but
also between widely divergent taxon (for example, bacteria
to trees). GE also removes the potentially undesirable
effects of linked alleles which could be inadvertently
introduced to the progeny by conventional breeding
programs.
Currently, a number of genetically modified (GM)
agricultural crops (maize, cotton, soybean, canola, squash,
papaya and alfalfa, sugarbeet) have been globally com-
mercialized (James 2008). However, GM forest trees,
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excepting commercial plantations of poplars in China (Lida
et al. 2004; Ewald et al. 2006), are still undergoing con-
fined field trials in a number of countries in the world
(Robischon 2006; Kikuchi et al. 2008). The future pros-
pects for commercial plantations of GM trees are contro-
versial and remain uncertain as many biological and
regulatory issues still need to be resolved (Ahuja 2000,
2001; Strauss 2003; Bradford et al. 2005; Williams 2005;
Farnum et al. 2007; Brunner et al. 2007; Sederoff 2007). As
compared to agricultural crops, GE research in the forest
trees is still faced with technical problems and limitations.
Many of these problems relate to the feasibility of in vitro
regeneration systems and application of transformation
methodologies to commercially important forest trees.
From the beginning of GE, in forest trees in the 1980s, only
a handful of genotypes (mostly of academic interest), that
could be easily regenerated and transformed, have been the
target of GE research. This was a necessary development
for the understanding of basic processes of transformation,
integration, and expression of transgenes in forest trees
(Charest and Michel 1991; Fladung et al. 1997; Tzfira et al.
1998; Ahuja 2000; Peña and Séguin 2001; Walter et al.
2002; Campbell et al. 2003; Tang and Newton 2003).
However, after almost two decades of GE research, only in
recent years attempts have been made to include selected
genotypes in forest trees (Meilan et al. 2004; Boerjan
2005), and that too on a limited scale.
The target traits (Table 1) that are commercially
important in forest tree domestication include accelerated
growth, increased height, wood properties, and adaptation
to environmental stresses, including drought and salt
tolerance (Campbell et al. 2003; Busov et al. 2005; Sedjo
2006). All these are quantitative traits controlled by hun-
dreds of genes. In addition, if all these polygenes act
additively and each has a small effect, then the transfer of
just a few genes by GE might have little impact on a
quantitative trait. However, recent research suggests that it
might be possible to have large effects on quantitatively
inherited traits by modifying just one gene by GE (for
example, lignin modification; Baucher et al. 2003). The
most common approach is to alter the expression of a
native gene controlling a quantitative trait, either by down-
regulating or up-regulating its expression. Down-regulation
Table 1 Target traits for GE in forest trees (modified from Sedjo 2006)
Wood quality traits Environmental adaptability
Increased wood density for improved Drought tolerance, Salt tolerance
timber strength
Reduced lignin for reduced pulping costs Cold tolerance
Reduced juvenile wood Pest resistance (insects/microbes)
Improved fiber characteristics Bioremediation
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Trees (2009) 23:1125–1135
can be accomplished by a variety of approaches, including
anti-sense suppression or RNA interference technologies
(Baulcombe 2004; Kusaba 2004). Up-regulation is some-
what difficult, requiring introducing a new copy of a target
gene under the control of a strong and/or inducible pro-
moter. The expression of a native gene is not affected by
this approach and, therefore, confounds the desired effect
on the phenotype. A more directed approach involving site-
directed homologous recombination (Kumar and Fladung
2001) to replace a native gene with the engineered gene is
still a long way from being developed in forest trees.
Forest trees have long generation cycles and their veg-
etative phases extend from one to several decades. Genetic
and phenotypic stability of transgenic trees are important
considerations for subsequent large-scale plantation of GM
trees (Ahuja 1988, 1997, 2000; Hawkins et al. 2003;
Hoenicka and Fladung 2006a; Brunner et al. 2007). In
addition, issues regarding escape of transgenes from GE
trees and their effects on the feral tree populations
acquiring the transgenes must also be addressed before
commercialization of GM forest trees (Brunner et al. 2007;
Farnum et al. 2007). In our opinion, stability of transgene
expression and escape of transgenes are important aspects
of GE in long-lived forest trees that will ultimately deter-
mine the future of GM tree in commercial forestry. In this
paper, we discuss stability and escape of the transgenes in
the forest trees.
Stability of transgene expression
One of the major concerns of GE in forest trees is the
stability of transgene expression in long-lived forest trees.
And longevity of an organism raises a number of questions
and concerns that must be addressed before commerciali-
zation of GM trees. Transgene instability has been widely
reported in herbaceous plants and forest trees. Variation in
gene expression may be caused by tissue culture-associated
somaclonal variation (Larkin and Scowcroft 1981; Ahuja
1987, 1998), integration patterns and copy number of the
transgene, and inactivation/silencing of the transgene in the
host genome (Finnegan and McElroy 1994; Meyer and
Saedler 1996; Ahuja and Fladung 1996; Fladung et al.
Silvicultural features
Accelerated growth rate
Improvement of tree architecture (tree form,
stem form, branching)
Flowering control (reproductive sterility)
Herbicide resistance
Trees (2009) 23:1125–1135 1127

1997; Stam et al. 1997; Ahuja 1997; Fladung 1999; Fagard
and Vauchert 2000; Cervera et al. 2000; Kumar and
Fladung 2001, 2002, 2004; Butaye et al. 2005; Wagner
et al. 2005; Hoenicka and Fladung 2006a). Variation in
transgene expression is not uncommon in the primary
transformants in plants, and is undesirable as it may
requires screening a large number of transformants in order
to select transgenic lines with acceptable levels of trans-
gene expression (Birch 1997; Bhat and Srinivasan 2002;
De Bolle et al. 2003). Therefore, one of the major
challenges facing transgenic research is to develop
cost-effective techniques to produce a high proportion of
transformants with little inter-transformant variation, and
transgenic plants showing desired phenotype with stable
transgene expression (Butaye et al. 2005). In this section,
we examine the stability of transgene expression in the
commercially important genes in forest trees.
A number of economically important genes (Table 2)
(for example, herbicide resistance, insect and disease
resistance, reduced lignin, and growth traits) have been
transferred to produce transgenic plants in several forest
tree species. These include (1) herbicide resistance genes
(aroA, BAR, CP4) in Populus (Fillatti et al. 1987; Donahue
et al. 1994; Meilan et al. 2002; Li et al. 2008a, b), Euca-
lyptus (Harcourt et al. 2000), Pinus radiata and Picea abies
(Bishop-Hurley et al. 2001; Charity et al. 2005); (2) insect
resistance gene (Bt) in Populus (Leple et al. 1995; Wang
et al. 1996; Meilan et al. 2000; Hu et al. 1999; Yang et al.
2003); Pinus radiata (Grace et al. 2005), Pinus taeda
(Tang and Tian 2003) and Picea glauca (Lachance et al.
2007); (3) bacterial and fungal resistance genes (D4E1,
ChitIV, STS, ESF39A, ech42) in hybrid poplar (Populus
tremula 9 P. alba) (Mentag et al. 2003), in Betula
(Pasonen et al. 2004), in Populus (Seppänen et al. 2004), in
Ulmus Americana (Newhouse et al. 2007), and in Picea
mariana and hybrid poplar (Populus nigra 9 P. maxi-
mowiczii) (Noël et al. 2005); (4) stress tolerance gene
(CaPF1) in Pinus strobus (Tang et al. 2007) and salt
tolerance gene (codA) in Eucalyptus (Yu et al. 2009); and
(5) lignin modification genes (CAD, 4Cl, COMT, CAld5H)
in Populus (Hu et al. 1999; Pilate et al. 2002; Baucher et al.
2003; Li et al. 2003; Halpin et al. 2007; Hancock et al.
2007) and Betula pendula (Tiimonen et al. 2005). In most
of these short-term studies there was a fairly stable and
predicable expression of transgenes in the selected trans-
genic trees under greenhouse and confined field trials.
However, there was variation in transgene expression
between transformants and consequently a large number of
transgeneic lines were scored for selection of relatively
stable translines. Therefore, the current focus of plant
transformation is toward fine tuning of transgene expres-
sion and stability in the transgenic forest trees.
Detailed field studies, ranging from 3 to 8 years, have
been carried out with GM poplars and white spruce.
Transgenic poplars have shown relatively stable expression
of the herbicide resistance genes up to 8 years under con-
fined field trials (Meilan et al. 2000, 2002; Li et al. 2008a,
b). Transgenic white spruce (Picea glauca) carrying the Bt
gene also showed a continued insecticidal activity in the
needles against budworm under confined field trials for
5 years (Lachance et al. 2007). Generally, stable expres-
sion was detected in those transgenic lines carrying one to

Table 2 Promoters and coding

Species Recombinant Gene Expression Reference
regions of some commercially
Promoter Codera
important recombinant genes
transferred in trees Populus (CaMV) 35S Bt Insect resistance Leple et al. (1995)
(CaMV) 35S AS4CL1 Lignin modification Hu et al. (1999)
(CaMV) 35S ASCAD Lignin modification Pilate et al. (2002)
(FMV) 34S CP4 Herbicide resistance Meilan et al. (2002)
(Populus) PTD DTA Reproductive sterility Skinner et al. (2003)
a
(CaMV) 35S prxC1a Improved height growth Kawaoka et al. (2003)
Coding sequences of Bt from
(CaMV) 35S GS1 Improved height growth Jing et al. (2004)
Bacillus thuringiensis; CP4
from Agrobacterium (CaMV) 35S AaXEG2 Improved height growth Park et al. (2004)
tumefaciens strain CP4; ChitIV (CaMV) 35S STS Fungal resistance Seppänen et al. (2004)
from sugar beet; BAR from Eucalyptus (CaMV) 35S Bt, BAR Insect and herbicide resistant Harcourt et al. (2000)
Streptomyces hydroscopicus;
CaPF1 from Capsicum annuum; (CaMV) 35S codA Salt tolerance Yu et al. (2009)
codA from Arthrobacter Betula (CaMV) 35S COMT Lignin modification Tiimonen et al. (2005)
globformis; DTA from Dianthus (CaMV) 35S ChitIV Fungal resistance Pasonen et al. (2004)
chinensis; CAD from Populus
Pinus (CaMV) 35S BAR Herbicide resistance Charity et al. (2005)
tremuloides; GS1 (glutamine
synthetase) from Pinus, (CaMV) 35S Bt Insect resistance Grace et al. (2005)
AaXEC2 from Aspergillus; (CaMV) 35S CaPF1 Freeze tolerance Tang et al. (2007)
prxC1a from horseradish; and Picea (CaMV) 35 Bt Insect resistance Lachance et al. (2007)
AS denotes antisense orientation

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fewer copies of the transgene (Brunner et al. 2007; Li et al.
2008a, b). However, there were exceptions, where one or
few copies of the transgene were not always associated
with stable expression of the transgene, as was reported in
the antifungal activity transgene (stilbenes, pinosylvin
synthase, STS) in Populus (Seppänen et al. 2004). All these
genes involved in herbicide resistance (aroA, BAR, CP4),
insect resistance (Bt), and fungal resistance (STS) are
dominant gain-of-function genes, and there is little infor-
mation for their effects on plant growth and development.
Although short-term and mid-term studies with reduced
lignin, herbicide, and insect resistance GE trees, in
particular poplars, have been encouraging with regard to
stability of transgene expression (Brunner et al. 2007;
Li et al. 2008a, b), long-term confined field trails would be
necessary for evaluating the continued stable transgene
expression in the forest trees, in particular conifers.
In addition to herbicide resistance, pest resistance, and
lignin reduction, genes impacting growth traits (Table 3)
have also been introduced into Populus. Over-expression
of horseradish peroxidase gene (prxC1a) (Kawaoka et al.
2003), pine cytosolic glutamine synthetase (GS1) gene
(Jing et al. 2004), and Aspergillus xylogluconase
(AaXEG2) gene (Park et al. 2004) resulted in increase in
height growth and stem diameter in Populus. These are
interesting developments in the GE research on growth
traits in forest trees. Although, stable growth increase was
reported over a 3-year period in confined field trials in GM
poplars carrying GS1 transgene (Jing et al. 2004), it would
be necessary to investigate long-term effects of these
over-expressing exogenes on the genetic and phenotypic
stability and adverse effects, if any, in the GE forest trees.
Transgene stability in space and time
An important aspect of transgene expression relates to
functional utility of a transgene in time and space in the
forest tree genome (Ahuja 2000). For example, herbicide
tolerance transgene may remain active throughout the
major part of the life of an annual crop, so that it has a

Table 3 Global area of Biotech

Rank Country Area (millions GM crops
Crops in 2007 (data from James
of hectares)
2008)
1. USA 62.5 Soybean, maize, cotton, canola,
squash, papaya, alfalfa, sugarbeet
2. Argentina 21.0 Soybean, maize, cotton
3. Brazil 15.8 Soybean, maize, cotton
4. India 7.6 Cotton
5. Canada 7.6 Canola, maize, soybean, sugarbeet
6. China 3.8 Cotton, tomato, poplar, petunia,
papaya, sweet pepper
7. Paraguay 2.7 Soybean
8. South Africa 1.8 Maize, soybean, cotton
9. Uruguay 0.7 Soybean, maize
10. Bolivia 0.6 Soybean
11. Philippines 0.4 Maize
12. Australia 0.2 Cotton, canola, carnation
13. Mexico 0.1 Cotton, soybean
14. Spain 0.1 Maize
15. Chile \0.1 Maize, soybean, canola
16 Colombia \0.1 Cotton, carnation
17. Honduras \0.1 Maize
18 Burkina Faso \0.1 Cotton
19. Czech Republic \0.1 Maize
20 Romania \0.1 Maize
21 Portugal \0.1 Maize
22 Germany \0.1 Maize
23 Poland \0.1 Maize
24 Slovakia \0.1 Maize
25 Egypt \0.1 Maize
Total area 125 (million hectares)
1 hectare = 2.47 acres

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Trees (2009) 23:1125–1135
competitive edge over the weeds when sprayed with an
herbicide. On the other hand, the utility of the herbicide-
tolerant transgene would be required only in the first few
years of the tree growth, when an herbicide is sprayed to
kill the competing weeds. After that initial growth period,
the product of the herbicide tolerance transgene, although
constitutively expressed, may not be required in the
absence of an herbicide. What will be the fitness cost of the
herbicide tolerant transgene during the next 10–50 years
life of the transgenic trees remains unknown. The trans-
genes for lignin modification, insect and disease tolerance
are required for the entire life of a tree, and their stable
functionality and expression would be of paramount
importance to the survival of the tree. On the other hand,
GE for reproductive sterility would require the activation
of floral ablation and/or male or female sterility trans-
gene(s) after an extended vegetative phase of a tree. The
question whether these tissue-specific transgenes for
reproductive sterility remain inactive/silent during the
extended vegetative phase in the tree genome remains to be
fully investigated. Studies with floral ablation genes have
shown that there were growth abnormalities in transgenic
birch (Lemmetyinen et al. 2004; Lännenpää et al. 2005)
and poplar (Wei et al. 2006, 2007) due to the leaky
expression of the floral ablation transgenes under confined
greenhouse and field conditions.
The transgenes involved in a qualitative traits (for
example, herbicide and pest resistance) are dominant
exogenes and transgenic plants with relatively stable
transgene expression have been produced in forest trees
(Hoenicka and Fladung 2006a; Brunner et al. 2007).
However, commercially important target traits in forest
tree, including accelerated growth, increased wood density,
and drought and cold tolerance (Table 1), are quantitative
traits controlled by a large number of genes. Most of genes
involved in these quantitative traits will affect growth and
development, and stability of transgene expression in such
traits would be necessary throughout the life of a transgenic
tree.
As trees grow, they increase in size, and complexity
with their long age, and changes in gene expression for
fast-growth and development may occur during seasonal
cycles. Transgenes involved in growth and adaptation may
also cause unintended pleiotropic effects on growth, and
may become vulnerable to gene silencing due to methyl-
ation of the promoter element during the extended vege-
tative phase of the transgenic trees. Therefore, promoter
fidelity and stability of transgene expression have to be
viewed at several different levels in space and time in the
long-lived forest trees (Ahuja 2000; Brunner et al. 2007).
Transgene expression may vary depending on the type
of promoter used to drive its expression in a transgenic
plant. Some promoters, for example, the most widely used
1129
promoter 35S from Cauliflower mosaic virus (CaMV),
seem to function well in most herbaceous plants and forest
tree species, and regulates a high expression of the
recombinant gene. Other heterologous promoters from
bacteria and plants also function well in transgenic forest
trees. Recent studies suggest that level of transgene
expression may be stabilized/enhanced in transgenic plants
by including (1) genome-guided transgenes (GGT), using
native or homologous genes and promoters from related
species (Strauss 2003); (2) incorporation of matrix attach-
ment regions (MARs) to flank the recombinant genes
(Allen et al. 2000; Butaye et al. 2004; Halweg et al. 2005;
Wei et al. 2006); (3) site-specific integration of a single
transgene by recombinant-directed Cre/lox transformation
(Ow 2002; Srivastava et al. 2004; Butaye et al. 2005; Luo
et al. 2007); and (4) engineering novel traits in plants
through RNA interference (RNAi) in transgenic plants
(Mansoor et al. 2006). However, the application of most of
these techniques to minimize variation in transgeneis
expression is still in experimental stages in the forest trees.
Therefore, it is important to optimize gene transfer tech-
nologies that produce preferentially stable expression of
transgenes involved in both qualitative and quantitative
traits in the forest trees.
Transgene escape
Gene transfer through pollen routinely occurs between
domesticated plants and their wild relatives (Ellstrand et al.
1999; Chapman and Burke 2006). Cross-pollination
between commercial oilseed rape and its wild relatives
(Stewart et al. 2003), and cultivated sunflower and wild
sunflower (Burke et al. 2002) can occur at considerable
distances, and pollen-mediated gene flow from perennial
bentgrass can occur at a distance of 21 km (Watrud et al.
2004). In spite of regulatory oversights on the field trials of
transgenic crops in confined locations, before commer-
cialization, there is evidence to suggest that transgenes can
go wild (Baack 2006). In 2003, herbicide-tolerant trans-
genic perennial creeping bentgrass (Agrostis stolonifera)
was grown on 162 ha of control district in central Oregon,
USA. Bentgrass provides a good turf for golf courses.
Despite the necessary precautions during harvest and seed
collection in the wind-pollinated bentgrass, there was
transgene escape through pollen. Herbicide-tolerant trans-
gene was found in a small number of natural bentgrass
plants (9 out of 20,400 sampled) up to 3.8 km down wind
of control area (Reichman et al. 2006). This is not an
absolute number of transgenic bentgrass outside the control
area, as Reichman et al. (2006) only surveyed the publi-
cally owned portion (10%) of the suitable habitat; 90% of
the potential habitat occurs on private lands. Bentgrass is
perennial and this raises the possibility that transgene flow
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might continue for many years in wild bentgrass popula-
tions, regardless of presence or absence of herbicide.
Clearly, transgenes cannot be kept on leash, and transgene
escape is a virtual reality from transgenic plantations, and it
is unlikely that transgenes can be retracted once they are
out of the bottle (Marvier and Von Acker 2005; Chapman
and Burke 2006). The spread of transgenic pollen/small
seed is an important reminder concerning transgene con-
tainment. Transgenic corn and canola seeds lost during
harvest/transportation have been reported to appear, one or
more years following planting of transgenic crops, on
agricultural lands or roadside (Baack 2006). If transgenes
can escape from transgenic crops to non-transgenic popu-
lations of the same species, or to related weedy species,
what would be the consequences of transgene dispersal in
forest trees?
One major concern with commercialization of GM trees
is that gene flow from transgenic tree plantations may have
negative impacts on natural forest populations. Both pollen
and seed can be dispersed long distance (many kilometers)
by wind from forest stands resulting in considerable gene
flow to the neighboring forest tree populations (Schuster
and Mitton 2000; Williams and Davis 2005; Slavov et al.
2009). If a transgene-carrying genome was to escape from
GM tree plantation and become established in the wild
feral tree populations, it could conceivably displace the
native forest tree genotype or lead to maladaptation (White
et al. 2007). Transgene fitness in the feral native tree
populations acquiring transgenes is an important issue in
forest trees (Farnum et al. 2007; Smouse et al. 2007).
Because of long generational cycles, field trial of GM trees
to assess transgene escape to conventional populations, and
their hybrids are not practical. The alternative would be to
develop and exploit simulation models to gain insight in
the gene flow in forest trees.
Models on gene flow and their implications
for transgenic forest trees
Models of pollen and seed dispersal have been proposed in
forest trees (Nathan et al. 2002; DiFazio et al. 2004;
Kuparinen and Schurr 2007, 2008). Pollen and seed dis-
persal is a highly stochastic process, and stochastic models
on gene flow can show different results depending on the
life history and environmental variables. Although several
different types of models have been used for simulating
gene flow across landscapes, we discuss two that take into
short-distance as well as long-distance dispersal. Based on
large field studies, genetic analysis, pollen dispersal, a
simulation model so-called STEVE (simulation of trans-
gene effects in variable environment) was used to monitor
gene flow across landscapes in poplar (DiFazio et al. 2004).
This model explored the potential escape of transgenes
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Trees (2009) 23:1125–1135
from genetically engineered poplar plantations into wild
populations of native poplar (DiFazio et al. 2004; Burczyk
et al. 2004). This model has provided some useful insights
into the process at play in transgene flow in trees. Reducing
the relative fertility levels in transgenic trees by moderate
amounts (to 90%) greatly reduced the gene flow, with
substantial transgenic competitive advantage. On the other
hand, transgenic fertility below 1% had little effect on
transgenic gene flow to the neighboring populations.
The second spatially explicit model AMELIE (A
Modeling framEwork for popuLatIon gEnotype dynamics)
formulates a framework that links the dynamics of the
populations and the genotypes for the study of transgenic
spread from GM trees to a conventional forest (Kuparinen
and Schurr 2007). This flexible model takes into consider-
ation life histories, reproductive systems, demographic and
environmental parametrics. According to this model,
transgene spread depends on the interplay of the initial
genotype of the GM crop (allelic heterozygosity or reces-
sivity) at the transgenic locus, population dynamics, and the
local environmental conditions. Additionally, the same
authors (Kuparinen and Schurr 2008) assessed the risk of
gene flow from genetically modified trees carrying a miti-
gation transgene. They examined long-term spread of
transgenes from GM tress carrying two traits: increased
growth rate and reduced fecundity (Kuparinen and Schurr
2008). According to this model the risk assessment of
breakup of transgene from the mitigation transgene requires
input from genetics, tree life history, pollen escape, local
dynamics, and dispersal of GM and conventional forest tree
populations. Based on these parameters, the model predicts
the necessary guidelines for the management of GM tree
populations. However, field experimental data would still
be necessary to verify the validity of STEVE, AMELIE or
other population genetics models of transgene flow in forest
trees. In the final analysis, the benefits of GE must outweigh
the risks in the long-lived forest trees for commercialization
of GM trees. Therefore, it would be desirable to have
containment measures in place before release of transgenic
trees in marketplace. In addition to invasiveness potential of
GM trees, other ecological risks, for example, horizontal
gene transfer, transgenic pollen allergenicity in humans,
and effect on animals and other plants and the ecosystem
must also be fully assessed (Käppeli and Auberson 1998;
Wolfenbarger and Phifer 2000; van Frankenhuzen and
Beardmore 2004; Jaffe 2004; Heinemann and Traavik 2004;
Hoenicka and Fladung 2006b).
Transgene containment
Several molecular approaches have been proposed to
impede transgene escape (Daniell 2002; Lu 2003; Brunner
Trees (2009) 23:1125–1135
et al. 2007; Luo et al. 2007). These include (1) genetic
engineering of reproductive sterility either by ablation of
floral organs and/or transgenic male sterility (Strauss et al.
1995; Skinner et al. 2003); (2) site-specific DNA excision
of transgene mediated by Cre/loxP recombination system
(Ow 2002; Gilbertson 2003); (3) transgene suppression via
RNA interference (Watson et al. 2005; Li et al. 2008c); (4)
targeting transgenes into chloroplast or mitochondrial
genomes to reduce transgene escape via pollen (Daniell
et al. 2005); and (5) transgene mitigation by tandomly
linking the primary transgene with a mitigator gene that
reduces the fitness of hybrids between transgenic plants and
their wild relatives (Gressel 1999; Al-Ahmad et al. 2006).
Although these strategies have been discussed for the
genetic containment of transgenes in the forest plantations
(Brunner et al. 2007), it may not be entirely possible to
achieve 100% reproductive sterility to stop the escape of
transgenic pollen and seed from the transgenic forest trees.
The methodology for transfer of transgenes to chloroplast
is also not full proof, as low levels of paternal inheritance
may occur in plants (Ruf et al. 2007). Although most of
angiosperm tree species (for example, Populus, Eucalyp-
tus, Quercus) show maternal inheritance of chloroplast
genome, most conifers (for example, Pinus, Pseudotsuga,
Larix, Sequoia) exhibit a strictly paternal inheritance of the
chloroplast (White et al. 2007). Therefore, engineering
transgenes into chloroplast genome may be an option for
preventing transgene escape in angiosperm trees (Okumura
et al. 2006), but it may not be practical in conifers. The
mitigation transgene approach may also have a drawback.
If the mitigation transgene is not tightly linked to the pri-
mary transgene, and hybridization between transgenic and
conventional populations occurs, it may cause a breakage
of the linkage between the primary and mitigation trans-
gene, resulting in conventional genotypes that express the
primary transgene (Chapman and Burke 2006; Kuparinen
and Schurr 2008). Although transgenic reproductive
sterility and RNA interference approaches are being
extensively experimented in Populus (Skinner et al. 2003;
Wei et al. 2007; Brunner et al. 2007; Li et al. 2008c), the
other molecular approaches still remain untested in the
forest trees. The main drawback in these containment
approaches would be the unintended pleiotropic effects of
reproductive sterility transgenes on growth, and imprecise
control by mitigation/excision/repression approaches to
silence/eliminate the transgenes before the initiation of the
reproductive phase in the long-lived forest trees.
Prospects of GM trees in forestry
Although a large number of confined field trials on GM tree
species (both angiosperms and conifers) have been
1131
established world-wide, there are almost no commercial
plantations of GM forest trees. The only exception is
China, where insect-resistant GM poplars have been com-
mercialized (Lida et al. 2004; Ewald et al. 2006; James
2008). On the other hand, agricultural GM crop plants have
been commercialized in many countries more than a
decade ago. Ever since the first release of a GM crop, the
‘‘Flavrsavr’’ tomato that had delayed ripening characteris-
tics, in the United States in 1994, the global area of GM
crops have increased from 1.7 million hectares in 1996 to
125 million hectares (*74-fold increase) in 2008.
Currently, the global status of approved GE food and fiber
crops as of 2008 (Table 3) stands as follows: (1) GM crops
are planted on 125 million hectares over 25 countries with
more than half (62.5 million hectares) in the United States;
(2) GM crops include maize, soybean, cotton, canola,
squash, papaya, tomato, sweet pepper, alfalfa; carnation,
petunia, sugarbeet, and poplar; (3) Target traits for GM
crops include herbicide resistance, insect resistance and
virus resistance; and (4) GM crops generated an annual
global market of $10 billion (James 2008).
In spite of commercial release of these GM agricultural
crops and huge world market, there are still obstacles to
testing and deployment of GM forest trees. How to address
the transgenic escape/flow problem in forest trees? It seems
that there might be two different options to deal with this
problem: (1) biosafety approach, and (2) ecological
approach (Williams 2005). The first premise relies on the
effective biosafety protocols, ensuring complete reproduc-
tive sterility in the GM trees (Strauss et al. 1995; Brunner
et al. 2007) that would be required before commercial release
of GM trees. The ecological approach assumes that escape of
transgenes is inevitable, and therefore, minimizing the risks
of invasiveness (for example, viability, fertility) of offspring
from GM trees and feral tree populations, by using low-risk
genome-guided transgenes (Strauss 2003), would be an
option, where transferred traits are functionally equivalent to
those produced by conventional breeding (Strauss 2003;
Strauss et al. 2004; Williams and Davis 2005).
The commercialization of GM forest trees is still in the
distant future, because the GE research has not progressed
as far as the crop plants, and there are a number of unre-
solved biosafety, environmental and regulatory obstacles
(Jaffe 2004; van Frankenhuzen and Beardmore 2004;
Hoenicka and Fladung 2006b; Finstad et al. 2007; Groover
2007; Sederoff 2007). These concerns are based on the
endogenous behavior of the transgene (stability, and
interaction with other genes in the host genome in space
and time), and exogenous effects of the transgene (dis-
persal of pollen, seed and vegetative propagules) on the
feral forest tree plantations and the ecosystem. These bio-
safety and regulatory concerns must be addressed before
the commercialization of GM trees.
123
1132
Acknowledgments I thank the Institute of Forest Genetics, USDA
Forest Service, and the Department of Plant Sciences, University of
California, Davis, for facilities. I also thank David Neale for helpful
suggestions on the manuscript.
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