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

� 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

(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,

Communicated by F. Canovas.

M. R. Ahuja (&)

60 Shivertown Road, New Paltz, NY 12561, USA

e-mail: mrahuja@hotmail.com

123

Trees (2009) 23:1125–1135

DOI 10.1007/s00468-009-0362-8

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; Pena and Seguin 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

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.

Table 1 Target traits for GE in forest trees (modified from Sedjo 2006)

Wood quality traits Environmental adaptability Silvicultural features

Increased wood density for improved

timber strength

Drought tolerance, Salt tolerance Accelerated growth rate

Reduced lignin for reduced pulping costs Cold tolerance Improvement of tree architecture (tree form,

stem form, branching)

Reduced juvenile wood Pest resistance (insects/microbes) Flowering control (reproductive sterility)

Improved fiber characteristics Bioremediation Herbicide resistance

1126 Trees (2009) 23:1125–1135

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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 (Seppanen et al. 2004), in

Ulmus Americana (Newhouse et al. 2007), and in Picea

mariana and hybrid poplar (Populus nigra 9 P. maxi-

mowiczii) (Noel 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

regions of some commercially

important recombinant genes

transferred in trees

a Coding sequences of Bt from

Bacillus thuringiensis; CP4from Agrobacteriumtumefaciens strain CP4; ChitIVfrom sugar beet; BAR from

Streptomyces hydroscopicus;

CaPF1 from Capsicum annuum;

codA from Arthrobacterglobformis; DTA from Dianthuschinensis; CAD from Populustremuloides; GS1 (glutamine

synthetase) from Pinus,

AaXEC2 from Aspergillus;

prxC1a from horseradish; and

AS denotes antisense orientation

Species Recombinant Gene

Promoter CoderaExpression Reference

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)

(CaMV) 35S prxC1a Improved height growth Kawaoka et al. (2003)

(CaMV) 35S GS1 Improved height growth Jing et al. (2004)

(CaMV) 35S AaXEG2 Improved height growth Park et al. (2004)

(CaMV) 35S STS Fungal resistance Seppanen et al. (2004)

Eucalyptus (CaMV) 35S Bt, BAR Insect and herbicide resistant Harcourt et al. (2000)

(CaMV) 35S codA Salt tolerance Yu et al. (2009)

Betula (CaMV) 35S COMT Lignin modification Tiimonen et al. (2005)

(CaMV) 35S ChitIV Fungal resistance Pasonen et al. (2004)

Pinus (CaMV) 35S BAR Herbicide resistance Charity et al. (2005)

(CaMV) 35S Bt Insect resistance Grace et al. (2005)

(CaMV) 35S CaPF1 Freeze tolerance Tang et al. (2007)

Picea (CaMV) 35 Bt Insect resistance Lachance et al. (2007)

Trees (2009) 23:1125–1135 1127

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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 (Seppanen 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

Crops in 2007 (data from James

2008)

1 hectare = 2.47 acres

Rank Country Area (millions

of hectares)

GM crops

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)

1128 Trees (2009) 23:1125–1135

123

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; Lannenpaa 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

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

Trees (2009) 23:1125–1135 1129

123

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

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 (Kappeli 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

1130 Trees (2009) 23:1125–1135

123

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

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.

Trees (2009) 23:1125–1135 1131

123

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.

References

Ahuja MR (1987) Somaclonal variation. In: Bonga JM, Durzan DJ

(eds) Cell and tissue culture in forestry. vol 1. Martinus Nijhoff

Publishers, Dordrecht, pp 272–285

Ahuja MR (1988) Gene transfer in forest trees. In: Hanover JE,

Keathley DE (eds) Genetic manipulation of woody plants.

Plenum Press, New York, pp 25–41

Ahuja MR (1997) Transgenes and genetic instability. In: Klopfenstein

NB, Chun WYW, Kim M-S, Ahuja MR (eds) Micropropagation

and genetic engineering and molecular genetics of Populus.

Technical Report RM-GTR-297, USDA Forest Service, Rocky

Mountain Research Station, Fort Collins, pp 90–100

Ahuja MR (1998) Somaclonal genetics of forest trees. In: Jain SM,

Brar DS, Ahloowalia BS (eds) Somaclonal variation and induced

mutations in crop improvement. Kluwer, Dordrecht, pp 105–121

Ahuja MR (2000) Genetic engineering in forest trees: state of the art

and future perspectives. In: Jain SM, Minocha SC (eds)

Molecular biology of woody plants. vol. 1. Kluwer, Dordrecht,

pp 31–49

Ahuja MR (2001) Recent advances in molecular genetics of forest

trees. Euphytica 121:73–195

Ahuja MR, Fladung M (1996) Stability and expression of chimeric

genes in Populus. In: Ahuja MR, Boerjan W, Neale DB (eds)

Somatic cell genetics and Molecular genetics of trees. Kluwer,

Dordrecht, pp 89–96

Al-Ahmad H, Dwer J, Moloney M, Gressel J (2006) Mitigation of

establishment of Brassica napus transgenes in volunteers using a

tandem construct containing a selectively unfit gene. Plant

Biotecnol J 4:7–21

Allen GC, Spiker S, Thompson WF (2000) Use of matrix attachment

regions (MARs) to minimize transgene silencing. Plant Mol Biol

43:361–376

Baack EJ (2006) Engineered crops: transgenes go wild. Curr Biol

16:R583–R584

Baucher M, Halpin C, Petit-Conil M, Boerjan W (2003) Lignin:

genetic engineering and impact on pulping. Crit Rev Biochem

Mol Biol 38:305–350

Baulcombe D (2004) RNA silencing in plants. Nature 431:356–363

Bhat SR, Srinivasan S (2002) Molecular and genetic analysis of

transgenic plants: considerations and approaches. Plant Sci

163:673–681

Birch RG (1997) Plant transformation: problems and strategies for

practical application. Annu Rev Plant Physiol Mol Biol 48:297–

326

Bishop-Hurley SL, Zubkiewicz RJ, Grace LJ et al (2001) Conifer

genetic engineering: transgenic Pinus radiata (D.Don.) and

Picea abies (Karst.) plants are resistant to the herbicide Buster.

Plant Cell Rep 20:235–243

Boerjan W (2005) Biotechnology and domestication of forest trees.

Curr Opin Biotechnol 16:159–166

Bradford KJ, Deynze AV, Gutterson N, Parrott W, Strauss SH (2005)

Regulating transgenic crops sensibly: lessons from plant breed-

ing, biotechnology and genomics. Nat Biotechnol 23:439–444

Brunner AM, Li J, DiFazio SP, Schevchenko O, Montgomery BE,

Mohamed R, Wie H, Ma C, Elias AA, Van Wormer K, Strauss

SH (2007) Genetic containment of forest plantations. Tree Genet

Genomes 3:75–100

Burczyk J, DiFazio SP, Adams WT (2004) Gene flow in forest trees:

how far do genes really travel? For Genet 11:1–14

Burke JM, Gardner KA, Rieseberg LH (2002) The potential for gene

flow between cultivated and wild sunflower (Helianthus annuus)

in the United States. Am J Bot 89:1550–1552

Busov VB, Brunner AM, Meilan R, Filichkin S, Ganio L, Gandhi S,

Strauss SH (2005) Genetic transformation: a powerful tool for

dissection of adaptive traits in trees. New Phytol 167:9–18

Butaye KJM, Goderis IJWM, Wouters PFJ, Pues JMTG, Delaure SL,

Boekaert WF, Depicker A, Cammue BPA, De Bolle MFC (2004)

Stable high-level transgene expression in Arabidopsis thalianausing gene silencing mutant and matrix attachment regions. Plant

J 39:440–449

Butaye KMJ, Cammue BPA, Delaure SL, De Bolle MFC (2005)

Approaches to minimize variation in transgene expression in

plants. Mol Breed 16:79–91

Campbell MM, Brunner AM, Jones HM, Strauss SH (2003) Forestry’s

fertile crescent: the application of biotechnology to forest trees.

Plant Biotechnol J 1:141–154

Cervera M, Pina JA, Juarez J, Navarro L, Pena L (2000) A broad

exploration of transgenic citrus: stability of gene expression and

phenotype. Theor Appl Genet 100:670–677

Chapman MA, Burke JM (2006) Letting the gene out of the bottle:

populations genetics of genetically modified crops. New Phytol

170:429–443

Charest PJ, Michel MF (1991) Basics of plant genetic engineering and

its potential applications to tree species. Information Report

Pl-X-104. Petawawa National Forestry Institute, Forestry

Canada, pp 1–48

Charity JA, Holland L, Grace LJ, Walter C (2005) Consistent and

stable expression of the nptII, uidA and bar genes in transgenic

Pinus radiata after Agrobacterium tumefaciens-mediated trans-

formation using nurse cultures. Plant Cell Rep 23:606–616

Daniell H (2002) Molecular strategies for gene containment in

transgenic crops. Nat Biotechnol 20:581–586

Daniell H, Kumar S, Dufourmantel N (2005) Breakthrough in

chloroplast genetic engineering of agronomically important

crops. Trends Biotechnol 23:238–245

De Bolle MFC, Butaye MMJ, Couke WJW, Goderis IJWM, Wouters

PFJ, van Boxel N, Brockaert WF, Cammue BPA (2003) Analysis

of the influence of promoter elements and matrix attachment

region on the inter-individal variation of transgene expression in

populations of Arabidopsis thaliana. Plant Sci 165:169–179

DiFazio SP, Slavov GT, Burczyk J, Leonardi S, Strauss SH (2004)

Gene flow from tree plantations and implications for transgenic

risk assessment. In: Walter C, Carson M (eds) Plantation forest

biotechnology for the 21st Century. Research Signpost, Trivan-

drum, pp 405–422

Donahue RA, Davis TD, Riemenschneider DF, Michler CH, Carter

DR, Marquardt PE, Sankhala D, Haissig BE, Isebrands JG

(1994) Growth, photosynthesis, and herbicide tolerance of

genetically modified hybrid poplar. Can J For Res 24:2377–

2383

Ellstrand NC, Prentice HC, Hancock JF (1999) Gene flow and

introgression from domesticated plants into their wild relatives.

Annu Rev Ecol Syst 30:539–563

Ewald D, Hu J, Yang M (2006) Transgenic forest trees in China. In:

Fladung M, Ewald D (eds) Tree transgenesis: recent develop-

ments. Springer, Berlin, pp 25–45

Fagard M, Vauchert H (2000) (Tans)gene silencing in plants: how

many mechanisms? Annu Rev Plant Physiol Plant Mol Biol

51:167–194

Farnum P, Lucier A, Meilan R (2007) Ecological and population

genetics research imperatives for transgenic trees. Tree Genet

Genomes 3:119–133

Fillatti JJ, Selmer J, McCown B, Haissig B, Comai L (1987)

Agrobacterium mediated transformation and regeneration of

Populus. Mol Gen Genet 206:192–199

1132 Trees (2009) 23:1125–1135

123

Finnegan J, McElroy D (1994) Transgene inactivation: plants fight

back. Biotechnology 12:883–888

Finstad K, Bonfils AC, Shearer W, Macdonald P (2007) Trees with

novel traits in Canada: regulation and related scientific issues.

Tree Genet Genomes 3:135–139

Fladung M (1999) Gene stability in transgenic aspen (Populus). I.

Flanking DNA sequences and T-DNA structure. Mol Gen Genet

260:1097–1103

Fladung M, Kumar S, Ahuja MR (1997) Genetic transformation with

different chimeric gene constructs: transformation efficiency and

molecular analysis. Transgenic Res 6:111–121

Gilbertson L (2003) Cre-lox recombination: cre-active tools for plant

biotechnology. Trends Biotechnol 21:550–555

Grace LJ, Charity JA, Gresham B, Kay N, Walter C (2005) Insect

resistance transgenic Pinus radiata. Plant Cell Rep 24:103–111

Gressel J (1999) Tandem constructs preventing the rise of super

weeds. Trends Biotechnol 17:361–366

Groover AT (2007) Will genomics guide a greener forest biotech-

nology? Trends Plant Sci 12:234–238

Halpin C, Thain SC, Tilston EL, Guiney E, Lapierre C, Hopkin DW

(2007) Ecological impacts of trees with modified lignin. Tree

Genet Genomes 3:101–110

Halweg C, Thompson WF, Spiker S (2005) The Rb7 matrix

attachment region increases the likelihood and magnitude of

transgene expression in tobacco cells: a flow cytometric study.

Plant Cell 17:418–429

Hancock JE, Loya WM, Giardina CP, Li L, Chiang VL, Pregitzer KS

(2007) Plant growth, biomass partitioning and soil carbon

formation in response to altered lignin biosynthesis in Populustremuloides. New Phytol 173:732–742

Harcourt RL, Kyozuka J, Floyd RB, Bateman KS, Tanaka H,

Decroocq V, Llewellyn DJ, Zhu X, Peacock WJ, Dennis ES

(2000) Insect- and herbicide-resistant transgenic eucalypts. Mol

Breed 6:307–315

Hawkins S, Leple JC, Cornu D, Jouanin L, Pilate G (2003) Stability of

transgene expression in poplar: a model forest tree species. Ann

For Sci 60:427–438

Heinemann JA, Traavik T (2004) Problems in monitoring horizontal

gene transfer in field trials of transgenic plants. Nat Biotechnol

22:1105–1109

Hoenicka H, Fladung M (2006a) Genomic instability in woody plants

derived from genetic engineering. In: Fladung M, Ewald D (eds)

Transgenesis: recent developments. Springer, Berlin, pp 301–321

Hoenicka H, Fladung M (2006b) Biosafety in Populus spp. and other

forest trees: from non-native species to taxa derived from

traditional breeding and genetic engineering. Trees 20:131–144

Hu W-J, Harding SA, Lung J, Popko JL, Ralph J, Stokke DD, Tsai C-

J, Chiang V (1999) Repression of lignin biosynthesis promotes

cellulose accumulation and growth in transgenic trees. Nat

Biotechnol 17:808–812

Jaffe G (2004) Regulating transgenic crops: a comparative analysis of

different regulatory processes. Transgenic Res 13:5–19

James C (2008) Global status of commercialized Biotech/GM Crops

2008. The International Service for the Acquisition of Agri-

biotech Applications (ISAAA Brief # 39), Ithaca, NY.

http://www.isaaa.org

Jing ZP, Gallardo F, Pascual MB, Sampalo P, Romero J, Torres de

Navarra A, Canovas FM (2004) Improved growth in a field trial

of transgenic hybrid poplar overexpressing glutamine synthetase.

New Phytol 164:137–145

Kappeli O, Auberson L (1998) How safe is safe enough in plant

genetic engineering? Trends Plant Sci 3:276–281

Kawaoka A, Mutsanga E, Endo S, Kondo S, Yoshida K, Shinmyo A,

Ebinuma H (2003) Ectopic expression of a horseradish perox-

idase enhances growth rate and increase oxidative stress in

hybrid aspen. Plant Physiol 132:1177–1185

Kikuchi A, Watanabe K, Tanaka Y, Kamada H (2008) Recent progress

on environmental biosafety assessment of genetically modified

trees and floricultural plants in Japan. Plant Biotechnol 25:9–15

Kumar S, Fladung M (2001) Controlling transgene integration in

plants. Trends Plant Sci 6:156159

Kumar S, Fladung M (2002) Transgene integration in aspen:

structures of integration sites and mechanisms of T-DNA

integration. Plant J 31:543–551

Kumar S, Fladung M (2004) Stability of transgene expression in aspen. In:

Kumar S, Fladung M (eds) Molecular genetics and breeding of forest

trees. Food Products Press, Binghamton, pp 293–308

Kuparinen A, Schurr F (2007) A flexible modeling framework linking

the spatio-temporal dynamics of plant genotypes and popula-

tions: applications to gene flow from transgenic forests. Ecol

Modell 202:476–486

Kuparinen A, Schurr F (2008) Assessing the risk of gene flow from

genetically modified trees carrying mitigation transgenes. Biol

Invasions 10:281–290

Kusaba M (2004) RNA interference in crop plants. Curr Opin

Biotechnol 15:139–143

Lachance D, Hamel L-P, Pelltier E, Valero J, Bernier-Cardou M,

Chapman K, Van Frankenhuyzen K, Seguin A (2007) Expres-

sion of a Bacillus thuringiensis cry1Ab gene in transgenic white

spruce and its efficacy against the spruce budworm (Choristone-ura fumiferana). Tree Genet Genomes 3:153–167

Lannenpaa M, Hassinen M, Ranki A, Holtta-Vuora M, Lemmetyinen

J, Keinonnen K, Sopanen T (2005) Prevention of flower

development in birch and other plants using a BPFULLI::BARN-ASE construct. Plant Cell Rep 24:69–78

Larkin PJ, Scowcroft WR (1981) Somaclonal variation—a novel

source of variability from cell cultures for plant improvement.

Theor Appl Genet 60:197–214

Lemmetyinen J, Keinonen K, Sopanen T (2004) Prevention of

flowering of a tree, silver birch. Mol Breed 13:243–249

Leple J, Bonadebottino M, Augustin S, Pilate G, Letan VD,

Delplanque A, Cornu D, Jouanin L (1995) Toxicity to Chryso-mela tremulae (Coleoptera, chrysomelidae) of transgenic poplars

expressing a cysteine proteinase-inhibitor. Mol Breed 1:319–328

Li L, Zhou Y, Sun J, Marita JM, Ralph J, Chiang VL (2003)

Combinatorial modification of multiple lignin traits in tree

through multigene cotransformation. Proc Natl Acad Sci USA

100:4939–4944

Li J, Brunner AM, Meilan R, Strauss SH (2008a) Stability of

transgenes in trees: expression of two reporter genes in poplar

over two field seasons. Tree Physiol 29:299–312

Li J, Meilan R, Ma C, Barish M, Strauss SH (2008b) Stability of

herbicide resistance over eight years of coppice in field-grown,

genetically engineered poplars. West J Appl For 23:89–93

Li J, Brunner AM, Schevchenko O, Meilan R, Ma C, Skinner JS,

Strauss SH (2008c) Efficient and stable transgene suppression

via RNAi in field-grown poplars. Transgenic Res 17:679–694

Lida W, Yifan H, Jianjun H (2004) Transgenic forest trees for insect

resistance. In: Kumar S, Fladung M (eds) Molecular genetics

and breeding of forest trees. The Haworth Press, Binghamton,

pp 243–261

Lu B-R (2003) Transgenic containment by molecular means—is it

possible and cost effective? Environ Biosafety Res 2:3–8

Luo R, Duan H, Zhao D, Zheng X, Deng W, Chen Y, Stewart CN,

McAvoy R, Jiang X, Wu Y, He A, Pei Y, Li Y (2007) ‘‘GM-

gene-deletor’’: fused loxP-FRT recognition sequences dramati-

cally improve the efficiency of FLP or GRF recombinase on

transgene excision from pollen and seed of tobacco plants. Plant

Biotechnol J 5:263–274

Mansoor S, Amin I, Hussain M, Zafar Y, Briddon RW (2006)

Engineering novel traits in plants through RNA interference.

Trends Plant Sci 11:559–565

Trees (2009) 23:1125–1135 1133

123

Marvier M, Von Acker RC (2005) Can crop transgenes be kept on a

leash? Front Ecol Environ 3:99–106

Meilan R, Ma C, Cheng S, Eaton JA, Miller LK, Crocket RP, DiFazio

SP, Strauss SH (2000) High levels of roundup and leaf beetle

resistance in genetically engineered hybrid cottonwoods. In:

Blattner KA, Johnson JD, Baumgartner DM (eds) Hybrid poplars

in the Pacific Northwest: culture, commerce and capability.

Washington State University, Pullman, pp 29–38

Meilan R, Han K-H, Ma C et al (2002) The CP4 transgene provides

high levels of tolerance to Roundup herbicide in field-grown

hybrid poplars. Can J For Res 32:967–976

Meilan R, Ellis D, Pilate G, Bruner AM, Skinner J (2004)

Accomplishments and challenges in genetic engineering of

forest trees. In: Strauss SH, Bradshaw HD (eds) The Bioengi-

neered Forest Challenges for Science and Society. Resources for

the Future, Washington, DC, pp 36–51

Mentag R, Luckevich M, Morency MJ, Seguin A (2003) Bacterial

disease resistance of transgenic hybrid poplar expressing the

synthetic antimicrobial peptide D4E1. Tree Physiol 23:405–411

Meyer P, Saedler H (1996) Homology-dependent gene silencing in

plants. Annu Rev Plant Physiol 47:23–48

Nathan R, Katul GG, Horn HS, Thomas SM, Oren R, Avissar R,

Pacala SW, Levin SA (2002) Mechanisms of long-distance

dispersal of seeds by wind. Nature 418:409–413

Newhouse AE, Schrodt F, Liang H, Maynard CA, Powell WA (2007)

Transgenic American elm shows reduced Dutch elm disease

symptoms and normal mycorrhizal colonization. Plant Cell Rep

26:977–987

Noel A, Levasseur C, Le VQ, Seguin A (2005) Enhanced resistance to

fungal pathogens in forest trees by genetic transformation of

black spruce and hybrid poplar with a Trichoderma harzianumendochitinase gene. Physiol Mol Plant Pathol 67:92–99

Okumura S, Sawada M, Park YW, Hayashi T, Shimamura M, Takase

H, Tomizawa K (2006) Transformation of poplar (Popolus alba)

plastids and expression of foreign proteins in trees. Transgenic

Res 15:637–644

Ow DW (2002) Recombinase-directed plant transformation for the

post-genomic era. Plant Mol Biol 48:163–200

Park YW, Baba K, Furuta Y, Lida I, Sameshima K, Arai M, Hayashi T

(2004) Enhanced growth and cellulose accumulation by over-

expression of xylogluconase in poplar. FEBS Lett 564:183–187

Pasonen H-L, Seppanen S-K, Degefu Y, Rytkonen A, Von Weiss-

enberg K, Pappinen A (2004) Field performance of chitinase

transgenic silver birches (Betula pendula): resistance to fungal

disease. Theor Appl Genet 109:562–570

Pena L, Seguin A (2001) Recent advances in genetic transformation

of trees. Trends Biotechnol 12:500–506

Pilate G, Guiney E, Holt K, Petit-Conil M, Lapierre C, Leple J-C,

Pollet B, Mila I, Webster EA, Marstorp HG, Hopkins DW,

Jouanin L, Boerjan W, Schuch W, Cornu D, Halpin C (2002)

Field and pulping performances of transgenic trees with altered

lignification. Nat Biotechnol 20:607–612

Reichman JR, Watrud LS, Lee EH, Burdick CA, Bollman MA, Strom

MJ, King GA, Mallory-Smith C (2006) Establishment of

transgenic herbicide resistant creeping bentgrass (Agrostisstolonifera L.) in non-agronomic habitats. Mol Ecol 15:4243–

4255

Robischon M (2006) Field trials with transgenic trees- state of art and

development. In: Fladung M, Ewald D (eds) Tree transgenesis:

recent developments. Springer, Berlin, pp 3–23

Ruf S, Karcher D, Bock R (2007) Determining the transgene

containment level provided by chloroplast transformation. Proc

Natl Acad Sci USA 104:6998–7002

Schuster WSF, Mitton JB (2000) Paternity and gene dispersal in

limber pine (Pinus flexilus James). Genet Soc Great Br 84:348–

361

Sederoff R (2007) Regulatory science in forest biotechnology. Tree

Genet Genomes 3:71–74

Sedjo RA (2006) Towards commercialization of genetically engi-

neered forests: economic and social considerations. Resources

for the Future, Washington, DC, pp 1–50

Seppanen S-K, Syrjala L, Von Weissenberg K, Teeri TH, Paajanen L,

Pappinen A (2004) Antifungal activity of stilbenes in vitro

bioassays and in transgenic Populus expressing a gene encoding

pinosylvin synthase. Plant Cell Rep 22:584–593

Skinner JS, Meilan R, Ma C, Strauss SH (2003) The Populus PTDpromoter imparts floral-predeterminant expression and enables

high levels of floral-organ ablation in Populus, Nicotiana and

Arabidopsis. Mol Breed 12:119–132

Slavov GT, Leonardi S, Burczyk J, Adams WT, Strauss SH, DiFazio

SP (2009) Extensive pollen flow in two ecologically contrasting

populations of Populus trichocarpa. Mol Ecol 18:357–373

Smouse PE, Robledo-Arnuncio JJ, Gonzalez-Martınez SC (2007)

Implications of natural propagule flow for containment of

genetically modified trees. Tree Genet Genomes 3:141–152

Srivastava V, Ariza-Netto M, Wilson AJ (2004) Cre-mediated site-

specific gene integration for consistent transgene expression in

rice. Plant Biotech J 2:169–179

Stam M, Mol JNM, Kooter JM (1997) The silence of genes in

transgenic plants. Ann Bot 79:3–12

Stewart CN, Halfhill MD, Warwick SI (2003) Transgene introgres-

sion from genetically modified crops to their wild relatives. Nat

Rev Genet 4:806–817

Strauss SH (2003) Genomics, genetic engineering, and domestication

of crops. Science 300:61–62

Strauss SH, Rottmann WH, Brunner AM, Sheppard LA (1995)

Genetic engineering of reproductive sterility in forest trees. Mol

Breed 1:5–26

Strauss SH, Brunner AM, Busov VB, Ma C, Meilan R (2004) Ten

lessons from 15 years of transgenic poplar research. Forestry

77:455–465

Tang W, Newton RJ (2003) Genetic transformation of conifers and its

application in forest biotechnology. Plant Cell Rep 22:1–15

Tang W, Tian Y (2003) Transgenic loblolly pine (Pinus taeda L.)

plants expressing a modified d-endotoxin gene from Bacillusthuringiensis with enhanced resistance to Dendrolimus punctatusWalker and Crypyothelea formosicola Staud. J Exp Bot 54:835–

844

Tang W, Newton RJ, Li C, Charles TM (2007) Enhanced stress

tolerance in transgenic pine expressing the pepper CaPF1 gene

is associated with the polyamine biosynthesis. Plant Cell Rep

26:115–124

Tiimonen H, Aronen T, Laakso T, Saranpaa P, Chiang V, Ylioja T,

Roininen H, Haggman H (2005) Does lignin modification affect

feeding preference or growth performance of insect herbivores in

transgenic silver birch (Betula pendula Roth)? Planta 222:699–

708

Tzfira T, Zuker A, Altman A (1998) Forest tree biotechnology:

genetic transformation and its application to future forests.

Trends Biotechnol 16:439–446

van Frankenhuzen K, Beardmore T (2004) Current status and

environmental impacts of transgenic forest trees. Can J For

Res 34:1163–1180

Wagner A, Phillips L, Narayan RD, Moody JM, Geddes B (2005)

Gene silencing studies in the gymnosperm Pinus radiata. Plant

Cell Rep 24:95102

Walter C, Charity J, Grace L, Hofig K, Moller R, Wagner A (2002)

Gene technologies in Pinus radiate and Picea abies: tools for

conifer biotechnology in the 21st century. Plant Cell Tissue

Organ Cult 70:3–12

Wang G, Castiglione S, Chen Y, Han Y, Tian Y, Gabriel DW, Han Y,

Mang K, Sala F (1996) Poplar (Populus nigra L) plants

1134 Trees (2009) 23:1125–1135

123

transformed with a Bacillus thuringiensis toxin gene: insecticidal

activity and genomic analysis. Transgenic Res 5:280–301

Watrud LS, Lee EH, Fairbrother A, Burdick C, Reichman JR,

Bollman M, Storm M, King G, Van de Water Pk (2004)

Evidence for landscape-level pollen-mediated gene flow from

genetically modified creeping bentgrass with CP4 EPSPS as a

marker. Proc Natl Acad Sci USA 101:14533–14538

Watson JM, Fusaro AF, Wang M, Waterhouse PM (2005) RNA

silencing platforms in plants. FEBS Lett 579:5982–5987

Wei H, Meilan R, Brunner AM, Skinner JS, Ma C, Strauss SH (2006)

Transgene sterility in Populus: expression properties of the

poplar PTLF, Agrobacterium NOS and two minimal 35S

promoters in vegetative tissues. Tree Physiol 26:401–410

Wei H, Meilan R, Brunner AM, Skinner JS, Ma C, Gandhi HT,

Strauss SH (2007) Field trial detect incomplete barstar attenu-

ation of vegetative cytotoxicity in Populus trees containing a

poplar LEAFY promoter::barnase sterility transgene. Mol Breed

19:69–85

White TL, Adams WT, Neale DB (2007) Forest genetics. CABI

Publishing, Cambridge

Williams CG (2005) Framing the issues on transgenic forests. Nat

Biotechnol 23:530–532

Williams CG, Davis BH (2005) Rate of transgenic spread via long-

distance dispersal in Pinus taeda. For Ecol Manage 217:95–102

Wolfenbarger LL, Phifer PR (2000) The ecological risks and benefits

of genetically engineered plants. Science 290:2088–2093

Yang MS, Lang LS, Gao BJ, Wang JM, Zheng JB (2003) Insecticidal

activity and transgene expression stability of transgenic hybrid

poplar clone 741 carrying two insect-resistance genes. Silvae

Genet 52:197–201

Yu X, Kikuchi A, Matsunga E, Nanto K, Sakurai N, Suzuki H,

Shibata D, Shimada T, Wanatabe KN (2009) Establishment of

the evaluation system of salt tolerance on transgenic woody

plants in the special netted-house. Plant Biotechnol 26:135–142

Zobel BJ, Talbert BJ (1984) Applied forest tree improvement. Wiley,

New York

Trees (2009) 23:1125–1135 1135

123