Tree genetic engineering and applications to sustainable forestry ...

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Dec 11, 2012 (4 years and 8 months ago)

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Tree genetic engineering and
applications to sustainable forestry
and biomass production
Antoine Harfouche
1
,Richard Meilan
2
and Arie Altman
3
1
Department of Forest Environment and Resources,University of Tuscia,Via S.Camillo de Lellis,Viterbo 01100,Italy
2
Department of Forestry and Natural Resources,Purdue University,715 West State Street,West Lafayette,IN 47907,USA
3
The Robert H.Smith Institute of Plant Sciences and Genetics in Agriculture,The Hebrew University of Jerusalem,P.O.Box 12,
Rehovot 76100,Israel
Forest trees provide raw materials,help to maintain
biodiversity and mitigate the effects of climate change.
Certain tree species can also be used as feedstocks for
bioenergy production.Achieving these goals may re-
quire the introduction or modified expression of genes
to enhance biomass production in a sustainable and
environmentally responsible manner.Tree genetic engi-
neering has advanced to the point at which genes for
desirable traits can now be introduced and expressed
efficiently;examples include biotic and abiotic stress
tolerance,improved wood properties,root formation
and phytoremediation.Transgene confinement,includ-
ing flowering control,may be neededto avoid ecological
risks and satisfy regulatory requirements.This and sta-
ble expression are key issues that need to be resolved
before transgenic trees can be used commercially.
Transgenic technologies to accelerate the
domestication of forest trees
Forests help to maintain biodiversity,protect land and
water resources,and mitigate climate change and increas-
ing CO
2
levels.Forests also provide social and environ-
mental benefits,in addition to supplying a wide range of
commercial products.There is increasing interest in the
use of fast-growing,short-rotation forest trees as a second-
generation bioenergy crop.However,the increased de-
mand for forest products is accompanied by rapid defores-
tation.There is an urgent need for reforestation and the
establishment of dedicated plantations.Conventional sil-
viculture and breeding techniques alone are no longer
sufficient to meet these requirements because of long
juvenile periods and the absence of native plant genes
needed to impart commercially important traits.Recent
advances in forest tree molecular biology,including gene
discovery,transcript profiling,genome sequencing and
genetic mapping,have paved the way to genetic engineer-
ing of trees for high-yielding,clonal plantations.
Tree genetic engineering,inadditionto other biotechnol-
ogies,might result,for example,in increased wood quality
and improved growth;this could lead to higher yields from
forest plantations,thereby reducing the need to cut natural
stands and sustaining native species [1].Likewise,geneti-
cally modified (GM) trees might lead to affordable biofuel
production and could thus alleviate competition for water
and other resources that might otherwise be used for food
and feed crops [2].Trees genetically engineered for pest
resistance might restore certain native tree species,such as
American chestnut (Castanea dentata),American elm
(Ulmus americana) and ash (Fraxinus spp.),that are either
critically endangered or have been lost from our forests
owing to the introduction of exotic pests [3].It has also been
suggested that trees engineered for delayed flowering will
lead to increased biomass production [4].Other benefits
include tolerance to arid,saline or cooler conditions,decon-
taminationof sitesthroughphytoremediationandenhanced
carbon-sequestration capacity.
Because they are only partially domesticated,forest
trees are amenable to genomic studies to elucidate the
relationship between genotypic and phenotypic diversity
in their populations.However,genomics resources for
forest trees are still underdeveloped and would benefit
from considerable investment [5].The application of tar-
geting-induced local lesions in genomes (TILLING) repre-
sents another gene discovery tool to facilitate tree
improvement [6].Recently,association genetics used to
study traits that control lignin and cellulose biosynthesis
in poplar has shown that a forward genetics approach can
be used to discover naturally occurring allelic variation in
genes associated with desirable traits [7].
Some of these issues have been discussed in previous
reviews on forest tree biotechnology [8–10].Here,we de-
scribe major recent scientific discoveries,emphasizing key
targets for genetic engineering and the regulatory implica-
tions of their use.We also provide a brief selective reviewof
field trials,recent progress in the development of GMtrees
worldwide and the process of tree genetic engineering for
product development.
Current status of tree genetic engineering
Gene transfer methods in forest tree species
Procedures for genetic transformation of forest trees
differ little from those for other plant species and are
mainly confined to the use of Agrobacterium,with a few
reports onparticle bombardment-mediatedtransformation.
Differentiation of transformed cells is a prerequisite to
Review
Corresponding author:Altman,A.(altman@agri.huji.ac.il).
0167-7799/$ – see front matter ￿ 2010 Elsevier Ltd.All rights reserved.doi:10.1016/j.tibtech.2010.09.003 Trends in Biotechnology,January 2011,Vol.29,No.1
9
obtaining transgenic plants and two systems are being used
in forest trees:organogenesis and embryogenesis.Such
transformation procedures,including the use of selectable
markers and screening methods,are well established.The
major obstacles to efficient production of transgenic trees
are:(i) difficulties in plant regeneration from Agrobacter-
ium-infectedorparticle-bombardedexplants;(ii) incomplete
development beyond the in vitro stage of rooted plants for
establishing field trials;and (iii) transgene instability dur-
ing the long lifespan of forest trees,including transgene
silencing and somaclonal variation.
Achievements inforest treegenetic engineering,especial-
ly poplars (Populus spp.),conifers (mainly Pinus spp.) and
eucalypts (Eucalyptus spp.),have already beensummarized
[8,11–17].We highlight here two recent exemplary reports.
The first is a novel approachusing Agrobacterium-mediated
insitubudtransformation,whichwas demonstratedrecent-
ly in Populus cathayana with efficiency of up to 2.24%[18].
This approach might be applicable to other species or culti-
vars that are not amenable to tissue culture.In addition,an
efficient Agrobacterium-mediated transformation protocol
for a difficult-to-transformcommercial hybrid poplar (Popu-
lus nigra ￿Populus maximowiczii) has beendeveloped[19].
By omitting thidiazuron (a common plant growth regulator)
fromthe regenerationmedium,the percentage of non-trans-
formed regenerated plants was minimized,resulting in an
up to 30-fold increase in the number of transgenic plants.
Transgene stability in forest trees
A fewstudies have begun to address transgene stability in
trees.Instability is common during in vitro culture and is
less common during early field screening of GM trees.In
poplar,a glyphosate tolerance transgene,CP4,was stable
for over 8 years under field conditions and nearly all
instability was observed during in vitro growth only.
CP4 was isolated from the Agrobacterium strain CP4
and encodes an enzyme that has a low affinity for the
herbicide glyphosate [20].
Transgene expression was analyzed in transformed
eastern white pine (Pinus strobus) plants harboring differ-
ent numbers of T-DNAinsertions [21].Post-transcriptional
gene silencing was mostly observed inlines withmore than
three inserts.In situ hybridization demonstrated that
silenced lines had two or more T-DNA insertions in the
same chromosome.There were,however,no differences in
shoot differentiation and development between transgenic
lines with multiple T-DNA inserts and those with fewer.
A recent study described RNA interference (RNAi) sta-
bility in field-grown poplars [22].Hybrid poplar clones (P.
tremula ￿P.tremuloides and P.tremula ￿P.alba) con-
taining the BAR gene were retransformed with four types
of intron-containing hairpin RNA(hpRNA) constructs that
contained inverted repeats targeting promoter or coding
sequences.Only RNAi that was directed at the coding
sequence was highly efficient at gene suppression.The
RNAi was stable over 3 years and matrix attachment
regions (MARs) had no discernible influence on suppres-
sion efficiency or stability,suggesting that RNAi can be
highly effective for functional genomics of woody peren-
nials.Ina follow-up study [23],transgene expression levels
in poplar were remarkably stable over several years,but
were not associated with the presence of MAR elements.
MARelements,however,have tended to reduce variability
in expression among transformants,to coordinate the
expression in linked transgenes,and to improve the struc-
ture and integrity of transgenic loci.
Transgenic tree product development
The public has a general awareness of genetic engineering;
however,a deeper understanding of transgenic tree prod-
uct development beyond proof of concept,including the
different phases of transgenic tree development and fac-
tors that can affect development expenses,is often lacking.
These are described in Box 1.
State of field trials of transgenic trees worldwide
Field testing is imperative for the development of useful,
durable and safe transgenic trees.A search of publicly
accessible databases worldwide revealed more than 700
field trials with GMwoody perennials [10].However,more
field trials are needed to provide science-based assessment
of the value and environmental safety of GM trees [9].
Table 1 and Table 2 list a variety of traits whose manipu-
lation could lead to the development of superior forest-tree
genotypes.Box 2 describes worldwide efforts to study and
promote transgenic forest trees.
Key targets for tree genetic engineering
Enhanced tolerance to abiotic stress:drought,salinity
and high and low temperature
Drought,which is often associated with osmotic or salinity
stress,is a major factor involved in decreases in forest
productivity.Enhancing drought and salinity tolerance is
of particular importance when reforesting marginal arid
and semi-arid areas,which are prone to degradation.
Molecular control of plant response to abiotic stress is
complex,usually involving coordinated expression of sever-
al genes.The use of known abiotic-stress-associated genes
fromother species [24] to enhance tolerance in forest trees
has been limited.However,recent studies in genomics,
transcriptomics andproteomics inseveral forest treespecies
[5,25],as well as release of the draft Eucalyptus grandis
genomic sequence (www.eucagen.org),have provided new
tools for improving abiotic stress tolerance in trees.
Overexpression of a pepper ERF/AP2 transcription fac-
tor,CaPF1,in eastern white pine resulted in a significant
increase in tolerance to drought,freezing and salt stress
[26].The increased tolerance was associated with poly-
amine biosynthesis.Moreover,overexpression of the cho-
line oxidase (codA) gene from Arthrobacter globiformis
resulted in increased tolerance to NaCl in several lines
of Eucalyptus globulus [27].Interestingly,overexpression
of a manganese superoxide dismutase (SOD) gene from
Tamarix androssowii in a hybrid poplar (Populus davidia-
na ￿Populus bolleana) resulted in enhanced SODactivity
on exposure to NaCl,along with a remarkable increase in
growth [28].Likewise,the ThCAP gene fromT.hispida led
to greater resistance to low temperatures when expressed
in transgenic poplar (P.davidiana ￿P.bolleana) [29].
In another study,ectopic expression of AtCBF1,a mem-
ber of the C-repeat binding factor family of transcription
factors,significantly increased freezing tolerance in non-
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Trends in Biotechnology January 2011,Vol.29,No.1
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acclimated leaves and stems of transgenic poplar [30].In
addition,the tomato jasmonic ethylene responsive factor
(JERF) gene that encodes an ERF-like transcription factor
was successfully expressed in a hybrid poplar (P.
alba ￿Populus berolinensis).In the presence of up to
300 mM NaCl,the transgenic plants were significantly
taller and more tolerant of higher salinity levels than
the controls [31].
Another gene affecting plant tolerance to salt is SP1,
which encodes a chaperone-like boiling-stable protein.SP1
was cloned fromP.tremula and its expression was induced
by salt,cold,heat and desiccation stress [32].Some of these
Box 1.Transgenic tree product development
Because of their long juvenile periods,forest trees are much more
difficult to domesticate than annual,herbaceous agronomic crops
and tree breeding is therefore more complex and capital- and time-
intensive.Genetic engineering provides an alternative strategy for
domesticating trees.
Figure I shows the five phases of the transgenic tree development
cycle,the time-scale fromgene discovery or licensing to final product
development,and estimated costs associated with the process in the
USA.The patent application process for newly discovered intellectual
property starts in the early stages of product development and needs
to be comprehensive.The final product can be patented along with
enabling technologies and genetic constructs,which might include
components other than coding sequences (e.g.promoters,selectable
markers,terminators).The developer should discuss the concept with
regulators early in the process to ensure that the necessary data are
collected and that appropriate safeguards are in place.The regulators
must determine whether the transgenic plants will have any negative
impact on the environment.In addition to obtaining permission to
performfield tests,it might be necessary to obtain a permit to import
materials that will be transformed.Finally,a petition for non-
regulated status must be submitted before the transgenic plants
can be commercialized.This petition must contain data demonstrat-
ing that the transgenic plants are safe.The time-cost estimates
shown for the USA are based on case studies (Kannan Grant,former
Associate Vice Chancellor for the Office of Technology Development
at the University of Nebraska–Lincoln,personal communication) that
involved extensive communication with members of the manage-
ment team for several plant biotechnology companies and on
published estimates for the development,in this case,of insect-
resistant maize and costs associated with its regulatory approval
[77].
Various factors can affect development expenses,including:(i) the
number of genes stacked;(ii) the gene source (in-house or licensed);
(iii) the institution developing the product (public or private);(iv) the
ease of genetic transformation;(v) the extent of regulatory oversight;
and (vi) the need to conduct extensive environmental risk assessment
before applying for non-regulated status.The average probability of
success is low in the discovery phase,but improves as product
development advances toward the final phase.A better under-
standing of the process will enable scientists,institutions,technology
licensors,federal regulators and investors to value and develop tree
biotechnology products.
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Time (years)
FDA
EPA
Deregulation
USDA/APHIS
Regulatory process
Patent-filing process
FTO analysis
Genetic engineering process
Phase IV
Regulatory
approval
Phase III
Advanced
development
Phase II
Early
development
Phase I
Proof of
concept
Discovery
and FTO
Estimated costs (% of TC)
• Identify genes
with potentially
valuable traits
in trees
• Identify the
genotypes to be
transformed
• Make licensing
arrangement to
obtain FTO
• Test gene
configurations in
core genotypes to
screen for desired
performance
• Meet with federal
regulators to
discuss the
research; studies
to generate
adequate data
• Choose best
events
• Conduct preliminary
field tests, on small-
scale, at different
locations, and under
different
environments
• Select successful
candidates for
commercial
production
• Generate relevant
data for regulatory
approval process
• Demonstrate durability
of gene-of-interest
• Generate relevant data
for regulatory approval
process
• Complete petition
for non-regulated
status
• Commercial
market launch
3-5% of TC
7-10% of TC
10-15% of TC
20-30% of TC
30-40% of TC
Estimated product development time:
10-13 years
Estimated total costs (TC):
$70-100M USD
0 1 2 3 4 5 6 7 8 9 10 11 12 13
TRENDS in Biotechnology
Figure I.The path frombasic research to the market,indicating howto add useful traits to trees-of-interest.The product development process for transgenic plants has
five main phases,beginning with gene discovery and licensing arrangements to provide freedomto operate (FTO),followed by proof of concept and early and advanced
product development.The process concludes with regulatory approval.The U.S.Department of Agriculture’s (USDA) Animal and Plant Health Inspection Service
(APHIS),the Food and Drug Administration (FDA),and the Environmental Protection Agency (EPA) coordinately regulate GM plants.
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findings are discussed in a comprehensive review of the
poplar response to excess salt [33].The gene encoding a
novel PeSP1 protein from Populus euphratica,which is
emerging as a model tree for studying salt tolerance,is an
ortholog of a gene from salt-sensitive P.tremula.It was
upregulated on NaCl treatment,but its expression under
non-stress conditions was considerably higher in the salt-
tolerant than in the salt-sensitive species.Interestingly,
PeSP1undergoesapost-translational modificationonstress
via sumoylation at specific sites (Figure 1) (A.Altman,
unpublished data).The SUMOylation motifs were found
to be conserved in most of paralogous SP1 proteins found in
other species (e.g.Arabidopsis,rice,P.tremula,and the
bacterium Rhodopirellula baltica).The effect of PeSP1 on
salt tolerance is being studied.
Trees engineered for increased abiotic stress tolerance
might not only promote survival during acute or chronic
stress,but also sustain productivity under moderate
stress.Genes derived from halophytes might offer oppor-
tunities for imparting greater tolerance,but it remains to
be seen whether they will be useful under moderate or
extreme stress.
Enhanced resistance to biotic stress:insects and disease
Genetically engineered insect resistance can be environ-
mentally beneficial because of the reduced need for syn-
thetic insecticides.Available technologies include genes
encoding Bacillus thuringiensis (Bt) toxins and proteinase
inhibitors.In 2002,insect-resistant black poplar (P.nigra)
containing Bt genes was approved for commercialization
by the Chinese Gene Security Committee [34].Hybrid
triploid poplars [(Populus tomentosa ￿P.bolleana) ￿P.
tomentosa] transformed with a cowpea trypsin inhibitor
gene (CpTI) exhibited resistance to three defoliating
insects:forest tent caterpillar (Malacosoma disstria),gyp-
sy moth (Lymantria dispar) and willow moth (Stilpnotia
candida) [35].
Forest trees play host to a wide range of fungal,bacterial
and viral pathogens.Trees engineered for disease resis-
tance can provide both environmental and commercial
benefits.Enhanced disease resistance has been achieved
using a variety of genes derived from plants and micro-
organisms,with varying degrees of success.For example,
Chinese white poplar (P.tomentosa) expressing a chitinase
gene from Beauveria bassiana (Bbchit1) exhibited in-
creased resistance to a pathogenic fungus (Cytospora chry-
sosperma) [36].Moreover,2-year-old poplars expressing
anti-microbial peptides have shown high resistance in leaf-
disc assays and Septoria musiva cankers have been less
frequent on field-grown transgenic trees [37].
Encouraging progress has been made toward the resto-
ration of threatened forest trees whose populations have
Table 2.Number of field trials with transgenic trees in Europe,1991-present
a
Genetically
engineered tree
Country Traits introduced into trees Number of
field trials
Number of
institutions
Public Private
Poplar
Belgium Altered wood composition 1 1
France Altered wood properties;sterility;lignin modification;herbicide tolerance 12 11 1
Germany Phytoremediation;herbicide tolerance;canopy form 5 5
Sweden Faster growth;insect resistance 2 2
Norway Growth 1 1
Spain Faster growth 1 1
United Kingdom Altered lignin biosynthesis 2 2
Poland Growth 1 1
Eucalyptus
Spain Marker system evaluation 1 1
United Kingdom Gene stability evaluation 1 1
Norway spruce,
Scots pine and
silver birch
Finland Fungal and insect resistance;sterility 5 5
Total
32 27 5
a
Data are taken from the website of the EC Joint Research Centre (http://gmoinfo.jrc.ec.europa.eu),and are current as of September 15,2010.
Table 1.Permits for field trials with transgenic trees in the USA,1985-present
a
Genetically
engineered tree
Traits introduced into trees
Number of permits
for field trials
Number of
institutions
Issued Pending Public Private
Poplar
Insect and bacterial resistance;herbicide tolerance;altered branching;
bud dormancy;wood development;floral development;flowering time;
gibberellin;lignin content and composition;light response;female and
male sterility;improved digestibility;phytoremediation
26 1 16 11
Eucalyptus
Altered lignin biosynthesis;growth rate;sterility;cold tolerance 7 7
Pine
Altered growth rate 3 1 4
White spruce
Insect resistance 1 1
American chestnut
Fungal resistance;herbicide tolerance 1 1 2
American elm
Disease resistance 1 1 2
Walnut
Herbicide tolerance;flowering control 5 5
Sweetgum
Fungal,viral and insect resistance 3 1 2
Papaya
Viral resistance;delayed fruit softening 5 5
Total
52 4 32 24
a
DataaretakenfromtheUSDA-APHIS’ BiotechnologyRegulatoryServiceswebsite(http://www.aphis.usda.gov/brs/status/relday.html),andarecurrent asof September 15,2010.
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been devastated by fungal pathogens [38].For example,
elmand chestnut have been transformed with anti-fungal
genes to impart resistance to the chestnut blight fungus
(Cryphonectria parasitica) and the fungus Ophiostoma
ulmi [39].English elm (Ulma procera) plantlets trans-
formed with anti-fungal genes are now being tested for
their ability to resist Dutch elmdisease fungi (O.ulmi and
O.novo-ulmi) [40].Despite these successes,the lack of
field-test results (Box 2) suggests that durable resistance
remains to be proven.Transgenic American elm has been
produced with a gene encoding a cationic anti-microbial
peptide,ESF39 [41].American elm trees expressing a
transgene encoding the synthetic anti-microbial peptide
ESF39A,driven by a vascular promoter from American
chestnut,exhibited milder Dutch elm disease symptoms
[42].However,the trees tested were too young to ensure
that resistance is stable.
A more recent attempt to enhance resistance to C.
parasitica in American chestnut involved overexpression
of a potential anti-fungal gene,oxalate oxidase (OxO),
derived fromwheat [43].The enzyme encoded by this gene
metabolizes oxalic acid.Because C.parasitica infection
leads to necrosis as a result of exposure to oxalic acid,as
with S.musiva,it is assumed that overexpression of this
gene in chestnut stems will confer resistance to the blight.
These preliminary results suggest that increasing dis-
ease resistance might require a variety,and perhaps a
combination,of transgenic products.Testing for disease
resistance ina natural setting is imperative andmulti-year
field trials (Tables 1 and 2) will be needed to verify the
durability of resistance against ever-evolving pathogen
populations.
Enhanced root development
Vegetative propagation via stem cuttings could help to
meet the increasing demand for products derived fromtree
plantations.Cuttings fromsome species are relatively easy
to root,whereas others are recalcitrant.The benefits of
genetically engineered trees will be more fully realized by
improving root formation.
Enhanced root development has been achieved by mod-
ifying the expression of genes involved in the biosynthesis
of plant hormones.In plants,gibberellin (GA) responses
are mediated by proteins containing a functional DELLA
domain,including GA insensitive (GAI),repressor of GA1
(RGA) and RGA-like1 (RGL1) [44].Overexpression of the
Arabidopsis thaliana GAI and RGL1 genes in poplar
resulted in greatly enhanced root development in trans-
genic plants grown in vitro.Roots of transgenic poplars
showed two- to sixfold greater fresh weight than wild-type
Box 2.State of field trials of transgenic trees worldwide
Although one of the first releases of GM trees was a field trial of
herbicide-tolerant poplar in Belgium in 1988 [78],China and the USA
are the only countries known to have commercially used transgenic
trees:insect-resistant poplar in China,and virus-resistant papaya
(Carica papaya) in the USA.In both the USA and Europe,the highest
number of transgenic tree field trials has been with poplar,which is
the focus of most tree genetic engineering research.Poplar is one of
the few trees that is easily amenable to genetic transformation and in
vitro regeneration.In many cases it can be propagated vegetatively
and is capable of rapid growth,usually reaching 4–6 mwithin 2 years.
Field trials in other developed countries involve poplar,white spruce
(Picea glauca) and black spruce (Picea mariana) in Canada,eucalyptus
in Australia,eucalyptus and pine in New Zealand,poplar and birch
(Betula spp.) in Russia,and papaya in Japan.
Field trials reported in China and parts of the developing world (e.g.
Indonesia,Chile,Brazil,India and South Africa) have involved poplar,
eucalyptus,pine and papaya.Initially,the predominant engineered
traits included those that yield a relatively quick return on investment,
such as tolerance to herbicides and biotic and abiotic stresses,
improved growth and altered wood properties.Recently,added-value
traits,such as bioenergy production,bioremediation,carbon seques-
tration,bio-pulping and species restoration,have been tested,
particularly by industry.
Widespread small-scale field tests are generally conducted by
academic institutions,although a few companies have conducted
large-scale,multi-site field trials.The latter have mainly been
performed in the USA,Brazil and New Zealand.Although academic
institutions have laboratories and controlled environments for
producing and testing transgenic trees,the infrastructure needed to
conduct field tests often requires the involvement of the private
sector.Despite the focus on forest trees in this review,papaya was
included because,to date,it is one of only two trees deregulated in
the USA.Transgenic HoneySweet plum(Prunus domestica),which is
highly resistant to the plum pox virus,was the second tree to be
deregulated.Even though American chestnut is now largely absent
from the landscape,it was one of the largest and fastest-growing
forest trees in eastern North America,and was important for both its
nuts and timber.American chestnut and papaya were devastated by
disease.Salvation of these and other threatened and introduced trees
suggests that,in the long run,genetic engineering might be useful for
promoting sustainability and maintaining biodiversity.
Table I lists the key companies involved in producing GM trees.
Some of these companies are developing transgenic trees through
collaboration with academic institutions.A particularly noteworthy
example is work led by ArborGen to engineer eucalyptus for cold
tolerance and sterility.Other partnerships are attempting to enhance
wood quantity and quality.A majority of field trials have occurred in
the model tree poplar,which can be viewed as a business decision.
The products currently under development hold promise for boosting
the yield of forest plantations to offer a sustainable supply of
feedstock to meet future energy needs and the demand for wood
products while preserving biodiversity by minimizing the cutting of
natural forest populations and rescuing endangered forest species
(e.g.American chestnut).
Table I.Key companies developing improved forest trees through genetic engineering
Company Research and development focus
ArborGen
a
(USA)
Genetic engineering of eucalyptus,pine and poplar for improved wood properties and cold tolerance
GenFor
b
(Chile)
Genetic engineering of pine for insect,fungal resistance,and improved wood quality
Nippon Paper (Japan)
Genetic engineering of eucalyptus for easier paper and pulp manufacturing
Scion (New Zealand)
Genetic engineering of pine for improved wood properties
Suzano Pulp and Paper (Brazil)
Genetic improvement of eucalyptus for increased biomass,faster growth,and improved digestibility
SweeTree Technologies (Sweden)
Genetic engineering of poplar,eucalyptus and spruce for increased fiber,biomass growth,
and improved wood properties
a
ArborGen is a joint venture between International Paper Company (USA) MeadWestvaco (USA) and Rubicon Limited (New Zealand) (www.arborgen.com).
b
GenFor is a joint venture between Silvagen (Canada),Interlink (USA) and Fundacio´ n Chile.
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plants.Furthermore,the roots of RGL1 and GAI transgen-
ic poplars were thick and ruddy and had an abundance of
lateral roots.
With the advent of RNAi technology,another strategy
has become available for improved rooting.Root develop-
ment was considerably improved in cuttings of a poplar
hybrid(P.tremula ￿P.alba) that was transformed withan
RNAi construct for an A.thaliana ABC transporter
(AtMRP5) gene (Figure 2) (R.Meilan,unpublished data).
Ordinarily,cuttings from the genotype used can only root
after hormonal treatment and extensive misting.
Improved wood properties
The development of bioenergy crops has attracted much
attention in the GM tree community.Certain forest tree
species can be grown as second-generation bioenergy crops
on marginal land [45].For competitive lignocellulosic feed-
stocks,their conversionefficiencies will needto be improved
[46].There have been two recent advances in this regard.
First,in an attempt to reduce or eliminate the need for
pretreatment,sixgenes involvedinligninbiosynthesis were
downregulated using anti-sense technology [47].This study
demonstrated that suppression of genes encoding enzymes
that act early in the biosynthetic pathway is the most
effective strategy for reducing lignin content.A second
approach involved altering lignin monomeric composition.
Inmost woodyspecies,ligninis mainlycomposedof guaiacyl
(G) and syringyl (S) units [48].The availability of woody
biomass with high-S lignin increases the yield of biofuel per
unit land area because of an increase in conversion efficien-
cy.Overexpression of the F5Hgene under control of the A.
thalianaC4Hpromoter led to poplar withlignincomprising
approximately 97.5%S [46].This finding might ultimately
lead to more affordable biofuels.
Phytoremediation of environmental pollutants
Phytoremediation involves the use of plants to remove
contaminants fromthe environment.Because this technol-
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]
Figure 2.Rooting is a limiting factor for a significant number of woody species.The photograph shows the rooting ability of transgenic poplar using an RNAi construct for
an Arabidopsis thaliana ABC transporter (AtMRP5) gene (left) compared to non-transgenic plants (right).The non-transgenic cuttings were dipped into a commercially
available root-induction product,Rootone
1
.The difficult-to-root transgenic poplar produced more roots in mediumwithout auxin (indole-3-acetic acid,IAA) than the non-
transgenic cuttings that received auxin treatment (R.Meilan,unpublished data).
[(
)
T
D
$
F
I
G
]
Figure 1.A 12.4-kDa novel protein (PeSP1) of P.euphratica forms a ring-shaped
dodecamer that is sumoylated on salt stress.Under salt stress,PeSP1 was restricted
to and localized in the plasmalemma and the nuclear membrane of P.euphratica
(unlike its cytosolic distribution without salt).This is due to salt-induced sumoylation
of specific sites of the complex PeSP1.The figure shows the crystal structure of the
dodecamer SP1 in which two SUMOylation motifs (indicated by circles and arrows)
are located in the external loop of every monomer of the SP1 complex [32].
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Trends in Biotechnology January 2011,Vol.29,No.1
14
ogy is less costly,less invasive and more esthetic,it has
many advantages over traditional engineering-based
methods.Phytoremediation plantings can provide addi-
tional environmental benefits,such as a means to seques-
ter carbon,control erosion,produce biofuel or fiber [49],
maintainwildlife habitats andcreate buffers against noise,
garbage and dust [50].Many (but not all) poplars are
riparian species that exhibit fast growth,deep and exten-
sive root systems,anda highdemandfor water,all of which
are desirable characteristics for phytoremediation.
Eastern cottonwood (Populus deltoides) has been trans-
formed with the merA and merB genes [51],which encode
organomercury lyases.Transgenic plants grown in vitro
were highly resistant to phenylmercuric acetate and were
able to detoxify organic mercury compounds considerably
faster than non-transgenic controls or plants containing
either of the two transgenes alone.Only shoots trans-
formed with both genes rooted in mercury-containing me-
dia.Based on a previous report of poplar containing only
merA[52],it is assumed that when grown in the absence of
mercury,the merA/merBpoplar will not performas well as
wild-type trees.
A poplar hybrid (P.tremula ￿P.alba) has been trans-
formed with an E.coli gene encoding g-glutamylcysteine
synthetase (g-ECS),GSH1,overexpression of which led to
increased glutathione S-transferase activity inchloroplast-
expressing lines.When leaf discs from these transgenic
plants were subjected to various concentrations of zinc,
they behaved similarly to wild-type trees.However,trees
expressing g-ECS in their cytosol accumulated significant-
ly more cadmium,chromiumand copper than wild-type or
chloroplast-expressing lines [53].
Enhanced herbicide resistance
Engineering of herbicide resistance in forest trees might
lead to better tree growth,lower total herbicide use,in-
creased reliance on environmentally benign active ingre-
dients,and soil and moisture conservation.Herbicide
resistance is normally governed by a few genes,many of
which have been inserted in trees,as demonstrated with
CP4 [20] and glutamine synthetase [54] genes in poplars.
Flowering control as a means of transgene confinement
in forest trees
Flowering control in transgenic forest tree species is one
means of preventing transgene spread in the environment.
Although no reliable method for engineering sterility in
trees has been fully developed,efforts are focused on three
major strategies:(i) dominant negative mutations (DNM);
(ii) RNAi;and (iii) tissue-specific ablation.Using the last
approach,it was shown that the PTLF gene promoter from
Populus trichocarpa is strongly expressed in developing
male and female inflorescences and is therefore a good
candidate for complete sterility [55].However,in a related
field study,there was incomplete attenuation of vegetative
cytotoxicity in poplar trees containing a PTLF::barnase
cassette and the barstar gene driven by various promoters
[56].Using DNM,it was shown that the A.thaliana MALE
STERILITY1 (MS1) gene encodes a PHD-type transcrip-
tion factor that regulates pollen and tapetal development
[57].Phylogenetic analysis revealed six MS1 homologs in
poplar.One member of this gene family might be poten-
tially useful for engineering of male sterility.
A poplar ortholog of CENTRORADIALIS (PopCEN1),a
gene that plays a key role inmaintaining trees ina juvenile
state,has been downregulated using RNAi [58].When
poplars harboring an RNAi construct were grown under
field conditions,four of the most strongly silenced lines
produced inflorescences or floral buds within 2 years of
planting,which was several years earlier than for wild-
type trees.Although overexpression of PopCEN1 resulted
in an almost complete absence of flowering,it was also
detrimental to shoot phenology and crown architecture.It
was suggested that PopCEN1 might be useful for prevent-
ing the transition to inflorescence development via floral or
inflorescence meristem-predominant promoters [58].
Recently,two AGAMOUS-like MADS-box genes,
EgAGL1 and EgAGL2,were isolated from flower buds of
eucalyptus.These genes were strongly expressed in flower
buds and consequently might regulate stamen and carpel
formation [59].They are considered candidates for engi-
neering of sterile eucalyptus.
A system for inducing male sterility in Monterey pine
(Pinus radiata) is based on overexpression of the stilbene
synthase gene,STS,under the control of a P.radiata male-
cone-specific promoter inthe anthers of tobacco plants [60].
This overexpression resulted in near-complete male steril-
ity (98–99.9%) in 70% of transformed tobacco plants.The
pine promoter-STS construct might be useful for various
gymnosperms [60].
Future regulatory needs and recommendations
Tree genetic engineering provides an opportunity for sus-
tainable production of forest products.However,the devel-
opment of GM trees could be hindered by regulatory and
social hurdles.Responsible use and development,as well
as science-based oversight of GM tree technologies,are
essential for regulatory and public acceptance.Consumer
acceptance of and willingness to eat food containing GM-
derived products was dependent on explaining the reason
for the modification and other non-scientific factors,such
as knowledge and trust [61].Given this,greater transpar-
ency and outreach could lead to greater public acceptance
of GMtrees destined for pulp and bioenergy production,for
example,thus reducing impacts on native forests.Howev-
er,transgene flow remains a major concern.Because the
plastid genome is inherited maternally,plastid transfor-
mation reduces the risk of gene flow [62].Even though a
plastid transformation system has been developed for
poplar,improvements are still needed [63].
It is now clear that multiple transgene containment
strategies will probably be needed to tackle biosafety con-
cerns for forest trees.Although the use of male-sterile
genotypes or transgenic mitigation tools might at least
partially achieve confinement,the best solution might
simply be to remove transgenes from pollen [64].For
example,site-specific recombination systems could facili-
tate highly efficient excision of transgenes from pollen of
hybrid aspen (P.tremula ￿P.tremuloides) [65].Genetic
modification of trees via cisgenesis is another potentially
useful strategy [66].Even if regulations for GMplants are
changed to exempt this practice,trees transformed with
Review
Trends in Biotechnology January 2011,Vol.29,No.1
15
cisgenes should still be tested to confirm that they do not
contain any foreign genetic material [67].Other potential
strategies,such as zinc-finger nucleases,could also be used
for transgene excision [68].Zinc-finger nucleases can be
used to make specific changes in the plant genome [69].
Although researchers continue to improve the safety of
transgenic trees,global harmonization of regulations is
needed.Consistent data collection and testing procedures
and information exchange will help to remove artificial
trade barriers,expedite the adoption of GM crops,foster
technology transfer,and protect developing countries from
exploitation [70].Ultimately,acceptance of science-based
approaches should advance research and development,
instill confidence,and bring the benefits of GM products
to consumers.
Barriers to adoption of GM forest trees
Although the use of GM forest trees in dedicated planta-
tions is moving closer to reality,concerns have been raised
about the safety of the genes used,the potential impact of
transgenic out-crossing withsexually compatible wild rela-
tives,and the possible impact on non-target organisms
[71,72].There is also concern about horizontal gene trans-
fer (HGT).Thus far,HGT studies have shown that the
likelihood of shifts in natural soil microorganism commu-
nities owing to the emergence of resistant bacterial strains
is almost nil [73].In addition,no changes in the ectomycor-
rhizal fungal community structure were found after trans-
genic poplars were used in the field for 8 years [74].
Likewise,no unintended impacts of transgenic pine trees
were observed for above-ground invertebrate communities
over a period of 2 years [75].Transgene escape through
vegetative propagation is another acknowledged risk con-
nected with GE trees [76],but this can be addressed.
To move this technology forward,these concerns must
be appropriately addressed through extensive field testing
of GMtrees (Table 1 and Table 2) and review of the safety
data submitted to the appropriate governmental agencies
before deregulationand large-scale use.Recently,indepen-
dent efforts have sought to establish new regulations and
strategies to safely use transgenic trees internationally.In
the USA,APHIS conducted a programmatic environmen-
tal impact statement of its regulatory system,which led to
substantial modification of this framework (available at
www.aphis.usda.gov/publications/biotechnology).Europe-
an Cooperation in Science and Technology (COST) action
FP0905 (www.cost-action-fp0905.eu) on the biosafety of
transgenic trees was also initiated.
However,the call for caution in how we use transgenic
trees should not result in a moratoriumon field studies.On
the contrary,rigorous studies are needed to test hypothe-
ses and develop strategies that provide a balance between
costs and benefits.Energy security andour future supply of
wood and fiber will depend on science providing the tools
for efficient and sustainable production of forest resources.
Acknowledgements
AH is supported by the Brain Gain Program (Rientro dei cervelli) of the
Italian Ministry of Education,University and Research (MIUR).A
research grant to AA by the Italian Ministry of the Environment and
Territories is gratefully acknowledged.The authors apologize to their
colleagues whose work was not cited owing to space limitations.
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