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Chapter7
Agrobacterium-mediated Transformation of Populus
Species1
Mee-Sook Kim, Ned B. Klopfenstein, and Young Woo Chun
Introduction
Although molecular biology of woody plants is a relatively young field, it offers considerable potential for breeding and selecting improved trees for multiple purposes.
Conventional breeding programs have produced improved growth rates, adaptability, and pest resistance;
however, tree improvement processes are time consuming because of the long generation and rotation cycles of
trees (Dinus and Tuskan this volume; Leple et al. 1992).
Genetic engineering of trees helps to compensate for conventional breeding disadvantages by incorporating known
genes into specific genetic backgrounds. Since the first
successful plant transformation was reported in 1983
(Herrera-Estrella et al. 1983; Murai et al. 1983), several
nonsexual gene transfer methods were developed for important agronomic crops and forest tree species. These
methods include biolistics (microprojectile bombardment),
electroporation, and Agrobacterium-mediated transformation. Biolistics and electroporation are discussed by Charest
et al. (this volume). This chapter focuses on Agrobacteriummediated gene transfer methods, which are widely-used
for plant transformation of broad-leaved, woody plants
because of their versatility and efficient application
(Brasileiro et al. 1991; Chun 1994; Han et al. 1996; Leple et
al. 1992).
· Agrobacterium spp. are soil bacteria tJ:tat naturally infect
many dicotyledonous and gymnospermous plants predis-
, Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds.
Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997.
Micropropagation, genetic engineering, and molecular biology
of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO:
U.S. Department of Agriculture, Forest Service, Rocky Mountain
Research Station. 326 p.
posed by wounding (Perani et al. 1986). Infection by A.
tumefaciens causes crown gall disease (figure 1), whereas
A. rhizogenes causes hairy root disease. In addition to its
chromosomal DNA, Agrobacterium contains 2 other genetic
components that are required for plant cell transformation; T-DNA (transferred DNA) and the virulence (vir) region, which are both located on the TI (tumor-inducing) or
Ri (root-inducing) plasmid (Zambryoski et al. 1989). The
T-DNA portion of the A. tumefaciens TI plasmid or the A.
rhizogenes Ri plasmid is transferred to the nucleus of a host
plant where it integrates into the nuclear DNA genetically
transforming the recipient plant. A region of the 1i plasmid outside the T-DNA, referred to as the wirulence region, carries the vir genes. Expression of vir genes occurs
during plant cell infection and is a prerequisite for the subsequent transfer of the T-DNA. Agrobacterium chromosomal regions are involved in attachment of Agrobacterium
to plant cells. The T-DNA of A. tumefaciens contains auxin
{iaaH, iaaM) and cytokinin (IPT) synthesis genes
(Zambryoski et al. 1989). These genes are referred to as
oncogenes and are responsible for tumor induction. In A.
rhizogenes, T-DNA contains multiple rol genes that induce
root formation (Zambryoski et al. 1989). The T-DNA also
encodes several genes responsible for the synthesis of compounds called opines, which are metabolic substrates for
the bacteria (Nester et al. 1984). Efficient transfer ofT-DNA
is facilitated by 24-base pair direct repeats at the T-DNA
borders. Genes within the T-DNA can be replaced with
genes of interest without affecting transfer efficiency (Han
et al. 1996; Jouanin et al. 1993).
Members of the genus Populus have a small genome size,
short rotation cycle, fast growth rate, and the capacity for
vegetative propagation. In addition, Populus spp. demonstrate developmental plasticity to tissue culture manipulations. These traits and susceptibility to
Agrobacterium-mediated transformation and techniques to
regenerate transgenic trees make Populus a suitable mod~I
system for genetic engineerin? of deciduou~ trees.~ th1s
chapter, we describe the ma1n Agrobacterzum-med1ated
transformation procedures developed for Populus andreview the results obtained using several Populus species.
51
Section II Transformation and Foreign Gene Expression
for infection and transformation; 2) infection: wounded starting explants are co-cultiva ted wi th an Agrobacteriz1111 strain
containing co-integra te or binary vectors; 3) selection: after
removal of residual Agrobacterium, transformed cells are selected for subsequent regeneration into transgenic p lants (figure 3); 4) regeneration: transformed cells are regenerated
during or after the selection period (figures3 and 4); and 5) confirmation: the presence or function of transgenes in the genome
of transgenic p lants is confirmed using molecular techniques
such as polymerase chain reaction, Southern hybridization,
northern hyb rid ization, western blotting, enzyme-linked
immunosorbent assay (ELISA), or enzyme activity assays.
Transgenes
Figure 1. Crown gall produced by Agrobacterium
tumefaciens stra in A281 infection of hybrid
poplar (Populus alba x P. grandidentata) stem
after approximately 1 0 weeks.
Gene Transfer to Populus Species
Populus has been known as a natu ral host for Agrobacteriu111
for many yea rs. DeCleene a nd De Ley (1976) cite early literature tha t suggests the susceptibi lity of 3 Populus species to
infection by A. tu111ejaciens. The presence of T-ON A sequences
in gall and root tissue confirmed Populus as a host fo r A.
tu111ejaciens and A. rhizogenes (Parsons e t al. 1986; Pythoud et
a l. 1987). These early pa thogenicity studies of Agrobacteriu111
provided the basis for its use as a tool to tra nsfer foreign
genes into the poplar genome.
The process fo r prod ucing transgenic pop la r plants includes 5 main components (figure 2): 1) initia tion: starting
exp lants (host species/genotype/ tissue type) a re selected
52
Several silviculturally usefu l genes have been isolated and
used for Agrobacterium-mediated transforma tion of Populus.
A table listing genes used in Populus transformation (Chun
1994) was updated fo r this chap ter (table 1). These genes
include the: 1) mutant aroA gene, which encodes glyphosate
tolerance via a 5-enolpyruylshikimate-3-phospha te synthase
(EPSP) tha t is less sen sitive to the herbicide g lyphosa te
(Donahue et al. 1994; Fillatti et al. 1987); 2) bar gene encoding the enzyme phosph inotricin acetytransferase (PAT) that
inactivates the herbicide phos.phinotricin (glufosinate) (De
Block 1990; Devillard 1992); 3) mutant crs1-1 gene from a
chlorsulfuron-herbicide-resistant line of Arabidopsis thaliana
(Brasileiro et a l. 1992); 4) OCI (oryzasta tin), a cysteih proteinase inhibitor, and PIN2 (proteinase inhibitor II), a trypsin /
chymotryp sin inhibitor gene for pest resistance (Heuchelin
et al. 1997 this volume; Klopfenstein et al.1991, 1993, 1997;
Leple et al. 1995); and 5) insecticidal protein genes from Bacillus thuringiensis (Bt) (H owe et al. 1994). O ther studies have
focused on transgene regulation (Chun and Klopfenstein
1995; Con fa lonieri et a l. 1994; Kajita et al. 1994; Klopfenstein
et a l. 1991; Lep le e t al. 1995; ilsson et al. 1992) and developmental influences (Ah uja and Fladung 1996; Charest et al.
1992; Ebinuma e t al. 1992; N ilsson e t a l. 1996a, 1996b;
Schwa rtzenberg et a l. 1994; Sundberg et a l. th is volume;
Tuominen e t al. 1995; Weigel and Tilsson 1995).
Transgene Copy Number
Few s tud ies have reported the copy nu mber of inserted
transgenes by Agrobacteriu111-mediated tra nsforma tion on
Populus species. Transgenic microshoots of hyb rid aspen (P.
alba x P tremula) contained from 1 to 3 copies of the inserted
foreign bar genes (De Block 1990); whereas, in vitro plants (P
tre111ula x P alba) regenerated from transformed roots contained 1 copy of the bar gene (Devillard 1992). Only a single
copy of the chloramphenicol acetyltransferase (CAT) gene
was inserted into the genome of transgenic hybrid poplar
(P alba x P. grandidentata) (Klopfenstein et al. 1991). In addition, 1 to 4 copies of crs1 -1 gene had been inserted per hy-
US DA Forest S ervice Ge n. Tech. Rep. RM-GTR-297. 1997.
Agrobacterium-mediated Transformation of Populus Species
~ Wounding
INITIATION
•••
t
Field Test
~
.:!: Dark Conditions
'I=
=I'
'I =
I'
Co-cultivation with
A. tumefaciens or
A. rhizogenes
CONFIRMATION
(e.g .. Southern blot. PCR.
northern hybridization .
western blot. ELISA.
and/or enzyme activity assay)
Greenhouse
Growth
Preculture (CIM or SIM)
INFECTION
...
/
.:!: Secondary selection and
regeneration to avoid
chimeric transformants
'
In vitro propagation
I
Decontamination
_:!: preselective culture
SELECTI ON
REGENERATION
'I.Je *'
I'
Selection of transformed
cells
:!: Additional selection for root
formation in selective media
Figure 2. The primary steps for Agrobacterium-med iated transformation of Populus species. CIM=callus inducing medium;
SIM=shoot inducing medium.
brid aspen (P. tremula x P alba) genome (Brasilciro ct a l. 1992).
Also, Howe ct al. (1991) showed that the number of inserted
0 A copies ranged from 1 to 10 after the maize transposable
element Ac (Activator) was transferred into hybrid poplar (P
alba x P grmzdidentata). However, it is unknown if all inserted
gene copies were expressed (Chun 1994; Leple et al. 1992).
Agrobacterium-mediated
Transformation
Host Species/Genotype/Tissue Type
A prerequisite for any genetic transformation work using Agrobacterium is the ability of the bacterium to infect
the plant of interest. The effect of 2 Agrobacterium
tumefaciens strai ns, A281 and A348, on infection of P.
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
triclzocarpa x P. del/aides (Parsons et al. 1986) was studied
and add itional information was gathe red on the effect of
popla r genotypes (Charest et a l. 1992). Prev ious s tudies
s howed significan t differences among the gen o types
w ith in species and the clones with in ge n otype
(Confa lonie ri et al. 1994; De Block 1990; Ri emenschneider
1990). A differential response of Leuce (currently termed
Populus) section culti va rs to infection by A. tumefaciens
was described by Nesme et a!. (1987), and s usceptibility
of aspen cu ltivars to A. tumefaciens was correlated to cytokinin sensitivi ty by Benedd ra eta!. (1996). In add ition, intra- and inter-specific hyb rid poplars coming fro m Aigeiros
or Tacamahaca sections differed in s uscep tibility to A .
lumefaciens C58 strain (Riemenschneider 1990).
It is criti cal to select appro p ria te starting mate ria ls
(o r explants) fo r Agrobacterium-mediated transformation . Po tential ly, exp lant materia l can be derived from
seed ling, leaf, in te rn ode, petio le, root, call us, or other
cells, tissues, and organs. In vitro cu ltured leaves and
internodes (stems) have been u sed most often to trans-
53
Section II Transformation and Foreign Gene Expression
Figure 3. Regeneration of a transformed shoot on selective medium. After co-cultivation of hybrid poplar
(Populus alba x P grandidentata) leaf pieces
with Agrobacterium tumefaciens containing
NOS-NPT/1 a nd PIN2 -CAT genes, transformants
were selected on Murashige and Skoog (MS)
{1962} regeneration medium s upplemented with
40 1-1g/ml kanamycin.
form many Populus s pecies. G reenwood stem internode sections of P. tremuloides are the most susceptible
to tumor fo rm ation a nd leaf disks are the leas t susceptible (Kubisiak et a l. 1993). Leple e t a l. (1992) showed
that inte rnode explants of P. tremula x P. alba produced
more trans formed calli than leaf explan ts.
A su spensio n culture transformatio n system for inserting genes into pop lar might offer severa l advantages inclu ding: 1) the ability to screen large numbers
of potentially transform ed cells; 2) effecti ve inhibitio n
of residu al Agrobacterium following co-cultivation; and
3) hig h trans formation freque n cies d u e to rapi dl y dividing s uspension cultures that may be ame nab le to
s table integra tion of foreign D A (Howe e t al. 1994).
Howe ver, it is freque ntl y unknown w hi ch cell type
w ithin an expla nt is the m ost transformable or the mos t
capab le of regenerating into a ferti le plant. The s mall
amount of availab le d a ta indicates that the mos t regenerable cells do no t necessarily correspond with the most
transform ab le cells (De Block 1993).
54
Figure 4. Secondary selection of transformants occurred
on Mu rashige and Skoog (MS) rooting medium
containing 20 1-1g/ml kanamyci n. Rooted plantlets
of transgenic hybrid poplar (Populus alba x P
grandidentata) were propagated in vitro
(Klopfenstein et a l. 1991 ).
Agrobacterium Strain
To assure high infectivity levels for effective tra nsformation, the most s uitable Agrobacterium s train should be
determined for each host species I geno typ e I tissue. Generally, tree species respond better to the nopaline strains
than octopine s trains of A. tumefaciens (Ahuja 1987). Most
transgenic poplars have been produced u sing nopaline
s trai ns of Agrobacterium (Han e t a l. 1996). The p lasmid
rather than the chromosomal background was the most
critical determ inant for infection (Kubisiak et al. 1993).
However, influence of plas mid type on infection levels has
varied w ith host species/genotype/tissue type (Kubisiak
et al. 1993).
Two designed vector systems are u sed in Agrobacteriummediated transformation: 1) co-integrate: T-D A includes
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
Agrobacterium-mediated Transformation of Populus Species
Table 1. Transformation research using Agrobacterium-mediated transformation systems with Populus species.
Transgenes 1
Bacterial
spp.3
Reference
T-DNA 2
T-DNA
bar, NPT/1
GUS, NPT/1
A.t.
A.r.
A.t.
A.t.
Parsons et al. 1986
Pythoud et al. 1987
De Block 1990
Wang et al. 1994
aroA, NPT/1
CAT, NPT/1
aroA, NPT/1
Ac, Bt, HPT, NPT/1
PIN2, NPT/1
A.t.
A.t.
A.t.
A.t.
A.t.
Fillatti et al. 1987
Klopfenstein et al. 1991
Donahue et al. 1994
Howe et al. 1994
Klopfenstein et al. 1997
P. alba x P. glandulosa
T-DNA
A.r.
Chung et al. 1989
P. davidiana
T-DNA
A.r.
Lee et al. 1989
P. tomentosa
CAT, NPT/1
A.t.
Wang et al. 1990
P.
P.
P.
P.
P.
P.
bar, NPT/1
GUS, NPT/1, T-DNA
crs1-1, NPT/1
bar, NPT/1
GUS, NPT/1
IPT, NPT/1
T-DNA
PIN2, NPT/1
T-DNA
iaaM, GUS, MPT/1
prxA 1, GUS, NPT/1
GR, NPT/1
A.t.
A.t.
A.t.
A.r.
A.t.
A.t.
A.t.IA.r.
A.t.
A.t.IA.r.
A.t.
A.t.
At.
De Block 1990
Brasileiro et al. 1991
Brasileiro et al. 1992
Devillard 1992
Leple et al. 1992
Schwartzenberg et al. 1994
Charest et al. 1992
Heuchelin et al. 1997
Charest et al. 1992
Ebinuma et al. 1992
Kajita et al. 1994
Endo et al., this volume
luxF2, HPT, NPT/1
OC/, NPT/1
OCI, NPT/1
iaaH, iaaM, HPT, NPT/1
LFY, NPT/1
Ac, ro/C, NPT/1
GUS, HPT
ro/C, NPT/1
phyA, phyB, NPT/1
A.t.
A.t.
A.t.
A.t.
A.t.
A.t.
A.t.
A.t.
A.t.
Nilsson et al. 1992
Leple et al. 1995
Leple et al. 1995
Tuominen et al. 1995
Weigel and Nilsson 1995
Ahuja and Fladung 1996
Nilsson et al. 1996a
Nilsson et al. 1996b
Sundberg et al., this volume
P. tremuloides
P. tremuloides
T-DNA
GUS, NPT/1
A.t.
A.t.
Kubisiak et al. 1994
Tsai et al. 1994
P. nigra
P. nigra
GUS, HPT, NPT/1, T-DNA
GUS, NPT/1, T-DNA
A.t.
A.t.
Confalonieri et al. 1994
Confalonieri et al. 1995
P. tremula
Ac, roiC, NPT/1
A.t.
Ahuja and Fladung 1996
P. deltoides
P. deltoides
T-DNA
GUS, NPT/1
A.t.
A.t.
Riemenschneider 1990
Dinus et al. 1995
Species
P. trichocarpa x P.
P. trichocarpa x P.
P. trichocarpa x P.
P. trichocarpa x P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
alba x
alba x
alba x
alba x
alba x
deltoides
deltoides
deltoides
deltoides
P. grandidentata
P. grandidentata
P. grandidentata
P. grandidentata
P. grandidentata
alba x P. tremula
tremula x P. alba
tremula x P. alba
tremula x P. alba
tremula x P. alba
tremula x P. alba
deltoides x P. nigra
deltoides x P. nigra
nigra x P. maximowiczii
sieboldii x P. grandidentata
sieboldii x P. grandidentata
sieboldii x P. grandidentata
tremula x
tremu/a x
tremula x
tremula x
tremula x
tremula x
tremu/a x
tremula x
tremula x
P.
P.
P.
P.
P.
P.
P.
P.
P.
tremu/oides
tremu/oides
tremu/oides
tremu/oides
tremu/oides
tremu/oides
tremuloides
tremuloides
tremu/oides
Ac (Activato,~transposable element from maize; aroA=bacterial5-enolpyruvylshikimate-3-phosphate synthase chimeric gene;
bar=phosphinotricin acetyltransferase gene; Bt=endotoxin gene from Bacillus thuringiensis; CAT=chloramphenicol
acetyltransferase gene; crs 1-1=mutant acetolactate synthase gene; GR=glutathione reductase gene; GUS=~-glucuronidase gene;
HPT=hygromycin phosphotransferase gene; iaaH=agrobacterial indoleacetamide hydrolase gene; iaaM=agrobacterial tryptophan
monooxygenase gene; /PT=agrobacterial isopentenyltransferase gene; LFY=flower-meristem-identity gene; luxF2=1uciferase
gene; NPT//=neomycin phosphotransferase gene; OC/=cystein proteinase inhibitor g~ne; phyA, phyB=phytochrome ge~es;
P/N2=wound-inducible potato proteinase inhibitor II gene; prxA 1=peroxidase gene; and ro/C=one of the genes responsible for
hairy root disease, caused by the Agrobacterium rhizogenes
2
Transferred DNA
3 A.t.=Agrobacterium tumefaciens; A.r.=Agrobacterium rhizogenes
1
USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.
55
Section II Transformation and Foreign Gene Expression
gene(s) of interest with a selectable marker gene instead
of oncogenes on the Ti-plasinid; and 2) binary: T-DNA is
located on a separate vector plasmid instead of the Ti-p lasmid. T-DNA also includes the gene(s) of interest and selectable marker gene (Walkerpeach and Velten 1994}. No
recombination event is necessary for the binary vector system, unlike the co-integrate vector system. Overall, A.
tumefaciens strains C58, A281, EHA101, and LBA4404 were
commonly used with binary vectors for transformation of
many poplars and seem to generate suitable transformation efficiencies (Brasileiro et al. 1991, 1992; Confalonieri
et al. 1994, 1995; De Block 1990; Ebinuma et al. this volume; Howe et al. 1994; Kajita et al. 1994; Klopfenstein et
al. 1991, 1993, 1997; Leple et al. 1992, 1995; Nilsson et al.
1992; Schwartzenberg et al. 1994; Sundberg et al. this volume; Tuominen et al. 1995).
Transformation Procedures
Several factors should be considered to improve transformation efficiency such as the Agrobacterium inoculum
titer, vir inducer, selectable marker system, and in vitro tissue culture manipulation techniques. Optimal results were
obtained by dipping initial host explants into a bacterial
suspension (5 to 6 x 108 cells/ ml) for 20 min to 4 h, then cocultivating them for 24 to 72 h on a liquid or semisolid
regeneration medium that contained plant growth regulators such as benzyladenine (BA}, 2,4-dichlorophenoxyacetic acid (2,4-D}, naphthaleneacetic acid (NAA}, or
thidiazuron (TDZ) (Confaloniei et al. 1994; Wang et al.
1994).
Acetosyringone (AS) and hydroxy-acetosyringone (OHAS) elicited the expression of Agrobacterium vir region genes
(Stachel et al. 1985). AS and OH-AS occur specifically in
exudates of wounded and metabolically active plant cells
and perhaps allow Agrobacterium to recognize susceptible
cells (Stachel et al. 1985). Transformation efficiency could
be increased during co-cultivation by using a vir region
inducer such as AS (10 to 200 ~M) (Confalonieri et al. 1995;
Howe et al. 1994; Kubisiak et al. 1993; Nilsson et al. 1992;
Weigel and Nilsson 1995).
A practical selectable marker system is essential to obtain high efficiency transformations while avoiding
nontransformed plants that escape selectioh (Leple et al.
1992). Selectable marker genes used for Populus transformation have encoded traits such as hygromycin resistance
(hygromycin phosphotransferase; HPT), neomycin resistance (neomycin phosphotransferase II; NPTII),
phosphinotricin (glufosinate) resistance (phosphinotricin
acetyltransferase; bar), and chlorsulfuron resistance (mutant acetolactate synthase; crs1-1). Because the NPTII gene
has been frequently employed in several woody plants
including Populus species to select transformants (table 1),
kanamycin is one of the most commonly used antibiotics
56
for a transformation selection system. Even modest kanamycin concentrations (10 mg/1) can inhibit regeneration
of untransformed hybrid poplar (P. alba x P. grandidentata)
(Chun et al. 1988). Culture on nonselective medium (without selective antibiotics) for 2 days to 2 weeks before transfer to a selective medium (with selective antibiotics) has
been used to obtain higher transformation frequencies
(Charest et al. 1992; Dinus et al. 1995; Tuominen et al. 1995;
Wang et al. 1994).
The transfer of explants to light conditions after decontamination using cefotaxime (250 to 500 mg/1) and/or
carbenicillin (250 to 500 mg/1), a preculture (shoot-inducing or callus-inducing medium induding BA, 2,4-D, NAA,
or TDZ) period before Agrobacterium-mediated infection,
or a prolonged infection period can enhance transformation frequencies dramatically (Confalonieri et al. 1994,
1995; De Block 1993; Leple et al. 1992; Schwartzenberg et
al. 1994; Tsai et al. 1994). Several studies demonstrate that
the Agrobacterium plasmid, explant type, in vitro techniques,
and use of a vir region inducing compound can substantially influence stable transformation frequency
(Confalonieri et al. 1994, 1995; De Block 1990; Kubisiak et
al.1993).
Reporter genes used to detect transgene expression have
included CAT, rl-glucuronidase (GUS), and luciferase
(luxF2) genes (table 1). To date, GUS has been used most
often and has been effective as a reporter gene in poplar
(Jouanin and Pilate this volume; Pilate et al. this volume).
Use of luxF2 as a reporter allows in vivo monitoring of gene
expression by nondestructive imaging (Nilsson et al. 1992;
Schneider et al. 1990). Inhibitors present in poplar leaf extracts can interfere with CAT-activity assays reducing the
advantage of CAT as a reporter gene in poplar (Klopfenstein
et al. 1991).
Limitations and Prospects
Although transformation technology has reached a relatively advanced level, many variables exist that can interfere with the generation of stable transformed plants that
express transgenes in a predictable manner (Ahuja this
volume; De Block 1993). Recently, there have been several
papers about the quantitative and qualitative instability
of transgenes in primary transformed plants and subsequent generations (reviewed by De Block 1993; Ahuja this
volume). Agrobacterium-mediated transformation is believed to result in random integration of transgenes into
the genome causing high variation in quantitative and
qualitative expression levels of transgenes in primary
transformants and I or subsequent generations. However,
an Agrobacterium-mediated system is a desirable method
USDA Forest Service Gen. Tech: Rep. RM-GTR-297. 1997.
Agrobacterium-mediated Transformation of Populus Species
to transform Populus because it is relatively inexpensive,
easy to use, can produce an acceptable transformation rate,
and transfers a limited copy number of transgenes.
Acknowledgments
This paper was supported in part by the USDA Forest
Service, funds from contract #DOE OR22072-17 with the
Consortium for Plant Biotechnology Research, Inc., and
the Biotechnology Graduate Research Associateship program of the Center for Biotechnology, University of Nebraska-Lincoln. Use of trade names in this paper does not
constitute endorsement by the USDA Forest Service.
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