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This file was created by scanning the printed publication. Errors identified by the software have been corrected; however, some errors may remain. 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. Literature Cited Ahuja, M.R. 1987. Gene transfer in forest trees. In: Hanover, J.W.; Keathley, D.E., eds. Genetic manipulation of woody plants. New York: Plenum Press: 25-41. Ahuja, M.R.; Fladung, M. 1996. Stability and expression of chimeric genes in Populus. In: Ahuja, M.R.; Boerjan, W.; Neale, D.B., eds. Somatic cell genetics and molecular genetics of trees. Dordrecht, The Netherlands: Kluwer Academic Publishers: 89-96. Beneddra, T.; Picard, C.; Petit, A.; Nesme, X. 1996. Correlation between susceptibility to crown gall and sensitivity to cytokinin in aspen cultivars. Phytopathology. 86: 225-231. Brasileiro, A.C.M.; Leple, J.C.; Muzzin, J.; Ounnoughi, D.; Michel, M.F.; Jouanin, L. 1991. An alternative approach for gene transfer in trees using wild-type Agrobacterium strains. Plant Molecular Biology. 17: 441-452. Brasileiro, A.C.M.; Tourneur, C.; Leple, J.C.; Combes, V.; Jouanin, L. 1992. Expression of the mutant Arabidopsis thaliana acetolactate synthase gene confers chlorsulfuron resistance to transgenic poplar plants. Transgenic Research. 1: 133-141. Charest, P.J.; Stewart, D.; Budicky, P.L. 1992. Root induction in hybrid poplar by Agrobacterium genetic transformation. Can. J. For. Res. 22: 1832-1837. Chun, Y.W. 1994. Application of Agrobacterium vector systems for transformation in Populus species. In: Kim, Z.5.; Hattemer, H.H., eds. Conservation and manipulation of genetic resources in forestry. Seoul: Kwang Moon Kag Publishing Co.: 206-218. Chun, Y.W.; Klopfenstein, N.B. 1995. Organ specific gene expression of the nos-NPTII gene in transgenic hybrid poplar. Journal of the Korean Forestry Society. 84:77-86. USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. Chun, Y.W.; Klopfenstein, N.B.; McNabb, H.S., Jr.; Hall, R.B. 1988. Transformation of Populus species by an Agrobacterium binary vector system. Journal of the Korean Forestry Society. 77: 199-207. Chung, K.H.; Park, Y.G.; Noh, E.R.; Chun, Y.W. 1989. Transformation of Populus alba x P. glandulosa by Agrobacterium rhizogenes. Journal of the Korean Forestry Society. 78: 372-380. Confalonieri, M.; Balestrazzi, A.; Bisoffi, S. 1994. Genetic transformation of Populus nigra by Agrobacterium tumefaciens. Plant Cell Reports. 13: 256-261. Confalonieri, M.; Balestrazzi, A.; Bisoffi, S.; Cella, R. 1995. 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