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

posed by wounding (Perani et al. 1986). Infection by A. Introduction 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 Although molecular biology of woody plants is a rela­ components that are required for plant cell transforma­ tively young field, it offers considerable potential for breed­ tion; T-DNA (transferred DNA) and the virulence (vir) re­ ing and selecting improved trees for multiple purposes. gion, which are both located on the TI (tumor-inducing) or Conventional breeding programs have produced im­ Ri (root-inducing) plasmid (Zambryoski et al. 1989). The proved growth rates, adaptability, and pest resistance; T-DNA portion of the A. tumefaciens TI plasmid or the A. however, tree improvement processes are time consum­ rhizogenes Ri plasmid is transferred to the nucleus of a host ing because of the long generation and rotation cycles of plant where it integrates into the nuclear DNA genetically trees (Dinus and Tuskan this volume; Leple et al. 1992). transforming the recipient plant. A region of the 1i plas­ Genetic engineering of trees helps to compensate for con­ mid outside the T-DNA, referred to as the wirulence re­ ventional breeding disadvantages by incorporating known gion, carries the vir genes. Expression of vir genes occurs genes into specific genetic backgrounds. Since the first during plant cell infection and is a prerequisite for the sub­ successful plant transformation was reported in 1983 sequent transfer of the T-DNA. Agrobacterium chromo­ (Herrera-Estrella et al. 1983; Murai et al. 1983), several somal regions are involved in attachment of Agrobacterium nonsexual gene transfer methods were developed for im­ to plant cells. The T-DNA of A. tumefaciens contains auxin portant agronomic crops and forest tree species. These {iaaH, iaaM) and cytokinin (IPT) synthesis genes methods include biolistics (microprojectile bombardment), (Zambryoski et al. 1989). These genes are referred to as electroporation, and Agrobacterium-mediated transforma­ oncogenes and are responsible for tumor induction. In A. tion. Biolistics and electroporation are discussed by Charest rhizogenes, T-DNA contains multiple rol genes that induce et al. (this volume). This chapter focuses on Agrobacterium­ root formation (Zambryoski et al. 1989). The T-DNA also mediated gene transfer methods, which are widely-used encodes several genes responsible for the synthesis of com­ for plant transformation of broad-leaved, woody plants pounds called opines, which are metabolic substrates for because of their versatility and efficient application the bacteria (Nester et al. 1984). Efficient transfer ofT-DNA (Brasileiro et al. 1991; Chun 1994; Han et al. 1996; Leple et is facilitated by 24-base pair direct repeats at the T-DNA al. 1992). borders. Genes within the T-DNA can be replaced with · Agrobacterium spp. are soil bacteria tJ:tat naturally infect genes of interest without affecting transfer efficiency (Han many dicotyledonous and gymnospermous plants predis- 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. demon­ strate developmental plasticity to tissue culture manipu­ lations. These traits and susceptibility to Agrobacterium-mediated transformation and techniques to , Klopfenstein, N.B.; Chun, Y. W.; Kim, M.-S.; Ahuja, M.A., eds. regenerate transgenic trees make Populus a suitable mod~ I Dillon, M.C.; Carman, R.C.; Eskew, L.G., tech. eds. 1997. system for genetic engineerin? of deciduou~ trees.~ th1s Micropropagation, genetic engineering, and molecular biology of Populus. Gen. Tech. Rep. RM-GTR-297. Fort Collins, CO: chapter, we describe the ma1n Agrobacterzum-med1ated U.S. Department of Agriculture, Forest Service, Rocky Mountain transformation procedures developed for Populus andre­ Research Station. 326 p. view the results obtained using several Populus species.

51 Section II Transformation and Foreign Gene Expression

for infection and transformation; 2) infection: wounded start­ ing explants are co-cultiva ted with an Agrobacteriz1111 strain containing co-integrate or binary vectors; 3) selection: after removal of residual Agrobacterium, transformed cells are se­ lected for subsequent regeneration into transgenic plants (fig­ ure 3); 4) regeneration: transformed cells are regenerated during or after the selection period (figures3 and 4); and 5) con­ firmation: the presence or function of transgenes in the genome of transgenic plants is confirmed using molecular techniques such as polymerase chain reaction, Southern hybridization, northern hyb ridization, western blotting, enzyme-linked immunosorbent assay (ELISA), or enzyme activity assays.

Transgenes Several silviculturally usefu l genes have been isolated and used for Agrobacterium-mediated transformation of Populus. A table listing genes used in Populus transformation (Chun 1994) was updated fo r this chapter (table 1). These genes include the: 1) mutant aroA gene, which encodes glyphosate tolerance via a 5-enolpyruylshikimate-3-phosphate synthase (EPSP) that is less sensitive to the herbicide glyphosate (Donahue et al. 1994; Fillatti et al. 1987); 2) bar gene encod­ ing the enzyme phosphinotricin acetytransferase (PAT) that inactivates the herbicide phos.phinotricin (glufos inate) (De Block 1990; Devillard 1992); 3) mutant crs1-1 gene from a chlorsulfuron-herbicide-resistant line of Arabidopsis thaliana (Brasileiro et al. 1992); 4) OCI (oryzastatin), a cysteih pro­ teinase 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; Figure 1. Crown gall produced by Agrobacterium Leple et al. 1995); and 5) insecticidal protein genes from Ba­ tumefaciens strain A281 infection of hyb rid cillus thuringiensis (Bt) (Howe et al. 1994). Other studies have poplar (Populus alba x P. grandidentata) stem focused on transgene regulation (Chun and Klopfenstein after approxi mately 1 0 weeks. 1995; Confa lonieri et al. 1994; Kajita et al. 1994; Klopfenstein et al. 1991; Leple et al. 1995; ilsson et al. 1992) and develop­ mental influences (Ah uja and Fladung 1996; Charest et al. 1992; Ebinuma e t al. 1992; Nilsson e t a l. 1996a, 1996b; Schwartzenberg et al. 1994; Sundberg et al. this volume; Tuominen et al. 1995; Weigel and Tilsson 1995). Gene Transfer to Populus Species Transgene Copy Number Populus has been known as a natural host for Agrobacteriu111 fo r many years. DeCleene and De Ley (1976) cite early litera­ Few s tud ies have reported the copy number of inserted ture tha t suggests the susceptibility of 3 Populus species to transgenes by Agrobacteriu111-mediated transformation on infection by A. tu111ejaciens. The presence of T-ON A sequences Populus species. Transgenic microshoots of hybrid aspen (P. in gall and root tissue confirmed Populus as a host fo r A. alba x P tremula) contained from 1 to 3 copies of the inserted tu111ejaciens and A. rhizogenes (Parsons et al. 1986; Pythoud et foreign bar genes (De Block 1990); whereas, in vitro plants (P a l. 1987). These early pathogenicity studies of Agrobacteriu111 tre111 ula x P alba) regenerated from transformed roots con­ provided the basis fo r its use as a tool to transfer foreign tained 1 copy of the bar gene (Devillard 1992). Only a single genes into the poplar genome. copy of the chloramphenicol acetyltransferase (CAT) gene The process fo r prod ucing transgenic pop lar plants in­ was inserted into the genome of transgenic hybrid poplar cludes 5 main components (figure 2): 1) initiation: starting (P alba x P. grandidentata) (Klopfenstein et al. 1991). In addi­ explants (host species/genotype/tissue type) are selected tion, 1 to 4 copies of crs1 -1 gene had been inserted per hy-

52 USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. Agrobacterium-mediated Transformation of Populus Species ... ~ Wounding Field Test CONFIRMATION ••• (e.g .. Southern blot. PCR. INITIATION ~ northern hybridization . .:!: Dark Conditions western blot. ELISA. t and/or enzyme activity assay) 'I= =I' Greenhouse Preculture (CIM or SIM) Growth

INFECTION 'I = I' Co-cultivation with A. tumefaciens or / In vitro propagation A. rhizogenes .:!: Secondary selection and regeneration to avoid chimeric transformants I Decontamination _:!: preselective' culture SELECTI ON REGENERATION 'I.Je *' I' :!: Additional selection for root Selection of transformed formation in selective media cells

Figure 2. The primary steps for Agrobacterium-mediated 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). triclzocarpa x P. del/aides (Parsons et al. 1986) was studied Also, Howe ct al. (1991) showed that the number of inserted and additional information was gathe red on the effect of 0 A copies ranged from 1 to 10 after the maize transposable poplar genotypes (Charest et al. 1992). Previous studies element Ac (Activator) was transferred into hybrid poplar (P showed significant differences among the geno types alba x P grmzdidentata). However, it is unknown if all inserted w ithin species and the clones within gen otype gene copies were expressed (Chun 1994; Leple et al. 1992). (Confa lonieri et al. 1994; De Block 1990; Ri emenschneider 1990). A differential response of Leuce (currently termed Populus) section cultivars to infection by A. tumefaciens was described by Nesme et a!. (1987), and susceptibility of aspen cultivars to A. tumefaciens was correlated to cyto­ Agrobacterium-mediated kinin sensitivi ty by Beneddra eta!. (1996). In addition, in­ Transformation tra- and inter-specific hybrid poplars coming from Aigeiros or Tacamahaca sections differed in susceptibility to A . lumefaciens C58 strain (Riemenschneider 1990). It is critical to select approp ria te starting materia ls Host Species/Genotype/Tissue Type (or explants) fo r Agrobacterium-mediated transforma­ A prerequisite for any genetic transformation work us­ tion . Potentially, explant materia l can be derived from ing Agrobacterium is the ability of the bacterium to infect seed ling, leaf, inte rnode, peti ole, root, callus, or other the plant of interest. The effect of 2 Agrobacterium cells, tissues, and organs. In vitro cultured leaves and tumefaciens strains, A281 and A348, on infection of P. internodes (stems) have been used most often to trans-

USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. 53 Section II Transformation and Foreign Gene Expression

Figure 3. Regeneration of a transformed shoot on selec­ tive medium. After co-cultivation of hybrid poplar (Populus alba x P grandidentata) leaf pieces with Agrobacterium tumefaciens containing NOS-NPT/1 and PIN2-CAT genes, transformants were selected on Murashige and Skoog (MS) {1962} regeneration medium supplemented with 40 1-1g/ml kanamycin. Figure 4. Secondary selection of transformants occurred on Mu rashige and Skoog (MS) rooting medium containing 20 1-1g/ml kanamycin. Rooted plantlets of transgenic hybrid poplar (Populus alba x P grandidentata) were propagated in vitro form many Populus species. G reenwood stem intern­ (Klopfenstein et a l. 1991 ). ode sections of P. tremuloides are the most susceptible to tumor fo rmation and leaf disks are the least suscep­ tible (Kubisiak et al. 1993). Leple et a l. (1992) showed that internode explants of P. tremula x P. alba produced Agrobacterium Strain more trans formed calli than leaf explants. A suspension culture transformation system for in­ To assure high infectivity levels for effective transfor­ serting genes into poplar might offer several advan­ mation, the most suitable Agrobacterium s train should be tages including: 1) the ability to screen large numbers determined for each host species I genotype I tissue. Gen­ of potentially transformed cells; 2) effective inhibition erally, tree species respond better to the nopaline strains of residual Agrobacterium following co-cultivation; and than octopine strains of A. tumefaciens (Ahuja 1987). Most 3) hig h trans formation frequencies d ue to rapidly di­ transgenic poplars have been produced using nopaline viding s uspension cultures that may be amenable to strains of Agrobacterium (Han e t al. 1996). The p lasmid stable integration of foreign D A (Howe et al. 1994). rather than the chromosomal background was the most However, it is freque ntly unknown w hich cell type critical determinant for infection (Kubisiak et al. 1993). within an explant is the m ost transformable or the mos t However, influence of plasmid type on infection levels has capable of regenerating into a fertile plant. The small varied with host species/genotype/tissue type (Kubisiak amount of available d ata indicates that the most regen­ et al. 1993). erable cells do no t necessarily correspond with the most Two designed vector systems are used in Agrobacterium­ transformable cells (De Block 1993). mediated transformation: 1) co-integrate: T-D A includes

54 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.

Bacterial Species Transgenes 1 spp.3 Reference 2 P. trichocarpa x P. deltoides T-DNA A.t. Parsons et al. 1986 P. trichocarpa x P. deltoides T-DNA A.r. Pythoud et al. 1987 P. trichocarpa x P. deltoides bar, NPT/1 A.t. De Block 1990 P. trichocarpa x P. deltoides GUS, NPT/1 A.t. Wang et al. 1994 P. alba x P. grandidentata aroA, NPT/1 A.t. Fillatti et al. 1987 P. alba x P. grandidentata CAT, NPT/1 A.t. Klopfenstein et al. 1991 P. alba x P. grandidentata aroA, NPT/1 A.t. Donahue et al. 1994 P. alba x P. grandidentata Ac, Bt, HPT, NPT/1 A.t. Howe et al. 1994 P. alba x P. grandidentata PIN2, NPT/1 A.t. Klopfenstein et al. 1997 P. alba x P. glandulosa T-DNA A.r. Chung et al. 1989 davidiana P. T-DNA A.r. Lee et al. 1989 P. tomentosa CAT, NPT/1 A.t. Wang et al. 1990 P. alba x P. tremula bar, NPT/1 A.t. De Block 1990 P. tremula x P. alba GUS, NPT/1, T-DNA A.t. Brasileiro et al. 1991 P. tremula x P. alba crs1-1, NPT/1 A.t. Brasileiro et al. 1992 P. tremula x P. alba bar, NPT/1 A.r. Devillard 1992 P. tremula x P. alba GUS, NPT/1 A.t. Leple et al. 1992 P. tremula x P. alba IPT, NPT/1 A.t. Schwartzenberg et al. 1994 P. deltoides x P. nigra T-DNA A.t.IA.r. Charest et al. 1992 P. deltoides x P. nigra PIN2, NPT/1 A.t. Heuchelin et al. 1997 P. nigra x P. maximowiczii T-DNA A.t.IA.r. Charest et al. 1992 P. sieboldii x P. grandidentata iaaM, GUS, MPT/1 A.t. Ebinuma et al. 1992 P. sieboldii x P. grandidentata prxA 1, GUS, NPT/1 A.t. Kajita et al. 1994 P. sieboldii x P. grandidentata GR, NPT/1 At. Endo et al., this volume P. tremula x P. tremu/oides luxF2, HPT, NPT/1 A.t. Nilsson et al. 1992 P. tremu/a x P. tremu/oides OC/, NPT/1 A.t. Leple et al. 1995 P. tremula x P. tremu/oides OCI, NPT/1 A.t. Leple et al. 1995 P. tremula x P. tremu/oides iaaH, iaaM, HPT, NPT/1 A.t. Tuominen et al. 1995 P. tremula x P. tremu/oides LFY, NPT/1 A.t. Weigel and Nilsson 1995 P. tremula x P. tremu/oides Ac, ro/C, NPT/1 A.t. Ahuja and Fladung 1996 P. tremu/a x P. tremuloides GUS, HPT A.t. Nilsson et al. 1996a P. tremula x P. tremuloides ro/C, NPT/1 A.t. Nilsson et al. 1996b P. tremula x P. tremu/oides phyA, phyB, NPT/1 A.t. Sundberg et al., this volume P. tremuloides T-DNA A.t. Kubisiak et al. 1994 P. tremuloides GUS, NPT/1 A.t. Tsai et al. 1994 P. nigra GUS, HPT, NPT/1, T-DNA A.t. Confalonieri et al. 1994 P. nigra GUS, NPT/1, T-DNA A.t. Confalonieri et al. 1995 P. tremula Ac, roiC, NPT/1 A.t. Ahuja and Fladung 1996 P. deltoides T-DNA A.t. Riemenschneider 1990 P. deltoides GUS, NPT/1 A.t. Dinus et al. 1995 1 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

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 for a transformation selection system. Even modest kana­ of oncogenes on the Ti-plasinid; and 2) binary: T-DNA is mycin concentrations (10 mg/1) can inhibit regeneration located on a separate vector plasmid instead of the Ti-p las­ of untransformed hybrid poplar (P. alba x P. grandidentata) mid. T-DNA also includes the gene(s) of interest and se­ (Chun et al. 1988). Culture on nonselective medium (with­ lectable marker gene (Walkerpeach and Velten 1994}. No out selective antibiotics) for 2 days to 2 weeks before trans­ recombination event is necessary for the binary vector sys­ fer to a selective medium (with selective antibiotics) has tem, unlike the co-integrate vector system. Overall, A. been used to obtain higher transformation frequencies tumefaciens strains C58, A281, EHA101, and LBA4404 were (Charest et al. 1992; Dinus et al. 1995; Tuominen et al. 1995; commonly used with binary vectors for transformation of Wang et al. 1994). many poplars and seem to generate suitable transforma­ The transfer of explants to light conditions after decon­ tion efficiencies (Brasileiro et al. 1991, 1992; Confalonieri tamination using cefotaxime (250 to 500 mg/1) and/or et al. 1994, 1995; De Block 1990; Ebinuma et al. this vol­ carbenicillin (250 to 500 mg/1), a preculture (shoot-induc­ ume; Howe et al. 1994; Kajita et al. 1994; Klopfenstein et ing or callus-inducing medium induding BA, 2,4-D, NAA, al. 1991, 1993, 1997; Leple et al. 1992, 1995; Nilsson et al. or TDZ) period before Agrobacterium-mediated infection, 1992; Schwartzenberg et al. 1994; Sundberg et al. this vol­ or a prolonged infection period can enhance transforma­ ume; Tuominen et al. 1995). tion 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 Transformation Procedures the Agrobacterium plasmid, explant type, in vitro techniques, and use of a vir region inducing compound can substan­ Several factors should be considered to improve trans­ tially influence stable transformation frequency formation efficiency such as the Agrobacterium inoculum (Confalonieri et al. 1994, 1995; De Block 1990; Kubisiak et titer, vir inducer, selectable marker system, and in vitro tis­ al.1993). sue culture manipulation techniques. Optimal results were Reporter genes used to detect transgene expression have obtained by dipping initial host explants into a bacterial included CAT, rl-glucuronidase (GUS), and luciferase suspension (5 to 6 x 108 cells/ ml) for 20 min to 4 h, then co­ (luxF2) genes (table 1). To date, GUS has been used most cultivating them for 24 to 72 h on a liquid or semisolid often and has been effective as a reporter gene in poplar regeneration medium that contained plant growth regu­ (Jouanin and Pilate this volume; Pilate et al. this volume). lators such as benzyladenine (BA}, 2,4-dichlorophenoxy­ Use of luxF2 as a reporter allows in vivo monitoring of gene acetic acid (2,4-D}, naphthaleneacetic acid (NAA}, or expression by nondestructive imaging (Nilsson et al. 1992; thidiazuron (TDZ) (Confaloniei et al. 1994; Wang et al. Schneider et al. 1990). Inhibitors present in poplar leaf ex­ 1994). tracts can interfere with CAT-activity assays reducing the Acetosyringone (AS) and hydroxy-acetosyringone (OH­ advantage of CAT as a reporter gene in poplar (Klopfenstein AS) elicited the expression of Agrobacterium vir region genes et al. 1991). (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; Limitations and Prospects Howe et al. 1994; Kubisiak et al. 1993; Nilsson et al. 1992; Weigel and Nilsson 1995). Although transformation technology has reached a rela­ A practical selectable marker system is essential to ob­ tively advanced level, many variables exist that can inter­ tain high efficiency transformations while avoiding fere with the generation of stable transformed plants that nontransformed plants that escape selectioh (Leple et al. express transgenes in a predictable manner (Ahuja this 1992). Selectable marker genes used for Populus transfor­ volume; De Block 1993). Recently, there have been several mation have encoded traits such as hygromycin resistance papers about the quantitative and qualitative instability (hygromycin phosphotransferase; HPT), neomycin resis­ of transgenes in primary transformed plants and subse­ tance (neomycin phosphotransferase II; NPTII), quent generations (reviewed by De Block 1993; Ahuja this phosphinotricin (glufosinate) resistance (phosphinotricin volume). Agrobacterium-mediated transformation is be­ acetyltransferase; bar), and chlorsulfuron resistance (mu­ lieved to result in random integration of transgenes into tant acetolactate synthase; crs1-1). Because the NPTII gene the genome causing high variation in quantitative and has been frequently employed in several woody plants qualitative expression levels of transgenes in primary including Populus species to select transformants (table 1), transformants and I or subsequent generations. However, kanamycin is one of the most commonly used antibiotics an Agrobacterium-mediated system is a desirable method

56 USDA Forest Service Gen. Tech: Rep. RM-GTR-297. 1997. Agrobacterium-mediated Transformation of Populus Species

to transform Populus because it is relatively inexpensive, Chun, Y.W.; Klopfenstein, N.B.; McNabb, H.S., Jr.; Hall, R.B. easy to use, can produce an acceptable transformation rate, 1988. Transformation of Populus species by an and transfers a limited copy number of transgenes. Agrobacterium binary vector system. Journal of the Ko­ rean Forestry Society. 77: 199-207. Chung, K.H.; Park, Y.G.; Noh, E.R.; Chun, Y.W. 1989. Trans­ formation of Populus alba x P. glandulosa by Agrobacterium rhizogenes. Journal of the Korean Forestry Society. 78: Acknowledgments 372-380. Confalonieri, M.; Balestrazzi, A.; Bisoffi, S. 1994. Genetic This paper was supported in part by the USDA Forest transformation of Populus nigra by Agrobacterium Service, funds from contract #DOE OR22072-17 with the tumefaciens. Plant Cell Reports. 13: 256-261. Consortium for Plant Biotechnology Research, Inc., and Confalonieri, M.; Balestrazzi, A.; Bisoffi, S.; Cella, R. 1995. the Biotechnology Graduate Research Associateship pro­ Factors affecting Agrobacterium fumefaciens-mediated gram of the Center for Biotechnology, University of Ne­ transformation in several black poplar clones. Plant Cell, braska-Lincoln. Use of trade names in this paper does not Tissue and Organ Culture. 43: 215-222. constitute endorsement by the USDA Forest Service. De Block, M. 1990. Factors influencing the tissue culture and the Agrobacterium tumefaciens-mediated transforma­ tion of hybrid aspen and poplar clones. Plant Physiol­ ogy.93: 1110-1116. De Block, M. 1993. The cell biology of plant transform·a­ Literature Cited tion: Current state, problems, prospects and the impli­ cations for the plant breeding. Euphytica. 71: 1-14. DeCleene, M.; DeLay, J. 1976. The host range of crown gall. Ahuja, M.R. 1987. Gene transfer in forest trees. In: Hanover, The Botanical Review. 42: 389-466. J.W.; Keathley, D.E., eds. Genetic manipulation of woody Devillard, C. 1992. Genetic transformation of aspen plants. New York: Plenum Press: 25-41. (Populus tremula x Populus alba) by Agrobacterium Ahuja, M.R.; Fladung, M. 1996. Stability and expression rhizogenes and regeneration of plants tolerant to herbi­ of chimeric genes in Populus. In: Ahuja, M.R.; Boerjan, cide. C. R. Acad. Sci. Paris, t. 314, serie III: 291-298. W.; Neale, D.B., eds. Somatic cell genetics and molecu­ Din us, R.J .; Stephens, C.J .; Chang, S. 1995. Agrobacterium lar genetics of trees. Dordrecht, The Netherlands: tumefaciens-mediated transformation of eastern cotton­ Kluwer Academic Publishers: 89-96. wood (Populus deltoides). In: Proceedings of the interna­ Beneddra, T.; Picard, C.; Petit, A.; Nesme, X. 1996. Correla­ tional poplar symposium: Poplar biology and its tion between susceptibility to crown gall and sensitiv­ implications for management and conservation; 1995 ity to cytokinin in aspen cultivars. Phytopathology. 86: August 20-25; Seattle, WA, U.S.A. Seattle, WA, U.S.A.: 225-231. University of Washington: 42. Abstract. Brasileiro, A.C.M.; Leple, J.C.; Muzzin, J.; Ounnoughi, D.; Donahue, R.A.; Davis, T.D.; Michler, C.H.; Michel, M.F.; Jouanin, L. 1991. An alternative approach Riemenschneider, D.E.; Carter, D.R.; Marquardt, P.E.; for gene transfer in trees using wild-type Agrobacterium Sankhla, N.; Sankhla, D.; Haissig, B.E.; Isebrands, J.G. strains. Plant Molecular Biology. 17: 441-452. 1994. Growth, photosynthesis, and herbicide tolerance Brasileiro, A.C.M.; Tourneur, C.; Leple, J.C.; Combes, V.; of genetically modified hybrid poplar. Can. J. For. Res. Jouanin, L. 1992. Expression of the mutant Arabidopsis 24: 2377-2383. thaliana acetolactate synthase gene confers chlorsulfuron Ebinuma, H.; Wabiko, H.; Ohshima, K.; Hata, K.; Sano, H. resistance to transgenic poplar plants. Transgenic Re­ 1992. The genetic engineering of poplar trees.- A first search. 1: 133-141. practical application of a homology-based interaction Charest, P.J.; Stewart, D.; Budicky, P.L. 1992. Root induc­ (Matzke effect) for prot.ection against the plant disease. tion in hybrid poplar by Agrobacterium genetic trans­ In: Proceedings, 1992 Fifth international conference on formation. Can. J. For. Res. 22: 1832-1837. biotechnology in the pulp and paper industry; Tokyo: Chun, Y.W. 1994. Application of Agrobacterium vector sys­ Uni Publishers Co., LTD.: 467-472. tems for transformation in Populus species. In: Kim, Z.- Fillatti, J.J.; Sellmer, ].; McCown, B.; Haissig, B.; Comai, L. 5.; Hattemer, H.H., eds. Conservation and manipulation 1987. Agrobacterium mediated transformation and regen­ of genetic resources in forestry. Seoul: Kwang Moon Kag eration of Populus. Mol. Gen. Genet. 206: 192-199. Publishing Co.: 206-218. Han, K.-H.; Gordon, M.P.; Strauss, S.H. 1996. Cellular and Chun, Y.W.; Klopfenstein, N.B. 1995. Organ specific gene molecular biology of Agrobacterium-mediated transfor­ expression of the nos-NPTII gene in transgenic hybrid pop­ mation of plant and its application to genetic transfor­ lar. Journal of the Korean Forestry Society. 84:77-86. mation of Populus. In: Stettler, R.F.; Bradshaw, H.D., Jr.;

57 USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. Section II Transformation and Foreign Gene Expression

Heilman, P.E.; Hinckley, T.M., eds. Biology of Populus Agrobacterium rhizogenes. Res. Rep. Inst. For. Gen. Ko­ and its implications for management and conservation. rea. 25: 149-153. Ottawa, Ontario, Canada: NRC Research Press: 201-222. Leple, J .C.; Bonade-Bittino, M.; Augustin, S.; Pilate, G.; Chapter 9. Dumanois Le Tan, V.; Delplanque, A.; Cornu, D.; Herrera-Estrella, L.; Depicker, A.; Van Montagu, M.; Schell, Jouanin, L. 1995. Toxicity to Chrysomela tremulae (Co­ J. 1983. Expression of chimeric genes transferred into leoptera: Chrysomelidae) of transgenic poplars express­ plant cells using a Ti-plasmid-derived vector. Nature. ing a cysteine proteinase inhibitor. Molecular Breeding. 303: 209-213. 1: 319-328. Heuchelin, S.A.; McNabb, H.S., Jr.; Klopfenstein, N.B. 1997. Leple, J.C.; Brasileiro, A.C.M.; Michel, M.F.; Delmotte, F.; Agrobacterium-mediated transformation of Populus X Jouanin, L. 1992. Transgenic poplars: expression of chi­ euramericana 'Ogy' using the chimeric CaMV 35S-pin2 meric genes using four different constructs. Plant Cell gene fusion. Can. J. For. Res. in press. Reports. 11: 137-141. Howe, G.T.; Goldfarb, B.; Strauss, S.H. 1994. Agrobacterium­ Murashige, T.; Skoog, F. 1962. A revised medium for rapid mediated transformation of hybrid poplar suspension growth and bio assays with tobacco tissue culture. cultures and regeneration of transformed plants. Plant Physiol. Plantarum. 15: 473-497. Cell, Tissue and Organ Culture. 36: 59-71. Murai, N.; Sutton, D.W.; Murray, M.G.; Slighton, J.L.; Merlo, Howe, G.T.; Strauss, S.H.; Goldfarb, B. 1991. Insertion of D.J.; Reichert, N.A.; Sengupta-Gopalan, C.; Stock, C.A.; the maize transposable element Ac into poplar. In: Barker, R.F.; Kemp, J.D.; Hall, T.C. 1983. Phaseolin gen~ Ahuja, M.R., ed. Woody plant biotechnology. New York: from bean is expressed after transfer to sunflower via tu­ Plenum Press: 283-294. mor-inducing plasmid vectors. Science. 222: 476-482. Jouanin, L.; Brasileiro, A.C.M.; Leple, J.C.; Pilate, G.; Cornu, Nesme, X.; Michel, M.F.; Digat, B. 1987. Population het­ D. 1993. Genetic transformation: a short review of meth­ erogeneity of Agrobacterium tumefaciens in galls of ods and their applications, results and perspectives for Populus L. from a single nursery. Appl. and Envir. forest trees. Ann. Sci. For. 50: 325-336. Microb. 53: 655-659. Kajita, S.; Osakabe, K.; Katayama, Y.; Kawai, S.; Matsumoto, Nester, E.W.; Gordon, M.P.; Amasino, R.M.; Yanofsky, M.F. Y.; Hata, K.; Morohoshi, N. 1994. Agrobacterium-medi­ 1984. Crown gall: A molecular and physiological analy­ ated transformation of poplar using a disarmed binary sis. Annu. Rev. Plant Physiol. 35: 387-413. vector and the overexpression of a specific member of a Nilsson, 0.; Alden, T.; Sitbon, F.; Little, C.H.A.; Chalupa, family of poplar peroxidase genes in transgenic poplar V.; Sandberg, G.; Olsson, 0. 1992. Spatial pattern of cau­ cell. Plant ?cience. 103: 231-239. liflower mosaic virus 35S promoter-luciferase expres­ Klopfenstein, N.B.; Allen, K.K.; Avila, F.J.; Heuchelin, S.A.; sion in transgenic hybrid aspen trees monitored by Martinez, J.; Carman, R.C.; Hall, R.B.; Hart, E.R.; enzymatic assay and non-destructive imaging. McNabb, H.S., Jr. 1997. Proteinase inhibitor II gene in Transgenic Research. 1: 209-220. transgenic poplar: Chemical and biological assays. Bio­ Nilsson, 0.; Little, C.H.A.; Sandberg, G.; Olsson, 0. 1996a. mass and Bioenergy. in press. Expression of two heterologous promoters, Klopfenstein, N.B.; McNabb, H.S., Jr.; Hart, E.R.; Hall, R.B.; Agrobacterium rhizogenes rolC and cauliflower mosaic Hanna, R.D.; Heuchelin, S.A.; Allen, K.K.; Shi, N.-Q.; virus 355, in the stem of transgenic hybrid aspen plants Thornburg, R.W. 1993. Transformation of Populus hy­ during the annual cycle of growth and dormancy. Plant brids to study and improve pest resistance. Silvae Molecular Biology. 31: 887-895. Genetica. 42: 86-90. Nilsson, 0.; Moritz, T.; Sundberg, B.; Sandberg, G.; Olsson, Klopfenstein, N.B.; Shi, N.Q.; Kernan, A.; McNabb, H.S., 0. 1996b. Expression of the Agrobacterium rhizogenes rolC Jr.; Hall, R.B.; Hart, E.R.; Thornburg, R.W. 1991. gene in a deciduous tree alters growth and develop­ Transgenic Populus hybrid expresses a wound-induc­ ment and leads to stem fasciation. Plant Physiology. 112: ible potato proteinase inhibitor II - CAT gene fusion. 493-502. Can. J. For. Res. 21: 1321-1328. Parsons, T.J.; Sinkar, V.P.; Stettler, R.F.; Nester, E.W.; Gor­ Kubisiak, T.L.; Mohn, C.A.; Fumier, G.R.; Hackett, W.P.; don, M.P. 1986. Transformation of poplar by Riu, K.-Z. 1993. Increasing the frequency of Agrobacterium tumefaciens. Biotechnology. 4: 533-536. Agrobacterium mediated transformation of Populus Perani, L.; Radke, S.; Wilke-Douglas, M.; Bossert, M. 1986. tremuloides. In: Mohn, C. A., compiler. Proceedings of the Gene transfer methods for crop improvement: Introduc­ second Northern Forest Genetics Association confer­ tion of foreign DNA into plants. Physiol. Plantarum. 68: ence; 1993 July 29-30; Roseville, MN, U.S.A. Dept. of 566-570. Forest Resources, University of Minnesota, St. Paul, MN, Pythoud, F.; Sinkar, V.P.; Nester, E. W.; Gordon, M.P. 1987. U.S.A.: Northern Forest Genetics Association: 189-198. Increased virulence of Agrobacterium rhizogenes con­ Lee, B.S.; Youn, Y.; Lee, S.K.; Choi, W.Y.; Kwon, Y.J. 1989. ferred by the vir region of pTiBo542: application to ge­ Transformation of Populus davidiana Dode by netic engineering of poplar. Biotechnology. 5: 1323-1327.

58 USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997. Agrobacterium-mediated Transformation of Populus Species

Riemenschneider, D.E. 1990. Susceptability of intra- and Walkerpeach, C.R.; Velten, J. 1994. Agrobacterium-mediated inter-specific . hybrid poplars to Agrobacterium gene transfer to plant cells: co-integrate and binary vec­ tumefaciens strain C58. Phytopathology. 80: 1099-1102. tor systems. In: Gelvin, S.B.; Schilperoort, R.A., eds. Plant Schneider, M.; Ow, D.W.; Howell, S.H. 1990. The in vivo molecular biology manual. Dordrecht, The Netherlands: pattern of firefly luciferase expression in transgenic Kluwer Academic Publishers: B1: 1-19. plants. Plant Molecular Biology. 14: 935-947. Wang, S.-P.; Xu, Z.-H.; Wei, Z.-M. 1990. Genetic transfor­ Schwartzenberg, K.; Doumas, P.; Jouanin, L.; Pilate, G. 1994. mation of leaf explants of Populus tomentosa. Acta Enhancement of the endogenous cytokinin concentra­ Botanica Science. 32: 172-177. tion in poplar by transformation with Agrobacterium T­ Wang, Y.C.; Tuskan G.A.; Tschaplinski T.J. 1994. DNAgene ipt. Tree Physiology. 14:27-35. Agrobacterium tumefaciens-media ted transformation Stachel, S.E.; Messens, E.; Van Montagu, M.; Zambryski, P. and regeneration of Populus. In: Michler, C.H.; 1985. Identification of the signal molecules produced Becwar, M.R.; Cullen, D.; Nance, W.L.; Sederoff, R.R.; by wound plant cells which activate the T-DNA trans­ Slavicek, J.M., eds. Applications of biotechnology to fer process in Agrobacterium tumefaciens. Nature. 318: tree culture, protection, and utilization; 1994 Octo­ 624-629. ber 2-6; Bloomington, MN, U.S.A. Gen. Tech. Rep. Tsai, C.-J.; Podila, G.K.; Chiang, V.L. 1994. Agrobacterium­ NC-175. St. Paul, MN, U.S.A.: U.S. Department of mediated transformation of quaking aspen (Populus Agriculture, Forest Service, North Central Forest tremuloides) and regeneration of transgenic plants. Plant Experiment Station: 22. Abstract. Cell Reports. 14: 94-97. Weigel, D; Nilsson, 0. 1995. A developmental switch suf­ Tuominen, H.; Sitbon, F.; Jacobsson, C.; Sandberg, G.; ficient for flower initiation in diverse plants. Nature. Olsson, 0.; Sundberg, B. 1995. Altered growth and wood 377: 495-500. characteristics in transgenic hybrid aspen expressing Zambryski, P.; Tempe, J.; Schell, J. 1989. Transfer and func­ Agrobacterium tumefaciens T-DNA indoleacetic acid-bio­ tion ofT-DNA genes from Agrobacterium Ti and Ri plas­ synthetic genes. Plant Physiology. 109: 1179-1189. mids in plants. Cell. 56: 193-201.

59 USDA Forest Service Gen. Tech. Rep. RM-GTR-297. 1997.

L