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Micropropagation, Genetic Engineering, and Molecular Biology of Populus

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Micropropagation, Genetic Engineering, and Molecular Biology of Populus 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).
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