agrobacterium-mediated transformation of populus species · 2012-10-19 · nonsexual gene transfer...
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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.
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This file was created by scanning the printed publication.Errors identified by the software have been corrected;
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Section II Transformation and Foreign Gene Expression
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 natural host for Agrobacteriu111 for many years. DeCleene and 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 for 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 transfer foreign genes into the poplar genome.
The process for prod ucing transgenic pop lar plants includes 5 main components (figure 2): 1) initia tion: starting explants (host species/genotype/ tissue type) a re selected
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for infection and transformation; 2) infection: wounded starting explants are co-cultiva ted with 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
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 for this chap ter (table 1). These genes include the: 1) mutant aroA gene, which encodes glyphosate tolerance via a 5-enolpyruylshikimate-3-phosphate synthase (EPSP) tha t is less sensitive to the herbicide g lyphosa te (Donahue et al. 1994; Fillatti et al. 1987); 2) bar gene encoding the enzyme phosphinotricin 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) (Howe et al. 1994). O ther studies have focused on transgene regulation (Chun and Klopfenstein 1995; Confa lonieri et a l. 1994; Kajita et al. 1994; Klopfenstein et a l. 1991; Leple 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; Nilsson e t a l. 1996a, 1996b; Schwartzenberg 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 number of inserted transgenes by Agrobacteriu111-mediated transformation on Populus species. Transgenic microshoots of hybrid 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-
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Agrobacterium-mediated Transformation of Populus Species
••• ... ~Wounding
INITIATION ~ .:!: Dark Conditions
Field Test
t CONFIRMATION
(e.g .. Southern blot. PCR. northern hybridization . western blot. ELISA.
'I= =I' Preculture (CIM or SIM)
Greenhouse Growth
and/or enzyme activity assay)
'I = I' INFECTION
/ Co-cultivation with A. tumefaciens or A. rhizogenes .:!: Secondary selection and
In vitro propagation
I ' regeneration to avoid chimeric transformants
SELECTION REGENERATION
Decontamination _:!: preselective culture
'I.Je *' I' Selection of transformed cells
:!: Additional selection for root formation in selective media
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). 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 abi lity of the bacterium to infect the plant of interest. The effect of 2 Agrobacterium tumefaciens strains, 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 showed significan t differences among the geno types w ith in species and the clones with in gen otype (Confa lonie ri et al. 1994; De Block 1990; Riemenschneider 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 cu ltivars to A. tumefaciens was correlated to cytokinin sensitivi ty by Benedd ra eta!. (1996). In addition, intra- and inter-specific hybrid poplars coming from Aigeiros or Tacamahaca sections differed in suscep tibility to A . lumefaciens C58 strain (Riemenschneider 1990).
It is criti cal to select approp ria te starting materia ls (or 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 used most often to trans-
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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 and PIN2-CAT genes, transformants were selected on Murashige and Skoog (MS) {1962} regeneration medium supplemented with 40 1-1g/ml kanamycin.
form many Populus species. G reenwood stem internode sections of P. tremuloides are the most susceptible to tumor fo rm ation and 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 explants.
A suspension culture transformation system for inserting genes into pop lar might offer severa l advantages including: 1) the ability to screen large numbers of potentially transformed cells; 2) effective inhibition of residual Agrobacterium following co-cultivation; and 3) hig h trans formation frequencies d ue to rapi dl y dividing s uspension cultures that may be amenable to s table integration of foreign D A (Howe e t al. 1994). However, it is freque ntly unknown w hi ch cell type within an explant is the m ost transformable or the mos t capab le of regenerating into a ferti le plant. The small amount of avai lab le d a ta indicates that the most regenerable cells do no t necessarily correspond with the most transformable cells (De Block 1993).
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Figure 4. Secondary selection of transformants occurred on Murashige 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 (Klopfenstein et a l. 1991 ).
Agrobacterium Strain
To assure high infectivity levels for effective transformation, the most suitable Agrobacterium s train should be determined for each host species I genotype 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 using nopaline s trains of Agrobacterium (Han e t a l. 1996). The p lasmid rather than the chromosomal background was the most critical determinant for infection (Kubisiak et al. 1993). However, influence of plasmid type on infection levels has varied w ith host species/genotype/tissue type (Kubisiak et al. 1993).
Two designed vector systems are used in Agrobacteriummediated transformation: 1) co-integrate: T-D A includes
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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 P. trichocarpa x P. deltoides T-DNA2 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 P. davidiana 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
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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
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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.
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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