Plant Cell Reports

Plant Cell Reports (1996) 1 5 : 6 5 3 - 6 5 7

9 Springer-Verlag 1996

Production of fertile transgenic peanut (Arachis hypogaea L.) plants using Agrobacterium tumefaciens Ming Cheng 1, Robert L. Jarret 2, Zhijian Li t, Aiqiu Xing 1, and James W. Demski 1 1 D e p a r t m e n t o f Plant Pathology, Georgia Station, University o f Georgia, 1109 Experiment Street, Griffin, Georgia 30223, U S A 2 U S D A / A R S , Plant Genetic Resources, Georgia Station, 1109 Experiment Street, Griffin, Georgia 30223, U S A Received 17 August 1995/Revised version received 8 N o v e m b e r 1995 - C o m m u n i c a t e d by G. C. Phillips

Abstract. Fertile transgenic plants of peanut (Arachis hypogaea L. cv. New Mexico Valencia A) were produced using an Agrobacterium-mediatedtransformation system. Leaf section explants were inoculated with A. tumefaciens strain EHA105 harboring the binary vector pBI121 containing the genes for /3-glucuronidase (GUS) and neomycin phosphotransferase II (NPTII). Approximately 10% of the shoots regenerated on selection medium were GUS-positive. Five independent transformation events resulted in the production of 52 fertile transgenic peanut plants, On average, 240 d were required between seed germination for explant preparation and the production of mature T 1 seed by T O plants. Molecular analysis of transgenic plants confirmed the stable integration of the transgenes into the peanut genome. GUS expression segregated in a 3:1 Mendelian ratio in most T 1 generation plants.

Abbreviations: GUS,/3-glucuronidase; NPTII, neomycinphosphotransferase II; MS medium, Murashige and Skoog medium (1962); BA, Nt-benzyladenine; NAA, 1-naphthaleneacetic acid.

Introduction

Agrobacterium-mediatedplant transformation is one of the most effective methods for the genetic engineering of dicotyledonous (Gasser and Fraley 1989) and monocotyledonous (Gould et al. 1990, Chart et al. 1993, Hiei et al. 1994) plants. The transfer of foreign genes into the genome of peanut ealli was previously achieved when seedling hypocotyl explants were co-cultivated with Agrobacterium (Laeorte et al. 1991, Mansur et al. 1993, Franklin et al. 1993). More recently, primary transgenic peanut plants were recovered from cultured seedling explants using Agrobacterium (Cheng et al. 1994, Eapen and George 1994). Transgenic peanut plants have also been produced by microprojectile bombardment (OziasCorrespondence to." J. W. Demski

Akins et al. 1993) and ACCELL technology. In all previous instances, fertile transgenic progeny were reported only when ACCELL technology was used (Brar et al. 1994). This study describes a protocol for the transformation of peanut and the production of fertile transgenic peanut plants by Agrobacterium-mediatedtransformation of leaflet explants. This protocol allows the production of mature transgenic Tz seed in about 240 d.

Materials and Methods Plant materials. Seed of Arachis hypogaea L. cv. NM Valencia A was kindly provided by Borden Peanut Company, Portales, New Mexico.

Basal medium, explant preparation, Agrobacterium culture and transformation. The basal medium (MSB) was composed of MS salts, B5 vitamins (Gamborg et al. 1968), 3% (w/v) sucrose, and 0.8% (w/v) Bacto-agar. The pH of the medium was adjusted to 5.8 prior to autoclaving. Leaf section explants were prepared according to Cheng et al. (1992). Agrobacterium turaefaciens strain EHA105 (Hood et al. 1986) harboring the binary plasmid pBI121 was used in all stable transformation experiments. This plasmid contained the GUS reporter gene (Jefferson 1987) driven by the CaMV 35S promoter, and the NPTII gene under the control of the nopaline synthase gene promoter (Fig. 2). Agrobacterium cultures were grown overnight at 27~ in liquid YEP medium (pH 5.2) containing 20 mg L 1 rifampicin and 50 mg L 1 kanamycin, to late log phase (ODt0o = 1.0-1.5). Bacteria were collected by centrifugation and resuspended in fresh LB medium supplemented with wounded tobacco leaf extract (20:1, v/v), prepared as described below. Wounded tobacco leaf extract was prepared using in vitro grown plants of Nicotiana tabacum (cv. Xanthi). Individual leaves were harvested, wounded by cutting fully expanded leaves into 0.3 x 0.3 cmz pieces, and incubated them overnight on MS basal medium. Extracts were prepared by grinding 2 g of leaf pieces in 2 ml sterile water and centrifuging the slurry at 1,000 x g at room temperature. The supernatant was removed and used to treat the Agrobacterium. Freshly prepared leaf sections of peanut were placed in the treated Agrobacterium solution for 10 rain, and then evacuated briefly. Agrobacterium-inoculatedexplants were blotted dry, and incubated on 25 ml of shoot regeneration (SR) medium (MSB supplemented with 1 nag L -1 NAA and 25 mg L 4 BA) in 20 X 100 mm Petri dishes for 2 d at

654 28~ in darkness. Explants were transferred to fresh SR medium supplemented with 300 mg L"~ claforan, and cultured at a 16 h photoperiod with a light intensity of 60 t~mol ra~ sL, for an additional 5d.

Plant regeneration and detection of transformedplants. Previously inoculated explants were transferred to SR medium supplementedwith 150 mg L~ kanamycin and 300 mg L~ claforan. Explants exhibiting shoot primordium formation after 30 d were subculturedto MSB with 1 mg L-~ NAA, I0 nag L 1 BA, 100 nag L~ kanamycin and 300 nag L~ claforan (SE medium) for an additional 2 months. GUS-positiveshoots were isolated from the original explants and transferred to MSB medium containing 0.5 mg L1 BA for further shoot development and proliferation. Shoots were rooted on R medium as described by Cheng et al. (1992). Putative transgenic shoots were screened for kanamycin resistance based on the ability of leaf segment explants from these shoots to produce callus on MSB supplemented with 2 mg La NAA, 0.5 mg Lt BA and 200 mg L~ kanamycin (C medium). The occurrence of callus on leaf sections within 4 weeks after transfer to C medium was taken as an indication of antibiotic resistance and transformation. Root formation on R medium supplemented with 50 mg L~ kanamycin within 21 d was taken as a further indication of transformation.

GUSexpressionassay. A binary vector carrying the GUSInt fusion gene was used to monitor transient GUS expression after Agrobacterium inoculation (Vancanneyt et al. 1990). Histochemical analyses of GUS expression were performed on tissues as described by Jefferson (1987). Tissue was stained for 3 - 5 h at 37~ Chlorophyll, if present, was removed by soaking the tissues in 95% ethanol after staining. Fluorometric GUS assays were conducted on leaf tissues using a TKO 100 Mini-Fluorometer (Hoefer Scientific Instruments). Southern blot analysis. Total genomic DNA was isolated from peanut leaves (Rogers and Bendich 1985), digested for 5 h with HindIII or EcoRI, fractionated on 1% agarose gel and blotted onto nylon membrane according to Southern (1975). Membranes were probed with a nicktranslated 32P-dATP900 bp fragment of the CaMV 35S promoter isolated from pBI121. The 35S promoter fragment was isolated from pBI121 following HindlII and BamHI digestion. Following overnight hybridization at 65~ membranes were washed for 30 rain each in 2 X SSC, 0.1% SDS; 1 X SSC, 0.1% SDS; 0.1X SSC, 1% SDS at 65~ wrapped in plastic wrap and subjected to autoradiography for 48 h at -70~

Results and Discussion Previous studies with A. hypogaea have demonstrated that young leaf tissues or zygotic embryos can be induced to regenerate plants via direct organogenesis or somatic embryogenesis (McKently et al. 1991, Cheng et al. 1992, Chengalrayan et al. 1994). The plant regeneration system previously reported by Cheng et al. (1992), that utilizes seedling leaf sections as an explant source, is suitable for peanut transformation mediated by Agrobacterium and is similar to transformation systems described for other crops (Horsch et al. 1985).

Development of a transformation protocol In preliminary experiments (data not shown), several vir gene induction treatments were evaluated for their ability

to stimulate Agrobacterium virulence (Okker et al. 1984, Schafer et al. 1987). In agreement with the report o f Mansur et al. (1993), co-cultivation o f explants with Agrobacterium previously treated with acetosyringone (Stachel et al. 1985) did not result in the production o f stable transgenic shoots. Transient GUS expression derived from the GUSInt fusion gene was enhanced in leaf segments co-cultivated for 2 d in SR medium with Agrobacterium previously treated with wounded tobacco leaf extract. GUS expression in treated explants increased proportionately with increasing periods o f co-cultivation and post co-cultivation, without selection. A 2 d cocultivation and 5 d post co-cultivation period was used in all subsequent transformation experiments. The transfer of T - D N A is mediated by the expression o f virulence genes (Zambryski 1992). Transcription o f the vir region is induced by various phenolic compounds, which are released by wounded plant host cells such as tobacco (Stachel et al. 1985, 1986). Inclusion o f acetosyringone in the co-cultivation medium has been used to enhance vir gene expression and increase the transformation efficiency o f various species including mustard (Hadfi and Bastschauer 1994), orange (Kaneyoshi et al. 1994), apple (James et al. 1993), pea (Davies et al. 1993), and rice (Hiei et al. 1994). Incorporation of acetosyringone in our Agrobacterium cultures did not significantly increase transient GUS gene expression. However, when wounded tobacco leaf extract was included in the Agrobacterium cultures, transient GUS gene expression was significantly increased. These results suggested that more than a single phenolic compound or signal molecule may be involved in vir induction and the gene delivery process. Selection on kanamycin (150 mg L -1) facilitated the screening of regenerants and resulted in a relatively small population of putative transformants for further evaluation. All GUS-positive shoots rooted in R medium containing 50 mg L -1 kanamycin. About 70% o f the leaf sections isolated from GUS-positive shoots formed callus on C medium containing 200 mg L ~ kanamycin. Table 1 summarizes the results of 5 independent transformation experiments. T h e frequency o f transformed fertile plants was 0.2 % - 0.3 % o f the leaf explants inoculated.

Expression and inheritance of the GUS gene in transgenic plants Endogenous GUS-like activity was not detected in leaf segments from non-transformed control plants. In contrast, high levels of GUS expression were observed in the leaf tissue of primary transformants (Fig. 1A). GUS activity was expressed in various organs and tissues o f both the regenerated To plants and their T1 progeny (Fig. 1, B-F). However, the intensity of GUS expression varied among the primary transformants as determined by both histochemical and fluorometrie assays (data not shown).

655

Figure. 1. GUS expression in transgenic peanut plants. A) GUS expression in leaf tissue from To transgenic plant 17-1. B) GUS expression in floral tissues of To plant 1-13. C) GUS expression in seed coat of To plant 1-13. D) GUS expression in cotyledonary tissues of seed from plant 1-13. (left, negative control). E) GUS expression in leaf tissues of transgenie T1 plant 17-2-2. F) GUS expression in stem tissues of transgenic TI plant 12-1-3.

656 Table 1. Transformation of peanut using Agrobacterium, summary of 5 independant replications .

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1 2 3 4 5 .

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325 583 370 315 332 .

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Leaf Responsive Shoots To plantsa/ sections explants after regenerated after indepeninoculated 30 d selection 90 d selection dent events

Replication .

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35 62 12 32 40 .

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9 15 3 12 10 .

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18/1 20/2 0/0 10/1 4/1 .

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fragment in DNA isolated from all GUS-positive To or T~ plants. Copy number reconstructions indicated that T1 plant 17-1-1 contained one copy of the GUS gene, while TOplant 1-13 and its progeny 1-13-1 contained at least 2 copies of the GUS gene. Southern blot analysis of additional GUS-positive TO and T~ plants confirmed that the CaMV 35S and GUS coding sequences were integrated into the peanut genome (data not shown).

Based on GUS reporter gene expression.

A 3:1 segregation ratio for GUS expression was observed in the progeny of To plants 1-4, 1-5,1-9, 12-1, 17-1 and 17-2 (Table 2). However, abnormal segregation ratios were observed in the progeny of TOplants 1-10, 113, and 1-45. These results suggest that TOplants 1-10, 113, and 1-45 may be chimeric. In addition, it is likely that transgene suppression or inactivation is accountable for a portion of the variability in GUS expression observed in some transgenic plants. The co-suppressive effects of multiple copies of a transgene were previously reported by Lima et al. (1990). Table 2. Inheritance of GUS activity in progeny of To transgenic peanut plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

TO Plant

No. ofT1 plants tested GUS+

GUS-

3:1 Segregation" x2 P

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1-4 1-5 1-9 1-I0 1-13 1-45 12-1 17-1 17-2

14 8 18 12 20 13 28 24 8

12 7 14 7 3 0 20 19 6

2 1 4 5 17 13 8 5 2

0.38 0.90 0.07 1.77 38.4 39.0 0.19 0.22 0.00

0.54 0.35 0.79 0.18 0.01 0.01 0.66 0.64 1.00

Goodness-of-fit to 3:1 ratio, df = 1.

Molecular DNA analysis DNA isolated from TO (1-13) and T 1 plants (1-13-1 and 17-1-1) derived from two independent TO(1-13 and 17-1) plants were analyzed for the presence of the CaMV 35S promoter region by Southern blot hybridization (Fig. 2). The CaMV 35S probe did not hybridize to the intact genomic DNA extracted from non-transformed plants (lanes 6 and 9). In contrast, a strong hybridization signal was observed in those lanes containing uncut DNA from the transformed TO 1-13 and T1 1-13-1 plants. This suggested integration of the CaMV 35S sequences into the peanut chromosomal DNA, in both TOand T 1 plants. Hybridization of the CaMV 35S promoter to HindIII and EcoRI digested genomic DNA from To plant 1-13 and from T1 plants 1-13-1 and 17-1-1 is shown in Fig. 2. The probe hybridized strongly to the 3.1 kb 35S-GUS-NOS3'

H

B

Production of fertile transgenic peanut (Arachis hypogaea L.) plants using Agrobacterium tumefaciens.

Fertile transgenic plants of peanut (Arachis hypogaea L. cv. New Mexico Valencia A) were produced using an Agrobacterium-mediated transformation syste...
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