Plant Cell Reports

Plant Cell Reports (1994) 13:582-586

9 Springer-Verlag 1994

Agrobacterium tumefaciens mediated gene transfer in peanut (Arachis hypogaea L.) Susan Eapen and Leela George Plant Biotechnology Section, Bhabha Atomic Research Centre, Trombay, Bombay - 400085, India Received 26 August 1993/Revised version received 18 March 1994 - Communicated by G. C. Phillips

ABSTRACT. Transgenic peanut plants were produced using Agrobacterium mediated gene transfer. Primary leaf explants of peanut were co-cultivated with Agrobacterium tumefaciens LBA 4404 harbouring the binary plasmid pBI 121 (conferring [3-glucuronidase activity and resistance to kanamycin) and cultured on regeneration medium supplemented with kanamycin to select putatively transformed shoots. They were rooted and plants were transferred to soil. Stable integration and expression of the transgenes were confirmed by NPT II assay, Southern blot hybridization and GUS assay. Abbreviations: BA, 6-benzyladenine; GUS, 13-glucuronidase; IAA, indole-3-acetic acid; NAA, a-naphthaleneacetic acid; NOS, nopaline synthase; NPT II, neomycin phosphotransferase II; SDS, Lauryl sulfate INTRODUCTION Peanut, Arachis hypogea L., has long been the focus of conventional plant breeding efforts because of its importance as a source of high quality oil and protein. Although conventional methods have led to the improvement of this crop, recent genetic engineering techniques can be used as an additional tool for introduction of agronomically useful traits into established cultivars. Modification of peanut using genetic engineering techniques would facilitate rapid development of new varieties with traits such as disease resistance or seed quality improvement. Agrobacterium mediated transformation has been used to develop transgenic leguminous crops such as mothbean (Eapen et aL 1987), soybean (Hinchee et al. 1988), pea (De Kathen and Jacobsen 1990; Puonti-Kaerlas et al. 1990) and Vicia narbonensis (Pickhardt et al. 1991). Although the susceptibility of peanut to Agrobacterium has been demon-

Correspondence to: S. Eapen

strated earlier (Dong et al. 1990; Lacorte et al. 1991; Mansur et al. 1993), transgenic plants have not yet been reported. In the present communication, we report regeneration of primary transgenic plants from leaf discs of peanut cocultivated with Agrobacterium tumefaciens. MATERIAL AND METHODS Plant material. Seeds of peanut (Arachis hypogaea L. fastigata type) cv TAG-24 (Nuclear Agriculture Division, BARC) were sterilised with 0.1% mercuric chloride for 7-8 min, followed by thorough rinsing with sterile water. Culture conditions and transformation. The seeds were germinated on MS basal medium (Murashige and Skoog 1962). Leaf discs were isolated from 9-10 d-old seedlings._Agrobacterium tumefaciens strain LBA 4404 (Hoeckema et al. 1983) containing the binary plasmid pBI 121 was used, which included the GUS reporter gene (Jefferson et al. 1987) driven by the CaMV 35S promoter and the neomycin phosphotransferase II (NPT I~ gene under the control of the nopaline synthase promoter (Fig. 1). Explants were treated with 48 h old bacterial suspension (grown on LB broth, approximately 108 cells/ml) in plastic petri dishes for 5 min and then co-cultivated for 3 d on MS (Murashige and Skoog 1962) agar medium containing 2 mg/l BA and 0.1 mg/l IAA. After co-cultivation, the explants were washed thoroughly with distilled water, blotted dry on a filter paper, and transferred to fresh medium of the same composition but supplemented with 500 mg/l carbenicillin and 50 mg/l kanamycin sulfate. After 3-4 weeks, explants showing regeneration were transferred to medium supplemented with 1 mg/! BA, 0.1 mg/1 IAA, 75 mg/l kanamycin sulfate and 250 mg/1 carbenicillin. Well developed shoots were excised and cultured on medium supplemented with 0.1 mg/l of BA + kanamycin sulfate (100 rag/l) for 30-40 d and transferred to half-strength MS medium containing 0.2 mg/l NAA for induction of roots. Plants with well developed roots were transplanted to paper cups and later to pots in the glasshouse. GUS assay. GUS enzyme activity in regenerated shoots was assayed histochemically in unfixed material (Jefferson 1987; Stompe 1992). The material was incubated in potassium ferricyanide (5 raM), potassium ferrocyanide (5 mM), 5-bromo-4-chloro-3-indolyl B-D

583

~:NP__T Nos I Pro [

II (Kan R) ~ H Nos lCaMV 35S Ter ! Pr~

Pstl

Nos Te r

Pstl

Fig. 1. T DNA region of pBI 121 plasmid showing NPT I1 and g u s A genes. The 1.9 kb Pst 1 fragment was used as one of the probes for Southern hybridization.

Hindlll

Bglll

Ncol;Xbal;BamHI

Sacl

EcoRI

35S-35S

I 620 bp

44 bp

2-.7 kbp

Ncol

250 bp ~

Fig. 2. pBI 426 plasmid. The Hin dl]l/Eco R1 fragment (3.7 kb) from this plasmid was used as one of the probes in Southern blots with genomic DNA from the transformed plants. glucuronide (0.3 %/v), sodium phosphate buffer (0.1 M pH 7.0), Triton-100 (0.06 % v/v) at 37 ~ C overnight and the tissue was cleared in 70 % alcohol before observation. N P T II assay. The expression of the NPT 1I gene was confirmed by measuring the NPT II activity in the regenerated plants by in situ gel assay (Reiss et al. 1984) as modified by Van den Broeck et al. (1985). Southern blot hybridization. DNA was isolated from 1-2 g of leaf tissues from putatively transformed plants grown in pots by the method of Della Porta et al. (1983), processed by phenol: chloroform: isoamyl alcohol treatment and the DNA precipitated with sodium acetate and ethanol. For Southern hybridization 10-15 ixg of DNA was digested overnight with Pst-1, electrophoresed in 0.8 % agarose gel and blotted onto positively charged nylon membrane as described by Sambrook et aL (1989). For isolation of the probe, plasmid pBI 426 (Plant Biotechnology Institute, NRC, Canada) having GUS: NPT II fusion gene (Datla et al. 1990) with a double CaMV 35S promoter and NOS-terminator was restriction digested with Hin dl/I-Eco RI (Fig. 2) and the corresponding 3.7 kb fragment was used as the probe. In another set of experiments, the 1.9 kb Pst-1 fragment isolated from pBI 121 (Fig. 1) was used as the probe. DNA isolated from different plants were used for hybridization with the two different probes. The labelled probe was prepared by random priming using 32P dATP and 32P dCTP (BR1T, Bombay). Hybridization was carried out at 42 ~ C for 48 h and blots washed thrice in solution containing NaCI, Tris and EDTA for 5 min each at room temperature. This was followed by two washes of 30 min duration at 65 ~ C in the same solution, but supplemented with SDS. The membrane was washed twice at room temperature in solution containing Tris, NaC1 and EDTA for 30 rain, and finally exposed to X-ray film at -70 ~ C with two intensifying screens for 2 d for autoradiography.

RESULTS AND DISCUSSION Leaf discs co-cultivated with A. tumefaciens were cultured on MS me(tim supplemented with BA (2 rag/l) + IAA (0.1 rag/l) + kanamycin (50 mg/l). Shoot buds developed from the cut end of the leaf discs at the region of the mid-vein (Fig. 3A). An average of 6.7 % of leaf discs regenerated shoot buds in presence of kanamycin (Table I). The control explants did not show any shoot regeneration in kanamycin supplemented medium while on a medium without kanamycin 33 % of explants produced shoot buds (Table 1, Fig. 3A). The putative transformed shoots were transferred to medium supplemented with BA (1 mg/l) + IAA (0.1 mg/l) + kanamycin sulfate (75 mg/1) for another 3 weeks (Fig. 3B), and later maintained on BA (0.1 rag/l) + kanamycin sulfate (100 mg/1) until they attained a height Table 1. Frequency of putatively transformed shoot regeneration from peanut leaf discs, co-cultivated with A. tumefaciens and cultured on MS + BA (2 rag/l) + IAA (0.1 mg/l) + kanamycin sulfate (50 mg/l). No. of No. of Shoot Average n explants explants induction of shoot cultured regenerating frequency buds/cultur shoots (%) +SE Control

66

22

33.3

7.0 + 1.8

Control Expt. 1 Expt. II Expt. 1II Expt. IV

96 260 192 304 206

0 20 11 20 15

0 7.6 5.7 6.5 7.2

0 1.4 + 0.1 1.3 +_ 0.2 1.3 +_ 0.1 1.3 + 0.2

No co-cultivation, no kanamycin selection. ** No co-cultivation, kanamycin selection *

584

Fig. 3A. Regeneration of shoots from leaf discs of peanut cultured on BA (1 rag/l) + IAA (0.1 mg/l), at the end of 3 weeks alter cultul'e induction. a. Control leaf discs (without co-cultivation with Agroabacterium) showing regeneration of shoot buds. b. Control leaf discs (without co-cultivation with Agrobacterium) showing complete suppression of regeneration in the presence of 50 mg/l kanamycin sulfate. c. Leaf discs co-cultivated with A. tumefaciens and grown on kanamycin sulfate (50 mg/1). One of the leaf discs is showing shoot regeneration. Fig. 3B. A putatively transformed peanut shoot growing on BA (1 rag/l) + NAA (0.1 mg/l) + kanamycin sulfate (75 mg/l). Fig. 3C. A putatively transformed peanut plant with well developed roots. Fig. 3D. A putatively transformed peanut plant growing in pot 4 months alter transfer. Fig. 3E. Histochemical location of GUS activity in regenerating shoots alter infection of leaf explants with A. tumefaciens LBA 4404/pBI 121. Dark areas represent the blue coloured tissues.

of 2-3 cms. Shoots were then transferred for rooting to half-strength MS medium supplemented with NAA (0.2mg/l) (Fig. 3C). Forty plants were selected and these were transferred to soil. The survival rate was only 50 % (Fig. 3D). Although the plants flowered, only 3 plants set seeds which were shriveled in nature. Histochemical assay of selected shoots for GUS activity demonstrated the presence of blue coloured cells typical of the gus A gene (Fig. 3E). However, only 6/15 shoots tested were GUS positive. Leaf tissues from control plants did not show GUS activity. Expression of

NPT II marker was studied in shoots selected for kanamycin resistance. The selected shoots proved positive for NPT II assay (Fig. 4A). Genomic DNA from 15 regenerated plants was isolated, digested with Pst-1 and hybridized with the Hin dIII-Eco R1 fragment of NPT II-GUS fusion gene (Fig. 2) from plasmid pBI 426. Hybridization was observed in all the transformed plants, but not in DNA from the control plant (Fig. 4B). Transformed plants showed a faint 1.9 kb fragment, which is the expected size of NPT II gene from genomic DNA digested with Pst-I (Fig.

585

+ Fig. 4A. NPT II activity in transformed peanut plant. Lane 1 is positive control which was an extract from progeny of a transformed kan R tobacco germinated on kanamycin supplemented medium. Lane 2 is an extract from non-transformed regenerated peanut plant and Lane 3 from a transformed peanut plant. Fig. 4B. Southern blot analysis of DNA from transformed peanut plants. Total genomic DNA was digested with restriction endonuclease Pst I, separated by agarose gel electrophoresis, blotted onto a nylon membrane and hybridized with Eco Rl/Hin dill fragment from plasmid pBI 426 having NIT 1] and GUS fusion genes. Lane C - Control (non-transformed) regenerated peanut plant. Lanes 1-7 - DNA from transformed plants. Faintly hybridizing bands are seen at 1.9 kb in the transformed lanes. Higher molecular weight fragments are seen in the transformed lanes, but not in the control. The variations i n hybridization intensity in the positive lanes are due to unequal DNA loading.

4B). In addition, the genomic DNA of transformed plants also contained additional high molecular weight fragments ranging in size from 8 to 2.5 kb. Because the probe contained both NPT II and GUS genes, these higher molecular weight fragments may represent hybridization to the gus A gene. When Southern hybridization was carded out using the Pst-I fragment from pBI 121 as a probe, the expected 1.9 kb fragment was observed in the genomic DNA of transformed plants (Fig. 5). No high molecular weight fragment was observed in this case. However, genomic DNA from different plants were used for hybridization with the two (pBI 426 and pBI 121) probes.

Fig. 5. Southern blot analysis of DNA from transformed peanut plant using 1.9 kb Pst I probe from PBI 121. C Control (non-transformed) peanut plant. T Transformed plant showing a band at the expected 1.9 kb site.

In the present study, expression of GUS activity in the regenerated shoots was not directly correlated with kanamycin resistance. The lack of expression of the gus A gene in kanamycin resistant shoots may be due to the alteration or loss of gus A gene. Ying et al. (1992) using the same plasmid in Carthamus, and Ottaviani et al. (1993) in potato, found expression of gus A gene only in some of the kanamyein resistant calli. In mothbean, only 23 % of the kanamycin resistant protoclones showed the expression of the co-transferred nopaline synthase gene (Eapen et al. 1987). The copy number and location of insertion and subsequent rearrangements can significantly affect expression level of the gene (Battraw and Hall 1990). Rearrangements could result in the loss of part or all of the coding sequence. Methylation of the GUS reporter gene is also known to alter gene expression in potato (Ottaviani et al. 1993). In peanut, primary leaf tissues are known to directly differentiate shoot buds (Seitz 1987; McKently et al. 1991; Eapen and George 1993). In the present study, we have used primary leaf explants for obtaining peanut transformants, similar to model plants like tobacco, petunia and tomato (Horsch et al. 1985). In other grain legumes such as mothbean, mesophytl protoplasts (Eapen et al. 1987) have been used to obtain transgenic plants, while cotyledonary nodes have been used in soybean (Hinchee et al. 1988). Shoot tips and epicotyl segments were used in K narbonensis (Pickhardt et al. 1991) and pea (Puonti Kaerlas et al. 1990, De Kathen and Jacobsen 1990) for Agrobacterium mediated gene transfer. Transgenic plants have been obtained by direct DNA transfer using particle gun bombardment in soybean (McCabe et

586 al. 1988) while PEG-mediated and electroporation techniques were used in mothbean (Kohler et al. 1987 a,b).

Particle bombardment technique has recently been evaluated for transformation of beans, peanuts and other grain legumes as well (Yang 1993). In peanut, whole embryos have been used for particle gun bombardment (Sclmall and Weissinger 1993) and GUS positive loci were obtained. To our knowledge, the present study represents the first report of transgenic plants of peanut. In the present study, 20 transgenic plants were grown in pots. Although all of them have flowered, so far only 3 plants have set seeds. These seeds are shriveled and have not shown germination. Many of the control leaf regenerants also showed poor seed-setting. Further studies are in progress to obtain a large number of transgenic plants, so that at least some of them will produce viable seeds for studies on heritability. ACKNOWLEDGEMENTS. The authors thank Dr. CR Bhatia, Secretary, DBT and Dr. PS Rao for their keen interest in the work; PVAL Ratnakar, P Viegas, AS Bhagwat and HS Mishra for help during various stages of experiments and for useful discussion; SV Pawar and RM Mudliar for photography. We are grateful to NRC, Canada for the plasmid pBI 426.

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