Plant Cell Reports

Plant Cell Reports (1996) 15:980-985

9 Springer-Verlag 1996

Genetic transformation of cotyledon explants of cowpea (Vigna unguiculata L. Walp) using Agrobacterium tumefaciens B. Muthukumar ~, M. Mariamma ~, K. Veluthambi 2, and A. Gnanam 3 1 Department of Plant Science, School of Biological Sciences, Madurai Kamaraj University, Madurai - 625 021, India 2 School of Biotechnology, Madurai Kamaraj University, Madurai - 625 021, India 3 Pondicherry University, Pondicherry - 605 014, India Received 29 March 1995/Revised version received 26 March 1996 - Communicated by G. C. Phillips

Abstract. Mature de-embryonated cotyledons with intact proximal end of Vigna unguiculata were cultured on B5 basal medium containing varying concentrations of BAP. Thirty-six percent of the explants produced shoots on B5 medium supplemented with 8x 10-6 M BAP. Cotyledon explants were pre-incubated for 24 h, inoculated with A. tumefac&ns pUCD2614 carrying pUCD2340, co-cultivated for 48 h and transferred to hygromycin-B (25 rag/l) containing shoot induction medium. Approximately 15-19% of the explants produced shoots on the selection medium. The elongated shoots were subsequently rooted on B5 basal medium containing hygromycin. The transgenic Plants were later established in pots. The presence of hpt gene in the transgenic plants was confirmed by Southern blot hybridization. Abbreviations : BAP: 6-Benzylaminopurine; 2,4-D: 2,4dichlorophenoxyacetic acid; hpt: h y g r o m y c i n phosphotransferase; IAA: Indole-3-acetic acid; NAA: 1-naphthaleneacetic acid. Keywords: D e - e m b r y o n a t e d c o t y l e d o n s - shoot regeneration - co-cultivation - Vigna unguiculata

Introduction Cowpea, Vigna unguiculata (L.) Walp, is an important grain legume. Gene manipulation techniques for improvement of cowpea have met with little success because an efficient and reproducible regeneration and transformation method has not yetbeen developed.

ofAgrobacteriurn - mediated gene transfer and in vitro plant regeneration has become imperative if we are to employ genetic manipulation of this important food crop in the future. Successful transformation and regeneration was accomplished in a few grain legumes using cotyledons and embryonal axes as explants (Hinchee et al. 1988; Fontanna et al. 1993; Schroeder et aL 1993). But no report is available on transformation and regeneration from such explants in cowpea. In the present report, we describe the conditions for effective transformation and regeneration of primary transformants in cowpea using cotyledons as explants.

Materials and Methods Plant materials. Seeds o f cowpea [Vigna unguiculata (L.) Walp] cv. C-152 (obtained from SPIC, Madras) were surface sterilized for 60 s with 70% ethanol and 10 min with 0.1% mercuric chloride followed by several rinses with sterile deionized water.

Culture conditions and regeneration. The seeds were germinated on B5 basal medium containing 1% agar (Gamborg et al. 1968). Mature de-embryonated cotyledon explants with the intact proximal end were excised from 2-3 d old seedlings and cultured on B5 basal medium supplemented with 2-12 • 10 -6 M BAP. All media were solidified with 0.8% agar (Qualigens Fine Chemicals, Bombay) and the medium pH was adjusted to 5.7 before autoclaving. The cultures were incubated at 25-+2~ under white fluorescent light (80-90 ~tE/m2/s) with 18 h photoperiod in 90 • 15 mm Petri dishes. The regenerated shoots (2 cm in length) were excised and rooted on B5 basal medium. Rooted plants were then transferred to pots filled with autoclaved sand and vermiculite mix ( l : l v/v) for gradual hardening, and were finally transferred to soil in pots. Histological analysis. The differentiated shoot buds were histologically analysed following the method o f Johansen (1940).

Bacterial strains. A mini T-binary vector pUCD2340 (Zyprian and Kado

Successful shoot regeneration of cowpea from cultured tissues has been reported only from shoot and root apices (Subramaniam et al. 1968; Kartha et al. 1981). Leaf explants of cowpea have been shown to be susceptible to A. tumefaciens transformation (Garcia et al. 1986, 1987). Even though shoot regeneration was reported from leaf explants of cowpea (Pandey and Bansal 1989), regeneration of transgenic plants using such explants has not been reported. Therefore, an integrated effort to standardize the protocols Correspondence to: B. M u t h u k u m a r

1990), comprising T-DNA border sequences from nopaline type Ti plasmid pTiC58 flanking a chimaeric hygromycin phosphotransferase (hpO gene (Fig. 3) for selection of transformed plant cells, was introduced into LBA4301 harbouring high copy stable virulence helper A. tumefaciens plasmid pUCD2614 (Rogowsky et al. 1990) using pRK2013 by triparental mating. All the strains were kind gifts from Prof. Kado (University of California, Davis, USA). TheAgrobacterium cells were grown at 28~ in 10 ml liquid YEP medium (An et al. 1988) containing 20 mg/I each of neomycin and gentamycin. The bacterial ceils were washed and resuspended in fresh YEP medium before inoculation.

981 Transformation and selection. Cotyledon explants were preincubated in shoot regeneration medium (B5 + 8 xl0 -6 M BAP) for different time periods (0, 24, 48, 72, 96 and 120 h) and infected with bacterial suspension (approximately 5 xl08 cells/ml as measured by optical density at 600 nm) for 2 min in small Petri dishes. The infected cotyledon explants were then blotted dry using sterile Whatman 1 filter paper and co-cultivated on the same medium for different time periods (24, 48, 72, 96 and 120 h). After co-cultivation, explants were washed with sterile deionized water several times, blotted dry using sterile Whatman 1 filter paper, and transferred to shoot induction medium (B5 + 8 xl0 -6 M BAP) containing 25 rag/1 hygromycin-B and 250 mg/l eefotaxime. Putative transgenie shoots were rooted in the presence o fhygromycin (25 mg/l) and later established in pots.

proximal end of the cotyledon. Cotyledons with intact proximal end had earlier been shown to be the potential source for plantlet regeneration in legumes such as Pisum sativam (Ozcan et al. 1992), Cajanus cajan (Mehta and Mohan Ram 1980), Glycine max (Cheng et al. 1980), Vigna mungo (Gill et al. 1987), Vigna radiata (Gulati and Jaiwal 1990), Arachis hypogaea (McKently et aL, 1990) and Phaseolus vulgaris (Franklin et al. 1991). But in most of the above observations shoot regeneration was from the cotyledonary node.

DNA isolation and Southern hybridization. Total genomic DNA was isolated from young shoots (4-5 g) of transgenic plants and from untransformed control plants grown in test-tubes according to the procedure described by Rogers and Benedich (1988). Eight to 10 tig of restriction digested total DNA from each sample was electrophoresed on 1% agarose gel. The conditions for Southern blotting and hybridization were similar to those described by Maniatis et aL (1982). The hybridization p robes prepared from the plasmid pUCD2340 were: PstI fragment of hpt gene (2.2 kb), PstI-NotI overlapping fragment of right border of T-DNA (2.5 kb), and HindlII-NotI non T-DNA fragment (2.23 kb) (Fig. 3). The probes were radiolabelled with tx-32P dCTP by Nick translation technique (Amersham

The explants showed bud formation when cultured on B5 basal medium containing 8 x 10 -6 M BAP. When the BAP concentration was higher than 1.5 x l 0 -5i M, regeneration decreased (Table 1), which indicated that lower concentrations of BAP induced better regeneration of shoot buds. Multiple shoot regeneration with minimal callus production facilitated plantlet regeneration in the shortest time. The regenerated plants were ready for transplantation to pots within 60-80 d of culture. Eleven plants survived out of 25 plants transferred to pots, for a survival rate of 44%. Seven plants set seeds (Fig. 1B) but only about 4% of the seeds showed germination. Additional work is needed to improve the fertility of regenerated cowpea plants as well as viability of progeny.

ki0. Results and Discussion

Regeneration, Mature de-embryonated cotyledons excised from 2-3 d old in vitro grown seedlings were cultured on B5 basal medium containing 2-12 x 10 -6 M BAP. Shoot formation was observed from the callus formed at the proximal end of the cotyledon explants within 15-20 d of culture (Fig. 1A). Nearly 36% of the explants produced Table 1. Shoot regeneration from mature cotyledon explants ofcowpea cv. C-152 after 4-5 weeks in culture on B5 basal medium supplemented with varying concentrations of BAP. Concentration of BAP (x 10-6 M) 2 4 5 6 8 10 12

% Cotyledons showing Mean no. of shoots/ shoot induction -+ SE cotyledon -+ SE 2.9 -+ 0.2 7.5 -+ 04 18.4 -+ 0.5 27.5 +- 0.9 36.0 -+ 0.2 31,2 -+ 0.5 26.1-+ 0.3

1.9 -+ 0.5 2.1 -+ 0.3 4.3 -+ 0.6 4.0 -+ 0.2 5.3 -+ 0.7 3.7-+ 0.l 1.0-+ 0.2

Each value represents mean-+ SE of 4 replicates each with 50-60 explants.

shoots on B5 medium containing 8 x 10-6 M BAP (Table 1). In total 50:60 shoots were produced. The morphology of the regenerated shoots was normal. The time taken for cotyledonary explants to produce complete plantlets was 4-6 weeks. Shoots always arose from the minimal callus produced at the proximal end and were never observed to develop from the distal end. Excised whole cotyledons with the intact proximal end without cotyledonary node tissue regenerated well in our studies apparently by adventitious shoot organogenesis (Fig. 2). When the cotyledons were sliced into two equal parts by longitudinal or transverse cuts, the proximal end of longitudinally sliced halves showed better regeneration than the transverse halves containing

Transformation. D e v e l o p i n g an Agrobacterium based transformation system for cowpea involved identifying conditions to induce Agrobacterium to transform without subsequently interfering with the regeneration of transformed cells. Transformation rate was calculated based on the number of explants which regenerated shoots on selection medium containing 25 mg/l of Hygromycin-B relative to the total number of explants co-cultivated. The effect of parameters such as pre-incubation period and cocultivation time on transformation frequency were evaluated. Cotyledon explants were pre-incubated for 0, 24, 48, 72, 96 and 120 h and transformation frequency was calculated by their shoot-forming ability in the selection medium. Pre-incubation for 24 h resulted in 17% of the explants producing shoots on the selection medium. If the cotyledon explants, after excision from the seedlings, were immediately co-cultivated without pre-incubation, only 10% of the explants produced shoots in the selection medium. Pre-incubation of cotyledons for 48, 72, 96 and 120 h produced 12, 8, 5 and 2% shoots, respectively (Fig. 4), in selection medium. Recent success in the production of transgenic plantsusingAgrobacterium in soybean (Hinchee et al. 1988), pea ( D e k a t h e n and J a c o b s o n 1990; Puonti-Kaerlas et al. 1990, 1992; Schroeder et al. 1993) and chick pea (Fontanna et al. 1993) were based on explants like embryonal axes, epicotyls and cotyledons. Even though Garcia et al. (1986) was able to show that cowpea plants are amenable to Agrobacterium-mediated transformation, no transgenic plants were recovered from the transformed tissues which were derived from leaves. From our results it

982

Fig. 1. Regenerationof shoots from cotyledonexplants of cowpea. (A) Callus and shoot formationafter 15-20 d of culture on/35 basal mediumcontaining 8 x 10.-6M ]3AP. Scale bar = 0.5 cm. (]3) Establishedregeneratedcowpeaplant showingpod development. is apparent that transgenic cowpea plants can be routinely produced using cotyledon explants, An important factor which enhanced the transformation efficiency of cowpea was the 24 h preculture of the explants, which probably served to reduce wound stress and increased the number of competent cells at the wound site (Schmidt and Willmitzer 1988; McHughen et al. 1989; Akama et al. 1992).

plants survived out of 17 plants established in pots, for a survival rate of only 35%. Even though 4 plants set seeds, none of them showed germination.

Analysis of the transformed plants. The integration of hpt gene in the DNA of transgenic plants was confirmed by

Another factor which can improve the transformation frequency is the optimization of the co-cultivation time. Pre-incubated c o t y l e d o n explants inoculated with A. tumefaciens were co-cultivated for 24, 48, 72, 96 and 120 h, After a 48 h co-cultivation period about 19% of the c o t y l e d o n explants produced shoots on hygromycin containing medium, while cotyledons co-cultivated for 24, 72, 96 and 120 h, respectively, produced 12, 14, 8 and 2% shoots on selection medium (Fig. 5). Longer than 72 h co-cultivation time resulted in bacterial overgrowth which reduced the transformation frequency. Most transformation experiments involve the use of a 2 or 3 d co-cultivation time, as in tobacco (Horsch et al. 1995) and leaf explants of cowpea (Garcia et al. 1986).

Establishment of transgenic plants. After optimizing the p a r a m e t e r s described, transformation was routinely performed to obtain transgenic plants. Cotyledons with intact p r o x i m a l end from 2-3 d old seedlings were preincubated for 24 h, inoculated with 5 • 108 cells/ml of bacterial suspension (120 s), co-cultivated for 48 h and transferred to hygromycin containing shoot induction medium (]35 + 8 • 10 -6 BAP + 25mg/l Hygromycin-B). Approximately 15-19% of the explants produced shoots on selection medium. The elongated shoots were subsequently rooted on B5 basal medium containing hygromycin (25 mg/l). Later, complete plantlets were transferred to pots. Six

Fig. 2. Histologicalanalysisof the cotyledonexplantsof cowpea(cv.C152). Cross section of shoot bud organisation and emergenceof shoots after 20 d of culture in shootinduction medium(135+ 8 x 10-6 M BAP). Bar represents0.2 mm. Southern blot hybridization analysis. Total DNAs isolated from transgenic plants were digested with PstI and probed with 2.2 kb PstI fragment of pUCD 2340 containing hpt gene. DNA isolated from transgenic shoots hybridized with PstI fragment and showed the signal at the expected size of 2.2 kb, indicating that the T-DNA of pUCD2340 was present

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Genetic transformation of cotyledon explants of cowpea (Vigna unguiculata L. Walp) using Agrobacterium tumefaciens.

Mature de-embryonated cotyledons with intact proximal end of Vigna unguiculata were cultured on B5 basal medium containing varying concentrations of B...
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