Plant Cell Reports (1996) 16:32-37 Plant Cell Reports © Springer-Verlag1996 Efficient transgenic plant regeneration through Agrobacterium-mediated transformation of Chickpea (Cicer arietinum L.) Sanchayita Kar *, Tony M. Johnson **, Pritilata Nayak, and S.K. Sen Plant Molecular& Cellular Genetics & Centre for Plant Molecular Biology,Bose Institute, P1/12, C.I.T. SchemeVII-M, Calcutta - 700 054, India * Present address." Department of Biochemistry& Molecular Biology,Box 9650, Mississipi State University, MS 39762, USA ** Present address: Department of Biochemistry,Collegeof Biological Sciences, 140 Gortner Laboratory, 1479 Gortner Avenue, St. Paul, MN 55108-1022, USA Received 6 July 1995/Revised version received 24 March 1996 - Communicatedby W. A. Parrott ABSTRACT: Three genotypes of chickpea ICCV-1, ICCV-6 and a Desi (local) variety were tested for plant regeneration through multiple shoot production. The embryo axis was removed from mature seeds, the root meristem and the shoot apex were discarded. These explants were cultured on medium containing MS macro salts, 4X MS micro salts, B5 vitamins, 3.0 mg/1 BAP, 0.004 mg/1 NAA, 3% (w/v) sucrose and incubated at 260C. The explants were transformed with Agrobacterium tumefaciens strain LBA4404 with binary vector pBI121 containing the uidA and nptlI genes. Multiple shoots were repeatedly selected with kanamycin. The selected kanamycin resistant shoots were rooted on MS medium supplemented with 0.05 mg/1 IBA. The presumptive transformants histochemically stained positive for GUS. Additionally, nptlI assay confirmed the expression of nptH in kanamycin resistant plants. Transgenic plants were transferred to soil and grown in the green house. Abbreviations: BAP- 6-benzylamino purine; 2,4-D - 2,4- dichlorophenoxy acetic acid; IAA- Indole acetic acid; IBA-Indole butaric acid; NAA-Naphthalene acetic acid ~TRODUCTION Chickpea (Cicer arietinum L.) is an important grain legume which has worldwide acceptance as a major source of protein for human as well as animal consumption. Much of its grain productivity is adversely affected by damages caused by a lepidopteran pest, the pod borer (Heliothis sp.). Raising insect resistant chickpea lines through conventional breeding is not feasible due to the nonavailability of any germplasm showing resistance against the damage caused by this insect (van der Have, 1970). Thus, establishment of insect resistant transgenic lines of chickpea is considered as a potential improvement strategy. One of the prerequisites for successful gene transfer to plants is the availability of a suitable protocol for transformation which is compatible with in vitro plant regeneration methods of the target plant species. In vitro plant regeneration in chickpea has been reported through organogenesis from shoot meristems (Bajaj and Dhanju, 1979; Sharma et al., 1979; Kartha et al., 1981), immatta'e cotyledons (Shri and Davis, 1992) and through embryogenesis from immature cotyledons (Sagare et at., 1993) and leaflet callus (Bama and Wakhlu, 1993, 1994; Kumar et al., 1994). Furthermore, Agrobacterium - mediated transformation in a number of legumes has been previously reported (Mariotti et aL, 1984; Owens and Cress, 1985; Jensen et aL, 1986; Bercetche et at., 1987; Hussey et aL, 1989). There are reports on transgenic plant production of other grain legumes (Vigna aconitifolia , Eapen et aL, 1987; GIycine max, Hinchee et al., 1988; Phaseolus, Mariotti et al., 1989; Pisum sativum, DeKathen and Jacobsen, 1990; Puonti-Kaerlas et al., 1990, 1992; Schroeder et al., 1993; Arachis hypogea, McKently et al., 1995). Recently, Fontana et al. (1993) have reported successful transformation of chickpea through Agrobacterium mediated genetic transformation. That report dealt with only one local variety. Though Islam et al (1994) have tested susceptibility of chickpea to Agrobacterium infection, reproducible protocols crossing the genotype barrier for gene transfer in chickpea have yet to be designed. In this communication, we report transgenic plant production of three genotypes of chickpea using the Agrobacterium - mediated gene transfer technique. This has been achieved through development of an efficient method of plant regeneration through multiple shoot formation. We believe that the reported method of plant regeneration in chickpea would be effective for other gene delivery methods, including the particle gun delivery system. Correspondence to." S. K. Sen