Agrobacterium-mediated genetic transformation of pigeon pea (Cajanus cajan (L.) Millsp.) using embryonal segments and development of transgenic plants for resistance against Spodoptera

Efficient transformation of embryonal segments of pigeon pea (Cajanus cajan (L.) Millsp.) was obtained by using Agrobacterium tumefaciens strain GV2260 harboring a modified binary vector pPK202 carrying the marker gene neomycin phosphotransferase II (npt II) and a synthetic cry I E-C gene under a constitutive 35 S promoter. Shoots developed on Agrobacterium treated explants were selected on MS medium supplemented with 2.0 mg l−1 BAP, 250 mg l−1 cefotaxime and 75 mg l−1 kanamycin. Elongated kanamycin resistant shoots were subsequently rooted on MS medium supplemented with 1.0 mg l−1 NAA and later transferred to sterile vermiculite followed by transfer to the transgenic green house. Integration of T-DNA into nuclear genome of transformed plants and its sexual transmission to the progeny of the transgenic plants were confirmed by PCR amplification of 700 bp npt II fragment and Southern blot hybridization analysis using the PCR amplified npt II fragment as probe. In vitro insect bioassay using Spodoptera litura larvae of first and second instar stages on T1 and T2 plants shows that the expression of the synthetic cry I E-C in transgenic pigeon pea plants confers protection against the insect larvae. Western analysis showed a 71.5 kDa band confirming the presence of cry I E-C protein in the T1 and T2 transgenic plants. This protocol allows effective transformation and quick regeneration of insect resistant transgenic plants of pigeon pea.