Protocol for efficient plant regeneration and Agrobacterium tumefaciens mediated genetic transformation of pigeonpea [Cajanus cajan (L.) Millsp.]

Abstract

A simple, efficient, reproducible and genotype independent high frequency plant regeneration protocol has been developed from cotyledonary node explants from 12-d-old in vitro raised pigeonpea seedlings cultured on shoot induction medium [Murashige and Skoog (MS) medium + 2.0 mg L-1 benzyladeninej. Shoot-buds originated from the cut ends of the cotyledonary node explants and multiple adventitious shoots developed from 80% of the explants. They elongated rapidly on shoot elongation medium comprising the MS medium supplemented with 0.5 mg L-1 gibberellic acid-A, rooted on MS medium supplemented with 0.5 mg L-1 Indole butyric acid (IBA). The survival rate of the in vitro regenerated plantlets was over 70%. The cotyledonary node explants were co-cultivated with Agrobactcrlum tumefaclens strain C-58 harboring the binary plasmid, pCAMBIA1301 [conferring glucuronldase (GUS) activity and resistance to hygromycinj, cultured on selection medium containing hygromycin to select putatively transformed shoots and rooted. About 24 putative TO transgenic plants have been produced and the stable expression and Integration of the transgenes was confirmed by GUS assay, PCR and Southern blot hybridization with a transformation efficiency of over 45%.