Agrobacterium-mediated genetic transformation of groundnut (Arachis hypogaea L.): An assessment of factors affecting regeneration of transgenic plants

Transgenic groundnut (Arachis hypogaea L.) plants were produced efficiently by inoculating different explants with Agrobacterium tumefaciens strain LB A4404 harbouring a binary vector pBI121 containing uidA (GUS) and nptll (ne omycin phosphotransferase) genes. Genetic transformation frequency was foun d to be high with cotyledonary node explants followed by 4 d cocultivation. This method required 3 days of precultivation period before cocultivation with Agrobacterium. A concentration of 75 mg/l kanamycin sulfate was added to regeneration medium in order to select transformed shoots. Shoot regener ation occurred within 4 weeks; excised shoots were rooted on MS medium cont aining 50 mg/l kanamycin sulfate before transferring to soil. The expressio n of GUS gene (uidA gene) in the regenerated plants was verified by histoch emical and fluorimetric assays. The presence of uidA and nptll genes in the putative transgenic lines was confirmed by PCR analysis. Insertion of the nptll gene in the nuclear genome of transgenic plants was verified by genom ic Southern hybridization analysis. Factors affecting transformation effici ency are discussed.