Agrobacterium tumefaciens-mediated high frequency genetic transformation of an Indian cowpea (Vigna unguiculata L. Walp.) cultivar and transmission of transgenes into progeny

A reproducible Agrobacterium tumefaciens-mediated genetic transformation system for the production of fertile transgenic plants of cowpea (Vigna unguiculata) that transmits transgenes into progeny in Mendelian fashion has been developed. The cotyledonary node explants excised from 4-d-old seedlings, raised in vitro on medium containing salts of Murashige and Skoog's and vitamins of Gamborg's media (MBM) and BAP (10 mu M), were co-cultured with Agrobacterium strain EHA105 harboring a binary vector pCAMBIA2301 that carried beta-glucuronidase (uidA) interrupted with an intron and neomycin phosphotransferase (nptII) genes as a reporter and a selectable marker, respectively. The green shoots recovered from Agro-infected explants on selection medium (MBM containing BAP 5 mu M, kanamycin 85 mg l(-1) and cefotaxime 500 mg l(-1)) were rooted on MS supplemented with IBA (2.5 mu M) and kanamycin (10 mg l(-1)). The rooted shoots that were found positive for the nptII gene by polymerase chain reaction (PCR) were established in soil and grown to maturity to collect the seeds. The integration of nptII gene into the cowpea genome was confirmed by Southern analysis. GUS activity was detected in vegetative and reproductive organs of T-0 plants and T-1 seedlings. The transgenes inherited in Mendelian fashion in T-1 progeny as detected by PCR. The transformation efficiency was 0.76% and 20-23 weeks were required from seed to seed generation time. This protocol can be used to introduce agronomically desired genes in cowpea for its genetic improvement. (c) 2006 Elsevier Ireland Ltd. All rights reserved.