Tissue Culture- and Selection-Independent Agrobacterium tumefaciens -Mediated Transformation of a Recalcitrant Grain Legume, Cowpea ( Vigna unguiculata L. Walp)

A simple and generally fast Agrobacterium -mediated transformation system with no tissue culture and selection steps has been developed for the first time in a recalcitrant food legume, cowpea. The approach involves wounding of 1-day-old germinated seeds with a needle or sonication either alone or in combination of vacuum infiltration with A. tumefaciens EH105 (pCAMBIA2301) carrying a β-glucuronidase (GUS) gene ( uidA ) and a neomycin phosphotransferase ( nptII ) gene for stable transformation. Sonicated and vacuum infiltrated seedlings showed the highest transient GUS activity in 90% of the explants. The sprouted co-cultured seeds directly established in soil and without selection were allowed to develop into plants which on maturity produced T 0 seeds. The presence of the alien genes, nptII and uidA in T 0 plants and their integration into the genome of T 1 plants were confirmed by polymerase chain reaction (PCR) and Southern blot analyses, respectively. The transgenes were inherited in the subsequent T 2 generation in a Mendelian fashion and their expression was confirmed by semi-quantitative PCR. The transformation frequency of 1.90% was obtained with sonication followed by vacuum infiltration with Agrobacterium . This approach provides favorable circumstances for the rapid meristem transformation and likely makes translational research ease in an important recalcitrant food legume, cowpea.