Agrobacterium tumefaciens-mediated genetic transformation of mungbean (Vigna radiata L. Wilczek) - a recalcitrant grain legume

Agrobacterium-mediated transformation of Vigna radiata L. Wilczek has been achieved. Hypocotyl and primary leaves excised from 2-day-old in-vitro grow n seedlings produced transgenic calli on B-5 basal medium supplemented with 5 x 10(-6) M BAP, 2.5 x 10(-6) M each of 2,4-D and NAA and 50 mg l(-1) kan amycin after co-cultivation with Agrobacterium tumefaciens strains, LBA4404 (pTOK233), EHA105 (pBin9GusInt) and C58C1 (pIG121Hm) all containing beta - glucuronidase (gusA) and neomycin phosphotransferase II (nptII) marker gene s. Transformed calli were found resistant to kanamycin up to 1000 mg(.)l(-1 ). Gene expression of kanamycin resistance (nptII) and gusA in transformed calli was demonstrated by nptII assay and GUS histochemical analysis, respe ctively. Stable integration of T-DNA into the genome of transformed calli o f mungbean was confirmed by Southern blot analysis. Transgenic calli could not regenerate shoots on B-5 or B-5 containing different cytokinins or auxi ns alone or in combination. However, for the first time, transformed green shoots showing strong GUS activity were regenerated directly from cotyledon ary node explants cultured after co-cultivation with LBA4404 (pTOK233) on B -5 medium containing 6-benzylaminopurine (5 x 10 (7) M) and 75 mg l(-1) kan amycin. The putative transformed shoots were rooted on B-5 + indole-3-butyr ic acid (5 x 10-6 M) within 10-14 days and resulted plantlets subsequently developed flowers and pods with viable seeds in vitro after 20 days of root induction. The stamens, pollen grains and T(o)eeds showed GUS activity. Mo lecular analysis of putative transformed plants revealed the integration an d expression of transgenes in T-o plants and their seeds. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.