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

Abstract

Agrobacterium-mediated transformation of Vigna radiata L. Wilczek has been achieved. Hypocotyl and primary leaves excised from 2-day-old in-vitro grown seedlings produced transgenic calli on B 5 basal medium supplemented with 5×10 −6 M BAP, 2.5×10 −6 M each of 2,4-D and NAA and 50 mg l −1 kanamycin after co-cultivation with Agrobacterium tumefaciens strains, LBA4404 (pTOK233), EHA105 (pBin9GusInt) and C58C1 (pIG121Hm) all containing β-glucuronidase ( gusA) and neomycin phosphotransferase II ( nptII) marker genes. 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, respectively. Stable integration of T-DNA into the genome of transformed calli of mungbean was confirmed by Southern blot analysis. Transgenic calli could not regenerate shoots on B 5 or B 5 containing different cytokinins or auxins alone or in combination. However, for the first time, transformed green shoots showing strong GUS activity were regenerated directly from cotyledonary node explants cultured after co-cultivation with LBA4404 (pTOK233) on B 5 medium containing 6-benzylaminopurine (5×10 −7 M) and 75 mg l −1 kanamycin. The putative transformed shoots were rooted on B 5+indole-3-butyric acid (5×10 −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 0 seeds showed GUS activity. Molecular analysis of putative transformed plants revealed the integration and expression of transgenes in T 0 plants and their seeds.