Optimization of parameters for Agrobacterium mediated transformation of black gram (Vigna mungo L. Hepper) using cotyledon explants

Abstract

Agrobacterium tumefaciens strain LBA4404 harbouring binary vector pCAMBIA 2301, which contains a neomycin phosphotransferase gene (nptII) and a β-glucuronidase (GUS) gene (uid A) was used for transformation of Vigna mungo cotyledon derived calli. Wounding of explants before infection, osmotic effects of infection and cocultivation media had an effect on the competence of the tissue as well as transforming ability of Agrobacterium cells. Transient GUS expression studies revealed that a cell density of 10⁸ cells/ml, 100 μM acetosyringone and 330 μM cysteine were effective in increasing the transformation frequency and obtaining stable transformants with a 3.8% transformation efficiency. IBA pulse treatment was effective in root induction of kanamycin selected putative transformants. Molecular analysis using polymerase chain reaction (PCR) of nptII gene confirmed the transgenic nature of T₀ transformants.

Key words: Black gram, genetic transformation, Agrobacterium tumefaciens, cotyledon.