GENETIC TRANSFORMATION OF BRASSICA JUNCEA WITH ANTIMICROBIAL WASABI DEFENSIN GENE

Abstract

Brassica juncea is one of the most important oilseed crop. An efficient and reproducible protocol was developed for Agrobacterium-mediated transformation of Brassica juncea variety NIFA RAYA with Wasabi defensin gene to produce transgenic plants. This gene was isolated from the leaves of Wasabia japonica. The expression of Wasabi defensin gene; encoding antimicrobial protein, in plasmid pEKH-WD is driven by the constitutive 35S promoter. The role of a number of factors such as choice of explants, the age of explants, different ratios of growth regulators, various concentrations of growth hormones and chemicals which can directly or indirectly influence the process of transformation was evaluated. Hypocotyls and cotyledons from 4-7 days old seedlings, when used as explants for transformation, were found to be the best in terms of producing transgenic calli and shoots. Regeneration of the explants on solidified MS plates supplemented with different hormone ratios and concentrations showed that medium containing 2 mgL-1 BAP and 0.2 mgL-1 NAA was the best for callus initiation. Whereas, 3 mgL-1 BAP and 0.3 mgL-1 NAA was found to be the best hormone combination for callus formation. For shoot regeneration 3 mgL-1 BAP and 0.5 mgL-1 NAA suplemented with 20 µM AgNO3 was the best combination. The transgenic nature of the transformed callus and regenerated shoots was confirmed via PCR analysis by using Wasabi defensin gene specific primers against their isolated genomic DNA.