Feasible regeneration and agro bacterium-mediated transformation of Brassica juncea with Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene

Abstract

In the present study an effort has been made to optimize the in vitro regeneration protocol for Agrobacterium-mediated transformation of Brassica juncea, because of its importance as oilseed crops. The highest callus induction frequency of 87% was observed on MS (Murashige and Skoog, 1962) medium supplemented with 4 µM 6-benzyladenine (BA) after four weeks of culture period. Subculturing of organogenic calli in MS media with a similar hormonal composition resulted in shoot organogenesis after six weeks of culture cultivation. The highest shoot induction frequency (92%) was recorded on MS medium containing 4 µM BA in combination with 1 µM of α-naphthalene acetic acid (NAA). Further, well-developed roots were formed in MS media augmented with 6 µM of Indole acetic acid (IAA) in combination with 1 µM Kinetin (Kn). Cotyledon explants were exploited in vitro for the successful transformation of B. juncea. A binary vector comprised of the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene under the transcriptional control of a glycinin promoter and with a basta selection marker was introduced into A. tumefaciens strain GV3101 via electroporation. EaDAcT gene is responsible for unusual triacylglycerol’s production where the sn-3 position is esterified with acetate instead of the long-chain fatty acid found in the triacylglycerol’s. The highest regeneration frequency (100%) of transgenic shoots was observed on MS medium supplemented with 4 µM BA plus 1 µM NAA in the presence of 25 mg l−1 basta and 160 mg l−1 timintin. The efficiency of stable transformation was found to be approximately 7% in the transgenic plants. Moreover, the transformed regenerated shoots were confirmed by PCR analysis using EaDAcT gene-specific primers. Keywords: Brassica juncea, Callus, Agrobacterium tumefaciens, Transgenic plants, Biodiesel