PJB-2017-527
In vitro regeneration and agro bacterium-mediated transformation of Brassica juncea with EaDAcT gene
Ijaz Naeem
Abstract
Brassica juncea is an important oilseed plant that can be used as a potential low-cost biodiesel crop. In the present study an effort has been made to optimize the in vitro growth condition for efficient regeneration and Agrobaterium mediated transformation of Brassica juncea. Indirect regeneration was observed in cotyledon explants with highest callus induction frequency (87%) observed at MS medium supplemented with 4 �M 6-benzyladenine (BA) after four weeks of culture. Sub culturing of organogenic calli in MS media with similar hormonal composition resulted in shoot organogenesis after six weeks of culture. Highest shoot induction frequency (92%) was recorded on MS medium containing 4 �M BA in combination with 1 �M of ?-naphthalene acetic acid (NAA). Further, well developed roots were formed in MS media augmented with 6 �M of Indole acetic acid (IAA) in combination with 1 �M Kinetin (Kn). Cotyledon explants were exploited in vitro for successful transformation of B. juncea. A binary vector comprised of Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene under the transcriptional control of a glycinin promoter and basta selection marker was introduced into A. tumefaciens strain GV3101 via electroporation. Highest regeneration frequency (100%) of transgenic shoots was observed on MS medium supplemented with 4 �M BA plus 1 �M NAA in the presence of 25 mgl-1 basta and 160 mgl-1 timintin. Transgenic nature of the transformed regenerated shoots was confirmed via PCR analysis using EaDAcT gene specific primers against their isolated genomic DNA.
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