Development of Genetically Engineering Mustard (Brassica Juncea) for Low Viscosity Biodiesel Production

Abstract

Indian mustard (Brassica juncea L.) is a member of the Brassicaceae family that is now becoming a global crop because of its drought tolerance, high oil content, short growing season and the ability to grow on low quality soils. The present study was aimed to reduce the viscosity of Brassica juncea oil by altering the fatty acid profile, via developing efficient and simple Agrobacterium mediated based tissue culture and floral dip methods of plant transfermation. For optimization of regeneration protocol, cotyledon explants of the Brassica juncea CV- Raya Anmol were cultured on MS medium supplemented with different concentrations of 6-Benzylaminopurine (BAP), (2, 4, 6, 8, and 10 µM) alone and in combination with 1µM of Gibberellic acid (GA3) and Naphthaleneacetic acid (NAA) separately. Maximum calli production (87%) was observed on MS medium containing 4 µM BAP only. The highest shoot induction efficiency (92%) was observed on medium supplemented with BAP (4 µM) and NAA (1 µM). For rooting, Indole-3-acetic acid (IAA) (6µM) and Kinetin (Kin) (1µM) was found as the best combination. A binary vector containing the EaDAcT (E.alatus diacylglycerol acetyltransferase) gene under the transcriptional control of a glycinin promoter and with a basta selection marker, was introduced into A. tumefaciens strain GV3101 via electroporation. An efficient tissue culture and In planta transformation was developed for B. juncea. In tissue culture transformation, cotyledon explants were used while for floral dip, plants at early stage of flowering with and without vacuum desiccator were used. The basta resistant putative transgenic plants were selected and further confirmed by PCR. The vacuum infiltration was found more efficient (0.85%) as compared to the normal floral dip method (0.15%).The developed transgenic B. juncea seeds showed altered TAG fatty acid composition with enhanced level of oleic acid (from 41% to 63%) and reduced eurcic acid level (from 13.7% to 2.53%), which is an ideal composition of fatty acids in oil to be used as biodisel. The developed protocols could be used to accumulate unusual acTAG in B. juncea seed, providing a direct way of biodiesel production from plant oil. Such results will be useful for the development of B. juncea as an alternative source of energy.