Genetic transformation of safflower (Carthamus tinctorius L.) and Arabidopsis for increased oil content

Abstract

India attained self-sufficiency in edible oilseed production, thanks largely to the technology mission on oilseeds. But, in the last decade again it has become a net importer of edible oil. The reversal in the trend could be associated with stagnation in the productivity, increasing population size and increase in the per capita consumption of oil. Apart from the conventional technologies that would help in improving productivity, biotechnology offers new avenues to address the stagnant edible oilseed production in India. India is the largest producer of safflower in the world with the highest acerage of 4.3 lakh hectares. Safflower produces oil rich in polyunsaturated fatty acids (Linoleic acid 78%) and is considered as a healthy cooking medium. Oil level in safflower cultivars ranges from 28-32% while the genetic potential is more than 40%. There are many successful reports of increasing the oil content through transgenic approach though most of them are not commercialized and are attempted mainly in Arabidopsis and Brassica. In most of these cases, attempts have been made to over express a single gene to elevate the levels of oil content while it has been opined that expression of multiple genes might actually lead to tangible increase in the oil content. Present study aims at assessing the effect of simultaneous expression of genes, glycerol-3-phosphate acyltransferase (GPAT9) and diacyl glycerol acyl transferase (DAGAT1), encoding the two rate limiting enzymes of oil accumulation pathway in increasing the oil content in safflower. The proposed work involves developing a set of three plant transformation vectors with the chosen genes, validating the constructs in the model dicot Arabidopsis thaliana and then using them for transforming safflower for increasing the seed oil content. Plant Transformation vectors carrying the genes (GPAT9 and DAGAT1), either singly or together were developed. Initially all the component sequences were isolated from their respective source and cloned into InsT/A vector and sequenced. Later they were assembled in pRT100 and subcloned into binary vector pCAMBIA 1300. The binary constructs were then mobilized into Agrobacterium tumefaciens strain LBA4404 and GV3101 to facilitate plant transformation. Arabidopsis was transformed with these constructs independently and the transgenic plants were identified and advanced to the T3 generation. T3 lines carrying the transgene(s) in homozygous condition have been obtained. Oil analysis would be carried out with the seeds of these homozygous lines to validate the effect of introduced gene cassettes on seed oil content. Putative safflower transgenic shoots have been obtained with the double gene cassette. These shoots have been grafted onto control root stocks, out of 50 grafted shoots only 6 survived in the green house but none of them had set seeds. Alternatively, in planta transformation was carried out with double gene construct and concoction of single gene constructs independently, T2 progeny were analysed but none of them were PCR positive. In planta transformation has been attempted with modified protocol and analysis of the progeny obtained is in progress.