Different screening protocol for Pisum sativum varieties for identification of Agrobacterium mediated transformation efficiency

Abstract

A number of abiotic stresses affect the plant growth and yield. Out of them, heat stress is a major abiotic stress which results drastic reduction of yield in grain legumes including peas. Increasing global warming day by day and uncontrolled human growth now directs to develop heat resilient varieties through transformation of pea. As many type of varieties available around the world, but it first of all becomes very important to identify that which variety support transformation because many pea varieties behave as recalcitrant crops like rice, cotton, Medicago. In this project, total 22 pea varieties, they are screened out through transformation by different protocols like transformation pf pea (a) in callus derived from mature embryo axis (b) in direct embryo (c) in half – cut embryo. Transformation in callus derived from mature embryo of 22 variety was transformed with observed that by doing GUS assay, proper concentration of acetosyringone (200microM and sonication for 4 sec with approx. 6 min vacuum infiltration showed great improvement in the number of GUS foci per explant from 1.0 to 38.6. T0 transgenic plants showed Mendelian inheritance pattern of transgene segregation in T1 progeny. After GUS assay, to know the stable integration of gene of interest into pea is determined by PCR, RT-PCR, and Southern hybridization of transgenic plants. The results show an efficient and easy transformation technique of pea for producing transgenic plants with high transformation frequency. These findings will surely enhance the development of resistant pea plants with desired traits against abiotic stress agrobacterium tumefaciens strain EHA harbouring pcambia2301 plasmid containing gene of GusA reporter construct for simple and easy analysis of desired gene function or to confirm the transformation in somatic embryos by GUS assay. Various factors affecting transformation efficiency were established; it is optimized that when 10 days old MEA derived callus is transformed at cell density at 0.3 OD600 with a proper co cultivation time(48h) along with 20 C temperature show efficient and maximum