Generation of Transgenic Medicago Sativa Resistant to Biotic and Abiotic Stresses by Expressing “Osmotin-Chitinase” Gene Chimera.

Generation of transgenic Medicago sativa resistant to biotic and abiotic stresses by expressing “osmotin-chitinase” gene chimera JAHNAVI R. KANCHARLA*, AJAY JAIN, SHIVENDRA V. SAHI Department of Biology, Western Kentucky University, 1906 College Height Blvd# 11080, Bowling Green, KY 42101 Medicago is a commonly used forage crop and often susceptible to various biotic and abiotic stresses. Combined expressions of chitinases and plant defense proteins such as osmotin have been shown to confer enhanced resistance to various stress responses. Therefore, in the present study we use this strategy of expressing “osmotin-chitinase” gene chimera for generating transgenic Medicago sativa that could potentially confer resistance to different biotic and abiotic stresses. To determine the Medicago cultivar that would be best suited for regeneration and transformation, initially we tested the per cent seed germination of several cultivars of Medicago (M. sativa ssp. sativa, M. sativa ssp. falcata, M. sativa ssp. caerulea, M. truncatula,  and M. Rugosa). Among these, M. sativa ssp. sativa, M. truncatula and M. Rugosa showed good per cent germination (50-80%) over a period of 2 weeks. Therefore these three cultivars were used subsequently for transformation and regeneration experiments. Different explants (cotyledons, hypocotyls, petioles) were used for inducing calli. Agrobacterium tumefaciens  strains (AGL1 and EHA 105), transformed with gene chimera osm-chi cloned into binary vector pBTEX with nptII as a selection marker,  were co-cultured with calli generated from different explants. Transformed calli were grown on callus inducing medium containing kanamycin (50 mg/L) and carbenicillin (350 mg/L). Transformed shoots were grown on the root inducing medium for developing into plantlets. At present the transformed seedlings have been transferred to the green house to allow them to grow to maturity for subsequent molecular and morphophysilogical studies for the confirmation of osm-chi gene chimera in the transgenic and their degree of resistance to various biotic and abiotic stresses.