GENERATION of Transgenic Medicago Sativa Overexpressing “Osmotin” GENE Under Constitutive Promoter.

GENERATION OF TRANSGENIC MEDICAGO SATIVA OVEREXPRESSING “OSMOTIN” GENE UNDER CONSTITUTIVE PROMOTER

Jahnavi Reddy Kancharla, Kenneth S. Sajwan, Shivendra V.Sahi                 

Medicago is widely used as a forage crop. It is often susceptible to various pathogenic infections and exhibits low growth in drought and extreme climatic conditions. In our study efforts are underway to develop a strategy for over expressing “Osmotin-Chitinase” gene chimera for generating transgenic Medicago that could potentially confer resistance to different stresses. Percent seed germination of several cultivars of Medicago (M. sativa ssp. sativa, M. sativa ssp. falcata, M. sativa ssp. caerulea, M. truncatula, and M. Rugosa) was tested to determine the cultivars with good percent germination. Among these, M. sativa ssp. sativa showed an average of 80% germination over a period of two weeks and was subsequently selected for use in transformation and regeneration experiments. Different explants (cotyledons, hypocotyls, petioles) were tested for callus induction. Among these, hypocotyl explants showed good percent callus formation. Agrobacterium tumefaciens EHA105 strain was transformed with Escherichia coli harboring gene chimera osm-chi cloned into binary vector pBTEX with nptII as a selection marker and was co-cultivated with calli generated from hypocotyl explants. Transformed calli were grown on callus inducing medium containing kanamycin (50 mg/L). Screening of the positive transgenics was performed using PCR. Molecular and morphophysilogical studies would be carried out for the confirmation of osmotin gene in the transgenics. Transformed shoots would be grown on the root inducing medium for developing into plantlets and would then be transferred to the green house and later tested for their degree of resistance to various biotic and abiotic stresses.