Development of Transgenic Medicago Harboring cry2A Gene From the Bacillus Thuringiensis to Confer Resistance towards Insects.

Medicago, the fourth most widely grown crop in the United States, is an important forage crop species. However, the crop is often prone to destruction by insects; thus, there is a need to develop transgenics expressing potent insecticidal traits. The cry2A gene from the Bacillus thuringiensis (Bt) bacterium induces the production of insecticidal protein and therefore provide an attractive strategy for developing insect-resistant alfalfa by incorporating this gene into the plant through Agrobacterium-mediated transformation. Hypocotyls from 2-3 week old seedlings of M.rugosa, M.falcata and M.sativa sp. sativa were excised and transferred to callus-induction medium (CIM). The induced callus was transformed with Agrobacterium tumefaciens (AGL1 strain) harboring cry2A in binary vector pAUIIA. To induce the vir gene in the Agrobacterium, acetosyringone was added to the medium with appropriate antibiotic selection. The potential transgenic plants were placed on a nutrient media containing ticarcillin and kanamycin, for the elimination of Agrobacterium and for the selection of plants that displayed transgenic qualities, respectively. To initiate shooting in the transformed calli, they were transferred to regeneration medium (Gamborg’s B-5). Among the three genotypes evaluated for their regeneration and transformation efficacy, a high value (40-50%) was observed for M.sativa sp. sativa followed by relatively lower values of 35-45% and 30-40% for M.rugosa and M. falcata, respectively. Southern blot and Real time PCR analysis would be carried out for subsequent confirmation of the transgenics obtained from the different species of Medicago. Upon confirmation, the transgenics would be tested under field conditions for their ability to withstand damage form insects.