DEVELOPMENT OF DISEASE RESISTANT TRANSGENIC PLANTS THROUGH TI PLASMID MEDIATED GENE TRANSFER

1. The appropriate gene construct is inserted within the T-DNA of a disarmed Ti plasmid. The recombinant vector is placed in Agrobacterium, which is co-cultured with the plant cells or tissues to be transformed for about 2 days.

2. In general the transgene construct involves a selectable reporter gene (Bacterial neo gene), the presence of which confers resistance to kanamycin.

3. During the leaf disc-Agrobacterium co-culture, aceto syring one released by plant cells induces the vir genes which bring about the transfer of recombinant T-DNA into many of the plant cells. The T-DNA would become integrated into the plant genome, and the transgene would be expressed. As a result, the transformed plant cells would become resistant to kanamycin.

4. After 2 days, the leaf discs are transferred onto a regeneration medium containing appropriate concentrations of kanamycin and carbenicillin. Kanamycin allows only transformed plant cells to divide and regenerate shoots in about 3-4 weeks, while carbenicilin kills Agrobacterium cells. The shoots are separated, rooted and finally transferred into soil.