<i>Agrobacterium-mediated </i>Transformation of Lucerne (<i>Medicago sativa </i>Linn.): Optimizing Biological and Physical Parameters

Abstract

An optimized protocol for the transformation of Indian cultivars of lucerne (<i>Medicago sativa </i>Linn.) using <i>Agrobacterium tumefaciens </i>has been developed. It involved a selectable marker gene encoding hygromycin phosphotransferase (<i>hpt </i>II) which confers resistance to the

antibiotic hygromycin, together with a reporter gene (<i>uid </i>A) encoding P-D glucuronidase (GUS); cotyledonary and hypocotyl explants from <i>in vitro </i>grown 5 days-old seedlings

raised from mature seeds of genotype L-2 and IL-75 as target tissues, and co-cultivation of explants with <i>A. tumefaciens </i>strain LBA-4404 harbouring plasmid pCAMBIA-1301.

Cotyledonary explant was found to be more responsive for transformation. Bacterial density of 5×10⁹ cells/ml and co-cultivation for three days were found to be most effective for stable GUS expression in 91 % of the hygromycin-resistant calli.