Somatic embryogenesis and Agrobacterium mediated genetic transformation in Indian accessions of lucerne (Medicago sativa L.)

Plant regeneration in lucerne (Medicago sativa) is highly genotype dependent and inspite of considerable information on in vitro regeneration in this species the Indian genotypes have not been investigated so far. A system of recurrent somatic embryogenesis (RSE) was established for the first time in Indian accessions of lucerne and utilized for Agrobacterium mediated genetic transformation. Seeds, hypocotyls and cotyledons of LLC-3, C-10, A-3 and IL-75 accessions and ovary explants of 5 somaclones of LLC-3 were used and globular shaped somatic embryos were observed in all of the cultures. All the developmental stages of somatic embryogenesis were observed in ovary culture of one somaclone only. These somatic embryos have been undergoing cycles of RSE and the system has perpetuated for 24 months. For Agrobacterium mediated genetic transformation, this system of RSE required creation of injury in individual somatic embryos for enhanced transformation efficiency that limited its utilization for large-scale transformation experiments. An efficient system to overcome the requirement of creation of injury was developed in which pre-culture of somatic embryos on 2,4-D supplemented medium prior to their co-cultivation with Agrobacterium and supplementation of 2,4-D in co-cultivation medium could significantly improve the transformation efficiency.