AGROBACTERIUM-RHIZOGENES MEDIATED TRANSFORMATION OF THE FORAGE LEGUMES MEDICAGO-SATIVA AND ONOBRYCHIS-VICIIFOLIA

Three cultivars of M. sativa and one cultivar of O. viciifolia were evaluated for their response to inoculation with A. rhizogenes strain A4T (containing pRiA4b). A cultivar-dependent response was observed in M. sativa with 94%, 25%, and 4% of infected stem explants producing transformed roots in the cultivars Vertus, Regen-S, and Rangelander, respectively. In O. viciifolia cv. Hampshire Giant, an explant-dependent response was observed with 78% and 50% of seedling cotyledon and hypocotyl explants responding, respectively. Leaf explants failed to produce transformed roots. Transformed roots showed plagiotropic and negatively geotropic growth on hormone-free agar MS medium. Production of transgenic shoots from O. viciifolia root cultures occurred spontaneously. Recovery of transgenic plants from M. sativa cv. Rangelander was achieved by transfer of callus (induced on UM medium containing 2.0 mg dm-3 2,4-D and 0.25 mg dm-3 kinetin) to MS medium containing 0.5 mg dm-3 BAP and 0.05 mg dm-3 NAA. Cultured roots of both species synthesized opines (agropine and mannopine). Extensive morphological variation was observed in plants of M. sativa (clone Al) and O. viciifolia (clone A4T1) established in the glasshouse. DNA sequences homologous to TL-DNA and TR-DNA were present in root clones and regenerated plants.