Rapid transformation and regeneration of alfalfa (Medicago falcata L.) viadirect somatic embryogenesis

Two simple, rapid and efficient protocols for the regeneration of transform ed tetraploid lines of alfalfa (Medicago falcata L.) have been developed an d compared. Leaf explants from embryogenic lines 47/1-150 and 47/1-5 were i noculated with Agrobacterium tumefaciens containing constructs carrying the nptII selectable marker gene and promoter:gusA gene fusions under the cont rol of the CaMV 35S or Arabidopsis cdc2a, CycB1 and CycA2 promoters. In the first regeneration system (the MSH system), inoculated leaf explants were incubated on MS medium supplemented with 2,4-D and kinetin and then subcult ured onto plant growth regulator-free MS medium in order to induce direct s omatic embryogenesis. In the second regeneration system (the B5h system), t he inoculated explants were incubated on B5h medium to induce indirect prod uction of somatic embryos via embryogenic callus. In both systems, an effec tive kanamycin selection regime was employed and was maintained when the em bryos were subcultured onto a recovery medium (Boi2Y) to promote further em bryo development. The use of Boi2Y medium was particularly important for sh ortening the regeneration time and promoting a higher frequency of healthy plantlet production from the somatic embryos. The mature somatic embryos we re finally transferred to plant growth regulator-free MS medium for plantle t formation. Transgenic plantlets were produced within 10-14 weeks in the M SH system and 12-16 weeks in the B5h system. The MSH system appears to be t he fastest transformation system reported for leguminous species to date. C onfirmation of transformation was obtained using a re-callusing assay on ka namycin and subsequent Southern blot hybridisation and PCR analysis. The ab ility to induce expression of GUS activity in leaf explants containing the cell division cycle gene promoter:gusA constructs by 2,4-D treatment also p roved to be a reliable indicator of transformation.