A novel system for rapid high frequency somatic embryogenesis in Medicago sativa

Abstract

A novel, genotype dependent system for rapid high frequency somatic embryogenesis in Medicago sativa L. was developed in which the first embryos are visible as early as 15 days after the explant (hypocotyl, petiole, leaf) is put into culture. The simplest method involves culture of the explants on a single Murashige and Skoog (MS) medium supplemented with 2 g l−1 casein hydrolysate, 9 μM 2,4‐dichlorophenoxyacetic acid (2,4‐D) and 1.2 μM kinetin. An efficient two‐step, two‐medium system was developed to allow separation of the induction and differentiation phases. The explants are cultured on MS with 22.6 μM 2,4‐D and 4.7 μM kinetin (induction medium) for 10 days and then on basal MS for 20 days. Embryo yields and embryo conversion to plantlets were strongly dependent on the 2,4‐D and kinetin concentrations in the induction medium. Both petiole and leaf explants were highly embryogenic and very little callus proliferation occurred when this method was used. Selected clones from three ssp. falcata‐based M. sativa cultivars showed a response very similar to the highly regenerable falcata clone F1.1, but it was not possible to produce large numbers of somatic embryos in tissue cultures of cv. Regen S, which is used in most M. sativa tissue culture research, with this procedure. These results suggest that there are two distinct developmental pathways for somatic embryogenesis in M. sativa, with Regen S cultures requiring extensive dedifferentiation during a prolonged callus phase, while the genotypes described in this report have no such requirement.