Maturation of alfalfa ( Medicago sativa L.) somatic embryos by abscisic acid, sucrose and chilling stress

Abstract

The vigor of seedlings from dry somatic embryos of alfalfa ( Medicago sativa L.) is considerably less than the vigor of those from truw seeds, hypothetically as the result of low levels of storage proteins, an inhibitory effects of the ABA in the maturation medium, or injury from the desiccation process. In an attempt to increase vigor, three different treatments, abscisic acid (ABA), osmotic stress (0.4 M sucrose) and chilling (4°C), were applied during the final stage of embryo maturation as signals to induce the expression of desiccation tolerance in the developing embryos. Somatic embryos were induced on medium containing 2,4-D and were transferred to hormone-free medium where they developed to the torpedo stage. Storage protein synthesis occurred after transfer of the embryos to maturation phase I medium containing 146 mM sucrose and 50 mM glutamine. Subsequently, in maturation phase II, somatic embryos were transferred to fresh medium, and exposed to one of the above inductive treatments for 3, 12 or 19 days. Somatic embryos were then dried at a relative humidity of 43%. Desiccated embryos were rehydrated by placing them directly on agar medium with no added nutrients and scored for quality based on germination after 7 days and leaf development (conversion) after 14 days. The highest somatic embryo quality was obtained after 3 days on maturation medium with sucrose or ABA; longer durations reduced both germination and conversion. In contrast, 19 days of maturation at 4°C was required for the somatic embryos to express significant levels of desiccation tolerance. A positive correlation was observed between the embryo's total protein content and their quality. SDS-PAGE indicated that the low molecular weight (LMW) proteins in the S-1 fraction (low salt soluble) and the S-2 (high salt soluble) storage protein, medicagin, declined in the somatic embryos after 3 days on maturation medium containing ABA or sucrose, whereas their levels continually increased during the chilling treatment. These results are discussed in relation to somatic embryo quality, desiccation tolerance and accumulation of storage proteins during the maturation of alfalfa somatic embryos.