Mechanisms Of Somatic Embryogenesis And Morphogenesis Of Xanthoceras Sorbifolia Bunge

Abstract With seed embryos of Xanthoceras sorbifolia Bunge as explants, variouscultural factors suitable for the somatic embryogenesis of X. sorbifolia. were obtained using solid and liquid suspension culture techniques. A stable somatic embryo culture system for X. sorbifolia was established. With the first way to get the somatic embryos of X. sorbifolia as the research objective, the mechanisms of morphogenesis of somatic embryos of X. sorbifolia were discussed from the following four aspects: histological observation, ultracytochemical localization of Ca²+, isozyme spectra and specific proteins. The results are as follows:1. The somatic embryogenesis of X. sorbifolia could be obtained through the following two pathways:The first way: The non-embryogenic callus (NEC) was induced by culturing the seed embryos of X sorbifolia on a medium of B₅ + 2,4-D 1 mg·L^-1 + sugar 20 g·L^-1 + agar 6 g·L^-1, and A-type embryogenic callus (EC) was obtained by a following culture on a medium of B₅ + 2,4-D 0.5 mg·L^-1 + sugar 20 g·L^-1 + agar 6 g·L^-1. On the medium of B₅ + 6-BA 1 mg·L^-1 + NAA 0.25 mg·L1 + sugar 20 g·L1, we got globular embryoid, heart shaped embryoid and A-type torpedo embryoid with obvious radicales. The torpedo embryoid developed into A-type cotyledonary embryoid and regenerative plants finally on the medium of B₅ + 6-BA 0.5mg·-L^-1 + NAA 0. Smg·L1 + sugar 20 g·L1.The second way: The NEC was developed on a medium of B₅ + 2,4-D 1 mg·L^-1 + sugar 20 g·L^-1 + agar 6 g·L^-1, and B-type EC formed on a medium of B₅ + 6-BA 0.5 mg·L^-1 + NAA 0.5 mg·L^-1 + 2,4-D 0.5 mg·L^-1 + sugar 20 g·L^-1 + agar 6 g·L^-1. Then globular embryoid, heart shaped embryoid and B-type torpedo embryoid without obvious radicales shaped but having two cotyledons on a medium of B₅ + 6-BA 0.5 mg·L^-1 + NAA 0.5 mg·L^-1+ sugar 20 g·L1. The radicals, some tender leaves, and finally regenerative plants, developed.2. The spatial and temporal distribution dynamics of Ca²⁺during the morphogenesis of somatic embryos of X. sorbifolia show that there is a lower deposit of Ca²⁺, which is distributed mainly in cell walls and intercellular spaces of NEC, whereas the deposit ofCa²⁺in embryogenic cells increases obviously, which is distributed mainly in plasma membranes, vacuole membrane, plasmid, cell walls and intercellular spaces. The deposit of increases significantly in the outmost cells of radicale tips where the outer cell walls thicken, indicating that Ca²⁺ a kind of signal molecule participates in the thickening and construction of cell walls. This is one of the innovations of this study.3. The experiment results on isozyme spectra of POD, SOD, amylase, EST, and ATPase during the morphogenesis of somatic embryos of X. sorbifolia show that the spectrum bands of these five isozymes increase with the development of somatic embryos, and some specific bands at the developmental stages were found, which could act as the biochemical markers during the somatic embryogenesis and development of X. sorbifolisa. The dynamic study of SOD isozyme spectra is another innovation of this study.4. The protein analyses by 2-dementional electrophoresis indicate that NEC has a lowest amount and expression of proteins; some new proteins could be found at stages of EC, globular embryoid, torpedo embryoid, cotyledonary embryoid and regenerative plant. The specific proteins at stages of EC, torpedo embryoid and regenerative plant are of 23.0, 27.1 and 23.15 kD, respectively, while cotyledonary embryoid has specific proteins of 25.1 and 26.2 kD. Above are some new findings in this study.