Somatic Embryos Maturation And Germination Of Quercus Variabilis BI

Quercus variabilis is an important cork sources species in China. It is very important to quickly propagation excellent clones, genetic improvement and gene resource preservation through somatic embryogenesis plant regeneration for this species. Immature zygotic embryos were used as explants to induce somatic embryos, this paper investigated the synchronous control of somatic embryogenesis and regeneration ability maintenance and proliferation. Several factors influencing somatic embryos maturation and germination were analyzed, such as light, sucrose concentration, hormone, activated charcoal, genotype and immature zygotic embryos development stage. Meanwhile the relationship between endogenous sucrose as well as starch concentration and maturation of somatic embryos in the mature process was studied . The main results were described as follows: 1)Initial inducing embryogenic callus and globular embryos had super regeneration capacity than other stage embryos. By combination 6-BA and NAA in medium, somatic embryos regeneration capacity would be prolonged and prevented from decline or lose after subculture several times. But 6-BA and NAA concentration could not be over 10 mg/L and 0.25 mg/L respectively . 2) Synchronization of somatic embryogenesis would realizated by adding different concentration 2,4-D in medium. Explants tended toward embryogenic callus with high 2,4-D concentration, contrarily being somatic embryos. Identical cotyledonary, heart-shape, globular-shape and embryogenic callus could be obtained by adding 0.5,1.0,1.5, 3.0 mg.L-1 2,4-D on MS medium individually. 3) Appearance of mature Somatic embryos would be described as cotyledons thick, milky or ivory white, pale yellow or green, hypolotyl developed into radicle and about 5-8mm long in diameter. Many factors affected somatic embryos maturation. High sucrose concentration was favorable to maturation, but increased the production of abnormal mature embryos. Somatic embryos would matured in proper ABA concentration. Quality would decrease once ABA concentration over 0.5 mg.L-1. Combination of cytokinins and auxin did not marked improve somatic embryos normal maturation, furthermore prevented their maturation in a higher concentration level. Prolong culture time(2 monthes) in mature medium could be helpful for somatic embryos maturation. Although AgNo3 , activated charcoal and PVP benefit maturation of somatic embryos, the effects were no as good as high sucrose or ABA treatment. Sorbitol and CH had no effect on somatic embryos maturation. Somatic embryos maturation needed proper light condition. When zygotic embryos development stage near maturation the somatic embryos induced from which would mature easily. , We selected superior mature medium and culture condition as fellow: by adding 0.5mg/L ABA and 0.5mg/LPVP together in basal medium with 0.5% sucrose and cultured in light for 2 months. Using this protocol somatic embryos maturation rate would keep steady about 70%. 4) There were the same change trend of the endogenous total sugars and sucrose content change in somatic embryos maturation. While globural stage had high concentration, then decreased, and the lowest level appeared in heart-shape stage. The highest the endogenous total sugars and sucrose content appeared cotyledonary stage. . But starch concentration changed specially. From glublar-shape to cotyledonary stage, concentration of starch accumulation increased continually. By adding higher concentration of exogenous sucrose in basal medium would increase the endogenous total sugar , source and starch concentration clearly. The endogenous total sugar, source and starch concentrations in mature somatic embryos were 1/2, 1/3 and 1/13 of that in mature zygotic embryos respectively. From this we can draw a conclusion that insufficient store substance accumulation should be one of the most important causes in maturation of Q. variabilis. 5) Somatic embryos maturation was closely related to germination. Fully mature somatic embryos could germinated on any medium, and germination rate could reach over 70%. Because most regenerated plantlets only had roots elongation, someone even developed lateral roots but without shoots sprouting. This phenomenon decreased plantlet conversion rate of Q. variabilis seriously. Normally maturation somatic embryos plantlet conversion rate was only 25%, while abnormal ones only 5%.