关键词: 日本落叶松, 胚性胚柄团, 体细胞胚发生, 细胞增殖, 外源激素

Abstract:

Objective: Genetic improvement using somatic embryogenesis of larch can improve the environmental adaptability and the wood yield of larch. Exogenous hormones are an important factor affecting the somatic embryogenesis of larch, so we explored the effects of exogenous hormones on the different stages of somatic embryogenesis of Larix kaempferi, in order to improve the quality of embryos obtained through somatic embryogenesis and to accelerate the genetic transformation of larch. Method: The delay phase, logarithmic phase and plateau phase of the growth curve were first determined. Then, according to the growth curve, orthogonal design was used to investigate the proliferation and somatic embryogenesis of proembryogenic masses at 2, 4-D 0.15, 0.30, 0.50 mg·L^-1, 6-BA 0, 0.15, 0.30 mg·L^-1 and ABA 0, 0.50, 1.00 mg·L^-1 concentrations on day 7, 14 and 21, factorial design was used to investigate the suspension cell growth and somatic embryogenesis at 2, 4-D 0.15, 0.30, 0.50 mg·L^-1 and 6-BA 0, 0.15, 0.30 mg·L^-1 concentrations on day 7, 14 and 21, and the optimum hormone concentrations of the two cultures were determined. Finally, the effects of GA₃ or IAA on somatic embryogenesis were studied using a somatic embryogenic induction medium without GA₃ or IAA as the control. Result: The results showed that day 7, 14 and 21 were in the delay phase, logarithmic phase and plateau phase of proembryogenic mass proliferation and suspension cell growth, respectively. Then, the effects of 2, 4-D, 6-BA, and ABA on proembryogenic mass proliferation were proved to be obvious(P < 0.05), and the optimal hormone combination comprised 2, 4-D 0.15 mg·L^-1 and ABA 0.50 mg·L^-1. The effects of 2, 4-D and 6-BA on suspension cell growth was dramatic(P < 0.05), and the optimal hormone combination comprised 2, 4-D 0.15 mg·L^-1 and 6-BA 0.15 mg·L^-1. Finally, we found that the effects of GA₃ and IAA on somatic embryogenesis were all significant(P < 0.05), and the mature somatic embryo number increased significantly when 10-20 mg·L^-1 GA₃ or IAA was added, whereas the higher concentrations of GA₃ or IAA significantly inhibited it. Conclusion: Exogenous hormones can effectively regulate the cell proliferation. The optimal hormone combination for proembryogenic mass proliferation comprised 2, 4-D 0.15 mg·L^-1 and ABA 0.50 mg·L^-1, and the optimal hormone combination for suspension cell growth comprised 2, 4-D 0.15 mg·L^-1 and 6-BA 0.15 mg·L^-1. In addition, 10-20 mg·L^-1 GA₃ or IAA could significantly increase the number of mature cotyledon embryos of Larix kaempferi. The results laid a foundation for large-scale propagation and genetic modification of larch.

Key words: Larix kaempferi, proembryogenic mass, somatic embryogenesis, cell proliferation, exogenous hormones