外源激素对日本落叶松体细胞胚发生不同阶段的影响

Effects of Exogenous Hormones on Different Stages of Somatic Embryogenesis of Larix kaempferi

【目的】探究外源激素对日本落叶松体细胞胚发生不同培养阶段的影响,优化日本落叶松的体细胞胚发生条件并提高体细胞胚的质量,为研究外源激素调控落叶松体细胞胚发生的生理机制提供数据支持,为落叶松的规模化繁育和遗传改良奠定基础。【方法】首先确定生长曲线的延迟期、对数期和平台期,然后根据生长曲线,采用正交设计研究胚性胚柄团在2,4-D 0.15、0.30、0.50 mg·L^(-1)、6-BA 0、0.15、0.30 mg·L^(-1)和ABA 0、0.50、1.00 mg·L^(-1)浓度下培养7天、14天、21天的鲜质量增长情况和14天的体胚发生能力,采用析因设计研究悬浮细胞在2,4-D 0.15、0.30、0.50 mg·L^(-1)和6-BA 0、0.15、0.30 mg·L^(-1)浓度下培养7天、14天、21天的细胞鲜质量增长情况和14天的体胚发生能力,结合鲜质量增长和体胚发生能力2种结果确定胚性胚柄团增殖和悬浮细胞生长的最佳激素浓度。最后,以不含GA3、IAA的体细胞胚诱导培养基为对照,分别研究了GA3和IAA 0、10、20、30、40 mg·L^(-1)对体细胞胚发生的影响。【结果】7、14、21天分别处于日本落叶松胚性胚柄团增殖和悬浮细胞生长的延迟期、对数期和平台期。在胚性愈伤组织增殖阶段,2,4-D、6-BA、ABA对胚性胚柄团增殖和体胚发生能力的影响显著(P<0.05),最佳的激素浓度是2,4-D 0.15 mg·L^(-1)+ABA0.50 mg·L^(-1);在悬浮培养阶段,2,4-D、6-BA组合对悬浮细胞生长和体胚发生能力的影响显著(P<0.05),最佳激素浓度是2,4-D 0.15 mg·L^(-1)+6-BA 0.15 mg·L^(-1);GA3、IAA对成熟子叶胚的数量的影响均显著(P<0.05),10~20 mg·L^(-1) GA3或IAA时成熟子叶胚数量显著升高,而更高的GA3或IAA浓度将会抑制体胚发生。【结论】外源激素可有效调控日本落叶松的体细胞胚发生,适宜增殖阶段胚性胚柄团增殖和体胚发生能力保持的激素组合为2,4-D 0.15 mg·L^(-1)+ABA 0.50 mg·L^(-1),适宜悬浮培养阶段细胞生长和体胚发生能力保持的激素组合为2,4-D 0.15 mg·L^(-1)+6-BA 0.15 mg·L^(-1),添加GA3或IAA 10~20 mg·L^(-1)可以显著提高成熟子叶胚的数量。

【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.