铁观音茶树体胚发生及其内源激素变化

Somatic embryogenesis and the changes of endogenous hormones in Camellia sinensis ‘Tieguanyin’

以乌龙茶品种铁观音茶树的未成熟胚为供试材料,建立高效的茶树体胚发生再生体系,同时采用高效液相色谱法（HPLC）分析茶树体胚发生过程中各个不同发育阶段的内源激素含量变化趋势.结果显示,诱导茶树体胚发生的培养基为改良MS培养基＋2 mg/L苄基腺嘌呤（BA）＋0.2 mg/L吲哚丁酸（IBA）＋800 mg/L甜菜碱,体胚增殖系统维持途径的培养基为改良MS培养基＋3 mg/L肉桂酸＋20 g/L蔗糖＋10 g/L山梨醇.当3%蔗糖与2%山梨醇组合使用时,子叶形胚的诱导率明显提高.将诱导产生的子叶形胚转接至添加5%蔗糖与5%聚二乙醇（PEG）的无激素MS培养基中,经过40 d左右培养,体胚能正常成熟.成熟体胚接种在附加2 mg/L BA与0.1 mg/L IBA的MS培养基上可以萌发成苗.在体胚发生早期,玉米素（ZT）和赤霉酸（GA3）含量均是先下降,在子叶形胚时期降至最低值,到体胎发育后期的成熟胚与萌发阶段呈明显上升趋势;脱落酸（ABA）含量由球形胚到子叶形胚阶段一直呈现下降趋势,到成熟胚阶段时,出现一个明显的峰值;球形胚中的吲哚乙酸（IAA）含量显著高于其他阶段,之后迅速降低,波动变化.ABA/IAA的比值先增大,然后减小;ABA/GA3的比值体胚发育前期维持在较高水平,到成熟萌发阶段下降;球形胚阶段IAA/ZT的比值高于其他各个阶段.本研究表明茶树不同发育阶段胚胎的体胚诱导率不同,与生理状态及其内源激素动态变化有关;同时茶树中内源激素含量和比例在体细胞胚胎发育过程中有显著的变化,这种变化在不同胚胎发育阶段起着特定的调节作用.

In this study, the immature embryos of oolong tea cultivar ‘Tieguanyin＇ were used to establish an efficient regeneration system of tea somatic embryogenesis. The high performance liquid chromatography（HPLC） method analyzed the trend changes in endogenous hormone content during different developmental stages of tea somatic embryogenesis. The results showed that the medium to induce somatic embryogenesis was modified MS medium supplemented with 2 mg/L benzylaminopurine（BA）, 0.2 mg/L indole-3-butytric acid（IBA）, and 800 mg/L betaine, and the medium for sustaining the somatic embryo proliferation system was modified MS medium supplemented with 3 mg/L cinnamic acid, 20 g/L sucrose, and 10 g/L sorbitol. The percentage of cotyledon embryos increased when 3% sucrose was used in combination with 2% sorbitol. The induced cotyledon embryos were transferred to the medium supplemented with 5% sucrose and 5% polyethylene glycol（PEG） without phytohormones, and the somatic embryos became mature after 40 d of culture. Mature embryos were induced to germinate and regenerate on the MS medium supplemented with 2 mg/L BA and 0.1 mg/L IBA. During the early somatic embryogenesis stage, the content of zeatin（ZT） and gibberellic acid（GA3） first decreased and reached the lowest value of the cotyledon embryo period, and the content of ZT showed a significant upward trend in the mature embryo and germination embryo period during the post-embryonic developmental stage. The content of abscisic acid（ABA） decreased from the globular embryos to the cotyledon embryos and reached a peak during the mature embryos stage. The content of indole-3-acetic acid（IAA） in the globular embryo was significantly higher than that in the other stages, and then decreased rapidly and showed small fluctuations. The ratio of ABA/IAA first increased and then decreased. During the early somatic embryogenesis stage, the ratio of ABA/GA3 was maintained at a high level and decreased during the mature embryo and germination embryo period. The ratio of IAA/ZT in the globular embryos was higher than that in the other stages. The present study showed that the induction rate of embryos during different developmental stages was different, which was related to the physiological state and dynamics of the endogenous hormones in the tea cultivar ‘Tieguanyin.＇ In addition, the content and proportion of endogenous hormones changed significantly during the development of somatic embryos. These changes play a specific regulatory role during different stages of embryonic development in the tea plant.