侯辛辛 陈健妙 高兴 赖杭桂 王健 胡新文 吴友根

摘要：【目的】建立海南地區油茶未成熟胚再生体系，为油茶组培快繁和遗传转化研究提供技术支持。【方法】以海南本地油茶合子胚为外植体，对其进行初代诱导、分化和增殖培养，筛选出最适于诱导分化的外植体胚发育时期，明确植株再生途径，从而建立分生结节组织植株再生体系。【结果】（1）盛花期后240～300 d的油茶幼胚在改良MS+激动素（KT）2.0 mg/L+萘乙酸（NAA）0.2 mg/L+玉米素（ZT）5.0 mg/L培养基上，初代诱导率在90.0%以上，以分生结节组织为主;（2）分生结节组织的适宜增殖培养基为改良MS+KT 1.0～2.0 mg/L+NAA 0.2 mg/L，增殖系数约4.6;（3）未成熟胚分生结节组织的分化培养基为改良MS+6-苄氨基嘌呤（6-BA）2.0 mg/L+NAA 0.2 mg/L+吲哚-3-乙酸（IAA）0.2 mg/L+ZT 5.0 mg/L+酸水解酪蛋白（CH）400.0 mg/L，平均每块分生结节组织诱导不定芽11.3个，不定芽以无糖暴露培养生根方式生根，移栽成活率为91.6%;（4）分生结节组织在改良MS+6-BA 2.0 mg/L+NAA 0.2 mg/L+ZT 5.0 mg/L+CH 400.0 mg/L上以分化体细胞胚状体为主，每块分生结节分化6.5个体细胞胚状体，具双子叶的体细胞胚状体在改良MS+6-BA 0.2 mg/L+IAA 2.0 mg/L+赤霉酸（GA3）0.5 mg/L+CH 200.0 mg/L培养基上有效成熟和萌发，萌发率为72.1%，胚芽和胚根有效伸长形成体胚再生植株，移栽成活率为94.7%。【结论】以油茶未成熟合子胚为外植体，通过分生结节组织方式增殖，可经体细胞胚状体发生和不定芽发生两种途径成功建立海南油茶未成熟胚分生结节植株再生体系。

关键词： 油茶;未成熟胚;分生结节组织;不定芽;体细胞胚状体;海南

Abstract：【Objective】To establish immature zygotic embryos regeneration system of Camellia vietnamensis， which provided technical support for the research on rapid propagation and genetic transformation of C.vietnamensis. 【Method】 Hainan native C. vietnamensis zygotic embryos were used as explants， zygotic embryos were induced to differentiate and proliferate to select the most suitable stage for inducing differentiation. Meanwhile， and the proliferation and plant regene-ration pathways were determined， so as to establish the regeneration system of meristematic nodules induced from immature zygotic embryos of C. vietnamensis. 【Result】（1）When the immature embryo at 240-300 d after full flowering stage cultured at the modified MS+kinetin（KT）2.0 mg/L+naphthaleneacetic acid（NAA） 0.2 mg/L+zeatin（ZT）5.0 mg/L me-dium， the initial induction rate of the immature embryo was more than 90.0% and the meristematic nodules were majority. （2）The suitable medium for the proliferation of meristematic nodule tissues was the modified MS+1.0-2.0 mg/L KT+0.2 mg/L NAA， and the proliferation coefficient was approximately 4.6. （3）The differentiation medium for immature embryo meristematic nodules was modified MS + 2.0 mg/L 6-benzyl adenine（6-BA）+0.2 mg/L NAA+0.2 mg/L indole-3-acetic acid（IAA）+ZT 5.0 mg/L and the average adventitious bud number was 11.3. Adventitious shoots rooted by sugar-free exposed cultivation， and plant transplant survival rate was 91.6%. （4）The meristematic nodule cultured at the modified MS+2.0 mg/L 6-BA+0.2 mg/L NAA+ZT 5.0 mg/L mainly differentiated into somatic embryo with the average somatic embryo number of 6.5. Somatic embryos with dicotyledonate were effectively germinated and developed into plantlets on modified MS+0.5 mg/L 6-BA+2.0 mg/L IAA+0.5 mg/L gibberellic acid（GA3）+CH 200.0 mg/L medium. The germination rate was 72.1%， the germ and the radicle were effectively extended to form a somatic embryo regeneration plant and plant transplant survival rate was 94.7%. 【Conclusion】With the immature zygotic embryos as explants，  two regeneration systems of meristematic nodules in Hainan native C. vietnamensis are established through somatic embryogenesis and bud generation pathways respectively after proliferation of meristematic nodules.

0 引言

【研究意义】油茶（Camellia oleifera）是山茶科（Theaceae）山茶属（Camellia）植物中油脂含量较高的植物总称，生长在我国南方地区的高山及丘陵地带，是我国特有的一种纯天然高级油料，与橄榄、油棕和椰子并称为世界四大木本油料植物（庄瑞林，2008;杨枝林等，2017;龚洪恩等，2019）。……