抑制‘白花油茶’叶片外植体褐化和诱导愈伤组织的研究

Study on Browning of Explant and Induction of Callus from Camellia oleosa Rehd Leaves

以‘白花油茶’Camellia oleosa Rehd叶片作为材料研究愈伤组织诱导过程中降低外植体的褐化现象和提高诱导率的方法。对‘白花油茶’嫩枝弱光培养,用维生素C漂洗外植体,培养基中加入维生素C和活性炭来抑制外植体的褐化现象,对基本培养基种类、激素浓度、琼脂浓度和糖类进行单因素实验和不同组合试验,以外植体的存活率、愈伤组织诱导率及其生长状况为指标,优化了‘白花油茶’叶片愈伤组织诱导的条件。结果表明,水中弱光培养枝条1~2 d,0.3 g·L^(-1)的维生素C溶液漂洗外植体2~3 min,培养基中添加1.0 g·L^(-1)活性炭和0.3 g·L^(-1)维生素C等都可以显著减少外植体的褐化,从而提高外植体的存活率。优化的培养基成分为基本培养基WPM,包含1.0 mg·L^(-1) 6-BA和1.5 mg·L^(-1) 2,4-D,15.0 g·L^(-1)蔗糖和15.0 g·L^(-1)葡萄糖,6.5 g·L^(-1)琼脂,1.0 g·L^(-1)活性炭和0.3 g·L^(-1)维生素C等能够提高外植体的愈伤组织的诱导率,减少‘白花油茶’外植体褐化现象,适当的培养条件能够快速有效地诱导出有活性的愈伤组织,为再生‘白花油茶’植株打下了基础。

Camellia oleosa Rehd leaves and branches were collected during March and June of 2016 at demonstration base in Hunan University of Science and Engineering for tissue culture with leaves as explant.Experiments were carried out with different culture medium,different hormones with different concentration,different agar concentration and sugar.The result demonstrated that leaves from water cultured branches for 1-2 days,explant washed by 0.3 g·L^（-1） vitamin C for 2-3 minutes,in culture media added with 1.0 g·L^（-1 ）activated carbon and 0.3 g·L^（-1） vitamin C could reduce browning of explant.The optimal culture medium for inducing callus was WPM,including 1.0 mg·L^（-1） 6-BA＋1.5 mg·L^（-1） 2,4-D＋15.0 g·L^（-1） sucrose＋15.0 g·L^（-1） glucose＋6.5 g·L^（-1 ）agar＋1.0 g·L^（-1） activated carbon＋0.3 g·L^（-1） vitamin C.