巴戟天离体再生及农杆菌介导的遗传转化

目的 :在巴戟天离体再生的基础上 ,建立一套根癌农杆菌转化的有效方法 ,为巴戟天新品种选育、引入外源目的基因奠定基础。方法 :以巴戟天带节茎为材料 ,农杆菌菌株为EHA101,含有质粒载体pGA482GG ,质粒上插入了GUS、NPTⅡ基因。结果 :MT基本培养基附加BA 1mg·L^-1,外植体出芽率最高 (97.8%) ,BA浓度升高 ,出芽率下降。外植体在培养基上的接种方式 ,以竖放为好 ,出芽所需的时间短且出芽率高。根的诱导 ,以MT附加 0.2～0.5mg·L^-1NAA效果较好 ,生根率可达 80.0%以上。巴戟天带节茎进行遗传转化 ,卡那霉素作为选择试剂 ,浓度为 50mg·L^-1。头孢霉素用作抑菌剂 ,浓度为 300mg·L^-1。经感染的外植体 ,在选择培养基上筛选 1.5个月后 ,抗性芽发生率为 14.8% ,GUS基因表达检测结果 ,抗性植株中 ,GUS反应呈阳性所占比例为 22.2%。结论 :通过探讨巴戟天的离体培养及根癌农杆菌介导的遗传转化 ,获得了较高的离体再生频率 ,建立了有效的遗传转化系统。

In Vitro Culture and the Agrobacterium -mediated Genetic Transformation of Morinda officinalis

OBJECTIVE: To establish an effective system for the Agrobacterium-mediated genetic transformation of M. officinalis, for laying a foundation for the improvement of breeds and introduction of foreign objective genes. METHOD: The explants used for culture were the nodular stem segments from M. officinalis. Agrobacterium tumefaciens strain was EHA101, containing vector plasmid pGA482GG. The GUS gene and NPT II gene were introduced into the plasmid. RESULT: MT basal medium with BA 1 mg.L-1 was effective to inducing the direct shoot formation, and the frequency of shoot formation was 97.8%. As BA concentrations increased, the ability of shoot formation decreased. The explants oriented with their apical ends protruding from the medium produced more shoots than when they were placed with their basal end upright or were placed horizontally. The optimal rooting medium for regenerating shoots was MT basal medium supplemented with 0.2 to 0.5 mg.L-1 NAA, and a root induction rate over 80.0% was observed. The selection pressure for kanamycin was 50 mg.L-1. Cefotaxime was used as antibiotics, and the concentration was 300 mg.L-1. After 1.5 months, 14.8% resistant shoots were emerged from the explants. Histochemical GUS assay showed that 22.2% of the resistant plants were GUS-positive. CONCLUSION: Plant regeneration system and Agrobacterium-mediated genetic transformation have been established for M. officinalis in vitro.