提高油菜游离小孢子胚诱导频率的研究

Studies on Improving the Frequency of Isolated Microspore-derived Embryos in Brassica napus L.

以11个甘蓝型油菜基因型为材料,采用NLN培养基进行游离小孢子培养,对如何提高可培养的基因型范围和产胚率进行了研究。结果表明,11个油菜基因型中有10个基因型可以诱导出胚,培养成功率达90.9%,表明采用NLN培养基进行游离小孢子培养油菜基因型范围比较宽,但各基因型间小孢子胚产量差别很大,每花蕾产胚量为0.08~3.53个,TR4和TR9两个基因型每花蕾产胚可达3.23,3.53个。以TR4和TR9两个基因型为试材,进一步改进培养基和培养方法,采用NLN培养基中添加激素和活性炭方法,可大大提高产胚率,产胚量分别达到7.11和10.05个/蕾;接种后,小孢子经33℃高温预处理可显著影响产胚量。子叶形小孢子胚在光下适当培养后转入B5＋BA 0.2 mg/L＋NAA 0.02 mg/L继代培养基上,大多数胚能长成绿芽,B5＋6-BA 0.2 mg/L＋3%蔗糖＋1%琼脂培养基有利于小孢子胚长成植株。

The application of microspore culture technique was restricted because of its low frequency of embryogenesis. Two methods of enhancing the frequency of embryogenesis were employed in the study, namely, activated charcoal and 6-BA treatment in NLN-13 media. Eleven Brassica napus genotypes（TRl-11 ） were used for isolated microspore culture. Among the 11 genotypes, 10 produced microspere-derived embryos. The production of microspore-derived embryos varied remarkably among these varieties. Embryo yield per bud is from 0.08 to 3.53. TR4 and TR9 were the best genotypes for microspere culture and their frequency of embryogenesis is 3.23 and 3.53, respectively. When treated with 0.5 mg/L activated charcoal after adding 0.2 mg/L 6-BA in NLN-13 media,TR4 and TR9 produced 7. 11 and 10.05 embryos per bud respectively, 2.88 and 6.52 higher than CK. Average embryo yield increased after pretreated in 33℃ for 24 hours. Microspore-derived embryos on B5 medium with 6-BA 0.2 mg/L and 3% Sucrose and 1% agar was good for regenerated plants. When microspore embryoid developed into the globular stage and early torpedo stage, adding fresh medium and transferring the cultures under shaking would greatly improve the synchronization of embryo development and reduce the abnormal embryoids.