北美蓝云杉体细胞胚发生技术研究

Somatic embryogenesis of Picea pungens Engelmann

研究北美蓝云杉体细胞胚发生技术,对该树种的胚胎发生机理和建立相关的快繁技术体系等具有一定的理论意义。以成熟胚为外植体,分别对基本培养基、生长调节剂种类和浓度对其体细胞胚发生各阶段的影响进行了研究,同时观察体细胞胚发生过程的形态特征。结果表明:1)胚性愈伤组织诱导培养基,首先在DCR培养基+2,4-D5.0mg/L+6-BA4.0mg/L+KT2.0mg/L诱导15d后,之后转入DCR培养基+2,4-D1.0mg/L+6-BA0.5mg/L+KT0.5mg/L,胚性愈伤诱导率最高为45.3%;2)胚性愈伤组织保持与增殖培养基为1/2LM培养基+2,4-D1.0mg/L+6-BA0.5mg/L+KT0.5mg/L+PEG40001.0g/L,胚性愈伤组织增殖率最高为102.27%;3)IBA可促进成熟体细胞胚的诱导,以1/2LM+IBA0.5mg/L+ABA2.0mg/L(以上4种培养基均附加蔗糖30g/L,谷氨酰胺450mg/L,水解酪蛋白500mg/L,琼脂5.0g/L,pH=5.8)的体细胞胚诱导率最高,为38.10%;4)在1/2MS+1.0g/L活性炭的培养基上,体细胞胚萌发,子叶生长但胚根发生较少。北美蓝云杉体细胞胚诱导过程中出现严重的畸形胚现象及生根难的问题,今后有待进一步研究。

Somatic embryogenesis is critical for understanding the mechanism of embryo development and clonal propagation. In this study,we investigated the optimal culture medium and growth regulator concentration for the in vitro somatic embryogenesis of Picea pungens,using mature embryos as explants. The mature embryos were first cultured on DCR medium containing 2,4-D 5.0 mg/L＋ 6-BA 4.0 mg/L＋ KT 2.0 mg/L for fifteen days,then transferred to DCR medium supplemented with 2,4-D 1.0 mg/L＋ 6-BA 0.5 mg/L＋ KT 0.5 mg/L,on which the highest induction frequency of embryogenic callus was up to 45.3%. Then the embryogenic callus were subcultured and proliferated on 1/2 LM culture medium with 2,4-D 1.0 mg/L＋ 6-BA 0.5 mg/L＋ KT 0.5 mg/L＋ PEG4000 1.0 g/L,with the highest proliferation rate reaching 102.27%. During the induction of somatic embryos,addition of IBA 0.5 mg/L＋ ABA 2.0 mg/L to the 1/2 LM culture medium resulted in the highest induction frequency of 38.10%. All the above-mentioned culture media contained glutamine 450 mg/L,casein hydrostat 500 mg/L,sucrose 30 g/L and agar 5.0 g/L with the pH value of 5.8. Regenerated somatic embryos could germinate and grow on 1/2 MS culture medium with AC 1.0 g/L,but fewer roots were produced. Further research is needed to improve the regeneration frequency of somatic embryos and the root generation.