葡萄未成熟胚诱导体细胞胚发生和植株再生与遗传鉴定  

Somatic embryogenesis from immature zygotic embryos and genetic fidelity identificating of regenerated plants of grapevine

作　　者：孙兴民[1] 余智莹[1] 张萌[1] 董瑞奇[1] 陶建敏[1] 

机构地区：[1]南京农业大学园艺学院,江苏南京210095

出　　处：《南京农业大学学报》2012年第3期13-18,共6页Journal of Nanjing Agricultural University

基　　金：国家948项目(2011-G28);江苏省农业科技自主创新资金项目(cx(10)110);江苏省农业资源开发局项目(2012-kj59)

摘　　要：以二倍体和四倍体葡萄杂交获得的未成熟合子胚为材料,诱导体细胞胚发生。采用的初生胚诱导培养基(SE培养基)为:NN69+1.80 mg.L-1NAA+0.4 mg.L-1水解酪蛋白+2 g.L-1活性炭+30 g.L-1蔗糖+6 g.L-1琼脂;次生胚诱导培养基分别为:3/4MS+0.35 mg.L-1IBA+30 g.L-1蔗糖+6 g.L-1琼脂(扩繁培养基)和MS+1.5 g.L-16-BA+0.2 g.L-1IBA+30 g.L-1蔗糖+6 g.L-1琼脂(胚状体萌发培养基)。结果表明:初生胚诱导率为11.11%和16.67%,体细胞胚在扩繁培养基上再生植株。利用流式细胞仪和SSR标记检测源自同一未成熟合子胚(胚珠)的不同株系的遗传稳定性,结果显示,所测植株均为三倍体,且都来自杂交所得的未成熟合子胚。本研究所建立的体细胞胚诱导方法及遗传稳定性检测技术,能够为植物材料保存、扩繁,转基因应用和研究植物发育及极性建成等提供途径。Somatic embryogenesis and plant regeneration were successfully established on Nitsch and Nitsch（NN69）medium from immature zygotic embryos（IZEs）of interploid crossing 2004-6-12 ×＇Aki Queen＇.The optimum hormone combinations was NN69＋1.80 mg·L-1 NAA＋0.4 mg·L-1 casein hydroly sate＋2 g·L-1 activated carbon＋30 g·L-1 sucrose＋6 g·L-1 agar（SE medium）for primary somatic embryogenesis directly from IZEs,with the frequency varying from 11.11% to 16.67% among IZEs of different sampling period after pollination.And importantly,somatic embryogenesis and normal germinating occurred synchronically.While the optimum medium for secondary somatic embryogenesis were 3/4MS＋0.35 mg·L-1 IBA＋30 g·L-1 sucrose＋6 g·L-1 agar（multiplication medium）and MS＋1.5 g·L-1 6-BA＋0.2 g·L-1 IBA＋30 g·L-1 sucrose＋6 g·L-1 agar（somatic embryo germination medium）.After subcultured on germination medium,somatic embryos converted into normal plantlets.The analysis of DNA content and genetic fidelity determined by flow cytometry and SSR of the regenerated plantlets clearly indicated that the four plantlets were triploid and the normal germinated plant and the somatic embryogenesis-derived plants came from the same immature zygotic embryos.We concluded that somaclonal variation was almost absent in our grapevine plant regeneration system,which was useful in research of transformation and embryo development and so on.

关 键 词：葡萄 未成熟合子胚 体细胞胚发生 流式细胞仪 SSR 

分 类 号：S663.1[农业科学—果树学]