A circulatory system useful both for long-term somatic embryogenesis and genetic transformation in Vitis vinifera L. cv. Thompson Seedless
A circulatory system useful both for long-term somatic embryogenesis and genetic transformation in Vitis vinifera L. cv. Thompson Seedless
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DOI：
10.1007/s11240-014-0471-y
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发表时间：
2014-03
期刊：
影响因子：
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通讯作者：
Q. Zhou;Lingmin Dai;Siyan Cheng;Jing He;Dan Wang;Jianxia Zhang;Yuejin Wang
中科院分区：
文献类型：
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作者：
Q. Zhou;Lingmin Dai;Siyan Cheng;Jing He;Dan Wang;Jianxia Zhang;Yuejin Wang
为了建立有效的功能验证和品种抗性改良的再生体系，通过葡萄二次胚胎发生的循环周期，建立了一个可用于胚性培养保持和转化的长期体系。CV。汤普森·塞德斯。对3种培养基及5种体细胞胚胎在二次胚胎发生中的作用进行了评价。鱼雷阶段和子叶中期的体细胞胚胎诱导效果最好，再诱导率约为80%。在该系统中产生的胚性愈伤组织、原胚团和SE可繁殖3年以上，均可用于农杆菌介导法转化。在此基础上，对不同的转基因选择制度进行了比较。在共培养后4周添加卡那霉素对胚胎恢复效果最好。通过在两种培养基上交替培养，提高了植株转化率：一种含有0.2 mg的L−1BA，另一种含有0.25 mg的L−1激动素。为了进一步验证该系统的有效性，将中国野生葡萄泛素连接酶基因(VpPUB23)导入Thompson Seedless进行功能鉴定。在获得的351株转基因植株中，与非转基因植株相比，高表达VpPUB23的植株对白粉病的抗性降低。
To establish an efficient regeneration protocol for functional validation and variety resistance improvement, a long-term system that useful for embryogenic culture maintenance and transformation was developed through recurrent cycles of secondary embryogenesis fromVitis viniferaL. cv. Thompson Seedless. Three media and five types of somatic embryo in secondary embryogenesis were evaluated. Somatic embryos (SE) in the torpedo and mid-cotyledonary stages gave the best embryogenic responses with re-induction rates of about 80 %. Embryogenic callus, proembryonic masses and SE produced in the system, could be propagated for over 3 years and all proved competent forAgrobacterium-mediated transformation. Based on this system, different transgenic selection regimes were compared. Addition of kanamycin at 4 weeks after co-cultivation was optimal for embryo recovery. Plant conversion was improved by alternating culture on two media: one containing 0.2 mg l−1BA and the other 0.25 mg l−1kinetin. To further test the efficiency of the system, a ubiquitin ligase gene (VpPUB23) from Chinese wildVitis pseudoreticulatawas transferred into Thompson Seedless for functional evaluation. Of the 351 transgenic plants obtained, those overexpressingVpPUB23exhibited decreased resistance to powdery mildew compared with non-transgenic plants.