Development of an efficient regeneration and Agrobacterium-mediated transformation system in crab apple (Malus micromalus) using cotyledons as explants
Development of an efficient regeneration and Agrobacterium-mediated transformation system in crab apple (Malus micromalus) using cotyledons as explants
复制标题
使用子叶作为外植体开发海棠 (Malus micromalus) 的高效再生和农杆菌介导的转化系统
DOI：
10.1007/s11627-013-9544-6
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发表时间：
2014-03-01
影响因子：
2.6
通讯作者：
Zhang, Zhihong
中科院分区：
文献类型：
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作者：
Dai, Hongyan;Li, Wenran;Zhang, Zhihong
苹果已成为蔷薇科遗传和基因组研究的典范种，但以离体叶片为外植体，通过农杆菌介导转化获得转基因苹果植株的难度较大。本研究以小苹果（Malus micromalus）子叶为外植体，建立了一种高效的再生和农杆菌介导的转化体系。从离体培养10-14 d的成熟胚苗上摘取的微苹果草近端子叶适合作为外植体进行再生和农杆菌介导的转化。子叶外植体在再生培养基上与农杆菌EHA105、pCAMBIA2301共培养3 d。在含20mg /L卡那霉素的选择性再生培养基上培养子叶外植体产生抗卡那霉素芽。在农杆菌悬浮液或共培养培养基中添加乙酰丁香酮对卡那霉素抗性芽的再生有轻微的促进作用。卡那霉素抗性芽的外植体最大比例为11.7%。通过β-葡萄糖醛酸酶活性的组织化学分析和新霉素磷酸转移酶II基因的聚合酶链反应扩增，证实了推定转化的植株。该转化系统还可以恢复从胚芽发育的非转化等基因对照，因此适合于苹果的功能基因组学研究。
Apple has become a model species for Rosaceae genetic and genomic research, but it is difficult to obtain transgenic apple plants by Agrobacterium-mediated transformation using in vitro leaves as explants. In this study, we developed an efficient regeneration and Agrobacterium-mediated transformation system for crab apple (Malus micromalus) using cotyledons as explants. The proximal cotyledons of M. micromalus, excised from seedlings that emerged from mature embryos cultured for 10–14 d in vitro, were suitable as explants for regeneration and Agrobacterium-mediated transformation. Cotyledon explants were cocultivated for 3 d with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA2301 on regeneration medium. Kanamycin-resistant buds were produced on cotyledon explants cultured on selective regeneration medium containing 20 mg/L kanamycin. Acetosyringone supplemented in the Agrobacterium suspension or in the cocultivation medium slightly enhanced the regeneration of kanamycin-resistant buds. The maximum percentage of explants with kanamycin-resistant buds was 11.7%. The putative transformed plants were confirmed by histochemical analysis of β-glucuronidase activity and the polymerase chain reaction amplification of the neomycin phosphotransferase II gene. This transformation system also enables recovery of nontransformed isogenic controls developed from embryo buds and is therefore suitable for functional genomics studies in apple.