Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes
Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes
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DOI：
10.3791/60782
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发表时间：
2020-02-01
影响因子：
1.2
通讯作者：
Wang, Kan
中科院分区：
文献类型：
--
作者：
Masters, Alicia;Kang, Minjeong;Wang, Kan
这里展示的是使用形态发生基因Baby boom（Bbm）和Wuschel 2（Wus 2）的农杆菌介导的玉米自交系遗传转化的详细方案。Bbm受玉米磷脂转移酶基因（Pltp）启动子调控，Wus 2受玉米生长素诱导型（Axigl）启动子控制。将在转移DNA（T-DNA）上携带这些形态发生基因和农杆菌毒力（vir）基因的额外拷贝的农杆菌菌株用于感染玉米未成熟胚外植体。体细胞胚在受感染胚的盾片上形成，可以通过除草剂抗性选择并萌发成植株。构建到DNA构建体中的热激活cre/IoxP重组系统允许在转化过程的早期阶段从玉米基因组中去除形态发生基因。使用该方案，W22、B73和Mo 17分别可以实现约14%、4%和4%的转化频率（每100个感染胚胎的独立转基因事件的数量）。
Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axigl) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/IoxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.