Protocol: Streamlined sub-protocols for floral-dip transformation and selection of transformants in Arabidopsis thaliana
Protocol: Streamlined sub-protocols for floral-dip transformation and selection of transformants in Arabidopsis thaliana
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DOI：
10.1186/1746-4811-5-3
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发表时间：
2009-01-27
期刊：
影响因子：
5.1
通讯作者：
Davis, Seth J.
中科院分区：
文献类型：
--
作者：
Davis, Amanda M.;Hall, Anthony;Davis, Seth J.
转化子的产生和鉴定对于许多基因功能的研究是必不可少的。在拟南芥中，一种称为花浸的革命性方案是现在最广泛使用的转化方法。尽管它很强大，但它涉及许多相对耗时费力的步骤，包括操作根癌农杆菌培养物和选择具有抗生素选择标记的植物系的无菌程序。此外，在要引入多个转基因的情况下，通过多代连续转化来实现这一点显著增加了所需的时间。为了规避这些瓶颈，我们开发了三个简化的子协议。首先，我们发现A. thaliana可以通过直接浸入A.根癌农杆菌培养物补充有表面活性剂，消除了将培养基更换为缓冲溶液的需要。接下来，我们举例说明A.具有双重转化事件的拟南芥品系可以通过简单地将花浸入两种A.根瘤菌培养物具有不同的转化载体。最后，我们报告了一种在层析砂上进行种子选择的替代方法，该方法不需要对种子进行表面消毒。这些子协议可以单独使用或组合使用，节省时间和金钱，并减少污染的可能性。
Generating and identifying transformants is essential for many studies of gene function. In Arabidopsis thaliana, a revolutionary protocol termed floral dip is now the most widely used transformation method. Although robust, it involves a number of relatively time-consuming and laborious steps, including manipulating an Agrobacterium tumefaciens culture and aseptic procedures for the selection of plant lines harboring antibiotic-selection markers. Furthermore, where multiple transgenes are to be introduced, achieving this by sequential transformations over multiple generations adds significantly to the time required. To circumvent these bottlenecks, we have developed three streamlined sub-protocols. First, we find that A. thaliana can be transformed by dipping directly into an A. tumefaciens culture supplemented with surfactant, eliminating the need for media exchange to a buffered solution. Next, we illustrate that A. thaliana lines possessing a double-transformation event can be readily generated by simply by floral-dipping into a mixture of two A. tumefaciens cultures harboring distinct transformation vectors. Finally, we report an alternative method of transformant selection on chromatography sand that does not require surface sterilization of seeds. These sub-protocols, which can be used separately or in combination, save time and money, and reduce the possibility of contamination.