Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method
Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method
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DOI：
10.1038/nprot.2006.97
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Chua, Nam-Hai
中科院分区：
文献类型：
--
作者：
Zhang, Xiuren;Henriques, Rossana;Chua, Nam-Hai
在过去的二十年里，几个实验室的共同努力已经导致了农杆菌介导的拟南芥转化的各种方法的发展。其中，花浸法是生产转基因拟南芥植株最简便、应用最广泛的方法。在这种方法中，雌性配子的转化是通过简单地将发育中的拟南芥花序浸入含有0.01 - 0.05% (vol/vol) Silwet L-77的5%蔗糖溶液中几秒钟完成的，重悬的农杆菌细胞携带要转移的基因。经过处理的植物被允许播种，然后将其镀在选择性培养基上以筛选转化体。在2-3个月内，至少可以获得1%的转化频率和至少几百个独立的转基因系，仅从两盆渗透植物（每盆20 - 30株）中产生。在这里，我们描述了我们实验室中用于拟南芥转化的花浸法的常规方案。转基因拟南芥植株可以在大约3个月内获得。
Collective efforts of several laboratories in the past two decades have resulted in the development of various methods for Agrobacterium tumefaciens - mediated transformation of Arabidopsis thaliana. Among these, the floral dip method is the most facile protocol and widely used for producing transgenic Arabidopsis plants. In this method, transformation of female gametes is accomplished by simply dipping developing Arabidopsis inflorescences for a few seconds into a 5% sucrose solution containing 0.01 - 0.05% (vol/vol) Silwet L-77 and resuspended Agrobacterium cells carrying the genes to be transferred. Treated plants are allowed to set seed which are then plated on a selective medium to screen for transformants. A transformation frequency of at least 1% can be routinely obtained and a minimum of several hundred independent transgenic lines generated from just two pots of infiltrated plants (20 - 30 plants per pot) within 2-3 months. Here, we describe the protocol routinely used in our laboratory for the floral dip method for Arabidopsis transformation. Transgenic Arabidopsis plants can be obtained in approximately 3 months.