Floral dip:: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana
Floral dip:: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana
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DOI：
10.1046/j.1365-313x.1998.00343.x
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发表时间：
1998-12-01
期刊：
影响因子：
7.2
通讯作者：
Bent, AF
中科院分区：
文献类型：
--
作者：
Clough, SJ;Bent, AF
农杆菌真空渗入法使拟南芥的转化成为可能，而不需要植物组织培养或再生。在本研究中，对该方法进行了评价，并开发了一种经过大幅修改的转化方法。劳动密集型的真空渗透过程被消除，有利于简单地浸渍发育中的花组织到含有根癌农杆菌，5%蔗糖和500微升/升的表面活性剂Silwet L-77的溶液中。蔗糖和表面活性剂是成功的花浸法的关键，植物接种时，许多未成熟的花蕾和少数角果是本生产转化后代的最高速率。在植物组织培养基中，激素苄氨基嘌呤和pH调节是不必要的，并且农杆菌可以在一定范围的细胞密度下施用于植物。反复施用农杆菌提高了转化率和转化体的总产量约2倍。接种后覆盖植株1天以保持湿度也使转化率提高了两倍。通过该方法可转化多种生态型。改进后的方法将有助于拟南芥的高通量转化，如T-DNA基因标记，定位克隆，或靶向基因置换的尝试。
The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method, plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.