Clough S J, Bent A F
Department of Crop Sciences, University of Illinois at Urbana-Champaign 61801, USA.
Plant J. 1998 Dec;16(6):735-43. doi: 10.1046/j.1365-313x.1998.00343.x.
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.
农杆菌真空渗透法使得无需植物组织培养或再生就能转化拟南芥成为可能。在本研究中，对该方法进行了评估，并开发了一种经过大幅改进的转化方法。省去了耗费人力的真空渗透过程，代之以将发育中的花组织简单浸泡在含有根癌农杆菌、5%蔗糖和每升500微升表面活性剂Silwet L - 77的溶液中。蔗糖和表面活性剂对花浸法的成功至关重要。在有大量未成熟花芽且角果较少时接种的植株产生转化后代的比率最高。无需植物组织培养基、激素苄氨基嘌呤和pH调节，农杆菌可以以一系列细胞密度应用于植株。重复应用农杆菌可使转化率和转化体的总产量提高约两倍。接种后覆盖植株1天以保持湿度也可使转化率提高两倍。多种生态型都可用此方法转化。这种改进的方法应有助于为诸如T - DNA基因标签、图位克隆或靶向基因替换尝试等工作进行拟南芥的高通量转化。
