An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol.
An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol.
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DOI：
10.1186/1746-4811-2-16
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发表时间：
2006-10-24
期刊：
影响因子：
5.1
通讯作者：
Somssich IE
中科院分区：
文献类型：
--
作者：
Logemann E;Birkenbihl RP;Ülker B;Somssich IE
农杆菌真空（Bechtold等1993）和花浸（Clough和Bent 1998）是产生转基因拟南芥植物的非常有效的方法。这些方法允许植物转化而不需要组织培养。在液体培养基中生长的大量细菌培养物对于这两种转化方法都是必需的。由于需要昂贵的大型振动器和有限的空间，这限制了在给定时间内可以完成的转换数量。此外，起始这些液体培养物所必需的来自固体培养基的细菌菌落通常不能以如此大的体积生长。因此，常常错过植物材料转化的最佳阶段，需要种植新的植物材料。为了避免与大型细菌液体培养物相关的问题，我们研究了在平板上生长的细菌是否也适合于植物转化。我们在这里证明，即使在4°C下储存一周后，在平板上生长的细菌也可以以类似的效率用于转化植物。这使得更容易同步农杆菌和植物进行转化。对除草剂选择后存活的T1植物进行DNA凝胶印迹分析，证明存活的植物确实是转基因的。简化的方法与以前报道的协议一样有效，并显着减少了工作量，成本和时间。此外，该协议还降低了涉及转基因生物的大规模污染的风险。最重要的是，使用此修改后的协议，每天可以执行更多的独立转换。
The Agrobacterium vacuum (Bechtold et al 1993) and floral-dip (Clough and Bent 1998) are very efficient methods for generating transgenic Arabidopsis plants. These methods allow plant transformation without the need for tissue culture. Large volumes of bacterial cultures grown in liquid media are necessary for both of these transformation methods. This limits the number of transformations that can be done at a given time due to the need for expensive large shakers and limited space on them. Additionally, the bacterial colonies derived from solid media necessary for starting these liquid cultures often fail to grow in such large volumes. Therefore the optimum stage of plant material for transformation is often missed and new plant material needs to be grown. To avoid problems associated with large bacterial liquid cultures, we investigated whether bacteria grown on plates are also suitable for plant transformation. We demonstrate here that bacteria grown on plates can be used with similar efficiency for transforming plants even after one week of storage at 4°C. This makes it much easier to synchronize Agrobacterium and plants for transformation. DNA gel blot analysis was carried out on the T1 plants surviving the herbicide selection and demonstrated that the surviving plants are indeed transgenic. The simplified method works as efficiently as the previously reported protocols and significantly reduces the workload, cost and time. Additionally, the protocol reduces the risk of large scale contaminations involving GMOs. Most importantly, many more independent transformations per day can be performed using this modified protocol.
影响因子：
7.2
作者：
Clough, SJ;Bent, AF
通讯作者：
Bent, AF
影响因子：
7.4
作者：
Desfeux, C;Clough, SJ;Bent, AF
通讯作者：
Bent, AF
影响因子：
16
作者：
Gómez-Gómez, L;Boller, T
通讯作者：
Boller, T