The floral inoculating protocol: a simplified Arabidopsis thaliana transformation method modified from floral dipping
The floral inoculating protocol: a simplified Arabidopsis thaliana transformation method modified from floral dipping
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DOI：
10.5511/plantbiotechnology.27.349
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发表时间：
2010-01-01
影响因子：
1.6
通讯作者：
Narusaka, Yoshihiro
中科院分区：
文献类型：
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作者：
Narusaka, Mari;Shiraishi, Tomonori;Narusaka, Yoshihiro
根癌农杆菌介导的浸花法是目前应用最广泛的拟南芥遗传转化方法。“浸花”过程中，A。将拟南芥花蕾浸入农杆菌细胞悬浮液中需要在液体培养基中生长的大量细菌培养物、大型振荡器和离心机以及用于它们的实验空间。这些因素限制了一次可以发生的转换数量。我们确定A.可以用105 μ l的农杆菌细胞悬浮液在花蕾中转化拟南芥，从而避免使用大体积的农杆菌培养物。使用这种改进的方案，我们从每个含有3A的盆中每次转化获得15-50个转基因植株。拟南芥植物。该方案可用于后续分析。这种简化的方法，没有花浸渍，这需要大量的农杆菌培养，提供了有效的转化，如以前报道的协议。这种方法减少了所需的工作量、成本、时间和空间。此外，这个修改的协议的一个重要方面是，它允许同时执行许多独立的转换。
The floral dip protocol mediated by Agrobacterium tumefaciens is the most widely used transformation method for Arabidopsis thaliana. The "floral dip" process in which A. thaliana flower buds are dipped in an Agrobacterium cell suspension requires large volumes of bacterial cultures grown in liquid media, large shakers and centrifuges, and experimental space for them. These factors limit the number of transformations that can occur at once. We established that A. thaliana can be transformed by inoculating 5 mu l of Agrobacterium cell suspension in flower buds, thus avoiding the use of large volumes of Agrobacterium culture. Using this modified protocol, we obtained 15-50 transgenic plants per transformation from each pot containing 3 A. thaliana plants. The protocol is satisfactory to be used for subsequent analyses. This simplified method, without floral dipping, which requires large volumes of Agrobacterim culture, offers as efficient a transformation as previously reported protocols. This method reduces the required workload, cost, time, and space. Furthermore, an important aspect of this modified protocol is that it allows many independent transformations to be performed at once.