Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method
Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method
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DOI：
10.1104/pp.123.3.895
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发表时间：
2000-07-01
期刊：
影响因子：
7.4
通讯作者：
Bent, AF
中科院分区：
文献类型：
--
作者：
Desfeux, C;Clough, SJ;Bent, AF
农杆菌介导的拟南芥转化的花浸法允许有效的植物转化而不需要组织培养。为了便于与其他植物物种一起使用，我们研究了这种方法的机制。在人工异交实验中，将根癌农杆菌应用于花粉供体植物不产生任何转化的后代，而将农杆菌应用于花粉受体植物以0.48%的比率产生转化体。具有在35 S、LAT 52或ACT 11启动子控制下携带gusA（编码β-葡萄糖醛酸酶[GUS]）的T-DNA的农杆菌菌株显示GUS活性递送至发育中的胚珠，而未观察到花粉或花粉管的GUS染色。来自同一种荚的转化子含有独立的T-DNA整合事件。在拟南芥花中，雌蕊群发育为开放的花瓶状结构，在开花前约3d融合形成封闭的子房室。与此相关，我们发现农杆菌感染的时机是至关重要的。只有在开花前5天或更长时间施用农杆菌，才能获得转化体并观察胚珠和胚囊的GUS染色。一个高6倍的转化率，获得了一个CRABS-CLAW突变体，保持开放的雌蕊群。我们的研究结果表明，胚珠是生产性转化的花浸法的网站，并进一步表明，农杆菌必须交付到内部的发展雌蕊室关闭之前，如果要实现有效的转化。
The floral-dip method for Agrobacterium-mediated transformation of Arabidopsis allows efficient plant transformation without need for tissue culture. To facilitate use with other plant species, we investigated the mechanisms that underlie this method. In manual outcrossing experiments, application of Agrobacterium tumefaciens to pollen donor plants did not produce any transformed progeny, whereas application of Agrobacterium to pollen recipient plants yielded transformants at a rate of 0.48%. Agrobacterium strains with T-DNA carrying gusA (encoding beta-glucuronidase [GUS]) under the control of 35S, LAT52, or ACT11 promoters revealed delivery of GUS activity to developing ovules, whereas no GUS staining of pollen or pollen tubes was observed. Transformants derived from the same seed pod contained independent T-DNA integration events. In Arabidopsis flowers, the gynoecium develops as an open, vase-like structure that fuses to form closed locules roughly 3 d prior to anthesis. In correlation with this fact, we found that the timing of Agrobacterium infection was critical. Transformants were obtained and GUS staining of ovules and embryo sacs was observed only if the Agrobacterium were applied 5 d or more prior to anthesis. A 6-fold higher rate of transformation was obtained with a CRABS-CLAW mutant that maintains an open gynoecium. Our results suggest that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure if efficient transformation is to be achieved.