An improved method for Preparing Agrobacterium cells that simplifies theArabidopsis transformation.pptVIP

An improved method for Preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol Background: 1.The Agrobacterium vacuum (Bechtold et al 1993) ,and floral-dip (Clough and Bent1998 ,who eliminated the vacuum-infiltration step) are very efficient methods for generating transgenic Arabidopsis plants. 2.优点： These methods allow plant transformation without the need for tissue culture. 3.缺陷：⑴Large volumes of bacterial cultures grown in liquid media 。⑵This limits the number of transformations and limited space on them. ⑶. the bacterial colonies often fail to grow in such large volumes. ⑷Therefore the optimum stage of plant material for transformation is often missed and new plant material needs to be grown. 4.Clough and Bent(2002) `s find ⑴several other variables also thought to be important for the efficiency of transformation. ⑵the MS salts, hormones and other constituents make no difference, that the density of the bacteria is not critical, and again that use of vacuum basically has no effect on transformation efficiency as long as a certain amount of surfactant is present. ⑶ plant health and the developmental state of the flowers were found to be the most important factors. Here we describe a modified version of the Agrobacterium mediated transformation that does not require growth of large bacterial liquid cultures. Instead, plants are transformed with bacteria grown on plates. We demonstrate that this method is as efficient and reliable as the previous methods in generating transgenic plants. Methods The simplified transformation method modified from the protocol by Clough and Bent - Grow healthy Arabidopsis plants until they are flowering(see Figure 1A). Optional: Clip first bolts to encourage proliferation of many secondary bolts. Optimal plants have many immature flower clusters and only few fertilized siliques, although a range of plant stages can be successfully transformed. - Transform the DNA construct into the