Studies On Transformation Of Wheat Mediated With Agrobacterium Without Tissue Culture

Agrobacterium tumefaciens has been widely utilized as a vector for plant genetic transformation, particularly on dicotyledonous plants. The last few years, the transformation of Monocotyledonous plants mediated with Agrobacterium has made good progress. There have been some routine method in transforming the rice and corn, in contrast, the wheat transformation are developing slowly, although the researcher has made breakthrough in using Agrobacterium to transform wheat since 1997. One of the main reason is the tissue culture for the wheat is very difficult and with the restriction to the different genotype, another one, the window of the reception material is so narrow that make there is no enough material for a long-time research.We select two stage in the time of wheat growing, that is the stage of seedling and the stage of flowering, to develop the method to transform wheat without tissue culture. We use the floral dip method to deal with the flower of wheat and get the result that when the first spikelet in the middle of the whole ear begin pollination and until it become a half granule, we can get high efficiency of the GUS activity if we perform the inoculation in this course. The method that deal with the seedling is the first attempt in wheat transformation, and the difficulty is mainly because the Agrobacterium can not transfer into the inner of the seedling and the vir gene can not express efficiently, to resolve the problem we use the surfactants and vacuum infiltration to help the Agrobacterium to transfer into the inner tissue, and add AS to help vir gene express.The frequency of GUS activity in the method of floral dip reach 61%, deal with the seedling we get the frequency of 22% for the GUS activity In the apex meristem of the seedling, slice the stained material and we observe the inner tissue also get blue.We also develop a new screen system in using hygromycin to screen the transformant of wheat, in the method we can easily observe the difference, and is also easy to perform, using this method we have screen 6 plants, while it's regretful that we haven't detect the integration of T-DNA when perform the PCR in the later-grown leaf of the 6 selected plant.