Establishment And Application Of The "Clean" Fragment Transformation System

Wheat is one of major world crops,as such is a primary target for improvement of agronomic characteristics by genetic engineering. The goal of genetic manipulation is to achieve stable transgenic plants with improvement of argronomical characteristics, and to minimise its potentical threat to health an environment. Transformation via particle bombardment has been the predominated method in wheat genetic manipulation. In routine transformation, the whole plasmid is transfered to the regenerable materials of wheat to recover stable transgenic plants resulted in the integration of vector backbone sequence into the host genome along with the genes of interests. The vector backcone sequence is not only superfluous but also has the tendency to promote arrangement by offering the homogenous sequences or hot-spots. In the present investigation, several gene cassettes were simultaneously transferred into different wheat varieties,and the stable transgenic plants have been obtained. The"clean"fragment transformation was efficiently established and optimized using linear gene cassettes lacking vector backbone sequence. This technique removed the vector backbone sequences from transgenic plants,and was used to improve the important agronomically characteristic of crops.Firstly, we used the scorable and selective remarker genes (gus and bar) to develop and optimise the"clean"fragement transformation procedure. Stable transgenic plants were recovered from the immature endosperms with transgenes on the circular whole plasmid(s) or linear gene cassettes lacking vector backbone sequences from the same plasmids, conrresponding to promoters, open reading frames and terminaters. The efficiency of transformation using gene cassettes was twice of that using whole plasmid(s). Southern-blot results showed 1-3 hybridizing bands in transgenic plants carrying gene cassettes. Expression analysis indicated gene cassettes expressed stably and segregated abiding Mendelian law in subsequent generations. Secondly, the optimization of the transformation procedure using gene cassettes can increase the efficiency of transformation typically, and thus gene cassettes were able to such efficiently integrated with the host genome as the whole plasmid(s). Finally, this"clean"fragment transformation was respectively utlilized to transform two kinds of HMW-GS genes 1Ax1 and 1Dx5 into the host genome. SDS-PAGE showed that amout of HMW glutenin subunit was increased in the stable transgenic plants.