Optimization Of Soybean Transformation System And Study On Shoot Regeneration From Soybean Root By Over-expressing AtWUSCHEL Gene

Soybean is one of the most important oil crops. Soybean seeds provide excellent source of protein for human and livestock nutrition. It is hard for traditional breeders to further improve soybean quality and production and to control the diseases and insects of soybean plants due to the limited germplasm resources. Biotech breeding has great advance in broadening germplasm resources. Stable transformation efficiency is needed for genetic modification of soybean. However soybean has been regarded as recalcitrant to transformation for many years. Increasing soybean transformation efficiency is of great importance for biotech breeding. Here, some conditions affected Agrobacterium tumerfaciens-mediated soybean transformation were evaluated. And in order to induce shoots from the isolated soybean roots, the goal of which was to use Agrobacterium rhizogenes-mediated transformation in soybean, AtWUS gene was expressed in soybean plants. The results were as follows:1. Soybean germination time affected contamination rate though no significance difference in transformation frequency (TF) between one day germination and five days germination. The explants prepared from one day germination seeds had less contamination than those from five days. Wounding too many times to the cotyledonary nodes for agrobacterium infection is not favourable for shoot elongation. It would produce hard callus on the wounding surface and impaired shoots regeneration. The TF was usually low if wounding too many times. Applying AgNO3 in Shoot Inducing or Shoot Elongation medium could delay leaf senescence and increased shoot elongation number. High moisture condensation on petri-dish lid could cause leaf chlorosis and death, which can impair plant growth. Poor health state of the plants caused a low survival rate when they were transferred to soil. Lights turned on inter-layer on the shelf and avoiding of cool air to blow directly onto the petri-dish could reduce moisture condensation under the petri-dish lids. The sensitivity to 6-BA of different soybean cultivars is variant. Zigongdongdou induced less shoots when used the same concentration of 6-BA as Jilinxiaoli No.1 and Williams 82. Varying from some plants, applying Silwet in the infection medium could not increase soybean TF.2. AtWUS gene was cloned into a plant expression vector and transformed into soybean. Heritable transgenic plants that over-expressing AtWUS gene were obtained. Although most of the transgenic plants had normal phenotype and grew well in To, the heredity rate of the AtWUS gene was low from To to T1. And some of the transgenic soybean plants, just as the other plants expressing WUS gene, showed abnormal phenotype, such as growing slowly, having dark green leaves, the edge of the leaves turned out to the abaxial, cluster of small pods which can not produce normal seeds and supernumerary carpels. Increased expression of WUS induced the over-expression of CLV3 gene in transgenic soybean plants.