Studies On Transforming Antisense Phospholipase D_γ (PLD_γ) Gene Into White Clover

White clover (Trifolium repens) is one of the most important legume forage plants. As a forge crop, it plays an important role in improving natural environment and conserving water and soil. The young shoots of White clovers were used as explants of transformation and several factors such as hormone concentration, explant sort, and illumination intensity were investigated to optimize the regeneration system in vitro. The antisense of phospholipase Dγ(anti-PLDγ) gene was introduced into the shoots of White clovers by the Agrobacerium tumefaciens-mediated transformation. In this study, we have established the genetic transformation system of the White clover which is very helpful for improvement of the White clover cultivar. The experimental results showed that different culture mediums can greatly affect on White clovers growth, differentiation, regeneration and root growth. In our experimental conditions, the best medium for shoot elongation is L2 supplemented with 0.25mg/L 6-BA and 94.7umol/L PIC; for shoot regeneration is MS supplemented with 0.2mg/L IBA and 1.2mg/L KT; for root differentiation is 1/2 MS supplemented with 0.2mg/L IBA and 0.8mg/L KT. The effects caused by other physical factors, such as pH, ager concentration, temperature and humidity also have been analyzed. The sensitivity of explants to Cefotaxine and Kanamycin were tested separatly in order to decide a reasonable selective pressure for transformation. The results showed that 300 mg/L Cefotaxine could inhibit Agrobacterium...