Studies On Genetic Transformation Of Wheat With Shoot Apical Meristem

There were many successful reports about the genetic transformation on wheat, but still it was considered a relatively difficult fields in the this aera. Systematic studies were carried out on the genetic transformation of wheat shoot apex, via bombardment the agrobacterium-mediated transformation methods. The main results were as follows:1. A technical system for transforming wheat shoot tips by particle bombardment was established preliminarily, which including the exposure of shoot tips by breaking off the coleoptile with forceps and blade, bombarment at 1,100 psi and 9cm as the optimized parameters, ect. The GUS assay show that the transient expression rate was 7.1% on average. A total of 802 T₀ spikes were harvested from the bombardments with the plasmid pABH9 carrying with badh/bar gene. Total 105 PPT resistant T₁ plants, coming from 42 T₀ plants, were obtained. PCR analysis showed that 6 T₁ plants were positive.2. The agrobacterium-mediated transformation system of wheat with shoot apex as explants, was also established preliminarily. A total of 221 T₀ plants were obtained, with agrobacterium C58 strain harboring pBIN438-BADH plasmid . Total 23 kanamycin resistant T₁ plants, corresponding to 6 T₀ plants, were obtained. PCR analysis showed that 2 plants from T₁ generation were positive.3. We developed a stainless steel brush to stab the shoot tips. The thread diameter of the brush is only 4～8μm. Several hundreds of wound points could be produced per operation. The brush not only could be used to operate delicate hurt, but also could be used to sweep off the remaining leavies around shoot tips. This has the practical value for improving the agrobacterium-mediated transformation and bombardment transforming method.