Preliminary Study Of Agrobacterium-mediated Transformation Method Of Wheat

Wheat, Triticum aestivum L., is of the utmost importance both nutritionally and economically in the world at large. The introduction of novel transgenes to produce better yielding and more resilient wheat crops depends on successful transformation techniques. Agrobacterium-mediated transformation and microparticle bombardment are the two widely used methods for wheat genetic transformation. So far, the majority of wheat genetic transformation that has been reported utilized microparticle bombardment. Agrobacterium tumefaciens is currently used, because it possesses several advantages over other forms of transformation including the ability to transfer large segments of DNA with minimal rearrangement, the precise insertion of transgenes resulting in fewer copies of inserted genes, and simple technology with lower cost. In this study, we present an Agrobacterium-mediated transformation method for wheat using freshly isolated immature embryos, precultured immature embryos, and mature embryos devied calli as explants.The main results are followings:1. By using Agrobacterium-mediated transformation method, freshly isolated immature embryos, precultured immature embryos, and mature embryos devied calli were transformed and 192 plantlets with high resistance to bialaphos were obtained. PCR analysis showed 19 plants were proved to be positive from 2 tested wheat varieties.2. Transformation efficiency via Agrobacterium was improved in several plant species by the use of heat and centrifugation treatments of the explants used for transformation. We analyzed, for the first time in wheat, effects of heat pretreatment and centrifugation of IEs prior to Agrobacterium inoculation. The most optimal treatment,43?for 10 min, cooling 1 min at 0?and centrifugation, yielded 1.5% stable transformation frequency.3. Modification of the ammonium nitrate content in the direct somatic embryogenesis induction medium can increase the number of primary embryos transitioning into shoot development (regeneration rate up to 43.4% vs control 34.7%).4. Osmotic treatment of mature embryos shown to be useful in Agrobacterium-mediated transformation of wheat (regeneration rate up to 39.8% vs control 32.5%).