| Wheat(Triticum aestivum L.) is one of the major cereal crops in the word. Due to its huge DNA and complex genetic background, genetic transformation and modification of wheat falls behind other crops. Therefore the establishment of wheat transformation system has great significance. Brachypodium distachyon(Brachypodium) is the first member of the pooideae subfamily to be sequenced and regarded as important model system for developing new energy and food crops in recent years. This study optimized the regeneration system of wheat and Brachypodium and then used two methods,Agrobacterium-mediated transformation and particle bombardment for transformation.The main content just as follows:1. Optimization of regeneration system of wheat tissue culture from mature embryoThe effects of different hormone combination on the callus induction and plant regeneration from mature embryos of two elite winter genotypes including Yangmai 158 and Zhengmai 9023 were studied. Although little difference was found in the ability of callus induction with the treatments, there were quite differences in the differentiation rate among different concentrations of these two auxins after transferring of the callus to the medium supplied with KT, the relative higher number of regenerated plants was observed at the callus induced with 4 mg/L Dicamba and further growth on the medium with 5mg/L KT in both genotypes. Compared with Zhengmai 9023, callus form mature embryos of Yangmai 158 had a higher plant regeneration rate.2. Preliminary study of wheat genetic transformationMature embryos of Yangmai 158 was transformated by two different methods. In Agrobacterium-mediated transformation system, different combinations of bacteria liquid concentrations and infection time were tested. The results showed that 6 resistant regeneration plantlets were obtained in treatment of bacteria liquid concentration OD600=0.8 and infection time 30 min, in other combinations had any regeneration plantlets appeared. In particle bombardment process, 4 combinations of parameters including target distance and acceleration pressure were tested. The results showed that had any regeneration plantlets were obtained in these treatments.3. Optimization of regeneration system of BrachypodiumIn paper, we studied the effects of different disinfection time and Cu SO4 on embryo culture from mature embryos of Brachypodium Bd21. The results showed that the seeds were thoroughly disinfected and got less damage after dipping into sodium hypochloritefor 15 min, the embryogenic callus induction efficiency were 89.3% higher than other treatments. The mature embryos were cultured on induction medium supplemented with0.6 mg/L Cu SO4, the embryogenic callus induction, embryogenic callus and regeneration efficiency were higher than control.4. Preliminary study of Brachypodium genetic transformationEmbryogenic callus from mature embryos of Bd21 was transformated by Agrobacterium-mediated transformation and particle bombardment. In Agrobacterium-mediated transformation system, different combinations of bacteria liquid concentration and infection time were tested. The results showed that 23 resistant regeneration plantlets were obtained in treatment of Agrobacterium concentration OD600=0.4 and infection time15 min, higher than other combinations. In particle bombardment process, different combinations of parameters including target distance and gold powder consumption were tested. The results showed that few regeneration plantlets were obtained in treatments of gold powder concentration for 1.0 mg/L and 12 cm target distance.5. Detection of the resistant plantsPCR was carried out to verify Hyg gene in resistant regeneration plantlets. The PCR detection in Agrobacterium-mediated transformation showed that 6 plants among them 5plants were positive ones in wheat genetic transformation and in Brachypodium genetic transformation showed that 48 plants among them 24 plants were positive ones. There were no positive plantlets obtained by particle bombardment method in both plants. |
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