Exploring Multigene Transformation In Brachypodium Distachyon To Creat Transgenic Plants With Hyperaccumulation Of Heavy Metals

Since contamination of soils by toxic heavy metals is a serious environmental problem, effective remediation methods are necessary. Phytoremediation, is an environment-friendly and ecologically responsible solar-driven technology with good public acceptance, which serves as a better solution to the problem. However, the hyperaccumulator on which hyperaccumulation based, is usually slow-growing and low-biomass. The weakness makes the technology time-consuming and restricts its applications. Therefore, research is conducted to genetically modify some suitable plants for better phytoremediation of heavy metals and other xenobiotics. Gramineae energy plants, such as Miscanthus sinensis, have the advantages of growing fast and high biomass output. If they can be turned into hyperaccumulator, it will show a broad prospect of applications. Meanwhile, Brachypodium distachyum, as a convenient model system for biofuel crops and Triticeae cereal crops, has many merits, for instance, small stature, short generation time, small genome, and available genome sequence draft. Since hyperaccumulation in plants is controlled polygenetically, the ability to manipulate multiple gene products is urgently required, especially, the improvement of capacity of transportation of heavy metals and stress tolerance.Therefore, the transformation technology system of Brachypodium distachyum is set up in this study firstly and the vectors based on the MultiRound Gateway system is used, which have different combinations of metal transporter genes and tolerance related genes in Thlaspi caerulescens and Arabidopsis thaliana (TcZNTl, AtCS, TcOPT3, AtHMA4, AtMTP1) to create transgenic plants by agrobacterium mediated transformation. Meanwhile, appropriate concentration of heavy metals (cadmium and lead) is been explored through the phenotype observation and some physiological index. The main results are obtained as follows:Immature embryo is used to drive combination embryogenic calli and the medium component and operational approach in tissue culturing and transformation system is improved in this study. Finally, three kinds of transgenic plants, G5, G4and G2, are obtained, which have the TcZNT1, AtCS and AtHMA4expressions in the T1generation, respectively. A hydroponic way is adopted to test the physiological responses of Brachypodium distachyum to heavy metals. It shows that even though50μM of Cadmium (Cd) already has heavy obvious damage to the plants; more attention should be paid to its biological availability improvement when Lead (Pb) is added. When the concentration of Cd is5μM, the distribution ratio of root and shoot is9:1. With the increase of the concentration of the treatment, the root-to-shoot transport ability of Cd is enhanced slightly, and the content of Potassium (K), as well as the content of Manganese (Mn) in root, decrease significantly. Under the treatments of5μM and50μM,99.9%of the Pb is accumulated in the root; the content of Phosphorus (P) in root increases with the increase of Pb concentration.