| Brachypodium distachyon is an emerging model plant for cereals due to its small size, low growth requirements, small and fully sequenced genome, diploid/tetraploid/hexaploid nature, transformable, closely related to cereal grasses especially wheat and barley. In this thesis, we described the establishment of an Agrobacterium-mediated transformation system for Brachypodium distachyon L. (ecotype Bd21). Using this transformation system, an ethanol inducible maize Ac/Ds transposon system was introduced into Brachypodium for a T-DNA/Ds tagging project and the preliminary data support that the vector system works probably well.1. Tissue culture and regeneration of immature embryos of Brachypodium distachyon L.We take the ecotype Bd21 as the material, which is cultured on MS basic media at 26±2℃and shoots are regenerated successfully from the calli of immature embryos of Brachypodium. we studied the tissue culture and regeneration ability of different size of immature embryo. The results indicated that, the smaller the immature embryos are, the better the embryogenic callus induction and regeneration are. The frequency of induction of immature embryo size <0.3mm is higher than 90%, but the size >0.7mm is only 20-25%. Both the compostion of media and the carbon source are the one of the immportant factors in tissue culture.We choose MS, LS, N6 media in our research in order to find the right media, and our results show no significant difference between the 3 media in embryogenic callus induction. The frequency of induction on these 3 media is 70-72%.We also test if there are different effects when use sucrose, maltose and glucose as carbon source respectively. We find that taking surose as carbon source perform better in embryogenic callus induction, which can reach 80% and taking maltose better in regeneration, which can reach 87%. We do differernt material sterilisation and the way of placement of immature embryo and the results show sterilization by 15% NaClO 5min and placing scutellum up is good in the induction.2. Establishment of genetic transformation system of Brachypodium distachyon. In this part, co-cultivation and selection are important. We choose solid MSAS media, sterile filter paper and liquid MSAS media as 3 variable filter and design 7 different ways of co-cultivation and we find only sterile filter paper and sterile filter paper+liquid MSAS could get high transformation efficiency, which are 58.1% and 60.4% respectively. We use the corresponding antibiotics to HPT gene and NPTII gene, and find homomycin and G418 can get good selection effect, to a level of 60-80%.3. GFP assay. After the transformation, we use 465nm laser to exam GFP in the transformed callus 72h later, and there is 80.6% callus showed GFP+; after 3 weeks there is 43.1% callus with strong GFP signal. Examnation on the whole transformed plant indicate that GFP expression is stable.4. Ethanol inducible epression of the Ac-transposase. The ethanol inducible Ac/Ds construct was introduced into Brachypodium genome via Agrobacterium-mediated transformation. When the transgenic plants were treated with ethanol, Ac-transposase was induced against the control plants.
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