Studies On The Transformation Of Miscanthus Sacchariflorus And Indica Rice With Exogenous Genes

In the first part of this thesis the biolistic bombardment transformation system for Miscanthus saccharjflorus was established and introduction of some agronomically useful genes was performed. In the second part of thesis the factors influencing the transfonnation efficiency of several indica rice varieties for both Agrobacterium and biolistic bombardment were investigated, and the insect and fungal resistant genes were introduced into several indica rice varieties. Establishment of biolistic bombardment transformation system for Miscanthus Saccha lorus and its transformation with exogenous genes Miscanthus saccharzflorus is one of the important commercial plants, it is not only an excellent raw material for papermaking and artificial board, also very important for sustaining ecological system. But it is extremely vulnerable to the insect attack. Therefore, any attempt that creates the insect resistant plants is encouraged. In this thesis we are attempting to improve the plant resistance to insect via biotechnology. As the first step the initiation and regeneration system for embryogenic callus of Miscanthus saccharjflorus were established at very high frequency. The result showed that the young inflorescence was the best explant for callus induction and easier differentiation into entire plantlets compared to other explants such as mature seeds, leaf, root etc. in Miscanthus saccharflorus. CC medium was the best basic medium for subculture of the callus amongst all the media tested.As the second step the selection scheme for tolerant callus of Miscanthus saccharflorus was established. The suitable selection scheme was culturing the callus on the medium containing 150-200 mg/L G4 18 or 20-30 mg/L Hyg or 10-20 mg(L Basta for 40-60 days. By using the particle bombardment transformation system potato proteinase inhibitor II (pinll) and pollen specific ribonuclease (barnase) genes were introduced into the callus of Miscanthus saccharflorus and 52 transgenic plants were obtained. PCR, Southern, and dot blot analysis showed that target genes were integrated into the genome of these transgenic plants, the further bioassays on resistance to insect and fertility for these transgenic plants are underway. Since the differentiation capacity of the embryogenic callus degenerated with period of subculture. The cryopreservation for embryogenic callus of Miscanthus saccharjflorus was also investigated. With treatment of desiccation and addition of protective agent (10 % DM50 + 0.5 M sorbitol) the callus cryopreserved in liquid nitrogen for 6 months still kept at high survival rate (68.4%) and regeneration frequency (78.3%), which makes all-year-around transformation possible. The optimization of biolistic bombardment and Agrobacleriwn transformation system for indica rice and their transformation with insect and fungal resistant genes Since the condition for initiation and regeneration of embryonic callus varied, the 14 different indica rice varieties were used in this thesis for investigation on these aspects in detail. The results showed that for most indica rice CC medium was the best for both callus initiation and subculture in which the browning of calli could be mitigated significantly. With supplement of 2.5-Smg/L absisic acid (ABA) the quality of calli can be improved and the rate of embryogenic calli were increased, the gelling agents also significantly influence the quality of calli which phytagel was the best among all the agars used. The selective efficiency and selective peri...