| Wheat is one of the main food crops in our country. Microprojectile bombardment and Agrobacterium-mediaied transformation are two widely used methods for integration of exogenous genes into the wheat genome. However, low transformation rates have limited the wide-scale adoption of these techniques in wheat breeding. This thesis investigates some of the main factors which influence wheat tissue culture and microprojectile bombardment, aiming to improve transformation efficiency and thus contribute to the wider exploitation of transgenic wheat in the future.The effects of 2,4-D, AgNO3, sucrose and CTK concentration and explant density on callus induction and regeneration were investigated using immature embryos or scutella of the widely grown local cultivars Zheng Mai 9023, Yu Mai 18, 98-161, Yu Mai 47 and Yu Mai 49, and model cultivars Bobwhite, Yang Mai 158. A single factor or multiple factor randomized blocks design was used as appropriate. In addition, effects of the length of culturing time on MSI medium before or after bombardment, size of gold particles, Hyg concentration, bombardment distance and chamber vacuum pressure on transformation efficiency were also investigated. Results showed that:1. Genotype and developmental stage of the explant were the two most crucial factors that influenced transformation efficiency;2. Of the cultivars tested Zheng Mai 9023 and 98-161 were best suited to microprojectile bombardment as they displayed a better response to tissue culture conditions than the model cultivars.3. The proportion of explants that sprouted in culture was high at low 2,4-D concentration (between 0 and 0.25 mg/L) for most tested cultivars and it was difficult to induce callus formation. The 2,4-D concentration giving the best response for induction of callus for different cultivars was between 0.5 and 1.0mg/L. The percentage of explants that regenerated shoots was highest when 2,4-D concentration was 0.5 mg/L for cultivars Zheng Mai 9023, Yu Mai 18 and Bobwhite. High concentrations of AgNO3 (10 mg/L) can restrain plumule growth, but also affects the physiological state of the callus and its regeneration potential. Whether AgNO3 should be added to a medium or not depends on the cultivars used. A sucrose concentration of 30g/L was best for root and seedling regeneration. Use of 2mg/L or 5mg/L of KT or Zt in the medium and explant density were found to have no significant effect on regeneration potential.4. Explants from field-grown material responded better to tissue culture conditions than corresponding explants from glasshouse-grown material, particularly with respect to regeneration potential, but field-grown material tended to have high rates of plumule growth. The percentage of explants which sprouted was higher when embryos were used as explants rather than scutella, but their regeneration potential was inferior to that of immature embryos. Results obtained using scutella from greenhouse-grown material as explants were good, so it was concluded that the explant of choice should be scutella.5. In the study of factors affecting wheat microprojectile bombardment, transformation efficiency was highest in all treatments when explants were precultured for seven days before bombardment. The optimum length of pre- and post-bombardment culture periods was found to be affected by genotype; transformation efficiency for Yang Mai 158 was highest when explants were cultured for five days after bombardment while transformation efficiency for Zheng Mai 9023 was highest when explants were precultured for three days. Length of the culture period before bombardment was generally found to be more important than length of that after bombardment. Selection of transformants was best at the highest Hyg concentration used (20mg/L). Gold particle size had no influence on transformation efficiency, but the efficiency of 1100psi acceleration pressure was higher than that of other bombardment pressures. The bombardment process and number of bombardments had major effects on callus... |
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