| Wheat scab caused by the fungus Fusarium graminearum is a destructive worldwide disease that widely happens in the warm and humid regions. In China, the disease is popular in the winter wheat region of the mid-lower reaches of the Yangtze River, and in recent years it's spreading to Huang-huai and Guan-zhong district. Wheat scab not only has caused huge losses to wheat yield, but also made infected seeds to produce mycotoxins, which is toxic to both human and the livestock. Breeding disease-resistant cultivars is the most effective measure for the disease control. Unfortunately, it is lack of wheat scab resistance sources. Therefore, transformation of wheat cultivars with scab resistance-related genes by genetic engineering technology will play an important role in wheat breeding for scab resistance.In this research, different combinations of the whole plasmid to conferring resistance gene and minimal gene cassettes containing scab resistance-related genes were co-bombarded into wheat cultivars Zheng 9023 and Yangmai 12 callus originated from precultured immature embryo. Then, using mannose and PPT as the selection agent, we compared the differences of the two genotypes in callus formation and regeneration efficiency. Some transgenic plants were regenerated with different foreign gene. The main results are follo wings:1. Callus growth of two wheat genotypes was significantly different:Zheng 9023 callus is dense, and the time of hypertonic may be extended. Yang 12 wheat callus is loose, and high osmotic treatment on callus for 20-24 h is appropriate. But the two genotypes have no significant difference in callus induction capacity, regeneration capacity and transformation efficiency, and both are the good receptors for wheat transformation.2.In our work, using different selection conditions, transgenic plants were screened and obtained in Zheng 9023 and Yangmai 12. The results of PCR analysis in To generation transgenic plants showed that, the positive plants for mannose selection were 31 and 10, for PPT selection were 26 and 32. The transformation efficiencies were 1.51%, 1.7%,2.18% and 2.78%, respectively. Southern blotting of T1 generation analysis confirmed that foreign genes had integrated into wheat genome, and the co-transformation with multiple minimal gene cassettes resulted in predominantly 'simple' integration patterns and produced low-copy-number transgenic plants. For co-transformation of Zheng 9023 with six genes,42.3% contained three or more different genes and 76.9% contained over two different genes. Furthermore, each integrated gene had the same probability of being inserted into wheat genome, and a similar result was found between integrated gene and selection marker gene. It indicated that co-transformation using multiple minimal cassettes was very effective.3. Mannose and PPT have different effects on callus formation and regeneration in wheat. The comparison of two selection system using Zheng 9023 callus as transformation receptor, the result showed:mannose as a positive selection agent had no effect on growth of transformation receptor, and callus browning was less. Transformation efficiency using mannose selection was 1.51%, a little lower than for PPT selection (2.03%), but the selection efficiency (7.1%) was significantly lower than for PPT selection (16.7%). Furthermore, we found that regeneration efficiency for mannose selection was significantly lower than for PPT selection in the early shoot regeneration. We considered that mannose selection during shoot regeneration was very efficient. |
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