Studies On Transformation Of Wheat With Genes Resistant To Disease And Pre-Harvest Sprouting

Wheat is one of the most important food crops in our country and world, wheat scab and pre-harvest viviparous (PHS) are worldwide disease damage and natural disaster individually in wheat production. In the middle and lower reaches of Yangtse River, wheat flowering, filling, pre-and post harvest always meet cloudy and rainy weather, resulted in scab and PHS frequently occurring, it caused serious wheat yield loss. Unfortunately, because of the lack of natural resistant resource in wheat and its relative species, the resistant wheat variety breeding progressed slowly. Using genetic engineering for crop genetic improvement research has become people's concern, wheat genetic transformation technology system also developed rapidly. In this study, cultivars Y12 and X25 as the recipient materials, the scab resistance-related genes were transferred into wheat; at the same time, the gene expression and trait performance of the transgenic wheat with PHS resistance gene were analysed and identified. The main results obtained were as follows:1. Using biolistics-mediated transformation methods, we transferred scab resistance-related genes into wheat cultivars Y12 and X25 callus that introduced from immature embryos. resistance-related genes and the gene combination were as follows:1) msrA1-D2 and pep3, pmi as marker genes; 2) D4E1, bar as marker gene. Several rounds of screening in the medium containing mannose, we obtained 7 transgenic X25 seedlings and 12 transgenic Y12 seedlings, their transformation frequency were 0.91% and 0.94% individually. By PPT selection, we obtained 13 transgenic Y12 seedlings, transformation frequency was 2.03%.2. In the T1 generation lines of transgenic wheat, the marker gene and the target gene had suffered different degrees of separation, the segregation ratios varied, but did not occur the phenomenon of gene completely lost.3. PCR identified 26 T3 generation lines of transgenic Z9023 wheat and 9 T3 generation lines of transgenic E13 wheat with Vpl gene from the maize. The results showed that these T3 progeny contained Vpl gene, and the Vpl gene had integrated into the wheat genome and can be inherited stably. 4. RT-PCR analysed the Vpl gene expression in the transgenic wheat lines. Extracted RNA from 20 d after flowering embryo in the T3 lines, then reversed transcription into cDNA, used Vpl gene specific primer analysis on the transcription level, showed that maize Vpl gene has expressed in transgenic wheat.5. Determination of seed germination showed that the almost all transgenic lines' germination index were significantly lower than non-transgenic control, that is the PHS resistance of genetically modified wheat had been improved significantly.These results provided basis for further cultruing new wheat varieties with scab and PHS resistance.