Studies On Transformation Of Wheat Cultivars Mediated By Biolistic Bombardment

Wheat is the main food crops in china.Wheat scab has caused huge not only yield losses,but also severe contamination of mycotoxins which is toxic to both human and the livestock.However,Wheat cultivars with immune or highly resistance to scab have not been found so far.Therefore,it is very important for wheat breeding resistance scab to transfer genes resistance scab into wheat by using transgenic technology.Establishing a highly efficient transformation system mediated by gene-gun is the basis for achieving this goal.In this research,three monocotyledon expression vectors containing ACE-D2, Rs-D2 and selectable marker PMI respectively together with other seven minimal cassette(contains promoter,open reading frame and terminator only) were co-bombarded into wheat Zheng9023 and Hual3 callus originated from precultured immature embryo.We observered the characters of Mannose selection system and analysed transgene integration trait,co-transformation frequency and separation of the law in the progeny in wheat co-transformed with multiple minimal cassettes.We also used BAR as a selectable marker gene for wheat transformation to investigate the effect of wheat geneotypes and bombardment times on transformation frequency.The results indicated that(1) mannose/PMI selection system was very effective. Transformation frequency and selection effiency for mannose selection were 0.98% and 50%respectively which were significantly higher than for PPT/BAR selection system(0.62%and 18.5%).(2)Southern blotting analyses showed that co-transformation with multiple mininal cassettes resulted in predominantly 'simple' integration patterns(1?3 copies),producing low-copy-number transgenic plants with a low frequency of transgene rearrangements.For transgenic plants bombarded with three genes,79.2%contained two different genes and 37.5%contained three different genes.The frequency of minimal cassette transformation was slightly higher than that of supercoiled plasmid cointegrate vectors.77.8%of transgenic plants bombarded with seven genes contained five to six transgenes and each gene had the same probability of being inserted into the wheat genome.We,therefore,concluded that gene transfer using multiple minimal cassettes was a feasible method for the production of transgenic plants containing several different transgenes.(3)Optimal bombardment times was 1.These results provided a good foundation for wheat genetic transformation by using transgenic technology.