| Plant gene engineering can reform the genetic traits orientationally, we can operate it accurately and scientifically at the genetic level, so it provides a new way for plant disease resistance breeding. Agrobacterium-mediated transformation has many merits. So it is one of the most important methods in genetic engineering. Cell suspension cultures of plant also has many merits, such as, it is homogeneous, grow fast and easy to control, in addition. Using cell suspension cultures can improve the transformation frequency and we can acquire the material whenever we want. Wheat, as one of the most important crop, the output of wheat decreases in the great degree because of Fusarium Head Blight(FHB) annually in the wheat growing area. Introducing alien genes that resistant to FHB into wheat by Agrobacterium tumefcaiens can obtain new cultivars that resistant to FHB efficiently. The establishment of wheat cell suspension cultures can also be beneficial for the transformation of wheat.In this study, we used the leaf base segments of Yangmai 158, Zhengmai 9023, Xiangmai 76 and Xiangmai 13 as the explant to induce calli, we obtained embryogenic calli by inducing and selecting the calli many times, through culturing these embryogenic calli in liquid medium, we obtained wheat cell suspension cultures. We used the calli precultured for 8 d of Yangmai 158 and Xiangmai 76 as the explant for transformation, transferred FHB resistant genes Ehc42, Chi, Hvglu and PPC-CHS3 by Agrobacterium tumefcaiens, used Bar gene and Pmi gene as selection markers to obtain resistant plants, through the PCR identification we obtained T0 transgenic plants. Through the molecular identifiaton of the offspring of transgenic plants, we confirmed that alien genes were integrated into the genomics of Yangmai 158 and Xiangmai 76, also the regularity of the deletion rate and the separation rate of target genes and marker genes has been analysised. We confirmed the resistance to FHB of transgenic wheat by infecting wheat spikes with the fungal conidia suspension. The main results are as follows:1. The establishment of wheat cell suspension cultures: Using the leaf segments of Yangmai 158, Zhengmai 9023, Xiangmai 76 and Xiangmai 13 as explants to induce calli, it is easiler for Xiangmai 13 and Zhengmai 9023 to obtain embryogenic calli than Yangmai 158 and Xiangmai 76. And we alse obtained cell suspension cultures of Xiangmai 13 and Zhengmai 9023, but Yangmai 158 and Xiangmai 76 did not. The results show that Xiangmai 13 and Zhengmai 9023 are more appropriate for the establishment of wheat cell suspension cultures.2. The optimization of infective parameter of Agrobacterium tumefcaiens: using the calli precultured for 8 d of Yangmai 158 as the explant for transformation, we have set 8 combinations of infective parameter, they are OD600=0.75, 30 min, OD600=0.75, 45 min, OD600=0.80, 30 min, OD600=0.80, 45 min, OD600=0.85, 30 min, OD600=0.85, 45 min, OD600=0.90, 30 min, OD600=0.90, 45 min. The results show that, the transformation frequency of the combination of OD600=0.85, 30 min is higher than others.3. The effect of different wheat cultivars on transformation frequency: we used the same infective parameter of Agrobacterium tumefcaiens to transform calli precultured for 8 d of Yangmai 158, Xiangmai 76 and Xiangmai 13, the results show that, the regeneration rate and transformation frequency of Yangmai 158 are all higher than Xiangmai 76 and Xiangmai 13, Yangmai 158 is more appropriate for Agrobacterium-mediated transformation than Xiangmai 76 and Xiangmai 13.4. The effet of different screening system on Agrobacterium-mediated transformation: using Bar gene and Pmi gene as marker genes, we analysised the difference of screening frequency and the growth situation between the two screening systems. The screening frequency of Pmi gene can reach 80%~100%, however, Bar gene screening system can only reach 3%. When using Pmi gene as marker gene, the transgenic plants grew quickly while the non-transformed plant did not. However when using Bar gene screening system, we have to subculture the resistant plants many times until we find the difference between the transgenic plants and non-transformed plants, so Pmi gene is more appropriate for Agrobacterium-mediated transformation than Bar gene.5. The transformation using Bar gene as marker gene: we transformed FHB resistant genes Ech42 gene and Chi gene to Yangmai 158, and use Bar gene as selection marker. We obtained 1 T0 trangenic plants for each gene, and their offspring showed the same regularity of the deletion rate and the separation rate of target genes and marker genes. As the generation of transgenic plants increases, the separation rate of target genes and marker genes increases and the deletion rate of target genes decreases. The target gene and marker gene all reserved in T4 generation of transforming Ech42 gene. Some of transgenic plants of transforming Chi gene in T4 generation still lost the target gene, but we obtained the transgenic plants that only have Chi gene. Through the identification by RT-PCR of these two lines in T4 generation, we confirmed that the alien genes had been integrated into wheat genome. And the transgenic plants transforming Chi gene in T4 generation inoculated with conidia had suppressed symptoms in contrast to the wild type, showing the transgenic lines had great resistance to FHB.6. The transformation using Pmi gene as marker gene: we transformed FHB resistant genes Hvglu gene and PPC-CHS3 gene to Yangmai 158 and Xiangmai 76, and use Pmi gene as selection marker. We obtained 4 T0 transgenic Yangmai 158 transforming Hvglu gene, 1 T0 transgenic Yangmai 158 transforming PPC-CHS3 gene and 3 T0 transgenic Xiangmai 76 transforming PPC-CHS3 gene. Through the identification by PCR, we confirmed that the alien genes had been integrated into wheat genome. And the transgenic plants transforming Hvglu gene inoculated with conidia had suppressed symptoms in contrast to the wild type, showing the transgenic lines had great resistance to FHB. |
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