Induction Of Meristem Through Immature Embryos And Transformation Of Wheat With GS5 And Ghd7

Wheat is one of the major food crops. Transgenic breeding techniques can purposefully improve wheat agronomic traits and make the high yield of wheat be a reality, these do great importance to the world’s increasing demand for food. In this study, induced cultivation of genotypes of non-callus meristem 15 wheat immature embryo were successfully realized. Particle bombardment and Agrobacterium-mediated were used to transform rice gene GS5 and Ghd7 to wheat genes. PCR and RT-PCR were used to detected the transgenic plants. T4 generation of GS5 transgenic lines’ agronomic traits were investigated and paraffin section to the broadening lines’ glumes and seeds had been observed. The main results were as follows: 1. 15 cultivars of wheat immature embryo can form meristem clusters, the meristem rates can reach over 84.6%, and the Long30 meristem clustering rate can reach up to 161.26%. Different cultivars had different meristem cluster rates, while the genotype was not the main reason of induction meristem. 2. Comparing the transformation of single gene and double genes, in certain cases of plasmid dosage and method, the increasing of transformation plasmid type didn’t affect the resistance rate, differentiation rate and regeneration rate of meristem. 3. The genetic transformation of particle bombardment in resistance rate, differentiation rate and regeneration rate were all higher than the Agrobacterium-mediated. The former’s resistance rate, differentiation rate and regeneration rate were respectively 14.99%, 31.38% and 16.48%, the latter were respectively 5.19%, 21.39% and 5.36%. 4. PCR was used to test the positive regeneration plants, using particle bombardment and Agrobacterium-mediated, respectively obtaining positive plants 1 and 8 GS5 gene regeneration strains, 1 and18 Ghd7 gene regeneration strains. GS5 or Ghd7 transgenic plants of Zheng9023 using Agrobacterium-mediated were bred to T2 generation, and the target gene were passed on to the offspring, and the positive rate of line Ghd7-3 reached to 100%. 5. By using Particle bombardment and Agrobacterium-mediated of GS5&Ghd7 genes, 189 and 5 resistant strains were gained respectively. PCR analysis showed that the two kinds of transformation methods both can get three types of positive transgenic plants one time, obtaining three types transgenic plants GS+Ghd7-, GS5-Ghd7+ and Ghd+GS5+. GS5+Ghd7+ plants get by particle bombardment were cultivated to T2 generation, Making the meristem as receptors to two gene genetic transformation be possible. 6. Through the investigation of the agronomic traits of 12 T4 generation of GS5 transgenic obtained by seniors, we found: different varieties showed difference in every agronomic trait, and different lines of the same variety also showed differences. 10 lines’ grain width were significantly broadened, and the most prominent was Zheng-2, the grain width broadened 12.04%, the grain length increased 2.24%, the length-width ratio reduced 9.17%, and the thousand kerner weight added 8.73 g.7. GS5 transgenic line were found GS5 gene expressed both in leaf and glume by RT-PCR. The glume was thickener, and the inner cell clusters were larger. The grain seed capsule was thickener, and the cell layers increased. So GS5 gene may broaden the grain width by regulating the morphology of inner glume and seed capsule.