Research On Regeneration Properties Of Culture System From Immature Embryos And Genetic Transformation Of Wheat By Biolistic Particle

Wheat is one of the most important food crops in China and the world.But common wheat is hexapliod, and the background of wheat is very complicated. So,wheat is the last one of the food crop which was transformed successfully.In wheat genetic transformation, immature embryo is the most application explant,but the regeneration rate was influenced by genotypes and the hormone ratio medium.Only a few highly regenerated varieties were used in wheat transformation,while a large quantity of elite cultivars can not be transformed successfully due to their poor regeneration property.This resulted in the transformation of wheat developing very slowly.Therefore selection of excellent receptor genotypes and optimization of wheat regeneration culture system from immature embryo is urgent in wheat transgenic work.The excellent wheat varieties planted in HuangHuai areas or shaanxi province in recent years,incluging zhengmai 9023,xiaoyan 22,xinong 2000,xinong 928 and so on, were selected as materials in this study to compare their regeneration properties system of wheat immature embryo tissue culture.On these basis,we do the following transgenic research: (1) Through the co-transformation by gene gun, we introduce the Zm - c1 or Zm - r (s) gene which control the corn anthocyanins synthesis and Bar gene into wheats, trying to get plants that excluding marker genes by offspring separation of transgenic plants; (2) Using the GFP gene as the safety marker genes, we transfer the ZmAL1 gene which can increase the volume of corn seed into wheat, in order to obtain safety transgenic wheat.The purpose of study is to obtain transgenic wheat with target genes,lay the foundation for functional identification of these genes in transgenic wheat.The main results obtained as follows:(1) Comparison of tissue culture regeneration properties of the immature embryo from different wheat.cultivarsThrough the immature embryo tissue culture of different wheat varieties ,we indicated that the induction rate of different genotypes of wheat varieties is higher, all above 95 percent, but callus quality is differences;The differentiation rate of different varieties of wheat genotypes is very different.The order of differentiation capacity of selected five varieties is: Xinong 928 > Zhengmai 9023 > Xinong 2000 > Xiaoyan 22 > Xinong 979.Considering both induction rate and the differentiation rate,Xinong 928 and Xinong 2000 are much better for plant regeneration.(2) Co-transformation of the corn Zm-c1 or Zm-r (s) and Bar gene by Biolistic Particle The genes transcription factors,Zm-c1 or Zm-r(s) which control the anthocyanins synthesis in corn gene was transformed into wheat varieties of xinong 2000, xinong 928 and xiaoyan 22 with bar as marker gene. there were 3 and 1 positive transgenic plants with the Zm-c1 gene in Xinong 2000 and Xinong 928, respectively .The transfermation ratio is respectively 0.23% and 0.08%;The respectively got there were thirteen and five transfegenic plants with Bar gene in Xinong 2000 and Xinong 928,respectively.The transfermation ratio is 0.98% and 0.42% ,respectively.With regards to transformation with the Zm-r(s) gene,we got 4 positive transgenic plants in Xinong 2000.The transfermation ratio is 0.13%.Fifteen transgenic plants with Bar gene were obtained in Xinong 2000.The transfermation ratio is 0.5%. All the transfer plants with the target gene contain the Bar gene.(3) Transformation of Wheat with ZmalI gene by Biolistic Particle and Molecular IdentificationThe over expressed vector pGlu-exbessa::ZmalI,which contained the fusion protein gene ZmalI and GFP controlled by the special glutenin promoter,was introduced into wheat by Biolistic Particle.Then 3 000 embryos of shaannong 138 were bombarded by Biolistic Particle, and 59 regenerated plants were obtained. 16 out of 59 were identified to be transgenic positive plants by PCR assay in the T0 plants.The transformation frequency was 0.53 %.10 of the 16 T0 transgenic plants generated seeds.We get 48 plants of T1 progency.PCR analysis indicated that 35 of the 48 T1 plants have the target gene.This implied that the target gene segragated in T1 plants.Seven of the 35 T1 plants were randomly selected to check the expression of the target in the kernels by performing the RT-PCR,The results showed that 6 of the 7 grains had the gene expression, indicating that the target genes in most seeds of T2 generation plant has been expressed.