Transformation Of Wheat With Genes Against Fusarium Head Blight And Hybrid Polymerization Of Transgenes In Wheat

The occurrence and damage of Fusarium head blight(FHB)are major problems in triticeae crop production in China.The main pathogen Fusarium graminearum for FHB has complexity interaction mechanisms with its host.Inadequate natural resistant germplasm constrains the research for resistance to FHB in wheat.The application of transgenic technology provides a new path for wheat FHB resistance breeding.Some key pathogenic genes of F.graminearum were previously selected by our laboratory.Xiangmai 76 has the character of good agronomic and production traits but highly susceptibility to FHB.This FHB susceptibility can be improved with the transgenic approach.In this study,we co-transformed F.graminearum RNAi constructs into Xiangmai 76 via particle bombardment.In addition,by means of multigene pyramiding breeding strategy,we created some pyramid transgenic lines which contain the genes that are involved in fengycin biosynthesis.The main results of this study are described as following:1.By co-transformation through particle bombardment,we transformed selectable marker gene Bar plasmid and linear gene expression cassette dubicmps-Chs3bAs2As3-nos into immature embryo calli of Xiangmai 76.The RNAi construct chs3b is originated from F.graminearum.Under the Bar/Bialaphos selection system,we obtained a transgenic plant that includes the marker gene Bar and the target gene Chs3bAs2As3.Now all of the transgenic plants are in the T2 generation.2.Using mannose as screening agent combined with particle bombardment we transformed RNAi constructs that target chitiase genes chs3b from F.graminearum.There are two combinations co-transformation into Xiangmai 76 immature embryo calli.These combinations comprise minimal gene expression cassettes dubi35s-Chs3bAs5As1-nos,dubicmps-Chs3bAs2As3-nos and Ubi-PMI-nos.After the analysis of PCRs,we obtained two transgenic plants containing genes Pmi,Chs3bAs5As1,and Pmi,Chs3bAs2As3,respectively.Now all of the transgenic lines are in the T2 generation.Besides,by means of multigene pyramiding breeding strategy,we generated four plants that have two constructs Chs3bAs5Asl and Chs3bAs2As3.3 We used multigene pyramiding approach to generate transgenic lines containing fengycin synthetase genes.Fengycin is lipopeptide produced by Bacillus spp.It has strong activity against filamentous fungi.Fengycin synthetase complex is an ordered system controlled by multiple genes(fenC,fenD,fenE,fenA,fenB,sfp,yczE).After analysis of the twenty transgenic lines from our laboratory by PCRs,we selected stably inherited lines to perform mutigene crossing.After the first round of hybridization,we obtained eight groups that contain female and male parental genes.According to the identification of the first round of hybridization,we selected pure lines as female to match seven hybrid groups to pyramid more genes.On the other hand,through the second round of hybridization,we obtained three transgenic plants that were confirmed to have all seven related genes.