The Initial Establishment Of Wheat Target Line For Site-specific Recombination System

Genetic engineering is one of important approaches for wheat breeding.However,the major problem of transgenic breeding is the random insertion of gene and multiple copies.The system of the site-specific recombinase mediated transformation could integrates the foreign gene in a specific location in which a recombinase could only recognizes a specific sequence.Resulting in single copy insertion after molecular screening.Moreover,genes could be continually stacked into the same location.Hence,reduced loci has greatly shortened the breeding process.To establish this system in wheat,the key step is to establish a target line which is obtained via traditional Agrobacterium-transformation approach.However,it is difficult for wheat to be transformed due to low transformation efficiency,genotype dependency and stringent experience requirement for a technicians.Therefore,the purpose of this study is to establish an Agrobacterium-mediated transformation system for wheat and applied it for the system of the site-specific recombinase mediated transformation thereafter.Firstly,the immature embryos of Fielder(Triticum aestivum L,cv.‘Fielder’)was used as explants to be infected by Agrobacterium,EHA105 containing a binary vector harboring,the DsRed as a reporter gene and a bar gene as a selection marker.After callus induction and PPT selection,the resistant calli were regenerated into plantlets.It took about 3 months to obtain the transgenic plants which showed DsRed expression and were also confirmed with PCR analysis.Secondly,with the above established transformation system,over 3000 immature embryos were transformed with Agrobacterium containing the construct pZH114 harboring the target site for later stacking.After selection and regeneration,39 transformation events with 93 transgenic plants were obtained with through PCR confirmation.Left border and right border which contained the target site were sequenced for its perfectness.32 transformation events with 68 transgenic plants had the perfect target site confirmed by sequencing to harbor the complete left and right borders including the complete target site.Hence,the Agrobacterium-mediated transformation system for wheat was established successfully and some putative target lines was obtained through thesystem in the present study.Those investigation laid a foundation for the site-specific integration research in wheat.