Establishment Of Highly Efficient Tissue Culture Systems And Btr2 Gene Editing Vector For Hordeum Brevisubulatum

[Hordeum brevisubulatium（Trin.）]is also called barley grass,wild rye,which is a perennial grass belonging to the genus Hordeum in the Poaceae.It has the characteristics of salt and alkali resistance,cold resistance,drought resistance and barren resistance,high nutritional value,good palatability and so on.It is an excellent grass species which can be used in mining good genes,improving ecological environment and feeding simultaneously.With the rapid development of biological technology,molecular design breeding and genetic engineering technology are playing an increasingly important role,which can not only shorten the breeding cycle of herbage,but also carry out directed genetic improvement of existing herbage varieties.If the above technology is to be used,a stable genetic transformation system must be established.In this study,Mengnong No.1 hybrid wild barley,an excellent grass seed bred in Inner Mongolia Agricultural University,was selected as the research object.An efficient tissue culture regeneration system and agrobacterium-mediated genetic transformation system were established,and a knockout vector was successfully constructed.This study laid a foundation for the study of gene function,molecular design breeding and the creation of new germplasm resources in wild barley.Specific research results are as follows:1.Wild barley seeds were used as explants for callus induction,and an efficient tissue culture and rapid propagation system was established:Callus induction medium was MS+5.0mg·L^-1 2,4-D+0.05 mg·L^-1 KT+1.0 g·L^-1CH+30.0 g·L^-1 sucrose+7.8.g·L^-1 Agar,The differentiation medium was MS+2.0 mg·L^-1 2,4-D+0.05 mg·L^-1 KT+50.0 g·L^-1 sucrose+3.0g·L^-1 Phytagel,and the rooting medium was 1/2 MS+15.0 g·L^-1 sucrose+5.0 g·L^-1 Agar.The average callus induction rate was 65.7%,the average differentiation rate was 70.0%,the highest rooting rate was 100%,and the rapid propagation coefficient was 35.2.Established a genetic transformation system mediated by Agrobacterium tumefaciens of Monong No.1 hybrid wild barley on the basis of the establishment of tissue culture and rapid propagation system.The screening concentration of hygromycin in wild barley under dark and light conditions was 15.0 mg·L^-1 and 5.0 mg·L^-1,respectively.p ANIC6B-WOX3a,an overexpression vector of WOX3a gene,was provided by our research group and transformed into Agrobacterium tumefaciens EHA105.The wild barley callus with strong differentiation ability was obtained by early screening of Agrobacterium containing the target vector.After co-culture for five days,callus screening,GUS staining verification,and PCR identification,transgenic positive callus were obtained.3.Btr2 was cloned by homologous sequence method to obtain the Montnon No.1 wild barley grain drop gene.The gene sequence length was 1069 bp.After series analysis,the target site（ACAGGCTGGCTATTTCGG）was designed between 648～978bp,and the p CXUN-Btr2 decidular gene editing vector was constructed and transferred to the callus line with high differentiation rate obtained by the previous screening.The follow-up experiment is ongoing.