Establishment Of Genome Editing System In Foxtail Millet Based On CRISPR/Cas9

Millet belongs to diploid plant.It is an earlier domesticated C4 crop,and it is also a good model crop.Millet has a long planting history in the whole world,especially in China,it plays an important role in miscellaneous grain cash crops in Shanxi Province.However,the research progress in crop improvement and other related aspects in millet is very outdated,owing to the obstacles in genetic transformation of millet.Genome editing is that a sequence-specific nuclease targets the targeted loci to produce DNA double-strand breaks,so that the targeted loci can be knocked out,replaced and inserted.Thus,mutation can be achieved without introducing foreign genes.Genome editing brings targeted mutations with high efficiency and convenience.In this experiment,3 sets of multiple genome editing system were constructed with CRISPR/Cas9 in Yugu 1.1.Establish a complete tissue-culturing system of millet.At the beginning of the experiment,we tried to induce callus from mature seeds.Six kinds of callus induction medium,CIM1,CIM2,CIM3,CIM4,CIM5,and CIM6,were used to induce callus with two kinds of millet,Yugu 1 and Jingu 21.Finally,Yugu 1 millet and CIM6 medium,which two can induce more embryogenic callus,were chosen to generate a large number of high-quality callus for further step.Then,the high-quality embryogenic callus was subcultured to PRM1 regeneration medium to generate bud differentiation.Next,we pinched the differentiated bud and subculture these bud to RM rooting medium to induce rooting.At last,the differentiated and regenerated complete plants were transplanted to the soil.All steps and operation above provide a reliable reference for further infecting callus to produce heritable mutant plants.2.We continuously tried and optimized the protoplast isolation and instantaneous transformation system based on wheat,rice and corn,which,these 3 and more plants,have stable protoplast isolation and instantaneous transformation system.As a result,a appropriate instantaneous protoplast transformation system of Yugu 1 millet was established,which laid solid foundation for constructing multiple genome editing system.3.In order to improve millet's agronomic characters,four genes concerning about sensory and flavor,Si Dof4,Si BADH2,Si GBSS1 and Si IPK1,were selected.Each gene selected two target sites,and each single target site was knockout by CRISPR/Cas9 genome editing.We picked out the targeted site with higher mutation efficiency in each target gene.Three perfect multiple genome editing systems,MCTU,PTG and Csy4,were constructed.Mutations were successfully produced in four genes mentioned above of Yugu 1.Single knockout and three sets of multiple genome editing systems,by comparison,it is found that although mutation efficiency of different methods is not the same,there is no significant distinction.And the mutation efficiency is concentrated among 30% to 40%.