| Barley is the fourth largest cereal crop in the world.It is widely cultivated because of its strong resistance and wide adaptability,because of its small genome it has become one of the model plants for genetic research of wheat.At present,there are few studies on the genetic modification of barley,and the limited genotypes are used.Therefore,the establishment of a commercial genetic transformation system for the promotion of barley varieties has great significance for the genetic improvement and gene function research of barley.In this study,a double-T-DNA vector p WMB123 was successfully transferred into barley by establishing an agrobacterium transformation system of a commercial barley variety Vlamingh,and stable genetically-transformed offspring were obtained.Transgenic plants without screening markers were obtained through isolation of offspring.At the same time,by designing universal sg RNA for Hv Nud gene which control barley naked phenotype and its homologous gene Ta Nud in wheat,were subjected to site-directed mutation to obtain gene editing mutants.The main results are summarized as follows:1.Through the optimization of the medium components and culture steps of the barley variety Vlamingh,it was found that in the callus differentiation stage,the addition of 1.0 mg / L KT,0.5 mg / L 6BA,and 0.05 mg / L NAA to the callus significantly promoted the callus.Differentiation.At the stage of differentiation and rooting,it was found that the rooting effect was best with SM1 medium(without other auxins)supplemented with 1.0 mg / L of IBA.The addition of 2.5 mg / L Cu SO4 to the medium significantly reduced the albinism of Barley transgenic plants.2.By constructing a double T-DNA vector containing both the target gene GUS and the marker gene Bar and transforming the excellent barley variety Vlamingh,14 transgenic plants were obtained in the T0 generation with a transformation efficiency of 10.14%.PCR,Bar strips,GUS staining and other tests confirmed that the Bar gene was contained in all T0 plants,and 10 of them contained the GUS gene.Four transgenic plants containing both the Bar gene and the GUS gene were selected and their offspring were tested.Two transgenic plants containing only the GUS gene and no Bar gene were screened from the BL-8 strain,the efficiency of production of marker-free barley was 6.9%.3.Three homologous alleles of Hv Nud in wheat(Traes CS7A02G376300,Traes CS7B02G277800 and Traes CS7D02G372700)were found by BLAST,and gene fragments were amplified by designing primer PCR.Sg RNA was designed in the same regions of four sequence.The CRISPR / Cas9 vector was constructed,and barley was transformed by the established genetic transformation system,and 10 transgenic plants were obtained,2 of which were edited.The wheat Fielder was also transformed,and 119 plants were obtained,of which 61 plants were edited,and there were 5 editing types in total.Five editing types,WL4,WL11,WL18,WL50,and WL56,were selected for progeny planting and hybridization.It is expected that a totally homozygous edited plant will be obtained. |
