雌雄青杨组织培养和PcXTH22基因过表达及敲除OA北大核心

To achieve genetic improvement of Populus cathayana Rehd through genetic engineering meth-ods,male and female poplar were used as materials to establish a systematic tissue culture regeneration.The optimal mediums for the differentiation,proliferation and rooting of female P.cathayana were MS+30 g/L sucrose+6 g/L agar+0.1 mg/L TDZ+0.3 mg/L IBA,MS+30 g/L sucrose+7 g/L agar+0.1 mg/L 6-BA+0.5 mg/L IBA,WPM+20 g/L sucrose+7 g/L agar+0.1 mg/L IBA+0.2 mg/L NAA;those for male P.cathayana were:WPM+30 g/L sucrose+6 g/L agar+0.1 mg/L TDZ+0.3 mg/L NAA,MS+30 g/L sucrose+7 g/L agar+1.0 mg/L 6-BA+0.1 mg/L NAA,WPM+20 g/L sucrose+7 g/L agar+0.2 mg/L IBA+0.02 mg/L NAA.Subsequently,the leaves of female P.cathayana after 1 month of sub-culture were used as genetic transformation receptor material for overexpression and knockout of the PcXTH22 gene.After infecting with Agrobacterium solution for 10～15 minutes,the leaf discs were co-cultured with Agrobacterium for 48 h and then transferred into mediacontaining 10 mg/L hygromycin and 200 mg/L temetine for selective culture.The obtained resistant plants were detected by PCR,and the trans-formation rate was almost 100%.Quantitative PCR showed that the transcriptional expression level of PcXTH22 in overexpressed strains increased by 40-824 times.Among the 5 CRISPR-Cas9-transformed lines,1 line was found to have frameshift mutation on both alleles and the protein translation was terminated prematurely,and the editing efficiency was 20%.Therefore,the study provides an excellent example of ge-netic improvement using our endemic native tree species.