The basic work for the system establishment about genetic transformation,cell fusion and regeneration is to formulate efficient protoplast isolation system.The influence of different factors such as enzyme liquid combination,mannitol concentration,enzymatic hydrolysis time and centrifugal speeds on the isolation and purification of protoplast from tartary buckwheat ‘YU 6-21’ mesophyll cells were studied.The results showed that the optimal enzyme solution for protoplast isolation was 1.5% cellulase R-10+0.5% macerozyme R-10+0.5 mol/L mannitol+20 mmol/L MES+20 mmol/L KCl+10 mmol/L CaCl₂+0.1% BSA.Fifth to sevevth true leaves,which were removed the lower epidermal layer with tape,were incubated in enzyme solution for 4 h at 25 ℃ in dark,and centrifugal speeds was 900 r/min for protoplast collection.The protoplast yield amounted to 6×10⁶/g fresh weight and the vitality was up to 90%;Dual-luciferase reporter vector was used as the reporter gene and protoplasts were regarded as the receptor to measure protoplast transformation efficiency,when the concentration of PEG4000 was 20% and transfection time was 20 min in dark,the high luciferase activities of Photinus pyralis and Renilla reniformis could be detected,indicating that dual-luciferase reporter vector can be successfully loaded into protoplasts.The study provide technical basis for tartray buckwheat protoplast transient expression system and genetic manipulation.