Abstract:

The optimal isolation conditions of protoplasts from young leaves of wine grape ‘GuiPu No.1’ was studied in this paper. The influence of some factors, such as enzyme composition, time of enzymatic hydrolysis, concentration of stabilizer, as well as speed and time of centrifugation, on isolation of protoplasts was comparatively analyzed, using young leaves of ‘GuiPu No.1’ aseptic seedling with 20-30 day as materials. The appropriate enzyme composition was 2%cellulase + 0.5%pectinase. The protoplast yield gradually increased with extension of enzymatic hydrolysis time. The appropriate enzymatic hydrolysis time was 8 hours for protoplast isolation from young leaves of ‘GuiPu No.1’. The protoplast yield significantly increased with rise of mannitol concentration in the range from 0.45~0.55 mol/L and the yield reached highest at mannitol concentration 0.55 mol/L 2.48×106 protoplast/(g?FW) and activity 79.78% The centrifugation speed of 1000 rpm and the centrifugation time of 6～8 min was appropriate for suspension purification of protoplast. The optimal enzyme solution for protoplast isolation was as follows: 2% Celluasw Onozuka R-10+0.5% PectolyaseY-23+25 mmol/L MES+0.55 mol/L Mannitol. Protoplasts with high yield and viability were obtained when incubation for 8 h. The centrifugation at 1000 r/min for 6-8 min in 30% sucrose was good for suspension purification of protoplast.