Effect of Several Enzyme Solutions on the Isolation and Culture of Grape Mesophyll Protoplasts

Viable mesophyll protoplasts were isolated from in vitro grown leaves of grapes (Vitis labruscana cv. Kyoho and V. vinifera cv. Cabernet Sauvignon). The best yield of mesophyll protoplasts (1.8 × 107 and 2.4 × 107 • g -1 leaf fresh weight, 'Kyoho' and 'Cabernet Sauvignon', respectively) was obtained in a combined enzyme solution of 0.5% Cellulase YC, 0.2% Macerozyme R -10 and 0.1% Driselase. More than 85% of the isolated protoplasts were viable. Most protoplasts regenerated the cell wall within first 5 days of culture. Cell division occurred after 5 to 10 days of culture in Gamborg's B5 liquid medium supplemented with 5μM 2, 4 - D, 2.5μM BA and 0.6 M sorbitol. In 'Kyoho', 12% of cell division frequency was found in the protoplasts isolated with 0.5% Cellulase YC, 0.2% Macerozyme R -10, 0.1% Driselase and 0.01% Pectolyase Y -23. In 'Cabernet Sauvignon', the frequency of cell division was low (1.4%) when the same enzyme solution was used for the protoplast isolation.