Abstract: To construct a protocol of isolation and purification of protoplasts from the seedling leaves of Bletilla striata. After a series of pretreating assay, the leaves were enzymatic hydrolyzed with mixed solution of mannitol, Cellulase R-10, Pectolyase Y-23 and Macerozyme R-10 for certain hours. Then the newly released protoplasts were purified through filtration and centrifugalization. Finally, the harvest protoplastswere detected in the survival rate by means of fluorescein diacetate (FDA). The preprocessing of seedlings living in dark field for 12 h and the leaves were immersed in 0.75 mol·L^-1 mannitol for 1 h was the most effective way for protoplasts isolation. The enzymes mixture of 0.75 mol·L^-1 mannitol+1.5% Cellulase R-10+0.4% Pectolyase Y-23+0.5% Macerozyme R-10 was the optimism composition of enzyme solution. The pH of 5.8 and 4 h enzyme dissolve with the revolving speed of 120 r·min^-1 at room temperature was the best environmental condition. Under this optimal condition, abundant of 4.72×10⁶ protoplasts with survival rate of 90.4% could be harvested from each gram leaf. This study gave a necessary help for protoplasts culture, cell fusion and genetic transformation of B.striata.

Key words: Bletilla striata, protoplast, isolation, purification, protocol