Abstract：In this study，plantlet bulblet of Longya Lily（Lilium brownii var. viridulum Baker）were used
as experimental material to optimize protoplasts preparation parameters by orthogonal experiment．Effects of
vibrating speed，enzymolysis time，centrifugal speed of harvesting protoplasts，and the sucrose concentration，
etc．factors influencing yield of preparing protoplasts were discussed．The results showed that the optimum
conditions were combined by 1.0% of cellulase，1.0% of pectinase enzymolysis subcultured for 40 d in vitro
regenerated seedling bulblet 3 h，50 r·min-1 vibrating speed of enzymolysis，700 r·min-1 centrifugal speed of
harvesting protoplasts，6 min centrifugation time，and 25% purified sucrose．With this combined conditions，
the per milliliter yield of protoplast isolated from per 1 g seedling bulblet could reach 6.60×106．
