Abstract: Experimental raw material of protoplast was obtained from embryo callus induction of Celastrus orbiculatus Thunb. under the condition of lower temperature at 4℃ and dark treatment for 24 h. The created culture system of protoplast originated from seedlings differentiation of callus regeneration after the culture of settling the shallow-layer solution and solid-liquid double layer culture as well as agarose-embedding based on MS medium. The results showed that: the lower temperature and dark treatment was beneficial to obtain protoplast with high yield and top quality; the optimum combination for the enzyme activity would be: 0.5% Cellulase+0.5% Pectinase+5 mg·L^-1 MES; the optimum time of enzymatic hydrolysis was 12 h; the optimum mannitol concentration was 13%; moreover, the optimum mode of enzymatic hydrolysis would be: standing for 12 h and oscillating for 0.5 h; meanwhile, better protoplast culture efficiency was based on the culture of settling the shallow-layer solution; the optimum differentiation medium of callus was MS+6-BA 2.0 mg·L^-1+IBA 0.1 mg·L^-1 and rooting medium could be 1/2 MS+NAA 0.1 mg·L^-1.

Key words: Celastrus orbiculatus Thunb., callus, protoplast culture, plant regeneration