Abstract:Calll were initiated from the seedling segment of Peucedanum praeruptorum Dunn and subcultured on the MS agar medium with 0.5 mg/L 2,4-D. Cell suspension culture with a lot of embryogenic cell clumps was obtained in liquid medium. Protoplasts were isolated from the cell clumps in enzyme mixture solution containing 1.5% Onozuka R-10, 0.3% Macerozyme R-10, 0.5% helicase, 5 mmol/L CaCl2 and 0.6 mol/L mannital, at pH 5.6 and shaking for 5- hours at 25℃. Helicase is necessary for isolation. After purified by washing, the protoplasts were cultured in liquid medium containing 1 mg/L 2,4-D +0.5 mg/L zeatin. First cell division was observed after four days. Large cell clumps were formed after thirty days. Microcalli of 1 mm in size was formed after about fifty days, and continued to grow on the MS solid medium containing 0.5 mg/L 2,4-D and 200 mg/L casein hydrolysate, and later differentiated into embryoids when transferred to MS agar medium with 0.1 mg/L zeatin. Eventually, embryoids developed into whole plantlets on the MS solid medium without phytohormones.