Abstract: Protoplasts were successfully isolated from somatic embryogenic cell suspension induced by pseudobulbils, shoots regeneration were achieved. Enzyme sort and concentration, treatment time and the culture time of cell suspensions had influence on protoplasts isolation. Enzyme solution contained 2.0% cellulase, 0.5% macerozyme, 5.0 mmol·L^-1 MES, 0.5 mol·L^-1mannitol, 0.5% CaCl2·2H2O. Optimum enzyme treatment time was 10 hours and cell suspensions subcultured for 3 days were adopted. The yield and viability were 26.67×10⁶ g-1 and 92.21%. Protoplasts were cultured on shallow liquid layers, where inositol was better than sucrose for promoting cells division and growth. Calli induced and proliferation medium was 1/2MS + 2, 4-D1.0 mg·L^-1 + EBR0.2 mg·L^-1 + 10 g·L-1 Ficoll + 500 mg·L-1 glutamine + 20 mg·L-1 glycine + 20 mg·L-1 aspartic acid. Differentiation medium was 1/2MS + 10 mg·L^-1 TDZ + 500 mg·L^-1 glutamine + 20mg·L^-1 glycine + 20 mg·L^-1 aspartic acid. Shoots regenerated on 1/2MS medium.

Key words: Rosa multiflora, protoplast, cell suspension culture, shoot regeneration