Saffron (Crocus sativus L.) protoplasts were isolated from the cells of a suspension culture or calli with a solution of Cellulase, Pectinase and Hemicellulase and embedded in Ca-alginate beads. They were cultured with or without nurse cells in MS medium supplemented with 2,4-D and 6-benzylaminopurine at 25°C. After several changes of medium, cell-clusters appeared on the surface of the Ca-alginate beads. The protoplasts without immobilization in Ca-alginate beads did not display cell division. Furthermore, growth of cell clusters in the medium with nurse cells was much better than in the medium without nurse cells. Then, the beads were transferred onto MS agar medium supplemented with 1-naphtalene acetic acid and 6-benzylaminopurine and cultured at 15°C or 20°C. After 3-4 months of culture, great calli were observed on the surface of the beads. The regenerated shoots were obtained approximately 6-7 months after protoplast isolation. The critical factors seem to be associated with the quality of the culture, enzyme composition and concentration used for protoplast isolation.