Abstract
A methods for growing of saffron (Croccus sativus L.) protoplasts immobilized in Ca-alginate is presented in detail. Protoplasts were isolated from the cells of the suspension culture (ISA and OGASAWARA 1988) with a solution of Cellulase "Onozuka" RS Pectolyase Y-23 and Driselase, and embedded in Ca-alginate beads. They were cultured with or without nurse cells in MS medium (MURASHIGE and SKOOG 1962) supplemented with 2, 4-D and Zeatin at 25°C. After several changes of medium, cell-clusters appeared on the surface of the Caalginate beads, although the protoplasts without immobilization in Ca-alginate beads did not display cell division. Growth of the cell clusters in the medium with nurse cells was much better than that in the medium without nurse cells. Then, the beads were transferred onto MS agar medium supplemented with 1-naphtaleneacetic acid and 6-benzylaminopurine, and subcultured at 15°C or 20°C. After 2 to 3 months of culture, shoots and roots regenerated from the callus tissue.
