Isolation and cultivation of protoplasts from morphogenetic callus cultures of Lilium

Protoplasts isolated from Lilium callus which was maintained on media containing 2% sucrose contained large deposits of starch granules and lysed during isolation and washing procedures. Stable protoplast preparations could be obtained from callus which had been subcultured on sucrose-free medium for 3 weeks. Maximum protoplast yield (1.5 × 106 per gram fresh weight) was obtained when KCl (0.3 M) was the osmotic stabilizer. Inclusion of CaCl2 (25 mM) and MgSO4 (25 mM) in the isolation and wash media decreased protoplast lysis. Viability of protoplasts isolated in the high salts medium was determined by their ability to accumulate sodium fluorescein in the cytoplasm. No cell-wall formation occurred when salts were used as the osmoticum in various culture media. Continuous light (5000 lx) was inhibitory to protoplast survival. When protoplasts were transferred, via a series of wash solutions, to culture media using sugars as the osmoticum and cultured in darkness, cell-wall formation was detected after 3 days and cell divisions after 21 days. Zeatin (10−6 M), was needed for cell-wall formation. Cell division was stimulated by a combination of zeatin (10−6 M), naphthaleneacetic acid (10−5 M), and 2,4-dichlorophenoxyacetic acid (10−7 M) in the basic nutrient medium.