Role of intracellular water retention strength in freezing tolerance of Chrysanthemum cinerariaefolium Vis. cell cultures

An efficient procedure using: sucrose as cryoprotectant to confer freezing tolerance on high-pyrethrin-producing cell lines of Chrysanthemum cineraria efolium was developed. The efficiency of sucrose depended on both its conce ntration in the pregrowth medium and on preculture duration. A low sucrose concentration (0.25 mol/L) induced insufficient dehydration of cells, and s o failed to prevent damage during freezing and prevented regrowth. The high cell dehydration obtained by incubating cells for 10 days in a medium supp lemented with 0.5 mol/L sucrose was essential for freezing tolerance, but w as insufficient to induce cell cryoprotection on its own. At this concentra tion, the pose-thaw viability rate increased with culture duration. Twenty days of pretreatment afforded a 57% viability rate, which was correlated wi th a different partitioning of water into unfrozen and frozen fractions, wi thout modifications in cell dehydration. To study the water sorption streng ths in pretreated cells, sorption isotherms were constructed. The effects o f sucrose concentration and preculture duration during pretreatments were i nvestigated. Sorption isotherms were analysed according to the D'Arcy and W att model. Sucrose acted by trapping intracellular water through modificati on of hydrogen and multimolecular linkages, and thus increased the unfrozen water fraction in cells.