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-pyrethrinproducing cell lines of Chrysanthemum cinerariaefolium was developed. The efficiency of sucrose depended on both its concentration in the pregrowth medium and on preculture duration. A low sucrose concentration (0.25 mol/L) induced insufficient dehydration of cells, and so failed to prevent damage during freezing and prevented regrowth. The high cell dehydration obtained by incubating cells for 10 days in a medium supplemented with 0.5 mol/L sucrose was essential for freezing tolerance, but was insufficient to induce cell cryoprotection on its own. At this concentration, the post-thaw viability race increased with culture duration. Twenty days of pretreatment afforded a 57% viability rate, which was correlated with a different partitioning of water into unfrozen and frozen fractions, without modifications in cell dehydration. To study the water sorption strengths in pretreated cells, sorption isotherms were constructed. The effects of sucrose concentration and preculture duration during pretreatments were investigated. Sorption isotherms were analysed according to the DʹArcy and Watt model. Sucrose acted by trapping intracellular water through modification of hydrogen and multimolecular linkages, and thus increased the unfrozen water fraction in cells.