Summary.Arabidopsis thaliana suspension cells were preserved in liquid nitrogen for over three years, using embedding of cells in calcium-alginate prior to subculture in sucrose-enriched medium, air-drying, and direct quenching in liquid nitrogen. Survival of cells reached 34%, yielding regrowth at the surface of all cryopreserved beads in less than 7 days. Following pretreatment and dehydration, the water content dropped from 2300% to 34% with respect to dry weight. Differential scanning calorimetry showed that glass transition occurred on cooling, followed by a slight crystallization event on rewarming. The survival of cells was independent of the cooling rate. The tolerance of the acute dehydration step increased progressively with sucrose pretreatment duration, indicating the requirement for adaptative cellular alterations. Ultrastructural studies revealed several changes in cells after sucrose pretreatment prolonged from 1 to 7 days: reversal of the initially plasmolyzed state, microvacuolation, numerous autophagic structures, scarcity of ribosomes, increase in number and size of starch grains. No cell division seemed to occur during this period. After air-drying and after a freeze-thaw cycle, followed by 24 h rehydration, regenerating cells had recovered a high level of ultrastructural organization and contained numerous polysomes suggesting an intense metabolic activity. Trehalose, a cryoprotective disaccharide not considered to be a metabolic substrate, yielded only 70% regrowth after freezing. Biochemical analysis showed that soluble sugars accumulated during the pretreatment, essentially sucrose or trehalose; the monosaccharide content also increased. In the light of these results, the action of sucrose in inducing freezing tolerance is discussed.

No abstract

No abstract

Résumé.-Les effets de la congélation et ceux des substances cryoprotectrices sur les cellules sont présentés en dégageant les aspects concernant les divers mécanismes qui entraînent la mort des cellules et ceux qui permettent leur résistance aux très basses températures. Les causes de la mortalité des cellules peuvent résulter d'un effet direct de la cristallisation intracellulaire ou d'un effet indirect dû à une cristallisation extracellulaire. La résistance des cellules aux très basses températures peut être acquise naturellement par un processus d'endurcissement ou artificiellement par l'apport de substances cryoprotectrices qui agissent au rùveau des cytomembranes et de la matrice cellulaire.Summary.-Effects of freezing and cryoprotectants on the animal and plant cells were studied in order to determine differents events leading to cell death and resistance to ultra-low temperatures. Cell injury depends on an intracellular cristallization (direct effect) or on an extracellular cristallization (indirect effect ). Plant cell resistance to ultra-low temperatures may be due to a cold harderùng process or to the use of cryoprotectants which act on different cytomembranes structures and cellular matrix.

No abstract