Effect of osmotic stress on tolerance of air-drying and cryopreservation of Arabidopsis thaliana suspension cells

Arabidopsis thaliana suspension cells were preserved in liquid nitrogen for over three years, using embedding of cells in calcium-alginate prior to su bculture in sucrose-enriched medium, air-drying, and direct quenching in li quid nitrogen. Survival of cells reached 34%, yielding regrowth at the surf ace of all cryopreserved heeds in less than 7 days. Following pretreatment and dehydration, the water content dropped from 2300% to 34% with respect t o dry weight. Differential scanning calorimetry showed that glass transitio n occurred on cooling, followed by a slight crystallization event on rewarm ing. The survival of cells was independent of the coding rate. The toleranc e of the acute dehydration step increased progressively with sucrose pretre atment duration, indicating the requirement for adaptative cellular alterat ions. Ultrastructural studies revealed several changes in cells after sucro se pretreatment prolonged from 1 to 7 days: reversal of the initially plasm olyzed 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-tha w cycle, followed by 24 h rehydration, regenerating cells had recovered a h igh level of ultrastructural organization and contained numerous polysomes suggesting an intense metabolic activity. Trehalose, a cryoprotective disac charide not considered to be a metabolic substrate, yielded only 70% regrow th after freezing. Biochemical analysis showed that soluble sugars accumula ted during the pretreatment, essentially sucrose or trehalose; the monosacc haride content also increased. In the light of these results, the action of sucrose in inducing freezing tolerance is discussed.