Importance of actin cytoskeleton behaviour during preservation of carrot cell suspensions in liquid nitrogen

Summary Conventional methods for preservation of suspended, highly vacuolated, plant cells in liquid nitrogen (LN) usually involve equilibration in molar concentrations of cryoprotective additives, followed by slow cooling to an intermediate subzero temperature (–40 °C), before quenching in LN. Cryomicroscopy was used to monitor the reversible protoplasmic shrinkage of cryoprotected carrot cells, caused by freeze-induced dehydration. Behaviour of actin filaments was analyzed by fluorescence microscopy after labelling with rhodarnine-conjugated phalloidin, in relation to the type of pretreatment and to survival and regrowth ability after preservation at — 196 °C. Loading with dimethylsulphoxide (Me₂SO, 5%) resulted in high survival rates (70%) and regrowth. After thawing, the actin filament (MF) abundance was reduced, but the structure and distribution of the remaining MFs seemed undisturbed. Higher Me₂SO concentrations caused further reduction of MFs, which appeared fragmented after thawing. MFs were maintained by pretreatment with 0.5 M sorbitol alone but carrot cells did not survive at — 196 °C. The same pretreatment, followed by incubation with cytochalasin D (10 M), which greatly reduced MFs, enabled plasmolyzed carrot cells to survive preservation in liquid nitrogen. Thus, after both Me₂SO and sorbitol plus cytochalasin D pretreatments, partial disruption of actin filaments seemed to accompany (Me₂SO) or promote (sorbitol plus cytochalasin D) freezing tolerance at extremely low temperatures.Abbreviations CD cytochalasin D - FDA fluorescein diacetate - LN liquid nitrogen - MF actin filament - Me₂SO dimethylsulphoxide