Cryoprotection in plant tissues related to reduced volume expansion of cryoprotectant solution

To clarify the mechanism of reduced volume expansion-related cryoprotection changes in solution volume during freezing using several types of cryoprotectant were investigated. The effect of each cryoprotectant solution on the survival of asparagus nodal segments cooled slowly (0.5°C/min) to -40°C was also examined. The ratio of the volume at -40°C to the volume at +20°C was used as an index for expansion, calculated as a ratio of the density at +20°C to the density at -40°C. Distilled, deionized water showed the largest volume change at a ratio of 1.094. The ratio gradually decreased with an increase in the molar concentration of cryoprotectant, with the magnitude of the change dependent on the nature of the cryoprotectant. Raffinose was the most effective in reducing volume expansion when compared with other cryoprotectants at a same concentration. Raffinose exhibited greatest cryoprotection in asparagus tissue at 0.6 M where the solution became saturated. Dimethyl sulfoxide (Me₂SO) at 1.69 M had the largest effect on cryoprotecting asparagus tissue. Furthermore, Me₂SO was also the most effective in reducing volume expansion among the group of cryoprotectants permeable to the plasma membrane. It is concluded that cryoprotection in tissues was closely related to reduced volume expansion especially at low concentration (≤1.0 M). Cryoprotectants of impermeable sugar group lost their cryoprotective effect at >1.0 M, which may due to severe dehydration and cell damage occurred in hypertonic solution. Useful cryoprotectants should be furnished with high ability of reducing volume expansion during freezing as well as low toxicity and high permeability for cells.