Abstract
When plant tissues were cryopreserved by utilizing a slow pre-freezing method, it was noticed that cotton plugs came out of the sample-containment straw during freezing only when distilled water was used as a freezing solution. This was likely due to a large ice expansion during freezing of distilled water. However, since plugs were remained on the straw when freezing with any cryoprotectant solution, it was assumed cryoprotectants might reduce ice expansion in the freezing solution. Furthermore, it seemed possible that cryoprotectants acted by reducing mechanical stress caused by extracellular ice crystals growing during the slow freezing procedure. In the present study, 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 frozen down slowly (0.5℃/min) to -40℃ was also examined. The ratio of the volume at -40℃ to the volume at +20℃ was used as an index for ice expansion, which could be calculated as a ratio of density at +20℃ to density at -40℃. Density at +20℃ was obtained by measuring both weight and volume of each solution. Density at -40℃ (r) was calculated by the following formula: r=w0^*r1/(w0-w1); rl, density of isooctane at -40℃ (known as 0.73999); w0, weight of an amount of solution in air; w1, weight of the same amount in isooctane at -40℃. Distilled 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. Raffmose was the most effective in reducing ice expansion when being compared with other cryoprotectants at a same concentration. Raffinose showed it's largest cryoprotection against asparagus tissue at 0.6M where the solution became saturated. Dimethylsulfoxide (DMSO) at 1.69M had the largest effect on cryoprotecting asparagus tissue. Furthermore, DMSO was also the most effective in reducing ice expansion among plasmamembrane-permeable groups of cryoprotectants.
