The cold tolerance and ice nucleation temperature of medaka embryos in three embryonic stages were investigated with different cryoprotectant treatments. The ice nucleation temperatures of the embryos in every stage showed a decreasing tendency by cryoprotectant treatment, in the order of trehalose, DMSO, and a combination of trehalose and DMSO. Further, the ice nucleation temperatures of the control embryos were depressed with the embryonic development. However, when the embryos were treated with the cryoprotectants, the stage dependency on the ice nucleation temperatures could not be recognized for 8 cell and optic bud stages, and only at the eyed stage the ice nucleation temperatures were significantly lower than those in early embryonic stages. On the other hand, the cold tolerance of the embryo at a temperature below 4℃, which was examined at the temperature range without occurrence of ice nucleation, became higher in every stage by cryoprotectant treatments. As for the stage dependency, it could be found that the cold tolerance of the embryos treated with cryoprotectants did not show a large difference among the stages, however, in the case of control embryo the cold tolerance at the exposure temperature below 4℃ showed an increasing tendency as the stage developed. As the conclusion, it was suggested that the lowering of the ice nucleation temperature of the embryos led to the increase in the survival rate of embryos at the cold temperature without ice nucleation, although other factors such as the development stage should be considered in.

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The suppression mechanism of ice crystal growth due to sugar which was stored in cells was investigated when plant tissues were encountered at low temperature. In this paper, the growth rate and the morphology of ice crystals grown in aqueous sucrose solution were measured and compared between them.

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The freezing and the melting processes of the solutions of trehalose and water were observed to know how trehalose protects the organisms from the freezing of their cellular water. The frozen specimens that were frozen rapidly at more than 10K supercooling showed the ramera structure composed of the ice phase and the trehalose rich phase. The specimens' surfaces were covered with the trehalose rich phase and very smooth. Melting of the specimens firstly began at the trehalose rich phase. The frozen specimen of 41.7wt% trehalose solution was soft enough to slide rapidly along the interface between the ice phase and the trehalose rich phase at -17℃.

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In-situ observations of an ice crystal growing in the solution of trehalose and water at various supercoolings were performed to investigate how trehalose suppresses the growth of ice crystals and protects organisms from the freezing of their cellular water. The growth rate along the a-axis of an ice crystal grown in a trehalose solution increased proportionally to the second power of the supercooling. The growth rate was suppressed to one forth when the concentration of trehalose was doubled. 20.8wt% trehalose solution suppressed the growth rate as well as 41.5wt% sucrose solution. Trehalose significantly suppressed the instability of ice crystals than sucrose. These results are considered to be related to the fact that a trehalose molecule binds more water molecules than does a sucrose molecule.

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The low-temperature thermal behaviour of aqueous alkali metal chloride-sugar solutions was investigated by differential scanning calorimetry (DSC). Glucose, galactose, mannose and fructose as a monosaccharide, trehalose as a disaccharide, raffinose as a trisaccharide, sodium chloride and potassium chloride as an alkali metal chloride were used. From the results, NaCl has a higher glass formation ability than KC1, glucose and galactose have a slight higher glass formation ability than mannose and fructose. The four monosaccharides have an almost same glass state stability. Trehalose and raffinose have the almost same glass formation ability and glass state stability as monosaccharides per a sugar ring.

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FTIR spectra are obtained for several different states of trehalose including dihydrate crystal, anhydrous form II, anhydrate crystal, dried melt, amorphous solid and aqueous solution. From the observation of the symmetric and antisymmetric stretch vibrations of the glycosidic linkage, it is found that this sugar takes at least three types of backbone conformation. The Ca symmetry structure of trehalose seems to have a feature that the sugar easily catches up water molecules. The sugar's conformation in anhydrous form II is shown to be 2. Next, the hygroscopic abilities of the anhydrate, form II and the amorphous solid are compared based on their IR spectra. Interestingly, form II alone is converted to the original dihydrate in a week under mild environmental conditions: relative humidity of 40% and room temperature. These results suggest the possibility that form II plays a role in avoiding the devitrification of the sugar glass. Finally we discuss the role of form II in preserving freeze-dried biomaterial.

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The stability of amorphous ATP, ADP and trehalose were examined from enthalpy relaxation process below the Tg by DSC. The enthalpy relaxation amount (ΔH) was obtained from the endothermic peak area on DSC heating curve that was appeared after aging for 2 to 48 hours below the Tg. The ΔH of every sample increased with aging time through the increasing rate showed significant variation depending on the kind of materials and aging temperature. The ΔH vs. aging time relation was analyzed to obtain a mean relaxation time constant τ by using Kohlrausch-Williams-Watts (KWW) equation. Then the aging temperature dependency of the τ was evaluated by Volgel-Fulcher-Tammann (VFT) equation. Judging from the obtained parameters in KWW and VFT equations, the amorphous state for ATP and ADP below Tg was suggested to be more stable than for trehalose and sucrose, and that the temperature dependency of the enthalpy relaxation rate below Tg were found to be non-Arrhenius type.

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It has been known that the dextran aqueous solution of high concentrations turns into a gelatinised state in the presence of highly-concentrated potassium ions, and that it is not gelatinised in the absence of potassium ions, or when their concentration is low. From the present study, It was found that the dextran-KCl aqueous solution, the concentration of which is not so high for both of dextran and KC1, turns into a gelatinised state as a result of the freeze-concentration. However, gelatinisation was reversible: the system gelatinised by the freeze-concentration treatment returned to the original solution state when it was kept at ambient temperatures.

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As possible cryoprotectants for cryopreservation of living plant cells at high pressures, supercooling behavior of aqueous solutions of DMSO, glycerol and ethylene glycol was investigated as a function of pressure. It is shown that glass formations of aqueous glycerol and ethylene glycol solutions are enhanced at high pressures. Glass transition temperature (T_g) increases with increasing pressure for all three solutions at R=3 (R=moles of water/moles of solute)..

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Miscibility of proteins and saccharides (monosaccharides to oligosaccharides) in aqueous frozen solutions was studied through thermal analysis to model freeze-dried protein formulations. Thermal transitions (glass transition temperature of maximally freeze-concentrated solutions: Tg's) of the frozen solutions showed varied solute miscibility depending on the combinations and concentration ratios. Many protein and saccharide combinations were freeze-concentrated into amorphous mixture phase in protein-rich to moderately saccharide-rich concentration ratios, whereas saccharide phase appeared besides the mixture phase above certain saccharide/protein concentration ratios. Some saccharide-rich combinations separated into individual chases in the freeze-concentrates.

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It is known that yeast cells which contain trehalose exhibit ethanol tolerance. The survival ratio against ethanol shock increases with an increase in trehalose content when its value is below 2.5 w% /dry solid. In the range of larger trehalose content, the survival ratio is saturated with a high value of 80-90 %. To explain such phenomena, we measured the amount of bound water in cells using thermogravimetry. Consequently, the amount of bound water steeply decreases when the trehalose content is less than2.5 w%, and is kept constant at a low level in more concentrated range. Thus, the tendencies of the survival and the bound water against torehalose concentration are similar to each other. On the basis of these results, we discuss the mechanism of ethanol tolerance induced by trehalose, and show that the water replacement mechanism, early proposed to explain the desiccation tolerance, are also hold in ethanol tolerance.

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To determine the contribution of various compatible solutes to an increase in freezing tolerance in detail, accumulation of sugars, proline and glycine betaine in leaves of three wheat cultivars was characterized and compared during cold acclimation. We have found that the concentration of sugars was much greater than that of proline or glycine betaine after cold acclimation and accumulation of proline occurred earlier than that of glycine betaine during the course of cold acclimation. There was a positive relationship between the extent of freezing tolerance and the sum of the concentration of sugars, proline and glycine betaine after cold acclimation, while the contribution of these three compatible solutes to the increased osmolality in leaves during cold acclimation seemed not to be correlated with freezing tolerance. These results suggest that the accumulation of compatible solutes has multiple roles in increasing the freezing tolerance in plants.

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The physiological changes in intact mume (Prunus mume Sieb. et Zucc.) seed were compared to fig (Ficus carica L.) drupelet which develop parthenocarpically with bearing seedless tissues by using ^1H-NMR spectroscopy. Water compartments shown as Lorentzian/Gaussian curves indicated that the mobile water components decreased in mume seeds inversely to that of fig drupelet during maturation. The high mobility of water in the immature mume seeds decreased with their development instead of diminished free water; consequently, oil accumulated. In contrast, free water in the fig drupelet tissues markedly increased with their development accompanying with sugar accumulation. Water in the tissues is present in several compartments, each showing different water molecule mobilities. Long component of the NMR relaxation times (T_1, T_2) derived from vacuolar water in mature seeds of mume fruit markedly decreased with lowering of water content while fig drupelet water ranging about 90% per fresh weight basis did not indicate obvious relationship to T_1 or T_2 compared to the seed. Thus, the water status is considered to reflect physiological changes such as that regarding cellular heterogeneity. The analysis of the water compartments, Lorentzian/Gaussian curve, provides a new concept of seed dehydration indicating contradictory water status in seedless tissues with maturation.

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Primary cultures of Lavandula hybrid and Lavandula stoechas were carried out. Adventitious shoots and callus with a high growth rate were obtained from the tissues of both species cultured on MS medium supplemented with appropriate concentrations of cytokinin and auxin. Subcultured callus cells of both species were cryopreserved in liquid nitrogen. Although cryopreserved cells died during the post-thaw culture on MS medium, recovery cultures were obtained on the medium from which ammonium ion was eliminated. All of the cells preserved by the vitrification method failed to recover from the frozen-thaw injury and died during the post-thaw culture.

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Changes in electrical and rheological properties of carrot cell tissues after freezing-thawing, cooking, and chloroform-vapor treatment were compared to clarify the mechanism of freezing injury. The electrical property was measured by impedance and the rheological properties were determined by the vibrating reed method. The change of rheological properties after chloroform-vapor treatment might be caused only by the loss of turgor pressure, while that after freezing-thawing seems to be caused by the injury of total cell tissues.

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Suspension-cultured cells of Arabidopsis thaliana were encapsulated in different gel beads; calcium alginate, K-carrageenan, agarose, agar, and cultured in media with high concentrations of sucrose. These cultured cells were dehydrated on silica gel and then cryopreserved in liquid nitrogen (LN_2). In the cells encapsulated in each gel beads, the higher rate of cell survival was observed after dehydration on silica gel as compared with those without encapsulation, suggesting some protective effects of the gels on cell dehydration. The culture of the cells in media with high concentrations of sucrose increased the rate of cell survival after cryopreservation in LN_2 with or without encapsulation. In the cells without encapsulation, however, the effect of sucrose in media was observed more clearly at higher concentrations than 0.75M

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