Supercooled water, aqueous solutions at low temperatures and their glassy states are closely related to cryopreservation of living cells, foods, and related materials. The studies we have conducted are mostly basic and relevant indirectly to cryo-biological technology. They are roughly divided into three groups: (1) The works intended to interpret anomalous properties of supercooled water, (2) Raman and DTA studies of aqueous solutions at low temperatures and their glassy states, and (3) Basic researches of cryo-preservation of plant-cells by vitrification. In the (1) group, an X-ray diffraction study of supercooled water was carried out and it is concluded that clathrate-like structures increase with decreasing temperature. The location of the second critical point of water is estimated to be T_<c2> < -100℃ and P_<c2> = 〜200MPa. In the (2) group, there are many Raman studies devoted to glassy aqueous solutions. It is found that ionization of sulfuric acid increases with decreasing temperature. In the DTA studies for supercooling behavior of aqueous solutions, it is confirmed that the linear relation between melting temperature (T_m) and homogeneous ice nucleation temperature (T_H) holds quite well for many aqueous solutions. In addition, the additivity rule is found to hold in the supercooling of aqueous solutions. In the (3) group, many measurements were made on the T_H values for various types of aqueous solutions as a function of pressure. From the observation that pressure accelerates supercooling and vitrification of an aqueous solution, we made preliminary experiments to preserve carrot cells at high pressure.

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Utilization of dry food and generation/conservation of body water plays a principal role in the tolerance of organisms against various environmental stresses such as aridity and high temperature. Here, I review two topics: evolution of dry bean use and bound water in stored bean pest. A large proportion of Callosobruchus seed beetle species are pests of stored, dry postharvest beans (i.e., seeds of legumes). However, the evolution of this feeding habit is poorly understood. We reconstructed the phylogeny of Callosobruchus and assessed which traits have been associated with the evolutionary origin or gain/loss of ability to use dry beans. Dry-bean use was phylogenetically constrained and associated with adaptation to arid climate. Thus, physiological adaptation to an arid climate renders beetles predisposed to become pests of dry beans. Body water conditions, associated with water conservation, at different life stages of insects under heat are yet to be understood. We compared loosely bound water (LBW) in seed beetles' body among life stages and between stored and non-stored bean pests at high temperatures, using ^1H-nuclear magnetic resonance spectroscopy. LBW was lowest in pupae. A non-stored bean pest retained higher LBW than a stored bean pest. I suggest an interpretation of the results.

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Freezing is one of extreme environments for organisms. On the other hand, freezing is useful strategy for storage of foods. The authors have studied on development of freezing tolerance of plants for storage and stable supply of foods. In order to understand the mechanisms of the freezing tolerance, low-temperature inducible genes were isolated from Chlorella, and genes responsible for synthesis of taurine and trehalose were isolated from common carp and tobacco plant, respectively. The cloned (LEA, FAD2, NTRC, PRX, CDO, CSD, TPS, TPP) genes were modified and respectively subcloned into expression vectors for expression in Saccharomyces cerevisiae. Freezing tolerance of the yeast cells expressing the genes was investigated to estimate functions of the expressed gene products.

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The cryobiology of mice has been investigated with the aim of identifying basic mechanisms of biological phenomena at cold temperatures and establishing cryotechnologies for mammalian cells. Recently, cryotechnologies have become essential to biological research using genetically engineered mice. Many cryopreserved embryos and sperm harvested from genetically engineered mice have been archived in mouse resource banks. Techniques of cryopreservation or cold storage of embryos or gametes are applied for the efficient production, preservation, and distribution of genetically engineered mice. Until now, we have studied the cryobiology of mouse embryos and gametes and developed various cryo- and related techniques to establish an efficient system of mouse resource bank. In this review, we describe the history and recent progress of the use of cryotechnologies for embryos and gametes stored in mouse resource banks.

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Water temperature directly affects the physiological phenomena of fish categorized into heterothermic animal. In particular, the water temperature has been known as a major factor that controls the reproductive phenomena, such as sexual differentiation, gametogenesis, spawning, etc. In the present paper, we describe about the effects of water temperature on the reproductive phenomena in fish.

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Seed germination, a crucial stage in a plant's life, is complicated by several factors, including plant hormones and environmental factors. Plant hormones such as gibberellins (GAs), abscisic acid (ABA), brassinosteroid and ethylene play key roles in seed germination. Reactive oxygen species (ROS) promote germination of seeds in several plant species. In contrast, antioxidants suppress the effect of ROS on seed germination. However, the mechanism underlying ROS-induced germination of seed has not been clarified. We therefore focus on relationships between ROS and plant hormones signaling in seed germination. After imbibition, ROS were produced in embryo and aleurone cells of barley seed and in embryonic axis of soybean seed. ROS produced in embryo of barley seed and embryonic axis of soybean seed regulated GA and ethylene biosynthesis, respectively. In aleurone cells of barley seed, ROS were produced by GA and were suppressed by ABA. ROS produced by GA in aleurone cells negatively regulate ABA signals and consequently promote production of a-amylase that acts as key enzyme in seed germination. Taken together, ROS produced after imbibition is likely to be involved in seed germination through the regulation of plant hormone biosynthesis and signaling.

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Low temperature have been broadly applied for storage technique of various vegetable and crops. Sweetpotato is one of the most popular crops grown in tropical and temperature regions. Because sweetpotato, originated from semi-tropical plants, is susceptible to cold stress, severe chilling stress by itself causes deterioration and/or irreversible damage in sweetpotato tuber tissues. However, mechanisms involved in physiological and biochemical changes of sweetpotato under chilling stress remain unclear. Thus, we focused on the gene expression profiles of cold stress-responsive transcriptional factors and carbohydrate metabolisms of sweetpotato in response to chilling stress. A sweetpotato homolog of Drought Responsive Element Binding factor (swDREB) is induced in sweetpotato within 6 h after treatment of chilling stresses. The expression of swDREB under chilling stress was maintained until 3 d. Chilling stress sequentially upregulated the expression of β-amylase (β-AMY) and trehalose-6-phosphate phosphatase (TPP). Increase of amylase activity and sugar content was also observed in sweet potato under chilling stress, These results suggest that swDREB mediates the expression of β-AMY and TPP via a cold stress-responsive transcription factor cascade, leading to degradation of starch in sweetpotato tuber and accumulation of maltose and trehalose.

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The effects of freeze-thaw cycles on the gelation of heat-induced gel from frozen surimi were studied. The rheological properties of the heated gel were measured by rheometer. The kinds of bands between the proteins of gel were estimated by solubilities of the gel for various solvents. Freeze-thaw cycles caused decreasing in breaking strength and breaking strain of the gel. The protein solubility rates of heated gel showed that rates of ionic bond and hydrogen bond were increased, whereas hydrophobic interaction, S-S bonding and more intensive bonding interaction were decreased by freeze-thaw cycles. From these results, it was suggested that the changes of protein solubility rates caused rheological property deterioration of the heated gel.

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Preservation and transportation of cultured plant cells after desiccation were attempted. Five different cells (Pogonatum inflexum, Polytrichum commune, Ceratopteris thalictroides, Daucus carota, Oryza sativa) were tested and Pogonatum cells appeared to be the most suitable for this study because Pogonatum cells showed the highest desiccation tolerance among the cells tested. Desiccated Pogonatum cells could be preserved over 6 weeks below 4℃ but could not survive at 26℃ after preservation for 7 days. Desiccated Pogonatum cells were transported by a commercially served system and then cultured for post-transport regrowth examination. Although the desiccated cells folded by aluminum foil could not survive after transportation, the cells transported in a cryotube were alive and re-growth was recognized.

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The seeds of the soybean Glycine max (L.) Merr. show high sensitivity to excessive water in the germination stage, and this susceptibility reduces germination rate, plant growth and yield. Therefore, it is important to determine the relationship between germination and water conditions. In this study, we investigated whether nuclear magnetic resonance (NMR) relaxation time (T_2) could be used to characterize germination in two soybean cultivars, 'Fukuyutaka' (yellow seed coat) and 'Wase-kurodaizu' (black seed coat), and one kidney bean cultivar, 'America-saito'. The germination index (GI) of 'Fukuyutaka' was the highest, followed by that of 'Wase-kurodaizu', and 'America-saito.' The water contents and T_2 values of long fraction in the two soybean cultivars increased faster than those in the kidney bean, and T_2 values more clearly explained the difference than water content. We also observed the digestion of seed storage substances. Starch granules were digested 72 hours after imbibition in the cotyledons of 'Fukuyutaka,' while still observed in those of 'Wase-kurodaizu' and 'America-saito.' Additionally, most oil bodies were digested in the cotyledons of 'Fukuyutaka,' while abundant oil bodies were still observed in those of 'Wase-kurodaizu' 72 hours after imbibition. This suggests that the difference in GI can be explained by water states indicated by NMR T_2 and the degradation of the storage substances of seeds after imbibition.

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Spirulina platensis NIES-46 was observed during freeze-thawing under an optical microscope. Based on the observation, viability of the algae was assessed after freeze-thawing for the purpose of establishment of cryopreservation protocol. It was considered that different results obtained with the different drying and cooling conditions can be explained by taking the trichome structure of the algae and the membrane permeation of dimethyl sulfoxide used as a cryoprotectant into consideration.

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The sleeping chironomid, Polypedilum vanderplanki, is the only insect known to be capable of anhydrobiosis. Recently, we discovered that Pv11 cells derived from this insect showed the capacity to survive desiccation. This finding allowed us to perform functional analysis of anhydrobiosis at the cellular level. Hence, we decided to investigate which genes were involved in anhydrobiosis using genome editing technology in Pv11 cells. In order to optimize the technology for non-model organisms like the sleeping chironomid, it is essential to select appropriate expression promoters. We isolated Pol II and Pol III type promoters from this insect for exogenous expression of Cas9 and a guide RNA, respectively, and confirmed their activity.

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