Gefriervorgange in lebendem Pflanzengewebe: der Beginn des intrazellularen Gefrierens

Highly sensitive temperature measurements by means of a thermistormade it possible to directly record individual crystallizationprocesses in petiolar tissue of Saintpaulia ionantha duringfast cooling. Supercooling to deep temperatures increases thefreezing velocity in the region of the so-called first freezingpoint. Extensive supercooling produces an increased amount ofice at the first freezing point, compared with less supercoolingof the same tissue. With the onset of intracellular freezing, ice formation in individualcells or cell complexes shows up as characteristic sharp peakssuperimposed on the curves. These peaks begin immediately afterthe first freezing point; intracellular freezing is thus indicatedto start at temperatures higher than the so-called second freezingpoint. The temperatures recorded by a probe are not equal tothe actual freezing temperatures of extracellular liquid orliving cells. They are influenced by loss of heat from samplesinto the surrounding air and by different supercooling of individualcells. (Received February 5, 1972; )     

细菌冰核提高印度谷螟过冷却点的研究

印度谷螟(Plodia interpunctella)是一种不耐结冰的昆虫,在冬季它通过降低过冷却 点以避免结冰。现已查明,冰核活性细菌能显著提高植物的过冷却点,导致许多作物在较高 的温度下发生霜冻害。本文也证明细菌冰核能显著提高印度谷螟虫的过冷却点。对照的平均过冷却点是-17.6℃;分别用0.1g和1g细菌冰核与1kg面粉混合后进行处理,平均过冷却点分别比对照提高了12.8℃和13.6℃。研究结果支持这样的观点：细菌冰核有可能成为一种在冬季使用的、杀灭不耐结冰害虫的生物制剂。     

Freezing Avoidance Mechanisms by Supercooling in Some Rhododendron flower Buds with Reference to Water Relations

Excised florets of some hardy Rhododendron species did not toleratefreezing at –5°C when ice-inoculated due to intracellularfreezing. Florets in intact December buds, however, could besupercooled to about –30°C. When flower buds of R.japonicum were slowly cooled with daily decrements of 5°Cto temperatures ranging from 0 to –20°C, the exothermtemperatures of the florets drastically decreased. This wasaccompanied by a decrease in water content of florets and peduncleand an increase in that of scales. The water in florets andthe peduncle is thought to migrate to scales and other tissuesduring the early stages of freezing; the dehydrated floret hasa lower freezing point which enhances its supercooling abilityand the dehydrated peduncle helps to maintain the supercooledstate of the florets. This hypothesis would explain the dependenceon the cooling rate of supercooling in Rhododendron flower buds.Water migration within flower buds was observed in other hardyRhododendron species with some variation in ice formation siteand the quantity of migrated water. The exotherm temperatureof excised florets was inversely proportional to their watercontent. Dehydration of flower buds by wind at 0°C alsoenhanced their supercooling ability. Mechanisms of freezingavoidance by supercooling in Rhododendron flower buds and therelationship of supercooling to freezing tolerance are discussed. ¹ Contribution No. 2254 from the Institute of Low TemperatureScience ² This is a revised form of the master's thesis of the seniorauthor (M.I.) which is cited in the present and previous papers(Sakai 1979a, b, etc.). (Received August 11, 1980; Accepted June 1, 1981)     

Plant Freezing and Damage

Imaging methods are giving new insights into plant freezingand the consequent damage that affects survival and distributionof both wild and crop plants. Ice can enter plants through stomataand hydathodes. Intrinsic nucleation of freezing can also occur.The initial growth of ice through the plant can be as rapidas 40 mm s^-1, although barriers can limit this growth. Onlya small fraction of plant water is changed to ice in this firstfreezing event. Nevertheless, this first rapid growth of iceis of key importance because it can initiate further, potentiallylethal, freezing at any site that it reaches. Some organs andtissues avoid freezing by supercooling. However, supercooledparts of buds can dehydrate progressively, indicating that avoidanceof freezing-induced dehydration by deep supercooling is onlypartial. Extracellular ice forms in freezing-intolerant as wellas freezing-tolerant species and causes cellular dehydration.The single most important cause of freezing-damage is when thisdehydration exceeds what cells can tolerate. In freezing-adaptedspecies, lethal freezing-induced dehydration causes damage tocell membranes. In specific cases, other factors may also causedamage, examples being cell death when limits to deep supercoolingare exceeded, and death of shoots when freezing-induced embolismsin xylem vessels persist. Extracellular masses of ice can damagethe structure of organs but this may be tolerated, as in extra-organfreezing of buds. Experiments to genetically engineer expressionof fish antifreeze proteins have not improved freezing toleranceof sensitive species. A better strategy may be to confer toleranceof cellular dehydration.Copyright 2001 Annals of Botany Company Freezing, dehydration, infrared video thermography, low temperature scanning electron microscopy, NMR micro-imaging     

Intracellular freezing of glycerolized red cells.

The response of glycerolized human red blood cells to freezing has been evaluated in terms of the thermodynamic state of the frozen intracellular medium. The physiochemical conditions requisite for intracellular freezing, characterized by the cooling rate and the degree of extracellular supercooling, are altered appreciably by the prefreezing addition of glycerol to the cells.Fresh human erythrocytes were suspended in an isotonic glycerol solution yielding a final cryophylactic concentration of either 1.5 or 3.0 m. Subsequently the cell suspension was frozen on a special low temperature stage, mounted on a light microscope, at controlled constant cooling rates with varying degrees of extracellular supercooling (ΔT_sc). The formation of a pure intracellular ice phase was detected by direct observation of the cells.The addition of glycerol produced several significant variations in the freezing characteristics of the blood. As in unmodified cells, the incidence of intracellular freezing increased with the magnitudes of both the cooling rate and the extracellular supercooling. However, the glycerolized cells exhibited a much greater tendency to supercool prior to the initial nucleation of ice. Values of ΔT_sc > ?20 °C were readily obtained. Also, the transition from 0 to 100% occurrence of intracellular ice covered a cooling rate spectrum in excess of 300 to 600 °K/min, as compared with 10 °C/min for unmodified cells. Thus, the incidence of intracellular ice formation was significantly increased in glycerolized cells.     

Change of supercooling capability in solutions containing different kinds of ice nucleators by flavonol glycosides from deep supercooling xylem parenchyma cells in trees

Deep supercooling xylem parenchyma cells (XPCs) in Katsura tree contain flavonol glycosides with high supercooling-facilitating capability in solutions containing the ice nucleation bacterium (INB) Erwinia ananas, which is thought to have an important role in deep supercooling of XPCs. The present study, in order to further clarify the roles of these flavonol glycosides in deep supercooling of XPCs, the effects of these supercooling-facilitating (anti-ice nucleating) flavonol glycosides, kaempferol 3-O-β-d-glucopyranoside (K3Glc), kaempferol 7-O-β-d-glucopyranoside (K7Glc) and quercetin 3-O-β-d-glucopyranoside (Q3Glc), in buffered Milli-Q water (BMQW) containing different kinds of ice nucleators, including INB Xanthomonas campestris, silver iodide and phloroglucinol, were examined by a droplet freezing assay. The results showed that all of the flavonol glycosides promoted supercooling in all solutions containing different kinds of ice nucleators, although the magnitudes of supercooling capability of each flavonol glycoside changed in solutions containing different kinds of ice nucleators. On the other hand, these flavonol glycosides exhibited complicated nucleating reactions in BMQW, which did not contain identified ice nucleators but contained only unidentified airborne impurities. Q3Glc exhibited both supercooling-facilitating and ice nucleating capabilities depending on the concentrations in such water. Both K3Glc and K7Glc exhibited only ice nucleation capability in such water. It was also shown by an emulsion freezing assay in BMQW that K3Glc and Q3Glc had no effect on homogeneous ice nucleation temperature, whereas K7Glc increased ice nucleation temperature. The results indicated that each flavonol glycoside affected ice nucleation by very complicated and varied reactions. More studies are necessary to determine the exact roles of these flavonol glycosides in deep supercooling of XPCs in which unidentified heterogeneous ice nucleators may exist.     

The Freezing Response of an Arctic Cushion Plant, Saxifraga caespitosa L.: Acclimation, Freezing Tolerance and Ice Nucleation

Cold hardiness in actively growing plants of Saxifraga caespitosaL., an arctic and subarctic cushion plant, was examined. Plantscollected from subarctic and arctic sites were cultivated ina phytotron at temperatures of 3, 9, 12 and 21 °C undera 24-h photoperiod, and examined for freezing tolerance usingcontrolled freezing at a cooling rate of 3–4 °C eitherin air or in moist sand. Post-freezing injury was assessed byvisual inspection and with chlorophyll fluorescence, which appearedto be well suited for the evaluation of injury in Saxifragaleaves. Freezing of excised leaves in moist sand distinguishedwell among the various treatments, but the differences werepartly masked by significant supercooling when the tissue wasfrozen in air. Excised leaves, meristems, stem tissue and flowerssupercooled to –9 to –15 °C, but in rosettesand in intact plants ice nucleation was initiated at –4to –7 °C. The arctic plants tended to be more coldhardy than the subarctic plants, but in plants from both locationscold hardiness increased significantly with decreasing growthtemperature. Plants grown at 12 °C or less developed resistanceto freezing, and excised leaves of arctic Saxifraga grown at3 °C survived temperatures down to about –20 °C.Exposure to –3 °C temperature for up to 5 d did notsignificantly enhance the hardiness obtained at 3 °C. Whenwhole plants of arctic Saxifraga were frozen, with roots protectedfrom freezing, they survived –15 °C and –25°C when cultivated at 12 and 3 °C, respectively, althougha high percentage of the leaves were killed. The basal levelof freezing tolerance maintained in these plants throughoutperiods of active growth may have adaptive significance in subarcticand arctic environments. Saxifraga caespitosa L., arctic, chlorophyll fluorescence, cold acclimation, cushion plant, freezing stress, freezing tolerance, ice nucleation, supercooling     

Metastable states of water and ice during pressure-supported freezing of potato tissue

Different ice modifications were obtained during freezing processes at several pressure levels from atmospheric pressure up to 300 MPa. In the pressure range between 210 and 240 MPa, a metastable ice I modification area was observed, as the nucleation of ice I crystals in the thermodynamically stable region of ice III was reached. A significant degree of supercooling was obtained before freezing the tissue water to ice III, which has to be considered when designing pressure-supported freezing processes. The effect of supercooling phenomenon on the phase transition time is discussed using a mathematical model based on the solution of the heat transfer governing differential equations. Phase transition and freezing times for the different freezing paths experimented are compared for the processes: freezing at atmospheric pressure, pressure-assisted freezing, and pressure-shift freezing. Different metastable states of liquid water are defined according to their process-dependent stability.     

Phylogenetic analyses in cornus substantiate ancestry of xylem supercooling freezing behavior and reveal lineage of desiccation related proteins

The response of woody plant tissues to freezing temperature has evolved into two distinct behaviors: an avoidance strategy, in which intracellular water supercools, and a freeze-tolerance strategy, where cells tolerate the loss of water to extracellular ice. Although both strategies involve extracellular ice formation, supercooling cells are thought to resist freeze-induced dehydration. Dehydrin proteins, which accumulate during cold acclimation in numerous herbaceous and woody plants, have been speculated to provide, among other things, protection from desiccative extracellular ice formation. Here we use Cornus as a model system to provide the first phylogenetic characterization of xylem freezing behavior and dehydrin-like proteins. Our data suggest that both freezing behavior and the accumulation of dehydrin-like proteins in Cornus are lineage related; supercooling and nonaccumulation of dehydrin-like proteins are ancestral within the genus. The nonsupercooling strategy evolved within the blue- or white-fruited subgroup where representative species exhibit high levels of freeze tolerance. Within the blue- or white-fruited lineage, a single origin of dehydrin-like proteins was documented and displayed a trend for size increase in molecular mass. Phylogenetic analyses revealed that an early divergent group of red-fruited supercooling dogwoods lack a similar protein. Dehydrin-like proteins were limited to neither nonsupercooling species nor to those that possess extreme freeze tolerance.     

Complex bud architecture and cell‐specific chemical patterns enable supercooling of Picea abies bud primordia

Bud primordia of Picea abies, despite a frozen shoot, stay ice free down to ?50 °C by a mechanism termed supercooling whose biophysical and biochemical requirements are poorly understood. Bud architecture was assessed by 3D—reconstruction, supercooling and freezing patterns by infrared video thermography, freeze dehydration and extraorgan freezing by water potential measurements, and cell‐specific chemical patterns by Raman microscopy and mass spectrometry imaging. A bowl‐like ice barrier tissue insulates primordia from entrance by intrinsic ice. Water repellent and densely packed bud scales prevent extrinsic ice penetration. At ?18 °C, break‐down of supercooling was triggered by intrinsic ice nucleators whereas the ice barrier remained active. Temperature‐dependent freeze dehydration (?0.1 MPa K^?1) caused accumulation of extraorgan ice masses that by rupture of the shoot, pith tissue are accommodated in large voids. The barrier tissue has exceptionally pectin‐rich cell walls and intercellular spaces, and the cell lumina were lined or filled with proteins, especially near the primordium. Primordial cells close to the barrier accumulate di, tri and tetrasaccharides. Bud architecture efficiently prevents ice penetration, but ice nucleators become active inside the primordium below a temperature threshold. Biochemical patterns indicate a complex cellular interplay enabling supercooling and the necessity for cell‐specific biochemical analysis.     

Characteristics and Importance of Heterogeneous ice Nuclei Associated With Citrus Fruits

The freezing behaviour and relative importance of heterogeneousice nuclei in affecting supercooling of Citrus sinensis fruitswere studied. The size of an ice nucleation active (INA) Pseudomonassyringae pool inhabiting fruits was positively correlated withthe nucleation temperature (NT) of the plant tissue, with amean of 369 log colony forming units (CFU) fruit^–1 atNT–25C. The INA bacterial pool was responsible for 23%of the nucleation events occurring at this temperature, and29% were attributed to an additional nucleating source. Thelatter was sensitive to bacterial ice nucleation inhibitors,it occupied a different microniche from that of P. syringaenuclei, yet was neither a fungus nor any of the bacterial strainsever reported as active. Treatment with an ice nucleation inactivebacterium antagonistic to INA bacteria, a lentil lectin, a protease,and guanidine reduced mean nucleation temperatures (MNT) offruits to –258, –266, –421, and–452C,respectively, compared to a MNT–167C for the controls.Thus, the citrus-associated nucleator apparently contained activeproteinaceous components but was void of carbohydrate-like groupsreportedly encountered at or near the bacterial ice nucleatingsite. Despite the different origins of citrus nuclei, bacterialnucleation inhibitors reduced nucleation in the field by 50%at NT––25C, an effect probably exerted throughthe proteinaceous site possessed by both nuclei. Key words: Citrus, ice nucleation, INA bacteria, supercooling     

Deep supercooling enabled by surface impregnation with lipophilic substances explains the survival of overwintering buds at extreme freezing

The frost survival mechanism of vegetative buds of angiosperms was suggested to be extracellular freezing causing dehydration, elevated osmotic potential to prevent freezing. However, extreme dehydration would be needed to avoid freezing at the temperatures down to ?45°C encountered by many trees. Buds of Alnus alnobetula, in common with other frost hardy angiosperms, excrete a lipophilic substance, whose functional role remains unclear. Freezing of buds was studied by infrared thermography, psychrometry, and cryomicroscopy. Buds of A. alnobetula did not survive by extracellular ice tolerance but by deep supercooling, down to ?45°C. An internal ice barrier prevented ice penetration from the frozen stem into the bud. Cryomicroscopy revealed a new freezing mechanism. Until now, supercooled buds lost water towards ice masses that form in the subtending stem and/or bud scales. In A. alnobetula, ice forms harmlessly inside the bud between the supercooled leaves. This would immediately trigger intracellular freezing and kill the supercooled bud in other species. In A. alnobetula, lipophilic substances (triterpenoids and flavonoid aglycones) impregnate the surface of bud leaves. These prevent extrinsic ice nucleation so allowing supercooling. This suggests a means to protect forestry and agricultural crops from extrinsic ice nucleation allowing transient supercooling during night frosts.     

Changes in supercooling and freezing processes accompanying leaf maturation in Buxus

The influence of maturity and size on freezing temperaturesof intact leaves of Buxus was investigated from the standpointof nucleation theory. During active leaf growth the extent ofsupercooling was dependent on the degree of leaf maturity, butwas unaffected by leaf size. After leaves were fully matured,the effect of leaf size predominated in supercooling. In general,the greatest supercooling was seen in semi-mature leaves; thisgradually decreased as maturity advanced. This change in supercoolingduring active leaf growth corresponded closely to changes inthe freezing pattern, especially to a gradual transition ofice nucleation sites to the apical part of the mid-vein. Thesetwo correlative changes are probably dependent on structuralchanges in tracheary elements that accompany maturation. Age-inducedchanges in supercooling in growing leaves are dependent on qualitativeaspects of nucleators, whereas size-induced changes in matureleaves are dependent on quantitative aspects of nucleators. (Received October 7, 1970; )     

Supercooling ability of Rhododendron flower buds in relation to cooling rate and cold hardiness

The freezing process and supercooling ability in flower budsof 11 native Rhododendron species were examined with referenceto the cooling rate and cold hardiness by differential thermalanalysis. The freezing patterns of the excised whole buds variedwith the season: in autumn, buds froze as whole units, whilein winter, freezing was initiated in the scales and propagatedto each floret. The supercooling ability of florets was enhancedduring winter. The freezing patterns in winter buds were stronglyinfluenced by the cooling rate (1 to 30°C/hr). Althoughthe first exotherm in scales occurred at –5 to –10°Gand was rate-independent, the occurrence of several floret exothermsshifted considerably to lower subzero temperatures at slowerrates. The most reliable cooling rate for testing maximum supercoolingability was l°C/hr. The exotherm in florets of hardier speciesoccurred at –20 to –25°C and at –7 to–20°C for less hardy ones, and were well correlatedwith their killing temperatures. Water relations within budtissues in response to freezing are briefly discussed. (Received June 26, 1980; )     

Supercooling ability of Rhododendron flower buds in relation to cooling rate and cold hardiness

The freezing process and supercooling ability in flower budsof 11 native Rhododendron species were examined with referenceto the cooling rate and cold hardiness by differential thermalanalysis. The freezing patterns of the excised whole buds variedwith the season: in autumn, buds froze as whole units, whilein winter, freezing was initiated in the scales and propagatedto each floret. The supercooling ability of florets was enhancedduring winter. The freezing patterns in winter buds were stronglyinfluenced by the cooling rate (1 to 30°C/hr). Althoughthe first exotherm in scales occurred at –5 to –10°Gand was rate-independent, the occurrence of several floret exothermsshifted considerably to lower subzero temperatures at slowerrates. The most reliable cooling rate for testing maximum supercoolingability was l°C/hr. The exotherm in florets of hardier speciesoccurred at –20 to –25°C and at –7 to–20°C for less hardy ones, and were well correlatedwith their killing temperatures. Water relations within budtissues in response to freezing are briefly discussed. (Received June 26, 1980; )     

A Cryomicroscopic Study of Cylindrocystis brebissonii De Bary and Two Species of Micrasterias Ralfs (Conjugatophyceae, Chiorophyta) during Freezing and Thawing

The changes in morphology of the unicellular algae Cylindrocystisbrebissonii and two species of Micrasterias during freezingand thawing were observed on a light microscope fitted witha temperature controlled stage. At slow rates of cooling extensiveshrinkage of the protoplast was observed. The response of thecell wall varied with cell-type. In C. brebissonii plasmolysiswas not observed and the cell wall and protoplast shrank together.In Micrasterias the cell wall did not contract and a distinctplasmolysis was observed. Following freezing to and thawingfrom –25?C cells of C. brebissonii were non-viable butremained osmotically responsive. Cooling at faster rates inducedintracellular ice formation in all cell-types. The criticalrate of cooling varied with cell-type and was determined bycell volume and suface area. Intracellular gas bubbles wereobserved during thawing following both rapid and slow cooling. Following cooling in dimethylsulphoxide cells of C. brebissoniiwere protected against freezing injury. The recovery on thawingfrom –196?C being determined by the rate of cooling, anoptimum rate of 1?C min^–1 was observed. During slow ratesof cooling (<2?C min^–1) cells remained unshrunken,at faster rates (10?C min^–1) the loss of cell viabilitywas related to osmotic shrinkage during cooling rather thanto nucleation of intracellular ice. Intracellular ice formationwas observed only following significantly faster rates of cooling(>20?C min^–1). Key words: Cylindrocystis, Micrasterias, cryomicroscopy, freezing injury     

Antifreeze glycoproteins: influence of polymer length and ice crystal habit on activity

The effect of the ice crystalline habit and the length of the polymer on the ability of the antifreeze glycoproteins (AFGP) from polar fish to depress the freezing temperature of water was investigated. The low-molecular-weight components of the glycoproteins, AFGP- 6–8, are inactive when a solution of such a sample is nucleated at ?6°C. A solution of large AFGP (1–4) is fully functional under the same conditions. The low-molecular-weight components differ from the height-molecular-weight components in that they contain some proline replacing the alanine in the Ala-Ala-Thr · disaccharide polymer unit. In the present experiments, antifreeze activity was examined in the presence of two different forms of ice crystal growth habits, and homodimders of AFGP 6 and 8 were prepared to investigate the function of polymer length and the on antifreeze activity at different degrees of supercooling. The results indicate that the ice crystal growth habit and the introduction of proline into the polymer unit may be responsible for the loss of activity at deep supercooling (?6°C) of AFGP 6–8. The loss in the ability of AFGP to depress the freezing temperature of water at deep supercooling is not solely due to polymer length, as carbodiimide-linked dimers of AFGP 6 do not function under these freezing conditions. A Model of antifreezing action based on Langmuirian adsorption of AFGP on the ice surface and direct competition between water and AFGP molecules for the incorporation sites in the ice crystal lattice is presented.     

Cryopreservation of primary cultures of mammalian somatic cells in 96-well plates benefits from control of ice nucleation

Cryopreservation of mammalian cells has to date typically been conducted in cryovials, but there are applications where cryopreservation of primary cells in multiwell plates would be advantageous. However excessive supercooling in the small volumes of liquid in each well of the multiwell plates is inevitable without intervention and tends to result in high and variable cell mortality. Here, we describe a technique for cryopreservation of adhered primary bovine granulosa cells in 96-well plates by controlled rate freezing using controlled ice nucleation. Inducing ice nucleation at warm supercooled temperatures (less than 5 °C below the melting point) during cryopreservation using a manual seeding technique significantly improved post-thaw recovery from 29.6% (SD = 8.3%) where nucleation was left uncontrolled to 57.7% (9.3%) when averaged over 8 replicate cultures (p < 0.001). Detachment of thawed cells was qualitatively observed to be more prevalent in wells which did not have ice nucleation control which suggests cryopreserved cell monolayer detachment may be a consequence of deep supercooling. Using an infra-red thermography technique we showed that many aliquots of cryoprotectant solution in 96-well plates can supercool to temperatures below −20 °C when nucleation is not controlled, and also that the freezing temperatures observed are highly variable despite stringent attempts to remove contaminants acting as nucleation sites. We conclude that successful cryopreservation of cells in 96-well plates, or any small volume format, requires control of ice nucleation.     

Seasonal variations in freezing curves of stem sections of Cornus stolonifera MICHX.I

Comparisons of freezing curves have been used to determine theviability of plant parts exposed to stress. To gain understandingof the natural seasonal variations in freezing curves, uniformtwig sections of red-osier dogwood (Cornus stolonifera MICHX.)were collected throughout the year from a single clone and subjectedto controlled freezing while the tissue temperature was recorded.The supercooling of samples ranges from –2 to –7,but the variation was random and unpredictable. There was noapparent relationship between supercooling and the season ofthe year or the hardiness of the tissue. The freezing pointdepression, as estimated by the temperature of the first freezingplateau, was always between –0.25 and –1.0 andbore no relationship to hardiness or season. The freezing curveswere basically of three types: Summer and winter curves withtwo distinct freezing points; Early autumn curves with 3distinctfreezing points and spring curves with one prominent first freezingpoint which tended to mask the second freezing point. ¹Scientific Journal Series paper No. 6628, Minnesota AgriculturalExperiment Station. This research was supported in part by agrant from the Louis W. and MAUD HILL Family Foundation. ²Present Address: Horticulture Department, University of Wisconsin,Madison, Wisconsin, U.S.A.     

Cryo-scanning electron microscopic study on freezing behaviors of tissue cells in dormant buds of larch (Larix kaempferi)

The freezing behavior of dormant buds in larch, especially at the cellular level, was examined by a Cryo-SEM. The dormant buds exhibited typical extraorgan freezing. Extracellular ice crystals accumulated only in basal areas of scales and beneath crown tissues, areas in which only these living cells had thick walls unlike other tissue cells. By slow cooling (5 °C/day) of dormant buds to −50 °C, all living cells in bud tissues exhibited distinct shrinkage without intracellular ice formation detectable by Cryo-SEM. However, the recrystallization experiment of these slowly cooled tissue cells, which was done by further freezing of slowly cooled buds with LN and then rewarming to −20 °C, confirmed that some of the cells in the leaf primordia, shoot primordia and apical meristem, areas in which cells had thin walls and in which no extracellular ice accumulated, lost freezable water with slow cooling to −30 °C, indicating ability of these cells to adapt by extracellular freezing, whereas other cells in these tissues retained freezable water with slow cooling even to −50 °C, indicating adaptation of these cells by deep supercooling. On the other hand, all cells in crown tissues and in basal areas of scales, areas in which cells had thick walls and in which large masses of ice accumulated, had the ability to adapt by extracellular freezing. It is thought that the presence of two types of cells exhibiting different freezing adaptation abilities within a bud tissue is quite unique and may reflect sophisticated freezing adaptation mechanisms in dormant buds.