An investigation of the frost hardiness of grapevine (Vitis vinifera) during bud break

The aim of this investigation was to assess ice nucleation and frost resistance of two varieties of grapevine (Siegrebbe and Madeleine Angevine) during bud burst under radiative freezing conditions analogous to those during Spring in the UK. During bud burst, grapevines were almost entirely resistant to freezing during frosts of less than -3°C by virtue of their ability to supercool. The risk of frost damage increased significantly as bud development progressed, and once buds had passed growth stage DS3 they became more sensitive to freezing and freezing damage was more extensive. The two varieties did not differ in frost resistance but, because of its earlier developing habit, variety Siegrebbe could be more prone to frost damage in the field. Buds were more prone to damage after freezing once bud burst had commenced and the damage could not be reversed by acclimating plants for periods of 7 to 21 days at 4°C in an 8 h photoperiod. Such acclimation appeared to predispose frozen buds to more extensive damage.     

Overwintering temperatures affect freezing temperatures of turions of aquatic plants

The effect of different overwintering temperatures (2.5 ± 1 °C in a refrigerator or outdoor natural overwintering on wet topsoil with weak frosts) on the freezing temperature and survival rate of turions of 10 aquatic plant species with different ecological traits (free-floating habit or bottom rooting) was studied using mini thermocouples. Dormant, non-hardened turions of 9 species exhibited freezing within a narrow temperature range of ?7.0 to ?10.2 °C, while Hydrocharis morsus-ranae froze at ?3.6 °C. The survival rate of the turions after the measurements was, however, very low (0–38%). In several species, the freezing temperature of turions at the beginning of germination was not significantly different (at p < 0.05) from the dormant ones. The mean freezing temperature of outdoor hardened turions of 6 species was within a very narrow range of ?2.8 to ?3.3 °C and was thus significantly higher by 4–7 °C (p < 0.0002) than that for the non-hardened turions. It is assumed that the freezing temperatures indicate freezing of the extracellular water. The hardened turions of all 7 species were able to survive mild winter frosts under the topsoil conditions at a rate of 76–100%. These characteristics suggest that the turions of aquatic species can be hardened by weak frosts and that their frost hardiness is based on the shift from frost avoidance in non-hardened turions to frost tolerance.     

The tolerance of black currant flowers to induced frosts

Flower buds on potted plants of 17 varieties of black currant were frosted to -3.3, -4.5 and -5.2 °C between the grape stage and full flower in 1979 and 1980. In all varieties more flower buds died after the -5.2 °C frosts and at full flower, and less after the -3.3 °C frosts and at the grape stage. Varieties related to Ben More and Ojebyn tolerated the -4.5 °C frosts until after first flower while Baldwin and Magnus became susceptible at the grape stage. Seabrooks Black, Greens Black and Ben Lomond and its relatives were intermediate. In both years flower buds tolerated frosts to similar growth stages but in 1980 the varieties flowered about 2 wk earlier than in 1979 and suffered more frost damage at full flower. The frosted plants had slightly larger fruits than the unfrosted ones in 1979. The immature fruit drop was similar in frosted and unfrosted plants in both years except when it was increased after -5.2 °C in 1980. It is pointed out that for reliable cropping, varieties should flower late as well as tolerate spring frosts and that tests of frost tolerance should be done for at least three growth stages.     

No increase in alpine snowbed productivity in response to experimental lengthening of the growing season

Climate change effects on snow cover and thermic regime in alpine tundra might lead to a longer growing season, but could also increase risks to plants from spring frost events. Alpine snowbeds, i.e. alpine tundra from late snowmelt sites, might be particularly susceptible to such climatic changes. Snowbed communities were grown in large monoliths for two consecutive years, under different manipulated snow cover treatments, to test for effects of early (E) and late (L) snowmelt on dominant species growth, plant functional traits, leaf area index (LAI) and aboveground productivity. Spring snow cover was reduced to assess the sensitivity of snowbed alpine species to severe early frost events, and dominant species freezing temperatures were measured. Aboveground biomass, productivity, LAI and dominant species growth did not increase significantly in E compared to L treatments, indicating inability to respond to an extended growing season. Edapho‐climatic conditions could not account for these results, suggesting that developmental constraints are important in controlling snowbed plant growth. Impaired productivity was only detected when harsher and more frequent frost events were experimentally induced by early snowmelt. These conditions exposed plants to spring frosts, reaching temperatures consistent with the estimated freezing points of the dominant species (～?10 °C). We conclude that weak plasticity in phenological response and potential detrimental effects of early frosts explain why alpine tundra from snowbeds is not expected to benefit from increased growing season length.     

The Influence of Season and Phenology on Freezing Tolerance in Silene acaulis L., a Subarctic and Arctic Cushion Plant of Circumpolar Distribution

Changes in the freezing tolerance for Silene acaulis L., a subarcticand arctic species of circumpolar distribution, were examinedto understand the extent of cold hardening and dehardening thatoccurs seasonally and with changes in plant phenology. Shootsof whole plants collected on a mountain ridge near Tromsø,Norway (69° N, 700 m above sea level) were frozen undercontrolled conditions at cooling rates of 3 to 4°C h^-1.The extent of freezing-induced injury was examined both by chlorophyllfluorescence and by visual inspection with a microscope. A freezingtolerance level of -30°C was observed in mid-winter, basedon a 50% lethal point for freezing injury. Loss of cold hardinesswas substantial in mid-summer, with freezing tolerance of -8·5to -9°C observed in mid-July. Plants still covered by snowin mid-July had a freezing tolerance of -12·5 to -13°C.The maintenance of a basic level of freezing tolerance throughoutthe summer may be adaptive in the northern latitude-regionsbecause of the occurrence of episodic frosts during the growingseason.Copyright 1993, 1999 Academic Press Silene acaulis L., Caryophyllaceae, freezing tolerance, chlorophyll fluorescence, cushion plant     

Frost in a future climate: modelling interactive effects of warmer temperatures and rising atmospheric [CO2] on the incidence and severity of frost damage in a temperate evergreen (Eucalyptus pauciflora)

Although plants are more susceptible to frost damage under elevated atmospheric [CO₂], the importance of frost damage under future, warmer climate scenarios is unknown. Accordingly, we used a model to examine the incidence and severity of frost damage to snow gum (Eucalyptus pauciflora) in a sub‐alpine region of Australia for current and future conditions using the A2 IPCC elevated CO₂ and climate change scenario. An existing model for predicting frost effects on E. pauciflora seedlings was adapted to include effects of elevated [CO₂] on acclimation to freezing temperatures, calibrated with field data, and applied to a study region in Victoria using climate scenario data from CSIRO's Global Climate Model C‐CAM for current (1975–2004) and future (2035–2064) 30 years climate sequences. Temperatures below 0 °C were predicted to occur less frequently while the coldest temperatures (i.e. those below ?8 °C) were almost as common in the future as in the current climate. Both elevated [CO₂] and climate warming affected the timing and rates of acclimation and de‐acclimation of snow gum to freezing temperatures, potentially reducing the length of time that plants are fully frost tolerant and increasing the length of the growing season. Despite fewer days when temperatures fall below 0 °C in the future, with consequently fewer damaging frosts with lower average levels of impact, individual weather sequences resulting in widespread plant mortality may still occur. Furthermore, delayed acclimation due to either warming or rising [CO₂] combined with an early severe frost could lead to more frost damage and higher mortality than would occur in current conditions. Effects of elevated [CO₂] on frost damage were greater in autumn, while warming had more effect in spring. Thus, frost damage will continue to be a management issue for plantation and forest management in regions where frosts persist.     

Effect of cholesterol-loaded cyclodextrins on bull and goat sperm processed with fast or slow cryopreservation protocols

Cholesterol-loaded cyclodextrins (CLC) added to the sperm before cryopreservation enhance sperm quality after freeze-thawing in several cold shock-sensitive species, including cattle and goats. However, all studies conducted to date have used conventional protocols, in which sperm are cooled slowly to 5°C before freezing. As cholesterol plays a significant role in sperm cold shock resistance, it is possible that CLC-treated sperm can withstand cooling damage when the sperm are not cooled slowly to 5°C before freezing. In this study, we determined whether CLC-treated goat (1 mg CLC/120×10⁶ sperm) and bull (2 mg CLC/120×10⁶ sperm) sperm quality, after thawing, was different for sperm frozen using conventional protocols (including a slow cooling phase to 5ºC) and protocols in which the sperm were frozen from room temperature, without cooling the sperm slowly to 5°C before freezing. CLC-treated sperm exhibited higher percentages of plasma membrane-intact sperm than control sperm when cryopreserved using conventional protocols. In addition, CLC treatment enhanced both sperm motility and plasma membrane integrity when sperm were frozen directly from room temperature. However, this treatment did not fully prevent the damage of the sperm after cooling rapidly and subsequent freezing, as the sperm quality was lower than that presented by the samples frozen using the conventional protocol. The results are promising, but studies to optimize the protocols for freezing sperm directly from room temperature need to be conducted, as well as studies to determine how cryopreserving sperm in this manner affects other sperm functions.     

Effects of growth stage and hardening conditions on the association between frost resistance and the expression of the cold-induced protein COR14b in barley

Sowing date, being determinant for growth stage, may play a decisive role in optimising freezing resistance of winter annual plants. In cereal species, in spite of the abundant literature analysing the factors responsible for the acquisition of frost resistance through the cold hardening process, the involvement of the growth stage per se, has been seldom considered, especially at the earlier vegetative phases. In this work the contribution of growth stage in determining resistance to freezing temperature has been analysed in field and growth chamber experiments using winter and spring barley cultivars exposed to different hardening conditions. Field damage was assessed twice during winter on plants sown at three different dates. In the growth chamber experiments several acclimation treatments at 11/7 and/or 3/1 °C (day/night) were simulated. In both field and laboratory experiments the development of cold acclimation was monitored by means of a COR14b specific antibody, since in previous studies the expression of COR14b was found genetically linked to frost resistance. The lowest resistance, found in the youngest plants and in spring cultivars, however, was not always associated with the lowest level of COR14b accumulation. COR14b accumulation correlated with frost resistance at the earlier field sampling date and in plants grown at 11/7 °C. In a following phase of the hardening process (second sampling in field and 4 weeks at 3/1 °C in growth chamber) the accumulation of COR14b was independent of plant stage and genotype, showing no association with freezing resistance. Results suggest that growth stage is crucial for the achievement of maximal resistance in barley, but not for COR14b expression.     

Spring emergence and canopy development strategies in miscanthus hybrids in Mediterranean,continental and maritime European climates

Due to its versatility and storability, biomass is an important resource for renewable materials and energy. Miscanthus hybrids combine high yield potential, low input demand, tolerance of certain marginal land types and several ecosystem benefits. To date, miscanthus breeding has focussed on increasing yield potential by maximising radiation interception through: (1) selection for early emergence, (2) increasing the growth rate to reach canopy closure as fast as possible, and (3) delayed flowering and senescence. The objective of this study is to compare early season re-growth in miscanthus hybrids cultivated across Europe. Determination of differences in early canopy development on end-of-year yield traits is required to provide information for breeding decisions to improve future crop performance. For this purpose, a trial was planted with four miscanthus hybrids (two novel seed-based hybrids M. sinensis × sinensis [M sin × sin] and M. sacchariflorus × sinensis [M sac × sin], a novel rhizome-based M sac × sin and a standard Miscanthus × giganteus [M × g] clone) in the UK, Germany, Croatia and Italy, and was monitored in the third and fourth growing season. We determined differences between the hybrids in base temperature, frost sensitivity and emergence strategy. M × g and M sac × sin mainly emerged from belowground plant organs, producing fewer but thicker shoots at the beginning of the growing season but these shoots were susceptible to air frosts (determined by recording 0°C 2 m above ground surface). By contrast, M sin × sin emerged 10 days earlier, avoiding damage by late spring frosts and producing a high number of thinner shoots from aboveground shoots. Therefore, we recommend cultivating M sac × sin at locations with low risk and M sin × sin at locations with higher risk of late spring frosts. Selecting miscanthus hybrids that produce shoots throughout the vegetation period is an effective strategy to limit the risk of late frost damage and avoid reduction in yield from a shortened growing season.     

Resistance of Phaseolus species to ice crystallization at subzero temperature

Dry bean ( Phaseolus vulgaris L.) cultivars possess little or no freezing tolerance and are killed at the temperature of ice formation in their tissues. An increase in frost tolerance by 2–3°C would expand dry bean production in the short growing seasons of the Canadian prairies and possibly to higher altitudes in the tropics where episodic frosts occur during the growing season. The objective of this study was to determine the differences in frost resistance of Phaseolus species in both controlled and field environments. Leaflets of dry bean cv. CDC Nighthawk, and wild relatives from the primary gene pool ( P. vulgaris var. mexicanus Freytag and P. vulgaris var. aborigineus (Burkart) Baudet) and the tertiary gene pool ( P. acutifolius var. tenuifolius A. Gray, P. filiformis Bentham, P. angustissimus A. Gray and P. ritensis M.E. Jones) were subjected to subzero temperatures with and without ice nucleation to determine the levels of tolerance and avoidance, respectively. The lethal temperature at which 50% of the leaflets were killed (LT₅₀) was 0.5–1°C lower for species of the tertiary gene pool compared to those from the primary gene pool. Leaflets of species from the tertiary gene pool were also characterized by extensive supercooling compared to leaflets of species from the primary gene pool. Resistance of Phaseolus species to spring and autumn frosts were determined on seedlings transplanted to the field. Phaseolus angustissimus , a species of the tertiary gene pool had the highest seedling survival in response to both autumn and spring frosts, when the minimum air temperatures were −5 and −7°C, respectively. Frost resistance of Phaseolus angustissimus , if successfully introgressed into bean germplasm, may enable the development of frost resistant dry bean cultivars.     

Viability and Morphology of rat heart cells after freezing and thawing of the whole heart

Intact adult rat hearts were cooled in the presence of 10% DMSO according to an external cooling program which approximated the optimal external three-step cooling program for the isolated adult heart cells: 20 min at ?20 °C, 0.2 °C/min from ?20 to ?25, ?30, or ?50 °C, and rapid cooling to ?196 °C. Following rapid thawing, cells were isolated after perfusion with a 0.1% collagenase solution. Only cells which originated from the free wall of the right ventricle could be isolated, even after cooling to ?20 °C. Most cells from hearts cooled to ?196 °C did not survive. When the third cooling step was omitted and the end temperature of the second cooling step was ?30 °C, 38% of the cells excluded trypan blue, 29% were morphologically intact, and 30% showed spontaneous contractions after thawing, expressed as percentages of the control, A much lower survival was found after cooling to ?50 °C.Histological and electron microscopical study of the heart immediately after thawing revealed no differences between hearts cooled to ?20, ?30, or ?196 °C. Also no marked differences were observed between the morphological integrity after freezing and thawing of the atrium, the left and right ventricle walls, and the ventricular septum. The survival data suggest the presence of nonmorphologically detectable alterations in cells frozen to ?196 °C, compared to cells frozen to ?30 °C. The morphological investigations indicate no essential differences in resistance of atrial and ventricular cells to the freezing process.Experiments involving neonatal rat hearts cooled to ?196 °C, according to the method which gave optimal preservation of the isolated cells, revealed that after thawing cells are present from which growing and contracting cultures can be derived. It appears that cells in the neonatal rat heart are more resistant to freezing to ?196 °C than cells in the adult rat heart.     

The freezing characteristics of cauliflower curd

Cultivar differences in frost resistance and the heritable nature of resistance were demonstrated using seedling cauliflower plants. Such cultivar differences were not however expressed in the curd. Selection for frost resistance in cauliflower should therefore use whole plant screening techniques. Curd material when frozen as isolated florets, supercooled over the range – 1°C to – 12°C and the mean freezing point of all curds tested was -6°C to -7.25°C (overall mean -6.44°C). Curd florets which supercooled but did not freeze were completely undamaged, whereas freezing always led to cell damage and death observed as water-soaking of the floret surface and measured using an electrical conductivity method. The large range of freezing points measured suggests a range of active ice nucleators either on or within the florets. When curds were frozen intact the ability of florets to supercool was severely restricted which was attributed to the seeding of freezing by the internal growth of ice crystals. A crop protection strategy needs to identify and control or modify warm temperature nucleators in cauliflower curd.     

The effect of cooling on mortality of the Asian weaver ant <Emphasis Type="Italic">Oecophylla smaragdina</Emphasis>

Cooling is an efficient method to immobilise insects for handling, but it is known that fast cooling to temperatures above freezing can cause chill injury and/or death. The mortality of Asian weaver ants, Oecophylla smaragdina, a tropical species that does not normally encounter near freezing temperatures, was investigated in experiments with cooling temperatures of 0 and 5°C and duration of cooling periods of 45 and 150 min. No significant difference in mortality was found between ants cooled to 5 and 0°C; however, cooling to these temperatures led to higher mortalities compared to non-cooled control ants. Following both 45 and 150 min of cold treatment, a significant excessive mortality of cooled ants (compared to uncooled ants) was observed, indicating that effects of cooling on mortality should be considered when performing studies on Asian weaver ants.     

Differences in frost hardiness of two Norway spruce morphotypes growing at Mt. Brocken, Germany

Norway spruce (Picea abies (L.) Karst.) exhibits strong ecotypic variation along altitudinal gradients in morphological traits, e.g. slenderness of crowns or arrangement of second-order branches. We were interested whether montane and lowland morphotypes differ in a key trait for the survival in cold environments, i.e. frost hardiness, and asked: (i) are montane morphotypes more resistant to frost damage and (ii) do they have a lower risk of frost damage by late frosts in spring than lowland morphotypes?We used the electrolyte leakage-method to measure frost hardiness on a monthly basis from October 2006 to May 2007 in stands of the montane and lowland morphotypes at Mt. Brocken in the Harz Mountains, Germany.LT₅₀ (i.e. the temperature that results in 50% of maximum electrolyte leakage) was assessed by freezing treatments in a frost chamber and was significantly influenced by morphotype, month and minimum ambient temperatures. LT₅₀ was significantly lower in the montane than in the lowland morphotype, with −107 °C and −49 °C, respectively. However, the interactions between morphotype with minimum ambient temperature or month were not significant. Thus, as frost hardiness of the two morphotypes responded to temperature in the same way, both morphotypes can be supposed to be exposed to the same risk of frost damage during hardening in autumn and dehardening in spring.     

Supercooling and heterogeneous nucleation of freezing in tissues of tender plants.

The extent to which tissue pieces of several frost tender plant species could be supercooled in the absence of external sources of ice nucleation was determined in a small cold chamber. A considerable range among plant species was revealed in their ability to supercool. This could be expressed as a differential nucleus spectrum that derives the minimum concentration of nucleating sites within the samples. French bean (Phaseolus vulgaris L.) was found to contain nucleators effective in the range ?4 to ?8 °C, whereas spring wheat (Triticum aestivum L.) contained sites active between ?8 and ?16 °C. The data indicate that these plant samples can supercool to temperatures below those normally injurious to them when they are frozen.     

N uptake and growth responses to sub-lethal freezing in the grass Poa pratensis L.

Background and aims

Climate warming has the potential to increase both the exposure and vulnerability of grass roots to frost in temperate regions by reducing snow cover and altering the timing of cold acclimation. Despite a strong research focus on the direct effects of freezing on grass mortality, the direct sub-lethal effects of freezing on grass performance have not been well-characterized. We examined sub-lethal responses of the grass Poa pratensis to variation in the timing, severity, rate and length of freezing.

Methods

We assessed short term root functional responses (¹⁵N uptake) and longer term plant growth responses to freezing administered both under controlled conditions in a refrigerated incubator, and in the field by manipulating snow and litter cover.

Results

In fall and spring, ¹⁵N uptake declined in response to 1?day of freezing down to ?10?°C or to 3?days of freezing at ?5?°C, whereas in winter, ¹⁵N uptake was insensitive to freezing. Long term growth responses were similar, with reduced growth only occurring for grasses frozen for 3?days at ?5?°C in spring, but not for grasses frozen in fall or winter. Snow and litter removal intensified soil freezing over winter, but did not significantly affect plant growth.

Conclusions

Our results demonstrate that while P. pratensis is relatively tolerant to frost damage over winter, it may be vulnerable to sub-lethal frost effects in fall, and particularly in spring. These sub-lethal effects occur at temperatures approximately 15–20?°C warmer than the published LT₅₀ values for this species.     

A microprocessor-controlled rate controller for use in cryopreservation

The design and operation of a microprocessor-controlled rate controller incorporated into a constant-rate cooling system used in the cryopreservation of cells is described. The controller differs from those currently available in that the actual temperature of the cell suspension being cooled is compared with a preselected ramp of the microprocessor. Differences between the two determine the opening of solenoid valves that permit entry of liquid N₂ vapors into the freezing chamber. The heat of fusion, which is released as extracellular ice crystallizes, automates the opening of additional solenoid valves. The rapid entry of N₂ vapors into the chamber cools the cell suspension, therefore restoring the programmed cooling rate. The functional recovery of murine splenic lymphocytes cooled at ?1.0 °C/min using this system exceeds the recoveries given in most other reports.     

Summer frost resistance and freezing patterns measured in situ in leaves of major alpine plant growth forms in relation to their upper distribution boundary

Summer frost resistance and ice nucleation temperatures for 33 alpine plant species were measured in situ to avoid the shortcomings of laboratory tests. Species were selected to investigate the relationship between plant stature and upper distribution boundary, and frost resistance and freezing patterns. The species tested in situ were on average 1.1 K (± 0.2, SE) frost hardier than in laboratory tests. Frost resistance (LT₅₀) ranged from ?4.5 to ?14.6 °C and appeared insufficient to protect against air temperature minima, corroborating reports of natural frost damage. All species tolerated extracellular ice formation (recorded at ?1.9 ± 0.2 °C; E1). Initial frost damage occurred at average temperatures 4.9 K below E1. In 64% of the species a second exotherm (E2) and frost damage were recorded between ?3.7 and ?9.4 °C. In the highest ranging species E2 was not detectable. Frost resistance increased with increasing upper distribution boundary (0.4 K per 100 m), corresponding well with the altitudinal decrease in air temperature minima. No relationship between plant stature and frost resistance was found. Graminoids were significantly frost hardier than other growth forms. Frost survival at high altitudes will depend not only on altitudinal increase in frost resistance but also on freezing avoidance strategies, snow cover protection and a high recuperation capacity.     

How endangered is sexual reproduction of high-mountain plants by summer frosts? Frost resistance, frequency of frost events and risk assessment

In temperate-zone mountains, summer frosts usually occur during unpredictable cold spells with snow-falls. Earlier studies have shown that vegetative aboveground organs of most high-mountain plants tolerate extracellular ice in the active state. However, little is known about the impact of frost on reproductive development and reproductive success. In common plant species from the European Alps (Cerastium uniflorum, Loiseleuria procumbens, Ranunculus glacialis, Rhododendron ferrugineum, Saxifraga bryoides, S. moschata, S. caesia), differing in growth form, altitudinal distribution and phenology, frost resistance of reproductive and vegetative shoots was assessed in different reproductive stages. Intact plants were exposed to simulated night frosts between ?2 and ?14 °C in temperature-controlled freezers. Nucleation temperatures, freezing damage and subsequent reproductive success (fruit and seed set, seed germination) were determined. During all reproductive stages, reproductive shoots were significantly less frost resistant than vegetative shoots (mean difference for LT₅₀ ?4.2 ± 2.7 K). In most species, reproductive shoots were ice tolerant before bolting and during fruiting (mean LT₅₀ ?7 and ?5.7 °C), but were ice sensitive during bolting and anthesis (mean LT₅₀ around ?4 °C). Only R. glacialis remained ice tolerant during all reproductive stages. Frost injury in reproductive shoots usually led to full fruit loss. Reproductive success of frost-treated but undamaged shoots did not differ significantly from control values. Assessing the frost damage risk on the basis of summer frost frequency and frost resistance shows that, in the alpine zone, low-statured species are rarely endangered as long as they are protected by snow. The situation is different in the subnival and nival zone, where frost-sensitive reproductive shoots may become frost damaged even when covered by snow. Unprotected individuals are at high risk of suffering from frost damage, particularly at higher elevations. It appears that ice tolerance in reproductive structures is an advantage but not an absolute precondition for colonizing high altitudes with frequent frost events.     

Cell preservation in a programmed cooling machine: the effect of variations in supercooling

When cells are cryopreserved in programmed cooling machines, they supercool to a variable and uncontrolled extent. Experiments were carried out with three cell-types (human peripheral lymphocytes, Chinese hamster lung fibroblasts, and mouse lymphoma cells) to determine whether there was any effect of supercooling on cell survival. Samples were cooled at 1 °C min^?1 in the presence of 12% v/v dimethyl sulphoxide (Me₂SO) to ?100 °C, and then thawed rapidly in a 37 °C water bath. There was no correlation between the extent of supercooling or the maximum cooling rate after freezing and cell survival, but the time taken for the sample temperature to return to the temperature at which freezing occurred did influence the survival of the two tissue culture cell lines. These results are interpreted on the basis of current theories according to which cells require sufficient time to lose water as they cool in order to avoid subsquent intracellular freezing, but must be cooled sufficiently rapidly to minimise solution effects. It is concluded that the variations in supercooling that occur in programmed cooling machines present no particular difficulties, providing appropriate cooling rates are chosen.