Freezing of water in dormant vegetative apple buds in relation to cryopreservation

Various empirical prefreezing protocols have been used to facilitate cryopreservation of dormant buds from woody plants. The objective of this research was to determine the quantity of water remaining in liquid phase, under different prefreezing conditions using pulsed nuclear magnetic resonance spectroscopy of dormant apple (Malus domestica Mill.) buds from three cultivars. During prefreezing, the quantity of water remaining in the liquid phase was less at −40°C<−30°C<−20°C for all cultivars tested. The prefreezing temperature had a greater influence on reducing the quantity of liquid water than the duration of prefreezing. Prefreezing to −40°C for 24 hours was optimal for `Patterson' and `McIntosh,' the hardiest cultivars, compared to −30°C for 24 hours with `Red Delicious.' Cryopreservation of dormant apple buds depends upon the quantity of liquid water during prefreezing, prior to immersion in liquid nitrogen, and upon the cultivar.     

Freezing Characteristics of Cultured Catharanthus roseus (L). G. Don Cells Treated with Dimethylsulfoxide and Sorbitol in Relation to Cryopreservation

The freezing behavior of dimethylsulfoxide (DMSO) and sorbitol solutions and periwinkle (Catharanthus roseus) cells treated with DMSO and sorbitol alone and in combination was examined by nuclear magnetic resonance and differential thermal analysis. Incorporation of DMSO or sorbitol into the liquid growth medium had a significant effect in the temperature range for initiation to completion of ice crystallization. Compared to the control, less water crystallized at temperatures below −30°C in DMSO-treated cells. Similar results were obtained with sorbitol-treated cells, except sorbitol had less effect on the amount of water crystallized at temperatures below −25°C. There was a close association between the per cent unfrozen water at −40°C and per cent cell survival after freezing for 1 hour in liquid nitrogen. It appears that, in periwinkle suspension cultures, the amount of liquid water at −40°C is critical for a successful cryopreservation. The combination of DMSO and sorbitol was the most effective in preventing water from freezing. The results obtained may explain the cryoprotective properties of DMSO and sorbitol and why DMSO and sorbitol in combination are more effective as cryoprotectants than when used alone.     

Freezing damage to isolated tomato fruit mitochondria as modified by cryoprotective agents and storage temperature

Isolated tomato (Lycopersicon esculentum var. Kc 146) fruit mitochondria could be stored successfully in the frozen state without a cryoprotective agent if the mitochondria were frozen quickly by immersion in liquid nitrogen and later thawed quickly at 30 C. Criteria of freezing damage were rate of respiration, adenosine diphosphate to oxygen ratio, and respiratory control ratio. Marked reduction in respiration and loss of respiratory control occurred when mitochondria were transferred from liquid nitrogen to −5, −10, or −18 C for 15 minutes prior to thawing at 30 C. Dimethylsulfoxide (5%) prevented freezing damage when mitochondria were incubated at −5 C but did not prevent freezing damage at −10 or −18 C. Isolated tomato mitochondria show promise as a model system for studying the nature of freezing damage and the mode of action of cryo-protective agents.     

Why is intracellular ice lethal? A microscopical study showing evidence of programmed cell death in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum

Background and Aims Conservation of the genetic diversity afforded by recalcitrant seeds is achieved by cryopreservation, in which excised embryonic axes (or, where possible, embryos) are treated and stored at temperatures lower than −180 °C using liquid nitrogen. It has previously been shown that intracellular ice forms in rapidly cooled embryonic axes of Acer saccharinum (silver maple) but this is not necessarily lethal when ice crystals are small. This study seeks to understand the nature and extent of damage from intracellular ice, and the course of recovery and regrowth in surviving tissues.Methods Embryonic axes of A. saccharinum, not subjected to dehydration or cryoprotection treatments (water content was 1·9 g H₂O g⁻¹ dry mass), were cooled to liquid nitrogen temperatures using two methods: plunging into nitrogen slush to achieve a cooling rate of 97 °C s⁻¹ or programmed cooling at 3·3 °C s⁻¹. Samples were thawed rapidly (177 °C s⁻¹) and cell structure was examined microscopically immediately, and at intervals up to 72 h in vitro. Survival was assessed after 4 weeks in vitro. Axes were processed conventionally for optical microscopy and ultrastructural examination.Key Results Immediately following thaw after cryogenic exposure, cells from axes did not show signs of damage at an ultrastructural level. Signs that cells had been damaged were apparent after several hours of in vitro culture and appeared as autophagic decomposition. In surviving tissues, dead cells were sloughed off and pockets of living cells were the origin of regrowth. In roots, regrowth occurred from the ground meristem and procambium, not the distal meristem, which became lethally damaged. Regrowth of shoots occurred from isolated pockets of surviving cells of peripheral and pith meristems. The size of these pockets may determine the possibility for, the extent of and the vigour of regrowth.Conclusions Autophagic degradation and ultimately autolysis of cells following cryo-exposure and formation of small (0·2–0·4 µm) intracellular ice crystals challenges current ideas that ice causes immediate physical damage to cells. Instead, freezing stress may induce a signal for programmed cell death (PCD). Cells that form more ice crystals during cooling have faster PCD responses.     

Stabilization of Frozen Lactobacillus delbrueckii subsp. bulgaricus in Glycerol Suspensions: Freezing Kinetics and Storage Temperature Effects

The interactions between freezing kinetics and subsequent storage temperatures and their effects on the biological activity of lactic acid bacteria have not been examined in studies to date. This paper investigates the effects of three freezing protocols and two storage temperatures on the viability and acidification activity of Lactobacillus delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol. Samples were examined at −196°C and −20°C by freeze fracture and freeze substitution electron microscopy. Differential scanning calorimetry was used to measure proportions of ice and glass transition temperatures for each freezing condition tested. Following storage at low temperatures (−196°C and −80°C), the viability and acidification activity of L. delbrueckii subsp. bulgaricus decreased after freezing and were strongly dependent on freezing kinetics. High cooling rates obtained by direct immersion in liquid nitrogen resulted in the minimum loss of acidification activity and viability. The amount of ice formed in the freeze-concentrated matrix was determined by the freezing protocol, but no intracellular ice was observed in cells suspended in glycerol at any cooling rate. For samples stored at −20°C, the maximum loss of viability and acidification activity was observed with rapidly cooled cells. By scanning electron microscopy, these cells were not observed to contain intracellular ice, and they were observed to be plasmolyzed. It is suggested that the cell damage which occurs in rapidly cooled cells during storage at high subzero temperatures is caused by an osmotic imbalance during warming, not the formation of intracellular ice.     

Factors Influencing the Induction of Freezing Tolerance by Abscisic Acid in Cell Suspension Cultures of Bromus inermis Leyss and Medicago sativa L

A 2-gram fresh weight inoculum of bromegrass (Bromus inermis Leyss. culture BG970) cell suspension culture treated with 7.5 × 10⁻⁵ molar abscisic acid (ABA) for 7 days at 25°C survived slow cooling to −60°C. Over 80% of the cells in ABA treated cultures survived immersion in liquid N₂ after slow cooling to −40 or −60°C. In contrast, a 6-gram fresh weight inoculum only attained a hardiness level of −28°C after 5 days of ABA treatment. Ethanol (2 × 10⁻² molar) added to the culture medium at the time of ABA addition, inhibited the freezing tolerance of bromegrass cells by 25°C. A 6-gram inoculum of both control and ABA treated bromegrass cells altered the pH of the medium more than a 2-gram inoculum. ABA inhibited the increase in fresh weight of bromegrass by 20% after 4 days. Both control and ABA (10⁻⁴ molar) treated alfalfa cells (Medicago sativa L.) grown at 25°C hardened from an initial LT₅₀ of −5°C to an LT₅₀ of −23°C by the third to fifth day after subculture. Thereafter, the cells dehardened but the ABA treated cells did not deharden to the same level as the control cells. ABA inhibited the increase in fresh weight of alfalfa by 50% after 5 days.     

Intracellular Freezing in the Infective Juveniles of Steinernema feltiae: An Entomopathogenic Nematode

Taking advantage of their optical transparency, we clearly observed the third stage infective juveniles (IJs) of Steinernema feltiae freezing under a cryo-stage microscope. The IJs froze when the water surrounding them froze at −2°C and below. However, they avoid inoculative freezing at −1°C, suggesting cryoprotective dehydration. Freezing was evident as a sudden darkening and cessation of IJs'' movement. Freeze substitution and transmission electron microscopy confirmed that the IJs of S. feltiae freeze intracellularly. Ice crystals were found in every compartment of the body. IJs frozen at high sub-zero temperatures (−1 and −3°C) survived and had small ice crystals. Those frozen at −10°C had large ice crystals and did not survive. However, the pattern of ice formation was not well-controlled and individual nematodes frozen at −3°C had both small and large ice crystals. IJs frozen by plunging directly into liquid nitrogen had small ice crystals, but did not survive. This study thus presents the evidence that S. feltiae is only the second freeze tolerant animal, after the Antarctic nematode Panagrolaimus davidi, shown to withstand extensive intracellular freezing.     

Survival of plant tissue at super-low temperatures v. An electron microscope study of ice in cortical cells cooled rapidly

Experiments were carried out with cortical cells in twig bark of mulberry trees in winter in order to clarify the mechanism of survival at super-low temperatures with rapid cooling and rewarming. Attention was given to the relation between the existence of intracellular ice crystals and survival.

Cortical cells were cooled rapidly by direct immersion into liquid nitrogen or isopentane cooled at various temperatures. After immersion, they were freeze-substituted with absolute ethanol at −78°. They were then embedded, sectioned and examined under the electron microscope for the presence and distribution of cavities left after ice removal.

Cells were found to remain alive and contain no ice cavities when immersed rapidly into isopentane baths kept below −60°. Those cells at intermediate temperatures from −20° to −45°, were almost all destroyed. It was also observed that many ice cavities were contained in the cells immersed rapidly into isopentane baths at −30°. The data seem to indicate that no ice crystals were formed when cooled rapidly by direct immersion into isopentane baths below −60° or into liquid nitrogen.

The tissue sections immersed in liquid nitrogen were rapidly transferred to isopentane baths at temperatures ranging from −70° to −10° before rapid rewarming. There was little damage when samples were held at temperatures below −50° for 10 minutes or below −60° for 16 hours. No cavities were found in these cells. Above −45°, and especially at −30°, however, all cells were completely destroyed even when exposed only for 1 minute. Many ice cavities were observed throughout these cells. The results obtained may be explained in terms of the growth rate of intracellular ice crystals.

     

Solubilization and Separation of Uridine Diphospho-d-glucose: beta-(1 --> 4) Glucan and Uridine Diphospho-d-glucose:beta-(1 --> 3) Glucan Glucosyltransferases from Coleoptiles of Avena sativa

The particulate glucan synthetase preparation isolated from a homogenate of oat coleoptiles at 4 C lost 65% of its original activity after 1 day when the UDP-d-glucose substrate concentration was 5 × 10⁻⁷m to 1.0 × 10⁻⁶m. Storage of the particulate enzyme at −20 C or in liquid nitrogen did not prevent the enzyme from losing its activity. Incorporation of 0.5% hovine serum albumin into the medium stabilized the particulate enzyme at 0 C for 6 days and for at least 2 weeks in liquid nitrogen.     

Two-Step Freezing Procedure for Cryopreservation of Rumen Ciliates, an Effective Tool for Creation of a Frozen Rumen Protozoa Bank

The present study aimed at the long-term storage of rumen protozoa as living cells in liquid nitrogen. The two-step or interrupted slow freezing procedure was used to cryopreserve six of the dominant species of rumen ciliates isolated from monofaunated animals, Dasytricha ruminantium, Entodinium caudatum, Epidinium ecaudatum caudatum, Eudiplodinium maggii, Isotricha prostoma, and Polyplastron multivesiculatum. We optimized the first step in the interrupted slow freezing procedure, from the extracellular ice nucleation temperature to the holding temperature, and studied the effects of the cooling rates on survival. In addition to the nature of the cryoprotectant (dimethyl sulfoxide), the equilibration temperature and equilibration time (25°C and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S. Kišidayová, J. Microbiol. Methods 22:185-192, 1995), we found that a holding temperature of −30°C, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2°C/min and 2.5°C/min, depending on the ciliate, and rumen juice as the freezing and thawing medium markedly improved the survival rate. Survival rates determined after 2 weeks in liquid nitrogen were 100% for Isotricha, 98% for Dasytricha, 85% for Epidinium, 79% for Polyplastron, 63% for Eudiplodinium, and 60% for Entodinium. They were not significantly modified after a period of 1 year in liquid nitrogen. Four of the five ciliate species cryopreserved for 8 months in liquid nitrogen successfully colonized the rumen when inoculated into defaunated animals. These results have made it possible to set up a bank of cryopreserved rumen protozoa.     

Influence of Storage at Freezing and Subsequent Refrigeration Temperatures on β-Galactosidase Activity of Lactobacillus acidophilus

The ability of three strains of Lactobacillus acidophilus to survive and retain β-galactosidase activity during storage in liquid nitrogen at −196°C and during subsequent storage in milk at 5°C was tested. The level of β-galactosidase activity varied among the three strains (0.048 to 0.177 U/10⁷ organisms). Freezing and storage at −196°C had much less adverse influence on viability and activity of the enzyme than did storage in milk at 5°C. The strains varied in the extent of the losses of viability and β-galactosidase activity during both types of storage. There was not a significant interaction between storage at −196°C and subsequent storage at 5°C. The strains that exhibited the greatest losses of β-galactosidase activity during storage in milk at 5°C also exhibited the greatest losses in viability at 5°C. However, the losses in viability were of much greater magnitude than were the losses of enzymatic activity. This indicates that some cells of L. acidophilus which failed to form colonies on the enumeration medium still possessed β-galactosidase activity. Cultures of L. acidophilus to be used as dietary adjuncts to improve lactose utilization in humans should be carefully selected to ensure that adequate β-galactosidase activity is provided.     

Survival of Plant Tissue at Super-Low Temperature VI. Effects of Cooling and Rewarming Rates on Survival

The survival rates of the cortical parenchymal cells of mulberry tree were determined as a function of cooling and rewarming rates. When cooling was carried out slowly at 1° to 15° per minute, all of the cells still remained viable even when rewarmed either rapidly or slowly. Survival rates gradually decreased to zero as the cooling rate increased from about 15° to 2000° per minute. In the intermediate cooling rates, when the cells were cooled at the rates lower than 14° per minute, from −2.2° to about −10°, these cells could survive subsequent rapid cooling and rewarming.

However, at cooling rates above 1000° per minute and with rapid rewarming, the effect of cooling rate reversed and survival increased, reaching a maximum at about 200,000° per minute. As the cooling rate increased above 15° per minute, survival rates became increasingly dependent on the rewarming rate, with rapid rewarming becoming less deleterious than slow rewarming.

The temperature range at which damage occurred during rewarming following removal from liquid nitrogen and in which growth rate of ice crystallization was greatest, was −30° to −40°. The survival rates even in the prefrozen cells at −30° decreased considerably by keeping them at −30° for 10 minutes after removal from liquid nitrogen. This fact indicates that intracellular freezable water remains to some degree even in the prefrozen cells at −30°. After removal from liquid nitrogen, all cells retained their viability, when they were passed rapidly through a temperature range between −50° and −2.5° within about 2 seconds, namely at the rates greater than 1000° per minute.

These observations are explained in terms of the size of the crystals formed within the cortical cells.

     

A Leech Capable of Surviving Exposure to Extremely Low Temperatures

It is widely considered that most organisms cannot survive prolonged exposure to temperatures below 0°C, primarily because of the damage caused by the water in cells as it freezes. However, some organisms are capable of surviving extreme variations in environmental conditions. In the case of temperature, the ability to survive subzero temperatures is referred to as cryobiosis. We show that the ozobranchid leech, Ozobranchus jantseanus, a parasite of freshwater turtles, has a surprisingly high tolerance to freezing and thawing. This finding is particularly interesting because the leach can survive these temperatures without any acclimation period or pretreatment. Specifically, the leech survived exposure to super-low temperatures by storage in liquid nitrogen (−196°C) for 24 hours, as well as long-term storage at temperatures as low as −90°C for up to 32 months. The leech was also capable of enduring repeated freeze-thaw cycles in the temperature range 20°C to −100°C and then back to 20°C. The results demonstrated that the novel cryotolerance mechanisms employed by O. jantseanus enable the leech to withstand a wider range of temperatures than those reported previously for cryobiotic organisms. We anticipate that the mechanism for the observed tolerance to freezing and thawing in O. jantseanus will prove useful for future studies of cryopreservation.     

Comparison of tissue preparation methods for assay of nicotinamide coenzymes

To prepare tissues for analysis of NAD⁺, NADH, NADP⁺, and NADPH, common practice is to freeze samples in liquid nitrogen, often followed by freeze-drying, before extraction in HCl or NaOH. With cucumber (Cucumis sativus L.) cotyledons, prefreezing in liquid nitrogen or slower freezing to −20°C yielded substantially lower values for NADH and NADPH than obtained from samples homogenized immediately in acid or base. Freeze-drying after freezing in liquid nitrogen generally caused even lower values of those coenzymes. We suggest that direct extraction is more likely to yield accurate results with cotyledons and other plant parts.     

Toxicity of Smoke to Epiphytic Ice Nucleation-Active Bacteria

Wheat straw smoke aerosols and liquid smoke condensates reduced significantly both the viability and the ice-nucleating activity of Pseudomonas syringae pv. syringae and Erwinia herbicola in vitro and on leaf surfaces in vivo. Highly significant reductions in numbers of bacterial ice nuclei on the surface of both corn and almond were observed after exposure to smoke aerosols. At −5°C, frost injury to corn seedlings colonized by ice nucleation-active bacteria was reduced after exposure to smoke aerosols. Effects on −9°C ice nuclei, although significant, were less than on ice nuclei active at −5°C. These results suggest that smoke from wildfires or smudge pots may reduce plant frost susceptibility and sources of ice nuclei important in other natural processes under some conditions.     

Freezing of water in red-osier dogwood stems in relation to cold hardiness

Studies of stem water in red-osier dogwood (Cornus stolonifera Michx.) using nuclear magnetic resonance spectroscopy indicated that most freezing occurs at temperatures above −30 C in cold-hardy and tender stems. Hardy and tender stems had about the same amount of unfrozen water at −40 C (0.28 gram of water per gram dry weight). When hardy stems were slowly cooled below −20 C, the temperature below which little additional freezing occurs, they survived direct immersion in liquid N₂ (−196 C). Fully hardy samples not slowly precooled to at least −15 C did not survive direct immersion in liquid N₂. The results support the hypothesis that cooling rate is an unimportant factor in tissue survival at and below temperatures where there is little freezable water.     

High Survivability of Cheese Whey-Grown Rhizobium meliloti Cells upon Exposure to Physical Stress

Whey, a by-product of the dairy industry, has been found to protect the rhizobia cells during freezing and thawing. Cells of rhizobia grown on whey sustained freezing better at −18°C than did cells grown on mannitol or sucrose. Suspensions of cells grown on whey or mannitol that were suspended in whey performed equally well at −18 and −80°C, with 94 and 100% survival, respectively. Whey-grown rhizobia in pellets withstood desiccation better than did their mannitol-grown equivalents. Rhizobia that were grown on whey and then inoculated onto commercial peat showed a survival rate of 100% after 23 weeks at −4°C. Whey-grown cells in peat performed better at various temperatures during storage, even when they were exposed to desiccation, than did mannitol-grown cells in peat. Whey, therefore, offers interesting possibilities as a Rhizobium protectant for the inoculum industry.     

Relationship between Freezing Tolerance of Root-Tip Cells and Cold Stability of Microtubules in Rye (Secale cereale L. cv Puma)

The response of cortical microtubules to low temperature and freezing was assessed for root tips of cold-acclimated and non-acclimated winter rye (Secale cereale L. cv Puma) seedlings using indirect immunofluorescence microscopy with antitubulin antibodies. Roots cooled to 0 or −3°C were fixed for immunofluorescence microscopy at these temperatures or after an additional hour at 4°C. Typical arrays of cortical microtubules were present in root-tip cells of seedlings exposed to the cold-acclimation treatment of 4°C for 2 days. Microtubules in these cold-acclimated cells were more easily depolymerized by a 0°C treatment than microtubules in root-tip cells of nonacclimated, 22°C-grown seedlings. Microtubules were still present in some cells of both nonacclimated and cold-acclimated roots at 0 and −3°C; however, the number of microtubules in these cells was lower than in controls. Microtubules remaining during the −3°C freeze were shorter than microtubules in unfrozen control cells. Repolymerization of microtubules after both the 0 and −3°C treatments occurred within 1 h. Root tips of nonacclimated seedlings had an LT-50 of −9°C. Cold acclimation lowered this value to −14°C. Treatment of 22°C-grown seedlings for 24 h with the microtubule-stabilizing drug taxol caused a decrease in the freezing tolerance of root tips, indicated by a LT-50 of −3°C. Treatment with D-secotaxol, an analog of taxol that was less effective in stabilizing microtubules, did not alter the freezing tolerance. We interpret these data to indicate that a degree of depolymerization of microtubules is necessary for realization of maximum freezing tolerance in root-tip cells of rye.     

Genetic inactivation of Par4 results in hyperactivation of NF-kappaB and impairment of JNK and p38

The Par4 gene was first identified in prostate cells undergoing apoptosis after androgen withdrawal. PAR4 was subsequently shown to interact with, and inhibit, atypical protein kinase C isoforms, functioning as a negative regulator of the NF-κB pathway. This may explain its pro-apoptotic function in overexpression experiments. To determine the physiological role of PAR4, we have derived primary embryonic fibroblasts (EFs) from Par4^−/− mice. We show here that loss of PAR4 leads to a reduction in the ability of tumour necrosis factor-α (TNF-α) to induce apoptosis by increased activation of NF-κB. Consistent with recent reports demonstrating the antagonistic actions of NF-κB and c-Jun amino-terminal kinase (JNK) signalling, we have found that Par4^−/− cells show a reduced activation of the sustained phase of JNK and p38 stimulation by TNF-α and interleukin 1. Higher levels of an anti-apoptotic JNK-inhibitor protein, X-chromosome-linked inhibitor of apoptosis, in Par4^−/− EFs might explain the inhibition of JNK activation in these cells.