Cryopreservation of in vitro-grown shoot-tips of hop (Humulus lupulus L.) using encapsulation/dehydration

 A cryopreservation procedure using encapsulation/dehydration was established for shoot-tips obtained from in vitro-grown shoots of hop. After dissection, shoot-tips were encapsulated in medium with alginate and 0.5 M sucrose. Optimal conditions consisted of preculture for 2 days in solid medium with 0.75 M sucrose, or in increasing sucrose concentrations, desiccation for 4 h with silicagel in a flow cabinet (16% water content) followed by rapid freezing and slow thawing. Shoot recovery after freezing 60 min in liquid nitrogen was around 80%. No phenotypical changes were observed in the recovered plants from cryopreserved shoot-tips growing in the field. Received: 20 April 1997 / Revised: 20 February 1998 / Accepted: 1 Dezember 1998     

The Effects of Water Deficits on Slices of Beetroot and Potato Tissue:3

The effects of osmotically induced water deficits on the metabolismof aged beetroot and potato discs were investigated. In thispaper the tissue-water relationships in two osmotica, mannitol,and Carbowax 1540, are described. Tissues in equilibrium with Carbowax solutions had lower freshweights than those in isotonic mannitol solutions, particularlyat water potentials below –0.7 J g^–1 with potatoand –2.0J g^– with beetroot. Potato discs killedby freezing and thawing lost water to Carbowax but not to mannitolsolutions. The extra effectiveness of Carbowax solutions inlowering fresh weight was attributed to an osmotic effect acrossthe cell wall. Carbowax was found to penetrate plasmolysed potatotissue, however, at a rate of about 3.0 mg (g fresh weight)^–1h^–. The extent to which water uptake occured on retransfer to waterwas unaffected by the nature of the soulte used, but dependon the degree of dehydration. The following phases were clearlydefined: (1) recovery to the fresh weight at full turgor, whenthe water potential of potato tissue was not reduced below –0.5J g^– and of beetroot below –1.2 J g^–1; (2)a declining degree of recovery with decreasing water potentialover the ranges –0.5 to –1.0 J g^–1 and –1.2to –3.0 J g^–1 for potato and beetroot, respectively,and (3)in potato, the absence of recovery of fresh weight followingreduction of the water potential below –1.0 J g^–1.     

冻融过程对荒漠短命植物种子萌发的影响

荒漠区冻融交替显著改变土壤温度和水分条件,并进一步影响荒漠植物种子萌发.为解析荒漠土壤冻融过程对植物种子萌发的影响,本研究以古尔班通古特沙漠4种典型短命植物[东方旱麦草(Eremopyrum orientale)、卵果鹤虱(Lappula patula)、尖喙拢牛儿苗(Erodiumoxyrrhychum)和条叶庭荠(...     

Root Growth and Water Uptake by Maize Plants in Drying Soil

Sharp, R. E and Da vies, W. J. 1985. Root growth and water uptakeby maize plants in drying soil.— J. exp. Bot. 36: 1441–1456. The influence of soil drying on maize (Zea mays L.) root distributionand use of soil water was examined using plants growing in thegreenhouse in soil columns. The roots of plants which were wateredwell throughout the 18 d experimental period penetrated thesoil profile to a depth of 60 cm while the greatest percentageof total root length was between 20–40 cm. High soil waterdepletion rates corresponded with these high root densities.Withholding water greatly restricted root proliferation in theupper part of the profile, but resulted in deeper penetrationand higher soil water depletion rates at depth, compared withthe well watered columns. The deep roots of the unwatered plantsexhibited very high soil water depletion rates per unit rootlength. Key words: Maize, roots, water deficit, soil water depletion     

The effects of freezing, storage, and thawing on cell compartment integrity and ultrastructure

 The effects of slow freezing and thawing on enzyme compartmentalization and ultrastructure were studied in rat liver slices frozen in dry ice, isopentane/ethanol-dry ice, or liquid nitrogen, and stored at –80°C for 1–14 days. Non-frozen slices served as controls. Frozen liver slices were thawed in a Karnovsky fixative and processed for transmission electron microscopy (TEM). After all freezing protocols, the outer zone of frozen-thawed tissue was ultrastructurally very similar to that of non-frozen liver. Towards the center of the tissue, the ultrastructure progressively deteriorated. Comparison with 50-μm cryostat sections prepared for TEM showed that thawing and not freezing is the detrimental step for fair preservation of ultrastructure. After thawing, homogenization, and differential centrifugation, distribution patterns of soluble marker enzymes were analyzed (cytosol, lactate dehydrogenase; mitochondrial matrix, glutamate dehydrogenase; lysosomes, acid phosphatase). The enzyme activities were not affected by storage for 2 weeks and the activity distributions showed that protein leakage from compartments was only minimally increased in frozen-thawed tissue compared with that from non-frozen tissue, irrespective of the method of freezing. In conclusion, fairly large tissue slices (20×5×3 mm) may be frozen and stored at –80°C for biochemical, ultrahistochemical or ultrastructural studies. For ultrastructural analysis, only the periphery of the tissue slice should be used. Accepted: 12 May 1997     

Quality improvements to mackerel (Scomber japonicus) muscle tissue frozen using a rapid freezer with the weak oscillating magnetic fields

Although freezing is the most popular long-term food preservation method, the formation of ice crystals during the freezing process often degrades the quality of the product. Recently, several reports have argued that oscillating magnetic fields (OMFs) may affect ice crystallization. In this paper, we investigated the effects of OMFs on fresh mackerel using the Cell Alive System® (CAS®) developed as an additional OMF generator for a rapid freezer. Mackerel fillets were frozen with home freezing (HF), air blast freezing without (ABF) or with CAS (ABF-CAS) (ABI Co. Ltd., Chiba, Japan), and stored them for 2 weeks in the frozen storage between −30 °C and −35 °C. We analyzed the tissue damages of thawed samples histologically. The OMFs has been shown to significantly inhibit tissue damage in mackerel tissue after freezing and thawing (especially, thawing in ice water). And it seems that OMFs suppressed the ice hole counts (p < 0.05), the mean size (p = 0.061), and the increase of interstitial area% (p < 0.05) after freezing/thawing. We also found that it is necessary to avoid re-crystallization during thawing to maintain the quality of the frozen product. The use of OMFs with rapid thawing has the potential to improve cryopreservation in the food industry as well as in the bioscience industry.     

Magnetic resonance imaging (MRI) of water during cold acclimation and freezing in winter wheat

Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) were used to analyse changes in the physical state of water in wheat crowns during cold acclimation and during the freezing/thawing cycle. Spectroscopically measured average spin-spin relaxation times (T₂) decreased during cold acclimation and increased when plants were grown at normal temperature. Spin-spin relaxation images whose contrast is proportional to T₂, times were calculated allowing association of water relaxation with regions of tissue in spin-echo images during acclimation and freezing. Images taken during freezing revealed nonuniform freezing of tissue in crowns and roots. Acclimated and non-acclimated wheat crowns were imaged during freezing and after thawing. Spin-echo image signal intensity and T₂ times decreased dramatically between -4°C and -8°C as a result of a decrease in water mobility during freezing. Images collected during thawing were diffuse with less structure and relaxation times were longer, consistent with water redistribution in tissue after membrane damage.     

Satiation-dependent, intra-cohort variations in prey size selection of young roach (Rutilus rutilus)

The tolerance to freezing and thawing of Leucodon sciuroides, a moss growing in mountainous areas of the Mediterranean (south-east Spain), was investigated by means of CO₂ gas exchange, modulated chlorophyll (Chl) a fluorescence and pigment analysis by high-performance liquid chromatography. Evidence is presented for freezing-induced decreases in CO₂ fixation that enhance non-radiative dissipation of absorbed light energy, a process which protects the photosynthetic apparatus. The photosynthetic apparatus of L. sciuroides remained fully recuperable after freezing, as indicated by the recovery of photosynthetic CO₂ fixation and Chl fluorescence parameters to pre-freezing values during thawing. The rapid recovery of photosynthesis activity during thawing in L. sciuroides suggests that this moss is capable of tolerating freeze-thaw cycles in a manner similar to that found at higher latitudes or in the Antarctic. The resistance of the photosynthetic apparatus of this moss to freezing might be achieved, at least partially, through the employment of dissipative pathways, such as non-radiative dissipation of absorbed light energy. Received: 4 June 1998 / Accepted: 15 February 1999     

Biochemical functionality and recovery of hepatocytes after deep freezing storage

Summary The present study was undertaken to define the conditions for optimal cryopreservation of hepatocytes. Two different freezing procedures were analyzed: a slow freezing rate (SFR) (−2° C/min down to −30°C and then quick freezing to −196° C) and a fast freezing rate (FFR) (direct freezing of tubes to −196° C: −39° C/min). Cells were frozen in fetal bovine serum containing 10% Dimethyl sulfoxide (DMSO). After rapid thawing at 37° C, followed by dilution and removal of the cryoprotectant, cells were plated and several parameters were followed as criteria for optimal cryopreservation of cells. The FFR cells showed no apparent ultrastructural damage after 24 h of culture. Plating efficiency and spreading were similar as controls. Gluconeogenesis from pyruvate and fructose, tyrosine amino transferase induction by glucagon and dexamethasone, urea production, and plasma protein synthesis of FFR cells were similar to those found in control cultures. The FFR procedure, in comparison to the SFR method, seemed to render the best preserved hepatocytes. The financial support for this work was from Fondo de Investigaciones Sanitarias de la Seguridad Social, Grants 41/82 and 48/82.     

Effect of cold acclimation on bulk tissue electrical impedance: I. Measurements with birdsfoot trefoil at subfreezing temperatures

The resistive and reactive components of electrical impedance were measured for birdsfoot trefoil (Lotus corniculatus L.) stems at freezing temperatures to −8°C. As temperature decreased the specific resistance at frequencies between 49 hertz and 1.11 megahertz of stems from cold acclimated plants increased more rapidly than from nonacclimated plants. This temperature dependence of specific resistance could be characterized by an Arrhenius activation energy; cold acclimated stems had a larger Arrhenius activation energy than nonacclimated stems. The low frequency resistance is believed to characterize the extracellular region of the stems and the high frequency resistance is believed to characterize the intracellular region of the stems. Cold acclimation increased the intracellular but not the extracellular resistance at nonfreezing temperatures. Cold acclimated stems were not injured by freezing to −8°C and thawing, but nonacclimated stems were injured by freezing to temperatures between −2.2 and −5.6°C and thawing. Injury to nonacclimated stems at freezing temperatures below −2.2°C was indicated by a decrease in the ratio of resistance at 49 Hz to that at 1.11 megahertz.     

Thermodynamic modeling and cryomicroscopy of cell-size, unilamellar, and paucilamellar liposomes

Cryomicroscope studies of large unilamellar liposomes indicate that liposomes are an excellent model for studying membrane response to freezing and thawing. Liposomes are attractive for such use because they can be custom-manufactured for a particular investigation. In addition, liposome responses to freezing and thawing mimic real cell behavior in a number of significant ways. Analogous behavior includes osmotic shrinkage at slow cooling rates, internal ice formation at fast cooling rates, comparable nucleation temperatures, and a variety of comparable thawing responses. Experimental determination has been made of the equilibrium osmotic properties and the nonequilibrium water transport properties of the egg lecithin liposomes used in the freezing studies. These properties have been used in a computer model to simulate volume changes resulting from water transport during freezing and thawing. Comparison between computer model predictions and experimental data for the liposome volume response during freezing indicates reasonable agreement whereas computer simulations of volume response during thawing do not match experimental data well.     

Brown bear sperm double freezing: Effect of elapsed time and use of PureSperm® gradient between freeze–thaw cycles

The use of sexed spermatozoa has great potential to captive population management in endangered wildlife. The problem is that the sex-sorting facility is a long distance from the semen collection place and to overcome this difficulty two freeze–thaw cycles may be necessary. In this study, effects of refreezing on brown bear electroejaculated spermatozoa were analyzed. We carried out two experiments: (1) to assess the effects of the two freezing–thawing cycles on sperm quality and to analyze three different elapsed times between freezing–thawing cycles (30, 90 and 180 min), and (2) to analyze the use of PureSperm between freezing–thawing cycles to select a more motile and viable sperm subpopulation which better survived first freezing. The motility, viability and undamaged acrosomes were significantly reduced after the second thawing respect to first thawing into each elapsed time group, but the elapsed times did not significantly affect the viability and acrosome status although motility was damaged. Our results with the PureSperm gradient showed higher values of viability in freezability of select sample (pellet) respect to the rest of the groups and it also showed a significant decrease in the number of acrosome damaged. In summary, the double freezing of bear semen selected by gradient centrifugation is qualitatively efficient, and thus could be useful to carry out a sex-sorting of frozen–thawed bear spermatozoa before to send the cryopreserved sample to a biobank. Given the low recovery of spermatozoa after applying a selection gradient, further studies will be needed to increase the recovery rate without damaging of the cell quality.     

Supercooling along an Altitudinal Gradient in Espeletia schultzii, a Caulescent Giant Rosette Species

Rada, F., Goldstein, G., Azocar, A. and Torres, F. 1987. Supercoolingalong an altitudinal gradient in Espeletia schultzii, a caulescentgiant rosette species.—J. exp. Bot. 38: 491–497. Tropical high Andes plants may be exposed to sub-zero temperaturesany night of the year. These plants have to rely on mechanismswhich protect them from these environmental conditions but atthe same time allow their growth and development. Supercoolinghas been found to be the principal avoidance mechanism in leavesof the caulescent giant rosette genus Espeletia in the Andes.We report here the differences in supercooling capacity andcold injury in several Espeletia schultzii populations growingalong an altitudinal gradient. The relationships between supercooling,water potential and leaf anatomy were also investigated. Thesupercooling capacity increased and injury temperature decreasedfrom lower to higher elevation populations. These changes maybe explained in terms of physiological, morphological and anatomicalcharacteristics of the leaves. Key words: Espeletia schultzii, supercooling, freezing avoidance mechanisms     

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     

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     

The concentration of cryoprotective lectins in mistletoe (Viscum album L.) leaves is correlated with leaf frost hardiness

The leaves of mistletoe (Viscum album L.) contain three galactose- and N-acetylgalactosamine-specific isolectin groups (ML I, II, III). The groups ML I and ML III showed strong cryoprotective activity during freezing and thawing of isolated spinach (Spinacia oleracea L.) thylakoid membranes, while ML II showed no such activity. The cryoprotective efficiency of the proteins was correlated with their relative hydrophobicity, as determined by a fluorescence titration assay. We found that the frost hardiness of mistletoe leaves was seasonally regulated under natural conditions. While leaves harvested in winter were not damaged by freezing to −20 °C, leaves harvested in July had already suffered 70% electrolyte leakage after freezing to −5 °C. Likewise, the amount of ML I and ML III varied during the year, with the highest amounts of these cryoprotective lectins in winter and early spring and the lowest amounts during the summer months. There was no comparable change in the amount of ML II. These data suggest that some lectins may play a role in the stabilization of cellular membranes under environmental stress conditions. Received: 18 December 1996 / Accepted: 29 March 1997     

A requirement for sucrose in xylem sap flow from dormant maple trees

The response of excised stem segments of several tree species to freezing and thawing cycles was studied. All species studied (Thuja occidentalis, Fagus grandifolia, and Betula papyrifera) except maple (Acer spp.) exuded sap while freezing and absorbed on thawing. Maple stems absorbed sap while freezing and exuded sap during the thaw only when sucrose was present in the vessel solution. Increased concentration of sucrose in the vessel sap led to increased exudation. In the absence of sucrose, maple stems absorbed sap on thawing. The presence of sucrose enhanced sap absorption during freezing cycles in maples. In general, large sugars, disaccharides and larger, could substitute for sucrose in the maple exudation response while sugar hexoses could not. The results are discussed in relation to the O'Malley-Milburn model (1983 Can J Bot 61: 3100-3106) of sap flow in maples.     

Cryosurgical technique: assessment of the fundamental variables using human prostate cancer model systems

Cryosurgery offers a promising therapeutic alternative for the treatment of prostate cancer. While often successful, complete cryoablation of cancerous tissues sometimes fails due to technical challenges. Factors such as the end temperature, cooling rate, duration of the freezing episode, and repetition of the freezing cycle have been reported to influence cryosurgical outcome. Accordingly, we investigated the effects of these variables in an in vitro prostate cancer model. Human prostate cancer PC-3 and LNCaP cultures were exposed to a range of sub-zero temperatures (−5 to −40 °C), and cells were thawed followed by return to 37 °C. Post-thaw viability was assessed using a variety of fluorescent probes including alamarBlue™ (metabolic activity), calceinAM (membrane integrity), and propidium iodide (necrosis). Freeze duration following ice nucleation was investigated using single and double freezing cycles (5, 10, and 20 min). The results demonstrated that lower freezing temperatures yielded greater cell death, and that LNCaP cells were more susceptible to freezing than PC-3 cells. At −15 °C, PC-3 yielded 55% viability versus 20% viability for LNCaP. Double freezing cycles were found to be more than twice as destructive versus a single freeze–thaw cycle. Both cell types experienced increased cell death when exposed to freezing temperatures for longer durations. When thawing rates were considered, passive (slower) thawing following freezing yielded greater cell death than active (faster) thawing. A 20% difference in viability between passive and active thawing was observed for PC-3 for a 10 min freeze. Finally, the results demonstrate that just reaching −40 °C in vitro may not be sufficient to obtain complete cell death. The data support the use of extended freeze times, multiple freeze–thaw cycles, and passive thawing to provide maximum cell destruction.     

A new technique for long preservation of 14C-labelled Cladocerans

Three ways of preserving labelled Cladocerans fed with ¹⁴C-Chlorella for 7.5–10 min were tested. Tracer leakage in 4% formalin at room temperature is rapid and extensive (half of the label was found in the animals after 1 hour of preservation). Even when individuals are frozen and sorting is made quickly in a liquid, losses nevertheless occur (substantial decrease of animal activity after only 4–5 min in the water in one of the two experiments performed). Results obtained after freezing in 4% formalin and sorting exactly 2 hours after thawing gave consistent losses: 16 separate experiments with Daphnia pulex, Ceriodaphnia spp. and Diaphanosoma brachyurum gave apparent filtering rates underestimated from 35% to 63% for freezing periods of up to 45 days. The good agreement in in situ community filtering rates between measured values and estimated ones from individual data confirmed the validity of a correction factor of x 2 applied to animals frozen in formalin.