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Seasonal changes in the freezing tolerance in situ were determined for Opuntia fragilis and Opuntia polyacantha native to southern Saskatchewan, Canada. Wintering terminal cladodes of O. fragilis and O. polyacantha survived −50 and −40 °C, respectively, without any apparent tissue injury. Approximately 50% of the cladodes of O. fragilis collected on either 10 October or 10 December 1985 tolerated −70 °C and immersion in liquid nitrogen (−196 °C) following slow prefreezing to −40 °C. These are the lowest values reported for the freezing tolerance of cacti as determined by regrowth and tissue necrosis. Newly formed cladodes of the same species did not tolerate −3.5 °C in June, but freezing tolerance increased from −7 to −50 °C from 6 September to 10 October. This increase in freezing tolerance was accompanied by a decrease in water content from 5.4 to 2.6 g H2O/g dry wt. and the cladodes were shrunken and reduced by approximately 30% in diameter. Although Opuntia is a succulent plant that avoids drought by storing water in the cladodes, during cold acclimation O. fragilis appears to shift to a more drought-tolerating strategy. In midsummer, the current year cladodes of O. fragilis tolerated a 60-min exposure to 50 °C, while the previous year cladodes tolerated 55 °C without any visible symptoms of injury. Temperature extremes of −30 and 51 °C were recorded at the microhabitats of O. fragilis. This study demonstrates that this species can acclimate to tolerate both the high and low temperature extremes experienced in its native habitat. Keywords: cacti, cold acclimation, freezing tolerance, heat tolerance, Opuntia, water relations.

Cryopreservation of a non-embryogenic bromegrass suspension culture was attempted using a slow prefreezing method (two-step method) and a vitrification method. We examined 17 solutions as cryoprotectants for the slow prefreezing method. The cryoprotectant (CSP1) comprising sucrose, dimethylsulfoxide (DMSO) and glycerol (10, 10, 5%, w/v) gave the best survival (> 80%) of bromegrass cells exposed to liquid nitrogen (LN) as determined by regrowth and fluorescent diacetate (FDA) staining. The optimum conditions were: exposure time to CSP1 (30 min), cooling rates (0.3–0.5°C/min) following ice-inoculation at −8°C, prefreezing temperature (−30 to −40°C). This protocol worked well when glass vials were used as containers and not plastic cryotubes. With CSP1, the slow dropwise addition of the protectant to the cells on ice that has frequently been used could be circumvented; direct addition of CSP1 to the cells at 25°C did not affect the survival. However, dilution of CSP1 by 5-fold with 3% sucrose following a freeze-thaw had to be done slowly (taking 15 min). The same cryoprotectant (CSP1) was also effective as a pretreatment for cryopreservation of bromegrass cells by vitrification. Following direct treatment with CSP1 at 25°C for 30 min, cells were incubated in a vitrification solution, PVS2 (Sakai et al., 1990) for about 2–4 min at 25°C prior to submersion in LN. This procedure gave 30–40% survival of bromegrass cells based on regrowth and FDA staining. Without the CSP1 pretreatment, bromegrass cells did not survive LN exposure by vitrification with PVS2. Preculture with abscisic acid or with high osmoticum (0.3–0.7 M sucrose) could not be used as a substitute for pretreatment with CSP1.

The charge-transfer (CT) complexes of a series of 4,4,5,5-tetramethylimidazolin-1-oxyls substituted with 4-(N-alkylpyridinium) groups at the 2-position with two radical anions, tetrafluorotetracyanoquinodimethanide (TCNQF4–) and hexacyanobutadienide (HCBD–) were prepared and their magnetic properties were investigated in the temperature range of 5 to 300 K. Ferromagnetic interaction was observed for the three CT complexes. Furthermore, the magnetic measurement at lower temperatures than 5 K showed that these CT complexes exhibited a ferromagnetic phase transition between 0.4 and 0.55 K. Consequently, the present CT complexes make a group of another type of organic ferromagnets without any magnetic metal elements.

Several viability assays were compared to determine the most sensitive and appropriate method for estimating the freezing, heat and salt tolerance of Bromus inermis Leyss cells cultured with or without 75 μM abscisic acid (ABA) for 4–7 days at 25°C. The sensitivity and reliability of individual viability tests depended on the type of stress applied and degree of injury. Regrowth, amino acid (AA) leakage and fluorescein diacetate (FDA) staining all gave comparable estimates of freezing tolerance. Triphenyl-tetrazolium chloride (TTC) reduction assays slightly overestimated freezing tolerance, but was most convenient. Polypeptide leakage from freeze-thawed cells, as determined by SDS-polyacrylamide electrophoresis (SDS-PAGE), revealed that protein leakage only occurred in ABA-treated cells frozen to −21°C or colder in contrast to control cells which leaked proteins following freezing to −5°C. The most sensitive test for assessing heat tolerance was regrowth, followed by FDA staining, when TTC. TTC tests overestimated heat tolerance compared with the other tests. The degree of overestimation was greater for heat tolerance estimates than for freezing tolerance estimates. It was partially improved by washing the cells prior to TTC assays. AA leakage tests were not appropriate for assessing heat tolerance, due to the erroneously high values of A280 readings obtained. For estimating salt tolerance, TTC assays were most convenient and were in close agreement with regrowth measurements whereas FDA staining tended to overestimate it. In general, while regrowth was most sensitive and reliable, TTC was most convenient. All viability assays consistently showed that ABA induced cross-adaptation to freezing, heat and salt stresses in bromegrass cells without a prior exposure to any of these stresses.

Summary The growth, freezing tolerance and two-dimensional protein profiles of bromegrass (Bromus inermisLeyss) cell cultures were investigated following repeated exposure to 75 μM abscisic acid (ABA) for 5 weeks. Following prolonged (36 d) exposure to ABA, freezing tolerance was enhanced and fresh-mass gain was reduced compared to a 10-d ABA treatment. Also, fifteen additional ABA-responsive proteins were detected after 5 weeks of ABA treatment that were not detected in cells treated for 10 d. In addition, intensity changes occurred in several ABA-responsive proteins that were detected after 10-d incubation periods. Two polypeptides (26 and 29 kD) initially responsive to ABA decreased over 5 weeks of treatment. Transfer of ABA-treated cells to culture media lacking ABA for 14 d resulted in a 22°C decrease in freezing tolerance and increased growth, but there was no detectable change in the ABA-responsive polypeptides. Abscisic acid treatment at 3°C increased freezing tolerance at an accelerated rate and to a greater level than 3°C alone, but less so than ABA treatments at 25°C. However, the same ABA-responsive polypeptides detected at 25°C were present in cells treated with ABA at 3°C. These observations suggest that synthesis and accumulation of ABA-responsive polypeptides are only one component involved in freezing tolerance and other factors must be involved.

Laminin alpha2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin alpha2 chain G domain using 110 soluble peptides by the peptide-coated plate and the peptide-conjugated Sepharose bead assays. Fourteen peptides showed cell attachment activity in either or both assays. Cell attachment to A2G94 (YFDGTGFAKAVG) was inhibited by anti-integrin beta1 antibody, suggesting that the peptide promotes an integrin beta1-mediated cell attachment. Five peptides promoted PC12 cell neurite outgrowth. Since A2G10 (SYWYRIEASRTG) promoted strong cell attachment in the bead assay but showed slight activity in the plate assay, we conjugated A2G10 to chitosan membranes which increase cell attachment activity of the peptides via conformational stability. A2G10-chitosan membrane promoted an integrin alpha6beta1-mediated cell attachment and spreading with well-organized actin stress fibers and neurite outgrowth. These active peptides are useful for evaluating the molecular mechanisms of laminin-receptor interactions.

Summary Leaves of Trachycarpus fortunei from palms growing in warm temperate regions of Europe and Japan as outdoor ornamentals were cooled down to low subfreezing temperatures, and the process of ice formation was followed using differential thermal analysis (DTA). Inactivation of photosynthetic function was determined by in vivo chlorophyll fluorescence, and the progress of injuries during freezing was assayed by viability tests after thawing. Ultrastructure of mesophyll cells was investigated by transmission electron microscopy. Silica incrustation of cell walls was documented by means of qualitative energy dispersive X-ray analysis (EDAX) on samples of Trachycarpus fortunei, Washingtonia filifera, and of the bamboo Pseudosasa japonica. DTA revealed typical freezing patterns, with a high temperature exotherm at about -6 to -8°C and a broad low temperature exotherm rising from about -13 to -14°C. As soon as the tissues froze, characteristic changes appeared in the kinetics of in vivo fluorescence. No irreversible inactivation of photosynthesis or damage was detectable at temperatures above appearance of the low exotherm. Within a short time after attainment of the exotherm peak, the leaf segments were found to be lethally damaged. Both of in vivo fluorescence and viability tests clearly indicated that Trachycarpus leaves remain in the persistent supercooled state down to quite low temperatures. The tensile state of water at threshold supercooling temperatures of Trachycarpus leaves was calculated according to the equation of Rajashekar and Burke (1982) and found to be about -14 MPa. An explanation for the permanent maintenance of such a large displacement from thermodynamic equilibrium could be that silica incrustation, as revealed by EDXA, favours supercooling by increasing the rigidity of the cell walls and/or resistance to the growth of ice crystals.

Somatic embryos of melon (Cucumis melo L.) were cryopreserved in liquid nitrogen (LN2) after controlled desiccation. Desiccation of embryos was carried out slowly under controlled relative humidity (RH) of 50-65 % after preculture with 10mg/l abscisic acid (ABA). Desiccated embryos were immersed directly into LN2. Of the total number of embryos 65 % survived storage in LN2 when desiccated at a RH of 60 %. Viability was affected mainly by the size of the embryos. The water content of the desiccated embryos was 11.8% at RH 60%, which was considered to be reasonably low to avoid freezing injury of the cells. This study demonstrates that the desiccation method enabled the cryopreservation of melon somatic embryos without the use of expensive facilities.

A cold-responsive chitinase gene, BiCHT1, was isolated from bromegrass (Bromus inermis) 'Manchar' suspension cells. BiCHT1 messenger RNA was detected at low levels in nonstressed bromegrass cells, whereas its accumulation was induced by incubation at 10 degrees C and 4 degrees C as detected by northern- and western-blot analyses. BiCHT1 was highly homologous to rye CHT9, known to encode an antifreeze protein. BiCHT1 was overexpressed in Escherichia coli and bromegrass cells using genetic transformation procedures. BiCHT1 products expressed in both systems had chitinase activity, but the expressed proteins did not affect the growth of ice crystals in any conditions tested. Besides cold stress, the expression of the BiCHT1 gene was up-regulated by exposure to 35 degrees C, but not by salt or osmotic stress, abscisic acid, or ethephon. BiCHT1 messenger RNA did not accumulate in response to methyl jasmonate and salicylic acid, but was slightly increased by prolonged culture at 25 degrees C and only transiently by chitin. Antifreeze activity detected in the culture medium was induced at 4 degrees C but only slightly at 10 degrees C. It was also induced by ethephon treatment, but not by abscisic acid, chitin, or prolonged incubation at 25 degrees C. The results of transgenics and expression analyses suggest that the BiCHT1 product is a major protein with chitinase activity secreted in the medium of cold-treated cells and is unlikely to be responsible for the antifreeze activity detected in the culture medium.

• Background and Aims Cryopreservation is a practical method of preserving plant cell cultures and their genetic integrity. It has long been believed that cryopreservation of plant cell cultures is best performed with cells at the late lag or early exponential growth phase. At these stages the cells are small and non-vacuolated. This belief was based on studies using conventional slow prefreezing protocols and survival determined with fluorescein diacetate staining or 2,3,5-triphenyltetrazolium chloride assays. This classical issue was revisited here to determine the optimum growth phase for cryopreserving a bromegrass (Bromus inermis) suspension culture using more recently developed protocols and regrowth assays for determination of survival. • Methods Cells at different growth phases were cryopreserved using three protocols: slow prefreezing, rapid prefreezing and vitrification. Stage-dependent trends in cell osmolarity, water content and tolerance to freezing, heat and salt stresses were also determined. In all cases survival was assayed by regrowth of cells following the treatments. • Key Results Slow prefreezing and rapid prefreezing protocols resulted in higher cell survival compared with the vitrification method. For all the protocols used, the best regrowth was obtained using cells in the late exponential or early stationary phase, whereas lowest survival was obtained for cells in the late lag or early exponential phase. Cells at the late exponential phase were characterized by high water content and high osmolarity and were most tolerant to freezing, heat and salt stresses, whereas cells at the early exponential phase, characterized by low water content and low osmolarity, were least tolerant. • Conclusions The results are contrary to the classical concept which utilizes cells in the late lag or early exponential growth phase for cryopreservation. The optimal growth phase for cryopreservation may depend upon the species or cell culture being cryopreserved and requires re-investigation for each cell culture. Stage-dependent survival following cryopreservation was proportionally correlated with the levels of abiotic stress tolerance in bromegrass cells.

1H-Nuclear magnetic resonance (NMR) microscopy was used to study freezing behavior in wintering leaf buds of Momi fir (Abies firma Sieb. et Zucc.) and Japanese red pine (Pinus densiflora Sieb. et Zucc.). The images acquired predominantly reflected the density of mobile (i.e., non-ice) protons mainly from unfrozen water. By comparing images taken at various subfreezing temperatures, we determined which tissues produced the high and low temperature exotherms detected by differential thermal analyses. Typical extra-organ freezing was successfully imaged in leaf buds of A. firma. The bud scales readily froze at -7 degrees C, but shoot primordia remained supercooled to -14 degrees C in December buds and to -21 degrees C in March buds. The size of supercooled shoot primordia was reduced with decreasing temperature, indicating a gradual decrease in water content of the shoot primordia. In contrast, the signal from shoot primordia of P. densiflora disappeared between -7 and -14 degrees C, corresponding to the high temperature exotherm at -8 degrees C, indicating extracellular freezing of the shoot primordia. The xylem and bark tissues readily froze at -7 degrees C in A. firma and between -7 and -14 degrees C in P. densiflora. We conclude that NMR microscopy can noninvasively provide more spatially specific information about freezing behavior in leaf buds than traditional methods such as differential thermal analysis. In particular, it allows the organized and harmonized freezing behaviors in complex organs to be visualized directly thereby revealing the diversity of mechanisms involved in freezing behaviors.

The effects of culture conditions on abscisic acid (ABA)-induced freezing tolerance were determined in smooth bromegrass (Bromus inermis Leyss cv. Manchar) cell suspension cultures. Bromegrass cultures initiated with 2g fr wt of cells achieved maximum freezing tolerances (greater than —32°C) at 25 to 30°C in the presence of 75 to 100^M ABA. High levels of freezing tolerance induced by ABA were correlated with high growth rates at 25 and 30°C. In control cells, incubation at 10°C induced optimum levels of hardiness with minimal growth. Prolonged exposure (6 weeks) of cells to 3°C, with or without ABA, increased freezing tolerance only by several degrees. Exogenous ABA concentrations greater than IOO^M were not inhibitory to growth. Repeated exposure to ABA, however, retarded growth and made the cells tolerant to temperatures below — 40°C. Removal of ABA from the medium resulted in dehardening of the cells both at 25 and 3°C. Nitrogen had a marginal effect on ABA-induced hardening at 25°C, but inhibited age-dependent hardening of control cell cultures. Light had no effect on the freezing tolerance of cultured cells. Addition of 10% sucrose, 30 min prior to freezing, to bromegrass cells treated with ABA for 4 days increased freezing tolerance more than 15°C. These observations are discussed in relation to the contrasting behaviour of the low temperature and photoperiod dependent cold acclimation of plants.

1H nuclear magnetic resonance (NMR) micro-imaging was used to study the freezing behaviour of wintering flower buds of Rhododendron japonicum (A. Gray) Suringer. Amulti-slice multi- echo pulse sequence was used to acquire images at different subfreezing temperatures. The images obtained predominantly reflected the density of mobile (i.e. non-ice) protons mainly from unfrozen water. By comparing these images taken at various subfreezing temperatures, we could determine which tissues produced high temperature exotherms and low temperature exotherms in differential thermal analyses. In flower buds of the cold-hardy R. japonicum, typical extra-organ freezing was successfully imaged. The scales readily froze at –7°C but some florets remained supercooled even at –21°C. The size of the supercooled florets was reduced with decreasing temperature which indicated a gradual decrease in floret water content. With decreasing temperature, there was a gradual decrease in the signal intensity of the flower bud axis including the peduncle and immature pith tissues, which implies either dehydration or partial freezing of these tissues. Deep supercooling in the entire mature pith tissues was also clearly visible in these images. Due to its non-invasive nature, NMR micro-imaging is a useful tool for studying freezing behaviours in various plant tissues, especially for imaging organised or harmonised freezing in complex organs as well as for clarifying the diversity and mechanisms involved in freezing behaviours.

Bromus inermis Leyss cell cultures treated with 75 micromolar abscisic acid (ABA) at both 23 and 3 degrees C developed more freezing resistance than cells cultured at 3 degrees C. Protein synthesis in cells induced to become freezing tolerant by ABA and low temperature was monitored by [(14)C]leucine incorporation. Protein synthesis continued at 3 degrees C, but net cell growth was stopped. Most of the major proteins detected at 23 degrees C were synthesized at 3 degrees C. However, some proteins were synthesized only at low temperatures, whereas others were inhibited. ABA showed similar effects on protein synthesis at both 23 and 3 degrees C. Comparative electrophoretic analysis of [(14)C]leucine labeled protein detected the synthesis of 19, 21 and 47 kilodalton proteins in less than 8 hours after exposure to exogenous ABA. Proteins in the 20 kilodalton range were also synthesized at 3 degrees C. In addition, a 31 kilodalton protein band showed increased expression in freezing resistant ABA treated cultures after 36 hours growth at both 3 and 23 degrees C. Quantitative analysis of [(14)C]leucine labeled polypeptides in two-dimensional gels confirmed the increased expression of the 31 kilodalton protein. Two-dimensional analysis also resolved a 72 kilodalton protein enriched in ABA treated cultures and identified three proteins (24.5, 47, and 48 kilodaltons) induced by low temperature growth.

Cold hardiness of leaf blades, leaf sheaths, culms, rhizomes, and leaf buds in wintering Sasa senanensis (Fr. et Sav.) Rehder, a dwarf bamboo, was studied paying special attention to the types of resistance mechanisms which were determined with differential thermal analysis. Coincidence of LT(25) (lethal temperature at which 25% of the tissues are injured) with the initiation temperature of LTE (low temperature exotherm) suggested that all of these tissues described above owe their cold hardiness mechanism mostly to deep supercooling. Deep supercooling in leaf blades was also substantiated with microscopic observations, suggesting that the units of supercooling were minute tissues compartmentalized by longitudinal and cross veins. It was also shown that cooling rates and storage of shoots at -5 degrees C for 1 to 5 days in the ice-inoculated state did not greatly affect the supercooling ability of leaf blades. Sasa senanensis seemed to exhibit a unique strategy against prolonged subzero temperature, and its leaves would be a good system for the study on mechanisms of deep undercooling in plants.

The relationship of the β-myrcene content with the susceptibility and/or resistance of Pinus species against the pine wood nematode was investigated. For this purpose, the β-myrcene content of the wood of one-year-old shoots was determined. The resistant trees were found to contain a very small amount of β-myrcene. On the other hand, the β-myrcene content of susceptible trees was comparatively high. This relationship was observed in both inter- and intraspecies and also between young and older trees of the same species, i.e., Pinus densiflora or P. thunbergii. The β-myrcene content can feasibly be used as an indicator for screening resistant Japanese red and black pine trees.

A new convenient assay method for nematode attractants was developed. Using this method, the attracting activity of volatile components in a pine, Pinus densiflora, was examined for the pine wood nematode, Bur saphelenchus xylophilus, which causes serious damage to pine trees in Japan. Among the volatile components, β-myrcene showed the strongest attracting potency. This compound was assumed to play an important role in the transmigration of the nematode from the sawyer to the pine tree and for the movement of the nematode inside the pine wood.

Cellular and extracellular protein profiles from Bromus inermis Leyss. cv Manchar cell suspension cultures cold hardened by low temperature and abscisic acid (ABA) treatment were analyzed by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Cellular proteins (25, 165, 190, and 200 kilodaltons) increased by low temperature growth and cellular proteins (20, 25, 28, 30, 32, 37, 40, 45, 200 kilodaltons) increased by exogenous ABA treatment were identified. Low temperature treatment inhibited the synthesis of a 22 kilodalton protein and ABA treatment resulted in the synthesis of two extracellular proteins (17 and 21 kilodaltons). Low temperature and ABA-induced hardening conditions increased or induced a 25 and a 200 kilodalton protein. The 25 and a 30 kilodalton protein previously shown to be enriched by ABA-induced hardening conditions at both 3 and 23 degrees C temperatures co-fractionated with the crude membrane fraction (30,000g sediment). The 200 kilodalton protein was detected in the 30,000g supernatant. Two-dimensional analysis of the crude membrane fraction resolved the 30 kilodalton protein band into a major polypeptide with an apparent isoelectric point of 6.85.

Elevation of venous pressure produces a rapid increase of capillary pressure to a level above the pressure in the veins (Landis and Pappenheimer, 1963). When capillary pressure is raised capillaries which were previously closed may open, resulting in an increase of oxygen transport to peripheral tissue. To examine this possibility we constructed a rapidly responding PO2 electrode using gold paste.