Plant Cold Hardiness Research Articles

Foliar applications improves grapevine plant cold hardiness

Understanding and enhancing the cold hardiness of vines is crucial for viticulture, as it directly impacts the sustainability and productivity of vineyards in diverse climatic regions. This study, therefore, meticulously examined grapevine responses to various treatments and sampling times, revealing nuanced dynamics in cold tolerance, physiological parameters, and growth outcomes. Our study found that treatments like K2SO4 significantly enhanced grapevine cold hardiness. Specifically, K2SO4 showed high R² values, indicating a substantial improvement in low-temperature hardiness for primary buds and in processes like bud burst and sprouting. Our results also revealed significant differences in cold hardiness across various treatments and sampling times, with K2SO4 leading to the highest LT50 value for primary buds. Additionally, in single-bud cuttings, K2SO4 application improved viability rates, root and shoot growth, and rooting rates, particularly observed in January. Growth parameters in single-bud cuttings further illustrated the impact of treatments, with the viability rate of primary buds being 61.5 % in the K2SO4 application and 62.0 % in the CaCl2 application. The highest root length of 2.5 cm and shoot length of 3.4 cm, along with a rooting rate of 61.0 %, root scale of 1.4, root number of 6.9, and basal callus rate of 24.6 % were obtained from the K2SO4 application. Our research found that the applications of K2SO4 and CaCl2 increased the cold hardiness of the buds and phloem tissue of the vine by approximately 1 °C compared to the control. It has been concluded that K2SO4 and CaCl2 applications during development can reduce the cold injury rate and economic losses in vines in areas with high risk of winter cold damaging events. The comprehensive findings of this study offer actionable insights that can significantly contribute to the refinement of vineyard management practices, promoting optimal grapevine performance and hardiness in the face of varying cold conditions.

Models for Estimating the Cold Hardiness of Sweet Cherry (Prunus avium cv. Sweetheart and Lapins) in Cold Climate Regions

Plant cold hardiness is a dynamic process, and seasonal changes occur through cold acclimation and deacclimation to help prevent lethal injury from the cold. Cold weather injury resulting from inadequate plant cold hardiness can result in significant economic losses to growers of perennial crops in temperate climates. The objective of the current study was to develop models that estimate the lethal temperature that causes 10%, 50%, and 90% mortality (LT10, LT50, LT90) to two cultivars of sweet cherry (Prunus avium) flower buds from the early fall through to spring. We parameterized regression models using lethal temperature data collected in the Okanagan Valley, British Columbia, Canada, over six seasons (2013–17, 2019–20, 2021–22) for ‘Sweetheart’ sweet cherry and three seasons (2013–15, 2016–17) for ‘Lapins’ sweet cherry. These models incorporate parameters that are based on equations that describe chill and heat accumulation that rely on measures of hourly air temperature. Model evaluation and validation using several seasons of lethal temperature data not included in model development were completed. Models for estimating the cold hardiness of sweet cherry showed good agreement between model lethal temperature predictions and observed values for both sweet cherry cultivars. In addition, an open-access, interactive, web-based application was developed to access the outputs of these models in real time for use by growers, researchers, and extension workers. These current models of sweet cherry cold hardiness have potential application for use as a decision support tool for cold damage management as well as crop site suitability modeling.

Cross‐disciplinary insights into the mechanisms of plant cold hardiness: From molecules to ecosystems

Cross‐disciplinary insights into the mechanisms of plant cold hardiness: From molecules to ecosystems

The effect of fertilization and irrigation treatments on cold hardiness of young peach plants

The effect of fertilization and irrigation treatments on cold hardiness of young peach plants

Identification and Characterization of Five Cold Stress-Related Rhododendron Dehydrin Genes: Spotlight on a FSK-Type Dehydrin With Multiple F-Segments.

Dehydrins are a family of plant proteins that accumulate in response to dehydration stresses, such as low temperature, drought, high salinity, or during seed maturation. We have previously constructed cDNA libraries from Rhododendron catawbiense leaves of naturally non-acclimated (NA; leaf LT50, temperature that results in 50% injury of maximum, approximately −7°C) and cold-acclimated (CA; leaf LT50 approximately −50°C) plants and analyzed expressed sequence tags (ESTs). Five ESTs were identified as dehydrin genes. Their full-length cDNA sequences were obtained and designated as RcDhn 1-5. To explore their functionality vis-à-vis winter hardiness, their seasonal expression kinetics was studied at two levels. Firstly, in leaves of R. catawbiense collected from the NA, CA, and de-acclimated (DA) plants corresponding to summer, winter and spring, respectively. Secondly, in leaves collected monthly from August through February, which progressively increased freezing tolerance from summer through mid-winter. The expression pattern data indicated that RcDhn 1-5 had 6- to 15-fold up-regulation during the cold acclimation process, followed by substantial down-regulation during deacclimation (even back to NA levels for some). Interestingly, our data shows RcDhn 5 contains a histidine-rich motif near N-terminus, a characteristic of metal-binding dehydrins. Equally important, RcDhn 2 contains a consensus 18 amino acid sequence (i.e., ETKDRGLFDFLGKKEEEE) near the N-terminus, with two additional copies upstream, and it is the most acidic (pI of 4.8) among the five RcDhns found. The core of this consensus 18 amino acid sequence is a 11-residue amino acid sequence (DRGLFDFLGKK), recently designated in the literature as the F-segment (based on the pair of hydrophobic F residues it contains). Furthermore, the 208 orthologs of F-segment-containing RcDhn 2 were identified across a broad range of species in GenBank database. This study expands our knowledge about the types of F-segment from the literature-reported single F-segment dehydrins (FSKn) to two or three F-segment dehydrins: Camelina sativa dehydrin ERD14 as F2S2Kn type; and RcDhn 2 as F3SKn type identified here. Our results also indicate some consensus amino acid sequences flanking the core F-segment in dehydrins. Implications for these cold-responsive RcDhn genes in future genetic engineering efforts to improve plant cold hardiness are discussed.

Grapevine leafroll disease alters leaf physiology but has little effect on plant cold hardiness.

Foliar sugar accumulation in grapevines with leafroll disease was correlated with lower photosynthesis, likely due to feedback inhibition. However, cold acclimation of dormant tissues remained unaffected by the virus status. Grapevine leafroll-associated viruses (GLRaV) contribute to losses in fruit yield and quality worldwide. Visually, leafroll disease symptoms appear similar to those associated with an imbalance in source/sink relations and a concomitant feedback inhibition of photosynthesis, which is often caused by an impasse in sugar translocation. In order to test this potential relationship and related physiological responses, leaf water status, gas exchange, non-structural carbohydrates, and dormant tissue cold hardiness were examined over 2years in healthy and GLRaV-3-infected, field-grown Merlot grapevines. Diurnal and seasonal changes in leaf water status and gas exchange were dominated by variations in water availability, temperature, and leaf age, while GLRaV-3 infection contributed less to the overall variation. By contrast, foliar carbohydrates increased markedly in infected plants, with starch accumulating early in the growing season, followed by soluble sugar accumulation, leaf reddening, and declining gas exchange. Photosynthesis correlated negatively with leaf sugar content. However, dormant-season cold hardiness of buds and cane vascular tissues was similar in healthy and infected vines. These findings support the idea that visible symptoms of grapevine leafroll disease are a consequence of carbohydrate accumulation which, in turn, may lead to feedback inhibition of photosynthesis. In addition, this study provided evidence that GLRaV-3 infection is unlikely to alter the susceptibility to moderate water deficit and winter damage in mature Merlot grapevines.

Thermal history parameters drive changes in physiology and cold hardiness of young grapevine plants during winter

Vitis vinifera is mainly cultivated in temperate areas, where seasons are well defined and winter conditions might be severe. To survive under these conditions during the dormant season, grapevines sense environmental parameters to trigger different protective mechanisms that lead to cold hardiness (CH). Crop yield and sustainability will be determined according to the level of CH reached in each organ. Moreover, different cultivars of V. vinifera exhibit different behavior throughout the dormant season, attaining a different status of CH. However, there is scarce information concerning how the same cultivar behaves under contrasting thermal environments. The aim of our research was to unveil how CH varies in trunks of the same cultivar under two contrasting environments and define which are the main thermal and biochemical parameters involved in this process. We submitted 2-year old plants of the same clone of cv. Malbec to two different thermal conditions: natural winter (control) and artificially warm winter (treatment). CH status, thermal and biochemical parameters in trunks were measured periodically over the dormant season, and this experiment was repeated for three years. Our results suggest that grapevine trunks subjected to a different environment reach dissimilar CH status, except at the end of winter. In addition, we determined that daily minimum temperature is the main thermal parameter that drives changes in CH. Also, we found that the total soluble sugars have the greatest relative weight in determining the CH compared with the other compounds evaluated. These results have practical implications in the establishment of vineyards for new growing regions. Moreover, with rising minimum temperature predicted by climate change scenarios, grapevines may be more vulnerable to cold events during the dormant season.

The Cold-Regulated Genes of Blueberry and Their Response to Overexpression of VcDDF1 in Several Tissues.

Expression of blueberry cold-regulated genes (VcCORs) could play a role in the variable cold hardiness of blueberry tissues. In this study, transcriptome comparisons were conducted to reveal expression of VcCORs in non-acclimated leaves, flower buds, and flowers of both non-transgenic and transgenic blueberries containing an overexpressed blueberry DWARF AND DELAYED FLOWERING gene (VcDDF1) as well as in fully chilled flower buds of non-transgenic blueberry. In non-transgenic blueberries, 57.5% of VcCOR genes showed differential expression in at least one of the three pairwise comparisons between non-acclimated leaves, flower buds, and flowers, and six out of nine dehydration-responsive element-binding factors showed differential expression. In addition, expression of VcDDF1 was not cold-inducible in non-transgenic blueberries and had higher expression in flowers than in leaves or non-acclimated flower buds. In transgenic blueberries, overexpression of VcDDF1 resulted in higher VcDDF1 expression in leaves than in flower buds and flowers. VcDDF1 overexpression enhanced expression of blueberry CBF1 and CBF3 in leaves and repressed expression of CBF3 in both flower buds and flowers. Overall, the results revealed tissue-specific expression patterns of VcCORs. The responses of VcCORs to overexpression of VcDDF1 suggest that it is possible to increase plant cold hardiness through overexpression of a non-cold-inducible gene.

Effects of drought stress on cold hardiness of non-acclimated viola ( Viola × wittrockiana ‘Iona Gold with Blotch’) in controlled conditions

Abstract Predicted increases in winter temperatures may negatively impact plant survival by preventing maximal cold acclimation prior to cold temperatures. Accordingly, research is needed to identify strategies that may help promote cold hardiness and increase freezing tolerance of cold season ornamental plants. Therefore, the objectives of this research were to study the effects of drought stress on freezing tolerance of Viola × wittrockiana Iona Gold with Blotch during cold season under non-cold acclimating (20 ± 1 °C) conditions and examine the physiological and biochemical changes in plants in response to freezing temperature. After being grown in the greenhouse, plants were first subjected to different levels of soil water availability including control (90%), 70% and 50% field capacity (FC). Then, some traits including proline, carbohydrate and chlorophyll were determined. Finally, plants were transferred to the Thermo Gradient Freezer with six freezing temperatures (0, −3, −6, −9, −12, and −15 °C), as well as 20 °C as the control treatment. Electrolyte leakage (EL) after temperature treatments, plant survival percentage (SU) and some traits related to regrowth after recovery period were determined. Result showed that drought stress increased some factors such as carbohydrate, proline and chlorophyll. Electrolyte leakage significantly increased by lowering temperature and increased by 46% at 15 °C compared to control. Plant survival was significantly affected by treatments so that lowering the temperature to −15 °C caused total mortality in all the plants of irrigation treatments. Plants under 70% FC at 0 °C had the highest increased reproductive component. Number of leaf and leaf area peaked under the moderate water deficit (70% FC) conditions at 0 °C. Plants under 70% FC at 0 °C had the highest increases (87, 134, 90 and 101%, respectively) in dry weights of vegetative, reproductive, root, and total dry weights compared to control. Drought stress can increase freezing tolerance of viola depending on temperature regime.

Extracellular ice management in the frost hardy horsetail Equisetum hyemale L.

Formation of extracellular ice at specific positions in the plant interior is a common and probably essential component of plant cold hardiness. Studies on extracellular freezing in spore-bearing plants are, however, scarce. In this study, extracellular ice formation in the cold hardy horsetail Equisetum hyemale L. is analyzed. Horsetails show an extensive system of intercellular air spaces which are probably crucial for internal ice storage during winter. Previous studies emphasized the spacious pith cavity as the main place for ice crystal growth. Shoots were studied during summer and in the frozen state in winter, after natural acclimatization, by using digital (incident light) microscopy, Scanning Electron Microscopy and Cryo Scanning Electron Microscopy. It was shown that the vallecular canals also contain a large share of ice bodies under freezing conditions. The vallecular canals, which are directly seated within the cortex and whose interior is directly connected to the cortex via gaps in the canal wall, were often and rapidly filled with ice. The pith cavity also contained ice, depending on the position along the shoot and the internode. The carinal canals contained almost no ice crystals. Furthermore, some ice crystals were detected in the intercellular spaces of the chlorenchyma and the substomatal chamber. The stomatal antechamber, however, was always ice-free, probably due to the presence of water-repellent wax crystals. The results of this study support available evidence for the crucial role of pre-existing extensive lacunae for extracellular ice formation in E. hyemale. Furthermore, the findings indicate that anatomical details of canal structure and position are important for the pattern of extracellular ice accumulation.

Overexpression of a peach CBF gene in apple: a model for understanding the integration of growth, dormancy, and cold hardiness in woody plants.

The timing of cold acclimation and deacclimation, dormancy, and budbreak play an integral role in the life cycle of woody plants. The molecular events that regulate these parameters have been the subject of much study, however, in most studies these events have been investigated independently of each other. Ectopic expression of a peach CBF (PpCBF1) in apple increases the level of both non-acclimated and acclimated freezing tolerance relative to the non-transformed control, and also inhibits growth, induces early bud set and leaf senescence, and delays bud break in the spring. The current study examined differences in the seasonal expression of genes (CBF, DAM, RGL, and EBB) that have been reported to be associated with freezing tolerance, dormancy, growth, and bud break, respectively, in the PpCBF1 T166 transgenic apple line and the non-transformed M.26 control. Results indicated that expression of several of these key genes, including MdDAM, MdRGL, and MdEBB was altered in transgenic T166 trees relative to non-transformed M.26 trees. In particular, several putative MdDAM genes, associated with the dormancy-cycle in other species of woody plants in the Rosaceae, exhibited different patterns of expression in the T166 vs. M.26 trees. Additionally, for the first time a putative APETALA2/Ethylene-responsive transcription factor, originally described in poplar and shown to regulate the timing of bud break, was shown to be associated with the timing of bud break in apple. Since the overexpression of PpCBF1 in apple results in a dramatic alteration in cold acclimation, dormancy, and growth, this transgenic line (T166) may represent a useful model for studying the integration of these seasonal life-cycle parameters.

Seasonal changes in ice nucleation activity in blueberry stems and effects of cold treatments in vitro

Ice nucleation activity (INA) of plant intrinsic origins is considered to play important roles in plant cold hardiness mechanisms. Yet, only a few studies have addressed the spatial and temporal localization of plant INA, how it is regulated and what its functional roles are. In our previous study (Kishimoto et al., 2014), we revised a test tube method and developed a highly reproducible assay for measuring INA of plant specimens and demonstrated that high INA occurred in the cell wall fraction of wintering bark tissues of blueberry stems and corresponded well to the freezing behavior (extracellular freezing) of the stem bark. Here, we followed precisely seasonal changes in the stem INA of two blueberry cultivars and alterations in the stem INA caused by artificial incubation at various low temperatures. INA of newly developed shoots was low but increased rapidly by July when the stem became seemingly matured, then gradually increased with the maximum in October or early November just before the first autumnal frost. Following the subsequent recurrent frosts, the stem INA gradually decreased. This tendency was consistent between the two cultivars differing in the level of cold hardiness. INA in the stems of September until February was increased by incubation at 0-7°C whilst decreased by freezing to lower temperatures. The in vitro results corroborate the seasonal changes in the stem INA in the field but the mechanisms remain to be investigated. The highest level of INA (expressed as the median ice nucleation temperature) observed with current year stems (7.5 mm-long) of Woodard in October of 2010-2013 was -0.9 ∼ -1.0°C when determined with 2mL assay system (-1.1 ∼ -1.3°C with 0.5mL system). This may likely be one of the highest INA of biological origins ever reported.

Cold hardiness in Arctic plants as a requirement of colonization of the post-glacial Arctic (Extended Abstract)

Cold hardiness in Arctic plants as a requirement of colonization of the post-glacial Arctic (Extended Abstract)

Understanding plant cold hardiness: an opinion

How plants adapt to freezing temperatures and acclimate to survive the formation of ice within their tissues has been a subject of study for botanists and plant scientists since the latter part of the 19th century. In recent years, there has been an explosion of information on this topic and molecular biology has provided new and exciting opportunities to better understand the genes involved in cold adaptation, freezing response and environmental stress in general. Despite an exponential increase in our understanding of freezing tolerance, understanding cold hardiness in a manner that allows one to actually improve this trait in economically important crops has proved to be an elusive goal. This is partly because of the growing recognition of the complexity of cold adaptation. The ability of plants to adapt to and survive freezing temperatures has many facets, which are often species specific, and are the result of the response to many environmental cues, rather than just low temperature. This is perhaps underappreciated in the design of many controlled environment experiments resulting in data that reflects the response to the experimental conditions but may not reflect actual mechanisms of cold hardiness in the field. The information and opinions presented in this report are an attempt to illustrate the many facets of cold hardiness, emphasize the importance of context in conducting cold hardiness research, and pose, in our view, a few of the critical questions that still need to be addressed.

提高植物抗寒性的机理研究进展

Low temperature stress is a worldwide major abiotic stress affecting plant yield and quality.Plant physiological ecology of cold hardiness research is a more active and fast growing field.In this paper,advances of study on the mechanism of improving plant cold hardiness was reviewed.Scientific research and production practice have showed that meteorological factors and/or factors of plant itself are the key factors affecting plant cold hardiness;the former is mainly temperature,photoperiod and water;while the latter is mainly plant genetic basis,growth period,development level and cell antioxidant capacity under low temperature stress.Ensuring fully expression of genes of plant cold hardiness is of important significance on improving plant cold hardiness.Genetic mechanism and regulation of plant cold hardiness are mainly through 5 paths to achieve,respectively: the rich diversity of low temperature induced plant proteins;low temperature transcription factor DREB/CBF can simultaneously control a plurality of expression of genes cold induced in plants;interactions of DREB/CBF and cofactor regulate expression of downstream genes;Ca2+,ABA and protein phosphorylation regulate gene expression induced by low temperature in the upstream;and the expression of genes of unsaturated fatty acid synthase.Genetic engineering to improve plant cold hardiness has been significant progress,but from the industrialization has still a lot of pioneering work to do.Currently,genetic engineering of improving plant cold hardiness are mainly through the introduction of regulatory genes and/or functional genes,the latter is mainly the introduction of genes related to osmotic stress resistance,genes of antifreeze proteins,genes of fatty acid desaturation metabolism key enzyme,genes of SOD and other antioxidant system component,as well as genes involved in the regulation of plant hormone.Agricultural and forestry technologies to improve plant cold hardiness have important practical value,many of them are of profound scientific mechanism,im which we focused on the mechanism of 5 main technologies improving plant cold hardiness,respectively: cold hardiness breeding,grafting,cold acclimation,the coupling of water and fertilizer and chemical induction.The prospect of future research on improving cold resistance of plant was discussed.

Physiological Research on Winter-hardiness: Deacclimation Resistance, Reacclimation Ability, Photoprotection Strategies, and a Cold Acclimation Protocol Design

Freezing is a major environmental stress during an annual cycle of overwintering, temperate-zone perennials. The timing and extent of seasonal cold acclimation (development of freezing tolerance in the fall) and deacclimation (loss of acquired freezing tolerance in response to warm temperatures) are of critical importance for winter survival, particularly in view of the climate change, i.e., unpredictable extreme weather occurrences. For example, plants may acclimate inadequately if exposed to a milder fall climate and may be damaged by sudden frosts. Alternatively, they may deacclimate prematurely as a result of unseasonable, midwinter warm spells and be injured by the cold that follows. Efficient cold acclimation ability, high deacclimation resistance, and efficient reacclimation capacity are, therefore, important components of winter survival in overwintering perennials. These components should be evaluated separately for a successful breeding program focused on improving winter-hardiness. Another layer of complexity that should be carefully considered is that endodormant status (shallow versus deep) of the reproductive/vegetative apices can significantly impact these components of winter-hardiness. Winter survival, especially by woody evergreens, requires tolerance of light stress, which can result in photo-oxidative damage at cold temperatures when biochemistry of photosynthesis is somewhat compromised but light harvesting is unaffected. Accumulation of Elips (early light-induced proteins) in overwintering evergreens during winter represents a relatively novel strategy to cope with such light stress, and investigations on the precise cellular mechanism and genetic control of this strategy deserve research in the future. Investigations into the mechanisms for cold acclimation use laboratory-based, artificial acclimation protocols that often do not closely approximate conditions that plants are typically exposed to in nature. To draw meaningful conclusions about the biology of cold acclimation and ultimately improve freeze resistance under field conditions, one should also include in cold acclimation regimens parameters such as exposure to subfreezing temperatures and realistic diurnal temperature fluctuations and light levels to simulate natural conditions. One of the main objectives of this article is to highlight two areas of research that we believe are important in the context of plant cold-hardiness but, so far, have not received much attention. These are: 1) to understand the biology of deacclimation resistance and reacclimation capacity, two important components of freeze-stress resistance (winter-hardiness) in woody perennials; and 2) to investigate the cellular basis for various strategies used by broad-leaved evergreens for photoprotection during winter. Our emphasis, in this context, is on a family of proteins, called Elips. The second objective of this article is to draw attention of the cold-hardiness research community to the importance of using realistic cold acclimation protocols in controlled environments that will approximate natural/field conditions to be better able to draw meaningful conclusions about the biology of cold acclimation and ultimately improve freeze resistance. Results from our work with Rhododendron (deciduous azaleas and broad-leaved evergreens), blueberry, and that of other researchers are discussed to support these objectives.

Deacclimation kinetics and carbohydrate changes in stem tissues of Hydrangea in response to an experimental warm spell

Temperate winters are becoming progressively milder due to climate warming, and temperature patterns are becoming increasingly irregular with risk of unseasonable warm spells. Warm spells may cause premature loss of plant cold hardiness and increase the risk of subsequent freezing injury. This study investigated the timing and rate of deacclimation and associated changes in soluble carbohydrates and water status in stems of Hydrangea macrophylla ssp. macrophylla (Thunb.) Ser. ‘Alma’ and Hydrangea paniculata Sieb. ‘Vanille Fraise’ in response to a simulated warm spell (22°C/17°C day/night). In H. macrophylla, deacclimation kinetics showed a sigmoid course with a short lag-phase followed by a fast deacclimation rate. In H. paniculata, the deacclimation pattern could not be determined precisely, but H. paniculata, the hardier genotype based on mid-winter freezing tolerance, deacclimated to a greater extent than H. macrophylla. These results imply that dehardening resistance is not related to mid-winter hardiness. In both species deacclimation was associated with rehydration and decreasing sugar levels, but species-specific quantitative and qualitative differences in the accumulation patterns of specific sugars were observed. In H. paniculata cold hardiness may be associated with 1-kestose, an oligofructan frequently associated with overwintering in herbaceous plants, but not previously related to freezing tolerance in woody perennials.

Plant Cold Hardiness: From the Laboratory to the Field

Plant Cold Hardiness: From the Laboratory to the Field

Plant cold hardiness: from the laboratory to the field

Plant cold hardiness: from the laboratory to the field

Class II chitinase accumulated in the bark tissue involves with the cold hardiness of shoot stems in highbush blueberry ( Vaccinium corymbosum L.)

To understand the development of cold hardiness in highbush blueberry plants ( Vaccinium corymbosum L.), we investigated seasonal changes in the protein compositions of current-year and overwintered shoots. Electrophoresis using SDS-polyacrylamide gels indicated that the amounts of a few proteins increased in autumn, in association with an enhancement of cold hardiness. Of these proteins, 65- and 60-kDa proteins were confirmed to be dehydrins by western blotting. While the levels of most of the accumulated proteins decreased in April, a 27-kDa protein maintained its level in the overwintered stem during spring. The amino acid sequence deduced from a cDNA for this protein showed significant similarities with known chitinases. The stem segments from overwintered shoots sampled in July showed higher cold hardiness against sub-zero temperature than those from current-year shoots, and the bark tissue of overwintered shoots had higher cold resistance than that of current-year shoots. The 27-kDa protein (BC27) was mostly found in the bark tissue of stems. Appreciable accumulation of this protein in floral buds was not detected. The purified BC27 protein exerted a cryoprotective effect on protoplasts subjected to a freeze–thaw treatment. These results suggest that the stems of highbush blueberry develop a system of cold hardiness different from that of the buds and apices, and that the accumulation of chitinase is involved in tolerance to low temperature in winter or unseasonably low temperature in spring.