Effects of cold acclimation on serum biochemical parameters and metabolite profiles in Schizothorax prenanti.

BACKGROUND: Environmental temperature is critical in regulating biological functions in fish. S. prenanti is a kind of cold-water fish, but of which we have little knowledge about the metabolic adaptation and physiological responses to long-term cold acclimation. RESULTS: In this study, we determined the physiological responses of S. prenanti serum after 30 days of exposure to 6℃. Compared with the control group, the levels of TC, TG, and LDL-C in the serum were significantly (P < 0.05) increased, and the level of glucose was significantly (P < 0.05) decreased under cold acclimation. Cold acclimation had no effect on the gene expression of pro-inflammatory factors and anti-inflammatory factors of S. prenanti. Metabolomics analysis by LC-MS showed that a total of 60 differential expressed metabolites were identified after cold acclimation, which involved in biosynthesis of amino acids, biosynthesis of unsaturated fatty acids, steroid degradation, purine metabolism, and citrate cycle pathways. CONCLUSION: The results indicate that cold acclimation can alter serum metabolites and metabolic pathways to alter energy metabolism and provide insights for the physiological regulation of cold-water fish in response to cold acclimation.

Ecophysiological responses of Ostreopsis towards temperature: A case study of benthic HAB facing ocean warming.

Reports of the benthic dinoflagellate Ostreopsis spp. have been increasing in the last decades, especially in temperate areas. In a context of global warming, evidences of the effects of increasing sea temperatures on its physiology and its distribution are still lacking and need to be investigated. In this study, the influence of temperature on growth, ecophysiology and toxicity was assessed for several strains of O. cf. siamensis from the Bay of Biscay (NE Atlantic) and O. cf. ovata from NW Mediterranean Sea. Cultures were acclimated to temperatures ranging from 14.5 °C to 32 °C in order to study the whole range of each strain-specific thermal niche. Acclimation was successful for temperatures ranging from 14.5 °C to 25 °C for O. cf. siamensis and from 19 °C to 32 °C for O. cf. ovata, with the highest growth rates measured at 22 °C (0.54-1.06 d-1) and 28 °C (0.52-0.75 d-1), respectively. The analysis of cellular content of pigments and lipids revealed some aspects of thermal acclimation processes in Ostreopsis cells. Specific capacities of O. cf. siamensis to cope with stress of cold temperatures were linked with the activation of a xanthophyll cycle based on diadinoxanthin. Lipids (neutral reserve lipids and polar ones) also revealed species-specific variations, with increases in cellular content noted under extreme temperature conditions. Variations in toxicity were assessed through the Artemia franciscana bioassay. For both species, a decrease in toxicity was observed when temperature dropped under the optimal temperature for growth. No PLTX-like compounds were detected in O. cf. siamensis strains. Thus, the main part of the lethal effect observed on A. franciscana was dependent on currently unknown compounds. From a multiclonal approach, this work allowed for defining specificities in the thermal niche and acclimation strategies of O. cf. siamensis and O. cf. ovata towards temperature. Potential impacts of climate change on the toxic risk associated with Ostreopsis blooms in both NW Mediterranean Sea and NE Atlantic coast is further discussed, taking into account variations in the geographic distribution, growth abilities and toxicity of each species.

Acclimation to warmer temperatures can protect host populations from both further heat stress and the potential invasion of pathogens.

Thermal acclimation can provide an essential buffer against heat stress for host populations, while acting simultaneously on various life-history traits that determine population growth. In turn, the ability of a pathogen to invade a host population is intimately linked to these changes via the supply of new susceptible hosts, as well as the impact of warming on its immediate infection dynamics. Acclimation therefore has consequences for hosts and pathogens that extend beyond simply coping with heat stress-governing both population growth trajectories and, as a result, an inherent propensity for a disease outbreak to occur. The impact of thermal acclimation on heat tolerances, however, is rarely considered simultaneously with metrics of both host and pathogen population growth, and ultimately fitness. Using the host Daphnia magna and its bacterial pathogen, we investigated how thermal acclimation impacts host and pathogen performance at both the individual and population scales. We first tested the effect of maternal and direct thermal acclimation on the life-history traits of infected and uninfected individuals, such as heat tolerance, fecundity, and lifespan, as well as pathogen infection success and spore production. We then predicted the effects of each acclimation treatment on rates of host and pathogen population increase by deriving a host's intrinsic growth rate (rm) and a pathogen's basic reproductive number (R0). We found that direct acclimation to warming enhanced a host's heat tolerance and rate of population growth, despite a decline in life-history traits such as lifetime fecundity and lifespan. In contrast, pathogen performance was consistently worse under warming, with within-host pathogen success, and ultimately the potential for disease spread, severely hampered at higher temperatures. Our results suggest that hosts could benefit more from warming than their pathogens, but only by linking multiple individual traits to population processes can the full impact of higher temperatures on host and pathogen population dynamics be realised.

Green/red light-sensing mechanism in the chromatic acclimation photosensor.

Certain cyanobacteria alter their photosynthetic light absorption between green and red, a phenomenon called complementary chromatic acclimation. The acclimation is regulated by a cyanobacteriochrome-class photosensor that reversibly photoconverts between green-absorbing (Pg) and red-absorbing (Pr) states. Here, we elucidated the structural basis of the green/red photocycle. In the Pg state, the bilin chromophore adopted the extended C15-Z,anti structure within a hydrophobic pocket. Upon photoconversion to the Pr state, the bilin is isomerized to the cyclic C15-E,syn structure, forming a water channel in the pocket. The solvation/desolvation of the bilin causes changes in the protonation state and the stability of π-conjugation at the B ring, leading to a large absorption shift. These results advance our understanding of the enormous spectral diversity of the phytochrome superfamily.

Low- and moderate-intensity aerobic exercise improves the physiological acclimatization of lowlanders on the Tibetan plateau.

This study investigates whether exercise as a strategy for improving physical fitness at sea level also offers comparable benefits in the unique context of high altitudes (HA), considering the physiological challenges of hypoxic conditions. Overall, 121 lowlanders who had lived on the Tibetan Plateau for >2 years and were still living at HA during the measurements were randomly classified into four groups. Each individual of the low-intensity (LI), moderate-intensity (MI), and high-intensity (HI) groups performed 20 sessions of aerobic exercise at HA (3680 m) over 4 weeks, while the control group (CG) did not undergo any intervention. Physiological responses before and after the intervention were observed. The LI and MI groups experienced significant improvement in cardiopulmonary fitness (0.27 and 0.35 L/min increases in peak oxygen uptake [ V ˙ $\dot{\mathrm{V}}$ O2peak], both p < 0.05) after exercise intervention, while the hematocrit (HCT) remained unchanged (p > 0.05). However, HI exercise was less efficient for cardiopulmonary fitness of lowlanders (0.02 L/min decrease in V ˙ $\dot{\mathrm{V}}$ O2peak, p > 0.05), whereas both the HCT (1.74 %, p < 0.001) and glomerular filtration rate (18.41 mL/min, p < 0.001) increased with HI intervention. Therefore, LI and MI aerobic exercise, rather than HI, can help lowlanders in Tibet become more acclimated to the HA by increasing cardiopulmonary function and counteracting erythrocytosis.

Photoacclimation and photophysiology of four species of toxigenic Dinophysis.

This study aimed to explore the effects of different light intensities on the ecophysiology of eight new Dinophysis isolates comprising four species (D. acuminata, D. ovum, D. fortii, and D. caudata) collected from different geographical regions in the US. After six months of acclimation, the growth rates, photosynthetic efficiency (Fv/Fm ratio), toxin content, and net toxin production rates of the Dinophysis strains were examined. The growth rates of D. acuminata and D. ovum isolates were comparable across light intensities, with the exception of one D. acuminata strain (DANY1) that was unable to grow at the lowest light intensity. However, D. fortii and D. caudata strains were photoinhibited and grew at a slower rate at the highest light intensity, indicating a lower degree of adaptability and tolerance to such conditions. Photosynthetic efficiency was similar for all Dinophysis isolates and negatively correlated with exposure to high light intensities. Multiple toxin metrics, including cellular toxin content and net production rates of DSTs and PTXs, were variable among species and even among isolates of the same species in response to light intensity. A pattern was detected, however, whereby the net production rates of PTXs were significantly lower across all Dinophysis isolates when exposed to the lowest light intensity. These findings provide a basis for understanding the effects of light intensity on the eco-physiological characteristics of Dinophysis species in the US and could be employed to develop integrated physical-biological models for species and strains of interest to predict their population dynamics and mitigate their negative effects.

The bioenergetics response of the coral Pocillopora damicornis to temperature changes during its reproduction stage.

Sexual reproduction of reef-building corals is vital for coral reef ecosystem recovery. Corals allocate limited energy to growth and reproduction, when being under environmental disturbance, which ultimately shapes the community population dynamics. In the present study, energetic and physiological parameters of both parental colonies and larvae of the coral Pocillopora damicornis were measured during their reproduction stage under four temperatures; 28 °C (low-temperature acclimation, LA), 29 °C (control temperature, CT), 31 °C (high-temperature acclimation, HA), and 32 °C (heat stress, HS). The results showed temperature changes altered the larvae release timing and fecundity in P. damicornis. Parental colonies exposed to the LA treatment exhibited reduced investment in reproduction and released fewer larvae, while retaining more energy for their development. However, each larva acquired higher energy and symbiont densities enabling survival through longer planktonic periods before settlement. In contrast, parental colonies exposed to the HA treatment had increased investment for reproduction and larvae output, while per larva gained less energy to mitigate the threat of higher temperature. Furthermore, the energy allocation processes restructured fatty acids concentration and composition in both parental colonies and larvae as indicated by shifts in membrane fluidity under adaptable temperature changes. Notably, parental colonies from the HS treatment expended more energy in response to heat stress, resulting in adverse effects, especially after larval release. Our study expands the current knowledge on the energy allocation strategies of P. damicornis and how it is impacted by temperature. Parental colonies employed different energy allocation strategies under distinct temperature regimes to optimize their development and offspring success, but under heat stress, both were compromised. Lipid metabolism is essential for the success of coral reproduction and further understanding their response to heat stress can improve intervention strategies for coral reef conservation in warmer future oceans.

Strategies of Molecular Signal Integration for Optimized Plant Acclimation to Stress Combinations.

Plant growth and survival in their natural environment require versatile mitigation of diverse threats. The task is especially challenging due to the largely unpredictable interaction of countless abiotic and biotic factors. To resist an unfavorable environment, plants have evolved diverse sensing, signaling, and adaptive molecular mechanisms. Recent stress studies have identified molecular elements like secondary messengers (ROS, Ca2+, etc.), hormones (ABA, JA, etc.), and signaling proteins (SnRK, MAPK, etc.). However, major gaps remain in understanding the interaction between these pathways, and in particular under conditions of stress combinations. Here, we highlight the challenge of defining "stress" in such complex natural scenarios. Therefore, defining stress hallmarks for different combinations is crucial. We discuss three examples of robust and dynamic plant acclimation systems, outlining specific plant responses to complex stress overlaps. (a) The high plasticity of root system architecture is a decisive feature in sustainable crop development in times of global climate change. (b) Similarly, broad sensory abilities and apparent control of cellular metabolism under adverse conditions through retrograde signaling make chloroplasts an ideal hub. Functional specificity of the chloroplast-associated molecular patterns (ChAMPs) under combined stresses needs further focus. (c) The molecular integration of several hormonal signaling pathways, which bring together all cellular information to initiate the adaptive changes, needs resolving.

Despite plasticity, heatwaves are costly for a coral reef fish.

Climate change is intensifying extreme weather events, including marine heatwaves, which are prolonged periods of anomalously high sea surface temperature that pose a novel threat to aquatic animals. Tropical animals may be especially vulnerable to marine heatwaves because they are adapted to a narrow temperature range. If these animals cannot acclimate to marine heatwaves, the extreme heat could impair their behavior and fitness. Here, we investigated how marine heatwave conditions affected the performance and thermal tolerance of a tropical predatory fish, arceye hawkfish (Paracirrhites arcatus), across two seasons in Moorea, French Polynesia. We found that the fish's daily activities, including recovery from burst swimming and digestion, were more energetically costly in fish exposed to marine heatwave conditions across both seasons, while their aerobic capacity remained the same. Given their constrained energy budget, these rising costs associated with warming may impact how hawkfish prioritize activities. Additionally, hawkfish that were exposed to hotter temperatures exhibited cardiac plasticity by increasing their maximum heart rate but were still operating within a few degrees of their thermal limits. With more frequent and intense heatwaves, hawkfish, and other tropical fishes must rapidly acclimate, or they may suffer physiological consequences that alter their role in the ecosystem.

Weaker photosynthetic acclimation to fluctuating than to corresponding steady UVB radiation treatments in grapevines.

The effects of transient increases in UVB radiation on plants are not well known; whether cumulative damage dominates or, alternately, an increase in photoprotection and recovery periods ameliorates any negative effects. We investigated photosynthetic capacity and metabolite accumulation of grapevines (Vitis vinifera Cabernet Sauvignon) in response to UVB fluctuations under four treatments: fluctuating UVB (FUV) and steady UVB radiation (SUV) at similar total biologically effective UVB dose (2.12 and 2.23 kJ m-2 day-1), and their two respective no UVB controls. We found a greater decrease in stomatal conductance under SUV than FUV. There was no decrease in maximum yield of photosystem II (Fv/Fm) or its operational efficiency (ɸPSII) under the two UVB treatments, and Fv/Fm was higher under SUV than FUV. Photosynthetic capacity was enhanced under FUV in the light-limited region of rapid light-response curves but enhanced by SUV in the light-saturated region. Flavonol content was similarly increased by both UVB treatments. We conclude that, while both FUV and SUV effectively stimulate acclimation to UVB radiation at realistic doses, FUV confers weaker acclimation than SUV. This implies that recovery periods between transient increases in UVB radiation reduce UVB acclimation, compared to an equivalent dose of UVB provided continuously. Thus, caution is needed in interpreting the findings of experiments using steady UVB radiation treatments to infer effects in natural environments, as the stimulatory effect of steady UVB is greater than that of the equivalent fluctuating UVB.

Resistance of Posidonia oceanica seedlings to warming: Investigating the importance of the lag-phase duration between two heat events to thermo-priming.

The increase of marine heat waves (MHWs) occurrence is exacerbated in Mediterranean Sea and temperature resilience-enhancing strategies on key species, such as the seagrass Posidonia oceanica, need to be investigated. "Priming" describes a stimulus that prepares an organism for an improved response to upcoming environmental changes by triggering a memory that remains during a lag-phase. The aim of this study, conducted in Sardinia (Italy), was to investigate whether the development of thermo-primed P. oceanica seedlings is affected by a field simulated MHW depending on the duration of the lag-phase. After the thermo-priming stimulus, seedlings had a 0, 7 or 14 days lag-phase and after that, for each lag-phase group, half of the seedlings experienced a simulated MHW (the other half served as controls). Some other seedlings did not experience either the priming stimulus or the lag-phase. Results did not show any evidence of a memory triggered by the priming stimulus, but they highlighted the importance of an acclimation phase before the highest temperature: seedlings that experienced a gradual increase of temperature had a higher number of leaves and shorter leaf necrosis length compared to seedlings that had a lag-phase between two heat events. Regardless the priming stimulus, MHWs slowed down the development of the leaf and root length. Considering the increase of temperature fluctuations, testing different intensities of priming and different length of lag-phase is necessary to provide information about the adaptive success of the species.

Alternative splicing plays a nonredundant role in greater amberjack (Seriola dumerili) in acclimation to ambient salinity fluctuations.

Alternative splicing (AS) is an important post-transcriptional mechanism for adaptation of fish to environmental stress. Here, we performed a genome-wide investigation to AS dynamics in greater amberjack (Seriola dumerili), an economical marine teleost, in response to hypo- (10 ppt) and hyper-salinity (40 ppt) stresses. Totally, 2267-2611 differentially spliced events were identified in gills and kidney upon the exposure to undesired salinity regimes. In gills, genes involved in energy metabolism, stimulus response and epithelial cell differentiation were differentially spliced in response to salinity variation, while sodium ion transport and cellular amide metabolism were enhanced in kidney to combat the adverse impacts of salinity changes. Most of these differentially spliced genes were not differentially expressed, and AS was found to regulate different biological processes from differential gene expression, indicative of the functionally nonredundant role of AS in modulating salinity acclimation in greater amberjack. Together, our study highlights the important contribution of post-transcriptional mechanisms to the adaptation of fish to ambient salinity fluctuations and provides theoretical guidance for the conservation of marine fishery resources against increasingly environmental challenges.

Climate change consequences on the systemic heart of female Octopus maya: oxidative phosphorylation assessment and the antioxidant system.

There is evidence that indicates that temperature modulates the reproduction of the tropical species Octopus maya, through the over- or under-expression of many genes in the brain. If the oxygen supply to the brain depends on the circulatory system, how temperature affects different tissues will begin in the heart, responsible for pumping the oxygen to tissues. The present study examines the impact of heat stress on the mitochondrial function of the systemic heart of adult O. maya. The mitochondrial metabolism and antioxidant defense system were measured in the systemic heart tissue of female organisms acclimated to different temperatures (24, 26, and 30°C). The results show that acclimation temperature affects respiratory State 3 and State 4o (oligomycin-induced) with higher values observed in females acclimated at 26°C. The antioxidant defense system is also affected by acclimation temperature with significant differences observed in superoxide dismutase, glutathione S-transferase activities, and glutathione levels. The results suggest that high temperatures (30°C) could exert physical limitations on the circulatory system through the heart pumping, affecting nutrient and oxygen transport to other tissues, including the brain, which exerts control over the reproductive system. The role of the cardiovascular system in supporting aerobic metabolism in octopus females is discussed.

Low frequency of the wild-type freezing-tolerance LsCBF7 allele among lettuce population suggests a negative selection during domestication and breeding.

KEY MESSAGE: Sustainable winter production in lettuce requires freezing tolerant varieties. This study identified a wild-type allele of LsCBF7 that could contribute to freezing tolerance improvement in lettuce. Lettuce is one of the most consumed vegetables globally. While ideally grown in 13-21 °C, its cultivation extends into winter in milder climates. However, occasional freezing temperatures can significantly reduce yields. Therefore, the development of freezing-tolerant lettuce varieties has become a long-term goal of lettuce breeding programs. Despite its significance, our understanding of freezing tolerance in lettuce remains limited. Plants have evolved a coping mechanism against freezing, known as cold acclimation, whereby they can increase freezing tolerance when pre-exposed to low nonfreezing temperatures. The CBF pathway is well-known for its central role in cold acclimation. Previously, we identified 14 CBF genes in lettuce and discovered that one of them, LsCBF7, had a loss-of-function mutation. In this study, we uncovered that accessions from colder regions carried the wild-type allele of LsCBF7 and this allele likely contributed to increased freezing tolerance, with 14% of the lettuce population carrying this allele. Interestingly, in wild lettuce (L. serriola) that is considered a progenitor of cultivated lettuce, this wild-type allele was much more common, with a frequency of 90%. This finding suggests that this wild-type allele may have undergone negative selection during the domestication or breeding of lettuce. Our data strongly indicate that this allele could be linked to early bolting, an undesirable trait in lettuce, which may have driven the negative selection. While this wild-type allele shows promise for improving freezing tolerance in lettuce, it is crucial to decouple it from the early bolting trait to fully harness its potential in lettuce breeding.

Heat acclimation improves exercise performance in hot conditions and increases heat shock protein 70 and 90 of skeletal muscles in Thoroughbred horses.

This study aimed to determine whether heat acclimation could induce adaptations in exercise performance, thermoregulation, and the expression of proteins associated with heat stress in the skeletal muscles of Thoroughbreds. Thirteen trained Thoroughbreds performed 3 weeks of training protocols, consisting of cantering at 90% maximal oxygen consumption (VO2max) for 2 min 2 days/week and cantering at 7 m/s for 3 min 1 day/week, followed by a 20-min walk in either a control group (CON; Wet Bulb Globe Temperature [WBGT] 12-13°C; n = 6) or a heat acclimation group (HA; WBGT 29-30°C; n = 7). Before and after heat acclimation, standardized exercise tests (SET) were conducted, cantering at 7 m/s for 90 s and at 115% VO2max until fatigue in hot conditions. Increases in run time (p = 0.0301), peak cardiac output (p = 0.0248), and peak stroke volume (p = 0.0113) were greater in HA than in CON. Pulmonary artery temperature at 7 m/s was lower in HA than in CON (p = 0.0332). The expression of heat shock protein 70 (p = 0.0201) and 90 (p = 0.0167) increased in HA, but not in CON. These results suggest that heat acclimation elicits improvements in exercise performance and thermoregulation under hot conditions, with a protective adaptation to heat stress in equine skeletal muscles.

Timing-specific parental effects of ocean warming in a coral reef fish.

Population and species persistence in a rapidly warming world will be determined by an organism's ability to acclimate to warmer conditions, especially across generations. There is potential for transgenerational acclimation but the importance of ontogenetic timing in the transmission of environmentally induced parental effects remains mostly unknown. We aimed to disentangle the effects of two critical ontogenetic stages (juvenile development and reproduction) to the new-generation acclimation potential, by exposing the spiny chromis damselfish Acanthochromis polyacanthus to simulated ocean warming across two generations. By using hepatic transcriptomics, we discovered that the post-hatching developmental environment of the offspring themselves had little effect on their acclimation potential at 2.5 months of life. Instead, the developmental experience of parents increased regulatory RNA production and protein synthesis, which could improve the offspring's response to warming. Conversely, parental reproduction and offspring embryogenesis in warmer water elicited stress response mechanisms in the offspring, with suppression of translation and mitochondrial respiration. Mismatches between parental developmental and reproductive temperatures deeply affected offspring gene expression profiles, and detrimental effects were evident when warming occurred both during parents' development and reproduction. This study reveals that the previous generation's developmental temperature contributes substantially to thermal acclimation potential during early life; however, exposure at reproduction as well as prolonged heat stress will likely have adverse effects on the species' persistence.

Acclimation to higher temperature and antioxidant supplemented diets improved rainbow trout (Oncorhynchus mykiss) resilience to heatwaves.

Coldwater species are challenged with increasing water temperatures and fluctuations over their upper thermal limits. This study evaluated the potential of acclimation to higher temperature and dietary antioxidants capacity to mitigate the adverse effects of heat shocks in rainbow trout. To this end, rainbow trout fingerlings were acclimated at optimal (14 °C) and high (20 °C) temperatures and fed on selenium (5 mg/kg) and polyphenol (2 g/kg) supplemented diets for 60 days and then were exposed to heat shocks by increasing water temperature up to 30 °C. Growth performance, survival rate, haemato-immunological parameters, and expression of HSP70α, HSP70ß, HSP90ß, and IL-1ß genes were measured to evaluate the hypothesises. The rainbow trout acclimated to 20 °C and fed on antioxidants supplemented diets showed a significantly higher aftershock survival rate. Moreover, fish acclimated to higher temperature showed higher red blood cell counts as well as serum total protein and albumin during the acclimation trial and heat shocks phase. Acclimation to higher temperature and feeding on antioxidants remarkably enhanced fish immune and antioxidant capacity in comparison to fish adapted to cold water and fed on the basal diet measured by improved respiratory burst and lysozyme activities and upregulation of IL-1ß expression during exposure of fish to heat shocks. Furthermore, fish acclimated to higher temperature, especially those fed on antioxidant supplemented diets, showed lower expression levels of HSPs genes during the heat shock phase, indicating that high heat shocks were less stressful for these fish in comparison to cold water acclimated fish. This finding was also supported by lower cortisol levels during heat shocks in fish acclimated to higher temperature. In conclusion, the results of this study indicated that acclimation to higher temperature and/or fed on diets supplemented by selenium and polyphenol, can help to mitigate the adverse effects of the heat shock in rainbow trout.

Micropropagation and cryopreservation of the rare endemic Colchicum figlalii germplasm.

BACKGROUND: The natural population of Colchicum figlalii (Varol) Parolly and Eren grows in a narrow area of serpentine rock clearings at an altitude of 1900-2100 m in Southwestern Anatolia (Sandras Mountain, Mugla, Turkey). The species is regarded as endangered according to the IUCN Red List Categories. OBJECTIVE: To develop an optimum procedure for in vitro propagation and cryopreservation of germplasm of this rare endemic. MATERIALS AND METHODS: A total of 281 bulbs were used as in vitro culture starting material and after surface sterilization, clean material was obtained from 157 of them. Woody Plant Medium (WPM), Olive Medium (OM), and Murashige and Skoog medium (MS) were used for in vitro culture establishment. RESULTS: The maximum regeneration rate (~67.3%) was obtained after four weeks of incubation on OM. The calli were successfully induced by using OM supplemented with 10.7 uM NAA from leaves of in vitro grown C. figlalii bulbs. A PVS2-vitrification procedure was used for cryopreservation of C. figlalii callus tissue. After cryo-storage, the best result for regeneration (66.7%) was obtained from calli treated with PVS2 for 75 min before plunging into liquid nitrogen. All rooted seedlings derived from cryopreserved calli were successfully acclimatized to greenhouse conditions. CONCLUSION: This study is an effective reference for future long-term conservation of similar species that are difficult to cryopreserve. Doi.org/10.54680/fr24410110412.