Molecular Mechanisms Underlying Freezing Tolerance in Plants: Implications for Cryopreservation

Abstract

Cryopreservation is a crucial technique for the long-term ex situ conservation of plant genetic resources, particularly in the context of global biodiversity decline. This process entails freezing biological material at ultra-low temperatures using liquid nitrogen, which effectively halts metabolic activities and preserves plant tissues over extended periods. Over the past seven decades, a plethora of techniques for cryopreserving plant materials have been developed. These include slow freezing, vitrification, encapsulation dehydration, encapsulation–vitrification, droplet vitrification, cryo-plates, and cryo-mesh techniques. A key challenge in the advancement of cryopreservation lies in our ability to understand the molecular processes underlying plant freezing tolerance. These mechanisms include cold acclimatization, the activation of cold-responsive genes through pathways such as the ICE–CBF–COR cascade, and the protective roles of transcription factors, non-coding RNAs, and epigenetic modifications. Furthermore, specialized proteins, such as antifreeze proteins (AFPs) and late embryogenesis abundant (LEA) proteins, play crucial roles in protecting plant cells during freezing and thawing. Despite its potential, cryopreservation faces significant challenges, particularly in standardizing protocols for a wide range of plant species, especially those from tropical and subtropical regions. This review highlights the importance of ongoing research and the integration of omics technologies to improve cryopreservation techniques, ensuring their effectiveness across diverse plant species and contributing to global efforts regarding biodiversity conservation.

1. Introduction

The diversity of life on Earth, both in terrestrial and aquatic ecosystems, is declining at an alarming rate [1,2]. The current extinction event is the sixth in the last 550 million years and may be the most severe in the planet’s history. It may exceed the scale of the previous five major extinctions in regards to the number of species affected [3,4]. Natural populations are exposed to many threats, including those arising from climate change, pollution, the emergence of novel pathogens, pests, invasive species, habitat loss, and over-harvesting [5,6,7]. Two principal strategies have been developed to address these challenges and ensure biodiversity preservation, i.e., in situ and ex situ. The former aims to protect species within their natural habitats, while the latter is focused on preserving species outside their natural habitats. Plant ex situ conservation includes preserving plant specimens in several collections, such as botanical gardens, germplasm banks (which preserve seeds, pollen, tissues, and organs), and field collections of vegetatively propagated crop species [8,9,10].

Cryopreservation is one of the most recent techniques for ex situ biodiversity conservation. The term “cryopreservation” describes a process whereby biological material is frozen and stored at extremely low temperatures [11,12]. This method employs liquid nitrogen (LN), which enables the maintenance of plant material at exceedingly low temperatures, specifically at −196 °C in liquid and at −150 °C in vapor over liquid. The utilization of LN presents many benefits, including relatively low cost, chemical inertness, accessibility, and a high degree of independence from electricity [11]. In the context of cryogenic conditions, the reduction or cessation of kinetic energy and molecular motion in biological systems has the effect of weakening transport and enzymatic reactions, which in turn results in the slowing of the aging process [13]. Nevertheless, the cryopreservation protocol must be optimized case-by-case, depending on the species and type of explant to be frozen. This is due to the necessity of implementing an effective method to prevent ice crystals from forming from the water in the cells. The formation and growth of ice crystals intracellularly cause cell membrane rupture, leading to loss of function and ultimately, to cell death [14]. Advances in mammalian systems in recent years have influenced the development of cryopreservation strategies for plant cells and organs [15].

Cryopreservation protocols have been developed for many species, primarily those of economic importance [16,17,18,19]. Nevertheless, disparities in tolerance to cryopreservation have been identified, as evidenced by the differential capacity for post-cryopreservation regeneration [20,21,22]. These observations suggest the existence of molecular-level mechanisms underlying freezing tolerance and metabolic resuscitation after thawing. This paper provides a comprehensive overview of this field’s current state of knowledge.

2. Overview of Cryopreservation Techniques

Since the initial paper on the cryopreservation of plant material was published in 1956, many techniques and protocols have been developed, offering an array of methods for effectively storing plant germplasm [23].

2.1. Slow Freezing

One of the most traditional cryopreservation techniques is slow freezing, also called controlled-rate freezing. This method has been extensively employed for the conservation of plant genetic resources. This method gradually cools the plant material at a rate of no more than 2 °C/min until the temperature reaches −40 °C, after which it is immersed in LN [18]. Consequently, at the outset of the process, the cells are subcooled instead of frozen. The cytoplasm remains unfrozen due to the concentration of solutes, and the cell wall protects the cell membrane from damaging ice crystals [24]. As the temperature decreases, the proportion of the extracellular solution that undergoes ice formation increases, leading to an elevation in the osmotic pressure gradient. This is counterbalanced by water migration from the cells into the intercellular spaces, where it freezes. Under optimal conditions, most of the water from the cells is removed, preventing the formation of intracellular ice crystals following subsequent immersion in LN [25]. Several controlled-rate freezing techniques have been developed to date [26]. This technique is effective for winter dormant buds, shoot tips from temperate and subtropical plants, undifferentiated cell suspensions, and calluses [15,27,28,29]. Modifying this method by pre-treatment of the plant material with cryoprotectant solutions allows its application to a broader range of species and improves regeneration efficiency after cryopreservation [13,18,30]. The utilization of standard operating procedures, programmed cooling rates, large-scale batch processing, and efficient use of working time are among the key benefits of this method [28]. However, there are significant drawbacks. Freezing rates require precise control through sophisticated and costly programmable freezers, and the concentration of cryoprotectant agents must also be carefully regulated [31]. Additionally, some cryoprotectant agents can be toxic to specific plant tissues, necessitating the development of protocols tailored to the species and tissues [32].

Cryopreservation of Dormant Buds

Cryopreservation of dormant buds is a protocol based on slow freezing. Its history dates back to the 1960s [33]. Since then, the protocol has been continuously modified and improved [27,34,35,36]. Its uniqueness lies in the fact that this method can be applied to preserving trees and shrubs native to temperate climates that undergo natural dormancy during winter [16]. Exposure to photoperiod and/or low temperatures induces this dormant phase, and plants must demonstrate moderate to high winter hardiness to be eligible for cryopreservation [37]. The protocol takes advantage of the natural acclimation of buds on the parent plant and the physical dehydration and slow freezing of the nodal section of the branch. The buds are collected and subjected to artificial acclimatization to cold conditions in the winter months. They are then dried in air, placed in cryovials, and exposed to slow freezing. Following thawing and rehydration of the buds, they are directly grafted onto rootstocks [38]. The absence of an in vitro culture stage in the protocol results in a notable reduction in the overall length of the process, a diminished risk of infection, and a decrease in associated costs [37]. In addition, this method does not utilize cryoprotectants and allows for the preservation of large amounts of material with minimal effort. However, it requires a programmable freezer that precisely controls the initial phase of slow freezing. Another problem is that genotypes within a species have different levels of freeze tolerance, resulting in different success rates [36,39].

2.2. Vitrification

Another cryopreservation method is vitrification. This methodology was developed and first implemented in the 1990s [40,41]. In general, vitrification can be defined as the kinetic transformation of a substance from a liquid to a glassy state [42]. During cryopreservation, biological materials are treated with a combination of highly concentrated penetrating and nonpenetrating cryoprotectants at 0 °C. This is followed by immersion in LN, which facilitates the rapid cooling process. During this phase, the viscous cryoprotectant solidifies into metastable glass, avoiding the formation of crystals [28]. Cryoprotectants enhance the viscosity of cells, thereby inhibiting the formation of ice crystals both within and outside the cell. This reduces the risk of damage to cellular organelle structures, as ice formation is prevented [28]. Cell membranes are protected by a gelatinous fluid that forms a glassy state, which enables cells to survive at cryogenic temperatures [43]. Furthermore, vitrification solutions assist in preventing additional lethal water loss and enhance the regeneration percentage after cryopreservation [44]. The high viscosity of this glass hinders the progression of chemical reactions that rely on molecular diffusion. Consequently, its formation impedes metabolic activity, leading to its stability over time [45]. One of the most notable benefits of vitrification is its extensive applicability to a diverse range of plant species and tissue types, including shoot tips and somatic and zygotic embryos [46,47,48]. Moreover, the technique is straightforward and necessitates minimal expenditure on costly equipment, conferring notable benefits in regards to cost-effectiveness and feasibility. The most substantial drawback is the toxicity of cryoprotectants, as previously discussed.

2.3. Encapsulation Dehydration

Simultaneously, encapsulation dehydration technology was developed in the 1990s [49]. It combines synthetic seed production and dehydration technologies [50]. Explants are encapsulated in gel beads, usually calcium alginate, placed in a high-sucrose liquid medium for osmotic dehydration, partially dried over silica gel or in a laminar flow chamber for physical dehydration, and placed in LN [51]. Depending on the species, desiccated capsules are either directly immersed in LN or subjected to a slow-freezing process [52]. Enclosing the explants in capsules allows them to survive dehydration. Most of the frozen water is removed from the cells during this process. During immersion in LN, vitrification of the dissolved internal substances occurs, preventing ice crystallization and resulting in a high percentage of regeneration after thawing [53]. This method is widely applicable. It can be adapted to different species and explant types. It exhibits low toxicity compared to that of other methods. Encapsulation protects explants from mechanical damage by providing an additional layer. However, the encapsulation process requires precise control of the degree of dehydration. It must be optimized and is labor- and time-consuming, mainly when used for extensive germplasm collections [51].

2.4. Encapsulation–Vitrification

The increased interest in using cryopreservation methodologies has resulted in continuous development of new techniques. One such technique is encapsulation–vitrification, which combines the encapsulation dehydration and vitrification protocols. This procedure was developed by Tannoury et al. in 1991 [54]. The encapsulation step protects explants during the procedure and reduces the harmful effects of both osmotic stress and cryoprotectant toxicity. A significant advantage of this method is that it allows cryopreservation through direct immersion in LN, eliminating the need to purchase programmable freezers [55]. This method also applies to explants from tropical or subtropical species, with which other cryopreservation methods often prove ineffective [16,56,57,58].

2.5. Droplet Vitrification

In 2000, Pennycooke and Towill published a subsequent cryopreservation protocol designated as droplet vitrification [59]. The method was based on the technique described by Kartha et al. for the cryopreservation of apical meristems in droplets of dimethyl sulfoxide (DMSO) [60]. It involves the placement of small explants, such as shoot tips or embryos, in a droplet of vitrification solution on a strip of aluminum foil, followed by incubation in the cryoprotectant mixture and rapid cooling by imbibition of liquid nitrogen [59]. The solution includes DMSO, ethylene glycol, glycerol, and MS medium with sucrose, placed as a droplet on the aluminum foil surface [61]. Rapid cooling and heating rates are achieved, reaching up to 130 °C per second, facilitated by the foil’s excellent thermal conductivity [62]. Moreover, explants are directly exposed to LN during cooling [16]. This method offers numerous advantages, including avoidance of explant manipulation during foil strip placement in the cryovial, which minimizes the risk of mechanical damage. By placing the explant directly into a drop of vitrification solution on the foil for dehydration, the exposure time to PVS2, which exhibits toxic properties, was significantly reduced. This method also ensures that the integrity of the explant is maintained and results in a high percentage of regeneration after thawing [62,63,64]. However, there are disadvantages, including the requirement for precise handling and extended time. Technical skills are required of the staff to ensure that the explants are not subjected to extensive exposure to PVS2 and at the same time, are not damaged or lost during handling or the application or removal of the vitrification solution [63,64].

2.6. Cryo-Plate Techniques

Recent advancements in cryopreservation have also centered around cryo-plates. The V cryo-plate method was developed by Yamamoto et al. in 2011 [65]. It combines encapsulation–vitrification and droplet vitrification methods using an aluminum cryo-plate with ten microwells. On the other hand, in 2013, Niino et al. introduced the D cryo-plate technique [66]. This method combines encapsulation and dehydration. There is a significant difference between the two protocols in regards to the time of treatment with the PVS solution. For the V cryo-plate method, the time is approximately four times shorter [66]. However, the D cryo-plate method, which uses physical dehydration, eliminates the risk of chemical stress and genetic alteration [38,67,68]. Depending on the species, its effectiveness is similar to or highly variable from that of other methods [69,70].

2.7. Cryo-Mesh Method

Funnekotter et al. developed the latest method, employing a stainless-steel cryo-mesh, in 2017 [71]. This method is analogous to the V-cryo plate method. The cryo-mesh facilitates rapid cooling and heating, provides uniform exposure to cryoprotectants, and minimizes the risk of mechanical damage to explants [71]. This approach offers a practical solution for the cryopreservation of fragile and tiny plant tissues.

Modern cryopreservation combines innovative and classical techniques to offer a wide range of solutions tailored to the specific needs of different plant species while minimizing the risk of damage and toxicity (

Table 1. Summary of cryopreservation techniques for the conservation of plant genetic resources.

Protocol	Description	Advantages	Disadvantages	Applications	References
Slow Freezing	Employs a gradual cooling rate (up to 2 °C per minute) followed by immersion in LN. Prevents intracellular ice formation.	Effective for winter dormant buds, shoot tips, and cell suspensions; low concentration of cryoprotectants; standardized procedures.	Requires costly programmable freezers and precise cryoprotectant regulation. Toxicity issues with some plant tissues.	Conservation of plant genetic resources, particularly for temperate and subtropical plants.	[18,24,25,26,28,31]
Cryopreservation of Dormant Buds	A protocol based on slow freezing, applicable to trees and shrubs in temperate climates that undergo natural dormancy.	No in vitro stage, reducing process length, infection risk, and costs. No toxic cryoprotectants.	Requires a programmable freezer. Different success rates across genotypes within a species due to variations in freeze tolerance.	Preservation of trees and shrubs from temperate climates.	[16,27,33,34,36,37]
Vitrification	Employs rapid cooling of biological material treated with cryoprotectants to prevent ice crystal formation by transforming liquid into a glassy state.	Extensive applicability to a diverse range of species and tissues. Cost-effective, simple, and minimal equipment requirements.	High toxicity of cryoprotectants.	Cryopreservation of shoot tips, somatic and zygotic embryos, and other plant tissues.	[28,40,42,43,44,45]
Encapsulation Dehydration	The explants are encapsulated in calcium alginate gel beads, dehydrated, and then cryopreserved in LN.	Low toxicity compared to that of other methods; protects explants from mechanical damage; adaptable to different species.	Labor- and time-consuming process. Requires precise control of dehydration.	Widely applicable across species; used for the cryopreservation of various explants.	[49,50,51,52,53]
Encapsulation–vitrification	Combines encapsulation dehydration with vitrification, providing protection during cryopreservation and reducing osmotic stress and cryoprotectant toxicity.	Allows cryopreservation without the need for programmable freezers; applicable to tropical and subtropical species.	Cytotoxicity; technical complexity.	Cryopreservation of explants, particularly from tropical or subtropical species.	[54,55,56,57,58]
Droplet Vitrification	Small explants are placed in a droplet of vitrification solution on aluminum foil and rapidly cooled by LN.	Reduces exposure to toxic cryoprotectants; minimizes mechanical damage; maintains explant integrity.	Requires precise handling and technical skill to avoid overexposure to cryoprotectants and mechanical damage.	Cryopreservation of apical meristems, shoot tips, and other small explants.	[59,60,61,63,64]
Cryo-plate Techniques	Uses aluminum cryo-plates with microwells for encapsulation–vitrification (V cryo-plate) or encapsulation dehydration (D cryo-plate).	Rapid cooling and reduced chemical stress in D cryo-plate method.	Varying effectiveness, depending on species.	Applicable to a wide range of plant tissues, depending on the species.	[65,66,67,68,69,70]
Cryo-mesh Method	Uses stainless-steel cryo-mesh for rapid cooling and heating, providing uniform exposure to cryoprotectants and minimizing mechanical damage.	Practical for fragile and tiny plant tissues.	The precision of this method requires considerable attention to detail in the manipulation of the sample.	Cryopreservation of fragile and tiny plant tissues.	[71]

). Despite the variety of available methods, the key challenges regarding controlling freezing conditions and optimizing protocols for individual species and tissues remain important for further research and technological improvement.

3. Cold Acclimatization

As mentioned previously, the cryopreservation procedures indicate that plant explants are subjected to many stressors during their application, with temperature being a principal contributing factor, alongside dehydration and chemical toxicity. Furthermore, oxidative and osmotic stress, disturbed ionic homeostasis, and disorders associated with changes in the cells’ physical and metabolic properties must also be considered [72]. Consequently, damage to plant explants occurs at the cellular level, leading to vacuolization, cell membrane rupture (including the nuclear membrane), cell lysis, and autophagy [73]. Therefore, it can be inferred that tolerance to freezing is linked to tolerance to all of these factors.

Plants native to temperate climates exhibit natural tolerance to freezing, as they undergo physiological and biochemical adaptation to cope with low temperatures and become dormant during winter [74]. This process, called cold acclimatization, is triggered after exposure to low but non-freezing temperatures [75]. Upon reception of a cold signal by plant cells, a plethora of physiological and metabolic alterations ensue, aiming to impede the formation of ice crystals within the cells. These alterations encompass modifications in the membrane lipid composition and the accumulation of cryoprotective molecules, such as soluble sugars, proteins, and other molecules that help stabilize cell structures and membranes. Substantial changes at the transcriptomic level are necessary for these alterations to manifest [76]. This adaptive capacity is not observed in plants from tropical and subtropical regions because they lack the genetic and physiological mechanisms to withstand freezing temperatures. Consequently, the cryopreservation of these plants is more challenging, yet not entirely impossible [64]. With the growing need for cryobank development, understanding the molecular basis associated with cryopreservation tolerance, demonstrated by the ability to survive and remain stable during storage, has become increasingly crucial.

In numerous cryopreservation procedures, plant material is subjected to cold pre-treatment, which enhances survival and regeneration following thawing. Cold acclimation pre-treatments stimulate a plant’s intrinsic defense mechanisms against the adverse effects of low temperatures [77]. This increases the content of starch grains, lipid bodies, sugars, dry matter, and phospholipids [78,79]. Changes in membrane lipid levels and composition may prevent freezing injury. After a relatively short acclimation period, improvements in cold tolerance have been observed in whole plants [78]. The beneficial impact of cold pre-treatment on donor plants has been documented in woody species, including Malus x domestica, Pyrus communis, and Morus [35,80,81,82]. Furthermore, the application of cold treatment has been proven to enhance the effectiveness of cryopreservation in plants derived from in vitro cultures, leading to improved survival rates in crops such as Solanum tuberosa, Humulus lupulus, and Allium sativum [83,84,85]. The benefits of cold pre-treatment have also been noted in isolated plant organs, such as apical meristems, in a range of species, including Vaccinium, Rubus, Pyrus, Tanacetum cinerariifolium, and Phoenix dactylifera [65,86,87,88,89]. Cold pre-treatment has also been beneficial for the cryopreservation of embryogenic calli [90,91]. Despite the documented benefits, the effectiveness of cold pre-treatment is not uniform across all plant species or even within different genotypes of the same species [21,92]. This variability presents a significant challenge in standardizing cryopreservation protocols, as the optimal conditions for cold pre-treatment, such as temperature, duration, and the specific developmental stage of the tissue, can vary widely between species. For example, while certain species may exhibit enhanced freezing tolerance following cold pre-treatment, others may not respond favorably or may even experience detrimental effects, emphasizing the need for species-specific optimization [93].

These findings highlight the significance of cold acclimatization as a critical factor contributing to freezing tolerance in numerous plant species. With the growing need for cryobank development, understanding the molecular basis associated with cryopreservation tolerance, demonstrated by the ability to survive and remain stable during storage, becomes increasingly crucial.

4. Molecular Basis of Freezing Tolerance

4.1. ICE–CBF–COR Cascade

Low temperatures induce both cold acclimation and dormancy. Thus, the two processes are interconnected, triggering the same cold-responsive (COR) pathway [94,95]. This pathway is part of the inducer of CBF expression—C-repeat binding factors—cold-regulated genes (ICE–CBF–COR) cascade [96,97]. The ICE–CBF–COR cascade represents a pivotal regulatory pathway in plant cold acclimation and freezing tolerance. This complex signaling cascade comprises three principal components that function synergistically to protect plants from cold stress [97]. Several physiological and biochemical changes are associated with cold acclimation, enhancing the freezing tolerance of plants following exposure to non-freezing temperatures.

4.1.1. Cold-Responsive (COR) Genes

Low temperatures induce both cold acclimation and dormancy. Thus, the two processes are interconnected, triggering the same cold responsive (COR) pathway [94,95]. This pathway is a part of the inducer of CBF expression—C-repeat binding factors—cold-regulated genes (ICE–CBF–COR) cascade [96,97]. Several physiological and biochemical changes are associated with cold acclimation, enhancing the freezing tolerance of plants following exposure to non-freezing temperatures. The COR pathway has been extensively studied in Arabidopsis, and over 200 COR genes have been identified [98]. However, this pathway is highly conserved among perennial plant species [95]. The COR pathway includes low-temperature induced (LTI), late embryogenesis proteins (LEA), ABA-inducible protein-coding (KIN1 and KIN2), responsive to desiccation (RD), and early dehydration-inducible (ERD) genes [99,100,101,102,103]. These genes are responsible for accumulating cytoprotective proteins, including antifreeze proteins, chaperones, functional proteins, kinases, and osmoregulators, including soluble sugars [104,105,106,107]. This results in increased freezing tolerance, achieved by stabilizing the osmotic potential of cells and repairing cold-damaged membranes [108]. The promoters of COR genes contain conserved C-repeat (CRT/DRE) cis-elements to which C-repeat binding factors (CBFs) bind [109]. Most known COR genes exhibit a typical structural pattern, with two flanking exons in the 5′ untranslated region (UTR) and 3′ UTR, as well as a central intron [110]. Specific COR genes play an essential role in maintaining the stability of various biomolecules, such as membrane phospholipids, proteins, and cytoplasmic proteins. This is achieved by regulating hydrophobic interactions, maintaining ion homeostasis, and scavenging ROS, depending on the prevailing temperature range, i.e., pre-hardening, hardening, or plant recovery [111,112,113].

4.1.2. CBF

The highly interconnected COR regulatory network is controlled by CBFs, which function as master transcription factors and are designated as dehydration-responsive element-binding proteins (DREBs) (Table 2) [114]. CBFs have been identified in many plant species and are members of the APETALA2/ethylene response factor (AP2/ERF) transcription factor family, one of the largest found in plants [115]. To ensure optimal plant growth, CBF expression must be maintained at a minimal level, without cold stress. However, following exposure to cold, CBFs are induced by transcription factors within 15 min, subsequently leading to the activation of downstream COR genes. CBFs regulate the majority of cold-responsive targets by binding to the DRE/CRT cis-element in the promoters of COR genes containing a conserved sequence (CCGAC) [116,117]. Prior research has demonstrated that individual proteins within the CBF family possess additional functions. For instance, in Arabidopsis, CBF4 plays a role in drought tolerance, and CBF2 functions as a negative regulator of CBF1 and CBF3 expression [118,119].

4.1.3. ICE

Two motifs, i.e., ICEr1 and ICEr2, which are essential for cold induction, have been identified in the promoters of CBFs [120]. These are the binding sites for the CBF expression 1–2 (ICE1-2) inducer, which encodes the MYC-type bHLH family of transcription factors. These proteins contain an essential DNA-binding domain and a helix–loop–helix motif that facilitates dimerization with other bHLH proteins. In addition to CBFs, ICE1 binds to the promoters of other COR genes, including galactinol synthase 3 (GolS3), KIN2, RCI2A, RCI2B, and COR413IM1. This direct regulation ensures a robust cold response via the activation of multiple pathways [121].

ICEs MYC transcription factors are activated by cold-dependent post-translational modifications [122]. Previous studies show that ICE1 is regulated by the high expression of osmotically responsive gene 1 (HOS1), which has a ubiquitin E3 ligase function and is a cold-induced negative regulator of CBFs and thus, COR [123]. In response to low temperatures, HOS1 is transported to the nucleus, where it ubiquitinates ICE1, thereby directing this transcription factor to proteasomal degradation [124]. Cold-activated open stomata kinase 1 (OST1) inhibits ICE1 degradation, which HOS1 mediates. It phosphorylates ICE1 and enhances its stability and transcriptional activity under cold stress conditions [125]. Moreover, ICE1 interacts with the transcription factor MYB15, leading to a decrease in its expression. MYB15 has been demonstrated to bind to the promoter regions of CBFs, thereby suppressing their expression and consequently, negatively regulating freezing tolerance in plants. These observations suggest that HOS1, ICE1, and MYB15 operate in a cascade to modulate CBFs expression, thereby controlling cold acclimatization [126]. Furthermore, the stabilization of ICE1 by SIZ1 has been demonstrated to affect CBF expression. SIZ1 is a SUMO E3 ligase that facilitates the conjugation of small ubiquitin-related modifiers (SUMO) with protein substrates, thereby serving as a post-translational regulator that reduces freezing tolerance [127].

4.2. Other TFs Regulating CBFs

Additionally, other transcription factors that regulate the expression of CBFs have been identified, including calmodulin-binding transcription activator 3 (CAMTA3), CESTA (CES), brassinazole-resistant 1/brassinosteroid insensitive 1-EMS-suppressor 1 (BZR1/BES1), circadian clock-associated 1/late elongated hypocotyl (CCA1/LHY), phytochrome-interacting factors (PIFs), ethylene insensitive 3 (EIN3), and suppressor of constant overexpression 1 (SOC1) [107,128,129,130,131,132].

4.2.1. CAMTA

CAMTA and Ca²⁺-regulated transcription factors have been shown to play a role in cold acclimation. CAMTA3 and other CAMTA family members (CAMTA1 and CAMTA2) undergo rapid induction in response to low temperatures. It has been demonstrated that CAMTA proteins can bind directly to the promoters of CBF genes (in particular, CBF1 and CBF2), thereby facilitating their transcription. This binding is crucial for the cold-induced accumulation of CBF transcripts, leading to COR genes expression that enhances frost tolerance in plants [133,134]. Although the precise mechanisms underlying CAMTA3-induced CBF transcription remain partially elucidated, CAMTA proteins are recognized as critical players in calcium signaling pathways [135]. Calcium ions (Ca²⁺) are perceived in response to cold stress, which leads to the activation of CAMTA3. This activation is postulated to link calcium signaling to the transcriptional regulation of CBF, thereby promoting cold acclimation. In particular, CAMTA3 has been shown to interact with specific DNA motifs, such as the rapid stress response element (RSRE), in the promoters of COR genes, thereby increasing their expression under cold conditions [135,136]. CAMTA and Ca²⁺-regulated transcription factors have been shown to play a role in cold acclimation, whereby they induce the expression of CBFs. However, the precise mechanism of action remains unclear [135]. CAMTA proteins are highly conserved across species. They are present in various organisms, from mosses to flowering plants. They have been identified in more than 40 plant species [137].

4.2.2. CCA1 and LHY

The transcription factors CCA1 and LHY are integral components of the circadian clock in plants and play an essential role in cold acclimation through the induction of CBF gene expression. CCA1 and LHY also induce the expression of CBF genes at low temperatures. Consequently, the expression of COR genes is enhanced, leading to increased cold tolerance [138]. The two transcription factors, CCA1 and LHY, possess the MYB motif and are involved in the circadian clock [139]. They positively regulate the expression of CBF1, CBF2, and CBF3. Genetic studies have revealed that plants with mutations in both genes (the cca1-11/lhy-21 double mutant) display a markedly reduced capacity to induce CBF genes in response to cold temperatures. This leads to a notable decline in COR expression and a subsequent reduction in freezing tolerance [138]. CCA1 and LHY cooperate with other cold-responsive elements, including ICE1 and CAMTA3, to enhance CBF expression in response to cold stress. As the temperature decreases, CCA1 and LHY accumulate at the CBF loci, facilitating their transcription. This interaction is particularly effective in the morning, when CCA1 and LHY levels are elevated, facilitating robust CBF activation. In contrast, as the evening progresses, CBF levels decline, leading to reduced CBF expression and an attenuated response to cold stress [138]. Their participation in cold acclimation and freezing tolerance has been documented in several plant species, including Arabidopsis, Oryza sativa, and Brassica oleracea [138,140,141].

4.2.3. SOC1

The SOC1 gene, encoding a MADS-box transcription factor, is a negative regulator of the expression of CBF genes. SOC1 binds to various forms of the CArG box located in the distal and proximal regions of CBFs, thereby inhibiting the transcription of CBF genes and negatively regulating the cold response [142]. Evidence for the direct repression of CBF by SOC1 is based on microarray analyses that have identified several COR genes (including COR15a, COR15b, KIN1, and KIN2) as targets of SOC1 regulation. In transgenic plants overexpressing SOC1, the transcription of these COR genes was significantly reduced. Conversely, loss-of-function mutations in SOC1 increased the transcription of these genes when they were exposed to cold conditions [142,143]. Research findings have demonstrated that SOC1 affects the kinetics of cold-inducible gene induction. To illustrate, experiments in which plants were exposed to cold temperatures revealed that SOC1 mutants exhibited a more pronounced induction of COR genes than that observed in wild-type plants. This indicates that SOC1 plays a pivotal role in mitigating the cold-induced expression of these genes, thus preventing excessive activation of the cold stress response, which can be deleterious to plant growth and development [142]. Notably, the negative regulation of CBFs by SOC1 is not significantly influenced by circadian rhythms, although the amplitude of its expression may exhibit variability. This suggests that SOC1 consistently regulates the cold response, regardless of the time of day, which is crucial for plants that experience temperature fluctuations. The ability of SOC1 to modulate CBFs expression in a circadian rhythm-independent manner highlights its significance in maintaining homeostasis during periods of low-temperature stress [142].

4.2.4. PIFs

PIFs represent a subfamily of basic helix–loop–helix (bHLH) transcription factors that play significant and multifaceted roles in plant responses to environmental stress, particularly in regards to cold acclimation, and have been shown to play both beneficial and detrimental roles in this process [144,145]. They are primarily known for their involvement in light signaling pathways. However, their dual role in regulating the cold stress response, mainly through their interaction with CBFs, demonstrates the complexity of their involvement in plant adaptation mechanisms [146,147]. PIFs, particularly PIF3, PIF4, and PIF7, have been identified as negative regulators of CBF gene expression under cold stress conditions. They bind directly to the CBF1, CBF2, and CBF3 promoter regions, thereby repressing transcription [144,145]. Therefore, it is evident that the repression of these pathways is crucial for preventing excessive activation of the cold acclimation pathway, which can lead to deleterious effects on growth and development. For example, under long-day conditions, PIF4 and PIF7 act as transcriptional repressors, thereby modulating the plant response to cold by controlling CBF expression. This mechanism allows plants to maintain growth while responding to cold stress, when necessary [147]. The regulatory function of PIFs in cold acclimation is also closely associated with their interaction with phytochrome B (phyB), a photoreceptor that mediates the response to light. Under unstressed conditions, phyB facilitates the degradation of PIFs, thereby relieving their inhibition of CBFs and enabling their acclimation to low temperatures. Conversely, in the presence of low temperatures, the stability of PIFs increases, resulting in their prolonged repression of CBFs expression. This dynamic balance between PIFs and phyB prevents plants from over-activating their responses to cold stress under non-stressful conditions, thereby optimizing their growth and development [145]. Notably, in response to cold stress, the interaction between CBF and PIF3 can stabilize phyB, a plant thermosensor. This stabilization enables phyB to regulate the expression of COR genes while concurrently suppressing the expression of PIF1, PIF4, and PIF5, which are associated with reduced cold tolerance. Consequently, the CBF-PIF3-phyB regulatory module is pivotal in mediating plant responses to cold stress, facilitating a balanced approach to stress adaptation and growth regulation [145].

4.2.5. CESTA and BZR1/BES1

CESTA (CES) is a basic helix–loop–helix brassinosteroid (BR)-dependent transcription factor that facilitates the constitutive expression of CBFs. The presence of brassinosteroids has been demonstrated to enhance CES’s capacity to stimulate CBF transcription, which is vital for activating downstream COR genes that assist plants in tolerating low temperatures. CES directly interacts with the promoter regions of CBF genes, including CBF1, CBF2, and CBF3, thereby enhancing their transcription during periods of cold stress. This binding event is crucial for the rapid induction of CBF, which occurs in response to low temperatures and represents a pivotal aspect of cold acclimation [148,149]. CES operates with other BR signaling pathway components, including BZR1/BES1. These transcription factors have also been identified as critical regulators of CBFs expression via binding to CBF promoters [150]. This observation lends further credence to a coordinated BR signaling network hypothesized to enhance cold tolerance [149]. Moreover, during the cold acclimation process, BR- and CES-dependent COR genes are regulated independently of the CBFs [148]. Furthermore, evidence suggests that BZR1/BES1 plays a role in upregulating CBFs expression within the BR signaling pathway [150].

4.2.6. EIN3

EIN3 is a critical transcription factor that mediates the ethylene signaling pathway in plants. It is pivotal in regulating CBF expression and modulating plant responses to cold stress [151]. EIN3 functions as a negative regulator of CBF expression within the ethylene signaling pathway [131]. It directly binds to the promoters of CBF genes, thereby inhibiting their transcription [152,153]. The suppression of CBF by EIN3 indicates that ethylene may play a role in modulating plants’ response to cold stress. Ethylene signaling, mediated by EIN3, could prioritize growth over stress resistance under certain conditions [154]. The ethylene pathway presents a contrasting scenario, wherein EIN1 suppresses CBFs expression [131].

4.3. MAPK Cascades

The mitogen-activated protein kinase (MAPK) cascade also regulates cold stress responses through a series of phosphorylation events dependent on Ca²⁺ influx into the cytosol [155]. In response to exposure to low temperatures, specific MAPK kinases, including MPK3, MPK4, and MPK6, undergo rapid activation and play pivotal roles in mediating cold responses [156]. The activation of MPK3 and MPK6 results in the phosphorylation of ICE1 and its subsequent degradation, ultimately reducing CBF expression and hypersensitivity to freezing. Therefore, the MKK4/5–MPK3/6 cascade negatively regulates the cold response. Mpk3 and mpk6 mutants exhibit elevated CBF gene expression and enhanced freezing tolerance [156]. It suggests that the normal function of these MAPKs is to suppress CBF expression during cold stress. This negative regulation is vital to prevent the over-activation of the cold acclimation pathway, which can harm plant growth and development. In contrast, the MEKK1–MKK2–MPK4 pathway positively affects cold response [110]. This pathway suppresses MPK3 and MPK6 activities, allowing for controlled CBF expression [110]. The interactions between these diverse MAPK pathways underscore the complex nature of the signaling networks involved in cold acclimation. This results in enhanced expression of CBF2 and subsequently, COR genes [155]. One component of this cascade is the transcription factor CAMTA3 [157].

The ICE–CBF–COR cascade is a critical regulatory mechanism that enables plants to adapt to cold stress and enhances freezing tolerance. Plants undergo physiological and biochemical changes through the coordinated action of ICE, CBF transcription factors, and COR genes (Figure 1). Protective proteins that stabilize cellular structures are produced. The involvement of other transcription factors, such as CAMTA3 and PIFs, highlights the complexity of this signaling pathway in the balance between cold tolerance and growth (Table 2). Overall, this signaling network is essential for cold acclimation. It ensures the survival and proper functioning of plants under low-temperature conditions.

Table 2. Transcription factors involved in cold acclimation.

Transcription Factor	Effect on Cold Acclimatization	Species	References
CBFs (DREBs)	positive regulator	A. thaliana Z. mays O. sativa	[114,115,116,117,118,119,158,159]
ICE1/ICE2	positive regulator	A. thaliana	[120,121,122]
HOS1	negative regulator	A. thaliana	[123,124]
OST1	positive regulator	A. thaliana	[125]
MYB15	negative regulator	A. thaliana	[126]
SIZ1	negative regulator	A. thaliana	[127]
CAMTA3	positive regulator	A. thaliana	[133,134,135,136,137]
CCA1/LHY	positive regulator	A. thaliana O. sativa B. oleracea	[138,139,140,141]
SOC1	negative regulator	A. thaliana	[142,143]
PIFs	positive/negative regulator	A. thaliana	[144,145,146,147]
CESTA	positive regulator	A. thaliana	[148,149]
BZR1/BES1	positive regulator	A. thaliana	[150]
EIN3	negative regulator	A. thaliana	[131,151,152,153,154]

Table 2. Transcription factors involved in cold acclimation.

Transcription Factor	Effect on Cold Acclimatization	Species	References
CBFs (DREBs)	positive regulator	A. thaliana Z. mays O. sativa	[114,115,116,117,118,119,158,159]
ICE1/ICE2	positive regulator	A. thaliana	[120,121,122]
HOS1	negative regulator	A. thaliana	[123,124]
OST1	positive regulator	A. thaliana	[125]
MYB15	negative regulator	A. thaliana	[126]
SIZ1	negative regulator	A. thaliana	[127]
CAMTA3	positive regulator	A. thaliana	[133,134,135,136,137]
CCA1/LHY	positive regulator	A. thaliana O. sativa B. oleracea	[138,139,140,141]
SOC1	negative regulator	A. thaliana	[142,143]
PIFs	positive/negative regulator	A. thaliana	[144,145,146,147]
CESTA	positive regulator	A. thaliana	[148,149]
BZR1/BES1	positive regulator	A. thaliana	[150]
EIN3	negative regulator	A. thaliana	[131,151,152,153,154]

4.4. Non-Coding RNAs

Non-coding RNAs (ncRNAs) constitute a family of RNAs that lack the protein-coding capacity. They generally assist in plant responses to environmental stressors and serve a pivotal function in regulating cold acclimatization and freezing tolerance [160]. They also play a crucial role in the pathways that regulate cold acclimation and freezing tolerance. The current classification of ncRNAs is based on transcript length and distinguishes between two broad categories: short non-coding RNAs (sRNAs) and long non-coding RNAs (lncRNAs).

4.4.1. lncRNAs

Long non-coding RNAs constitute a highly diverse group of transcripts exceeding 200 nucleotides in length, with nuclear and cytoplasmic localization. They lack the protein-coding capacity but act as riboregulators, influencing gene expression through transcriptional and post-transcriptional regulation [161].

The involvement of lncRNAs in regulating cold adaptation has been demonstrated; however, their mechanisms of action remain unclear [162]. Many lncRNAs are expressed in response to cold treatment [163,164]. Targets for lncRNAs are the genes encoding CBFs, LEAs, and WRKY transcription factors, among others [165]. SVALKA is one of the most extensively studied lncRNAs and provides a mechanism for tightly regulating CBF1. The transcription of SVALKA by RNA polymerase II results in a cryptic lncRNA that overlaps with CBF1 on the antisense strand called asCBF1. The transcription of asCBF1 leads to a polymerases II collision, which immediately restricts the production of full-length transcripts of CBF1. The SVALKA-asCBF1 cascade represents the mechanism that effectively regulates CBF1 expression [166]. In contrast, cold-induced lncRNA1 (CIL) has been identified as a positive regulator of cold stress in Arabidopsis. It regulates genes involved in reactive oxygen species (ROS) homeostasis, hormone signal transduction, and glucose metabolism, ultimately enhancing the tolerance of plants to cold [167]. Similarly, intergenic lncRNA XH123, described in cotton, has been demonstrated to function as a positive regulator of cold tolerance [168]. In the model plant Medicago truncatula, two lncRNAs, MtCIR1 and MtCIR2, are associated with the regulatory network of cold tolerance by regulating CBF genes. The functional characterization of MtCIR2, located in the CBF gene, demonstrated that it induces the accumulation of sugars and reduces polysaccharide content within the hemicellulose of the cell wall [169]. Moreover, lncRNAs have been identified in cassava and have been demonstrated to function as positive regulators of cold tolerance. Cold-responsive intergenic lncRNA 1 (CRIR1) regulates several cold-stress-related genes in a CBF-independent pathway, including abscisic stress-ripening protein (ASR) and galactinol synthase (GOLS), as well as the transcription factors MeNAC and MeNFYA. Additionally, CRIR1 has been observed to interact with the cold shock protein 5 (MeCSP5), which has been demonstrated to function as an RNA chaperone [170]. CSP proteins have been shown to prevent the formation of mRNA secondary structures at low temperatures, thereby facilitating translation [171]. Consequently, CRIR1 regulates the expression of COR genes and enhances their translational efficiency [170].

4.4.2. sRNAs

Small regulatory RNA molecules (sRNAs) are pivotal in regulating gene expression, primarily at the post-transcriptional level. Among them, microRNAs (miRNAs) deserve particular attention. As expected, they regulate low-temperature response, cold acclimation, and freezing tolerance. miRNAs regulate gene expression at the post-transcriptional level through the post-transcriptional degradation or translational repression of target messenger RNAs (mRNAs) [172]. In addition, they can induce epigenetic modifications, including DNA and histone methylation [173]. A considerable number of miRNAs have been shown to alter their expression in response to cold temperatures [174,175,176]. In Arabidopsis, the CBF-dependent cold tolerance signaling pathway is positively regulated by miR156, miR397, and miR394 [177,178,179]. However, in O. sativa, OsmiR319 acts as a positive regulator of CBFs, whereas OsmiR535 exerts a negative effect [180,181]. The upregulation of miR408 has been demonstrated to enhance low-temperature tolerance in Arabidopsis [182]. In response to cold stress, the downregulation of miR398 has been observed, leading to an increase in the expression of Cu/Zn superoxide dismutase (SOD), which detoxifies reactive oxygen species (ROS) in Triticum aestivum, Solanum lycopersicum, and Vitis vinifera [183,184,185]. Rice-specific OsmiR1425 has been demonstrated to regulate cold tolerance by modulating the levels of pentatricopeptide repeat (PPR) protein [186,187]. Overexpression of miR402, which targets the demeter-like protein 3(DML3) gene (i.e., 5-methylcytosine DNA glycosidase), a key enzyme involved in the control of DNA methylation status, has been observed to positively influence the Arabidopsis response under low-temperature conditions [174]. Cold-induced miR165/166 expression downregulation in Arabidopsis enhances cold and drought tolerance [188]. The upregulation of miR528 has been observed to enhance chilling tolerance in several plant species, including Arabidopsis, Pinus elliottii, and O. sativa. This phenomenon is thought to result from the indirect inhibition of MYB30 expression. MYB30, in turn, interacts with the β-amylase promoter, resulting in the repression of genes within the BMY family involved in the regulation of starch metabolism. This leads to the increased production of maltose, sucrose, and fructose [189]. It has been suggested that altering the expression levels of the microRNAs (miRNAs) associated with cold-induced genes is critical for successful cryopreservation [190].

4.5. Other Epigenetic Mechanisms

In addition to ncRNAs, the epigenetic regulation of cold stress is also influenced by mechanisms such as DNA methylation and histone modifications. In general, the term “DNA methylation” describes the transfer of a methyl group to the C5 position of a cytosine, which results in the formation of 5-methylcytosine. DNA methylation affects both the promoter regions of genes and their coding regions [191]. Modifying the DNA methylation pattern is a significant mechanism by which gene expression is regulated in response to cold treatment [192]. A reduction in total methylation levels was observed following cold treatment, indicating that cold-responsive genes in genotypes with increased tolerance to this abiotic factor may exhibit a higher potential for activation [193,194]. It has been demonstrated that both methylation and demethylation occur during cold adaptation. A total of 51 genes that exhibited both altered methylation levels and changes in expression following exposure to cold temperatures were identified in the rice genome. This group of genes includes those belonging to the ICE–CBF–COR pathway. One notable observation was the reduction in methylation at the promoter region of the open stomatal 1 (OST1) gene homolog, which interacts with and phosphorylates ICE1 and upregulates its expression [195]. Additionally, cold treatment induces demethylation and the elevated transcriptional activity of the ICE1 and CBF2 genes. This occurs in conjunction with the demethylation of the promoters of genes linked to DNA methylation and the subsequent induction of their expression [196,197,198,199]. The chromatin remodeler PICKLE (PKL) has also been shown to play a pivotal role in cold stress responses in Arabidopsis [200]. Furthermore, PKL functions in collaboration with photoperiod-independent early flowering 1 (PIE1), a SWR1 chromatin remodeling complex member, to deposit H3K27me3 histones at gene loci [201]. The application of cold reduced the levels of H3K27me3 histones in COR15 and galactinol synthase 3 (GOLS3) genes. This suggests that PKL modulates the response to cold stress by modifying histones in COR genes [202]. In addition, the promoters of several COR genes, including COR15 and COR47, are enriched for histone acetylation [203,204,205]. In O. sativa and Z. mays, histone acetylation is also induced in the DREB1 promoter [206,207]. Moreover, the deacetylation of lysine residues on histone subunits H3 and H4 and an increase in the presence of the non-canonical H3 subunit acetylated at the ninth lysine residue (H3K9ac) were observed in Z. mays. A decrease in the DNA methylation and dimethylation of H3K9 accompanied this reaction [206,208].

4.6. Antifreeze Proteins (AFPs)

Antifreeze proteins (AFPs) are specialized proteins, glycopeptides, and peptides that play a crucial role in the cryopreservation of plant tissues by binding to ice and inhibiting its formation and recrystallization, thereby protecting cells from freezing damage. In plants, AFPs have been identified in species that experience freezing conditions, in which they bind to the surface of ice crystals, thereby inhibiting their growth and preventing the formation of large, potentially damaging ice crystals [209]. As uncontrolled ice formation can lead to mechanical damage to cell structures, dehydration, and cell death, this ice-binding activity is essential during the cryopreservation process.

The principal mechanism by which AFPs protect plant cells consists of their interaction with the ice nuclei. AFPs adsorb on the surface of tiny ice crystals, thereby reducing the freezing point of water, a phenomenon known as thermal hysteresis. This binding effectively prevents the growth of ice crystals by creating a barrier that inhibits the addition of water molecules to the ice network [210]. Moreover, AFPs can modify the ice crystal morphology, facilitating the formation of smoother, less damaging shapes. This reduces the risk of physical damage to cell membranes and organelles [211]. It is especially beneficial during cryopreservation because it preserves the integrity of the cellular structure, which is critical for the viability of the tissues at the time of thawing.

In plants, the most common types of AFP proteins are pathogenesis-related (PR) proteins [212]. Chitinase-like proteins have been identified in several species, including Secale cereale, Solanum dulcamara, Chimonanthus praecox, Bromus inermis, Hevea brasiliensis, and Picea abies [213,214,215,216,217,218]. While thaumatin-like IBPs have been identified in T. aestivum, β-1,3 glucanase, and chitinase-like IBPs have been observed in Raphanus sativus [219,220,221]. Furthermore, IBPs displaying structural homology with polygalacturonase inhibitor proteins (PGIPs) were identified in Daucus carota and Hippophaes rhamnoideum [222,223,224]. In addition to identifying PR proteins in plants, other types of AFPs have been documented. Among these, STHP-64 has been identified in Solanum dulcamara, revealing sequence homology with the transcription factor WRKY [225]. Some dehydrins also exhibit AFP functions. For example, PCA60 has been identified in Prunus persica [226]. AFPs are efficiently secreted into the apoplastic space. Therefore, they are detected in the growth medium rather than in cells cultured in vitro [215,227].

4.7. Late Embryogenesis Abundant (LEA) Proteins

Late embryogenesis abundant (LEA) proteins are a group of proteins that play critical roles in plant tissue survival during cold and freezing exposure. They were first identified during the embryo maturation and desiccation processes [228]. LEAs are crucial stress-responsive proteins. They accumulate in response to desiccation, cold, and osmotic stress commonly encountered during cryopreservation [229]. Dehydrins (DHNs) belonging to the LEA family play a role in the anti-aggregation of enzymes and in protecting cell structures during cold stress and dehydration [230]. They are highly hydrophilic and intrinsically disordered, which allows them to remain soluble in a dehydrated state and to interact with various cellular components to protect them from damage [231,232]. Critical to their protective function during the freezing and thawing processes of cryopreservation is their ability to stabilize the cellular matrix in a glassy state, as well as proteins, membranes, and other structures under stress [232,233]. DHNs belong to the LEA II group and protect plant cells from dehydration damage caused by drought, cold, salinity, and osmotic stress [234,235]. During freezing, ice formation in the extracellular spaces leads to a decrease in the water potential. It causes water to move out of the cell, resulting in cellular dehydration. Dehydration can lead to membrane phase transitions, causing membranes to leak or fuse improperly, which can be fatal to cells. Dehydrins help prevent these deleterious effects by binding to membrane phospholipids, stabilizing the membrane structure, and preventing phase transitions that lead to leakage [236,237,238]. In addition, dehydrins can interact with lipid bilayers to form a protective layer around the membrane. This mitigates the effects of cold-induced dehydration [239]. Numerous DHNs associated with the cold response have been identified, including DHN24, P-80, LTI30, WCS120, and WCOR410 [236,240,241,242].

4.8. Chaperones

Molecular chaperones are essential proteins that assist in the folding, assembling, and stabilizing of other proteins, particularly under stress conditions. They are, therefore, critical for plant cryopreservation. Chaperones are a family of unrelated proteins that bind specifically to the surface of other proteins. This prevents the formation of non-functional structures resulting from abnormal interactions [243]. Chaperones include heat shock proteins (HSPs), which stabilize proteins and membranes. In addition, HSPs can participate in protein refolding [244]. Based on molecular weight, amino acid sequence homology, and function, five major classes of HSPs have been identified in plants. These classes include HSP100, HSP90, HSP70, HSP60, and the small heat shock proteins (sHSPs) [245,246]. Levels of HSP70, HSP90, chaperonin 20 (HSP60), and HSP17.4C1 (sHSP) are increased in Arabidopsis upon exposure to low temperatures. The overexpression of specific chaperones in genetically modified plants has increased their tolerance to cryogenic stress and survival after cryopreservation [247,248,249,250]. This group also includes RNA chaperones ubiquitous in all living organisms. They facilitate the proper folding of RNA molecules during RNA metabolism. In Arabidopsis, the RNA chaperone AtCSP2 plays a role in adapting to cold and future development [251]. In addition, the glycine-rich RNA-binding proteins AtGRP2 and AtGRP7 have been observed to provide cold and freezing tolerance to plants. They also exhibit RNA chaperone activity during cold adaptation [252,253].

4.9. Osmoprotectants

In response to low temperatures, plants accumulate a variety of soluble compounds. These include free sterols, sterol glucosides, acylated sterols, glucosides, arabinoxylans, cerebrosides, raffinose and other soluble sugars, amino acids (alanine, glycine, proline, and serine), polyamines, and betaines [254]. Raffinose family oligosaccharides (RFOs), including raffinose and stachyose, accumulate in dormant tissues. They provide tolerance to cold and drought stress [255]. In Castanea sativa, it has been shown that low temperature induces the upregulation of dual-specificity protein phosphatase 4 (DSP4). DSP4 most likely increases oligosaccharide synthesis during winter dormancy through starch dephosphorylation and degradation [256]. Genes encoding galactinol synthase (GolS), which catalyzes the first step in synthesizing RFOs, are upregulated in the dormant buds of woody perennials [257,258]. T. aestivum membrane sterols are essential in mitigating plant responses to low temperatures [259]. In response to abiotic stress, proline plays a role in osmotic regulation, as well as in the stabilization of membrane proteins, the induction of stress gene expression, and the scavenging of ROS. It also regulates cytosolic acidity, maintains the NAD/NADH ratio, increases photosystem II photochemical efficiency, and reduces lipid peroxidation [260]. Increased endogenous proline levels in response to cold stress have been observed in species that exhibit natural cold tolerance. The stabilization of transcriptional and translational mechanisms is likely facilitated by glycine betaine. There is also a correlation between its levels and cold tolerance [261].

5. Challenges and Future Directions

Cryopreservation offers significant potential for the long-term conservation of plant genetic resources. However, the natural diversity of species, with unique physiological, biochemical, and genetic characteristics, represents a significant challenge in standardizing cryopreservation protocols. It has been observed that some species, and even genotypes within a species, exhibit greater resilience to cryopreservation procedures, suggesting a reduced probability of survival following freezing and thawing. Notably, a lack of tolerance to cryopreservation is widespread among tropical and subtropical species with no natural tolerance to cold and desiccation. Moreover, the tissue’s developmental stage and physiological state at the time of cryopreservation significantly impact viability following thawing. Immature or unacclimated tissues are typically more vulnerable to damage. This highlights the necessity for precise timing and pre-treatment strategies, such as cold acclimation or the application of osmotic stress prior to cryopreservation. In contrast, the effective vitrification of tissues without producing the side effects associated with cytotoxicity represents a significant challenge, particularly in the case of large and complex explants such as buds, meristems, or embryos. Achieving consistent and reproducible results is also a significant challenge, particularly when scaling up protocols for implementation in gene banks or conservation programs. Variable and unpredictable cryopreservation efficiencies are influenced by several factors, including the genetic makeup of the material, handling of explants, expertise of the personnel involved, and equipment available in the cryopreservation laboratory.

In the future, the further development and integration of omics technologies with cryopreservation research can revolutionize the field by providing a holistic view of the biological processes involved. By combining data from genomics, transcriptomics, proteomics, and metabolomics, scientists can construct comprehensive plant cryotolerance models, leading to the identification of critical regulatory networks and molecular pathways. This system’s biological approach can uncover new targets for genetic engineering or chemical intervention, paving the way for developing next-generation cryopreservation techniques. Furthermore, the prediction of cryopreservation outcomes based on specific genetic, proteomic, or metabolic profiles could be facilitated by applying bioinformatics, machine learning, and deep neural networks to omics data. Predictive models can be developed to assess the cryotolerance of plant tissues before cryopreservation, allowing for the selection of the most appropriate methods and treatments for each species or genotype. Implementing such a customized approach to cryopreservation could significantly improve its efficiency.

6. Conclusions

Cryopreservation is an essential technique for the long-term conservation of plant genetic resources, and it provides a viable solution for preserving plant diversity in the face of global environmental challenges. This process depends on understanding and optimizing the molecular mechanisms underlying plant freezing tolerance. Key factors contributing to successful cryopreservation include cold acclimation, which enhances the survival of plant tissues during freezing, and the regulation of cold-responsive genes via pathways such as the ICE–CBF–COR signaling cascade. Transcription factors, non-coding RNAs, and epigenetic modifications refine the ability of plants to withstand cryogenic conditions.

However, the effectiveness of cryopreservation varies across species and genotypes, particularly in tropical and subtropical plants that lack natural cold tolerance. This variability highlights the need for species-specific protocols and pre-treatment strategies. The integration of advanced omics technologies promises to address these challenges by providing a comprehensive understanding of the biological processes involved, potentially leading to more effective and standardized cryopreservation methods.

In summary, cryopreservation is a powerful tool for plant conservation. However, its success depends on continued research and technological advances to overcome limitations and ensure its broad application to diverse plant species.