Method for preserving germplasm resources based on salt stress memory

Abstract

The application provides a germplasm resource preservation method based on salt stress memory, which is used for cryopreservation of in vitro cell lines of salt-tolerant plants, and comprises the following steps: S1, transferring the cell line into a liquid medium containing 45-55 mmol / L sodium chloride, and culturing at 23-27 DEG C for 42-54 h to perform salt stress memory pretreatment; S2, transferring the cell line after the salt stress memory pretreatment into an anti-freezing protective agent for equilibrium treatment for 15-30 min; the anti-freezing protective agent comprises the following components at the following concentrations: dimethyl sulfoxide 8-15 v / v%, ethylene glycol 5-10 v / v%, sucrose 0.3-0.5 mol / L, trehalose 2-3 w / v%, and calcium chloride 8-15 mmol / L; S3, reducing the temperature of the cell line after the equilibrium treatment to -30 DEG C to -50 DEG C at a rate of 0.5-3 DEG C / min, and then putting it into liquid nitrogen for preservation. The application activates the endogenous protection mechanism in the cell by salt stress memory pretreatment before freezing; and an exogenous physical and chemical barrier is constructed by the anti-freezing protective agent. A double synergistic protection system of endogenous defense and exogenous protection is constructed. The systemic stress defense state of the cell is established, and the survival rate of the preservation is improved.

Description

Technical Field

[0001] This application relates to the field of ultra-low temperature preservation technology for plant germplasm resources, and in particular to a method for preserving germplasm resources based on salt stress memory. Background Technology

[0002] Forest and grassland germplasm resources are important strategic resources for national ecological security and the material foundation for the sustainable development of forestry and grassland industries. Due to the increasing area of ​​saline-alkali land year by year, a large number of high-quality forest and grassland germplasm resources are facing survival threats. It is urgent to preserve saline-alkali resistant germplasm resources through germplasm resource conservation methods to support ecological restoration and the utilization of saline-alkali land.

[0003] Existing forest and grassland germplasm resources are preserved using cryopreservation technology. First, explants are induced into cell lines. Then, sucrose pre-culture is used to reduce intracellular free water content through hypertonic dehydration. After pretreatment, a cryoprotectant with dimethyl sulfoxide (DMSO) as the active component is used for equilibration. This cryoprotectant is designed based on an animal cell model, and its formulation consists of DMSO alone, or a combination of DMSO, ethylene glycol, and sucrose as the base components. After equilibration, a cooling process is performed, followed by liquid nitrogen preservation.

[0004] However, sucrose pretreatment is a physical dehydration process. Although it can reduce ice crystal damage by reducing intracellular free water, it cannot establish a systematic resistance defense state, resulting in insufficient cell tolerance under subsequent severe cryogenic freezing stress, and thus low survival rate. Summary of the Invention

[0005] The purpose of this application is to provide a germplasm resource preservation method based on salt stress memory, so as to establish a systematic stress resistance defense state of cells and improve the survival rate of cryopreservation.

[0006] To address the aforementioned technical problems, the embodiments of this application provide the following technical solutions: This application provides a germplasm resource preservation method based on salt stress memory for cryopreservation of salt-tolerant plant cell lines, comprising the following steps: S1 The cell line was transferred into a liquid culture medium containing 45-55 mmol / L sodium chloride and cultured at 23-27℃ for 42-54 h to perform salt stress memory pretreatment. S2. The cell line pretreated with salt stress memory is transferred into a cryoprotectant and equilibrated for 15-30 min. The cryoprotectant contains the following components at the following concentrations: dimethyl sulfoxide 8-15 v / v%, ethylene glycol 5-10 v / v%, sucrose 0.3-0.5 mol / L, trehalose 2-3 w / v%, and calcium chloride 8-15 mmol / L. S3 cools the equilibrated cell line to -30°C to -50°C at a rate of 0.5-3°C / min, and then stores it in liquid nitrogen.

[0007] In some embodiments, the method further includes the following steps prior to step S1: Plant explants were collected, disinfected, and then inoculated into an induction medium for callus induction culture. The callus tissue was sequentially transferred to selection media with sodium chloride concentrations of 45-55 mmol / L, 95-105 mmol / L and 145-155 mmol / L for stepwise selection and culture, with each stage lasting 14-21 days, to obtain salt-tolerant callus tissue. The salt-tolerant callus was proliferated and cultured to obtain the cell line.

[0008] In some embodiments, the salt stress memory preprocessing includes: S101 The cell line is transferred into the liquid culture medium containing 45-55 mmol / L sodium chloride and cultured at 23-27°C for 29-31 h; S102 The cell line treated in step S101 is cultured in the same culture medium at 6-12℃ for 10-18 h. S103 The cell line treated in step S102 is cultured in the same culture medium at 23-27°C for 3-5 h.

[0009] In some embodiments, the salt stress memory preprocessing includes: S111 The cell line was transferred into the liquid culture medium containing 45-55 mmol / L sodium chloride and 50-200 mmol / L osmotic adjustment inducer and cultured at 23-27°C for 22-30 h. S112 Add an oxidative stress inducer to the liquid culture medium after culturing in step S111 to a concentration of 50-200 μmol / L, and continue culturing at 23-27℃ for 20-24 h. The osmotic conditioning inducer includes at least one of sorbitol or mannitol; the oxidative stress inducer includes at least one of hydrogen peroxide or abscisic acid.

[0010] In some embodiments, the method further includes the following steps after step S1 and before step S2: The first DNA methylation level of the promoter region of a specific stress-response gene in the cell line before the salt stress memory pretreatment is obtained, and the second DNA methylation level of the promoter region of the specific stress-response gene in the cell line after the salt stress memory pretreatment is obtained; the specific stress-response gene includes at least one of a proline synthase gene, a trehalose synthase gene, or a stress protection protein gene. If the second DNA methylation level decreases to a preset threshold relative to the first DNA methylation level, it is determined that salt stress memory has been formed, and the process proceeds to step S2. If the second DNA methylation level does not decrease to the preset threshold relative to the first DNA methylation level, the salt stress memory pretreatment time is extended by 4-8 h, and / or the sodium chloride concentration is increased to 55-65 mmol / L.

[0011] In some embodiments, a mapping relationship between the second DNA methylation level and the concentration of the dimethyl sulfoxide in a baseline cell line is established in advance; In step S2, the concentration of dimethyl sulfoxide is determined by querying the mapping relationship based on the second DNA methylation level of the cell line.

[0012] In some embodiments, the mapping relationship is pre-established for cell lines of different plant species; and / or, the mapping relationship is pre-established for cell lines of different parts of the same plant species.

[0013] In some embodiments, step S2 further includes: When the second DNA methylation level is higher than the first threshold, the concentration of dimethyl sulfoxide in the cryoprotectant is increased; When the second DNA methylation level is lower than the second threshold, the concentration of dimethyl sulfoxide is reduced, while the concentrations of trehalose and sucrose are increased simultaneously; wherein, for every 1 v / v% decrease in dimethyl sulfoxide, the concentration of trehalose is increased by 0.5-1.5 w / v%, and the concentration of sucrose is increased by 0.05-0.15 mol / L; the second threshold is lower than the first threshold.

[0014] In some embodiments, each batch of the cell line is preserved in liquid nitrogen in the form of at least three backup samples; Following step S3, the method further includes: S4 Every 6 months, one of the backup samples is recovered and its third DNA methylation level is detected. If the third DNA methylation level is within a preset tolerance range relative to the second DNA methylation level, then the backup samples in this batch will continue to be stored. If the third DNA methylation level exceeds the preset tolerance range relative to the second DNA methylation level, other backup samples from the same batch are revived, subcultured for 2-3 generations in a proliferation medium containing 25-35 mmol / L sodium chloride, with each generation cultured for 14-21 days, and then re-preserved according to steps S1 to S3.

[0015] In some embodiments, a correspondence is established beforehand between the content of endogenous osmotic regulators in a baseline cell line and the concentration of the dimethyl sulfoxide; the endogenous osmotic regulators include at least one of proline or trehalose; In step S2, the content of the endogenous osmotic regulator in the cell line is detected, and the concentration of dimethyl sulfoxide is determined by looking up the corresponding relationship based on the content.

[0016] Compared to existing technologies, the germplasm resource preservation method based on salt stress memory provided in this application... Salt stress memory pretreatment with 45-55 mmol / L sodium chloride was used. This non-lethal salt stress served as the first messenger (signal trigger) for the isolated cell lines of salt-tolerant plants, activating the intracellular mitogen-activated protein kinase (MAPK) cascade signaling pathway, namely MAPKKK → MAPKK → MAPK, which amplifies and transmits signals through stepwise phosphorylation.

[0017] Once activated MAPKs enter the cell nucleus, they induce DNA demethylation in the promoter regions of specific stress-responsive genes, relieving the inhibition of transcription of these genes and thereby initiating the upregulation of expression of various downstream stress-resistance genes, including genes synthesizing osmotic regulatory substances and stress-protective protein genes.

[0018] Among the genes involved in the synthesis of osmotic regulators, upregulation of the Δ1-pyrroline-5-carboxylic acid synthase gene (P5CS gene) promoted proline synthesis, while upregulation of the trehalose-6-phosphate synthase gene (TPS gene) and the trehalose-6-phosphate phosphatase gene (TPP gene) promoted intracellular trehalose accumulation. For stress protection genes, upregulation of the dehydrin gene and the late embryogenesis-rich protein gene (LEA protein gene) increased the content of protective proteins.

[0019] This process enables cells to establish a "stress memory" state before entering the ultra-low temperature environment, significantly increasing intracellular osmotic regulatory substances and protective proteins, establishing a systemic anti-stress defense state for the cells, thereby greatly improving the cells' tolerance to subsequent severe freezing stress.

[0020] Trehalose molecules in cryoprotectants possess excellent water-retention capabilities. During cooling, they form a high-viscosity amorphous matrix on the cell membrane surface. This matrix stabilizes the water molecule layer on the membrane surface through a hydrogen bond network, effectively preventing the phospholipid bilayer from undergoing a phase transition from a liquid crystal phase to a gel phase under ultra-low temperature conditions. This avoids membrane structure collapse and permeability changes caused by membrane phase transitions, ensuring the integrity of the cell membrane during cooling and thawing. Meanwhile, Ca... 2+ Carrying a positive charge, it acts as a "stabilizer" for cell wall structure, combining with free carboxyl groups in the pectin layer of the cell wall to form an "egg-box" structure (calcium pectate), significantly enhancing the mechanical strength and rigidity of the cell wall and effectively resisting physical compression caused by ice crystals; simultaneously, Ca... 2+ Through electrostatic interactions, it forms ion bridges with negatively charged membrane phospholipid heads (such as phosphatidylserine), further enhancing the mechanical strength and stability of the cell membrane. On the other hand, Ca... 2+ Entering the cell as a second messenger, it activates the calcium signaling pathway and synergizes with the MAPK cascade signaling pathway to further enhance the expression of genes synthesizing osmotic regulators and stress-protective proteins. In other words, the physical barrier protection provided by trehalose in the cryoprotectant and the chemical structural stability provided by calcium chloride complement each other, achieving comprehensive reinforcement of the cell wall-cell membrane bilayer structure.

[0021] In summary, this invention activates endogenous cellular protective mechanisms before freezing through salt stress memory pretreatment, forming a pre-armed defense; and constructs an exogenous physicochemical barrier through cryoprotectants, achieving a relay reinforcement. This constructs a dual synergistic protection system of endogenous defense and exogenous protection. It achieves comprehensive protection of cells from the inside out, establishes a systemic stress-resistant defense state of cells, and significantly improves the survival rate of germplasm resources during cryopreservation. Attached Figure Description

[0022] The above and other objects, features, and advantages of exemplary embodiments of this application will become readily understood by reading the following detailed description with reference to the accompanying drawings. In the drawings, several embodiments of this application are illustrated by way of example and not limitation, with the same or corresponding reference numerals denoteing the same or corresponding parts, wherein: Figure 1 A schematic diagram illustrating the process of a germplasm resource preservation method based on salt stress memory is shown. Detailed Implementation

[0023] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

[0024] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application shall have the ordinary meaning as understood by one of ordinary skill in the art to which this application pertains.

[0025] like Figure 1 As shown, this application provides a method for preserving germplasm resources based on salt stress memory, used for the cryopreservation of isolated cell lines of salt-tolerant plants, comprising the following steps: S1 The cell line was transferred into a liquid culture medium containing 45-55 mmol / L sodium chloride and cultured at 23-27℃ for 42-54 h to perform salt stress memory pretreatment.

[0026] Specifically, the salt-tolerant plants described in this application include, but are not limited to, sheepgrass, alfalfa, crested wheatgrass, alkali grass, or wild barley. The in vitro cell line refers to a population of plant cells with vigorous division and totipotency obtained through tissue culture techniques, existing in the form of suspension cultured cells or callus tissue. In vitro cell lines have advantages such as consistent genetic background, rapid proliferation, and ease of handling, making them an important form of germplasm resource preservation. Cryopreservation refers to preservation in a liquid nitrogen (-196℃) environment, at which cell metabolic activity almost ceases, enabling long-term preservation of germplasm resources.

[0027] The liquid culture medium used in this application can be MS liquid medium, B5 liquid medium, N6 liquid medium, or other plant tissue culture liquid media known in the art. The liquid culture medium can be supplemented with suitable plant growth regulators (such as 2,4-dichlorophenoxyacetic acid, 6-benzylaminopurine, etc.) and carbon sources (such as sucrose, glucose, etc.) according to the cell line type and culture requirements. Liquid culture media can facilitate more uniform and rapid transmission of salt stress signaling molecules, which is beneficial for cells to respond quickly to salt stress signals.

[0028] The concentration of sodium chloride in the liquid culture medium can be 45 mmol / L, 50 mmol / L, or 55 mmol / L, etc.; the incubation temperature can be 23℃, 25℃, or 27℃, etc.; and the incubation time can be 42 h, 47 h, or 54 h, etc. In a preferred embodiment of this application, the concentration of sodium chloride in the liquid culture medium can be 50 mmol / L, the incubation temperature is 25±1℃, and the incubation time is 48 h.

[0029] This application uses 45-55 mmol / L sodium chloride for salt stress memory pretreatment. This non-lethal salt stress acts as the first messenger (signal trigger) of the isolated cell line of salt-tolerant plants, activating the intracellular MAPK cascade signaling pathway, namely the MAPKKK→MAPKK→MAPK cascade signaling pathway, which achieves signal amplification and transmission through stepwise phosphorylation.

[0030] Once activated MAPKs enter the cell nucleus, they induce DNA demethylation in the promoter regions of specific stress-responsive genes, relieving the inhibition of transcription of these genes and thereby initiating the upregulation of expression of various downstream stress-resistance genes, including genes synthesizing osmotic regulatory substances and stress-protective protein genes.

[0031] Among the genes involved in the synthesis of osmotic regulators, upregulation of the P5CS gene promoted proline synthesis, while upregulation of the TPS and TPP genes promoted the accumulation of intracellular trehalose. For stress protection genes, upregulation of the dehydrin and LEA protein genes increased the content of protective proteins.

[0032] This process enables cells to establish a "stress memory" state before entering the ultra-low temperature environment, significantly increasing intracellular osmotic regulatory substances and protective proteins, establishing a systemic anti-stress defense state for the cells, thereby greatly improving the cells' tolerance to subsequent severe freezing stress.

[0033] In some embodiments, the method further includes the following steps prior to step S1: Plant explants were collected, disinfected, and then inoculated into an induction medium for callus induction culture. The callus tissue was sequentially transferred to selection media with sodium chloride concentrations of 45-55 mmol / L, 95-105 mmol / L and 145-155 mmol / L for stepwise selection and culture, with each stage lasting 14-21 days, to obtain salt-tolerant callus tissue. The salt-tolerant callus was proliferated and cultured to obtain the cell line.

[0034] Specifically, explants can be collected from healthy, disease-free plants, including but not limited to young leaves, stem segments, petioles, or root tips. After collection, the explants are disinfected with ethanol or sodium hypochlorite, followed by rinsing 3-5 times with sterile deionized water to remove residual disinfectant. The disinfected explants are then ready for callus induction culture.

[0035] The induction medium can be based on MS medium, supplemented with 1.0-2.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5-1.0 mg / L 6-benzylaminopurine (6-BA), 30 g / L sucrose, and 7 g / L agar, and adjusted to pH 5.7-5.9. The callus induction culture conditions can be: temperature 24-26℃, light intensity 1500-2000 lux, and photoperiod 12 h / d.

[0036] After callus tissue has grown, a stepwise selection culture is performed. The callus tissue is cut into small pieces and successively transferred to selection media with sodium chloride concentrations of 45-55 mmol / L, 95-105 mmol / L, and 145-155 mmol / L for culture. In a preferred embodiment of this application, the sodium chloride concentrations for the three-stage gradient selection are 50 mmol / L, 100 mmol / L, and 150 mmol / L, respectively. The selection media formulation is the same as the induction media, except that corresponding concentrations of sodium chloride are added. The culture time for each stage is 14-21 days, for example, 14 days, 18 days, or 21 days, with 18 days being preferred. During the three-stage gradient selection process, after each stage, the surviving callus tissue must be removed from the old culture medium and transferred to freshly prepared selection media containing the sodium chloride concentration of the next stage. Through this gradient, non-lethal salt stress environment, poorly growing or browned sensitive cells are gradually eliminated, and salt-tolerant callus tissue with continuous vigorous growth is selected.

[0037] Finally, the salt-tolerant callus obtained through screening was cultured for proliferation to obtain the isolated cell line of the salt-tolerant plant.

[0038] Through the above three-stage gradient stress screening, cells with poor salt tolerance can be effectively eliminated, and salt-tolerant cell lines with stable genetic characteristics can be screened out. This ensures that the cell population has a uniform salt tolerance base before entering the salt stress memory pretreatment, thereby improving the effect of subsequent cryopreservation and batch-to-batch stability.

[0039] Furthermore, in some embodiments of this application, the salt stress memory preprocessing includes: S101 The cell line is transferred into the liquid culture medium containing 45-55 mmol / L sodium chloride and cultured at 23-27°C for 29-31 h. Preferably, the sodium chloride concentration in this stage is 50 mmol / L, the culture temperature is 25±1°C, and the culture time is 30 h. This stage is the salt stress induction stage, where non-lethal salt stress activates the intracellular MAPK cascade signaling pathway, initiating the expression of salt stress response genes.

[0040] S102 The cell line treated in step S101 is cultured in the same culture medium at 6-12°C for 10-18 h. Preferably, the culture temperature for this stage is 8°C and the time is 12 h. Since salt stress and low temperature stress share part of the MAPK signaling pathway, low temperature stress pretreatment can form a synergistic additive effect with salt stress signaling, further inducing the expression of cold response genes (COR genes), while maintaining and further enhancing the osmotic regulatory substances and protective proteins accumulated in the cells under low temperature conditions. Through the temporal superposition of salt stress and low temperature stress, the cell's stress memory is significantly enhanced, and the expression of cryogenic apoptosis genes is pre-regulated, making it less likely for cells to initiate programmed cell death during subsequent ultra-low temperature preservation.

[0041] S103. The cell line treated in step S102 is cultured in the same culture medium at 23-27°C for 3-5 hours. Preferably, the culture time in this stage is 4 hours. This stage provides the cells with a short buffer period, allowing them to adjust their metabolic state before entering the freezing process, gradually restore their metabolic activity, and simultaneously allow the expression products of stress memory-related genes (such as proline, trehalose, dehydrogenase, LEA protein, etc.) to accumulate and function fully.

[0042] The above-mentioned multi-stress synergistic pretreatment, through the temporal superposition of salt stress and low temperature stress, utilizes the cross-adaptation mechanism of plant cells to different abiotic stresses (i.e., training under one stress enhances tolerance to another stress), making the stress memory of cells more stable and lasting, and further improving the cells' tolerance to subsequent ultra-low temperature freezing stress, thereby obtaining a higher ultra-low temperature preservation survival rate.

[0043] Furthermore, in other embodiments of this application, the salt stress memory preprocessing includes: S111 The cell line was transferred into the liquid culture medium containing 45-55 mmol / L sodium chloride and 50-200 mmol / L osmotic inducer and cultured at 23-27°C for 22-30 h. During this stage, sodium chloride acts as a signal trigger to initiate a stress response, while the osmotic inducer simulates a drought or hyperosmotic environment, inducing cells to rapidly synthesize and accumulate osmotic substances such as proline and trehalose by lowering the water potential of the culture medium. This allows the cells to establish a preliminary osmotic defense state, laying the material basis for subsequent antifreeze treatment.

[0044] In step S112, an oxidative stress inducer is added to the liquid culture medium after culturing in step S111 to a concentration of 50-200 μmol / L, and the cells are cultured at 23-27°C for 20-24 h. The osmotic regulation inducer includes at least one of sorbitol or mannitol; the oxidative stress inducer includes at least one of hydrogen peroxide or abscisic acid. At this stage, the exogenously added oxidative stress inducer acts as a signaling molecule, mimicking the oxidative burst signal generated during cryopreservation, activating intracellular antioxidant enzyme systems (such as SOD, POD, and CAT) and related signal transduction pathways. This "pre-oxidative training" enables cells to establish oxidative stress memory, significantly improving their ability to cope with reactive oxygen species (ROS) attacks during subsequent cryopreservation and effectively preventing oxidative damage during freezing.

[0045] By employing multidimensional stress coupling pretreatment, which involves the temporal superposition of salt stress, osmotic stress, and oxidative stress, and utilizing the cross-communication mechanism between stress signals, comprehensive activation of different stress resistance pathways was achieved. During this process, cells not only accumulated a large amount of osmotic regulatory substances but also established a robust antioxidant defense system, making the cells' stress memory state more comprehensive and stable. This significantly improved the survival rate and regenerative growth capacity of germplasm resources after cryopreservation.

[0046] S2. The cell line pretreated with salt stress memory is transferred into a cryoprotectant and equilibrated for 15-30 min. The cryoprotectant contains the following components at the following concentrations: dimethyl sulfoxide 8-15 v / v%, ethylene glycol 5-10 v / v%, sucrose 0.3-0.5 mol / L, trehalose 2-3 w / v%, and calcium chloride 8-15 mmol / L.

[0047] In a preferred embodiment of this application, the concentrations of each component in the antifreeze are: dimethyl sulfoxide 10-14 v / v%, ethylene glycol 6-10 v / v%, sucrose 0.35-0.45 mol / L, trehalose 2-2.5 w / v%, and calcium chloride 9-12 mmol / L.

[0048] As a more preferred embodiment of this application, the concentrations of each component in the antifreeze are: dimethyl sulfoxide 12 v / v%, ethylene glycol 8 v / v%, sucrose 0.4 mol / L, trehalose 2 w / v%, and calcium chloride 10 mmol / L.

[0049] Specifically, in this composite cryoprotectant, dimethyl sulfoxide and ethylene glycol act as permeable protectants, rapidly penetrating into cells to lower the freezing point of intracellular fluid and reduce the formation of intracellular ice crystals. Sucrose acts as a non-permeable protectant, primarily providing extracellular osmotic pressure balance to prevent cell rupture due to drastic osmotic pressure changes during freezing. Trehalose and calcium chloride are specific components designed for the characteristics of the plant cell wall-cell membrane bilayer structure. Trehalose molecules have excellent water retention capacity; during cooling, they form a high-viscosity amorphous matrix on the cell membrane surface. This matrix stabilizes the water molecule layer on the membrane surface through a hydrogen bond network, effectively preventing the phospholipid bilayer from undergoing a phase transition from the liquid crystal phase to the gel phase under ultra-low temperature conditions. This avoids membrane structure collapse and permeability changes caused by membrane phase transitions, ensuring the integrity of the cell membrane during cooling and thawing. Meanwhile, calcium chloride... 2+ Carrying a positive charge, it acts as a "stabilizer" for cell wall structure, combining with free carboxyl groups in the pectin layer of the cell wall to form an "egg-box" structure (calcium pectate), significantly enhancing the mechanical strength and rigidity of the cell wall and effectively resisting physical compression caused by ice crystals; simultaneously, Ca... 2+ Through electrostatic interactions, it forms ion bridges with negatively charged membrane phospholipid heads (such as phosphatidylserine), further enhancing the mechanical strength and stability of the cell membrane. On the other hand, Ca... 2+ Entering the cell as a second messenger, it activates the calcium signaling pathway and synergizes with the MAPK cascade signaling pathway to further enhance the expression of genes synthesizing osmotic regulators and stress-protective proteins. In other words, the physical barrier protection provided by trehalose in the cryoprotectant and the chemical structural stability provided by calcium chloride complement each other, achieving comprehensive reinforcement of the cell wall-cell membrane bilayer structure.

[0050] After the above-mentioned balancing treatment with cryoprotectants, a full osmotic balance was achieved inside and outside the cells, each component of the cryoprotectant played its full role, and the cell membrane structure was effectively reinforced, preparing for subsequent programmed cooling preservation.

[0051] In summary, this invention activates endogenous cellular protective mechanisms before freezing through salt stress memory pretreatment, forming a pre-armed defense; and constructs an exogenous physicochemical barrier through cryoprotectants, achieving a relay reinforcement. This constructs a dual synergistic protection system of endogenous defense and exogenous protection. It achieves comprehensive protection of cells from the inside out, establishes a systemic stress-resistant defense state of cells, and significantly improves the survival rate of germplasm resources during cryopreservation.

[0052] Furthermore, in some embodiments, the method further includes the following steps after step S1 and before step S2: The first DNA methylation level of the promoter region of a specific stress-response gene in the cell line before the salt stress memory pretreatment is obtained, and the second DNA methylation level of the promoter region of the specific stress-response gene in the cell line after the salt stress memory pretreatment is obtained; the specific stress-response gene includes at least one of a proline synthase gene, a trehalose synthase gene, or a stress protection protein gene. If the second DNA methylation level decreases to a preset threshold relative to the first DNA methylation level, it indicates that salt stress has successfully activated DNA demethylation in the promoter region of a specific stress response gene, and then it is determined that salt stress memory has been formed, proceeding to step S2; If the second DNA methylation level does not decrease to the preset threshold relative to the first DNA methylation level, the salt stress memory pretreatment time is extended by 4-8 h, and / or the sodium chloride concentration is increased to 55-65 mmol / L. After the enhancement treatment, the DNA methylation level is re-tested until it decreases to the preset threshold. If the preset threshold is not reached after 3 enhancement treatments, the cell line is determined to be unsuitable for this method.

[0053] Specifically, for ease of distinction, this application refers to the methylation level obtained before pretreatment as the first DNA methylation level, and the methylation level obtained after pretreatment as the second DNA methylation level. The DNA methylation level can be detected using any method known in the art, such as methylation-sensitive amplification polymorphism (MPA), bisulfite sequencing, or bisulfite sequencing analysis.

[0054] The preset threshold can be set according to the tolerance and gene expression characteristics of the specific cell line. This preset threshold can be determined through preliminary experiments before the formal application of this method: Cell lines from the same source as the cell line to be preserved are used, and different salt concentrations and / or different treatment times are set for salt stress memory pretreatment. The DNA methylation level of the promoter region of the specific stress-response gene in each treatment group is detected, and subsequent cryopreservation is performed. The survival rate of each group is calculated. The methylation level decrease corresponding to a satisfactory survival rate (e.g., above 70%) is used as the preset threshold. For example, the preset threshold can be a reduction in the DNA methylation level of the promoter region of the specific stress-response gene to 60%-80% compared to the untreated control.

[0055] DNA methylation is an important epigenetic mechanism for the formation of stress memory in plants. Salt stress can induce DNA demethylation in the promoter regions of specific stress-responsive genes (such as P5CS and TPS), thereby relieving the inhibition of transcription of these genes and promoting the synthesis of osmotic regulatory substances such as proline and trehalose. DNA methylation levels are generally negatively correlated with gene expression; DNA demethylation can promote proline accumulation by upregulating P5CS gene expression. Therefore, by detecting changes in DNA methylation levels in the promoter regions of specific stress-responsive genes, it is possible to objectively and quantitatively determine whether salt stress memory has been successfully established. By introducing epigenetic indicators as a quality control method, precise monitoring and dynamic adjustment of the salt stress memory formation process have been achieved, ensuring that cells entering the cryopreservation process are in optimal physiological defense states, effectively improving the success rate and survival rate of cryopreservation.

[0056] Furthermore, in practical applications, when initially establishing a salt stress memory pretreatment process for a specific plant species and cell line, the aforementioned detection method can be used to determine the preset threshold and optimal pretreatment parameters (including sodium chloride concentration, treatment time, etc.) for that cell line. Once determined, subsequent routine preservation operations of homologous cell lines (i.e., cell lines of the same plant species and from the same source) do not require repeated DNA methylation detection; the determined process parameters can be directly used for salt stress memory pretreatment, significantly simplifying the operation process, increasing throughput, and reducing detection costs, thus facilitating the widespread application of this method in actual germplasm resource preservation. Only when the cell line source changes (e.g., changing the plant species, changing the explant sampling site, or the cell line undergoing excessive passages leading to phenotypic changes), should the preset threshold and pretreatment parameters be re-determined. When using the determined process parameters for routine preservation operations, step S1 is completed directly into step S2, without the need for the DNA methylation detection step between steps S1 and S2. This "one-time setup, multiple uses" process setting mode ensures both the scientific rigor and precision of the preservation process, while also taking into account the convenience and economy of practical operation, making this method more suitable for large-scale, routine germplasm resource preservation work.

[0057] In some embodiments of this application, a mapping relationship between the second DNA methylation level and the concentration of the dimethyl sulfoxide in a baseline cell line is established in advance; In step S2, the concentration of dimethyl sulfoxide is determined by querying the mapping relationship based on the second DNA methylation level of the cell line.

[0058] Furthermore, the mapping relationship is pre-established for cell lines of different plant species; and / or, the mapping relationship is pre-established for cell lines of different parts of the same plant species.

[0059] Specifically, the reference cell line refers to a standard cell line used to establish the mapping relationship between DNA methylation level and dimethyl sulfoxide concentration. This reference cell line should be consistent with the source of the cell line to be preserved, i.e., belong to the same plant species and originate from the same part of the plant.

[0060] The mapping relationship is established as follows: A baseline cell line is taken and subjected to salt stress memory pretreatment under different conditions (e.g., different sodium chloride concentrations and / or different treatment times) according to the aforementioned S1 step. The second DNA methylation level of the promoter region of the specific stress response gene under each condition is detected. Then, cryoprotectants are prepared with different concentrations of dimethyl sulfoxide (DMSO) (e.g., 5 v / v%, 8 v / v%, 10 v / v%, 12 v / v%, 15 v / v%) for cryopreservation experiments. The cryopreservation survival rate is used as an indicator to determine the optimal DMSO concentration corresponding to each methylation level range, thereby establishing the mapping relationship between the second DNA methylation level and the DMSO concentration. This mapping relationship can be pre-established for different plant species and / or different cell lines.

[0061] In step S2, the second DNA methylation level of the cell line to be preserved is compared with the mapping relationship to determine the dimethyl sulfoxide concentration corresponding to the cell line, thereby enabling the targeted formulation of cryoprotectants.

[0062] DNA methylation is an upstream regulatory event in the formation of salt stress memory, and its changes occur before the accumulation of osmotic regulators such as proline and trehalose. This application utilizes DNA methylation as a root marker of salt stress memory formation, directly establishing a correlation between DNA methylation and dimethyl sulfoxide (DMSO) concentration to guide the determination of DMSO dosage in cryoprotectants. Through this mapping relationship, the effect of salt stress memory pretreatment can be quantitatively translated into precise DMSO dosage, achieving precise and personalized cryoprotectant formulations, which helps to minimize DMSO cytotoxicity while ensuring protective efficacy.

[0063] In other embodiments of this application, step S2 further includes: When the second DNA methylation level is higher than the first threshold, it indicates that the salt stress memory pretreatment effect is weak, the cell's own accumulation of endogenous protective substances is insufficient, and its tolerance to ultra-low temperature freezing stress is limited. At this time, it is necessary to increase the concentration of dimethyl sulfoxide in the cryoprotectant to enhance exogenous chemical protection and compensate for the lack of the cell's own antifreeze ability.

[0064] The second threshold is lower than the first threshold. When the second DNA methylation level is lower than the second threshold, it indicates that salt stress memory has been fully established and the cell's self-defense capability is strong. At this time, the concentration of dimethyl sulfoxide can be reduced to decrease its chemical damage to the cell; at the same time, the concentrations of trehalose and sucrose can be increased.

[0065] Dimethyl sulfoxide (DMSO) primarily exerts its antifreeze effect by penetrating into the cell interior, while trehalose and sucrose mainly provide osmotic pressure balance and membrane stability extracellularly. When the concentration of DMSO decreases, the intracellular antifreeze potential weakens, necessitating an increase in the concentration of extracellular cryoprotectants to maintain osmotic pressure balance. Trehalose's excellent membrane stabilizing ability fills the protective gap, compensating for the reduced protective effect. Experimental data show that for every 1 v / v decrease in DMSO, the concentration of trehalose increases by 0.5-1.5 w / v%, and the concentration of sucrose increases by 0.05-0.15 mol / L. Within this range, a considerable level of vitrification protection and osmotic pressure balance can be maintained; insufficient supplementation leads to inadequate protection, while excessive supplementation can easily cause hyperosmotic damage. This ratio represents the optimal balance between reducing chemical toxicity risks and ensuring cryoprotection effectiveness.

[0066] This approach enables precise customization of cryoprotectant components based on the real-time status of epigenetic markers: enhancing chemical protection for cells with weak memory and reducing toxicity while strengthening biological protection for cells with strong memory. This personalized adjustment strategy effectively ensures cell survival rates under different conditions while minimizing the toxic effects of chemical reagents on highly resilient cells, significantly improving the recovery vitality of preserved germplasm resources.

[0067] Furthermore, in some other embodiments of this application, in order to conduct long-term quality monitoring of cell lines after cryopreservation and ensure the long-term stable preservation of germplasm resources, each batch of the cell lines is preserved in liquid nitrogen in the form of at least three backup samples. Following step S3, the method further includes: S4 Every 6 months, one of the backup samples is recovered and its third DNA methylation level is detected. If the third DNA methylation level is within a preset tolerance range relative to the second DNA methylation level, the salt stress memory state of this batch of cell lines is determined to be stable, and the backup samples of this batch continue to be preserved. The preset tolerance range refers to the interval within which the second DNA methylation level is allowed to fluctuate within a certain range, centered on the second DNA methylation level. The upper and lower limits of the specific range can be determined through prior experiments based on the specific plant species and cell lines. For example, the preset tolerance range can be ±10% to ±20% of the second DNA methylation level.

[0068] If the third DNA methylation level exceeds the preset tolerance range relative to the second DNA methylation level, it indicates that the cell line has undergone epigenetic drift and salt stress memory has declined during long-term cryopreservation. In this case, other backup samples from the same batch are taken for resuscitation and subcultured for 2-3 generations in proliferation medium containing 25-35 mmol / L sodium chloride, with each generation cultured for 14-21 days, to reactivate the cell's stress memory mechanism. Then, the cells are re-preserved according to steps S1 to S3.

[0069] Specifically, for ease of distinction, this application refers to the DNA methylation level detected after resuscitation as the third DNA methylation level.

[0070] Through the aforementioned periodic monitoring and memory refresh mechanism, long-term dynamic maintenance of germplasm resources in vitro can be achieved: backup samples with good memory status continue to be preserved, while backup samples with faded memory are restored to their stress resistance status through the "refresh-re-save" process, thereby ensuring that each batch of cell lines in the in vitro bank always maintains effective stress memory and high survival rate, and achieving true long-term stable preservation of germplasm resources.

[0071] In some embodiments, a correspondence is established beforehand between the content of endogenous osmotic regulators in a baseline cell line and the concentration of the dimethyl sulfoxide; the endogenous osmotic regulators include at least one of proline or trehalose; In step S2, the content of the endogenous osmotic regulator in the cell line is detected, and the concentration of dimethyl sulfoxide is determined by looking up the corresponding relationship based on the content.

[0072] Specifically, proline content can be determined using the acidic ninhydrin method. Trehalose can be determined using high-performance liquid chromatography.

[0073] The pre-establishment of the correspondence is as follows: a baseline cell line is taken, and cell samples with different contents of endogenous osmotic regulators are obtained by setting salt stress memory pretreatment of different intensities or durations; the contents of proline and / or trehalose in each sample are detected respectively; then, according to the content level of each sample, the samples are subjected to cryopreservation experiments with different concentrations of dimethyl sulfoxide cryoprotectant, and the optimal dimethyl sulfoxide concentration corresponding to each content range is screened with the cryopreservation survival rate as the indicator, thereby constructing the correspondence.

[0074] The content of endogenous osmotic regulators directly reflects the physiological defense state of cells after salt stress memory pretreatment. The more proline or trehalose accumulated intracellularly, the stronger the cell's own antifreeze and dehydration capacity, and the relatively lower its dependence on exogenous cryoprotectants. By establishing the correlation between this content and dimethyl sulfoxide concentration, precise customization of cryoprotectant formulations was achieved, effectively balancing cryoprotection effects and cell viability maintenance, and significantly improving the success rate of cryopreservation. This method is simple to operate and provides intuitive indicators.

[0075] This approach complements the aforementioned method for determining dimethyl sulfoxide concentration based on DNA methylation levels. DNA methylation is an upstream marker of stress memory formation, while endogenous osmotic regulators are the physiological expression of stress memory. Both provide a basis for the personalized formulation of cryoprotectants from different perspectives, allowing for flexible selection of one or a combination of both based on actual testing conditions and equipment.

[0076] S3 The cell line after equilibration is transferred into a cryotube and cooled to -30°C to -50°C at a rate of 0.5-3°C / min, and then stored in liquid nitrogen.

[0077] In a preferred embodiment of this application, the programmed cooling is performed at a rate of 0.5-2°C / min to reduce the temperature to -35°C to -45°C.

[0078] In a more preferred embodiment of this application, the programmed cooling is performed at a rate of 1°C / min to -40°C.

[0079] When needed, the cryovials can be removed from liquid nitrogen and quickly placed in a 40°C water bath to thaw for 2 minutes. After complete thawing, the cells can be transferred sequentially to culture media containing 0.3 mol / L sucrose and 0.15 mol / L sucrose to gradually remove the protective agent, and finally transferred to sucrose-free culture medium.

[0080] By precisely controlling the cooling rate, cells can complete sufficient osmotic regulation and dehydration under the action of the cryoprotectant. When the temperature drops to the preset range of -30℃ to -50℃, intracellular free water has flowed out to a moderate extent, and the cells are in a highly concentrated antifreeze state. At this time, the introduction of liquid nitrogen can achieve rapid vitrification, effectively avoiding physical damage to the cell membrane system caused by ice crystal recrystallization during the cooling process, thereby significantly improving the survival rate of cells after revival.

[0081] In some embodiments, the step of establishing an ex vivo library is also included: For each cell line preserved using the methods described in steps S1-S3, at least three backup samples shall be preserved in liquid nitrogen. Genetic stability of cell lines before and after preservation was assessed using RAPD molecular marker technology, with amplification pattern differences of less than 5%. The FDA-PI dual fluorescence staining method was used to detect cell viability of backup samples every 6 months. When the viability was below 70%, subculture was performed to update the cells.

[0082] Furthermore, the RAPD molecular marker technology uses 10-15 random primers for amplification, and each sample is detected three times.

[0083] Furthermore, the genetic stability test also includes morphological observation and physiological index measurement of the preserved cells; The morphological observations include microscopic observations and records of cell size, color, and cytoplasmic uniformity; The physiological indicators include cell membrane integrity and proline content. Cell membrane integrity was detected using the FDA-PI dual fluorescence staining method.

[0084] Example 1

[0085] Explant collection and disinfection: Young stem segments of Leymus chinensis were collected, soaked in 0.8% sodium hypochlorite solution for 10 minutes, and rinsed 4 times with sterile water. The disinfected explants were inoculated into induction medium and callus was induced at 25±2℃, light intensity of 1800 lux, and photoperiod of 12h / d.

[0086] Three-stage gradient screening: The induced callus tissues were sequentially transferred to screening media containing 50 mmol / L, 100 mmol / L, and 150 mmol / L sodium chloride for 18 days at each stage. The survival rates at each stage were 72%, 52%, and 58%, respectively, with an overall screening efficiency of 21.7%. The salt-tolerant callus tissues obtained through screening were then propagated in proliferation medium for 30 days to obtain cell lines.

[0087] Salt stress memory pretreatment: The cell line was transferred into liquid MS medium containing 50 mmol / L sodium chloride and cultured at 25±1℃ for 48 hours.

[0088] Cryoprotectant equilibration: Cell lines pretreated with salt stress memory were transferred to a compound cryoprotectant and equilibrated at room temperature for 20 minutes. The cryoprotectant formulation was: dimethyl sulfoxide 12 v / v%, ethylene glycol 8 v / v%, sucrose 0.4 mol / L, trehalose 2 w / v%, and calcium chloride 10 mmol / L.

[0089] Programmed cooling and liquid nitrogen preservation: The equilibrated cell lines were transferred into cryovials and cooled to -40°C at a rate of 1°C / min, and then stored in liquid nitrogen.

[0090] Thawing and recovery: Remove the cryovials from liquid nitrogen and quickly place them in a 40°C water bath for 2 minutes to thaw. After complete thawing, transfer the cells sequentially to culture media containing 0.3 mol / L sucrose and 0.15 mol / L sucrose to gradually remove the protective agent. Finally, transfer them to sucrose-free culture medium to complete the recovery process.

[0091] Indicator Testing: I. Detection of phosphorylation level in MAPK cascade signaling pathway Total cellular protein was extracted before and after salt stress memory pretreatment. Western blot analysis was used to detect the phosphorylation levels of MAPKKK, MAPKK, and MAPK using antiphosphorylated MAPKKK, antiphosphorylated MAPKK, and antiphosphorylated MAPK antibodies. Total MAPK protein was used as an internal control to analyze the relative changes in phosphorylation levels of each protein.

[0092] II. DNA Methylation Level Detection Genomic DNA was extracted from cells before and after pretreatment. Methylation analysis of the promoter regions of stress-responsive genes P5CS, TPS, TPP, dehydrin, and LEA was performed using bisulfite sequencing, and the DNA methylation level in each region was calculated.

[0093] III. Gene Expression Level Detection The expression levels of P5CS, TPS, TPP, dehydrin, and LEA genes were detected by real-time quantitative PCR (qRT-PCR) before and after pretreatment.

[0094] The test results for tests one through three are shown in Table 1.

[0095] Table 1

[0096] The phosphorylation levels of MAPK-level signaling pathways (MAPKKK, MAPKK, MAPK) were significantly upregulated (2.8-3.5-fold), indicating that 45-55 mmol / L sodium chloride acted as a signal trigger, successfully activating the core intracellular stress-resistance signaling pathway and achieving cascade amplification of the signal. The DNA methylation levels in the promoter regions of key stress-resistance genes (P5CS, TPS, etc.) decreased significantly (36%-45%). This demethylation process relieved the transcriptional repression of genes and is a key switch connecting external stress signals with intrinsic gene expression, verifying the existence of "changes in DNA methylation levels" as a regulatory hub.

[0097] With the release of transcriptional repression, the expression levels of downstream functional genes (P5CS, TPS, TPP, dehydrin, LEA) were simultaneously and significantly upregulated (1.9-3.2-fold). This indicates that the cells have successfully synthesized a large amount of stress-resistant substances and established a systemic defense state. This confirms that salt stress pretreatment successfully induced the establishment of "stress memory" in cells through the pathway of "signal recognition (MAPK phosphorylation) → epigenetic regulation (DNA demethylation) → functional expression (gene upregulation)," providing solid molecular biological evidence for the subsequent significant improvement in cryopreservation survival rate.

[0098] IV. Detection of Physiological and Biochemical Indicators Intracellular proline content was determined by acidic ninhydrin method before and after pretreatment, intracellular trehalose content was determined by high performance liquid chromatography, and cell membrane integrity was detected by FDA-PI dual fluorescence staining method.

[0099] The test results for 4 are shown in Table 2.

[0100] Table 2

[0101] The upregulation of the P5CS gene (a key enzyme in proline synthesis) and the TPS / TPP gene (a key enzyme in trehalose synthesis) observed in Table 1 was successfully translated into the accumulation of specific metabolites, as shown in Table 2. The proline and trehalose contents increased significantly by 48% and 47%, respectively, confirming that gene-level regulation has been implemented in the storage of functional substances. Proline and trehalose are important osmotic regulators and natural cryoprotectants in plant cells. The significant increase in their contents effectively enhanced the cell's water-holding capacity and membrane stability, directly supporting the increase in cell membrane integrity from 68% to 82%. This result explains why pretreated cells were better able to resist subsequent freezing stress.

[0102] V. Genetic stability testing Genetic stability of cell lines before and after preservation was tested using RAPD molecular marker technology. 10-15 primers were randomly selected and each sample was tested three times. The difference rate was calculated based on the amplification pattern. The difference rate of the amplification pattern was less than 5%, which indicates that cryopreservation did not cause significant changes in the genomic DNA level of the cell lines and that the genetic stability was good.

[0103] VI. Survival Rate Testing After resuscitation, cell viability was detected using the FDA-PI dual fluorescence staining method, and the percentage of surviving cells out of the total number of cells was calculated. The survival rate was found to be 82%, indicating that the ultra-low temperature preservation method based on salt stress memory described in this invention can effectively protect cell viability and achieve a high preservation survival rate.

[0104] Example 2

[0105] The difference between Example 2 and Example 1 lies in the salt stress memory pretreatment.

[0106] Cell lines that were not pretreated with salt stress memory served as a blank control group (0 mmol / L sodium chloride, cultured for 48 h). Experimental groups with different sodium chloride concentrations and different incubation times were set up, and the survival rate under each treatment was detected. The specific parameters and detection results for different treatments (each treatment corresponds to 9 sample sizes) are shown in Table 3.

[0107] Table 3

[0108] Compared with the blank control group (48%), the survival rate of all pretreated groups was significantly improved, confirming the effectiveness of the pretreatment. In terms of concentration, 50 mmol / L was the optimal point; too low a concentration (40 mmol / L) resulted in insufficient stress stimulation, while too high a concentration (60 mmol / L) may cause cell damage, leading to a decrease in survival rate. In terms of time, 48 h was the optimal culture time; too short a time (36 h) resulted in insufficient memory activation, while too long a time (60 h) led to diminishing returns due to cell fatigue. In summary, treatment with 50 mmol / L sodium chloride for 48 h was the optimal combination, under which the survival rate of cryopreservation could reach 82%.

[0109] Example 3

[0110] The difference between Example 3 and Example 1 is that after the salt stress memory pretreatment, a low temperature stress step was introduced, and recovery culture was carried out after the low temperature stress ended.

[0111] For the same batch of cell lines, salt stress pretreatment was first applied: the cell lines were transferred to liquid MS medium containing 50 mmol / L sodium chloride and cultured at 25±1℃ for 30 h. Subsequently, low-temperature stress was applied: different low-temperature stress temperatures (6℃, 8℃, 10℃) and treatment times (10 h, 12 h, 14 h) were set up as experimental groups. After the low-temperature stress was completed, the cells were restored to culture at 25±1℃ for 4 h.

[0112] The survival rate test results for each experimental group are shown in Table 4 (each experimental group corresponds to 9 sample sizes). Table 4

[0113] Compared with the single salt stress pretreatment in Example 1 (survival rate 82%), the survival rate of all experimental groups increased to 86%-88% after the introduction of the low temperature stress step, indicating that low temperature stress, as a second messenger, successfully induced the cells to produce a cross-adaptation response, further enhancing their antifreeze ability. Among them, the 8℃ / 12h treatment group reached the peak (88%).

[0114] Example 4

[0115] The difference between Example 4 and Example 1 is that different antifreeze components were used, and the survival rate of each component was tested. The specific parameters and test results for different components (each component corresponds to 9 sample sizes) are shown in Table 5.

[0116] Table 5

[0117] Table 5 shows that the survival rate of the conventional formula (dimethyl sulfoxide, ethylene glycol, sucrose) was 62%. Adding trehalose alone increased the survival rate to 72%, indicating that trehalose can protect cell membrane structure by forming an amorphous matrix. Adding calcium chloride alone increased the survival rate to 70%, indicating that Ca2+ can enhance membrane stability through ion bridging. When trehalose and calcium chloride were added simultaneously (i.e., the components of this invention), the survival rate significantly increased to 82%, 20 percentage points higher than the conventional formula, and this increase was greater than the combined effect of trehalose and calcium chloride alone (18 percentage points), indicating a synergistic effect between the two in cell membrane protection. The cryoprotectant containing trehalose and calcium chloride of this invention can effectively improve the survival rate during cryopreservation.

[0118] Example 5

[0119] Example 5 of this application uses the same technical solution as Example 1 to verify the cryopreservation of alfalfa and crested wheatgrass, respectively, with sheepgrass from Example 1 as a control, to examine the applicability of this method to different forest and grassland species. The survival rates of each species are shown in Table 6 (6 sample sizes for each species). Table 6

[0120] Table 6 shows that the survival rates of sheepgrass, alfalfa, and crested wheatgrass under the same cryopreservation method were all greater than 79%, indicating that the method described in this application has good applicability to the tested forest and grassland species and can effectively improve the survival rate of different forest and grassland germplasm resources under cryopreservation.

[0121] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preserving germplasm resources based on salt stress memory, used for the cryopreservation of isolated cell lines of salt-tolerant plants, characterized in that, Includes the following steps: S1 The cell line was transferred into a liquid culture medium containing 45-55 mmol / L sodium chloride and cultured at 23-27℃ for 42-54 h to perform salt stress memory pretreatment. S2. The cell line pretreated with salt stress memory is transferred into a cryoprotectant and equilibrated for 15-30 min. The cryoprotectant contains the following components at the following concentrations: dimethyl sulfoxide 8-15 v / v%, ethylene glycol 5-10 v / v%, sucrose 0.3-0.5 mol / L, trehalose 2-3 w / v%, and calcium chloride 8-15 mmol / L. S3 cools the equilibrated cell line to -30°C to -50°C at a rate of 0.5-3°C / min, and then stores it in liquid nitrogen.

2. The germplasm resource preservation method based on salt stress memory according to claim 1, characterized in that, The procedure preceding step S1 also includes: Plant explants were collected, disinfected, and then inoculated into an induction medium for callus induction culture. The callus tissue was sequentially transferred to selection media with sodium chloride concentrations of 45-55 mmol / L, 95-105 mmol / L and 145-155 mmol / L for stepwise selection and culture, with each stage lasting 14-21 days, to obtain salt-tolerant callus tissue. The salt-tolerant callus was proliferated and cultured to obtain the cell line.

3. The germplasm resource preservation method based on salt stress memory according to claim 1, characterized in that, The salt stress memory preprocessing includes: S101 The cell line is transferred into the liquid culture medium containing 45-55 mmol / L sodium chloride and cultured at 23-27°C for 29-31 h; S102 The cell line treated in step S101 is cultured in the same culture medium at 6-12℃ for 10-18 h. S103 The cell line treated in step S102 is cultured in the same culture medium at 23-27°C for 3-5 h.

4. The germplasm resource preservation method based on salt stress memory according to claim 1, characterized in that, The salt stress memory preprocessing includes: S111 The cell line was transferred into the liquid culture medium containing 45-55 mmol / L sodium chloride and 50-200 mmol / L osmotic adjustment inducer and cultured at 23-27°C for 22-30 h. S112 Add an oxidative stress inducer to the liquid culture medium after culturing in step S111 to a concentration of 50-200 μmol / L, and continue culturing at 23-27℃ for 20-24 h. The osmotic conditioning inducer includes at least one of sorbitol or mannitol; the oxidative stress inducer includes at least one of hydrogen peroxide or abscisic acid.

5. The germplasm resource preservation method based on salt stress memory according to claim 1, characterized in that, The steps following step S1 and before step S2 include: The first DNA methylation level of the promoter region of a specific stress-response gene in the cell line before the salt stress memory pretreatment is obtained, and the second DNA methylation level of the promoter region of the specific stress-response gene in the cell line after the salt stress memory pretreatment is obtained; the specific stress-response gene includes at least one of a proline synthase gene, a trehalose synthase gene, or a stress protection protein gene. If the second DNA methylation level decreases to a preset threshold relative to the first DNA methylation level, it is determined that salt stress memory has been formed, and the process proceeds to step S2. If the second DNA methylation level does not decrease to the preset threshold relative to the first DNA methylation level, the salt stress memory pretreatment time is extended by 4-8 h, and / or the sodium chloride concentration is increased to 55-65 mmol / L.

6. The germplasm resource preservation method based on salt stress memory according to claim 5, characterized in that, A mapping relationship between the second DNA methylation level and the concentration of the dimethyl sulfoxide in the baseline cell line is established in advance; In step S2, the concentration of dimethyl sulfoxide is determined by querying the mapping relationship based on the second DNA methylation level of the cell line.

7. The germplasm resource preservation method based on salt stress memory according to claim 6, characterized in that, The mapping relationship is pre-established for cell lines of different plant species; and / or, the mapping relationship is pre-established for cell lines of different parts of the same plant species.

8. The germplasm resource preservation method based on salt stress memory according to claim 5, characterized in that, Step S2 further includes: When the second DNA methylation level is higher than the first threshold, the concentration of dimethyl sulfoxide in the cryoprotectant is increased; When the second DNA methylation level is lower than the second threshold, the concentration of dimethyl sulfoxide is reduced, while the concentrations of trehalose and sucrose are increased simultaneously; wherein, for every 1 v / v% decrease in dimethyl sulfoxide, the concentration of trehalose is increased by 0.5-1.5 w / v%, and the concentration of sucrose is increased by 0.05-0.15 mol / L; the second threshold is lower than the first threshold.

9. The method according to claim 5, characterized in that, Each batch of the cell line is preserved in liquid nitrogen in the form of at least three backup samples; Following step S3, the method further includes: S4 Every 6 months, one of the backup samples is recovered and its third DNA methylation level is detected. If the third DNA methylation level is within a preset tolerance range relative to the second DNA methylation level, then the backup samples in this batch will continue to be stored. If the third DNA methylation level exceeds the preset tolerance range relative to the second DNA methylation level, other backup samples from the same batch are revived, subcultured for 2-3 generations in a proliferation medium containing 25-35 mmol / L sodium chloride, with each generation cultured for 14-21 days, and then re-preserved according to steps S1 to S3.

10. The method according to claim 1, characterized in that, A pre-established correspondence between the content of endogenous osmotic regulators in a baseline cell line and the concentration of the dimethyl sulfoxide is provided; the endogenous osmotic regulators include at least one of proline or trehalose. In step S2, the content of the endogenous osmotic regulator in the cell line is detected, and the concentration of dimethyl sulfoxide is determined by looking up the corresponding relationship based on the content.

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