Freeze protectant for endangered seed cryopreservation and its preparation and application method

Abstract

The application discloses an anti-freezing protective agent for endangered seed ultra-low temperature preservation and a preparation and application method thereof, and relates to the technical field of biological resource preservation. The anti-freezing protective agent is composed of a star-shaped copolymer, a transmembrane coordinator, an intracellular stable nucleus and a buffer solution, and forms a biomimetic multi-layer protection system. The preparation method comprises synthesizing each component and preparing a working solution. In application, two-step penetration loading, programmed cooling and rapid thawing are used to realize intracellular and extracellular synergistic vitrification. The application provides an ultra-low temperature preservation solution method which is specially used for endangered seeds and can systematically maintain the integrity of cell structure, in view of the applicability limitation and lack of systematic cell protection of the prior art.

Description

Technical Field

[0001] This invention relates to the field of biological resource preservation technology, and more specifically, to cryoprotectants for the cryopreservation of endangered seeds and their preparation and application methods. Background Technology

[0002] Cryopreservation (usually liquid nitrogen, -196℃) is a key technology for the long-term safe preservation of endangered plant germplasm resources. However, seeds face multiple stresses during cooling, preservation, and thawing, including ice crystal damage, cell dehydration, membrane system rupture, and oxidative stress, leading to loss of viability after thawing. Traditional antifreeze strategies, such as using high-concentration osmotic agents (e.g., dimethyl sulfoxide, glycerol) to achieve vitrification, while inhibiting ice crystal formation, are highly toxic to seed cells and easily cause damage due to osmotic pressure shocks. Existing technologies typically focus on providing extracellular protection or simple intracellular osmotic regulation, lacking the ability to synergistically regulate the intracellular and extracellular environments.

[0003] For example, Chinese patent CN117770305A discloses an antifreeze agent for frozen seafood, composed of sodium alginate, trehalose, and tea polyphenols, focusing on forming an ice coating and providing antioxidant protection on the food surface. However, this approach has a relatively simple composition and mechanism of action, primarily targeting the external protection of aquatic muscle tissue. It is difficult to apply to plant seeds with complex cellular structures and seed coat barriers, especially failing to achieve intelligent delivery of the protectant into embryonic cells and stable intracellular residence. Similarly, Chinese patent CN108359613A discloses an antifreeze agent for Cordyceps sinensis strains, containing various polymers, sugars, and surfactants. While this approach targets microorganisms, its protective mechanism provides a mixed extracellular environment without a transmembrane transport unit or a protective nucleus responsive to the intracellular microenvironment. For eukaryotic plant seed cells requiring precise regulation of intracellular vitrification, the protective specificity and efficiency are insufficient, and the complex formulation may have unknown effects on seed physiology.

[0004] In summary, existing technologies either have limitations in their applicability or lack a systematic and programmed protection design from extracellular to intracellular perspectives, making it difficult to meet the dual requirements of high efficiency and safety for cryopreservation of endangered seeds. Therefore, this paper proposes cryopreservation agents for endangered seeds and their preparation and application methods. Summary of the Invention

[0005] To overcome the above-mentioned defects of the prior art, embodiments of the present invention provide cryoprotectants for the cryopreservation of endangered seeds and their preparation and application methods, so as to solve the problems of insufficient applicability of existing cryopreservation technologies to endangered seeds, single protection mechanism, and lack of synergistic protection between cells and cells.

[0006] To achieve the above objectives, the present invention provides the following technical solution: an antifreeze agent for cryopreservation of endangered seeds, comprising an extracellular matrix, a transmembrane coordinator, an intracellular stabilizing nucleus, and a buffer solution; The extracellular matrix comprises a star-shaped copolymer in which the hydrophilic segments of polyethylene glycol and the hydrophobic segments of polycaprolactone form a thermosensitive structure. The terminal keratin-like peptide segments enhance its adhesion and film-forming properties on the seed surface, thereby guiding the formation of a stable amorphous protective matrix in the extracellular environment during the cooling process. The structure of the transmembrane coordinator conjugate is: transmembrane peptide-linker-sugar molecule, wherein the transmembrane peptide is used to temporarily anchor to the cell membrane, the linker is an hydrazone bond or a ketal bond to provide environmentally responsive dissociation, and the sugar molecule is trehalose or trehalose-6-phosphate to play a protective role in the cell. This structure enables the directional transport of sugar molecules into the cell during the pretreatment stage. The intracellular stable core is a microsphere with a core-shell structure. Its poly-N-isopropylacrylamide hydrogel core undergoes thermosensitive contraction during thawing to release the loaded histone deacetylase inhibitor. The intermediate layer formed by ethylene glycol monomethyl ether oligomer and lecithin serves as an intracellular vitrification promoting matrix. The outer lipid membrane, composed of phospholipids containing thioester bonds and cell-penetrating peptides, provides oxidative stress responsiveness and membrane fusion capability, together constructing multiple protective sites within the cell.

[0007] Furthermore, the number average molecular weight of the polyethylene glycol hydrophilic segments is between 2,000 and 5,000, and the number average molecular weight of the polycaprolactone hydrophobic segments is between 1,000 and 3,000. This molecular weight range enables the star copolymer to possess good water solubility, film-forming properties, and moderate hydrophobic aggregation ability at low temperatures.

[0008] Furthermore, the buffer solution is a mixture of dipotassium hydrogen phosphate and potassium dihydrogen phosphate in a weight ratio of 2:1 to 4:1, with a pH of 6.5 to 7.0. This condition is close to the physiological pH of seed cells and helps maintain the stability of cell membranes and proteins during the treatment process.

[0009] Furthermore, the star-shaped copolymer is present in the cryoprotectant at a mass percentage of 8% to 12%, the transmembrane coordinator is present in the cryoprotectant at a concentration of 0.2 mmol / L to 0.8 mmol / L, and the intracellular stabilizing nucleus is present in the cryoprotectant at a volume percentage of 1% to 3%. This concentration ratio ensures that each component performs its function while avoiding osmotic damage or toxicity due to excessive concentration.

[0010] Furthermore, the ethylene glycol monomethyl ether oligomer is a dimer or trimer, and its higher molecular weight and hydroxyl density compared to the monomer help to form a more stable, water-holding glassy structure within cells.

[0011] A method for preparing an antifreeze protectant for cryopreservation of endangered seeds includes the following steps: S1. The star copolymer is dissolved in the buffer solution at a low temperature of 2 to 8 degrees Celsius to obtain an extracellular matrix solution. The low temperature is intended to maintain the integrity of the star copolymer structure and prevent its premature aggregation. S2. At a temperature of 4 to 10 degrees Celsius, the transmembrane coordinator and the intracellular stable nucleus are added to the extracellular matrix solution and mixed evenly to obtain the cryoprotectant. This temperature range is beneficial for maintaining the activity of the transmembrane coordinator and the structural stability of the intracellular stable nucleus, and ensures that the three are evenly dispersed to form a homogeneous working solution.

[0012] A method for applying an antifreeze protectant for cryopreservation of endangered seeds includes the following steps: S1. Pretreatment and infiltration: The seeds are placed in the pretreatment solution and the antifreeze agent in sequence for gradient treatment, so that the protection system is loaded in an orderly manner from the outside to the inside; S2. Programmed cooling: The osmotically treated seeds are cooled from 0 degrees Celsius to -40 to -50 degrees Celsius at a rate of 0.5 to 1.5 degrees Celsius per minute. This rate allows moisture to seep out slowly and reduces thermal stress. The temperature was kept constant for 5 to 20 minutes within the critical temperature range of -10 to -20 degrees Celsius. This stage provided a matching time window for the dramatic increase in extracellular matrix viscosity and the adjustment of intracellular solution properties, promoting synergistic glass transition. S3. Ultra-low temperature storage: After cooling, the seeds are placed in liquid nitrogen or an environment below -80 degrees Celsius for storage, so that the seed cells enter a stable state of metabolic stagnation. S4. Rapid thawing: Place the stored seeds directly into a water bath at 35 to 42 degrees Celsius to thaw them, quickly bypassing the dangerous temperature zone of ice crystal recrystallization and restoring the cell's liquid environment.

[0013] Furthermore, in step S1, the pretreatment solution contains the star copolymer and the transmembrane coordinator, and the concentration of the star copolymer is 30% to 50% of the concentration of the cryoprotectant. The lower concentration of the star copolymer facilitates initial film formation and works together with the transmembrane coordinator to initiate cell membrane modification and preliminary permeation regulation, laying the foundation for the subsequent loading of high-concentration cryoprotectant.

[0014] Furthermore, in step S1, the treatment conditions for the seeds in the pretreatment solution are: temperature 2 to 6 degrees Celsius, time 2 to 4 hours. This low-temperature slow infiltration process promotes the effective binding and translocation of transmembrane coordinators. The treatment conditions in the cryoprotectant are: temperature 0 to 2 degrees Celsius, time 10 to 20 minutes. This short ice bath loading is designed to achieve high concentration filling of the cryoprotectant and localization of the intracellular stable nucleus, while inhibiting cellular metabolic activity.

[0015] Furthermore, step S4 is followed by a washing step using a resuscitation solution, which is a phosphate buffer containing 0.5 to 1.0 mmol / L adenosine. Adenosine, as an energy precursor, helps to provide initial energy for the early metabolic recovery of thawed seeds and assists in removing residual protective agents from the cell surface.

[0016] The technical effects and advantages of this invention are as follows: This invention achieves systematic protection of seed cells at a spatial level by constructing a biomimetic protection system composed of a star-shaped copolymer, a transmembrane coordinator, and an intracellular stable core. The star-shaped copolymer forms a thermosensitive matrix outside the cell, guiding the external solution to form an amorphous state during cooling through a phase transition, providing a stable physical buffer environment for the cells. The transmembrane coordinator, as a smart carrier, reversibly embeds into the cell membrane during the pretreatment stage, efficiently and controllably transporting small-molecule protective agents such as trehalose into the cell, coordinating the balance of osmotic pressure inside and outside the cell, and avoiding the rapid dehydration and damage caused by a purely hypertonic external solution. This delivery method helps improve the uniformity and safety of protective agent loading.

[0017] This invention further extends the protective function to the cell interior by introducing an intracellular stabilizing core with a core-shell structure. This stabilizing core not only serves as an additional vitrification site within the cell, promoting the generation of a homogeneous glassy state, but its outer responsive lipid membrane can also sense and respond to intracellular oxidative stress during the initial thawing phase, enabling the on-demand release of protective substances. Simultaneously, its thermosensitive hydrogel core can rapidly release active substances after thawing, aiding cell repair. This design helps to more comprehensively maintain the structural integrity of cell membranes and organelles throughout the entire cryopreservation period.

[0018] In terms of application methods, this invention employs a strategy combining two-step infiltration and programmed cooling. Gradient infiltration loading avoids the sudden impact of the cryoprotectant on the cells, allowing the protective system to assemble in an orderly manner. The specific isothermal holding phase during programmed cooling provides a time window for the extracellular matrix and intracellular solution to achieve dynamic matching of rheological and thermodynamic properties, reducing critical steps caused by micro-damage due to mismatch between internal and external contraction. The entire scheme closely integrates material design and application processes, contributing to improving the reliability and versatility of cryopreservation technology. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the preparation process of the antifreeze protectant of the present invention; Figure 2This is a flowchart of the application method of the antifreeze protectant of the present invention; Figure 3 This is a structural diagram of the antifreeze protectant of the present invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below. Experimental methods not specified with particular conditions in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, all reagents and materials used are commercially available.

[0021] The core of this technical solution lies in constructing a biomimetic multilayer protective structure for endangered seeds. Through the sequential action and synergistic cooperation of three core components (star copolymer, transmembrane coordinator, and intracellular stabilizing nucleus), the seed cells achieve a matched glass transition inside and outside the cells, thereby maintaining the integrity and viability of the cell structure at ultra-low temperatures.

[0022] Specifically, the star-shaped copolymer forms a matrix on the outside of the seed that can adjust its properties with temperature changes; the transmembrane coordinator is responsible for the intelligent transport of small molecule protectants into the cell during the pretreatment stage; and the intracellular stable nucleus is loaded with multifunctional active substances.

[0023] During programmed cooling, a specific temperature holding platform is set to dynamically match the rheological properties of the extracellular matrix solution with the physicochemical properties of the intracellular solution, ultimately guiding the formation of a stable glassy state both inside and outside the cells, thus maximally inhibiting ice crystal growth and freezing damage. During thawing, rapid warming prevents recrystallization, and a specific resuscitation solution assists cell repair.

[0024] The following examples illustrate in detail the preparation of each component, the formulation of the preservative, and the complete preservation method.

[0025] Example 1: Preparation of star copolymer SC1 This embodiment details the synthesis of star copolymers with terminal modifications of keratinoid peptide segments.

[0026] S1. Synthesis of hydrophilic segment precursor: Under nitrogen protection, 50.0 g of polyethylene glycol monomethyl ether with a number average molecular weight of 3500 was dissolved in 500 mL of anhydrous dichloromethane.

[0027] Add 2.5 g of succinic anhydride and 0.5 g of 4-dimethylaminopyridine catalyst to the solution and stir at 25 degrees Celsius for 24 hours.

[0028] After the reaction is complete, the reaction solution is slowly poured into 3000 ml of pre-cooled anhydrous diethyl ether to precipitate.

[0029] The white solid was collected by filtration, washed three times with diethyl ether, and dried to constant weight in a vacuum drying oven at 30 degrees Celsius to obtain a polyethylene glycol derivative with a carboxyl group at the end, denoted as intermediate product L1.

[0030] S2. Constructing a star-shaped core and connecting hydrophobic segments: Dissolve the intermediate product L1 (20.0 g) obtained in step S1 in 200 mL of anhydrous N,N-dimethylformamide.

[0031] Slowly add dropwise a mixed solution of ethylenediamine (0.6 g) and triethylamine (1.0 mL) dissolved in 50 mL of N,N-dimethylformamide to the solution and react at 25°C for 12 hours.

[0032] Subsequently, 15.0 g of isocyanate-terminated polycaprolactone (pre-dissolved in 100 mL of N,N-dimethylformamide) with a number average molecular weight of 2000 was slowly added dropwise to the above reaction system, and the reaction was continued at 40 degrees Celsius for 18 hours.

[0033] The reaction solution was transferred into a dialysis bag with a molecular weight cutoff of 8000, dialyzed with deionized water for 3 days, and then freeze-dried to obtain a white flocculent solid, which was designated as intermediate product L2.

[0034] S3. Terminal peptide modification: Dissolve the intermediate product L2 (5.0 g) obtained in step S2 in 50 mL of phosphate buffer at pH 7.4.

[0035] Add 50 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 30 mg of N-hydroxysuccinimide to the solution in sequence, and stir at room temperature for 30 minutes to activate.

[0036] Subsequently, a chemically synthesized keratinoid model peptide (amino acid sequence: VYPSDTG, 100 mg) was added, and the mixture was gently stirred at 25 degrees Celsius for 24 hours.

[0037] After the reaction was complete, the mixture was transferred to a dialysis bag with a molecular weight cutoff of 3500 and dialyzed with deionized water for 48 hours, with the water changed every 8 hours. The final solution was freeze-dried to obtain a star copolymer, denoted as star copolymer SC1.

[0038] Example 1a: Preparation of star copolymers SC2 and SC3 As an alternative implementation, the raw materials were modified to synthesize the product, following the steps of Example 1, to demonstrate the feasibility of achieving a specific molecular weight range.

[0039] Preparation of star copolymer SC2: Using polyethylene glycol monomethyl ether with a number average molecular weight of 2000 and polycaprolactone diol with a number average molecular weight of 3000 (pre-reacted with hexamethylene diisocyanate to convert to terminal isocyanate groups), other conditions were the same as in Example 1, to obtain star copolymer SC2.

[0040] Preparation of star copolymer SC3: Using polyethylene glycol monomethyl ether with a number average molecular weight of 5000 and polycaprolactone diol with a number average molecular weight of 1000 (pre-reacted with hexamethylene diisocyanate to convert to terminal isocyanate groups), other conditions were the same as in Example 1, to obtain star copolymer SC3.

[0041] Example 2: Preparation of transmembrane coordinator TC1 This embodiment details the synthesis of transmembrane coordinators with hydrazone bonds as linkers.

[0042] S1. Transmembrane peptide synthesis and purification: A transmembrane peptide with the amino acid sequence LFFLLFFF was synthesized using the fluorene methoxycarbonyl solid-phase peptide synthesis method with RinkAmideMBHA resin as a carrier.

[0043] After peptide chain assembly was completed, the peptides were treated at room temperature for 3 hours using a cleavage reagent consisting of trifluoroacetic acid, water and triisopropylsilane in a volume ratio of 95:2.5:2.5.

[0044] The reaction solution was precipitated with diethyl ether, washed by centrifugation, and then vacuum dried to obtain crude peptide. The crude peptide was dissolved in water containing 0.1% trifluoroacetic acid and purified using a high-performance liquid chromatography system equipped with a C18 reversed-phase column with acetonitrile-water (containing 0.1% trifluoroacetic acid) gradient elution. The target peak fraction was collected, lyophilized, and the pure transmembrane peptide was obtained, denoted as intermediate P1.

[0045] S2. Peptide aldehyde modification: The intermediate product P1 (0.1 mmol) was dissolved in 5 mL of anhydrous dimethyl sulfoxide, and 4-formylbenzoic acid (0.12 mmol), coupling agent 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (0.12 mmol), and N,N-diisopropylethylamine (0.24 mmol) were added. The reaction was carried out at 25°C for 6 hours.

[0046] After the reaction was complete, the solution was added dropwise to 50 mL of ice-cold ether to precipitate the solid. The solid was collected by centrifugation, yielding a peptide derivative with benzaldehyde groups at the ends, which was denoted as intermediate product P2.

[0047] S3. Hydrazine modification of sugar molecules: Weigh trehalose-6-phosphate (0.12 mmol) and dissolve it in 5 mL of a mixed solution of methanol and water (volume ratio 1:1).

[0048] Add 0.15 mmol of 4-hydrazinobenzoate, adjust the pH to 6.0 with triethylamine, and stir the reaction at room temperature for 12 hours.

[0049] After the reaction was completed, most of the solvent was removed by rotary evaporation in a 40°C water bath. The residue was separated by preparing a thin-layer chromatography plate with silica gel and using chloroform-methanol-water (volume ratio 65:25:4) as the developing solvent to obtain the product, which was denoted as intermediate product S1.

[0050] S4. Hydrazone bond assembly: Dissolve intermediate product P2 obtained in step S2 and intermediate product S1 obtained in step S3 in 2 mL of anhydrous dimethyl sulfoxide and mix.

[0051] Add 10 μL of glacial acetic acid as a catalyst to the mixed solution and stir at 25°C in the dark for 24 hours.

[0052] After the reaction was completed, the product was purified using the same high-performance liquid chromatography system and method as in step S1. The main peak fraction was collected, lyophilized, and a white solid was obtained, which was denoted as transmembrane coordinator TC1.

[0053] Example 2a: Preparation of transmembrane coordinator TC2 As an optional implementation, this embodiment describes the synthesis of transmembrane coordinators with ketal bonds as linkers.

[0054] Weigh out 0.12 mmol of trehalose and dissolve it in 5 mL of anhydrous N,N-dimethylformamide. Add 10 mg of p-toluenesulfonic acid monohydrate.

[0055] The intermediate product P2 (0.1 mmol) obtained in step S2 of Example 2 was dissolved in 2 mL of anhydrous N,N-dimethylformamide and then added to the above solution. The mixture was stirred and reacted at 40°C for 24 hours under nitrogen protection.

[0056] After the reaction was complete, the reaction solution was added dropwise to 50 mL of ice-cold diethyl ether to precipitate the solid, which was then collected by centrifugation. The crude product was purified using the same high-performance liquid chromatography conditions as in step S1 of Example 2, and after lyophilization, a transmembrane coordinator was obtained, denoted as transmembrane coordinator TC2.

[0057] Example 3: Preparation of intracellular stable nucleus MS1 This embodiment details the preparation of microspheres with a core-shell structure.

[0058] S1. Synthesis and sieving of thermosensitive hydrogel core: The monomer N-isopropylacrylamide (1.0 g), the crosslinking agent N,N'-methylenebisacrylamide (0.04 g) and the model drug trogostatin A (10 mg) were dissolved together in 20 ml of deionized water and bubbled with nitrogen for 30 minutes.

[0059] Subsequently, ammonium persulfate (2 mg) as the initiator and N,N,N',N'-tetramethylethylenediamine (4 μL) as the catalyst were added, and the mixture was allowed to stand at 25°C for 6 hours to polymerize.

[0060] The obtained hydrogel block was mechanically broken using a glass homogenizer, and then sieved through 200-mesh and 400-mesh stainless steel standard sieves in sequence to collect hydrogel particles with a particle size between 20-40 micrometers. The particles were then thoroughly washed with deionized water to obtain the drug-loaded thermosensitive hydrogel core, which was denoted as intermediate product G1.

[0061] S2. Construction and coating of liquid crystal intermediate layer: Weigh 0.4 g of ethylene glycol monomethyl ether trimer and 0.2 g of lecithin, and dissolve them together in 10 mL of chloroform.

[0062] The intermediate product G1 (0.5 g wet weight) obtained in step S1 was dispersed in the organic phase, and chloroform was slowly evaporated using a rotary evaporator at a water bath of 30 degrees Celsius and a rotation speed of 60 rpm for about 2 hours. The resulting particles coated with the intermediate layer were denoted as intermediate product M1.

[0063] S3. Assembly of the outer responsive lipid membrane: 0.3 g of phospholipid 1-palmitoyl-2-(thiosuccinate monoester)-sn-glycerol-3-phosphocholine containing a thioester bond was dissolved in 6 mL of anhydrous ethanol along with a common cell-penetrating peptide (representative sequence: TAT peptide, amino acid sequence: GRKKRRQRRR, 10 mg).

[0064] The intermediate product M1 obtained in step S2 was uniformly dispersed in 30 mL of phosphate buffer (concentration of 10 mmol / L, pH 7.4) at 55°C. Under vigorous magnetic stirring at 1000 rpm, the lipid ethanol phase was slowly added dropwise to the aqueous phase at a rate of 1 mL / min using a syringe pump.

[0065] After the addition was complete, the mixture was stirred at 55 degrees Celsius for 2 hours. After the reaction was completed, the suspension was centrifuged at 4 degrees Celsius and 10,000 rpm for 10 minutes to collect the solid particles. The particles were resuspended in phosphate buffer and washed twice under the same conditions to obtain the stable intracellular nucleus, which was designated as the stable intracellular nucleus MS1.

[0066] Example 3a: Preparation of intracellular stable nucleus MS2 As an optional implementation, referring to step S2 of Example 3, the ethylene glycol monomethyl ether trimer is replaced with an equal mass of ethylene glycol monomethyl ether dimer, and other conditions are exactly the same as in Example 3, to obtain an intracellular stable nucleus, denoted as intracellular stable nucleus MS2.

[0067] Example 4: Preparation of the working solution for the antifreeze protectant S1. Preparation of the extracellular matrix basal solution: Weigh 1.0 g of the star copolymer SC1 prepared in Example 1, and slowly add it to 9.0 g of pre-cooled phosphate buffer solution under a 4°C ice-water bath and magnetic stirring. This buffer solution is prepared by mixing dipotassium hydrogen phosphate and potassium dihydrogen phosphate at a weight ratio of 3:1, and the pH is adjusted to 6.8 using 1 mol / L sodium hydroxide solution. Continue stirring for 4 hours to obtain a homogeneous and clear solution, denoted as solution E1.

[0068] S2. Prepare standard working solution: Take 4.85 ml of solution E1 into a 10 ml centrifuge tube and keep the tube in a 4°C freezer.

[0069] Add 0.1 mL of a 10 mmol / L aqueous solution of transmembrane coordinator TC1, gently vortex to mix, and add 0.25 mL of a homogeneous phosphate-buffered dispersion of intracellular stable nucleus MS1, in which MS1 accounts for 20% of the total volume.

[0070] The antifreeze working solution was obtained by gently mixing the mixture on a horizontal shaker at 4 degrees Celsius and 30 revolutions per minute for 1 hour. This solution is denoted as working solution W1.

[0071] S3. Preparation of working solutions of various concentrations: Take 4.85 mL of solution E1, add 0.25 mL of TC1 aqueous solution with a concentration of 10 mmol / L, add 0.4 mL of MS1 dispersion (20%), mix well to obtain working solution W2.

[0072] Take 4.85 mL of solution E1, add 0.4 mL of TC1 aqueous solution with a concentration of 10 mmol / L, add 0.75 mL of MS1 dispersion (20%), mix well to obtain working solution W3.

[0073] S4. Preparation of pretreatment solution: Take 4 mL of solution E1, add 1 mL of phosphate buffer solution of the same pH, mix well, then add 0.2 mL of transmembrane coordinator TC1 aqueous solution with a concentration of 2 mmol / L, mix well to obtain the pretreatment solution, wherein the concentration of star copolymer SC1 is about 4% and the concentration of transmembrane coordinator TC1 is about 0.2 mmol / L.

[0074] Example 5: Cryopreservation of endangered plant seeds (taking yew seeds as an example) This embodiment demonstrates in detail the entire process of preserving seeds using the protective agent and method described in this solution.

[0075] S1. Seed pretreatment (moisture content adjustment): Select mature, disease-free yew seeds and place them in a constant temperature and humidity chamber to equilibrate for 14 days at 15 degrees Celsius and 80% relative humidity.

[0076] S2. Penetration Loading (Two-Step Method): S2.1 Preparation of pretreatment solution: Take 5 ml of the star copolymer pretreatment solution with a concentration of 4% prepared in step S4 of Example 4 for later use.

[0077] S2.2 First step of infiltration (low temperature slow infiltration): Immerse the seeds treated with S1 in the above pretreatment solution. Place the container in a 4°C biochemical incubator and allow it to infiltrate for 3.5 hours.

[0078] S2.3 Second step of infiltration (ice bath loading): Gently blot away excess pretreatment solution from the seed surface with sterile filter paper, and quickly transfer the seed to a container containing the antifreeze working solution W1 prepared in Example 4. Immerse the seed in an ice-water bath at 1 degree Celsius for 18 minutes.

[0079] S3. Programmed cooling: Transfer the seeds that have completed permeation loading, along with approximately 0.5 mL of working solution W1, into a 2.0 mL cryovial. Place the cryovial in the sample chamber of the programmed cooling apparatus.

[0080] Set the cooling program: start cooling from 1 degree Celsius at a rate of 1.0 degrees Celsius per minute; when the sample temperature reaches -15 degrees Celsius, the instrument automatically holds this temperature for 12 minutes; after the holding phase ends, continue cooling to -40 degrees Celsius at a rate of 1.0 degrees Celsius per minute.

[0081] S4. Ultra-low temperature storage: When the programmable temperature instrument shows that the sample temperature has reached -40 degrees Celsius, quickly take out the cryopreservation tube and immediately put it into the gas phase region of the liquid nitrogen Dewar flask for pre-cooling for 5 minutes, and then completely immerse it in liquid nitrogen for long-term storage.

[0082] S5. Rapid thawing and recovery: When viability testing is required, remove the cryovial from liquid nitrogen and immediately place it in a -35°C low-temperature alcohol bath for 2 minutes to equilibrate.

[0083] Then, quickly transfer the cryovials to a preheated 40-degree Celsius water bath and gently and quickly shake them up and down until the ice crystals inside the tubes completely disappear.

[0084] Immediately remove the seeds and place them in a petri dish containing resuscitation solution (phosphate buffer containing 0.8 mmol / L adenosine, pH 7.0). Gently rinse three times at 20°C for 5 minutes each time.

[0085] Example 6: Preservation Effect Verification Experiment To verify the effectiveness of this technical solution, the following comparative experimental group was set up, and the core test results are clearly presented in tabular form: Control group Z1: Yew seeds were not treated with any antifreeze and were directly subjected to the cooling, storage and thawing steps S3, S4 and S5 in Example 5.

[0086] Control group Z2: The working solution W1 was replaced with a conventional vitrification solution (composed of 30% glycerol, 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.4 mol / L sucrose dissolved in basal medium), and the same procedure as steps S2 to S5 in Example 5 was followed.

[0087] Experimental group E1: Strictly follow all the steps of Example 5 and use working solution W1.

[0088] Each treatment group was set up in 3 replicates, and 50 yew seeds with the same physiological state were used in each experiment. After thawing and recovery in step S5, all seeds from all groups were sown in sterilized seedling substrate and placed in a light incubator at 25 degrees Celsius with a light / dark cycle of 12 hours / 12 hours for a standard germination test.

[0089] Regular observations and records were kept, and the average germination rate of each group was calculated after 30 days. By comparing the average germination rate of experimental group E1 with control groups Z1 and Z2 after 30 days, the relative effectiveness of the antifreeze agent and method in maintaining seed viability after cryopreservation can be objectively evaluated. Specific test data are summarized in Table 1 below.

[0090] Table 1: Effects of different cryoprotection treatments on the germination rate of Taxus chinensis seeds after cryopreservation Note: Germination rates in the table are the mean ± standard deviation of three replicate experiments.

[0091] As shown in Table 1, under the same cryopreservation and thawing process, the average germination rate of seeds preserved using the cryoprotectant and method of this scheme (experimental group E1) was significantly higher than that of the unprotected control group (Z1) and the control group using conventional cryoprotectants (Z2). This comparative experiment directly demonstrates the positive effect of this technical scheme in effectively maintaining the viability of endangered seeds after cryopreservation.

[0092] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cryoprotectant for the cryopreservation of endangered seeds, characterized in that, It consists of the extracellular matrix, transmembrane coordinators, an intracellular stabilizing nucleus, and a buffer solution; The extracellular matrix comprises a star copolymer having a polyethylene glycol hydrophilic segment, a polycaprolactone hydrophobic segment, and a keratin-like peptide segment attached to the end of the segment. The structure of the transmembrane coordinator conjugate is: transmembrane peptide-linker-sugar molecule, wherein the linker is a hydrazone bond or a ketal bond, and the sugar molecule is trehalose or trehalose-6-phosphate; The intracellular stable nucleus is a microsphere with a core-shell structure, which consists of, from the inside out: a poly-N-isopropylacrylamide hydrogel core containing a histone deacetylase inhibitor, an intermediate layer formed by ethylene glycol monomethyl ether oligomer and lecithin, and an outer lipid membrane composed of phospholipids containing thioester bonds and cell-penetrating peptides.

2. The cryoprotectant for cryopreservation of endangered seeds according to claim 1, characterized in that, The number-average molecular weight of the polyethylene glycol hydrophilic segments is between 2,000 and 5,000, and the number-average molecular weight of the polycaprolactone hydrophobic segments is between 1,000 and 3,000.

3. The cryoprotectant for cryopreservation of endangered seeds according to claim 1, characterized in that, The buffer solution is a mixture of dipotassium hydrogen phosphate and potassium dihydrogen phosphate in a weight ratio of 2:1 to 4:1, with a pH value of 6.5 to 7.

0.

4. The cryoprotectant for cryopreservation of endangered seeds according to claim 1, characterized in that, The star copolymer is present in the cryoprotectant at a mass percentage of 8% to 12%, the transmembrane coordinator is present in the cryoprotectant at a concentration of 0.2 mmol / L to 0.8 mmol / L, and the intracellular stable nucleus is present in the cryoprotectant at a volume percentage of 1% to 3%.

5. The cryoprotectant for cryopreservation of endangered seeds according to claim 1, characterized in that, The ethylene glycol monomethyl ether oligomer is a dimer or a trimer.

6. A method for preparing an antifreeze protectant for cryopreservation of endangered seeds as described in any one of claims 1 to 5, characterized in that, Includes the following steps: S1. The star-shaped copolymer is dissolved in the buffer solution at a temperature of 2 to 8 degrees Celsius to obtain an extracellular matrix solution; S2. Under conditions of 4 to 10 degrees Celsius, the transmembrane coordinator and the intracellular stable nucleus are added to the extracellular matrix solution and mixed evenly to obtain the cryoprotectant.

7. A method for using the cryopreservation antifreeze agent for the cryopreservation of endangered seeds according to any one of claims 1 to 5, characterized in that, Includes the following steps: S1. Pretreatment and infiltration: The seeds are sequentially placed in the pretreatment solution and the antifreeze agent for treatment; S2. Programmed cooling: The seeds that have undergone osmosis treatment are cooled from 0 degrees Celsius to -40 to -50 degrees Celsius at a rate of 0.5 to 1.5 degrees Celsius per minute, and kept at a constant temperature within the range of -10 to -20 degrees Celsius for 5 to 20 minutes; S3. Ultra-low temperature storage: Store the cooled seeds in liquid nitrogen or an environment below -80 degrees Celsius. S4. Rapid thawing: Thaw the stored seeds directly in a water bath at 35 to 42 degrees Celsius.

8. The method of applying the antifreeze protectant according to claim 7, characterized in that, In step S1, the pretreatment solution contains the star copolymer and the transmembrane coordinator, and the concentration of the star copolymer is 30% to 50% of the concentration in the antifreeze agent.

9. The method of applying the cryoprotectant for cryopreservation of endangered seeds according to claim 8, characterized in that, In step S1: The treatment conditions for the seeds in the pretreatment solution were: temperature 2 to 6 degrees Celsius, time 2 to 4 hours; The treatment conditions in the antifreeze are: temperature 0 to 2 degrees Celsius, time 10 to 20 minutes.

10. The method of applying the cryoprotectant for cryopreservation of endangered seeds according to any one of claims 7 to 9, characterized in that, Step S4 is followed by a washing step using a resuscitation solution, which is a phosphate buffer containing 0.5 to 1.0 mmol / L adenosine.

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