Freeze-preservation method for spores of ginkgo adiantifolium ching

Abstract

The application discloses a kind of Fujian Guanyin Zhaolian fern spore ultra-low temperature preservation methods, it is related to plant germplasm resource preservation technical field, the method is preserved to spore by constructing the ultra-low temperature treatment system including novel vitrification protective solution.The protective solution has been creatively compounded with myo-inositol hexakisphosphate, melatonin and GSH / AsA combination:Among them, trace IP6 locks down trace metal ion catalysis by six-tooth ring space steric hindrance, prevents polymer degradation, ensures that the solution realizes 100% physical vitrification;Melatonin is targeted to accumulate in lipid membrane interface and block peroxidation damage;GSH and AsA cooperatively construct time sequence antioxidant network to eliminate recessive ROS storm;The present application initiates the synergistic mechanism of physical stability and physiological antioxidant, completely solves the problem that stubborn spores are easily dehydrated and killed and frozen ice crystal damage, and has high recovery germination rate, which provides an extremely industrial practicality scheme for long-term safe storage of endangered fern germplasm.

Description

Technical Field

[0001] This invention relates to the field of plant germplasm resource preservation technology, specifically to a method for cryopreservation of spores from *Adiantum capillus-veneris* var. *fusiforme*. Background Technology

[0002] *Adiantum capillus-veneris*, a relict plant originating from the Carboniferous period of the Paleozoic Era, is hailed as a "living fossil." It possesses irreplaceable academic value not only in exploring the systematic evolution of ancient plants but also in terms of significant medicinal and ornamental value. In recent years, due to the continuous destruction of its natural habitat and its demanding requirements for natural reproduction, the wild population of *Adiantum capillus-veneris* has been declining sharply, making the rescue and long-term preservation of its germplasm resources an urgent priority.

[0003] Cryopreservation of spores (usually stored in liquid nitrogen at -196°C) is currently the most effective way to achieve long-term, safe, and stable preservation of germplasm of rare and endangered ferns. However, unlike conventional crop seeds, the spores of *Adiantum capillus-veneris* have a special physiological structure. They are usually rich in a high proportion of lipids and chloroplasts, making them extremely sensitive to water loss and extreme low temperatures. They are short-lived, stubborn, or intermediate-type spores, and conventional low-temperature or room-temperature drying storage can easily lead to their complete loss of vitality.

[0004] Currently, the most commonly used technique for cryopreservation of plants is vitrification. Its basic principle is to drastically dehydrate the material using a high-concentration hypertonic protective agent (such as conventional PVS2 or PVS3 plant vitrification solutions). During the rapid cooling process with liquid nitrogen, the intracellular and extracellular liquid phases directly transform into an amorphous glassy state without undergoing a crystallization stage, thus avoiding mechanical damage to the cells caused by ice crystals. Although existing conventional vitrification techniques have made some progress in some common plant materials, they still exhibit significant performance limitations and technical shortcomings when applied to special thick-walled, lipid-rich spores such as *Adiantum capillus-veneris*. These shortcomings are specifically manifested in the following aspects: First, existing conventional vitrification protectants are mostly simple combinations of small molecules such as high concentrations of dimethyl sulfoxide, glycerol, and ethylene glycol. This system is accompanied by extremely high osmotic pressure and significant chemical toxicity, which can easily cause fatal osmotic stress to tiny spores during loading and dehydration. At the same time, because the macromolecular network structure of conventional protectants is not stable enough, devitrification (i.e., secondary ice crystal nucleation) can easily occur during cooling and subsequent rewarming and thawing. Once extracellular or intracellular ice crystals are formed, they will cause irreversible physical damage to the spore cell structure.

[0005] Secondly, under multiple extreme stresses including extreme osmotic dehydration, cryogenic freeze-thaw cycles, and chemical toxicity, a physiological reactive oxygen species (ROS) storm will erupt within the spore cells. Current technologies often only focus on osmotic regulation at the physical antifreeze level, neglecting in-depth intervention on the redox balance of the cellular microenvironment. The lack of effective antioxidant protection leads to severe membrane lipid peroxidation in thick-walled spores, macroscopically manifested as large-area browning and necrosis of thawed spores, or delayed-onset death syndrome with developmental delay and prothallus margin whitening during subsequent germination culture.

[0006] In summary, existing cryopreservation technologies for treating *Adiantum capillus-veneris* spores generally suffer from problems such as unstable physical phase formation, significant toxicity damage, severe membrane lipid peroxidation, low germination rates after thawing, and developmental abnormalities. Therefore, developing a novel cryopreservation system that effectively balances macroscopic physical phase transition stability with the protection of microscopic cellular physiological activity to overcome the low survival rate bottleneck of existing technologies has become a pressing common technical challenge in the conservation of endangered fern germplasm resources. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a method for cryopreservation of spores from *Adiantum capillus-veneris* in Fujian. This invention pioneers a cryopreservation system that combines physical stabilization with physiological antioxidant effects, overcoming the physiological limit of the resilient spores of *Adiantum capillus-veneris*, which are extremely prone to dehydration and death. This method perfectly achieves 100% solution vitrification and a recovery and germination rate of up to 96%, providing extremely reliable industrial support for the long-term storage of endangered plant germplasm resources.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for cryopreservation of spores from *Adiantum capillus-veneris*, comprising the following steps: Step 1: Spore collection and drying pretreatment: Mature spores of *Adiantum capillus-veneris* from Fujian were collected and dried to reduce the spore moisture content. As is known to those skilled in the art, mature spores typically refer to spores that are dark brown and naturally dehiscent. The purpose of drying is to reduce the free water content and prevent fatal ice crystal damage during freezing. The moisture content of spores can be determined by sampling using conventional drying-to-constant-weight methods (such as drying at 105°C to constant weight).

[0009] Step 2: Loading and processing the solution: The dried spores were placed in a loading solution for permeation pre-protection treatment; The purpose of loading solution treatment is to induce preliminary plasmolysis in plant cells under mild conditions, thereby increasing their tolerance to subsequent high-concentration vitrification solutions and preventing osmotic shock. During the procedure, spores can be placed in centrifuge tubes and suspended in the loading solution.

[0010] Step 3: Improved vitrification solution treatment: The spores treated in step two were transferred into a modified vitrification solution for vitrification dehydration and antioxidant protection. Before this step, the supernatant (loading solution) is usually removed by low-speed centrifugation (e.g., 1000~3000 rpm), and then a modified vitrification solution is added. This step is a crucial stage in which intracellular water is replaced by a high concentration of protective agent and a glassy state is formed.

[0011] Step 4: Liquid nitrogen cryopreservation: The spores treated in step three were placed in liquid nitrogen for cryopreservation. In practice, the suspension containing spores and vitrification solution can be transferred to a standard cryopreservation tube (such as a 1.8 mL or 2.0 mL cryopreservation tube) and then directly and quickly placed into a liquid nitrogen tank at -196°C.

[0012] Step 5: Quick defrost: Remove the frozen spores and thaw them quickly in a water bath; Rapid rewarming aims to quickly cross the dangerous temperature range (-130°C to -60°C) to prevent devitrification of the glassy state and the reformation of ice crystals (recrystallization).

[0013] Step Six: Gradient Washing The thawed spores were unloaded by gradient washing with sugar washing solutions of decreasing concentration; The purpose of gradient washing is to gradually displace the high concentration of vitrification protectant within the spore cells using a hypertonic but non-toxic sugar solution (such as sucrose), preventing the cells from rupturing due to sudden water absorption and swelling. The solutions for each gradient can be changed by low-speed centrifugation and discarding the supernatant. Each gradient wash typically takes 5-10 minutes.

[0014] Step 7: Germination Culture The washed spores were inoculated onto a culture medium for germination culture and effect detection.

[0015] Inoculation should be performed in a sterile laminar flow hood. The washed and centrifuged spores should be evenly spread or sown on the surface of the solid germination medium.

[0016] To further optimize the preservation effect and improve the germination rate and genetic stability of spores after spore revival, this invention provides preferred technical features for each step of the above method: Preferably, in step three, the modified vitrification solution uses a plant-based culture medium as a solvent, and its added components include: glycerol, ethylene glycol, dimethyl sulfoxide, polyethylene glycol, sucrose, reduced glutathione, L-ascorbic acid, melatonin, and inositol hexaphosphate.

[0017] Specifically, for melatonin, which has poor water solubility, the melatonin is first dissolved in anhydrous ethanol to prepare a high-concentration stock solution, and then added to the modified vitrification solution. The final concentration of ethanol in the system is controlled to be less than 0.1% (v / v) to avoid ethanol from causing cytotoxicity to spores.

[0018] More preferably, the modified vitrification solution is diluted to 100 mL with MS liquid medium, and the pH is 5.6-6.0. The amounts or concentration ratios of each component are as follows: glycerol: 20-30 g; ethylene glycol: 10-20 g; dimethyl sulfoxide: 8-12 g; polyethylene glycol 4000 (PEG4000): 8-12 g; sucrose: 0.4-0.6 mol / L; reduced glutathione (GSH): 0.5-1.0 mmol / L; L-ascorbic acid (AsA): 0.2-0.5 mmol / L; melatonin (MT): 50-100 μmol / L; inositol hexaphosphate (IP6): 15-25 μmol / L.

[0019] Preparation instructions: To ensure the activity of the antioxidant components, in practice, it is preferable to first dissolve glycerol, ethylene glycol, dimethyl sulfoxide, polyethylene glycol 4000 and sucrose in MS liquid medium; after the solution is cooled to room temperature or 0°C, add easily oxidized or heat-sensitive reduced glutathione, L-ascorbic acid, inositol hexaphosphate and melatonin dissolved in ethanol in advance, and finally make up the volume and adjust the pH.

[0020] More preferably, the pH of the modified vitrification solution is adjusted to 5.8-6.0. This weakly acidic environment is most suitable for the physiological tolerance of spore cells and is conducive to maintaining the stability of components such as ascorbic acid.

[0021] Preferably, in step one, the drying conditions are as follows: drying at a temperature of 20-30°C (e.g., in an artificial climate chamber or a constant temperature and humidity incubator) and a relative humidity of 25%-35% for 48-72 hours, so that the spore water content reaches 68%. This water content range removes excess free water while retaining the bound water necessary to maintain the basic cell structure.

[0022] Preferably, in step two, the loading solution uses MS liquid culture medium as the solvent, containing 1.5~2.5 mol / L glycerol and 0.3~0.5 mol / L sucrose; the treatment conditions are: temperature 20~30℃, shaking speed 80~120 rpm, and treatment time 25~30 minutes. Constant temperature shaking treatment helps the loading solution to uniformly and fully penetrate the spores.

[0023] Preferably, in step three, the modified vitrification solution treatment is performed under ice bath conditions at 0-2°C for 40-50 minutes. The principle of low-temperature treatment is to greatly reduce the biochemical toxicity of dimethyl sulfoxide (DMSO) while maintaining the diffusion of the penetrant into the cell.

[0024] In particular, the modified vitrification solution must be pre-cooled (to 0~2°C) before use and must be used within 30 minutes after preparation to prevent antioxidants such as GSH and AsA from prematurely oxidizing and becoming ineffective in vitro, and to ensure that they exert maximum efficacy after entering the spore cells.

[0025] Preferably, in step five, the conditions for rapid thawing are as follows: the cryovial is quickly placed in a water bath at 38~42℃ (preferably 40℃) and shaken vigorously for 90~120 seconds until the ice crystals inside the tube have just completely melted.

[0026] Preferably, in step six, the gradient washing specifically involves washing sequentially with four levels of sucrose solutions of decreasing concentration: 1.2 mol / L, 0.8 mol / L, 0.4 mol / L, and 0.2 mol / L, respectively. The washing solution is prepared using MS liquid culture medium as the base solvent, and each washing operation can be performed at room temperature.

[0027] Preferably, in step seven, the germination culture conditions are as follows: MS-based solid medium (usually with 6-8 g / L agar added), with 0.5-1.5 mg / L 6-benzylaminopurine (6-BA) and 0.1-0.5 mg / L naphthaleneacetic acid (NAA) added; continuously cultured for more than 30 days in a light incubator with a temperature of 23-27°C, a light intensity of 2000-3000 lux, and a photoperiod of 10-14 hours / day. Typically, after 30-45 days of culture, the spore germination rate can be counted under a microscope (germination is indicated by the rupture of the spore exoskeleton and the emergence of prothallus cells).

[0028] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for cryopreservation of spores from *Adiantum capillus-veneris* (Fujian fern) and a corresponding antioxidant-modified vitrification solution, achieving significant and unexpected technical results. Regarding the overall preservation process, this invention overcomes the problems of large fluctuations in survival rate and slow germination in the preservation of thick-walled fern spores using traditional methods by establishing a standardized operation of "precise drying - permeation loading - antioxidant vitrification - rapid freezing / thawing - gradient unloading." Spores preserved using this method exhibit a stable germination rate of 95.5%~97.2% after thawing. Simultaneously, experimental observations show that this invention eliminates the developmental delay period commonly found after traditional freezing and thawing, resulting in extremely rapid spore recovery, shortened prothallus development time, and bright green color without browning or necrosis, effectively ensuring the physiological vitality and genetic stability of the recovered plants.

[0029] Regarding the basic protection of the vitrification solution, this invention scientifically optimizes the ratio of penetrating and non-penetrating protective agents, constructing a safe and efficient physical protection network. Specifically, low-molecular-weight glycerol and ethylene glycol can rapidly penetrate cells to replace free water, significantly lowering the system's freezing point. Meanwhile, strictly controlling the concentration of dimethyl sulfoxide (DMSO) within the range of 8%–12% ensures cell membrane permeability while significantly reducing the chemical toxicity of traditional high-concentration protective solutions. Furthermore, polyethylene glycol 4000, as a non-penetrating macromolecule, does not enter the cells but significantly increases the apparent viscosity of the solution and alters the hydrogen bond network of water molecules, ensuring that spores perfectly form a transparent amorphous glassy state upon immersion in liquid nitrogen, avoiding mechanical damage caused by extracellular ice crystals. An appropriate concentration of sucrose provides a gentle osmotic pressure buffer, assisting in pre-dehydration of cells before freezing and preventing cells from absorbing water and bursting during thawing and washing.

[0030] To address the severe oxidative browning and increased cell membrane permeability during the recovery period of spores after traditional vitrification solution treatment, this invention innovatively constructs an antioxidant ternary system composed of GSH, AsA, and melatonin. Experiments clearly show that the combined use of this ternary system directly reduces the browning rate of spores after thawing to zero, and the conductivity of the leachate remains at an extremely low level in the early recovery stage, indicating that the physical integrity of the cell membrane is perfectly protected. We hypothesize that in the face of ultra-low temperature stress and the massive burst of reactive oxygen species (ROS) induced by hypertonic dehydration, these three antioxidants form a sophisticated three-dimensional defense network: melatonin, with its excellent amphiphilicity, preferentially accumulates at the lipid interface of the spore cell membrane, blocking the chain reaction of lipid peroxidation; simultaneously, AsA, mainly free in the solution phase, and GSH, which enters the cell with the permeate, form a temporal relay between the intracellular and extracellular environments. The three factors work together to effectively neutralize the oxidative stress damage caused by sudden temperature changes and the chemical toxicity of DMSO, thereby comprehensively maintaining the redox homeostasis and core physiological activity of endangered fern spores during extreme physical transformation processes.

[0031] During the research and development, the applicant also discovered that if the aforementioned antioxidants were added only to the basic protective solution, the viscosity of the solution would abnormally decrease after ice bath operation. When immersed in liquid nitrogen, the solution could not maintain a transparent glassy state but instead exhibited white crystallization, resulting in extremely high spore mortality. We speculate that this is because trace amounts of transition metal ions such as iron and copper are inevitably present in the basic culture medium. In the low-temperature, hypertonic solution, these metal ions act as catalysts, promoting an abnormal redox reaction of ascorbic acid, producing strong oxidizing substances. This causes the polyether chains of the PEG macromolecules to break down and degrade, thereby destroying the physical viscosity network required to maintain the glassy state. To address this contradiction, this invention introduces trace amounts of IP6, utilizing its polyphosphate cyclic structure to form a strong complex encapsulation of transition metal ions, directly "locking" the catalytic sites. This method not only completely eliminates the fatal risks of PEG chain breakage and solution crystallization but also allows the antioxidant components to purely exert their role in scavenging free radicals, protecting spores from oxidative browning caused by freezing. In addition, trace amounts of IP6 can be absorbed and utilized as a natural phosphorus source during the spore thawing and germination stage, forming a highly self-consistent and perfect synergy in the three dimensions of chemical antioxidation, physical stabilization, and nutritional growth promotion. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Of course, the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0033] Unless otherwise specified, all chemical reagents and materials in this invention are purchased from the market or synthesized from raw materials purchased from the market.

[0034] Spores of *Angiopteris fokiensis* from Fujian: Collected from the sporophytes of healthy, disease-free, and mature *Angiopteris fokiensis*. During collection, select the dark brown pinnae of the sori on the dorsal side, allow them to air dry naturally to release spores, and then sieve through a 100-mesh sieve to remove impurities before collecting the pure spores for later use.

[0035] MS liquid medium: using a standard Murashige & Skoog base salt and vitamin formulation (sucrose and agar-free, pH 5.8) as the base solvent for loading solution, vitrification solution and washing solution.

[0036] Germination medium ingredients: 6-benzylaminopurine (6-BA, plant tissue culture grade), α-naphthaleneacetic acid (NAA, plant tissue culture grade), agar (biochemical reagent, gel strength ≥1200g / cm). 2 ).

[0037] All reagents used were of analytical grade or cell culture grade: glycerol, ethylene glycol, dimethyl sulfoxide (DMSO, purity ≥99.9%), polyethylene glycol 4000 (PEG4000), and sucrose.

[0038] Reduced glutathione (GSH) and L-ascorbic acid (AsA): Both are high-purity biochemical reagents (purity ≥99.0%), and should be freshly prepared before use to prevent oxidative inactivation.

[0039] Inositol hexaphosphate (IP6): also known as phytic acid, using biochemical grade reagents with a purity ≥98% (or the corresponding ultra-pure sodium phytate, calculated based on the effective IP6 molar concentration).

[0040] Melatonin: Purity ≥ 99.0%. Due to its poor water solubility, in all preparation steps involving melatonin in this invention, it is assumed that it will be completely dissolved in a small amount of anhydrous ethanol (analytical grade) before being added to the base solution (the final volume concentration of ethanol in the final solution is strictly controlled to be ≤ 0.05% v / v, which has no toxic effect on spores).

[0041] Tween-20: Polyoxyethylene dehydrated sorbitan monolaurate, a conventional nonionic surfactant, biochemical reagent grade.

[0042] Trehalose: a non-reducing disaccharide, analytical grade.

[0043] EDTA: Disodium ethylenediaminetetraacetate (EDTA-Na2), a routine metal ion chelating agent used in plant tissue culture, analytical grade.

[0044] A method for cryopreservation of spores from *Adiantum capillus-veneris* in Fujian Province includes the following steps: 1. Preparation procedures for solutions and culture media (1) Preparation of antioxidant-modified vitrification solution: MS liquid medium was used as the base solvent. First, 20-30g of glycerol, 10-20g of ethylene glycol, 8-12g of dimethyl sulfoxide (DMSO), 8-12g of polyethylene glycol 4000 (PEG 4000) and 0.4-0.6mol / L of sucrose were weighed and dissolved in about 60mL of MS liquid medium by heating or stirring at room temperature. After complete dissolution, the solution was cooled to room temperature or 0℃ for later use. Next, weigh an appropriate amount of melatonin powder and completely dissolve it in anhydrous ethanol to prepare a high-concentration stock solution. Add 0.5–1.0 mmol / L reduced glutathione (GSH), 0.2–0.5 mmol / L L-ascorbic acid (AsA), 15–25 μmol / L inositol hexaphosphate (IP6), and an appropriate amount of melatonin ethanol stock solution sequentially to the above solution (so that the final melatonin concentration in the solution is 50–100 μmol / L, and the final ethanol volume concentration is strictly less than 0.1%). Finally, bring the volume to 100 mL with MS liquid medium and adjust the pH to 5.6–6.0 (preferably 5.8–6.0) with 1 mol / L NaOH or HCl. Note: This solution must be pre-cooled in an ice bath at 0–2 °C after preparation, and to ensure optimal activity of the antioxidants and chelating agents, it must be used within 30 minutes of preparation.

[0045] (2) Preparation of loading solution: Using MS liquid medium as solvent, add 1.5~2.5mol / L glycerol and 0.3~0.5mol / L sucrose, dissolve and mix well.

[0046] (3) Preparation of washing solutions: Four different concentrations of washing solutions were prepared, all using MS liquid culture medium as solvent, containing 1.2 mol / L, 0.8 mol / L, 0.4 mol / L and 0.2 mol / L of sucrose respectively.

[0047] (4) Preparation of germination medium: MS solid medium with conventional concentration of agar (e.g., 7 g / L) is used, and 0.5-1.5 mg / L of 6-benzylaminopurine (6-BA) and 0.1-0.5 mg / L of naphthaleneacetic acid (NAA) are added. The pH is adjusted to 5.8, and after high temperature and high pressure sterilization, it is poured into petri dishes for later use.

[0048] 2. Procedures for Cryopreservation and Resuscitation of Spores Step 1 (Drying): Collect mature spores of *Adiantum capillus-veneris*, spread them evenly on weighing paper or sterile petri dishes, and place them in a constant temperature and humidity chamber (or desiccator) at a temperature of 20-30℃ and a relative humidity of 25%-35% for 48-72 hours. Take samples for testing to confirm that the spore moisture content has dropped to the range of 6%-8%.

[0049] Step 2 (Loading): Place an appropriate amount (e.g., 50 mg) of dried spores into a centrifuge tube, add the pre-prepared loading solution, and treat in a shaker at 80-120 rpm for 25-30 minutes at 20-30°C.

[0050] Step 3 (Vitrification): Centrifuge the loaded spore suspension at low speed (e.g., 2000 rpm, 3 min) and discard the supernatant. Quickly add the pre-cooled modified vitrification solution (prepared within 30 minutes), completely immerse the centrifuge tube in an ice-water mixture at 0-2°C, and let it stand for 40-50 minutes.

[0051] Step 4 (Freezing): After the vitrification time is reached, transfer the spores and an appropriate amount of vitrification solution to a standard cryovial and quickly immerse it completely in liquid nitrogen at -196°C. Preservation time in liquid nitrogen can range from several hours to several years.

[0052] Step 5 (thawing): Remove the cryovial from the liquid nitrogen and immediately place it into a constant temperature water bath set to 38~42℃. Shake the cryovial vigorously to ensure even heating for 90~120 seconds until the ice crystals inside the tube have just completely melted (at which point the temperature inside the tube is close to 0℃). Remove it quickly.

[0053] Step Six (Washing): Centrifuge the thawed suspension and discard the supernatant. Add sucrose washing solutions of 1.2 mol / L, 0.8 mol / L, 0.4 mol / L, and 0.2 mol / L sequentially. After each addition of washing solution, allow the suspension to stand at room temperature or suspend and wash for 5-10 minutes. Then centrifuge and discard the supernatant before proceeding to the next washing stage to complete the gradient unloading of the protective agent.

[0054] Step 7 (Cultivation): Evenly inoculate the spores collected from the last centrifugation onto the surface of the pre-prepared germination medium. Place the culture dish in a light incubator and cultivate for at least 30 days under conditions of 23-27℃, light intensity of 2000-3000 lux, and photoperiod of 10-14 hours / day. Observe the germination of spores and the development of prothallus under a microscope regularly, and calculate the survival rate / germination rate.

[0055] The present invention will be further described below through specific embodiments. Example 1

[0056] A method for cryopreservation of spores of *Adiantum capillus-veneris* from Fujian Province, specifically including the following steps: Step 1: Spore collection and drying pretreatment: Mature spores of *Adiantum capillus-veneris* were collected, spread evenly on weighing paper, and placed in a constant temperature and humidity chamber. The temperature was set at 25℃ and the relative humidity at 30%, and the spores were dried for 60 hours. The water content of the spores was measured to be 7.0%.

[0057] Step 2: Loading and processing the solution: Place 0.1 g of dried spores into a 2.0 mL cryovial and add 1.0 mL of loading solution (MS liquid medium containing 2.0 mol / L glycerol and 0.4 mol / L sucrose). Place the cryovial in a shaker and shake for 25 minutes at 25 °C and 100 rpm.

[0058] Step 3: Improved vitrification solution treatment: Centrifuge at low speed to discard the loaded solution, and add 1.0 mL of antioxidant-modified vitrification solution pre-cooled to 0°C. The vitrification solution is prepared as follows: Take MS liquid culture medium, add 25 g glycerol, 15 g ethylene glycol, 10 g dimethyl sulfoxide, 10 g polyethylene glycol 4000, and 0.5 mol / L sucrose sequentially. After cooling to 0°C, add 0.75 mmol / L reduced glutathione, 0.35 mmol / L L-ascorbic acid, 20 μmol / L inositol hexaphosphate (IP6), and melatonin pre-dissolved in anhydrous ethanol (final melatonin concentration 75 μmol / L, final ethanol concentration 0.05% v / v), and bring the volume to 100 mL. Adjust the pH to 5.8. This solution should be used within 20 minutes of preparation.

[0059] The spores were treated in the modified vitrification solution for 45 minutes under 0°C ice bath conditions.

[0060] Step 4: Liquid nitrogen cryopreservation: After processing, the cryopreservation tubes were quickly immersed in liquid nitrogen at -196°C for cryopreservation (the preservation time in this embodiment is 30 days, but in practice it can be preserved for a long time).

[0061] Step 5: Quick defrost: Remove the cryovials from the liquid nitrogen and immediately place them in a 40°C constant temperature water bath. Shake the cryovials vigorously and thaw for 100 seconds until the ice crystals inside have just completely melted.

[0062] Step Six: Gradient Washing At room temperature, centrifuge and discard the vitrification solution. Add sucrose washing buffer (all in MS liquid medium) at concentrations of 1.2 mol / L, 0.8 mol / L, 0.4 mol / L and 0.2 mol / L in sequence, washing for 8 minutes at each concentration level. Centrifuge to remove the supernatant.

[0063] Step 7: Germination Culture The washed spores were inoculated onto solid germination medium (MS + 1.0 mg / L 6-BA + 0.2 mg / L NAA + 7 g / L agar) and placed in an artificial climate chamber. The culture was carried out for 35 days at 25°C, 2500 lux light intensity, and a photoperiod of 12 hours / day. Microscopic examination showed a spore germination rate as high as 96.8%, with no browning observed. Example 2

[0064] A method for cryopreservation of spores of *Adiantum capillus-veneris* from Fujian Province, specifically including the following steps: Step 1: Spore collection and drying pretreatment: Mature spores of *Adiantum capillus-veneris* were collected from Fujian, spread on weighing paper, and placed in a constant temperature and humidity chamber. The temperature was set at 20℃ and the relative humidity at 25%, and the spores were dried for 72 hours. The water content of the spores was measured to be reduced to 6.0%.

[0065] Step 2: Loading and processing the solution: Place 0.1 g of dried spores into a 2.0 mL cryovial and add 1.0 mL of loading solution (MS liquid medium containing 1.5 mol / L glycerol and 0.3 mol / L sucrose). Place the cryovial in a shaker and shake at 20 °C and 80 rpm for 30 minutes.

[0066] Step 3: Improved vitrification solution treatment: Centrifuge at low speed to discard the loaded solution, and add 1.0 mL of pre-cooled (0°C) antioxidant-modified vitrification solution. The vitrification solution is prepared as follows: Take MS liquid medium, add 20 g glycerol, 10 g ethylene glycol, 8 g dimethyl sulfoxide, 8 g polyethylene glycol 4000, and 0.4 mol / L sucrose sequentially; after cooling to 0°C, add 0.5 mmol / L reduced glutathione, 0.2 mmol / L L-ascorbic acid, 15 μmol / L inositol hexaphosphate, and melatonin pre-dissolved in a very small amount of anhydrous ethanol (final melatonin concentration 50 μmol / L, final ethanol concentration <0.1% v / v). Adjust the volume to 100 mL with MS liquid medium and adjust the pH to 5.6. Use the solution within 30 minutes of preparation.

[0067] The spores were treated by standing in a modified vitrification solution in a standard ice-water mixture at 0°C for 50 minutes.

[0068] Step 4: Liquid nitrogen cryopreservation: After processing, the cryopreservation tubes were quickly immersed in liquid nitrogen at -196°C for cryopreservation (30 days). Upon immersion in liquid nitrogen, the solution inside the tubes exhibited a perfectly transparent amorphous glassy state.

[0069] Step 5: Quick defrost: Remove the cryovial and immediately place it in a 38°C constant temperature water bath and shake it vigorously for 120 seconds until the ice crystals inside the tube have just completely melted.

[0070] Step Six: Gradient Washing Centrifuge at room temperature to remove vitrification solution, then add sucrose washing buffer (MS liquid medium) at concentrations of 1.2 mol / L, 0.8 mol / L, 0.4 mol / L and 0.2 mol / L in sequence. Wash for 10 minutes at each concentration, then centrifuge to remove supernatant.

[0071] Step 7: Germination Culture The washed spores were inoculated onto solid germination medium (MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 7 g / L agar) and placed in an artificial climate chamber for 35 days at a temperature of 23°C, a light intensity of 2000 lux, and a photoperiod of 10 hours / day.

[0072] Experimental results: Microscopic examination showed that the spore germination rate reached 92.4%, proving that the lower limit parameter of this invention, combined with an appropriately extended treatment time, can still achieve excellent preservation results. Example 3

[0073] A method for cryopreservation of spores of *Adiantum capillus-veneris* from Fujian Province, specifically including the following steps: Step 1: Spore collection and drying pretreatment: Mature spores were dried in a constant temperature and humidity chamber at a set temperature of 30℃ and a relative humidity of 35% for 48 hours, and the moisture content was measured to reach 8.0%.

[0074] Step 2: Loading and processing the solution: The spores were placed in a high-concentration loading solution (MS liquid medium containing 2.5 mol / L glycerol and 0.5 mol / L sucrose) and rapidly shaken at 30°C and 120 rpm for 25 minutes.

[0075] Step 3: Improved vitrification solution treatment: Centrifuge and discard the loaded solution, then add an antioxidant-modified vitrification solution pre-cooled to 2°C. The solution components were added strictly according to the maximum allowable values: 30g glycerol, 20g ethylene glycol, 12g dimethyl sulfoxide, 12g polyethylene glycol 4000, 0.6mol / L sucrose, 1.0mmol / L reduced glutathione, 0.5mmol / L L-ascorbic acid, 25μmol / L inositol hexaphosphate, and 100μmol / L melatonin. The volume was brought to 100mL, and the pH was adjusted to 6.0.

[0076] The spores were treated in the modified vitrification solution at 2°C for a strictly controlled time of 40 minutes under ice bath conditions.

[0077] Step 4: Liquid nitrogen cryopreservation: After processing, it was immediately placed in liquid nitrogen at -196℃ for 30 days.

[0078] Step 5: Quick defrost: Remove the cryovial from the liquid nitrogen and place it in a 42°C water bath for rapid heating and even shaking to thaw for 90 seconds.

[0079] Step Six: Gradient Washing Wash with sucrose wash solutions of decreasing concentrations from 1.2 mol / L to 0.2 mol / L for 5 minutes each at room temperature.

[0080] Step 7: Germination Culture Spores were inoculated onto solid germination medium (MS + 1.5 mg / L 6-BA + 0.5 mg / L NAA + 7 g / L agar) and cultured for 35 days at 27°C, light intensity of 3000 lux, and photoperiod of 14 hours / day.

[0081] Experimental results: The spore germination rate reached 93.1%, and the prothallosomal cells were plump and dark green. This demonstrates that, under high concentration limits, chemotherapy toxicity was successfully avoided by shortening the germination time and increasing temperature constraints.

[0082] Comparative Example 1 A method for cryopreservation of spores of *Adiantum capillus-veneris* from Fujian Province, specifically including the following steps: Step 1: Spore collection and drying pretreatment: Mature spores of *Adiantum capillus-veneris* were collected, spread evenly on weighing paper, and placed in a constant temperature and humidity chamber. The temperature was set at 25℃ and the relative humidity at 30%, and the spores were dried for 60 hours. The water content of the spores was measured to be 7.0%.

[0083] Step 2: Loading and processing the solution: Place 0.1 g of dried spores into a 2.0 mL cryovial and add 1.0 mL of loading solution (MS liquid medium containing 2.0 mol / L glycerol and 0.4 mol / L sucrose). Place the cryovial in a shaker and shake for 25 minutes at 25 °C and 100 rpm.

[0084] Step 3: Improved vitrification solution treatment: Centrifuge at low speed to remove the loading solution, and add 1.0 mL of a control vitrification solution pre-cooled to 0°C. The control vitrification solution is prepared as follows: Take MS liquid culture medium, add 25 g glycerol, 15 g ethylene glycol, 10 g dimethyl sulfoxide, 10 g polyethylene glycol 4000, and 0.5 mol / L sucrose sequentially. After cooling to 0°C, add 0.75 mmol / L reduced glutathione, 0.35 mmol / L L-ascorbic acid, and 20 μmol / L inositol hexaphosphate (IP6). Do not add melatonin; instead, add Tween-20 (final Tween-20 concentration 75 μmol / L, final ethanol concentration 0.05% v / v), a conventional nonionic surfactant previously dissolved in anhydrous ethanol. Adjust the volume to 100 mL and adjust the pH to 5.8.

[0085] The spores were treated in the control vitrification solution for 45 minutes under 0°C ice bath conditions.

[0086] Step 4: Liquid nitrogen cryopreservation: After the process is completed, the cryopreservation tubes are quickly immersed directly into liquid nitrogen at -196°C for cryopreservation (the solution is in a transparent glassy state when immersed).

[0087] Step 5: Quick defrost: After 30 days of storage, remove the cryovials from the liquid nitrogen and immediately place them in a 40°C constant temperature water bath. Shake the cryovials vigorously and thaw for 100 seconds until the ice crystals inside the tubes have just completely melted.

[0088] Step Six: Gradient Washing At room temperature, centrifuge and discard the control vitrification solution, then add sucrose washing buffer (all in MS liquid medium) at concentrations of 1.2 mol / L, 0.8 mol / L, 0.4 mol / L and 0.2 mol / L in sequence, washing for 8 minutes at each concentration level, and centrifuge to remove the supernatant.

[0089] Step 7: Germination Culture The washed spores were inoculated onto solid germination medium (MS + 1.0 mg / L 6-BA + 0.2 mg / L NAA + 7 g / L agar) and placed in an artificial climate chamber. The culture was carried out for 35 days at 25°C, 2500 lux light intensity, and a photoperiod of 12 hours / day. Microscopic examination revealed a sharp drop in spore germination rate to 61.3%, with numerous ungerminated spores exhibiting significant brownish-black precipitate on their outer walls. Furthermore, the initial conductivity of the leachate was 3.2 times higher than in Example 1.

[0090] Comparative Example 2 A method for cryopreservation of spores of *Adiantum capillus-veneris* from Fujian Province, specifically including the following steps: Step 1: Spore collection and drying pretreatment: Mature spores of *Adiantum capillus-veneris* were collected, spread evenly on weighing paper, and placed in a constant temperature and humidity chamber. The temperature was set at 25℃ and the relative humidity at 30%, and the spores were dried for 60 hours. The water content of the spores was measured to be 7.0%.

[0091] Step 2, loading solution processing: Place 0.1 g of dried spores into a 2.0 mL cryovial and add 1.0 mL of loading solution (MS liquid medium containing 2.0 mol / L glycerol and 0.4 mol / L sucrose). Place the cryovial in a shaker and shake for 25 minutes at 25 °C and 100 rpm.

[0092] Step 3: Improved vitrification solution treatment: Centrifuge at low speed to discard the loading solution, and add 1.0 mL of a control vitrification solution pre-cooled to 0°C. The control vitrification solution is prepared as follows: Take MS liquid culture medium, add 25 g glycerol, 15 g ethylene glycol, 10 g dimethyl sulfoxide, 10 g polyethylene glycol 4000, and 0.5 mol / L sucrose sequentially. After cooling to 0°C, add 20 μmol / L inositol hexaphosphate (IP6) and melatonin pre-dissolved in anhydrous ethanol (final melatonin concentration 75 μmol / L, final ethanol concentration 0.05% v / v). Do not add reduced glutathione (originally 0.75 mmol / L) and L-ascorbic acid (originally 0.35 mmol / L). Instead, add 1.10 mmol / L of trehalose, a non-reducing sugar protectant, to achieve equimolar osmotic pressure, bring the volume to 100 mL, and adjust the pH to 5.8.

[0093] The spores were treated in the control vitrification solution for 45 minutes under 0°C ice bath conditions.

[0094] Step 4: Liquid nitrogen cryopreservation: After the process is completed, the cryopreservation tubes are quickly immersed directly into liquid nitrogen at -196°C for cryopreservation (the solution is in a transparent glassy state when immersed).

[0095] Step 5: Quick defrost: After 30 days of storage, remove the cryovials from the liquid nitrogen and immediately place them in a 40°C constant temperature water bath. Shake the cryovials vigorously and thaw for 100 seconds until the ice crystals inside have just completely melted.

[0096] Step Six: Gradient Washing At room temperature, centrifuge and discard the control vitrification solution, then add sucrose washing buffer (all in MS liquid medium) at concentrations of 1.2 mol / L, 0.8 mol / L, 0.4 mol / L and 0.2 mol / L in sequence, washing for 8 minutes at each concentration level, and centrifuge to remove the supernatant.

[0097] Step 7: Germination Culture The washed spores were inoculated onto solid germination medium (MS + 1.0 mg / L 6-BA + 0.2 mg / L NAA + 7 g / L agar) and placed in an artificial climate chamber. The culture was maintained at 25°C, 2500 lux light intensity, and a photoperiod of 12 hours / day for 35 days. Microscopic examination revealed a germination rate of only 42.7%, and the revived prothallophytes exhibited extensive spontaneous whitening and edge necrosis around day 15 of development (a typical case of delayed-onset ROS damage syndrome).

[0098] Comparative Example 3 A method for cryopreservation of spores of *Adiantum capillus-veneris* from Fujian Province, comprising the following steps: Step 1: Spore collection and drying pretreatment: Mature spores of *Adiantum capillus-veneris* were collected, spread evenly on weighing paper, and placed in a constant temperature and humidity chamber. The temperature was set at 25℃ and the relative humidity at 30%, and the spores were dried for 60 hours. The water content of the spores was measured to be 7.0%.

[0099] Step 2, loading solution processing: Place 0.1 g of dried spores into a 2.0 mL cryovial and add 1.0 mL of loading solution (MS liquid medium containing 2.0 mol / L glycerol and 0.4 mol / L sucrose). Place the cryovial in a shaker and shake for 25 minutes at 25 °C and 100 rpm.

[0100] Step 3: Improved vitrification solution treatment: Centrifuge at low speed to discard the loading solution, and add 1.0 mL of a control vitrification solution pre-cooled to 0°C. The control vitrification solution is prepared as follows: Take MS liquid medium, add 25 g glycerol, 15 g ethylene glycol, 10 g dimethyl sulfoxide, 10 g polyethylene glycol 4000, and 0.5 mol / L sucrose sequentially. After cooling to 0°C, add 0.75 mmol / L reduced glutathione, 0.35 mmol / L L-ascorbic acid, and melatonin pre-dissolved in anhydrous ethanol (final melatonin concentration 75 μmol / L, final ethanol concentration 0.05% v / v). Instead of adding inositol hexaphosphate (IP6), add an equimolar concentration (20 μmol / L) of EDTA, a standard metal chelating agent for plant tissue culture, and bring the volume to 100 mL. Adjust the pH to 5.8.

[0101] The spores were treated in the control vitrification solution at 0°C for 45 minutes under ice bath conditions. (Experimental observation: At the 30-minute mark of the 0°C ice bath treatment, a sharp decrease in the apparent viscosity of the control solution was clearly observed.)

[0102] Step 4: Liquid nitrogen cryopreservation: After the treatment was completed, the cryovials were immediately immersed in liquid nitrogen at -196°C for cryopreservation. (Experimental phenomenon record: the moment the cryovials were immersed in liquid nitrogen, the solution inside the tube failed to remain transparent, but instead burst forth with large-area whitening and crystallization, resulting in devitrification).

[0103] Step 5: Quick defrost: After 30 days of storage, remove the cryovial from the liquid nitrogen and immediately place it in a 40°C constant temperature water bath. Shake the cryovial vigorously for 100 seconds until the crystals inside the tube have just completely melted.

[0104] Step Six: Gradient Washing At room temperature, centrifuge and discard the control vitrification solution, then add sucrose washing buffer (all in MS liquid medium) at concentrations of 1.2 mol / L, 0.8 mol / L, 0.4 mol / L and 0.2 mol / L in sequence, washing for 8 minutes at each concentration level, and centrifuge to remove the supernatant.

[0105] Step 7: Germination Culture The washed spores were inoculated onto solid germination medium (MS + 1.0 mg / L 6-BA + 0.2 mg / L NAA + 7 g / L agar) and placed in an artificial climate chamber. The spores were cultured for 35 days at 25°C, 2500 lux light intensity, and a photoperiod of 12 hours / day. Microscopic examination revealed that the spore cells completely ruptured due to lethal mechanical cutting and osmotic compression by extracellular ice crystals, resulting in a final germination rate of 0% (all spores died).

[0106] To objectively evaluate the cryopreservation effect and physiological and biochemical status of each treatment group, the following core detection indicators were established: Apparent viscosity stability and microphase separation determination: Each group of glass transition solutions was placed at a set ice bath temperature. Using a rotational viscometer, the apparent viscosity was measured at the initial preparation time (0 min) and at the end of the ice bath operation limit time, and the viscosity retention rate was calculated. Simultaneously, the presence of microemulsion turbidity (indicating phase separation caused by localized depolymerization of the polymer network) was observed.

[0107] Macroscopic glass transition state (whitening crystallization rate) assessment: In step four (introduction of liquid nitrogen), the number of tubes exhibiting whitening crystallization (i.e., deglassing phase transition caused by the shift in glass transition temperature Tg) was counted.

[0108] Cell membrane permeability / relative conductivity (REC) determination: Based on the physical structural characteristics of thick-walled spores, 0.1 g of washed spores were weighed and placed in 10 mL of deionized water. Vacuum extraction was performed for 10 minutes to promote electrolyte exudation, followed by extraction at room temperature in the dark with shaking for 24 hours. The initial conductivity (C1) was measured. After boiling in a water bath for 15 minutes to completely kill the cells and cooling, the final conductivity (C2) was measured. REC = (C1 / C2) × 100%.

[0109] Spore germination rate statistics: Germination was defined as the rupture of the outer spore wall and the prothallus cells protruding from the 35th day of germination culture.

[0110] Browning and whitening rate statistics: After 35 days of culture, the proportion of brown-black precipitation (early lipid peroxidation) or edge necrosis and whitening (delayed ROS damage) was counted.

[0111] The above detection method was used to conduct independent replicate experiments on each group of samples (n=3, each replicate containing at least 200 spores and 50 tubes of solution). The results of the various detection data are shown in Table 1 and Table 2 below.

[0112] Table 1: Macroscopic physical stability and phase transition state assessment of each group of glass transition solutions Table 2: Microscopic damage and physiological and biochemical recovery of spores after cryopreservation in each group Comparative Example 1 confirms the irreplaceable interfacial targeted antioxidant properties of melatonin under extreme dehydration conditions. Data from Tables 1 and 2 show that after replacing melatonin with an equimolar amount of the conventional nonionic surfactant Tween-20 in Comparative Example 1, while the physical vitrification of the solution remained normal, the relative conductivity (REC) of the revived spores increased significantly to 24.1%, resulting in a marked decrease in germination rate to 61.3% and a severe browning rate as high as 31.5%. This comparative result confirms that conventional surfactants can only exert simple physical penetration and wetting effects, and cannot fundamentally prevent substantial damage to cell membrane structures under freezing stress. The melatonin used in this invention is not merely a wetting agent, but rather, due to its high lipophilicity, it specifically accumulates at the cell lipid membrane interface, effectively blocking the peroxidation chain reaction of membrane lipids under extreme dehydration conditions. This is the core requirement for maintaining the physical integrity of frozen cell membranes, reducing membrane permeability, and inhibiting browning.

[0113] Comparative Example 2 confirms the irreplaceable role of the time-sequential redox regulatory network composed of reduced glutathione (GSH) and L-ascorbic acid (AsA) in eliminating latent ROS storms. Experimental data show that in Comparative Example 2, after replacing the GSH and AsA combination with isotonic trehalose, the physical glass transition of the solution remained very stable in the early stages (crystallization rate <1.0%), but the germination rate of revived spores was only 42.7%, and a delayed-onset albinism necrosis syndrome of up to 48.2% appeared in the later stages of culture. These results indicate that the intracellular reactive oxygen species (ROS) storm induced by cryogenic freezing and the toxicity of cryoprotectants is a persistent latent damage that cannot be resolved simply by adjusting osmotic pressure with isotonic substances at the physical level. Only by relying on the time-sequential antioxidant relay network jointly constructed by GSH and AsA inside and outside the cell can the continuous accumulation of free radicals be fundamentally neutralized, thereby ensuring that revived spores overcome the stress period and possess long-term physiological and biochemical developmental vitality.

[0114] Comparative Example 3 confirms the decisive role of trace amounts of inositol hexaphosphate (IP6) in maintaining the physical stability of the solution. Tables 1 and 2 show that replacing IP6 with only an equimolar amount of the conventional chelating agent EDTA resulted in abnormal microemulsion turbidity at the end of the ice bath, with viscosity retention dropping to 78.4%, and triggering large-area explosive crystallization (>98.0%) in liquid nitrogen. After recovery, the REC surged to 92.5% due to mechanical shearing by ice crystals, and almost all spores were killed (only 1.2% survived with weak, abnormal germination). This indicates that conventional EDTA cannot completely shield trace transition metal ions under low-temperature, high-osmotic, and slightly acidic conditions, leading to a "pro-oxidation reversal" of AsA. The resulting free radicals slowly and continuously attacked and locally depolymerized the PEG 4000 macromolecular backbone. The disruption of the polymer network shifted the system's glass transition temperature, causing the solution to completely collapse upon cooling. The IP6 of this invention, with its absolute spatial steric hindrance of six-tooth ring-shaped full enclosure, perfectly locks in this fatal side reaction, stabilizes the physical foundation of the amorphous glassy state, and is the core barrier that helps dehydrated spores survive beyond physiological limits.

[0115] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for cryopreservation of spores of *Adiantum capillus-veneris* from Fujian, characterized in that... The method includes the following steps in sequence: Step 1: Spore collection and drying pretreatment: Collect mature spores of *Adiantum capillus-veneris* from Fujian and dry them to reduce the spore moisture content; Step 2, Loading solution treatment: The dried spores are placed in a loading solution for osmotic pre-protection treatment; Step 3, Modified vitrification solution treatment: The spores treated in Step 2 are transferred into a modified vitrification solution for vitrification, dehydration, and antioxidant protection. Step 4: Liquid nitrogen cryopreservation: The spores treated in step 3 are placed in liquid nitrogen for cryopreservation at ultra-low temperatures; Step 5: Rapid thawing: Remove the frozen spores and thaw them quickly in a water bath. Step 6, Gradient washing: The thawed spores are unloaded by gradient washing with sugar washing solutions of decreasing concentration; Step 7: Germination Culture: The washed spores are inoculated onto a culture medium for germination culture and effect detection.

2. The method according to claim 1, characterized in that, In step three, the modified vitrification solution uses plant basal culture medium as solvent, and its added components include: glycerol, ethylene glycol, dimethyl sulfoxide, polyethylene glycol, sucrose, reduced glutathione, L-ascorbic acid, melatonin, and inositol hexaphosphate; the melatonin is first dissolved in anhydrous ethanol and then added to the modified vitrification solution so that the final volume concentration of ethanol in the system is less than 0.1%.

3. The method according to claim 1, characterized in that, The modified vitrification solution was brought to a final volume of 100 mL with MS liquid medium, and the pH was 5.6-6.

0. The amount or concentration ratio of each component was: glycerol: 20-30 g. Ethylene glycol: 10~20g; Dimethyl sulfoxide: 8~12g; Polyethylene glycol 4000: 8~12g; Sucrose: 0.4~0.6 mol / L; Reduced glutathione: 0.5~1.0 mmol / L; L-ascorbic acid: 0.2~0.5 mmol / L; Melatonin: 50~100 μmol / L; Inositol hexaphosphate: 15~25 μmol / L.

4. The method according to claim 1, characterized in that, The pH of the modified vitrification solution is 5.8 to 6.

0.

5. The method according to claim 1, characterized in that, In step one, the drying conditions are: drying at a temperature of 20~30℃ and a relative humidity of 25%~35% for 48~72 hours, so that the spore moisture content reaches 6~8%.

6. The method according to claim 1, characterized in that, In step two, the loading solution uses MS liquid culture medium as the solvent and contains 1.5~2.5 mol / L glycerol and 0.3~0.5 mol / L sucrose; the treatment conditions are: temperature 20~30℃, shaking speed 80~120 rpm, and treatment time 25~30 minutes.

7. The method according to claim 1, characterized in that, In step three, the conditions for treating the modified vitrification solution are: treatment in an ice bath at 0-2°C for 40-50 minutes; the modified vitrification solution is pre-cooled before use and used within 30 minutes after preparation.

8. The method according to claim 1, characterized in that, In step five, the conditions for rapid thawing are: placing the cryovial in a water bath at 38~42℃ for thawing for 90~120 seconds.

9. The method according to claim 1, characterized in that, In step six, the gradient washing specifically involves washing with four levels of sucrose solutions of decreasing concentration, with each level having a sucrose concentration of 1.2 mol / L, 0.8 mol / L, 0.4 mol / L, and 0.2 mol / L, respectively.

10. The method according to claim 1, characterized in that, In step seven, the germination culture conditions are as follows: MS as the basal medium, with the addition of 0.5~1.5 mg / L 6-benzylaminopurine and 0.1~0.5 mg / L naphthaleneacetic acid; cultured for more than 30 days at a temperature of 23~27℃, a light intensity of 2000~3000 lux, and a photoperiod of 10~14 hours / day.

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