Method for improving vitrification ultralow-temperature preservation effect of anthurium andraeanum calluses

Abstract

The invention discloses a method for improving the vitrification ultralow-temperature preservation effect of anthurium andraeanum calluses. The method comprises the following steps: S1, pre-culturing the anthurium andraeanum calluses; s2, carrying out permeation protection on the pre-cultured anthurium andraeanum calluses by using loading liquid; s3, sucking out the loading liquid, and carrying out dehydration treatment on the percolated and protected anthurium andraeanum callus by using vitrification liquid containing plum blossom dehydrin protein; and S4, carrying out cryopreservation on the dehydrated anthurium andraeanum calluses by using liquid nitrogen. According to the method, 10 [mu] mol / L plum blossom dehydrin protein is added into the vitrification solution to serve as an exogenous additive substance, so that the cell activity after ultralow-temperature cryopreservation of the vaccinium bracteatum calluses is effectively improved, and the ultralow-temperature preservation effect of multiple vaccinium bracteatum varieties can be obviously improved, including Guanghua Ziyun, Xiaolinglin, Xianwang, Jun Zhongjiang and the like.

Description

Technical Field

[0001] This invention relates to the field of plant tissue preservation technology, and more specifically, to a method for improving the vitrification cryopreservation effect of Anthurium callus. Background Technology

[0002] Wedding candles Anthurium andraeanum Anthurium (Lind.), also known as flamingo flower, is a species of anthurium belonging to the genus Anthurium in the family Araceae. Anthurium Anthurium, a perennial evergreen herbaceous plant, is one of the most ornamental varieties in the Anthurium genus. Its flowers and leaves are beautiful, a delicate red and tender green, with an upright red spathe and a golden spadix. Under suitable conditions, it can bloom year-round and remains evergreen, making it a highly valued ornamental plant and one of the world's most famous cut and potted flowers. With the continuous increase in new varieties and growing market demand, this flower has developed into an important variety in my country's ornamental horticulture and cut flower industry, highly favored by consumers and continuously exported to the international market, leading to a growing demand for Anthurium germplasm resources. However, traditional methods of preserving Anthurium germplasm resources require a large amount of manpower. The resources, materials, and land available for Anthurium are limited, and the demand for large-scale, long-term preservation cannot be met. Furthermore, the main propagation methods for Anthurium include seed propagation, division propagation, and tissue culture. However, due to limited seed production and the need for timely sowing, these methods are mostly used for breeding research. Division propagation is less efficient, leading to a constraint on seedling supply. Moreover, Anthurium germplasm resources contain rich genetic information, which is of great significance for the breeding of new varieties and genetic improvement. However, Anthurium has a complex genetic background, and international basic research capabilities are very weak, resulting in a lack of biological information. Therefore, protecting the genetic integrity of Anthurium germplasm resources has become an urgent task.

[0003] The main methods for cryopreservation of plant materials include vitrification, freeze-drying, rapid freezing, two-step freezing, slow freezing, and embedding dehydration preservation. These methods utilize different principles and effects to stabilize plant materials at low temperatures. Among them, vitrification has become an important method for modern germplasm preservation due to its simplicity and low equipment requirements. This technology was first proposed by Luyet in the 1930s, and after continuous exploration by scientists, it was successfully applied to plant material preservation in 1989 by Uragami et al. The core principle of vitrification is to treat the plant material with a high concentration of cryoprotectant and then rapidly transfer it to liquid nitrogen, causing the plant material to form an amorphous glassy structure during rapid cooling. This process effectively avoids cell damage caused by ice crystal formation, while maintaining cell structural integrity through a high-viscosity solution, avoiding osmotic pressure imbalance and pH fluctuations. Currently, cryopreservation technology has been successfully applied to more than 30 economic crops, including ginger, red cherry, pineapple, red taro, almond, wild strawberry, papaya, and prickly pear, providing an innovative solution for germplasm resource preservation and is a relatively ideal method among plant germplasm resource preservation methods.

[0004] The core of ultra-low temperature vitrification preservation lies in the use of a vitrification solution (PVS). This solution, scientifically formulated with a mixture of permeable and non-permeable preservatives, is rapidly cooled to its glass transition temperature, causing the water within the plant material to form a non-crystalline vitrified solid structure. This allows the plant to be preserved in this vitrified state at low temperatures for extended periods. In this ultra-low temperature vitrification preservation system, the growth processes and metabolic activities of all cells are essentially stagnant, ensuring the relative biological stability of the plant material. Compared to traditional ultra-low temperature preservation methods, water molecules do not undergo recombination or structural and volume changes, avoiding the mechanical and osmotic damage caused by ice crystal formation in traditional freezing methods. This ensures the viability of cells after recovery from culture and improves the survival rate of the plant material, thus guaranteeing cell integrity and regeneration capacity. Furthermore, vitrification ultra-low temperature preservation requires simple equipment, does not require expensive instruments, and has a straightforward operation procedure. In addition, ultra-low temperature preservation technology can significantly extend the preservation time of germplasm resources, reduce land area, lower preservation costs, and is unaffected by environmental factors. Moreover, cryopreservation methods have been extended to diverse in vitro culture systems such as suspension cells, shoot tips, and callus tissues, with plant shoot tip preservation covering more than 170 species.

[0005] Anthurium callus, an important material in plant tissue culture, possesses strong regeneration capacity and good genetic stability. Studies have analyzed the biological stability of regenerated plants after cryopreservation using molecular marker technology, showing no change in biological stability. Callus culture exhibits rapid propagation and a high proliferation coefficient, enabling large-scale production and the generation of numerous regenerated plants, providing a rich source of material for the preservation and utilization of Anthurium germplasm resources. However, callus is prone to increased cell mutation rate and decreased genetic stability during long-term subculturing. Vitrification cryopreservation saves space, avoids subculturing, and solves these problems. Therefore, researching a suitable vitrification cryopreservation system for Anthurium callus is crucial for maintaining its genetic stability and regeneration capacity.

[0006] Currently, there are not many species of Anthurium plants that can be vitrified at ultra-low temperatures, and the preservation effects vary among different species or varieties. Therefore, it is necessary to optimize different Anthurium ultra-low temperature preservation systems. Summary of the Invention

[0007] To address the aforementioned technical problems, the present invention aims to provide a method for cryopreservation of anthurium callus using vitrification. Based on existing anthurium in vitro culture systems and a preliminary laboratory-established cryopreservation system for anthurium callus using vitrification, the cryopreservation conditions for anthurium are further optimized.

[0008] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for improving the cryopreservation effect of vitrification on anthurium callus, comprising the following steps: S1. Pre-culture of Anthurium callus tissue; S2. Use loading solution to permeate and protect the pre-cultured Anthurium callus; S3. Remove the loading solution and dehydrate the protected anthurium callus with a vitrifying solution containing plum blossom dehydrating protein. S4. The dehydrated anthurium callus was cryopreserved using liquid nitrogen.

[0009] As some specific embodiments of the present invention, the anthurium callus is anthurium callus that has been subcultured 4-6 times with a basic culture medium and has good growth status, with each subculture lasting 30-45 days.

[0010] As some specific embodiments of the present invention, the basic culture medium is a 1 / 2MS solid culture medium containing 25-35 g / L sucrose, preferably 30 g / L.

[0011] As some specific embodiments of the present invention, in step S1, the culture medium used for pre-culture is 1 / 2 MS culture medium containing 0.4-0.6 mol / L sucrose, preferably 0.5 mol / L; the pre-culture temperature is 22-25℃, preferably 25℃; the conditions are dark culture, and the pre-culture time is 1-2 days, preferably 2 days.

[0012] Furthermore, the basal culture medium and / or pre-culture medium also contain 1 mg / L of TDZ and 1 mg / L of 2,4-D.

[0013] As some specific embodiments of the present invention, in step S2, the loading solution is a 1 / 2 MS culture medium containing 0.3-0.5 M sucrose and 1.8-2.2 M glycerol, preferably with a sucrose concentration of 0.4 M and a glycerol concentration of 2 M.

[0014] In some specific embodiments of the present invention, in step S2, the temperature for permeation protection using the loading fluid is room temperature, and the time is 55-65 min, preferably 60 min. The term "room temperature" as used in the present invention has a meaning known in the art, specifically referring to 20-30°C, preferably 20-25°C.

[0015] As some specific embodiments of the present invention, in step S3, the vitrification solution is a 1 / 2 MS culture medium containing 0.3-0.5 mol / L sucrose, 25-35% (w / v) glycerol, 10-20% (w / v) ethylene glycol and 10-20% (w / v) dimethyl sulfoxide, preferably a 1 / 2 MS culture medium containing 0.4 mol / L sucrose, 30% (w / v) glycerol, 15% (w / v) ethylene glycol and 15% (w / v) dimethyl sulfoxide.

[0016] As some specific embodiments of the present invention, the concentration of plum blossom dehydrin protein in the vitrifying solution is 8-12 μmol / L, preferably 10 μmol / L.

[0017] As some specific embodiments of the present invention, in step S3, the temperature of the dehydration treatment is 0-4 ℃ and the time is 55-65 min, preferably 0 ℃ and 60 min.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention effectively improves the cell activity of Anthurium callus after cryopreservation by adding 10 μmol / L of plum blossom dehydrin protein to the vitrification solution as an exogenous additive. It can also significantly improve the cryopreservation effect of multiple Anthurium varieties, including Pink Champion, Guanghua Ziyun, Xia Zhilin, Zong Wangguo, and Chun Zhiyun. Attached Figure Description

[0019] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 This is a schematic diagram of the Anthurium plant material and its callus in Example 1. In this diagram, A is a schematic diagram of the Anthurium variety 'Pink Champion', B is a schematic diagram of the young leaves of Anthurium, C is a schematic diagram of the callus after 14 days of induction, and D is a schematic diagram of the well-grown Anthurium callus block used for subsequent experiments. Figure 2 The graph shows the survival rate of anthurium callus under cryopreservation with different pre-culture times and loading solutions in Example 1. Figure 3 The graph shows the survival rate of eccentric anthurium callus after cryopreservation using PVS2 vitrifying solution with different exogenous substances added in Example 1. Figure 4 This is a schematic diagram showing the recovery culture of Anthurium callus after 7 days of cryopreservation using P-type dehydrating agent P as an exogenous substance added to vitrification solution PVS2 in Example 1. Detailed Implementation

[0020] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0021] Example 1 1. Experimental materials 1.1 Experimental Subjects The explant materials used in the experiment were tender, incompletely unfolded leaves of six varieties: Pink Champion, Guanghua Ziyun, Xia Zhilin, Zong Wangguo, and Chun Zhiyun.

[0022] 1.2 Experimental Reagents 1 / 2MS powder, sucrose, agar, thidiazuron (TDZ), 2,4-dichlorophenoxyacetic acid (2,4-D), glycerol (glycerol), dimethyl sulfoxide (Me2SO), ethylene glycol, triphenyltetrazolium chloride (TTC), 95% ethanol, liquid nitrogen, lotus dehydrin, plum dehydrin, dehydrin protein SK3 and Y2SK2.

[0023] 1.3 Experimental Instruments and Equipment Electronic balance (JA1003, Shanghai Liangping Instrument Co., Ltd.), large-scale light incubator (CMP0, Controlled Environments Limited), constant temperature water bath (DK-S 24, Shanghai Jinghong Experimental Equipment Co., Ltd.), ultra-clean workbench (BHC-1000ⅡA2B2, Shanghai Lichen Bangxi Instrument Technology Co., Ltd.), ultraviolet spectrophotometer (B269-291600, Shanghai Heshang Scientific Instrument Co., Ltd.), etc.

[0024] 1.4 Culture medium and solution formulation Basic culture medium: 1 / 2 MS + 30 g / L sucrose + 20 g / L agar powder + 1 mg / L TDZ + 1 mg / L 2,4-D; Pre-medium: 1 / 2 MS + 0.5 mol / L sucrose + 1 mg / L TDZ + 1 mg / L 2,4-D; Loading solution I: 1 / 2 MS + 0.4 mol / L sucrose + 2 mol / L glycerol; Loading fluid II (50% PVS2): prepared by mixing 1 / 2 MS liquid and PVS2 solution at a volume ratio of 1:1; Glass transition solution PVS2: 1 / 2 MS + 0.4 mol / L sucrose + 30% (w / v) glycerol + 15% (w / v) ethylene glycol + 15% (w / v) Me2SO; Washing buffer (Dilu): 1 / 2 MS + 1.2 mol / L sucrose; After the above culture media and solutions are prepared, they are all sterilized in an autoclave before use.

[0025] 2. Experimental Methods 2.1 Induction of Anthurium callus Six different varieties were cut from the greenhouse (e.g., Figure 1 As shown in A, the young leaves of the anthurium (such as the pink champion variety) Figure 1 (B), rinse with running water for 30 min, transfer to a laminar flow hood, rinse twice with sterile water → soak in 75% alcohol for 20 s → rinse three times with sterile water → disinfect with 1.3% sodium hypochlorite for 15 min → rinse four more times with sterile water → blot dry with filter paper, finally cut into small squares about 1 cm in length and width, inoculate onto 1 / 2 MS basal medium, transfer to an incubator for dark culture at 25℃ to obtain anthurium callus (e.g., ... Figure 1 As shown in C, this is the callus tissue after 14 days of induction. Subculture was performed every 30-45 days, for a total of 4-6 subcultures, to obtain well-grown anthropogenic callus tissue (e.g., ...). Figure 1As shown in D, the eccentric anthurium callus was cultured once every 30 days, for a total of 6 subcultures and 180 days, and was then subjected to subsequent vitrification and cryopreservation experiments.

[0026] 2.2 Vitrification Cryogenic Preservation Scheme (1) Pre-culture: Selected eccentric anthurium callus blocks with good growth and consistent physiological state, inoculated onto 1 / 2 MS solid medium with 0.5 mol / L sucrose concentration, and cultured in the dark at 25℃.

[0027] (2) Osmotic protection / loading: After the pretreated eccentric anthracis callus was cut into small pieces, it was placed in a 2 mL cryovial and 1.8 mL of loading solution I was added for osmotic protection. The mixture was treated at room temperature for 60 min. (3) Dehydration: After removing the loading liquid I, add 1.8 mL of glass transition liquid PVS2 to the cryovial and dehydrate at 0℃ for 60 min.

[0028] (4) Freezing and thawing: The cryovial was quickly immersed in liquid nitrogen and frozen for 1 hour, then thawed rapidly in a 40°C water bath for 90 seconds.

[0029] (5) Washing: Aspirate the glass transition liquid PVS2 and wash with washing solution at room temperature for 30 min, changing the washing solution every 10 min.

[0030] (6) Recovery culture: After the treated eccentric callus blocks were placed in a basic culture medium and recovered in a dark environment for 24 h, their relative survival rate was determined.

[0031] 2.3 Optimization of pre-incubation time and osmotic protection loading solution (1) Pre-culture: Selected callus blocks of eccentric anthurium with good growth and consistent physiological state after 6 subcultures (180 days) were inoculated onto pre-culture medium and cultured in the dark at 25℃ for 1 and 2 days. (2) Permeation protection / loading: After the pretreated anemonetic anemone callus blocks were cut into small pieces, they were placed in 2 mL cryovials and 1.8 mL loading solution (Ⅰ, Ⅱ) were added for permeation protection for 1 h.

[0032] Perform the remaining operations according to steps (3) to (6) in section 2.2. The entire vitrification cryogenic treatment experiment was repeated three times.

[0033] 2.4 Constructing different vitrification solutions by adding different exogenous substances to PVS2 The glass transition solution is prepared as shown in Table 1 below: Table 1. Glass Transition Solution Formulation Table

[0034] Follow these steps: (1) Pre-culture: Selected callus blocks of eccentric anthurium with good growth and consistent physiological state after 6 subcultures (180 days), inoculated onto pre-culture medium, and cultured in the dark at 25℃ for 2 days; (2) Permeation protection: After the pretreated partial embryonic anthracis callus was cut into small pieces, it was placed in a 2 mL cryovial and 1.8 mL of loading solution I was added for permeation protection for 60 min. (3) Dehydration: After removing the loading solution, add 1.8 mL of the glass transition solution with added exogenous substances as shown in Table 1 above to the cryovial and dehydrate at 0℃ for 60 min. Perform the remaining operations according to steps (4) to (6) in section 2.2. Each treatment experiment was repeated three times.

[0035] 3. Result Verification 3.1 Viability Validation Method – TTC Assay for Cell Viability The principle of the TTC method is that metabolically active cells convert water-soluble triphenyltetrazolium chloride (TTC) into a lipid-soluble red formazan derivative through a biocatalytic reduction reaction mediated by mitochondrial dehydrogenases. The absorbance (TTC value) of the supernatant is measured at a wavelength of 485 nm using a spectrophotometer. The TTC value represents the cryopreservation rate of cells from eccentric anthurium callus after vitrification and cryopreservation following various treatments. The procedure is as follows: (1) Prepare 0.8% TTC solution: Weigh 0.8g of triphenyltetrazolium chloride (TTC) and add it to 100 mL of 50 mmol / L phosphate buffer solution with pH 7.4; (2) Weigh about 50 mg of the sample and put it into a 10 mL centrifuge tube, and add 2 mL of 0.8% TTC solution; (3) Stain at 25℃ for 20 h; (4) Use a pipette to aspirate the TTC solution, and then rinse with sterile water 3 times; (5) Remove the distilled water, add 5 mL of 95% ethanol, and then place the centrifuge tube in an 80°C water bath for 50 min; (6) Take the supernatant and measure the absorbance at a wavelength of 485 nm using a spectrophotometer, with 95% ethanol as a blank control; (7) Calculate the survival rate: Survival rate (%) = (TTC value of treated sample / TTC value of untreated sample) × 100%.

[0036] Among them, the processed sample refers to the sample after the processed eccentric callus block has been placed in a basic culture and recovered in a dark environment for 24 hours.

[0037] Untreated samples refer to normally cultured embryogenic callus, that is, embryogenic callus that has not undergone any pre-culture or cryopreservation process.

[0038] 3.2 Verification Results 3.2.1 Validation results of survival rate after optimization of pre-incubation time and osmotic protection loading solution In the experiment in section 2.3, two levels of pre-incubation time were set, namely 1 and 2 days, and two levels of loading solution were also set, namely loading solution I and loading solution II. The survival rate verification results are shown in Table 2 below. Figure 2 As shown, Figure 2 Different lowercase letters on the middle column represent the differences between groups when the significance level is set to 0.05.

[0039] Table 2. Validation results of pre-incubation time and loading solution optimization for survival rate.

[0040] From Table 2 above and Figure 2 It can be seen that the cell viability did not change significantly when the pre-culture time was 1 day and the loading solution I was used, and when the pre-culture time was 2 days and the loading solution II was used. However, when the pre-culture time was 2 days and the loading solution I was used, the cell cryopreservation rate showed a significant difference, indicating that the degree of damage to the eccentric anthurium callus cells was significantly reduced under this treatment, which is conducive to cell survival. The results of this experiment show that the effect of pre-culture for 2 days and loading solution I is better.

[0041] 3.2.2 Verification results of constructing different vitrification solutions by adding different exogenous substances to PVS2 The experimental group with a pre-culture time of 2 days, loading medium I, and no addition of exogenous PVS2 vitrification solution was used as the control group (CK), and its cell viability was calculated to be 30.64%.

[0042] Table 1 shows the verification results of four different vitrification solutions constructed with different exogenous substances. Figure 3 And as shown in Table 3 below: Table 3 Verification Results of Glass Tempering Fluid Types

[0043] As shown in Table 3 above, the addition of endogenous resistance protein dehydrin to the vitrification solution PVS2 significantly improved the relative cell survival rate of cryopreserved Anthurium callus. In particular, the dehydrin protein PVS2-P, at 10 μmol / L, could increase the cell survival rate by 18.26% compared with the control group, which is a very significant improvement.

[0044] like Figure 3As shown, the different lowercase letters on each column represent the differences between groups when the significance level is set at 0.05. It can be seen that adding different exogenous substances to the vitrification solution during the dehydration stage significantly improved the cell vitrification rate of *Anthurium stenoptera* callus blocks after preservation using the ultra-low temperature vitrification method. In particular, treatment with 10 μmol / L *Pyrophyllum oxypetalum* P resulted in a cell viability rate as high as 48.90% after 1 day of recovery culture, and even higher after 7 days of recovery culture (e.g., ...). Figure 4 The diagram shows a partial embryonic callus after 7 days of recovery culture. The scale bar in the diagram represents 1 cm. It can be seen that some partial embryonic callus is in a dense yellow callus state and has activity (it can achieve normal proliferation with continued culture). The cell survival rate is still 31.56%, indicating that the optimal cryopreservation system for Anthurium 'Pink Champion' is a pre-culture time of 2 days, loading with loading solution I for 60 minutes, and dehydration treatment with vitrification solution PVS2-P at 0℃ for 60 minutes.

[0045] Example 2 The experiment of the Anthurium cultivar Pink Champion in Example 1 above under an optimized cryogenic preservation system was applied to four Anthurium cultivars: Guanghua Ziyun, Xia Zhilin, Zong Wangguo, and Chun Zhiyun.

[0046] All four varieties were pre-cultured for 2 days, loaded with medium I, and vitrified in PVS2 without the addition of exogenous substances as the control group (CK). The experimental group with the highest cell survival rate of the Pink Champion variety (with 10 μmol / L Plum Blossom Dehydrin P added to the vitrified in PVS2) was applied to all four Anthurium varieties. The survival rate was tested, and the results are shown in Table 4 below.

[0047] Table 4. Survival rate verification results of different Anthurium varieties.

[0048] As shown in Table 4 above, when the Guanghua Ziyun variety was dehydrated by adding 10 μmol / L Plum blossom dehydrating agent P to the vitrification solution PVS2, the cell viability increased by 5.13% compared with the control group after cryopreservation, which showed a significant difference. This indicates that the PVS2 of plum blossom dehydrating agent significantly improved the cryopreservation effect of the Guanghua Ziyun variety of Anthurium.

[0049] The relative cell survival rate of the Xia Zhi Lin variety was increased by 8.35% compared with the control group when treated with PVS2 of Plum Blossom Dehydrating Agent, showing a significant difference. For the Xia Zhi Lin variety, PVS2 of Plum Blossom Dehydrating Agent can also improve the cryopreservation effect of the Xia Zhi Lin variety anthurium.

[0050] Treatment of the *Anthurium arvense* cultivar with PVS2 of *Prunus mume* increased cell viability by 7.61% compared to the control group. Therefore, PVS2 of *Prunus mume* can also improve the cryopreservation effect of *Anthurium arvense* cultivar *Prunus mume*.

[0051] The cell viability of the Spring Rhyme variety treated with PVS2 of Plum Blossom Dehydrating Agent increased by 15.61% compared with the control group, which is a very significant difference. Therefore, PVS2 of Plum Blossom Dehydrating Agent can also improve the cryopreservation effect of Spring Rhyme variety Anthurium.

[0052] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for improving the vitrification effect of cryopreservation of Dianthus chinensis callus, characterized in that, The method comprises the following steps: S1, pre-culturing the callus of Magnolia liliiflora Desr; S2, using loading liquid to perform osmotic protection on the callus of Magnolia liliiflora Desr after pre-culturing; S3, sucking the loading liquid, and using vitrification liquid containing M. liliiflora dehydrin to perform dehydration treatment on the callus of Magnolia liliiflora Desr after osmotic protection; S4, using liquid nitrogen to freeze the callus of Magnolia liliiflora Desr after dehydration treatment.

2. The method of claim 1, wherein, The callus of Magnolia liliiflora Desr is well-grown callus of Magnolia liliiflora Desr after being subcultured for 4-6 times with a basic medium, and the time for each subculture is 30-45 days.

3. The method of claim 2, wherein, The basic medium is 1 / 2MS solid medium containing 25-35 g / L sucrose.

4. The method of claim 1, wherein, In step S1, the medium used for pre-culturing is 1 / 2MS liquid medium containing 0.4-0.6 mol / L sucrose, the pre-culturing temperature is 22-25℃, the pre-culturing condition is dark culture, and the pre-culturing time is 1-2 days.

5. The method of claim 4, wherein, The pre-culturing time is 2 days.

6. The method of claim 1, wherein, In step S2, the loading liquid is 1 / 2MS liquid medium containing 0.3-0.5 M sucrose and 1.8-2.2 M glycerol.

7. The method of claim 1, wherein, In step S2, the temperature for using the loading liquid to perform osmotic protection is room temperature, and the time is 55-65 min.

8. The method of claim 1, wherein, In step S3, the vitrification liquid is 1 / 2MS liquid medium containing 0.3-0.5 mol / L sucrose, 25-35%(w / v) glycerol, 10-20%(w / v) ethylene glycol and 10-20%(w / v) dimethyl sulfoxide.

9. The method of claim 1, wherein, In step S3, the concentration of M. liliiflora dehydrin in the vitrification liquid is 8-12 μmol / L.

10. The method of claim 1, wherein, In step S3, the dehydration treatment temperature is 0-4℃, and the time is 55-65 min.

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