Phalaenopsis Roche protocorm preservation method

Abstract

The invention relates to a preservation method of phalaenopsis Rosei protocorm. The preservation method comprises the following steps: obtaining the phalaenopsis Rosei protocorm; the protocorm is soaked in a pre-culture solution, pre-culture is conducted for a certain time, and the pre-culture solution comprises 0.25 M to 0.45 M saccharose; soaking the pre-cultured protocorm into a loading solution, and carrying out loading treatment for a certain time; soaking the loaded protocorm into a vitrification solution, and vitrifying for a certain time; carrying out ultra-low temperature treatment on the vitrified protocorm in an environment of-190 DEG C; and storing the protocorm subjected to the ultra-low temperature treatment. The overall trend of each physiological index of each key step of the Phalaenopsis Roche preserved by the method provided by the invention is beneficial to better growth and development of cells, and low-temperature freezing injury to the protocorm of the Phalaenopsis Roche caused by low-temperature stress can be effectively inhibited to a certain extent.

Description

Technical Field

[0001] The present invention relates to the field of ecology, and particularly to a method for preserving protocorms of Phalaenopsis lobbii. Background Art

[0002] Phalaenopsis has a peculiar flower shape, bright colors, and a flowering period of several months. It is an indispensable ornamental flower for festivals and has extremely high ornamental and economic value. Due to the overexploitation of Phalaenopsis plants by humans because of their excessive fame, and the serious destruction of their habitats, their distribution range has become smaller and their numbers have become fewer in the past decade. Many varieties are facing the threat of endangerment. Given the serious survival crisis faced by the Phalaenopsis population, it has currently been listed in the "Red List" by the International Union for Conservation of Nature and Natural Resources (IUCN).

[0003] Orchidaceae plants usually reproduce by division, but Phalaenopsis is a monopodial epiphytic orchid with very few lateral branches. Conventional asexual reproduction is difficult to implement and cannot be carried out on a large scale. Phalaenopsis plants can also be propagated by seeds, but their embryos are incompletely developed and there is no endosperm. They must symbiose with fungi in the natural environment, but their germination speed is very slow and it is difficult to meet the requirements of large-scale propagation. Using tissue culture technology, a large number of tissue culture seedlings can be obtained in a very short time, thus increasing the reproduction speed of Phalaenopsis.

[0004] The protocorm pathway is a special morphology unique to Orchidaceae plants during tissue culture and is also a main way for the rapid propagation of this type of plant. The sources of Phalaenopsis protocorms include explants such as stems, shoot tips, leaves, seeds, and flower stalks.

[0005] Phalaenopsis lobbii is a plant of the genus Phalaenopsis and is a rare and endangered species unique to China. It has been included in the "China Biodiversity Red List - Higher Plants Volume" (2013 - 9). Currently, this species has been included in the "IUCN Red List", and at the same time it is a wild plant with an extremely small population that China gives priority to saving and protecting. According to the "Implementation Plan for the Rescue and Protection of Wild Plants with Extremely Small Populations in China (2010 - 2015)" by the State Forestry Administration of China, the proportion of this species in the wild population is less than 50 plants, and in the "Implementation Plan for the Rescue and Protection Project of Wild Plants with Extremely Small Populations in Guangxi (2012)" it is less than 10. Therefore, the protection of the germplasm resources of Phalaenopsis lobbii is an urgent problem to be solved. Cryopreservation technology is one of the most effective methods for germplasm resource preservation.

[0006] In the prior art, there are studies on the preservation of germplasm resources using cryopreservation by vitrification and cryopreservation by droplet-vitrification. Cryopreservation by droplet-vitrification is similar to cryopreservation by vitrification. The biggest difference is that a high-concentration vitrification cryoprotectant is dropped onto an aluminum foil strip, and then the aluminum foil strip is transferred to a freezing tube for liquid nitrogen preservation. Based on cryopreservation by vitrification, cryopreservation by droplet-vitrification is an efficient cryopreservation method in which small droplets of PVS2 solution are placed on highly thermally conductive materials such as aluminum foil on the basis of cryopreservation by vitrification. The rapid freezing and rewarming during the freezing process are the keys to ensuring that the solutes in the sample and the solutes in the environment are completely vitrified and preventing problems such as rupture and recrystallization during the rewarming process. In the droplet technique, due to the use of aluminum foil with good thermal conductivity and the addition of a small-volume cryoprotectant to the outer packaging, the risk of intracellular freezing is greatly reduced.

[0007] However, cryopreservation by droplet-vitrification cannot completely solve the problem of cryopreservation of plants, such as cell dehydration caused by cytotoxicity during vitrification, and chemical poisoning or excessive osmotic damage caused by water loss. Different species, genera, and tissues to be preserved have different characteristics, such as environmental adaptability, cell water content ratio, the amounts of sugars, proteins, and free ions in cells, etc. Therefore, for different plant genera, more adaptable preservation methods are still needed. Summary of the Invention

[0008] In view of the technical problems existing in the prior art, the present invention provides a method for preserving protocorms of Phalaenopsis lobbii, comprising: obtaining protocorms of Phalaenopsis lobbii; immersing the protocorms in a pre-culture solution and pre-culturing for a certain period of time; wherein, the pre-culture solution includes 0.25M - 0.45M sucrose, such as 0.27 - 0.43M, 0.3 - 0.4M, or any sub-range or value therebetween; immersing the pre-cultured protocorms in a loading solution and performing a loading treatment for a certain period of time; immersing the loaded protocorms in a vitrification solution and vitrifying for a certain period of time; immersing the vitrified protocorms in small droplets containing the vitrification solution; performing cryopreservation on the small droplets containing the protocorms in an environment of -190°C to -220°C; and preserving the cryopreserved protocorms.

[0009] The preservation method as described above, wherein the diameter of the protocorms is 1 mm - 2 mm, such as 1.2 - 1.5 mm, 1.3 - 1.7 mm, 1.5 - 1.8 mm, or any sub-range or value therebetween.

[0010] The preservation method as described above, wherein the protocorm is obtained by germinating plant tissues or seeds for 58 - 62 days; preferably, the protocorm is obtained by germinating plant tissues or seeds for 60 days. Among them, the tissues include but are not limited to stems, shoot tips, leaves, flower stalks, etc.

[0011] The preservation method as described above, wherein the preculture solution contains 0.3M - 0.4M sucrose.

[0012] The preservation method as described above, wherein the loading solution contains (1 - 5) g / L of 1 / 2MS, (0.1 - 1) M / L of sucrose, (1 - 4) M / L of glycerol, and (50 - 200) mL / L of coconut milk; preferably, the loading solution contains 2.3 g / L of 1 / 2MS, 0.4 M / L of sucrose, 2 M / L of glycerol, and 100 mL / L of coconut milk.

[0013] The preservation method as described above, wherein the vitrification solution contains (1 - 5) g / L of 1 / 2MS without agar and sucrose, (100 - 500) mL / L of glycerol, (100 - 200) mL / L of ethylene glycol, (0.1 - 0.9) M / L of sucrose, and (100 - 200) mL / L of dimethyl sulfoxide; preferably, the vitrification solution contains 2.3 g / L of 1 / 2MS without agar and sucrose, 300 mL / L of glycerol, 150 mL / L of ethylene glycol, 0.4 M / L of sucrose, and 150 mL / L of dimethyl sulfoxide.

[0014] The preservation method as described above, wherein the vitrification time is 1.5 h - 3 h; preferably, the vitrification time is 2 - 2.5 h, such as 2.2 - 2.6 h, 2.3 - 2.8 h, 2.5 h or any sub - range or value therebetween.

[0015] The preservation method as described above, which includes cryopreserving the vitrified protocorm in an environment below - 190°C for about 0.5 - 2 h, such as about 1 - 2 h, 1 - 1.5 h, about 1 h, etc.

[0016] A method for rewarming cryopreserved protocorms, comprising: subjecting the cryopreserved protocorms obtained by the method of the present invention above to a constant temperature treatment at about 40 - 60°C for 75 - 120 s, such as 80 - 110 s, 95 - 115 s, 90 - 100 s, 90 s or any sub - range or value therebetween; preferably 90 s; and immersing the protocorms subjected to the constant temperature treatment into an unloading solution for unloading treatment.

[0017] The method as described above, wherein the immersion time of the protocorm in the unloading solution is 15 - 30 min, such as 16 - 25 min, 18 - 28 min, 20 min or any sub - range or value therebetween.

[0018] The method as described above, wherein the unloading solution comprises (1 - 5) g / L of 1 / 2MS, (0.5 - 2) M / L, and (50 - 150) mL / L of coconut milk; preferably, the unloading solution comprises 2.3 g / L of 1 / 2MS, 1.2 M / L, and 100 mL / L of coconut milk.

[0019] The vitrification method of small droplets in this application is simple to operate, and the survival rate of the protocorms of Phalaenopsis lobbii preserved is the highest. For each key step of the Phalaenopsis lobbii preserved by the method of this application, the overall trend of each physiological index is beneficial to the better growth and development of cells, and can effectively inhibit the low-temperature damage to the protocorms of Phalaenopsis lobbii caused by low-temperature stress to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Next, the preferred embodiments of the present invention will be further described in detail with reference to the drawings, wherein:

[0021] Figure 1 Showing the survival rate of the protocorms of Phalaenopsis lobbii determined by the TTC method under pre-culture with different sucrose concentrations according to an embodiment of the present invention;

[0022] Figure 2 Showing the TTC survival rate of the protocorms of Phalaenopsis lobbii under different PVS2 treatment times according to an embodiment of the present invention;

[0023] Figure 3 Showing the content of soluble sugar during the cryopreservation process of the protocorms of Phalaenopsis lobbii by the vitrification method of small droplets according to an embodiment of the present invention; wherein, F is the protocorm taken out of the bottle without any operation; CK is the protocorm taken out of the bottle and directly put into a cryotube, immersed in liquid nitrogen for 1 h and then taken out; PC is the protocorm after pre-culture; PC + LS is the protocorm after pre-culture - loading; PC + LS + PVS2 is the protocorm after pre-culture - loading - PVS2; PC + LS + PVS2 + UL is the protocorm after pre-culture - loading - PVS2 - liquid nitrogen cryopreservation - unloading; the same hereinafter;

[0024] Figure 4 Showing the content of soluble protein during the cryopreservation process of the protocorms of Phalaenopsis lobbii by the vitrification method of small droplets according to an embodiment of the present invention;

[0025] Figure 5 Showing the content of proline during the cryopreservation process of the protocorms of Phalaenopsis lobbii by the vitrification method of small droplets according to an embodiment of the present invention;

[0026] Figure 6 Showing the content of malondialdehyde during the cryopreservation process of the protocorms of Phalaenopsis lobbii by the vitrification method of small droplets according to an embodiment of the present invention;

[0027] Figure 7 Show the content of peroxidase during the cryopreservation of protocorms by the droplet vitrification method of Phalaenopsis lobbii according to an embodiment of the present invention;

[0028] Figure 8 Show the content of superoxide dismutase during the cryopreservation of protocorms by the droplet vitrification method of Phalaenopsis lobbii according to an embodiment of the present invention; and

[0029] Figure 9 Show the content of catalase during the cryopreservation of protocorms by the droplet vitrification method of Phalaenopsis lobbii according to an embodiment of the present invention. Detailed implementation manners

[0030] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0031] In the following detailed description, reference may be made to the various specification drawings that form a part of this application and illustrate specific embodiments of the application. In the drawings, like reference numerals generally describe substantially similar components in different figures. The various specific embodiments of the present application have been described in sufficient detail below to enable those of ordinary skill in the art with relevant knowledge and technology to implement the technical solutions of the present application. It should be understood that other embodiments may also be utilized or structural and logical changes may be made to the embodiments of the present application.

[0032] The proprietary terms involved herein have the following meanings:

[0033] The proprietary term "protocorm" refers to a special form unique to orchid plants during the tissue culture process, and is also a main way for the rapid propagation of this type of plants. The sources include explants such as stems, shoot tips, leaves, seeds, and flower stalks. Using protocorms for test-tube asexual propagation lays the foundation for the industrial production of Phalaenopsis.

[0034] The proper noun "germplasm resources" is also known as variety resources, genetic resources or gene resources. It refers to the general term for plants carrying various different genetic materials, including varieties or strains of cultivated, wild and artificially created plants. Among them, germplasm is the genetic material that is passed from parents to offspring through germ cells or somatic cells and determines the inherent biological traits. Germplasm resource preservation refers to the technology of storing plant germplasm under natural or artificially created suitable environmental conditions to maintain its vitality and heredity. In some embodiments, the germplasm resources can be protocorms, leaves, roots, shoot tips, etc. of plants. In other embodiments, the germplasm resources can be cells of plants.

[0035] The proper noun "preculture solution" refers to the treatment carried out before the cryogenic treatment of protocorms, which is used to improve the damage of cryogenic temperature to protocorm cells and meristems and increase the survival rate of protocorms. In some embodiments, the preculture solution contains sucrose and 1 / 2MS or is composed of them.

[0036] The proper noun "PVS2 (plant vitrification solution 2)" refers to plant vitrification solution 2, corresponding to PVS1 and PVS3, etc. It is made of sucrose, 1 / 2 MS, glycerol, ethylene glycol and dimethyl sulfoxide. PVS2 can effectively protect a variety of plant materials during cryopreservation, avoiding crystallization phenomenon and solute damage.

[0037] In some embodiments, the preparation method of 1 L PVS2 solution is as follows: Take 2.3 g of 1 / 2MS without agar and sucrose, add 300 mL of glycerol, 150 mL of ethylene glycol, 0.4M sucrose, make up the volume to 1000 mL with distilled water, adjust the pH to 5.8, put it into a high-temperature sterilization pot and sterilize at 121 °C for 20 min. After cooling, add 150 mL of filtered dimethyl sulfoxide (DMSO) and store it in a 4 °C refrigerator for standby (try to prepare it and use it immediately).

[0038] The proper noun "CK" refers to the control group, which here refers to the sample obtained by directly putting the protocorms into a cryotube, immersing them in liquid nitrogen for 1 h and then taking them out.

[0039] The proper noun "PC" refers to the protocorms after preculture.

[0040] The proper noun "loading" refers to the operation of mixing protocorms in a loading solution to avoid the damage of cells caused by drastic changes in osmotic pressure. In some embodiments, the loading solution is LS solution. In some embodiments, the loading solution contains 1 / 2MS, sucrose, glycerol and coconut milk.

[0041] In some embodiments, the method for preparing 1 L of loading solution is as follows: Take 2.3 g of 1 / 2 MS without agar and sucrose, add 0.4 M of sucrose, 2 M of glycerol, and 100 mL of coconut juice. Make up the volume to 1000 mL with distilled water, adjust the pH to 5.8, place it in an autoclave and sterilize at 121 °C for 20 min. After cooling, store it in a 4 °C refrigerator for later use.

[0042] The proprietary term "unloading" or "UL" refers to UnLoading. Unloading is the process of restoring the viability of cryopreserved protocorms. In some embodiments, the method for preparing 1 L of unloading solution is as follows: Take 2.3 g of 1 / 2 MS without agar and sucrose, add 1.2 M of sucrose and 100 mL of coconut juice. Make up the volume to 1000 mL with distilled water, adjust the pH to 5.8, place it in an autoclave and sterilize at 121 °C for 20 min. After cooling, store it in a 4 °C refrigerator for later use.

[0043] The proprietary term "coconut juice" refers to the juice inside the coconut fruit. According to an embodiment of the present application, coconut juice is added to the loading solution or the unloading solution to provide the nutrients required at each stage of the protocorm, including nitrogen source, vitamins, etc.

[0044] The proprietary term "soluble sugar" refers to soluble sugar, abbreviated as SS. Its content reflects the viscosity of the cytoplasm. The higher the content of soluble sugar, the higher the viscosity of the cytoplasm, indicating a higher freezing point and stronger water retention ability.

[0045] The proprietary term "soluble protein" refers to soluble protein, abbreviated as SP. A higher content of soluble protein in plants can generate substances such as proline (abbreviated as Pro) when plants are under low-temperature stress, thus alleviating low-temperature damage.

[0046] The proprietary term "malondialdehyde" is abbreviated as MDA, which is an important physiological and biochemical index of plants. It is a negative product of cell membrane oxidation, can reflect the degree of lipid peroxidation of cell membranes, and is one of the important indicators to measure whether plant tissues are damaged during cryopreservation. The higher the MDA content, the greater the damage to the cell membrane and the lower its cell activity.

[0047] The proprietary term "reactive oxygen species" is abbreviated as ROS, and its content is an important parameter affecting the activity rate of plants. Research shows that a higher ROS is the main reason for the decrease in the activity rate of plants after cryopreservation.

[0048] The proprietary terms "superoxide dismutase" is abbreviated as SOD, "peroxidase" is abbreviated as POD, and "catalase" is abbreviated as CAT. The antioxidant enzyme system formed by the cooperation of SOD, POD, and CAT can eliminate excessive reactive oxygen species in cells.

[0049] The term "ultra-low temperature" generally refers to a temperature below -190°C, such as the temperature of liquid nitrogen. In some embodiments, the ultra-low temperature is -196 to -210°C. In ultra-low temperature preservation, long-term preservation of genetic resources based on liquid nitrogen is not only simple and safe, but also greatly reduces preservation costs and saves storage space.

[0050] In the present application, the material selected for ultra-low temperature preservation of protocorms by the small droplet vitrification method is the protocorms of Phalaenopsis rothenbergii obtained after aseptic germination of seeds for 60 days, which have good growth, uniform size, and a diameter of about 1-2 mm.

[0051] Effects of pre-culture sucrose concentration and vitrification time on the survival rate of protocorms:

[0052] Successful cryopreservation must avoid lethal intracellular freezing when rapidly cooled in liquid nitrogen (Sakai and Yoshida 1967). Therefore, cells and tissues to be cryopreserved must be completely dried before being vitrified in liquid nitrogen. The vitrification (glass formation) process of cryopreservation does not require controlled freezing rates and can be cryopreserved by transferring cells and meristems directly to liquid nitrogen. Vitrification is replaced by a highly concentrated vitrification solution (removing all or most of the freezable cellular water at room temperature or 0°C). This method is critical for cryopreservation of macroscopic tissues or organs such as meristems and somatic embryos.

[0053] In the present application, the protocorms of Phalaenopsis rothenbergii were treated with a pre-culture solution of 0.4M sucrose concentration and cultured in a 120 rpm dark shaking incubator for two days, and a higher survival rate was shown in a PVS2 solution for 2 h. When the protocorms of Phalaenopsis rothenbergii were treated with a pre-culture solution of 0.3M sucrose concentration, the same 120 rpm dark shaking incubator was cultured for two days, and the PVS2 treatment group for 2.5 h showed a higher survival rate of up to 98.10%. Therefore, it can be considered that the 0.3M sucrose solution is the most suitable concentration for the pre-culture solution of Phalaenopsis rothenbergii.

[0054] Effects of cryopreservation steps of small droplet vitrification on the physiology of protocorms: Under stress, plant cells will undergo a series of physiological changes, which are to maintain the balance of cells to support their normal growth or development. Before ultra-low temperature, pre-culture and dehydration steps can greatly reduce the proportion of free water in cells, thereby avoiding damage to cell membranes and cell structures caused by intracellular freezing as much as possible. When plants are subjected to low temperature stress, POD, SOD, CAT, MDA, free proline, soluble protein and soluble sugar play an important role in maintaining balance.

[0055] However, there are differences in the mechanism of action among different species and different cold tolerances. Compared with normal protocorms (i.e., treatment group F), during pre-culture, the high-concentration sucrose solution in the pre-culture solution will reduce the water content in protocorm cells due to the change in the solution concentration gradient between cells, and the dehydration process will further reduce the water content of the cells.

[0056] Soluble proteins, soluble sugars, and free proline are all important osmotic adjustment substances, which can regulate cell osmotic potential and freezing point, and improve the cold resistance of plants. In Figure 3 the soluble sugar content of the protocorms of Phalaenopsis lobbii after PC treatment was significantly increased, and the soluble sugar content in each step of subsequent droplet vitrification was significantly higher than that of group F, indicating that droplet vitrification cryopreservation improved the cell permeability of the protocorms of Phalaenopsis lobbii and enhanced its stress resistance.

[0057] Figure 4 It also shows that the soluble protein content of the protocorms of Phalaenopsis lobbii after a series of cryopreservation steps such as PC by droplet vitrification is also significantly higher than that of group F.

[0058] Figure 5 It also shows that the Pro content is the highest after PC. Under plant stress conditions, the accumulation of proline will increase. It shows that the high-concentration pre-culture solution significantly reduces the water content in cells, increases the cell osmotic potential, and improves the low-temperature stress resistance of the protocorms of Phalaenopsis lobbii.

[0059] Malondialdehyde (MDA) is an important product of membrane lipid peroxidation reaction, and its content can intuitively reflect the peroxidation level of cell membranes. It can be seen from the results of the examples that from the start of PC treatment until UL, the MDA content in the protocorms of Phalaenopsis lobbii gradually increases, indicating that the cells are damaged to a certain extent.

[0060] POD belongs to one of the antioxidant enzyme systems. It can remove reactive oxygen in time to avoid the harm of oxidative stress to plants. SOD is an important antioxidant and anti-aging substance, and its mechanism is similar to the intracellular action of POD. As Figure 7 shown, the POD content after PC treatment is significantly higher than that of group F, reaches the highest after LS, and the POD content throughout the process is significantly higher than that of group F.

[0061] As Figure 8 shown, although the overall trend of SOD changes in this experiment, they are not significant. During the whole process of droplet vitrification cryopreservation, the highest value of SOD in the protocorms of Phalaenopsis lobbii reaches the peak after PVS2.

[0062] CAT is an enzyme scavenger, a conjugated enzyme with iron porphyrin as a cofactor. Under the action of active oxygen, it can promote H 2 O 2 Decomposes into molecular oxygen and water, removes hydrogen peroxide from the body, and protects cells from H 2 O 2 The toxicity is considered to be the body's resistance to H 2 O 2 It is an important pathway for toxicity and one of the key enzymes in the biological defense system. Figure 9 As shown in the figure, after ultra-low temperature preservation by small droplet vitrification, the content of CAT in the protocorms of Phalaenopsis roeblingii decreased, but recovered after LS. Compared with group F, the decrease was not significant. Therefore, it can be seen that the small droplet vitrification method effectively inhibited the responsiveness of the protocorms of Phalaenopsis roeblingii to H 2 O 2 The small droplet vitrification ultra-low temperature preservation method of the present invention greatly improves the low temperature resistance of Roche Phalaenopsis as a whole.

[0063] In some embodiments, the method for preserving the protocorm of Phalaenopsis rosenbergii comprises:

[0064] Obtain a protocorm of Phalaenopsis rothenbergii, wherein the protocorm of Phalaenopsis rothenbergii is obtained after germination of its seeds or culturing of its stems, stem tips, leaves, pedicels and other tissues for 58-62 days; further, in some embodiments, the germination or culturing time is about 60 days. In some embodiments, the diameter of the protocorm is 1 mm-2 mm, such as 1.2-1.5 mm, 1.3-1.7 mm, 1.5-1.8 mm or any sub-range or value therein.

[0065] Soaking the protocorm in a pre-culture solution for a certain period of time; wherein the pre-culture solution comprises 0.25M-0.45M sucrose, such as 0.27-0.43M, 0.3-0.4M or any sub-range or value therebetween;

[0066] The pre-cultured protocorms are immersed in a loading solution and subjected to loading treatment for a certain period of time; wherein the loading solution comprises (1-5) g / L 1 / 2MS, (0.1-1) M / L sucrose, (1-4) M / L glycerol, and (50-200) mL / L coconut water; preferably, the loading solution comprises 2.3 g / L 1 / 2MS, 0.4 M / L sucrose, 2 M / L glycerol, and 100 mL / L coconut water.

[0067] Immerse the processed original bulblets in the vitrification solution for a certain period of vitrification. Among them, the vitrification solution includes (1 - 5) g / L of 1 / 2MS without agar and sucrose, (100 - 500) mL / L of glycerol, (100 - 200) mL / L of ethylene glycol, (0.1 - 0.9) M / L of sucrose, and (100 - 200) mL / L of dimethyl sulfoxide. Preferably, the vitrification solution includes 2.3 g / L of 1 / 2MS without agar and sucrose, 300 mL / L of glycerol, 150 mL / L of ethylene glycol, 0.4 M / L of sucrose, and 150 mL / L of dimethyl sulfoxide. The vitrification time is 1.5 h - 3 h; preferably, the vitrification time is 2 - 2.5 h, such as 2.2 - 2.6 h, 2.3 - 2.8 h, 2.5 h, or any sub - range or value therebetween.

[0068] Put the original bulblets after being treated with the vitrification solution into an aluminum foil strip with small droplets of the vitrification solution, and put the aluminum foil strip with the original bulblets into a cryotube.

[0069] Ultra - low temperature treatment of the aluminum foil strip in an environment below - 190°C. In some embodiments, the ultra - low temperature is - 196°C to - 210°C.

[0070] Preserve the original bulblets after ultra - low temperature treatment.

[0071] A method for rewarming the original bulblets cryopreserved at ultra - low temperature, including: subjecting the ultra - low temperature - treated original bulblets obtained by the method of the present invention to a constant temperature treatment at about 40 - 60°C for 75 - 120 s, such as 80 - 110 s, 95 - 115 s, 90 - 100 s, 90 s, or any sub - range or value therebetween; preferably 90 s;

[0072] Immerse the original bulblets after constant temperature treatment in the unloading solution for unloading treatment. Among them, the unloading solution includes (1 - 5) g / L of 1 / 2MS, (0.5 - 2) M / L, and (50 - 150) mL / L of coconut milk; preferably, the unloading solution includes 2.3 g / L of 1 / 2MS, 1.2 M / L, and 100 mL / L of coconut milk. The immersion time of the original bulblets in the unloading solution is 15 - 30 min, such as 16 - 25 min, 18 - 28 min, 20 min, or any sub - range or value therebetween.

[0073] The technical solution of this application will be described below through specific experimental schemes. Those skilled in the art should understand that the following embodiments are only for illustrating the technical solution of this application and do not limit the protection scope of this application.

[0074] Example 1 Pre - culture of original bulblets

[0075] Select the pods of Phalaenopsis lobbii obtained by artificial pollination from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. After 60 days of aseptic germination, the protocorms of Phalaenopsis lobbii are obtained. In order to explore the most suitable experimental protocol for the droplet vitrification method of Phalaenopsis lobbii protocorms, in this example, the inventor changes the osmotic pressure of the protocorms by using gradient concentrations of sucrose in the pre-culture solution, in order to find the optimal protective concentration of the pre-culture solution for cryopreservation of Phalaenopsis lobbii by vitrification.

[0076] The pre-culture solution is prepared in 5 groups, as shown in Table 1.

[0077] Table 1 Different treatment groups of the pre-culture solution for Phalaenopsis lobbii protocorms

[0078]

[0079] In the conical flasks of the pre-culture solution, each flask is set with 50 mL of the pre-culture solution volume, and there are 5 groups of pre-culture solution treatments. Except for the third group which needs to make 24 flasks (see Table 2 for details), each treatment group is set with 4 flasks as 4 replicates. After preparation, it is placed in an autoclave for sterilization and cooling. In the laminar flow hood, use tweezers to pick out the eligible Phalaenopsis lobbii protocorms and knock them into the conical flasks containing the pre-prepared and sterilized pre-culture solution. About 50 protocorms are knocked into each conical flask. Ensure aseptic operation throughout the process. Place the conical flasks with protocorms in a shaker at 120 rpm and culture in the dark for 2 days.

[0080] Example 2 Process of the droplet vitrification method

[0081] In order to explore the most suitable experimental protocol for the droplet vitrification method of Phalaenopsis lobbii protocorms, in this example, the inventor finds the most suitable vitrification time point for Phalaenopsis lobbii protocorms by changing the treatment time gradient of the protocorms obtained by the method of Example 1 soaking in PVS2 solution, to prevent the protocorms from being poisoned. Fourteen groups of experiments are set in this step, see Table 2 for details:

[0082] Table 2 Different treatment time groups of PVS2 in the droplet vitrification method of Phalaenopsis lobbii protocorms

[0083]

[0084] In this example, the cryopreservation method of protocorm small droplet vitrification includes: placing the pre-culture solution containing protocorms after 2 days of pre-culture on a laminar flow bench, picking out the protocorms from the conical flask, putting them into a small beaker, pouring in the loading solution (2.3 g / L 1 / 2MS + 0.4 M / L sucrose + 2 M / L glycerol + 100 mL / L coconut milk), and treating on ice for 30 min. Suck out the loading solution, transfer to an ice box for operation, pour in PVS2, and treat for different times, namely 0 h, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h. Put the protocorms after PVS2 treatment into aluminum foil strips with small droplets of PVS2, put the aluminum foil strips with protocorms into cryotubes, fill them with liquid nitrogen, quickly cover the lids and put them into liquid nitrogen for treatment for 1 h. During thawing, thaw in a 40-degree water bath for 1.5 min. Then perform unloading. During unloading, pour in the unloading solution (2.3 g / L 1 / 2MS + 1.2 M / L + 100 mL / L coconut milk) and treat for 20 min.

[0085] In this example, the control group was to directly put the protocorms (without experiencing the pre-culture and small droplet vitrification process) into liquid nitrogen for preservation for 1 h, and then place them at room temperature for 20 min.

[0086] Example 3 Determination of cell viability by TCC method

[0087] Use the 1% 2,3,5-triphenyltetrazolium chloride (TTC) staining solution (SL7141) of Coolaber Company to detect the activity of protocorms of Rhynchostylis gigantea after cryopreservation. TTC is a fat-soluble photosensitive complex that reacts with respiratory enzymes in normal tissues and appears red.

[0088] Take the unloaded protocorms, roughly wipe the surface with filter paper to remove the possible unloading solution, then weigh them. Take 5 g for each repetition (ensure that the number of protocorms in each group repetition is more than 100 to ensure a large enough sample size), and put them into cryotubes. Pour the TTC staining solution into the cryotubes and store them at room temperature in the dark for 24 h, then observe the color change. Red is recorded as having activity, and other colors are not included in the survival rate statistics.

[0089] Example 4 Determination of physiological indexes during the cryopreservation of small droplet vitrification

[0090] To study the effects of each step during cryopreservation on the physiological state of protocorms, during the cryopreservation of protocorms by small droplet vitrification, the inventor took samples at the end of each step. Each physiological index of each treatment was measured 3 times repetitively.

[0091] Six groups of treatments were set up in this experiment, which involved the following samples: untreated protocorms (F), protocorms that were directly put into liquid nitrogen for 1 h without any treatment (CK), pre-cultured protocorms (PC), pre-cultured and loaded protocorms (PC+LS), pre-cultured, loaded and PVS2-treated protocorms (PC+LS+PVS2), pre-cultured, loaded, PVS2-treated and unloaded protocorms (PC+LS+PVS2+UL). This covered each key preservation step of the droplet-vitrification cryopreservation method. The treatment group with the highest survival rate after cryopreservation, which contained 0.3 M sucrose pre-culture solution and 2.5 h of PVS2 treatment, was selected to measure soluble sugar, soluble protein, superoxide dismutase, catalase, peroxidase, malondialdehyde, and proline.

[0092] 1. Determination of soluble sugar

[0093] The anthrone colorimetric method was used to determine soluble sugar. The plant soluble sugar content detection kit from Solarbio was used and the test was carried out according to the manufacturer's instructions. The soluble sugar content in the sample was calculated according to the following formula and the obtained curve:

[0094] Soluble sugar (mg / g) = ;

[0095] where, V1: volume of sample added, for example, V1 is 0.04 mL; V2: total volume of sample, for example, V2 is 10 mL; W: sample mass, g.

[0096] 2. Determination of soluble protein

[0097] The principle for the determination of soluble protein is: under alkaline conditions, proteins reduce Cu 2+ to Cu + , and Cu + forms a purple-blue complex with the BCA reagent. By measuring its absorbance at 562 nm and comparing it with the standard curve, the concentration of the protein to be measured can be calculated. Among common detergents, TritonX-100, Tween, etc. have no obvious effect on the measurement results, while chelating agents (EDTA, EGTA), reducing agents (DTT, mercaptoethanol), lipids, etc. have a greater impact. The BCA protein content determination kit from Solarbio was used and the detection was carried out according to the instructions of the manufacturer. The protein content was obtained from the standard curve.

[0098] 3. Determination of superoxide dismutase

[0099] The basic principle for the determination of superoxide dismutase (SOD) is: the reaction system of xanthine and xanthine oxidase will generate superoxide anions (O 2- ), and this O 2-It can reduce nitro blue tetrazolium to formazan, which appears blue at a wavelength of 560 nm, and blue light has absorption at 560 nm; SOD can effectively eliminate O 2- , thus effectively inhibiting the formation of formazan; over time, the darker the blue color of the reaction solution indicates that the activity of SOD gradually decreases; otherwise, the lighter the blue color of the reaction solution indicates that the activity of SOD gradually increases. The superoxide dismutase (SOD) activity assay kit from Solarbio was used, and the determination was carried out according to the instructions of the manufacturer. Calculate the SOD activity in the sample according to the following formula:

[0100] SOD activity (U / g mass) = [Inhibition percentage ÷ (1 - Inhibition percentage) × Vtotal reaction] / (W × Vsample ÷ Vtotal sample) × F = 10 × [Inhibition percentage / (1 - Inhibition percentage)] ÷ W × F;

[0101] Among them, Vtotal reaction: total reaction volume, 0.2 mL; Vsample: volume of the sample added to the reaction system, 0.02 mL; Vtotal sample: volume of the extraction solution added, 1 mL; W: sample mass, g; F: sample dilution factor.

[0102] 4. Determination of catalase

[0103] The principle of the catalase determination is that H 2 O 2 has a characteristic absorption peak at 240 nm, and CAT can decompose H 2 O 2 , causing the absorbance of the reaction solution at 240 nm to decrease with the reaction time. The CAT activity can be calculated based on the change rate of the absorbance. Use the catalase (CAT) activity detection kit from Solarbio and perform the test according to the manufacturer's instructions. Calculate the CAT activity in the sample according to the following formula: CAT (U / g mass) = {[ΔA × Vtotal reaction ÷ ( × d) × 10 6 / Vsample ÷ Vtotal sample × W} ÷ T = 764.5 × ΔA ÷ W;

[0104] Among them, Vtotal reaction: total volume of the reaction system, 2 × 10 -4 L; ε: molar extinction coefficient of H 2 O 2 , 43.6 L / mol / cm; d: optical path of the cuvette, 1 cm; Vsample: volume of the sample added, 0.01 mL; Vtotal sample: volume of the extraction solution added, 1 mL; T: reaction time, 1 min; W: sample mass, g; 10 6 : unit conversion factor, 1 mol = 10 6 μmol.

[0105] 5. Determination of peroxidase

[0106] The principle of peroxidase assay is that in the presence of hydrogen peroxide, POD can oxidize guaiacol to produce a tea-brown substance, which has the maximum light absorption at 470 nm. The peroxidase (POD) activity detection kit from Solarbio was used and the test was carried out according to the manufacturer's instructions. Calculate the POD activity in the sample according to the following formula:

[0107] POD (U / g mass) = (ΔA÷0.05)×[Vtotal reaction / (W×Vsample÷Vtotal sample)]÷T = 9800×ΔA÷W;

[0108] Among them, Vtotal reaction: the total volume of the reaction system, 0.245 mL; Vsample: the volume of the sample added, 0.005 mL; Vtotal sample: the volume of the extraction solution added, 1 mL; T: the reaction time, 1 min; W: the sample mass, g.

[0109] 6. Determination of malondialdehyde

[0110] The basic principle of malondialdehyde detection is that malondialdehyde (MDA) can undergo a condensation reaction with thiobarbituric acid (TBA) under high temperature and acidic environment to form a brown-red trimethine (3,5,5-trimethyl oxazole-2,4-dione), and its maximum absorption wavelength is 532 nm. The content of malondialdehyde in the sample was determined by colorimetry. The catalase (MDA) activity detection kit from Solarbio was used and the test was carried out according to the manufacturer's instructions. Calculate the MDA content in the sample according to the following formula:

[0111] MDA content (nmol / g mass) = [12.9×(Δ532 - Δ600) - 1.12×Δ450]×Vtotal / (W×Vsample÷Vextraction) = {5×[12.9×(Δ532 - Δ600) - 1.12×Δ450]}÷W;

[0112] Among them, Vtotal: the total volume of the reaction system, 5×10-4 L; Vsample: the volume of the sample added, 0.1 mL; Vextraction: the volume of the extraction solution added, 1 mL; W: the sample mass, g.

[0113] 7. Determination of proline

[0114] The measurement principle of proline is to extract proline (hereinafter referred to as Pro) with sulfosalicylic acid (SA), and after heating, Pro reacts with an acidic ninhydrin solution to produce red, and its absorption is measured at 520 nm. According to the instructions of the manufacturer, the proline (Pro) content detection kit from Solarbio was used for the test. The content of Pro in the test sample was calculated by the following formula:

[0115] Pro content (μg / g mass) = X × (Vextraction / W) = X / W;

[0116] Where, Vextraction: volume of extraction solution added, 1 mL; W: sample mass, g.

[0117] Among these substances, Pro is extracted with sulfosalicylic acid (SA), and after heat treatment, Pro reacts with the acidic ninhydrin solution to produce red color, and the absorbance is measured at 520 nm.

[0118] Example 5 Measurement Results

[0119] 1. Effect of sucrose concentration in pre - culture solution on the survival rate of protocorms

[0120] Figure 1 The survival rate of Phalaenopsis lobbii protocorms after pre - culture with different sucrose concentrations was determined by the TTC method according to Example 3 of the present invention.

[0121] Figure 1 The results show that under the condition of keeping the PVS2 treatment time at 2 h unchanged, five different sucrose concentrations for pre - culture have different effects on the TTC results of cryopreservation of Phalaenopsis lobbii protocorms. When the sucrose concentration is 0.2 M, the survival rate is the lowest, which is 61.01%; the survival rates at 0.3 M and 0.4 M sucrose concentrations are higher, which are 76.85% and 79.44% respectively.

[0122] From Figure 1 It can be seen that under the condition of controlling variables, the trend of TTC survival rate of the droplet vitrification method for Phalaenopsis lobbii protocorms decreases when the sucrose concentration is between 0.1 M - 0.2 M, increases when the sucrose concentration is between 0.2 M - 0.4 M, and shows a downward trend again after 0.4 M. It reaches the highest value at 0.4 M concentration. Thus, it can be known that when the pre - culture concentration is 0.4 M, the pre - culture effect on Phalaenopsis lobbii is the best.

[0123] 2. Effect of vitrification treatment time on the survival rate of protocorms

[0124] Figure 2 The TTC survival rate of Phalaenopsis lobbii protocorms with different PVS2 treatment times according to Example 2.

[0125] The results showed that the survival rate of protocorms of *Phalaenopsis lobbii* increased first and then decreased with the increase of PVS2 treatment time. The TTC result was the best at 98.10% when treated with PVS2 for 2.5 h. The survival rate was the lowest at 32.40% under the 0 h treatment. After the PVS2 treatment time reached 2.5 h, the survival rate decreased significantly with the increase of treatment time. This indicates that short-term vitrification treatment has little harm to the protocorms of *Phalaenopsis lobbii*, but long-term PVS2 treatment will cause great damage to the protocorms of *Phalaenopsis lobbii*, significantly reducing the survival rate. Figure 2 It is shown that under the premise of controlling variables, the dehydration effect of PVS2 treatment for 2.5 h on *Phalaenopsis lobbii* is the best.

[0126] 3. Physiological changes during the cryopreservation of protocorms by the small-droplet vitrification method

[0127] (1)Soluble sugar

[0128] Figure 3 It is the content of soluble sugar during the cryopreservation of protocorms of *Phalaenopsis lobbii* by the small-droplet vitrification method according to Example 2 of the present invention.

[0129] It can be seen from Figure 3 that the soluble sugar content in the CK group directly plunged into liquid nitrogen was significantly increased compared with that in the F group of protocorms growing normally. Among the CK and PC groups, the soluble sugar content in the protocorms of *Phalaenopsis lobbii* was the highest, 86.99 mg / g in the CK group and 88.24 mg / g in the PC group. Compared with the treatment group F, the soluble sugar content increased by at least 52.04 mg / g, and the difference was significant (P < 0.05). There was little difference in the soluble sugar content between the two treatment groups of PC and CK. This indicates that pre-culture treatment has little effect on the soluble sugar content compared with direct dry plunge.

[0130] However, adding LS, PVS2, and UL treatments after pre-culture all had an inhibitory effect on the soluble sugar content in the protocorms of *Phalaenopsis lobbii*. Among them, after the LS and PVS2 treatments, the soluble sugar content reached the lowest, 56.29 mg / g and 57.4 mg / g respectively.

[0131] (2)Soluble protein

[0132] Figure 4 It is the content of soluble protein during the cryopreservation of protocorms of *Phalaenopsis lobbii* by the small-droplet vitrification method according to Example 2 of the present invention.

[0133] It can be seen from Figure 4It can be seen that the soluble protein concentration in the CK group was significantly increased compared with that in the F group. The soluble protein content of the protocorms of Phalaenopsis lobbii reached the highest level in the CK group and after UL treatment, which were 0.130 mg / g and 0.126 mg / g respectively, and was at least 0.051 mg / g higher than that in the treatment group F, showing a significant difference (P < 0.05). There was a significant difference in the soluble protein concentration after CK and PC treatments, indicating that direct dry inoculation could increase the cell osmotic potential and promote the production of soluble proteins in the protocorms of Phalaenopsis lobbii. However, the soluble protein content gradually increased after LS and reached the lowest level at LS, which was 0.070 mg / g.

[0134] (3)Proline

[0135] Figure 5 It is the content of proline during the cryopreservation of protocorms of Phalaenopsis lobbii by the small droplet vitrification method according to Example 2 of the present invention.

[0136] From Figure 5 It can be seen that the content of proline (abbreviated as Pro) in the CK group was significantly lower than that in the F group. After PC treatment, the Pro content was significantly increased compared with that in the CK group, reaching 18.9 μg / g. With further treatment, the Pro content gradually decreased and reached the lowest level after UL, which was 8.96 μg / g, showing no significant difference from the F group. However, the Pro content in all experimental groups of the small droplet vitrification method treatment steps was higher than that in the F and CK groups.

[0137] (4)Malonicdialdehyde

[0138] Figure 6 It is the content of malonicdialdehyde during the cryopreservation of protocorms of Phalaenopsis lobbii by the small droplet vitrification method according to Example 2 of the present invention.

[0139] From Figure 6 It can be seen that the content of malonicdialdehyde (abbreviated as MDA) in the CK group was decreased compared with that in the F group, but there was no significant difference. It gradually increased from the PC treatment until after UL, and the MDA content after UL treatment reached the highest level, which was 231.92 nmol / g, and the lowest level in the treatment group was 45.49 nmol / g for PC.

[0140] (5)Peroxidase

[0141] Figure 7 It is the content of peroxidase during the cryopreservation of protocorms of Phalaenopsis lobbii by the small droplet vitrification method according to Example 2 of the present invention.

[0142] From Figure 7It can be seen that the content of peroxidase (abbreviated as POD) in the CK group decreased compared with that in the F group, but not significantly. The POD content of the protocorms of Phalaenopsis lobbii was the lowest after PC during the droplet-vitrification method process, which was 3859.33 U / g. After that, the POD content increased significantly. The POD content reached the highest after LS treatment, which was 8396 U / g. After that, the POD content in each treatment group showed a significant decreasing trend. However, the POD content in all groups increased significantly compared with that in the CK group, indicating that each step of the droplet-vitrification method promoted the production of POD in the protocorms of Phalaenopsis lobbii.

[0143] (6)Superoxide Dismutase

[0144] Figure 8 It is the content of superoxide dismutase during the cryopreservation of protocorms of Phalaenopsis lobbii by the droplet-vitrification method according to Example 2 of the present invention.

[0145] It can be seen from Figure 8 that the content of superoxide dismutase (abbreviated as SOD) in the CK group decreased compared with that in the F group, but not significantly. Each step of the cryopreservation by the droplet-vitrification method changed the SOD content of the protocorms of Phalaenopsis lobbii, but not significantly. The SOD content reached the highest at PVS2, which was 28.04 U / g; the SOD content was the lowest at PC, which was 2.51 U / g.

[0146] (7)Catalase

[0147] Figure 9 It is the content of catalase during the cryopreservation of protocorms of Phalaenopsis lobbii by the droplet-vitrification method according to Example 2 of the present invention.

[0148] It can be seen from Figure 9 that the treatment of the CK group decreased the content of catalase (abbreviated as CAT) in the protocorms of Phalaenopsis lobbii compared with that in the F group, but there was no significant difference. The CAT content after PC and PVS2 in the treatment group decreased significantly, and the lowest reached 258.37 U / g after PVS2. The decrease in other groups was not significant. It may be that the treatment process of the droplet-vitrification method has little effect on the production of CAT in the protocorms of Phalaenopsis lobbii.

[0149] The inventor applied tissue culture technology to aseptically sow the seeds of Phalaenopsis lobbii, obtaining a large number of protocorms of Phalaenopsis lobbii for further cryopreservation research. Further, the applicant explored that when using 1 / 2MS + sucrose + 100 mL / L coconut milk as the nutrient additive in the germination medium for Phalaenopsis lobbii seeds, the germination rate of Phalaenopsis lobbii seeds was the highest, the most protocorms of Phalaenopsis lobbii were obtained, and the germination rate at 60 days was as high as 87.13%. Orchidaceae plants grow slowly and have a long juvenile period, and it takes an average of 4 - 5 years to evaluate the flower quality of the offspring and obtain new seeds. Some native species exhibit complex reproductive processes (specific physiology of cross-pollination and seed germination), and these factors hinder their reproduction and preservation. Therefore, the materials of native Phalaenopsis are very precious, and the preservation method of the present invention is an effective way to protect native Phalaenopsis lobbii.

[0150] A cryoprotectant is a chemical that can reduce the damage to cells during the freezing process and thawing. The vitrified state has various benefits for dehydrated cells: it can inhibit water loss, crystallize salts and proteins into the cytoplasm, avoid changes in pH value caused by water removal, and avoid cell collapse when overly dehydrated. However, these cryoprotectants are toxic and may cause osmotic stress, resulting in cell death, or altering their morphogenetic responses in the culture (Sakai, 1995). To explore the effects of such cryoprotectants at the physiological level, the inventor conducted a series of physiological index detections on the protocorms of Phalaenopsis lobbii cryopreserved by the small-droplet vitrification method. After each key step of cryopreservation, the determinations of soluble sugar, soluble protein, superoxide dismutase, catalase, peroxidase, malondialdehyde, and proline were carried out. And the effects of each step of the small-droplet vitrification method cryopreservation on various physiological indexes were analyzed in detail. For example, during the vitrification dehydration treatment, the oxidative stress reached the highest level, leading to corresponding changes in physiological indexes caused by antioxidant reactions and the like.

[0151] Cryopreservation is a highly innovative preservation technology and a protocorm preservation technology with high safety and stability. This technology will provide an important technical means for the long-term, efficient, and safe preservation of rare Orchidaceae plant germplasm resources in China and for the conservation of numerous Orchidaceae plant germplasm resources. In the cryopreservation program of Orchidaceae plants, the small-droplet vitrification method cryopreservation technology is an improvement of the original cryopreservation technology, and this technology can enable Orchidaceae plants of different varieties to adapt to different preservation requirements. Therefore, cryogenic vitrification may be the best method for the preservation of Orchidaceae plant germplasm and explants (such as pollen, seeds, and protocorms).

[0152] In summary, the cryopreservation technology of Orchidaceae plants is still in its infancy and is a new topic worthy of attention. For specific Orchidaceae species, how to select different cryopreservation treatment methods remains to be further studied in the future. Based on the previous observation results, the types of currently commonly used cryopreservation technologies, and the inventors' research on different materials of various different native orchid species using different cryopreservation methods, the reactions of different native species vary greatly. Therefore, it is necessary to develop species-specific and more appropriate cryopreservation protocols for different orchids and different materials of orchids.

[0153] The above embodiments are only for illustrating the present invention and are not intended to limit the present invention. Those of ordinary skill in the relevant technical fields can still make various changes and modifications without departing from the scope of the present invention. Therefore, all equivalent technical solutions should also fall within the scope of the disclosure of the present invention.

Claims

1. A method for preserving the original corm of Phalaenopsis rothenbergii, comprising: Obtain the original corm of Phalaenopsis rothenbergii; Soaking the protocorm in a pre-culture solution for a certain period of time, wherein the pre-culture solution comprises 0.25M-0.45M sucrose; soaking the pre-cultured protocorms in a loading solution and subjecting them to loading treatment for a certain period of time; soaking the loaded protocorm into a vitrification solution and performing vitrification treatment for a certain period of time; The vitrified protocorms were immersed in a small drop of vitrification solution; Ultra-low temperature treatment of small droplets containing protocorms in an environment of -190°C to -220°C; as well as The cryogenically treated protocorm is stored.

2. The storage method according to claim 1, wherein: The diameter of the protocorm is 1 mm-2 mm.

3. The preservation method according to claim 1, wherein the protocorm is obtained from plant tissue or seeds after germination for 58-62 days.

4. The storage method according to claim 1, wherein the pre-culture solution comprises 0.3M-0.4M sucrose.

5. The storage method according to claim 1, wherein: The loading solution includes (1-5) g / L 1 / 2MS, (0.1-1) M / L sucrose, (1-4) M / L glycerol, and (50-200) mL / L coconut water.

6. The storage method according to claim 1, wherein: The vitrification solution includes (1-5) g / L 1 / 2MS without agar and sucrose, (100-500) mL / L glycerol, (100-200) mL / L ethylene glycol, (0.1-0.9) M / L sucrose, and (100-200) mL / L dimethyl sulfoxide.

7. The storage method according to claim 1, wherein the glass transition time is 1.5h-3h.

8. A method for rewarming cryopreserved protocorms, comprising: The cryogenically treated protocorm preserved according to any one of the preservation methods of claims 1 to 7 is subjected to a constant temperature treatment of 40-60° C. for 75-120 s; as well as The thermostatically treated protocorm is immersed in an unloading solution to perform an unloading treatment.

9. The method according to claim 8, wherein the protocorm is immersed in the unloading solution for 15-30 min.

10. The method according to claim 8 or 9, wherein the unloading solution comprises (1-5) g / L 1 / 2MS, (0.5-2) M / L, and (50-150) mL / L coconut water.

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