A method for preserving protocorms of Kingidium deliciosum

Abstract

The present invention relates to a method for preserving protocorms of Kingidium deliciosum, comprising: obtaining protocorms of Kingidium deliciosum; soaking the protocorms in a loading solution for loading treatment; soaking the loaded material in a vitrification solution for vitrification treatment; wherein the vitrification solution is PVS2 solution; the soaking time is 0-3 h; and cryopreserving the vitrified protocorms. The method for treating protocorms in this application is simple to operate, can greatly save costs, and the survival rate of the protocorms preserved by the method of this application is high.

Description

Technical Field

[0001] The present invention relates to the protection of plant protocorms, and particularly to a method for preserving the protocorms of Phalaenopsis deliciosa Rchb. f. Background Art

[0002] Orchidaceae Juss. plants are the second largest family among angiosperms after Asteraceae, and also the largest family among monocotyledons. There are about 800 genera and 28,000 species in the world, widely distributed in various terrestrial ecosystems except extreme regions. There are 171 genera and 1,247 species of Orchidaceae plants in China, making it one of the countries with the richest wild Orchidaceae plants. They are mainly distributed in regions such as Yunnan, Taiwan, Hainan, Guangdong, and Guangxi, with a series of evolutionary groups and rich geographical distribution types.

[0003] Phalaenopsis Blume plants have flower shapes similar to butterflies, and are popular among people because of their long flowering periods, rich flower colors, and numerous flowers. They have become one of the best-selling potted flowers in the world. Phalaenopsis Blume plants are perennial epiphytic herbs with well-developed fleshy roots, long and flat, attached to rock walls or tree barks. The leaves are flat and thick, elliptical or obovate-lanceolate. Phalaenopsis Blume plants prefer humid, high-temperature, semi-shaded environments, are not cold-tolerant and afraid of strong light and drought. Waterlogging at the roots will cause them to rot. Therefore, they are suitable to be planted in loose and well-drained bark pieces or sphagnum moss, and mostly grow on the trunks of tropical or subtropical forests at low altitudes.

[0004] The seeds of wild Orchidaceae plants need the participation of specific symbiotic fungi when germinating, have strict requirements for habitats, and strong dependence on other organisms such as pollinating animals. In recent years, due to climate change, the population of some species of Phalaenopsis Blume, such as Phalaenopsis deliciosa Rchb. f., has been continuously decreasing, increasing the risk of extinction. Therefore, the task of protecting its germplasm resources is extremely urgent.

[0005] On the other hand, the fruits of Orchidaceae plants such as Phalaenopsis deliciosa Rchb. f. contain tens of thousands of tiny seeds. The seeds only have an embryo and a seed coat, without endosperm. The seeds have no nutrients by themselves and cannot germinate independently. Under natural conditions, they need to rely on symbiotic fungi to provide nutrients to germinate. The germination conditions are relatively harsh, with strict requirements for the quality of natural habitats, resulting in a very low natural germination rate, even less than 2%. Under natural conditions, the enzyme activity in the seeds of Orchidaceae is relatively strong, and the seed vigor is difficult to maintain for a long time, and the lifespan is relatively short. A protocorm is a flat spherical object formed after the germination of orchid seeds, with adventitious roots growing at its base, and is morphologically similar to the proembryo formed by the embryo during seed germination. The protocorm can further grow into an orchid plant, and can also be cut into several small pieces to form several new protocorm clusters respectively.

[0006] It can be seen that the germination conditions of Orchidaceae seeds are demanding and their lifespan is short, resulting in difficulties in natural reproduction. Therefore, artificially preserving the protocorms and allowing them to grow into plants during the appropriate period can effectively protect their germplasm resources. Summary of the Invention

[0007] In view of the technical problems existing in the prior art, the present invention provides a method for preserving the protocorms of Kingidium wardianum, comprising: obtaining the protocorms of Kingidium wardianum; soaking the protocorms in a loading solution for loading treatment; soaking the loaded protocorms in a vitrification solution for vitrification treatment; wherein, the vitrification solution is PVS2 solution; the soaking time is 0 - 3 h; soaking the vitrified protocorms into small droplets containing the vitrification solution; and preserving the small droplets containing the protocorms in an ultra-low temperature environment of -190°C to -210°C.

[0008] For the method as described above, the ultra-low temperature environment is an environment with a temperature less than -190°C; more preferably, the ultra-low temperature environment is an environment with a temperature in the range of (-196 to -210)°C.

[0009] For the method as described above, the loading solution comprises (100 - 150) g / L sucrose, (1 - 5) g / L 1 / 2MS, (150 - 200) g / L glycerol; preferably, the loading solution comprises 137 g / L sucrose, 2.3 g / L 1 / 2MS, and 184 g / L glycerol.

[0010] For the method as described above, the loading treatment time is 20 - 40 min, preferably 30 min.

[0011] For the method as described above, the vitrification solution comprises (100 - 150) g / L sucrose, (1 - 5) g / L 1 / 2MS, (200 - 400) mL / L glycerol, (100 - 200) mL / L ethylene glycol, (100 - 200) mL / L dimethyl sulfoxide; preferably, the vitrification solution comprises 137 g / L sucrose, 2.3 g / L 1 / 2MS, 300 mL / L glycerol, 150 mL / L ethylene glycol, and 150 mL / L dimethyl sulfoxide.

[0012] For the method as described above, the protocorms are obtained by aseptic sowing of seeds; wherein, the time of tissue culture is not less than 30 days. For the method as described above, the time of tissue culture is 30 - 40 days, 40 - 50 days, 55 - 60 days.

[0013] For the method as described above, the diameter of the original bulb is not less than 0.5 mm; preferably, the diameter of the original bulb is 0.5 mm - 1 mm. For example, the diameter of the original bulb is 0.55 - 0.95 mm, 0.6 - 0.9 mm, 0.65 - 0.85 mm, 0.7 - 0.8 mm, or a sub-range or any value therein.

[0014] For the method as described above, the soaking time of the loaded original bulb in the vitrification solution is 0 - 3 h, for example 1 - 2 h, 0.5 - 2.5 h, 1.5 - 2 h, 0.8 - 2.2 h, 1.7 - 2.4 h, 0.7 h, 1.6 h, 2 h, 2.7 h, or a sub-range or any value therein.

[0015] For the method as described above, it further includes pre-culturing the original bulb, which includes: putting the original bulb into a pre-culture solution; sealing the pre-culture solution and the original bulb therein; and putting it into a shaker, shading and treating it at a rotation speed of (50 - 200) r / min for 1 - 3 days; preferably treating it for 2 days.

[0016] For the method as described above, wherein the environmental temperature for pre-culturing is room temperature; preferably, the pre-culturing temperature is 20 - 30 °C.

[0017] For the method as described above, wherein the pre-culturing time is 1 - 3 days, for example 1.5 - 2.5 days, 1.2 - 2.8 days, 2 - 3 days, 1.5 days, 2 days, 2.5 days, or a sub-range or any value therein.

[0018] For the method as described above, wherein the pre-culture solution includes (100 - 150) g / L sucrose, (1 - 5) g / L 1 / 2MS, (50 - 150) mL / L coconut milk; preferably, the pre-culture solution includes: 137 g / L sucrose, 2.3 g / L 1 / 2MS, 100 mL / L coconut milk.

[0019] For the method as described above, it further includes putting the vitrified original bulb into a droplet of the vitrification solution so that the vitrification solution completely wraps the original bulb.

[0020] According to another aspect of the present application, a method for rewarming the cryopreserved original bulb is provided, including: subjecting the cryopreserved original bulb obtained by the method as described above, for example, to a constant temperature treatment at (for example 60 °C) for 75 - 120 s (for example 80 - 115 s, 90 - 110 s, 100 - 105 s, 82 s, 90 s, 95 s, 103 s, or a sub-range or any value therein); and unloading the original bulb after the constant temperature treatment.

[0021] For the method as described above, the step of unloading the pre-cultured protocorms includes soaking the pre-cultured protocorms in an unloading solution for 15 - 30 min (such as 18 - 28 min, 20 - 26 min, 23 - 25 min, 17 min, 20 min, 22 min or a sub-range or any value therein).

[0022] For the method as described above, the unloading solution includes (350 - 500) g / L sucrose and (1 - 5) g / L 1 / 2MS; preferably, the unloading solution includes: 411 g / L sucrose and 2.3 g / L 1 / 2MS.

[0023] The method for treating protocorms of the present application is simple to operate and can greatly save costs. Moreover, the survival rate of the protocorms preserved by the method of the present application is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Next, the preferred embodiments of the present invention will be further described in detail with reference to the drawings, wherein:

[0025] Figure 1 shows a brief flow chart of droplet vitrification cryopreservation, rewarming, and viability detection of the protocorms of Cymbidium erythraeum according to an embodiment of the present invention;

[0026] Figure 2 shows an image of TTC staining of the protocorms of Cymbidium erythraeum after cryopreservation according to an embodiment of the present invention;

[0027] Figure 3 shows a bar chart of the survival rate of the protocorms of Cymbidium erythraeum after cryopreservation varying with the PVS2 soaking time according to an embodiment of the present invention;

[0028] Figure 4 shows the soluble sugar content of Cymbidium erythraeum after different treatments according to an embodiment of the present invention;

[0029] Figure 5 shows the soluble protein content of Cymbidium erythraeum after different treatments according to an embodiment of the present invention;

[0030] Figure 6 shows the proline (Pro) content of Cymbidium erythraeum after different treatments according to an embodiment of the present invention;

[0031] Figure 7 shows the malondialdehyde (MDA) content of Cymbidium erythraeum after different treatments according to an embodiment of the present invention;

[0032] Figure 8Shows the superoxide dismutase (SOD) content of Cymbidium erythraeum Lindl. after different treatments according to an embodiment of the present invention;

[0033] Figure 9 Shows the peroxidase (POD) content of Cymbidium erythraeum Lindl. after different treatments according to an embodiment of the present invention;

[0034] Figure 10 Shows the catalase (CAT) content of Cymbidium erythraeum Lindl. after different treatments according to an embodiment of the present invention. Detailed implementation manners

[0035] 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.

[0036] In the following detailed description, reference may be made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration specific embodiments in which the invention may be practiced. In the drawings, like reference numerals 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 be utilized or structural, logical or electrical changes may be made to the embodiments of the present application.

[0037] There are three common ways to preserve orchid germplasm resources: in-situ conservation, tissue culture subculture, and cryopreservation.

[0038] In-situ conservation, in the form of nature reserves or conservation points, protects the natural habitats of orchid plants and the ecosystems therein to the greatest extent, but is vulnerable to natural disasters or pests and diseases.

[0039] Tissue culture subculture and cryopreservation are off-site conservation methods, and in vitro conservation is carried out in the form of test-tube seedlings or cryopreservation in the germplasm resource preservation library. However, because the temperature, light, and humidity required by orchid plants are relatively strict, the construction of greenhouses requires a large amount of work, high costs, and high human and material resources. Moreover, tissue culture subculture is prone to variation, and the more times of operation, the more likely it is to be contaminated, resulting in material loss.

[0040] Ultra-low temperature preservation is to store materials in liquid nitrogen. The material metabolism and growth activities of the materials almost completely stop but still remain viable, and they can resume growth after thawing. Compared with the commonly used low-temperature preservation, ultra-low temperature preservation is more stable and can prevent material variation. The selected preservation materials should be both totipotent and capable of developing into complete plants. It is also necessary to ensure that the cell structure of the materials themselves is intact during liquid nitrogen freezing (-196°C) and is not damaged by the ultra-low temperature environment, so as to ensure the activity of the materials after preservation. There are 6 commonly used ultra-low temperature preservation methods, namely the stepwise cooling method, the rapid freezing method, the drying method, the vitrification method, the small droplet vitrification method, and the encapsulation-vitrification method. Multiple studies have shown that the small droplet vitrification method has better effects on the ultra-low temperature preservation of plant materials. Among them, Hayashi et al. [1] By comparing the effects of the vitrification method and the small droplet vitrification method on the ultra-low temperature preservation of carnation shoot tips, it was found that the survival rate of the vitrification method was above 80%, and the survival rate of the small droplet vitrification method was above 90%. However, during the ultra-low temperature preservation process, plant materials will inevitably be subjected to temperature stress and osmotic pressure changes. Li Xiaodan [2] Measured the MDA content of the embryogenic callus of Agapanthus praecox after ultra-low temperature preservation by the vitrification method, and found that the MDA content of the plant materials after preservation increased significantly, indicating that the ultra-low temperature preservation by the vitrification method will inevitably cause oxidative stress and apoptosis of plants. Therefore, considering species differences, the different frost resistances of plants and the morphological structures of plant tissues, conducting targeted research on the ultra-low temperature preservation method of the small droplet vitrification method suitable for specific materials can greatly improve the survival rate after freezing.

[0041] [1] Hayashi, Yang Hua, Han Jing, et al. Effects of Vitrification and Small Droplet Vitrification Methods on the Ultra-Low Temperature Preservation of Carnation Shoot Tips [J]. Journal of Anhui Agricultural Sciences, 2018, 46(19): 105-107+141. DOI: 10.13989 / j.cnki.0517-6611.2018.19.030.

[0042] [2] Li Xiaodan. Research on the Ultra-Low Temperature Preservation Technology of the Embryogenic Callus of Agapanthus praecox by Vitrification Method [D]. Northeast Forestry University, 2013.

[0043] This application uses Cymbidium erythraeum Lindl. as plant materials, and by analyzing the effects of the soaking time of PVS2 and the vitrification method of protocorms on ultra-low temperature preservation, it is expected to find the most suitable ultra-low temperature preservation technology for the protocorms of Cymbidium erythraeum Lindl., providing technical theory for protecting its germplasm resources. Subsequently, by measuring the physiological and biochemical indexes of the main steps in the most suitable ultra-low temperature preservation of the protocorms of Cymbidium erythraeum Lindl., analyzing their changing rules, and revealing the stress resistance principle, it provides a basis for optimizing the technology in the later stage.

[0044] This application uses Kingidium deliciosum as the test material and the controlled variable method to compare the survival rates of protocorms after cryopreservation with different PVS2 soaking times, so as to determine the cryopreservation method more suitable for Kingidium deliciosum and provide technical support for protecting the germplasm resources of Kingidium deliciosum. And measure the physiological and biochemical indexes of each important step in the optimal cryopreservation method of Kingidium deliciosum protocorms, so as to explore the response mechanism of Kingidium deliciosum under low temperature stress. The main research results are as follows:

[0045] Protocorms of Kingidium deliciosum with a size of 0.5 - 1 mm are suitable for cryopreservation by the small-droplet vitrification method with a PVS2 soaking time of 2 h, and the survival rate after preservation is 80.20%.

[0046] By comprehensively analyzing the physiological and biochemical indexes of protocorms of Kingidium deliciosum during cryopreservation, including pre-culture, PVS2 soaking, and post-thawing, it is found that the main reason for the decrease in protocorm activity is membrane lipid peroxidation. Based on the measurement results of soluble sugar, soluble protein, proline, and the antioxidant enzyme system, it can be known that each step before putting into liquid nitrogen provides support for protocorms to cope with cryogenic stress.

[0047] The professional terms involved in this application have the following meanings:

[0048] "Kingidium deliciosum" mentioned in this article refers to Phalaenopsis deliciosa Rchb. f. It was originally in the genus Kingidium. Due to its similar appearance and growth habits to the genus Phalaenopsis, it was incorporated into the genus Phalaenopsis. Its common name is Phalaenopsis appendiculata or Kingidium deliciosum. In recent years, it has been included in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Kingidium deliciosum grows on the trunks of mountain forests at an altitude of 450 - 1100 m and is distributed in Malaysia, Cambodia, Laos, etc. In China, it is mainly distributed in Hainan Province (Ledong Li Autonomous County, Changjiang Li Autonomous County, Sanya City). The plant of Kingidium deliciosum is relatively small, with papery leaves, oval-like, and slightly wavy edges. It has a paniculate inflorescence, and the flowering period is in July. The flower color shows a gradual change from purple to white from the center to the edge of the petals, and the root of the lip is light yellow. It is a high-quality native species of the genus Phalaenopsis in China and a good breeding material.

[0049] "Protocorm" mentioned in this article refers to the morphological form from the germination of orchid seeds to before becoming a seedling. It is the original spherical embryo formed by the swelling of the seed embryo, and its cells have high totipotency. Compared with the shoot tip tissue commonly used for cryopreservation of other plants, protocorms are easier to obtain and tougher, so they are commonly used for the preservation of germplasm resources of orchid plants.

[0050] As used herein, "germplasm resources" are also known as variety resources, genetic resources or gene resources, which refer to the general term of plants carrying various different genetic materials, including varieties or strains of various cultivated, wild and artificially created plants. Among them, germplasm is the genetic material that parents transmit 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 some other embodiments, the germplasm resources can be cells of plants.

[0051] As used herein, the "ultra-low temperature preservation technology" refers to the technology of storing plant materials in an environment below -190 °C and the plant materials still have activity after thawing. In some embodiments, the ultra-low temperature is -196 to -210 °C. In some other embodiments, the ultra-low temperature is the temperature of liquid nitrogen. The key to the ultra-low temperature preservation technology is to minimize the water content in cells and reduce the formation of ice crystals in cells, and two important factors, the use of cryoprotectants and the ultra-low temperature preservation method, need to be considered.

[0052] In some embodiments, the cryoprotectant is characterized by being easily soluble in water, non-toxic to cells, and easily removed from tissue cells. In some embodiments, the cryoprotectant can play the following protective roles on the protocorm tissue: generating strong hydration in the solution, increasing the viscosity of the solution, thereby reducing the rate of ice crystal formation and growth while the temperature drops; increasing the cell membrane permeability, accelerating the flow of water in the cell to the outside of the cell to freeze, thereby preventing damage caused by ice formation in the cell; preventing the toxicity of the "solution effect" before or during ultra-low temperature freezing; and directly or indirectly acting on the cell membrane to reduce the damage of ultra-low temperature to the cell membrane.

[0053] In some embodiments, the cryoprotectants can be divided into permeating type and non-permeating type. Among them, the permeating type cryoprotectant refers to the component that is easy to penetrate the cell membrane and enter the cell interior, thereby reducing the freezing point of the cell and increasing the cell membrane permeability to water. In some embodiments, the permeating type cryoprotectant can include glycerol, dimethyl sulfoxide, etc. The non-permeating type cryoprotectant generally refers to the component that cannot penetrate into the cell interior but is easily soluble in water and can dilute the concentration of extracellular electrolytes to reduce the damage of solutes to cells. In some embodiments, the non-permeating type cryoprotectant includes polyvinylpyrrolidone, sucrose, polyethylene glycol, dextran, etc.

[0054] In some embodiments, the loading solution (LS) and / or plant vitrification solution, such as PVS1, PVS2, PVS3, etc., mentioned herein all contain cryoprotectants.

[0055] As used herein, "loading" refers to the operation of immersing the original bulb in a loading solution to avoid cell damage caused by drastic changes in osmotic pressure. In some embodiments, the loading solution generally comprises 2 mol / L glycerol and 0.4 mol / L sucrose. In some embodiments, the loading solution comprises: 137 g / L sucrose, 2.3 g / L 1 / 2 MS, 184 g / L glycerol, or a combination thereof.

[0056] As used herein, "PVS2 (plant vitrification solution 2)" refers to plant vitrification solution 2, corresponding to PVS1 and PVS3, etc., which contains sucrose, 1 / 2 MS, glycerol, ethylene glycol, and dimethyl sulfoxide, or a combination thereof. PVS2 can effectively protect various plant materials during cryopreservation, avoiding crystallization and solute damage. In some embodiments, PVS2 comprises: 137 g / L sucrose, 2.3 g / L 1 / 2 MS, 300 mL / L glycerol, 150 mL / L ethylene glycol, and 150 mL / L dimethyl sulfoxide, or a combination thereof.

[0057] In some embodiments, "cryopreservation" is achieved by one or more of the methods such as the stepwise cooling method, rapid freezing method, drying method, vitrification method, small droplet vitrification method, and encapsulation vitrification method. Due to species differences or differences in the morphological structures of different tissues, it is necessary to precisely study cryopreservation methods for specific materials, thereby greatly improving the post-thaw survival rate.

[0058] As used herein, the "stepwise cooling method" refers to cooling the plant material from 0°C to -40°C at a cooling rate of 0.5 - 2°C per minute, and then immersing it in liquid nitrogen for further cooling to quickly reach -196°C. The process of stepwise cooling allows sufficient time for free water in the cells to precipitate outside the cells, forming ice extracellularly. This can effectively reduce the water content inside the cells and avoid cell damage caused by intracellular ice formation.

[0059] As used herein, the "rapid freezing method" refers to directly immersing the plant material in liquid nitrogen from 0°C or room temperature (25°C) to reach a state of -196°C at a rapid cooling rate of 1000°C per minute. During the freezing process, water molecules between plant cells cannot form ice crystals, thus avoiding the occurrence of freezing damage. Compared with the gradual slow freezing of the stepwise cooling method, this method is more rapid and direct.

[0060] As used herein, the "drying method" refers to culturing pre-preserved plant materials on a culture medium containing a high concentration of sugars, or drying them by silica gel water absorption and sterile air blowing to appropriately dehydrate the materials. During cryopreservation, if the water content of plant materials is too high, crystals will form in the plants, resulting in mechanical damage. However, if the water content is too low, the tissue cells of the plant materials will die due to water loss. Therefore, appropriate drying and dehydration can reduce damage to cells during preservation, improve the survival rate of materials, and enhancing the cryoresistance of cells by controlling the dehydration speed and degree.

[0061] As used herein, the "vitrification method" refers to the solidification process in which a liquid transforms into a non-crystalline (glass state). It involves immersing the materials in a vitrification solution, dehydrating the cells before plunging them into liquid nitrogen, and enhancing the viscosity of the protoplasts. During the rapid cooling process, the intracellular water molecules cannot be arranged in the state of ice crystals, thus avoiding mechanical damage caused by the freezing of free water and solute damage to some tissues caused by too slow freezing.

[0062] As used herein, the "small droplet vitrification method" refers to, after the plant materials are immersed in PVS2, first dropping PVS2 on an aluminum foil strip and then putting the pre-preserved materials into the PVS2 droplets. This can ensure that the materials enter the fully vitrified state more quickly when plunged into liquid nitrogen, and at the same time facilitate the thawing step after cryopreservation, reducing the damage caused by uneven temperature changes in plant cells.

[0063] As used herein, the "encapsulation-vitrification method" refers to a method that combines encapsulation and dehydration. Before cryopreservation, the plant materials are encapsulated, and by adding exogenous hormones, the plants can have a better environment and antioxidant effects, facilitating the subsequent penetration of PVS2 and avoiding a decrease in activity after cryopreservation due to uneven liquid exchange of the solution. The steps of the encapsulation-vitrification method can be: mixing the plant materials with a modified Knop medium containing 3% (w / v) sodium alginate and 0.2 mol / L sucrose at room temperature; dropping the mixture into a Knop culture solution containing 0.1 mol / L CaCl2 + 0.2 mol / L sucrose, ensuring that there is only one plant material in one encapsulation bead during encapsulation, gently shaking the mixture to prevent adhesion, and then fixing for 30 min; subsequently performing the subsequent cryopreservation steps. 2+ Mix the plant materials with a modified Knop medium containing 3% (w / v) sodium alginate and 0.2 mol / L sucrose at room temperature; drop the mixture into a Knop culture solution containing 0.1 mol / L CaCl2 + 0.2 mol / L sucrose, ensuring that there is only one plant material in one encapsulation bead during encapsulation, gently shaking the mixture to prevent adhesion, and then fixing for 30 min; subsequently performing the subsequent cryopreservation steps.

[0064] As used herein, "soluble sugar", "soluble protein", "proline", "malondialdehyde", "reactive oxygen species", "superoxide dismutase", "peroxidase", "catalase", etc. refer to the relevant physiological and biochemical indexes at each stage during the cryopreservation of plant materials. Their changes can reflect the strategies adopted by the plants to cope with stress at present, and can be used to infer the plant stress resistance mechanism, and can also measure whether the current cryopreservation steps are suitable for preserving the protocorms of this plant.

[0065] As used herein, "soluble sugar" refers to soluble sugar, abbreviated as SS, and its content reflects the thickness and adhesiveness of the cytoplasm. The higher the soluble sugar content, the higher the thickness and adhesiveness of the cytoplasm, indicating a higher freezing point and stronger water retention ability.

[0066] As used herein, "soluble protein" refers to soluble protein, abbreviated as SP. A higher soluble protein content in plants can generate substances such as proline (abbreviated as Pro) when the plants are coping with low-temperature stress, thereby alleviating low-temperature damage.

[0067] In some embodiments, by measuring the soluble sugar and soluble protein contents in plant tissues, the low-temperature tolerance of the plant body can be judged.

[0068] As used herein, "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 cell membrane lipid peroxidation, and is one of the important indicators for measuring 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.

[0069] As used herein, "reactive oxygen species" is abbreviated as ROS, and its content is an important parameter affecting the survival rate of plants. Research shows that a higher ROS is the main reason for the decrease in the survival rate of plants after cryopreservation.

[0070] As used herein, "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.

[0071] In some embodiments, the germplasm resources referred to herein are protocorms, which are obtained by aseptic sowing of seeds; wherein, the time of aseptic sowing is not less than 30 days. For example, the time of aseptic sowing is 30 - 40 days, 40 - 50 days, 55 - 60 days. In some embodiments, the diameter of the protocorm is not less than 0.5 mm; preferably, the diameter of the protocorm is 0.5 mm - 1 mm. For example, the diameter of the protocorm is 0.55 - 0.95 mm, 0.6 - 0.9 mm, 0.65 - 0.85 mm, 0.7 - 0.8 mm or a sub-range or any value thereof. In some embodiments, there may be a certain error in the range of the diameter involved herein, and this error is acceptable to those skilled in the art and has a negligible impact on the technical effect.

[0072] In some embodiments, the method for preserving the protocorm includes:

[0073] Pre-treat the protocorms. Put the protocorms into the pre-culture solution; wherein, the pre-culture solution comprises (100 - 150) g / L sucrose, (1 - 5) g / L 1 / 2MS, (50 - 150) mL / L coconut milk; preferably, the pre-culture solution comprises: 137 g / L sucrose, 2.3 g / L 1 / 2MS, 100 mL / L coconut milk. Seal the pre-culture solution and the protocorms therein; and place them in a shaker, shielded from light and adjust the rotation speed to (50 - 150) r / min for a certain period of time. Among them, the rotation speed of the shaker is 100 r / min. Among them, the environmental temperature for pre-culture is room temperature; preferably, the pre-culture temperature is 20 - 30 °C. Among them, the pre-culture time is 1 - 3 days, such as 1.5 - 2.5 days, 1.2 - 2.8 days, 2 - 3 days, 1.5 days, 2 days, 2.5 days or sub-ranges or any values therein.

[0074] Soak the pre-treated protocorms in the loading solution for loading treatment; in some embodiments, the loading solution comprises (100 - 150) g / L sucrose, (1 - 5) g / L 1 / 2MS, (150 - 200) g / L glycerol; preferably, the loading solution comprises 137 g / L sucrose, 2.3 g / L 1 / 2MS, 184 g / L glycerol. In some embodiments, the loading treatment time is 15 - 40 min, preferably 20 - 40 min, more preferably 25 - 35 min, and more preferably 30 min.

[0075] Soak the loaded protocorms in the vitrification solution for vitrification treatment so that the vitrification solution completely wraps the protocorms; wherein, the vitrification solution is PVS2 solution; the soaking time is 0 - 3 h, preferably the soaking time of the loaded protocorms in the vitrification solution is 1 - 2 h, 0.5 - 2.5 h, 1.5 - 2 h, 0.8 - 2.2 h, 1.7 - 2.4 h, 0.7 h, 1.6 h, 2 h, 2.7 h or sub-ranges or any values therein. The vitrification solution comprises (100 - 150) g / L sucrose, (1 - 5) g / L 1 / 2MS, (200 - 400) mL / L glycerol, (100 - 200) mL / L ethylene glycol, (100 - 200) mL / L dimethyl sulfoxide; preferably, the vitrification solution comprises 137 g / L sucrose, 2.3 g / L 1 / 2MS, 300 mL / L glycerol, 150 mL / L ethylene glycol, 150 mL / L dimethyl sulfoxide.

[0076] Perform cryogenic ((-196 to -210) °C) treatment on the vitrified protocorms and store the cryogenically treated protocorms.

[0077] Those skilled in the art should understand that the 1 h, 0.5 h, 2 h, 3 h, etc. mentioned herein all include errors within a certain range, which can be accepted by experimental personnel and the influence on experimental results can be ignored. In some embodiments, the error is within 0.1 h; in some embodiments, the error is within 0.05 h.

[0078] The present invention also provides a method for rewarming cryopreserved protocorms, including: subjecting the cryopreserved protocorms obtained by the method according to the present invention, for example, to a constant temperature treatment at 60°C for 60 - 120 s (such as 80 - 115 s, 90 - 110 s, 100 - 105 s, 82 s, 90 s, 95 s, 103 s or a sub-range or any value therein); and unloading the protocorms after the constant temperature treatment. The method for unloading protocorms includes soaking the protocorms after the constant temperature treatment in an unloading solution and soaking at room temperature for 15 - 30 min (such as 18 - 28 min, 20 - 26 min, 23 - 25 min, 17 min, 20 min, 22 min or a sub-range or any value therein). Among them, the unloading solution includes (350 - 500) g / L sucrose and (1 - 5) g / L 1 / 2MS; preferably, the unloading solution includes 411 g / L sucrose and 2.3 g / L 1 / 2MS.

[0079] and (1 - 5) g / L 1 / 2MS; preferably, the unloading solution includes: 411 g / L sucrose, 2.3 g / L 1 / 2MS.

[0080] The technical solution of the present application will be described below through specific examples:

[0081] Example 1: Preparation of cryopreservation solution

[0082] The solution ratio and method of this experiment are as follows:

[0083] Preculture solution (preculture): 137 g / L sucrose + 2.3 g / L 1 / 2MS + 100 mL / L coconut milk.

[0084] Loading solution (LS): 137 g / L sucrose + 2.3 g / L 1 / 2MS + 184 g / L glycerol.

[0085] Plant vitrification solution (PVS2): 137 g / L sucrose + 2.3 g / L 1 / 2MS + 300 mL / L glycerol + 150 mL / L ethylene glycol + 150 mL / L dimethyl sulfoxide.

[0086] Unloading solution (UL): 411 g / L sucrose + 2.3 g / L 1 / 2MS.

[0087] After the above solutions are prepared, adjust the pH value to pH = 5.8 with NaOH and HCl. LS and PVS2 containing glycerol are relatively viscous, and the pH adjustment process should be accompanied by rapid and uniform stirring to ensure accurate pH measurement. Put the above solutions (except PVS2) into an autoclave and sterilize for 20 min, then wait until it cools down to room temperature. Put PVS2 without dimethyl sulfoxide into the autoclave for sterilization. Subsequently, in a laminar flow hood, filter and sterilize dimethyl sulfoxide with a disposable syringe filter (PES membrane 0.22 μm Millipore.) and mix it with PVS2 (lacking dimethyl sulfoxide) that has cooled down to room temperature after autoclave sterilization, thus completing the preparation of PVS2. Put the above sterile solutions into a refrigerator at 4 °C for storage and wait for use.

[0088] Example 2: Cryopreservation of Protocorms of Cymbidium erythraeum Lindl.

[0089] Protocorms specifically refer to the morphological form from the germination of orchid seeds to before seedling formation. They are primitive globular embryos formed by the swelling of the seed embryo, and their cells have high totipotency. Compared with the shoot tips of other plants commonly used for cryopreservation, protocorms are easier to obtain and tougher, and can be used for the cryopreservation of orchid protocorms.

[0090] In this example, Cymbidium erythraeum Lindl. was planted in the orchid greenhouse of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. Mature fruits were obtained 3 months after artificial cross-pollination between different individuals. The method for obtaining its protocorms was as follows: Mix all the seeds in 1 fruit with 1 mL of sterile water and evenly sow them into 3 tissue culture flasks (2.3 g / L 1 / 2MS + 20 g / L sucrose + 100 mL / L coconut milk + 5 g / L carrageenan, pH 5.8). Culture in a tissue culture room at a culture temperature of 25 - 28 °C, a light intensity of 1500 - 2000 lx, and a light duration of 12 h / day. Approximately 30 days after sowing, it can be seen that the seeds swell and germinate into protocorms with a size of 0 - 0.5 mm. After 60 days, the diameter of the protocorms grows to 0.5 - 1 mm.

[0091] In this experiment, the droplet vitrification method was used to cryopreserve the protocorms. Figure 1 It is a brief flow chart for the droplet vitrification cryopreservation and viability detection of Cymbidium erythraeum Lindl. protocorms. As Figure 1As shown in the figure, the steps of cryopreservation of the protocorms of Kingidium deliciosum by the small droplet vitrification method are as follows: pre-culture (PC), soaking in the loading solution (LS), soaking in PVS2, making small droplets, and plunging into liquid nitrogen (LN). In order to detect the survival rate of the protocorms after preservation, the following steps are also carried out: soaking in the unloading solution (UL), and TTC test. Since the protocorms are more tender than seeds, liquid pre-culture needs to be carried out before officially entering the cryopreservation steps to ensure that the protocorms are prepared for the following series of steps. The main difference between the small droplet vitrification method and the vitrification method is that before plunging into liquid nitrogen, small droplets need to be made for the materials, and this step can better protect the materials when plunging into liquid nitrogen. In this experiment, the control variable method was used to study the cryopreservation of the protocorms of Kingidium deliciosum by selecting different diameters, PVS2 soaking times, vitrification methods and small droplet vitrification methods. Through the activity test after preservation, the most suitable cryopreservation method for the protocorms of Kingidium deliciosum was determined. All experimental operations were carried out in a laminar flow hood to ensure a sterile environment. The specific process is as follows:

[0092] ① Pre-culture: Put the pre-culture solution into a 150 mL conical flask, 50 mL per flask. Select protocorms of Kingidium deliciosum with diameters of 0 - 0.5 mm, 0.5 - 1 mm, and >1 mm respectively, and load about 50 protocorms into each flask. After attaching the sealing film to the bottle stopper, place it in a shaker at 100 r / min and pre-culture in the dark at room temperature for 2 d.

[0093] ② Loading: Put the pre-cultured protocorms into a 10 mL small beaker, add 5 mL LS, and soak at room temperature for 30 min.

[0094] ③ Vitrification: After loading, replace LS with 5 mL of 0℃ PVS2, then place the 10 mL small beaker on ice and soak for 0, 1, 2, 3 h respectively.

[0095] ④ Making small droplets: Prepare an aluminum foil strip with the same size as the cryotube, fold it in half to an angle of 60° (with the rough side facing in), and evenly drop 3 drops of PVS2 on the aluminum foil strip with a pipette, 20 μl per drop. Put the vitrified protocorms into the small droplets, about 3 - 5 in each drop, and ensure that the PVS2 droplets completely wrap the protocorms.

[0096] ⑤ Liquid nitrogen preservation: After filling the cryotube with liquid nitrogen, quickly put the prepared aluminum foil strip into the cryotube, cover the lid, and preserve for 1 h. The temperature of liquid nitrogen is -196℃.

[0097] ⑥ Unloading: Put all the aluminum foil strips after liquid nitrogen preservation into a 10 mL small beaker containing 10 mL UL, use forceps to stir the aluminum foil strip to make the small droplets fall off, take out the aluminum foil strip, and soak at room temperature for 20 min.

[0098] ⑦Activity test: After unloading, replace UL with 1 mL of 1% TTC, put it into a cryotube, and place it in the dark at room temperature for 24 h to wait for staining. After staining, count the total number of protocorms and the number of protocorms stained red (staining red indicates viability). Calculate the survival rate: Survival rate = (number of viable protocorms / total number of protocorms) × 100%.

[0099] Each test was repeated 3 times.

[0100] Control: Directly put the protocorms into liquid nitrogen for preservation for 1 h, and then place them at room temperature for 20 min.

[0101] Example 3: Determination of physiological and biochemical indexes during the cryopreservation of protocorms of Cymbidium erythraeum Lindl.

[0102] ① Selection of test samples: In this test, untreated protocorms (F), and 3 samples of protocorms of Cymbidium erythraeum Lindl. at different steps during the cryopreservation process in Example 2 were selected: protocorms after preculture (PC), protocorms after vitrification (PC + PVS2), and protocorms after unloading (PC + PVS2 + UL). A control (control check, CK) group was set: directly put the untreated protocorms into liquid nitrogen for preservation for 1 h, and then place them at room temperature for 20 min for sampling.

[0103] ② Determination of physiological and biochemical indexes: Seven physiological and biochemical indexes, namely soluble sugar (SS), soluble protein (SP), proline (Pro), malondialdehyde (MDA), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), were determined for the samples of the test group and the control. Sampling was repeated 3 times, and each sample was measured 3 times. The measured values were the corresponding contents, concentrations, and activities in fresh samples. All indexes were measured using kits produced by SolarbioLIFESCIENCES and an enzyme-labeling instrument (EPOCH).

[0104] The obtained data were used to draw tables with Excel 2016, process data with SPSS 25.0, and make pictures with Photoshop 2021.

[0105] 1. Selection of vitrification method for protocorms

[0106] As described in Example 2,protocorms of Cymbidium erythraeum Lindl. with a size of 0.5 - 1 mm were cryopreserved using the vitrification method and the small droplet vitrification method respectively. The soaking time in PVS2 was 2 h. After cryopreservation, the protocorms of Cymbidium erythraeum Lindl. were stained with TTC. By comparing the two methods, the cryopreservation method suitable for the protocorms of Cymbidium erythraeum Lindl. was found. The vitrification method in this experiment was different from the small droplet vitrification method in Example 2 in steps ④ and ⑤. The vitrified protocorms were directly put into a cryotube containing PVS2 solution and then plunged into liquid nitrogen for preservation. According to the formula: survival rate = (number of viable protocorms / total number of protocorms) × 100%, the survival rates of the two treatment methods were calculated.

[0107] The test results showed that the survival rate of the vitrification method was 52.59% ± 6.15%, while that of the small droplet vitrification method was 80.20% ± 2.22%. This indicated that the protocorms of Cymbidium erythraeum Lindl. were more suitable for cryopreservation by the small droplet vitrification method, and the survival rate was about 27.61% higher than that of the vitrification method.

[0108] 2. Effect of protocorm diameter on survival rate

[0109] As described in Example 2,protocorms with different diameters (0 - 0.5 mm, 0.5 - 1 mm, >1 mm) were cryopreserved using the small droplet vitrification method with a PVS2 soaking time of 2 h. According to the formula: survival rate = (number of viable protocorms / total number of protocorms) × 100%, the survival rates of protocorms with different diameters were calculated.

[0110] The results showed that the survival rate of 0 - 0.5 mm protocorms was 0, the survival rate of 0.5 - 1 mm protocorms was 80.20% ± 2.22%, and the survival rate of >1 mm protocorms was 29.30% ± 2.47%. This indicated that the protocorms of Cymbidium erythraeum Lindl. with a size of 0.5 - 1 mm were more suitable as cryopreservation materials, with the highest survival rate. Compared with the second - highest group, the survival rate was about 50.9% higher.

[0111] 3. PVS2 soaking time for cryopreservation of protocorms of Cymbidium erythraeum Lindl.

[0112] As described in Example 2,protocorms of Cymbidium erythraeum Lindl. with a size of 0.5 - 1 mm were selected and cryopreserved using the small droplet vitrification method with PVS2 soaking times of 0, 1, 2, and 3 h respectively, and one control (CK) was set.

[0113] Figure 2 The TTC staining results of the protocorms of Cymbidium erythraeum Lindl. after cryopreservation are given. Figure 2 B is Figure 2Magnified view of the square in Figure A, from which the difference in the staining degree of the original bulb can be clearly seen: green or a small part of red indicates that the non-viable original bulb will not be completely stained by TTC; while the viable original bulb will be stained red by TTC.

[0114] Figure 3 It is a bar graph showing the change in the survival rate of cryopreservation of the protocorms of Kingidium deliciosum with the soaking time of PVS2. From Figure 3 it can be seen that the survival rate of the control group (CK) is 0; as the soaking time of PVS2 increases, the survival rate of the protocorms shows a trend of first increasing and then decreasing. When the soaking time of PVS2 is 2 h, the survival rate reaches the peak, which is 80.20%. The survival rate when the soaking time of PVS2 is 1 h (49.6%) ranks second. The survival rate when the soaking time of PVS2 is 0 h (31.11%) is the lowest. Therefore, about 2 h is taken as the most suitable soaking time of PVS2 for the cryopreservation of protocorms.

[0115] 4. Determination of physiological and biochemical indexes of cryopreservation of protocorms of Kingidium deliciosum

[0116] For the 4 test samples described in ① above in this example: untreated protocorms (F), pre-cultured protocorms (PC), vitrified protocorms (PC + PVS2), unloaded protocorms (PC + PVS2 + UL), and the control (control check, CK) group, the following 7 physiological and biochemical indexes are determined: soluble sugar (SS), soluble protein (SP), proline (Pro), malondialdehyde (MDA), superoxide dismutase (SOD), peroxidase (POD), catalase (CAT).

[0117] (1) Soluble sugar, soluble protein, proline and malondialdehyde

[0118] Figure 4 and Figure 5 are the test results of soluble sugar and soluble protein of 5 test samples of Kingidium deliciosum. From Figure 4 and Figure 5 it can be seen that the change trends of soluble sugar and soluble protein are the same. During the cryopreservation process, they first increase and then decrease, and both increase significantly and reach the peak after soaking in PVS2, which are 105.56 mg / g and 15.13 mg / g respectively. After being stored in liquid nitrogen, the content decreases, but the content of soluble sugar and soluble protein is still significantly higher than that of the untreated protocorms, and the control content is significantly lower than the content of each step. Based on the above results, it shows that each step before putting into liquid nitrogen in this study is beneficial to the increase of the content of soluble sugar and soluble protein, and still maintains a relatively high content after cryopreservation.

[0119] Figure 6 and Figure 7 are the test results of proline and MDA of 5 Cymbidium erythraeum Lindl. test samples. As can be seen from the figure, proline first increases and then decreases during the ultra-low temperature process, significantly increases and reaches the peak after soaking in PVS2, which is 93.10 μg / g, showing a similar change trend to the contents of soluble sugar and soluble protein. The content decreases after being stored in liquid nitrogen, but it is still significantly higher than that of the untreated protocorms. MDA gradually increases during the ultra-low temperature storage process and is higher than the control in all cases, indicating that ultra-low temperature storage may cause certain stress to the protocorms.

[0120] (2)Antioxidant enzymes

[0121] Figure 8 and Figure 9 are the test results of SOD ( Figure 8 )and POD ( Figure 9 )of 5 Cymbidium erythraeum Lindl. test samples. As can be seen from Figure 8 , the SOD activity first increases and then decreases during the ultra-low temperature storage process, reaches the peak after soaking in PVS2, which is 163.11 U / g. The activity decreases after being stored in liquid nitrogen, but it is still significantly higher than that of the untreated protocorms, and the activity of the control group is significantly lower than that of other samples. As can be seen from Figure 9 , the activity of POD shows little change compared with the control in each step before being put into liquid nitrogen, and slightly increases after soaking in PVS2. The activity decreases after being stored in liquid nitrogen, which may be caused by the low temperature stress after being put into liquid nitrogen.

[0122] Figure 10 are the CAT test results of Cymbidium erythraeum Lindl. The CAT activity first increases and then decreases during the ultra-low temperature storage process, reaches the peak after pretreatment, which is 527.51 U / g. It may be stressed by PVS2 and liquid nitrogen in the subsequent steps, resulting in a gradual decrease in CAT, but the activity after ultra-low temperature storage is still higher than that of the control.

[0123] 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 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 protocorm of Cymbidium spicatum, comprising: Obtain the protocorm of the large-pointed capsule orchid; The protocorm is obtained from seeds after aseptic sowing; The diameter of the protocorm is 0.5 mm to 1 mm; Pre-culturing the protocorm comprises: The protocorm is placed in a pre-culture solution; wherein the pre-culture solution comprises (100-150) g / L sucrose, (1-5) g / L 1 / 2MS, and (50-150) mL / L coconut milk; the pre-culture solution and the protocorm therein are sealed; and the sealed material is placed in a shaker, shielded from light, and shaken at a speed of (50-200) r / min for 1-3 days; Immersing the pre-cultured protocorms in a loading solution for 20-40 min; wherein the loading solution comprises (100-150) g / L sucrose, (1-5) g / L 1 / 2MS, and (150-200) g / L glycerol; The loaded protocorm is immersed in a vitrification solution for vitrification treatment for 2-3 hours; wherein the vitrification solution is a PVS2 solution; the vitrification solution comprises (100-150) g / L sucrose, (1-5) g / L 1 / 2MS, (200-400) mL / L glycerol, (100-200) mL / L ethylene glycol, and (100-200) mL / L dimethyl sulfoxide; Immerse the vitrified protocorm into a small drop of vitrification solution, ensuring that the PVS2 drop completely encapsulates the protocorm; The small droplets containing the protocorms are stored in an ultra-low temperature environment of -190°C to -210°C.

2. The method according to claim 1, wherein the seeds are sown aseptically for no less than 30 days.

3. The method of claim 1, wherein the loading solution comprises 137 g / L sucrose, 2.3 g / L 1 / 2MS, and 184 g / L glycerol.

4. A method for thawing cryopreserved protocorms, comprising: Treating the cryogenically treated protocorm obtained by the method according to any one of claims 1 to 3 at a constant temperature of about 60° C. for 75 to 120 s; as well as Unloading of thermostatically treated protocorms.

5. The method according to claim 4, wherein the step of unloading the thermostatically treated protocorms comprises soaking the thermostatically treated protocorms in an unloading solution for 15-30 minutes; wherein: The unloading solution included (350-500) g / L sucrose and (1-5) g / L 1 / 2MS.

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