Preservation method of protocorm of cynanchum macrocephalum

Abstract

The invention relates to a preservation method of a Cymbidium macrocephalum protocorm. The preservation method comprises the following steps: obtaining the Cymbidium macrocephalum protocorm; soaking the protocorm into a loading solution for loading treatment; soaking the loaded material into a vitrification solution for vitrification treatment; wherein the vitrification solution is a PVS2 solution; the soaking time is 0-3 hours; and storing the vitrified protocorm at an ultralow temperature. The protocorm treatment method is simple to operate, and the cost can be greatly saved. And the survival rate of the protocorm preserved by the method provided by the invention is high.

Description

Technical Field

[0001] The invention relates to the protection of plant protocorms, in particular to a method for preserving the protocorms of Cymbidium spicatum. Background Art

[0002] Orchidaceae Juss. is the second largest family of angiosperms after Asteraceae and the largest family of monocots. There are about 800 genera and 28,000 species in the world, widely distributed in all terrestrial ecosystems except extreme areas. my country has 171 genera and 1,247 species of orchids, making it one of the countries with the richest wild orchids. They are mainly distributed in Yunnan, Taiwan, Hainan, Guangdong, Guangxi and other regions, with a series of evolutionary groups and rich geographical distribution types.

[0003] Phalaenopsis Blume plants have butterfly-like flower shapes, long flowering periods, rich colors, and a large number of flowers, making them popular among people. They have become one of the best-selling potted flowers in the world. Phalaenopsis plants are perennial epiphytic herbs with well-developed fleshy roots that are long and flat and attached to rock walls or tree barks. The leaves are flat and thick, and are oval or obovate-lanceolate. Phalaenopsis plants prefer a humid, hot, and semi-shady environment. They are not cold-resistant and are afraid of strong light and drought. Waterlogging at the roots will cause them to rot. Therefore, they should be planted in loose, well-drained bark blocks or sphagnum moss. They are mostly grown on tree trunks in tropical or subtropical forests at low altitudes.

[0004] The seeds of wild orchids require the participation of specific symbiotic bacteria when germinating, and they have strict requirements on habitats and are highly dependent on other organisms such as pollinators. In recent years, due to climate change, the population of some species of Phalaenopsis, such as the Phalaenopsis deliciosa Rchb. f., has continued to decline, increasing the risk of extinction. Therefore, the task of protecting its germplasm resources is imminent.

[0005] On the other hand, the fruits of orchids such as Orchidaceae contain tens of thousands of tiny seeds. The seeds only have embryos and seed coats, but no endosperm. The seeds themselves have no nutrients and cannot germinate independently. Under natural conditions, they need the help of symbiotic bacteria to provide nutrition to germinate. The germination conditions are relatively harsh and have strict requirements on the quality of natural habitats, resulting in an extremely low natural germination rate, even less than 2%. Under natural conditions, the enzyme activity in the seeds of the orchid family is strong, and the seed vitality is difficult to maintain for a long time, and the lifespan is short. The protocorm is a flat spherical object formed after the germination of orchid seeds. It has a false root at the base, which is morphologically similar to the protoembryo formed by the embryo when the seeds germinate. The protocorm can further grow into an orchid plant, and can also be cut into several small pieces to form several new protocorm clusters.

[0006] It can be seen that orchid seeds have strict requirements for germination and short lifespan, which makes their natural reproduction difficult. Therefore, artificial preservation of protocorms and growing them into plants at an appropriate time 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 proposes a method for preserving the protocorms of the Orchidaceae plant, comprising: obtaining the protocorms of the Orchidaceae; immersing the protocorms in a loading solution for loading treatment; immersing the loaded protocorms in a vitrification solution for vitrification treatment; wherein the vitrification solution is a PVS2 solution; the immersion time is 0-3 h; immersing 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] In the method 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 between (-196 and -210)°C.

[0009] The method as described above, wherein the loading solution comprises (100-150) g / L sucrose, (1-5) g / L 1 / 2MS, and (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] The method as described above, wherein the loading treatment time is 20-40 min, preferably 30 min.

[0011] The method as described above, wherein 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; 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] In the above method, the protocorm is obtained from seeds after aseptic sowing; wherein the time of tissue culture is not less than 30 days. In the above method, the time of tissue culture is 30-40 days, 40-50 days, 55-60 days.

[0013] In the method as described above, 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 any sub-range or value therein.

[0014] According to the method described above, the soaking time of the loaded protocorms in the vitrification solution is 0-3 h, such as 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 any subrange or value therein.

[0015] The method as described above further comprises pre-culturing the protocorm, which comprises: placing the protocorm in a pre-culture solution; sealing the pre-culture solution and the protocorm therein; and placing in a shaking incubator, shielding from light and treating at a rotation speed of (50-200) r / min for 1-3 days; preferably treating for 2 days.

[0016] In the method as described above, the pre-culture environment temperature is room temperature; preferably, the pre-culture temperature is 20-30°C.

[0017] The method as described above, wherein 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 any subrange or value therein.

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

[0019] The method as described above further comprises placing the vitrified protocorm into a drop of a vitrification solution, so that the vitrification solution completely wraps the protocorm.

[0020] According to another aspect of the present application, a method for relieving ultra-low temperature preserved protocorms is proposed, comprising: isothermal treating the ultra-low temperature treated protocorms obtained, for example, according to the method described above, 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 subrange or any value therein); and unloading the isothermal treated protocorms.

[0021] In the method as described above, the step of unloading the thermostatically treated protocorms comprises soaking the thermostatically treated protocorms in an unloading solution for 15-30 min (e.g., 18-28 min, 20-26 min, 23-25 ​​min, 17 min, 20 min, 22 min or a subrange or any value therein).

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

[0023] The original corm processing method of the present application is simple to operate and can greatly save costs. In addition, the original corm preserved by the method of the present application has a high survival rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The preferred embodiments of the present invention will be further described in detail below with reference to the accompanying drawings, wherein: Figure 1 A brief flow chart showing the ultra-low temperature storage, thawing and activity detection of small droplet vitrification of the protocorm of Orchidaceae according to one embodiment of the present invention is shown; Figure 2 An image of TTC staining of the protocorm of Orchidaceae after cryopreservation according to an embodiment of the present invention is shown; Figure 3 A bar graph showing the survival rate of the protocorms of Orchidaceae preserved at ultra-low temperatures according to an embodiment of the present invention as a function of the PVS2 immersion time is shown; Figure 4 The soluble sugar content of the large pointed capsule orchid after different treatments according to one embodiment of the present invention is shown; Figure 5 The soluble protein content of Cymbidium orchid subjected to different treatments according to one embodiment of the present invention is shown; Figure 6 The proline (Pro) content of the large-pointed sac orchid after different treatments according to one embodiment of the present invention is shown; Figure 7 The figure shows the malondialdehyde (MDA) content of the large-pointed sac orchid after different treatments according to one embodiment of the present invention; Figure 8 The figure shows the superoxide dismutase (SOD) content of the large pointed capsule orchid after different treatments according to one embodiment of the present invention; Fig. 9 The figure shows the peroxidase (POD) content of the large-pointed orchid after different treatments according to one embodiment of the present invention; Fig.10The graph shows the catalase (CAT) content of Cymbidium orchid after different treatments according to an embodiment of the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] In the following detailed description, reference may be made to the various specification drawings that are part of the present application and are used to illustrate specific embodiments of the present application. In the accompanying drawings, similar reference numerals describe substantially similar components in different figures. The various specific embodiments of the present application are described below in sufficient detail so that a person of ordinary skill in the art with relevant knowledge and skills in the art can implement the technical solutions of the present application. It should be understood that other embodiments may also be used or structural, logical or electrical changes may be made to the embodiments of the present application.

[0027] There are three common ways to preserve orchid germplasm resources: in situ protection, tissue culture subculture, and ultra-low temperature preservation.

[0028] In situ conservation, in the form of nature reserves or protection sites, protects the natural habitats of orchids and the ecosystems within them to the greatest extent possible, but they are vulnerable to natural disasters or pests and diseases.

[0029] Tissue culture subculture and cryopreservation are off-site preservation, and in vitro preservation is carried out in the form of test tube seedlings or cryopreservation in the germplasm resource preservation bank. However, because orchids require strict temperature, light, and humidity, the construction of a greenhouse is large, expensive, and requires high manpower and material costs. In addition, tissue culture subculture is prone to mutation, and the more operations are performed, the more likely it is to be contaminated, resulting in material loss.

[0030] Ultra-cryopreservation is to store materials in liquid nitrogen. The material metabolism and growth activities of the materials are almost completely stopped but they are still active. After thawing, they can resume activity and continue to grow. Compared with commonly used cryopreservation, ultra-cryopreservation is more stable and can prevent material mutation. The selected preservation material should be both omnipotent and able to develop into a complete plant. It is also necessary to ensure that the cell structure itself is intact during liquid nitrogen freezing (−196°C) and is not destroyed by the ultra-low temperature environment, so as to ensure the activity of the material after preservation. There are 6 commonly used ultra-cryopreservation methods, namely step-by-step cooling method, quick freezing method, drying method, vitrification method, small droplet vitrification method, and embedding vitrification method. Multiple studies have shown that the use of small droplet vitrification method for ultra-low temperature preservation of plant materials is more effective. Among them, Hayashida et al. [1] By comparing the effects of vitrification and small droplet vitrification on the cryopreservation of carnation stem tips, it was found that the survival rate of vitrification was over 80%, and the survival rate of small droplet vitrification was over 90%. However, during the cryopreservation process, plant materials are inevitably subject to temperature stress and osmotic pressure changes. [2] The MDA content of embryonic callus of Agapanthus nymphaeaceae after ultra-low temperature preservation by vitrification was measured, and it was found that the MDA content of plant materials increased significantly after preservation, indicating that ultra-low temperature preservation by vitrification will inevitably cause oxidative stress and cell apoptosis in plants. Therefore, considering the species differences, the freezing resistance of plants and the different morphological structures of plant tissues, targeted research on the ultra-low temperature preservation method of small droplet vitrification that is suitable for specific materials can greatly improve the survival rate after freezing.

[0031] [1] Lin Tian, ​​Yang Hua, Han Jing, et al. Effects of vitrification and small droplet vitrification on cryopreservation of carnation stem tips[J]. Anhui Agricultural Science, 2018, 46(19): 105-107+141. DOI: 10.13989 / j.cnki.0517-6611.2018.19.030. [2] Li Xiaodan. Research on cryopreservation technology of embryonic callus of Agapanthus nymphaeaceae by vitrification[D]. Northeast Forestry University, 2013. This application uses the large-pointed capsule orchid as the plant material, and analyzes the effects of the PVS2 immersion time and vitrification method of the protocorm on ultra-low temperature preservation, in order to find the most suitable ultra-low temperature preservation technology for the protocorm of the large-pointed capsule orchid, and provide a technical theory for protecting its germplasm resources. Subsequently, by determining the most suitable physiological and biochemical indicators for the main steps of the ultra-low temperature preservation of the protocorm of the large-pointed capsule orchid, analyzing their changing laws, revealing the principle of stress resistance, and providing a basis for later optimization of technology.

[0032] This application uses the large-pointed sac as the experimental material, and uses the controlled variable method to compare the survival rate of the protocorms after ultra-low temperature preservation with different PVS2 immersion times, so as to determine the ultra-low temperature preservation method that is more suitable for the large-pointed sac, and provide technical support for the protection of the germplasm resources of the large-pointed sac. And determine the physiological and biochemical indicators of each important step in the optimal ultra-low temperature preservation method of the protocorms of the large-pointed sac, so as to explore the response mechanism of the large-pointed sac under low temperature stress. The main research results are as follows:

[0033] The 0.5-1mm protocorms of Orchidaceae are suitable for ultra-low temperature preservation using the small droplet vitrification method with a PVS2 immersion time of 2h, and the survival rate after preservation is 80.20%.

[0034] Comprehensive analysis of the physiological and biochemical indices of the protocorms of the large-pointed sac orchid during cryopreservation, pre-culture, PVS2 immersion, and unloading revealed that the main cause of the decrease in protocorm activity was membrane lipid peroxidation. Based on the results of the determination of soluble sugars, soluble proteins, proline, and antioxidant enzyme systems, it was found that each step before immersion in liquid nitrogen provided support for the protocorms to cope with ultra-low temperature stress.

[0035] The professional terms involved in this application have the following meanings: The "large pointed capsule orchid" mentioned in this article refers to Phalaenopsis deliciosa Rchb. f., which was originally a genus of pointed capsule orchid (Kingidium). Because of its appearance and growth habits similar to those of Phalaenopsis, it was incorporated into the Phalaenopsis genus, commonly known as the bent stem callus orchid or large pointed capsule Phalaenopsis. 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). Large pointed capsule orchid grows on tree trunks in mountain forests at an altitude of 450-1100m. It is distributed in Malaysia, Cambodia, Laos and other places. In my country, it is mainly distributed in Hainan Province and other places (Ledong Li Autonomous County, Changjiang Li Autonomous County, Sanya City). Large pointed capsule orchid plants are small, with papery, elliptical leaves and wavy edges. Panicle inflorescence, flowering period in July, flower color changes 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 Phalaenopsis in my country and a good breeding material.

[0036] The "protocorm" mentioned in this article refers to the morphology of orchid seeds from germination to seedling formation. It is the original spherical embryo formed by the expansion of the seed embryo, and its cells are highly omnipotent. Compared with the stem tip tissues commonly used for cryopreservation of other plants, the protocorm is easier to obtain and tougher, so it is often used to preserve the germplasm resources of orchids.

[0037] The "germplasm resources" mentioned in this article are also called variety resources, genetic resources or gene resources. They refer to the general term for plants carrying various genetic materials, including cultivated, wild and artificially created varieties or strains of various plants. Among them, germplasm is the genetic material that parents pass to offspring through reproductive cells or somatic cells and determines the inherent biological traits. Germplasm resource preservation refers to the technology of storing plant germplasm under suitable environmental conditions, either natural or artificially created, to maintain its vitality and heredity. In some embodiments, germplasm resources can be the original corms, leaves, roots, stem tips, etc. of plants. In other embodiments, germplasm resources can be plant cells.

[0038] The "ultra-low temperature preservation technology" mentioned in this article refers to the technology of storing plant materials in an environment below -190°C, and the plant materials are still active after thawing. In some embodiments, the ultra-low temperature is -196 to -210°C. In other embodiments, the ultra-low temperature is the temperature of liquid nitrogen. The key to ultra-low temperature preservation technology is to reduce intracellular water as much as possible and reduce the formation of ice crystals in cells. Two important factors need to be considered: the use of protective agents and ultra-low temperature preservation methods.

[0039] In some embodiments, the protective agent is soluble in water, non-toxic to cells, and easily removed from tissue cells. In some embodiments, the protective agent can play the following protective effects on the tissue of the protocorm: produce strong hydration in the solution, increase the viscosity of the solution, thereby reducing the speed of ice crystal formation and growth when the temperature drops; increase the permeability of the cell membrane, accelerate the flow of water in the cell to the outside of the cell to freeze, thereby preventing damage to the cell caused by freezing; before or during ultra-low temperature freezing, prevent the toxicity of the "solution effect"; and directly or indirectly act on the cell membrane to reduce the damage to the cell membrane caused by ultra-low temperature.

[0040] In some embodiments, protective agents can be divided into permeable and non-permeable types. Among them, permeable protective agents refer to components that easily penetrate the cell membrane to enter the interior of the cell, thereby lowering the cell freezing point and increasing the permeability of the cell membrane to water. In some embodiments, permeable protective agents may include glycerol, dimethyl sulfoxide, etc. Non-permeable protective agents generally refer to components that cannot penetrate into the interior of the cell but are easily soluble in water, and can dilute the concentration of extracellular electrolytes to reduce solute damage to the cell. In some embodiments, non-permeable protective agents include polyvinyl pyrrolidone, sucrose, polyethylene glycol, dextran, etc.

[0041] In some embodiments, the loading solution (LS) and / or the plant vitrification solution described herein, such as PVS1, PVS2, PVS3, etc., contain a protective agent.

[0042] "Loading" as used herein refers to immersing the protocorm in a loading solution to avoid damage to the cells due to drastic changes in osmotic pressure. In some embodiments, the loading solution generally includes 2 mol / L glycerol and 0.4 mol / L sucrose. In some embodiments, the loading solution includes: 137 g / L sucrose, 2.3 g / L 1 / 2MS, 184 g / L glycerol or consists of them.

[0043] "PVS2 (plant vitrification solution 2)" as mentioned herein refers to plant vitrification solution 2, which corresponds to PVS1 and PVS3, etc., and comprises or consists of sucrose, 1 / 2 MS, glycerol, ethylene glycol and dimethyl sulfoxide. PVS2 can effectively protect a variety of plant materials during ultra-low temperature storage to avoid crystallization and solute damage. In some embodiments, PVS2 comprises or consists of: 137g / L sucrose, 2.3g / L 1 / 2MS, 300mL / L glycerol, 150mL / L ethylene glycol and 150mL / L dimethyl sulfoxide.

[0044] In some embodiments, "ultra-low temperature preservation" is achieved by one or more of the following methods: step-by-step cooling, rapid freezing, drying, vitrification, small droplet vitrification, embedding vitrification, etc. Due to species differences or differences in the morphological structure of different tissues, it is necessary to accurately study the ultra-low temperature preservation method for specific materials, thereby greatly improving the survival rate after freezing.

[0045] The "step-by-step cooling method" mentioned in this article 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 cooling it again by adding liquid nitrogen to quickly reach -196°C. The step-by-step cooling process allows the free water in the cells to have sufficient time to precipitate out of the cells and freeze outside the cells, which can effectively reduce the water content in the cells and avoid cell damage caused by intracellular freezing.

[0046] The "quick freezing method" mentioned in this article refers to directly putting plant materials from 0℃ or room temperature (25℃) into liquid nitrogen, so that it reaches a state of -196℃ at a rapid cooling rate of 1000℃ 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 step-by-step cooling method, this method is faster and more direct.

[0047] The "drying method" mentioned in this article refers to culturing the pre-preserved plant materials on a medium containing a high concentration of sugars, or absorbing water through silica gel and drying with sterile air to properly dehydrate the materials. During ultra-low temperature storage, excessive water content in plant materials will lead to crystallization in the plant body, causing mechanical damage, but too low water content will cause the tissue cells of the plant materials to die due to water loss. Therefore, proper drying and dehydration can reduce damage to cells during storage and increase the survival rate of materials. Controlling the dehydration speed and degree can enhance the cell's antifreeze ability.

[0048] The "vitrification method" mentioned in this article refers to the solidification process of liquid into amorphous (glassy) state. It is to immerse the material in a vitrification solution, dehydrate the cells before immersing in liquid nitrogen, and enhance the viscosity of the protoplasts. During the rapid cooling process, the water molecules in the cells cannot be arranged into ice crystals, thereby avoiding mechanical damage caused by the freezing of free water and solute damage to some tissues caused by slow freezing.

[0049] The "small droplet vitrification method" mentioned in this article means that after the plant material is soaked in PVS2, PVS2 is first dropped on an aluminum foil strip, and the pre-preserved material is placed in the PVS2 droplet. This ensures that the material enters a completely vitrified state more quickly when it is put into liquid nitrogen. It also facilitates the thawing step after ultra-low temperature storage and reduces damage caused by uneven temperature changes in plant cells.

[0050] The "embedding vitrification method" mentioned in this article refers to a method that combines embedding and dehydration. Before ultra-low temperature storage, the plant material is embedded and exogenous hormones are added to provide the plant with a better environment and antioxidant effect, which is convenient for the subsequent penetration of PVS2 and avoids uneven exchange of solution liquids, which leads to reduced activity after ultra-low temperature. The steps of the embedding vitrification method can be: at room temperature, the plant material is mixed with 3% (w / v) sodium alginate and 0.2 mol / L sucrose in a Ca-free solution. 2+ The modified Knop medium was mixed; the mixture was dropped into the Knop culture medium containing 0.1 mol / L CaCl2 + 0.2 mol / L sucrose, and when embedding, it was ensured that there was only one plant material in one embedding bead, and the mixture was gently shaken to prevent adhesion, and fixed for 30 minutes; then the subsequent ultra-low temperature storage step was carried out.

[0051] The "soluble sugar", "soluble protein", "proline", "malondialdehyde", "reactive oxygen", "superoxide dismutase", "peroxidase", "catalase" and so on mentioned in this article refer to the relevant physiological and biochemical indicators at each stage during the ultra-low temperature preservation process of plant materials. Their changes can reflect the strategies currently adopted by plants to cope with stress, and can be used to infer the plant's stress resistance mechanism. It can also be used to measure whether the current ultra-low temperature preservation steps are suitable for preserving the protocorms of the plant.

[0052] The "soluble sugar" mentioned in this article refers to soluble sugar, abbreviated as SS, and its content reflects the density and adhesion of the cytoplasm. The higher the soluble sugar content, the higher the density and adhesion of the cytoplasm, indicating that it has a higher freezing point and stronger water retention capacity.

[0053] The "soluble protein" mentioned in this article refers to soluble protein, abbreviated as SP. A higher soluble protein content in plants can produce substances such as proline (abbreviated as Pro) when plants cope with low temperature stress, thereby alleviating low temperature damage.

[0054] In some embodiments, the tolerance of a plant to low temperature can be determined by measuring the soluble sugar and soluble protein content of plant tissues.

[0055] The "malondialdehyde" mentioned in this article is abbreviated as MDA. It is an important plant physiological and biochemical indicator. It is a negative product of cell membrane oxidation and can reflect the degree of cell membrane lipid peroxidation. It is one of the important indicators to measure whether plant tissues are damaged during ultra-low temperature storage. The higher the MDA content, the greater the damage to the cell membrane and the lower the cell activity.

[0056] The "reactive oxygen" mentioned in this article is abbreviated as ROS, and its content is an important parameter affecting plant survival rate. Studies have shown that higher ROS is the main reason for the reduced survival rate of plants after ultra-low temperature preservation.

[0057] The "superoxide dismutase" mentioned in this article is abbreviated as SOD, the "peroxidase" is abbreviated as POD, and the "catalase" is abbreviated as CAT. The antioxidant enzyme system of SOD, POD and CAT that cooperates with each other can eliminate excessive active oxygen in cells.

[0058] In some embodiments, the germplasm resources described herein are protocorms, which are obtained from seeds after aseptic sowing; wherein, the time for aseptic sowing is not less than 30 days. For example, the time for 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 therein. In some embodiments, the range of diameters involved in this article may have a certain error, which is acceptable to those skilled in the art, and the impact on the technical effect is negligible.

[0059] In some embodiments, the method of preserving the protocorm comprises: Pretreating the protocorms. The protocorms are 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 water; preferably, the pre-culture solution comprises: 137 g / L sucrose, 2.3 g / L 1 / 2MS, and 100 mL / L coconut water. The pre-culture solution and the protocorms therein are sealed; and placed in a shaker, shielded from light and adjusted to a speed of (50-150) r / min for a certain period of time. The shaker speed is 100 r / min. The ambient temperature of the pre-culture is room temperature; preferably, the pre-culture temperature is 20-30°C. 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 any sub-range or value therein.

[0060] The pretreated protocorms are immersed in a loading solution for loading treatment; in some embodiments, the loading solution comprises (100-150) g / L sucrose, (1-5) g / L 1 / 2MS, and (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. 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.

[0061] The loaded protocorms are immersed in a vitrification solution for vitrification treatment, so that the vitrification solution completely wraps the protocorms; wherein the vitrification solution is a PVS2 solution; the immersion time is 0-3 h, and preferably the immersion 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 a subrange or any value 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, and (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.

[0062] The vitrified protocorms are treated at ultra-low temperatures (-196 to -210°C) and the ultra-low temperature treated protocorms are preserved.

[0063] 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 the experimenter and whose impact on the 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.

[0064] The present invention also provides a method for rewarming cryopreserved protocorms, comprising: treating the cryopreserved protocorms obtained by the method of the present invention at a constant temperature of 60°C for 60-120 s (e.g., 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 thermostatically treated protocorms. The method for unloading the protocorms comprises soaking the thermostatically treated protocorms in an unloading solution for 15-30 min (e.g., 18-28 min, 20-26 min, 23-25 ​​min, 17 min, 20 min, 22 min or a sub-range or any value therein) at room temperature. The unloading solution comprises (350-500) g / L sucrose and (1-5) g / L 1 / 2MS; preferably, the unloading solution comprises 411 g / L sucrose and 2.3 g / L 1 / 2MS.

[0065] and (1-5) g / L 1 / 2MS; preferably, the unloading solution comprises: 411 g / L sucrose, 2.3 g / L 1 / 2MS.

[0066] The technical solution of this application will be described below through specific embodiments: Example 1: Preparation of cryopreservation solution The test solution ratio and method are as follows: Preculture solution (preculture): 137g / L sucrose + 2.3g / L 1 / 2MS + 100mL / L coconut water.

[0067] Loading solution (LS): 137 g / L sucrose + 2.3 g / L 1 / 2MS + 184 g / L glycerol.

[0068] Plant vitrification solution (PVS2): 137g / L sucrose + 2.3g / L 1 / 2MS + 300mL / L glycerol + 150mL / L ethylene glycol + 150mL / L dimethyl sulfoxide.

[0069] Unloading solution (UL): 411 g / L sucrose + 2.3 g / L 1 / 2MS.

[0070] After the above solutions are prepared, use NaOH and HCl to adjust the pH value to pH=5.8. LS and PVS2 containing glycerol are relatively viscous, and the pH adjustment process should be stirred quickly and evenly to ensure accurate pH measurement. Put the above solutions (except PVS2) into the sterilizer, sterilize for 20 minutes, and wait to cool to room temperature. Put the PVS2 without dimethyl sulfoxide into the sterilizer for sterilization, and then filter and sterilize the dimethyl sulfoxide with a disposable syringe filter (PES membrane 0.22um Millipore.) in the clean bench, and mix it with the PVS2 (lacking dimethyl sulfoxide) that has been sterilized in the sterilizer and cooled to room temperature to complete the preparation of PVS2. Store the above sterile solution in a 4°C refrigerator and wait for use.

[0071] Example 2: Cryopreservation of the protocorm of Cymbidium spicatum Protocorms specifically refer to the morphology of orchid seeds from germination to seedling formation. They are primitive spherical embryos formed by the expansion of the seed embryo, and their cells are highly omnipotent. Compared with the stem tip tissues of other plants that are often used for cryopreservation, protocorms are easier to obtain and tougher, and can be used to preserve protocorms of orchids.

[0072] In this embodiment, the large-pointed capsule orchid was planted in the orchid greenhouse of the Vegetable and Flower Research Institute of the Chinese Academy of Agricultural Sciences, and mature fruits were obtained 3 months after artificial self-pollination. The method for obtaining the original corm is as follows: all seeds in one fruit are mixed with 1 mL of sterile water, and evenly sown into 3 tissue culture bottles (2.3g / L 1 / 2MS+20g / L sucrose+100mL / L coconut juice+5g / L carrageenan, pH 5.8). Cultivate in a tissue culture room at a culture temperature of 25-28°C, a light intensity of 1500-2000lx, and a light of 12h / day. About 30 days after sowing, it can be seen that the seeds swell and germinate into a protocorm of 0-0.5mm. After 60 days, the diameter of the original corm grows to 0.5-1mm.

[0073] In this study, the small droplet vitrification method was used to preserve the protocorms at ultra-low temperatures. Figure 1 The following is a simplified flow chart of cryopreservation of small droplets of vitrification and activity detection of protocorms of Orchidaceae. Figure 1As shown in the figure, the steps of ultra-low temperature preservation of protocorms of Orchidaceae by small droplet vitrification are: pre-culture (PC), soaking in loading solution (LS), soaking in PVS2, making small droplets, and immersing in liquid nitrogen (LN). In order to detect the survival rate of protocorms after preservation, the following steps are also performed: soaking in unloading solution (UL) and TTC test. Because protocorms are more tender than seeds, liquid pre-culture is required before formally entering the ultra-low temperature preservation step to ensure that the protocorms are prepared for the next series of steps. The main difference between the small droplet vitrification method and the vitrification method is that small droplets need to be made for the material before immersing in liquid nitrogen. This step can better protect the material when immersing in liquid nitrogen. This experiment uses the controlled variable method to select different diameters, PVS2 immersion time, vitrification method and small droplet vitrification method to study the ultra-low temperature preservation of protocorms of Orchidaceae. Through the activity test after preservation, the most suitable ultra-low temperature preservation method for protocorms of Orchidaceae is determined. All experimental operations are carried out on a clean bench to ensure a sterile environment. The specific process is as follows:

[0074] ① Pre-culture: Place the pre-culture solution in 150mL conical bottles, 50mL per bottle. Select protocorms of Cymbidium spicatum with diameters of 0-0.5mm, 0.5-1mm, and >1mm, and place about 50 protocorms in each bottle. After attaching sealing film to the bottle stopper, place it in a shaker at 100 r / min and pre-culture at room temperature for 2 days away from light.

[0075] ② Loading: Place the pre-cultured protocorms into a 10 mL beaker, add 5 mL LS, and soak at room temperature for 30 minutes.

[0076] ③Vitrification: After loading, replace LS with 5 mL 0℃ PVS2, and then place the 10 mL beaker on ice and soak for 0, 1, 2, and 3 h respectively.

[0077] ④ Make small droplets: prepare an aluminum foil strip of the same size as the cryotube, fold it in half to an angle of 60° (with the rough side facing inward), and use a pipette to evenly drop 3 drops of PVS2 on the aluminum foil strip, 20μl per drop. Place the vitrified protocorms into the small droplets, about 3-5 per drop, and ensure that the PVS2 droplets completely cover the protocorms.

[0078] ⑤ Storage in liquid nitrogen: After the cryotube is filled with liquid nitrogen, quickly put the prepared aluminum foil strip into the cryotube, cover it, and store it for 1 hour. The temperature of liquid nitrogen is -196℃.

[0079] ⑥ Unloading: Put all the aluminum foil strips after storage in liquid nitrogen into a 10mL small beaker containing 10mL UL, use tweezers to rinse the aluminum foil strips to make small droplets fall off, take out the aluminum foil strips, and soak them at room temperature for 20 minutes.

[0080] ⑦ Activity test: After unloading, replace UL with 1mL 1% TTC, place in a cryotube, and place at room temperature away from light for 24 hours, waiting for staining. After staining, count the total number of protocorms and the number of protocorms stained red (red staining indicates activity). Calculate the survival rate: survival rate = (number of active protocorms / total number of protocorms) × 100%.

[0081] Each experiment was repeated 3 times.

[0082] Control: The protocorms were directly placed in liquid nitrogen for 1 hour and then placed at room temperature for 20 minutes.

[0083] Example 3: Determination of physiological and biochemical indicators during cryopreservation of protocorms of Cymbidium spicatum ① Selection of test samples: This test selected untreated protocorms (F) and three samples of the protocorms of the large-pointed sac orchid at different steps in the cryopreservation process of Example 2: the protocorms after pre-culture (PC), the protocorms after vitrification (PC+PVS2), and the protocorms after unloading (PC+PVS2+UL). Set up a control (CK) group: the untreated protocorms were directly placed in liquid nitrogen for 1 hour, and then placed at room temperature for 20 minutes for sampling.

[0084] ② Determination of physiological and biochemical indicators: The samples of the test group and the control were tested for 7 physiological and biochemical indicators, including soluble sugar (SS), soluble protein (SP), proline (Pro), malondialdehyde (MDA), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). The sampling was repeated 3 times, and each sample was tested 3 times. The measured values ​​are the corresponding content, concentration, and activity in the fresh sample. All indicators were measured using the kit produced by SolarbioLIFESCIENCES and the microplate reader (EPOCH).

[0085] The obtained data were used to draw tables using Excel 2016, SPSS 25.0 for data processing, and Photoshop 2021 for image production.

[0086] 1. Selection of vitrification methods for protocorms As described in Example 2, the 0.5-1 mm protocorms of Cymbidium apex were cryopreserved using the vitrification method and the small droplet vitrification method, respectively, with the PVS2 immersion time being 2 hours. After the cryopreserved protocorms of Cymbidium apex were subjected to TTC staining, the two methods were compared to find a cryopreservation method suitable for the protocorms of Cymbidium apex. The vitrification method in this experiment is different from the small droplet vitrification method in Example 2 in steps ④ and ⑤. The vitrified protocorms are directly placed in a cryovial filled with PVS2 solution and stored in liquid nitrogen. The survival rates under the two treatment methods were calculated according to the formula: Survival rate = (number of active protocorms / total number of protocorms) × 100%.

[0087] The experimental results showed that the survival rate of the vitrification method was 52.59%±6.15%, while the survival rate of the small droplet vitrification method was 80.20%±2.22%, indicating that the protocorms of the large-pointed sac orchid are more suitable for ultra-low temperature preservation by the small droplet vitrification method, and the survival rate is about 27.61% higher than that of the vitrification method.

[0088] 2. Effect of protocorm diameter on survival rate As described in Example 2, protocorms of different diameters (0-0.5 mm, 0.5-1 mm, >1 mm) were cryopreserved using the small droplet vitrification method and the PVS2 immersion time was 2 h. The survival rate of protocorms of different diameters was calculated according to the formula: survival rate = (number of active protocorms / total number of protocorms) × 100%.

[0089] The results showed that the survival rate of 0-0.5mm protocorms was 0, the survival rate of 0.5-1mm protocorms was 80.20% ± 2.22%, and the survival rate of >1mm protocorms was 29.30% ± 2.47%. This shows that 0.5-1mm protocorms of Orchidaceae are more suitable as cryogenic materials, with the highest survival rate, which is about 50.9% higher than the second highest group.

[0090] 3. PVS2 immersion time for cryopreservation of protocorms of P. spicata As described in Example 2, 0.5-1 mm protocorms of Cymbidium spicatum were selected and cryopreserved using the small droplet vitrification method with PVS2 immersion times of 0, 1, 2, and 3 h, respectively, and a control (CK) was set up.

[0091] Figure 2 The results of TTC staining of protocorms of Cymbidium maximum after cryopreservation are given. Figure 2 B is Figure 2 The enlarged image in the box in Figure A clearly shows the difference in the staining degree of the protocorms: green or a small part of red indicates that the inactive protocorms will not be completely stained by TTC; while the active protocorms will be stained red by TTC.

[0092] Figure 3 This is a bar graph showing the survival rate of the protocorms of the large-pointed sac orchid preserved at low temperatures as a function of the PVS2 immersion time. Figure 3 It can be seen that the survival rate of the control group (CK) was 0; with the increase of PVS2 immersion time, the survival rate of the protocorm increased first and then decreased. When the PVS2 immersion time was 2 hours, the survival rate reached a peak of 80.20%. The survival rate of the PVS2 immersion time of 1 hour (49.6%) was second. The survival rate of the PVS2 immersion time of 0 hours (31.11%) was the lowest. Therefore, about 2 hours was the most suitable PVS2 immersion time for cryopreservation of protocorms.

[0093] 4. Determination of physiological and biochemical parameters of Cryopreservation of P. maxima protocorms For the four test samples described in ① above in this embodiment: untreated protocorms (F), pre-cultured protocorms (PC), vitrified protocorms (PC+PVS2), unloaded protocorms (PC+PVS2+UL), and control (control check, CK) group, the following seven physiological and biochemical indices were measured: soluble sugar (SS), soluble protein (SP), proline (Pro), malondialdehyde (MDA), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT).

[0094] (1) Soluble sugar, soluble protein, proline and malondialdehyde Figure 4 and Figure 5 The results are for the soluble sugar and soluble protein test of 5 samples of Cymbidium spicatum. Figure 4 and Figure 5 It can be seen that the change trend of soluble sugar and soluble protein is the same. They first increase and then decrease during the cryopreservation process. They both increase significantly and reach the peak after soaking in PVS2, which are 105.56 mg / g and 15.13 mg / g respectively. The content decreases after liquid nitrogen storage, but the soluble sugar and soluble protein content of the untreated protocorm are still significantly higher than that of the control, and the content of each step is significantly lower than that of each step. In summary, the results show that each step before the liquid nitrogen injection in this study is conducive to the increase of soluble sugar and soluble protein content, and after cryopreservation, the content is still relatively high.

[0095] Figure 6 and Figure 7The proline and MDA test results of 5 samples of P. cymbidium. As shown in the figure, proline first increased and then decreased during the ultra-low temperature process. After soaking in PVS2, it increased significantly and reached a peak of 93.10 μg / g, which is similar to the change trend of soluble sugar and soluble protein content. The content decreased after liquid nitrogen storage, but it was still significantly higher than that of untreated protocorms. MDA gradually increased during ultra-low temperature storage and was higher than the control. It can be seen that ultra-low temperature storage may cause certain stress to the protocorms.

[0096] (2) Antioxidant enzymes Figure 8 and Fig. 9 SOD of 5 samples of Cymbidium spicatum ( Figure 8 ) and POD ( Fig. 9 ) test results. Figure 8 It can be seen that the SOD activity first increased and then decreased during the cryopreservation process, reaching a peak of 163.11U / g after soaking in PVS2. The activity decreased after liquid nitrogen storage, but it was still significantly improved compared with the untreated protocorms. The activity of the control group was significantly lower than that of other samples. Fig. 9 It can be seen that the activity of POD in each step before being put into liquid nitrogen did not change much from that of the control, and there was a slight increase after being immersed in PVS2. The activity decreased after being stored in liquid nitrogen, which may be due to the low temperature stress after being put into liquid nitrogen.

[0097] Fig.10 This is the CAT test result of the large-pointed capsule orchid. The CAT activity first increased and then decreased during the cryopreservation process, reaching a peak of 527.51U / g after pretreatment. In the subsequent steps, it may be stressed by PVS2 and liquid nitrogen, causing the CAT to gradually decrease, but compared with the control, the activity after cryopreservation is still increased.

[0098] The above embodiments are only used to illustrate the present invention, but not to limit the present invention. Ordinary technicians in the relevant technical field 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 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; Pre-culturing the protocorm, comprising: 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; immersing the vitrified protocorm into a small drop of vitrification solution; 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 according to claim 1, wherein the diameter of the protocorm is 0.5 mm to 1 mm.

4. 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.

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

6. The method according to claim 5, 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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