Method for preserving orchid seeds

Abstract

The invention relates to a preservation method of orchid seeds. The preservation method comprises the following steps: soaking the orchid seeds into a loading solution; replacing the loading solution with a plant vitrification solution, and carrying out a vitrification reaction for 0.25-1.5 h; treating the vitrified seeds at an ultralow temperature; and storing the seeds subjected to the ultra-low temperature treatment. The orchid seeds preserved by the method have high survival rate and germination rate, the method is simple to operate, germplasm resource protection of orchid can be effectively promoted, and diversity of orchid is reserved.

Description

Technical Field

[0001] The present invention relates to the protection of plant germplasm resources, and particularly to a method for preserving seeds of Orchidaceae plants. Background Art

[0002] The fruits of Orchidaceae 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 bacteria to provide nutrients to germinate. The germination conditions are relatively harsh, and the requirements for the quality of the natural habitat are strict, 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 it is difficult to maintain the seed viability for a long time, and the lifespan is short. It can be seen that the germination conditions of Orchidaceae seeds are harsh and the lifespan is short, resulting in difficulties in their natural reproduction.

[0003] Therefore, there is an urgent need to find a method for preserving seeds of Orchidaceae plants to improve their survival rate and germination rate, thereby contributing to the protection of Orchidaceae plant germplasm resources. Summary of the Invention

[0004] In view of the technical problems existing in the prior art, the present invention provides a method for preserving seeds of Orchidaceae plants, comprising: soaking the seeds of Orchidaceae plants in a loading solution for 20 - 40 min, such as 22 - 35 min, 25 - 38 min, 28 - 32 min, 30 min or any value or range therebetween; replacing the loading solution with a plant vitrification solution and performing a vitrification reaction for 0.25 - 2.5 h; preferably, the time of the vitrification reaction is 0.5 - 1 h; more preferably, the time of the vitrification reaction is 0.35 - 1.4 h, 0.5 - 1 h, 0.5 - 1.3 h, 0.8 - 1.2 h or any value or sub-range therebetween; placing the vitrified seeds in an environment below -190 °C for ultra-low temperature treatment; and preserving the seeds after ultra-low temperature treatment.

[0005] The preservation method as described above further comprises: disinfecting the seeds of Orchidaceae plants.

[0006] In the preservation method as described above, the disinfection treatment uses alcohol and / or sodium hypochlorite solution.

[0007] In the preservation method as described above, the concentration of the sodium hypochlorite solution is 1.5 - 3%.

[0008] In the preservation method as described above, the loading solution comprises: (100 - 150) g / L or (0.3 - 0.5) M / L sucrose, (2 - 3) g / L 1 / 2MS, and (150 - 220) g / L or (1.5 - 2.5) M / L glycerol; preferably, the loading solution further comprises (80 - 120) mL / L coconut milk.

[0009] The preservation method as described above, wherein the loading solution comprises 137 g / L sucrose, 2.3 g / L 1 / 2MS, and 184 g / L glycerol.

[0010] The preservation method as described above, wherein the loading solution comprises 2.3 g / L 1 / 2MS, 0.4 M / L sucrose, 2 M / L glycerol, and 100 mL / L coconut juice.

[0011] The preservation method as described above, wherein the plant vitrification solution comprises: (100 - 150) g / L or (0.3 - 0.5) M / L sucrose, (2 - 3) g / L 1 / 2MS, (100 - 200) mL / L glycerol, (100 - 200) mL / L ethylene glycol; preferably, the plant vitrification solution further comprises (100 - 200) mL / L dimethyl sulfoxide. The preservation method as described above, wherein the plant 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. The preservation method as described above, wherein the plant vitrification solution comprises 2.3 g / L 1 / 2MS + 300 mL / L glycerol + 150 mL / L ethylene glycol + 0.4 M / L sucrose.

[0012] The preservation method as described above, wherein the seeds of Orchidaceae plants are cryopreserved at a temperature below -190°C (for example, in liquid nitrogen at -196°C to -210°C, such as in liquid nitrogen at -196°C) for 0.5 - 2 h, for example, 0.6 - 1.5 h, 0.8 - 1.7 h, 1 - 1.8 h, 1.2 - 1.5 h, or any value or sub-range therebetween. Preferably, the cryopreservation lasts for 1 h.

[0013] The preservation method as described above, wherein the cryopreserved seeds are stored for at least 100 days. The preservation method as described above, wherein the cryopreserved seeds are stored in an environment below -20°C, for example, in liquid nitrogen or in an environment of about -80°C for at least 100 days.

[0014] The preservation method as described above, wherein the Orchidaceae plants include plants of the genus Phalaenopsis. The preservation method as described above, wherein the plants of the genus Phalaenopsis include, but are not limited to, Kingidium deliciosum, Nothodoritis zhejiangensis, and Phalaenopsis lobbii.

[0015] A method for rewarming the seeds of an orchid plant as described above, including: subjecting the seeds obtained by the preservation method as described above to a constant temperature treatment at 40 - 60 °C for 60 - 120 s; soaking the seeds after the constant temperature treatment in a unloading solution and performing an unloading treatment for 15 - 30 min; wherein, the unloading solution includes: (360 - 450) g / L sucrose, (2 - 3) g / L 1 / 2MS; preferably, the unloading solution further includes (80 - 120) mL / L coconut juice.

[0016] In the method as described above, the unloading solution includes 411 g / L sucrose + 2.3 g / L 1 / 2MS. In the method as described above, the unloading solution includes 2.3 g / L 1 / 2MS + 1.2 M / L sucrose + 100 mL / L coconut juice.

[0017] In the method as described above, the unloading treatment time is 15 - 30 min, such as 18 - 25 min, 20 - 28 min, 20 - 25 min, 20 min or any value or sub - range therebetween.

[0018] A cultivation method for the seeds of an orchid plant preserved by any of the preservation methods as described above or the seeds of an orchid plant rewarmed by any of the methods as described above, including: sowing the seeds of an orchid plant preserved by any of the preservation methods as described above or the seeds of an orchid plant rewarmed by any of the methods as described above in a germination medium.

[0019] In the method as described above, the germination medium includes: (1 - 5) g / L 1 / 2MS; (10 - 30) g / L sucrose; 5 - 10 g / L carrageenan; and (50 - 150) mL / L coconut juice, or (10 - 40) g / L potato, or (10 - 30) g / L banana, or (10 - 30) g / L corn.

[0020] In the method as described above, the germination medium includes: 2.3 g / L 1 / 2MS; 20 g / L sucrose; 7 g / L carrageenan; and 100 mL / L coconut juice, or 30 g / L potato, or 30 g / L banana, or 20 g / L corn.

[0021] In the method as described above, it further includes first culturing the seeds in a dark environment for a certain period of time, such as 5 - 15 days, or 7 - 10 days, and then changing to a natural light cycle for cultivation, such as 12 h / d of light (light from Monday to Friday, no light on Saturday and Sunday).

[0022] The seeds of the orchid plant preserved by the method of the present invention have a very high survival rate and germination rate, and the method of the present invention is simple to operate, can effectively promote the protection of the germplasm resources of orchid plants, and retain the diversity of orchid plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Next, the preferred embodiments of the present invention will be further described in detail with reference to the accompanying drawings, where:

[0024] Figure 1 Shows the steps of cryopreserving seeds by vitrification according to an embodiment of the present invention;

[0025] Figure 2 Shows a cross-section of the fruit of Kingidium deliciosum according to an embodiment of the present invention;

[0026] Figure 3 Shows a microscopic photograph of TTC staining of Kingidium deliciosum seeds after cryopreservation according to an embodiment of the present invention;

[0027] Figure 4 Shows the survival rate of cryopreserved Kingidium deliciosum seeds according to an embodiment of the present invention;

[0028] Figure 5 Shows the germination rate of cryopreserved Neofinetia phalaenopsis seeds for 100 days according to an embodiment of the present invention; and

[0029] Figure 6 Shows the germination rate of Rhynchostylis retusa seeds in different germination media according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

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

[0031] In the following detailed description, reference may be made to the accompanying drawings that form a part hereof and that show, by way of illustration, specific embodiments in which the claimed subject matter may be practiced. In the drawings, like reference numerals generally refer to like components throughout the different views. The 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 practice 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.

[0032] The Orchidaceae plants involved in the present application may be common Orchidaceae plants or endangered Orchidaceae plants. For example, in some embodiments, the Orchidaceae plants may be plants of the genus Phalaenopsis, such as Kingidium deliciosum, Neofinetia phalaenopsis, Rhynchostylis retusa, etc.

[0033] As used herein, the "ultra-low temperature preservation technology" refers to a technology in which plant materials are stored in an environment below -190°C, such as stored in liquid nitrogen (-196°C), and the plant materials still have activity after thawing. 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.

[0034] In some embodiments, the ultra-low temperature preservation technology also includes the use of cryoprotectants. Cryoprotectants are characterized by being easily soluble in water, non-toxic to cells, and easily removed from tissue cells. In some embodiments, cryoprotectants can play the following protective roles on the tissues of germplasm resources:

[0035] Produce strong hydration in the solution, increase the viscosity of the solution, thereby reducing the rate of ice crystal formation and growth while 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 caused by ice formation in the cell; prevent the toxicity of the "solution effect" before or during ultra-low temperature freezing; and directly or indirectly act on the cell membrane to reduce the damage of ultra-low temperature to the cell membrane.

[0036] In some embodiments, cryoprotectants can be divided into permeating and non-permeating types. Among them, permeating cryoprotectants refer to components that are easy to penetrate the cell membrane and enter the cell interior, thereby reducing the freezing point of the cell and increasing the permeability of the cell membrane to water. In some embodiments, permeating cryoprotectants can include glycerol, dimethyl sulfoxide, etc. Non-permeating cryoprotectants generally refer to components that cannot penetrate into the cell interior but are easily soluble in water and can dilute the concentration of extracellular electrolytes to reduce the damage of solutes to cells. In some embodiments, non-permeating cryoprotectants include polyvinylpyrrolidone, sucrose, polyethylene glycol, dextran, etc.

[0037] In some embodiments, the loading solution (LS) and / or plant vitrification solution mentioned herein, such as PVS1, PVS2, PVS3, etc., all contain cryoprotectants.

[0038] As used herein, "loading" refers to the operation of mixing germplasm resources in a loading solution to avoid damage to cells caused by drastic changes in osmotic pressure. In some embodiments, the loading solution generally includes 2 mol / L glycerol and 0.4 mol / L sucrose.

[0039] As used herein, the full English name of "PVS2" is plant vitrification solution 2, that is, plant vitrification solution 2, corresponding to PVS1 and PVS3, etc., and is made of sucrose, 1 / 2 MS, glycerol, ethylene glycol, and dimethyl sulfoxide. PVS2 can effectively protect various plant materials during ultra-low temperature preservation, avoiding crystallization phenomena and solute damage.

[0040] In some embodiments, "cryopreservation" is achieved by one or more of the methods such as the stepwise cooling method, the rapid freezing method, the drying method, the vitrification method, the small droplet vitrification method, the encapsulation vitrification method, etc. Different species and different tissues have different morphological structures. Precisely studying the cryopreservation method for specific materials can greatly improve the post-thaw survival rate.

[0041] As used herein, the "vitrification method" refers to the solidification process in which a liquid transforms into a non-crystalline (glass state). Before being immersed in liquid nitrogen, the material is soaked in a vitrification solution to dehydrate the cells and enhance the viscosity of the protoplast. During the rapid cooling process, the intracellular water molecules cannot be arranged in the state of ice crystals, thus avoiding the mechanical damage caused by the freezing of free water and the solute damage to some tissues caused by too slow freezing.

[0042] To make the vitrification cryopreservation technology easier to understand, this experiment uses PS to draw a brief flow chart, as shown in Figure 1 . Figure 1 It is the steps of cryopreserving seeds by the vitrification method according to an embodiment of the present invention. In this application, the cryopreservation method for orchid seeds generally includes the following steps: soaking in a loading solution (LS), soaking in PVS2, and immersing in liquid nitrogen (LN) for preservation. Figure 1 It also shows steps such as rewarming the preserved seeds and soaking in an unloading solution (UL).

[0043] Furthermore, the cryopreservation method for orchid seeds includes:

[0044] Disinfection and sterilization: In some embodiments, the orchid seeds are placed in a disinfectant solution and soaked for a certain period of time. In some embodiments, the sterilization treatment steps use, for example, ethanol treatment, sodium hypochlorite solution treatment, etc. In some embodiments, the concentration of the sodium hypochlorite solution is 1.5 - 3%.

[0045] Loading: Put the seeds in the sterilized fruits into a container, add the loading solution LS, and soak for 20 - 40 min, such as 22 - 35 min, 25 - 38 min, 28 - 32 min, 30 min or any value or range therebetween. In some embodiments, one or more pods of seeds can be loaded into each container. In some embodiments, all or part of the seeds in one or more pods can be loaded into each container. In some embodiments, the container can be a test tube, an EP tube, a small beaker, etc. In some embodiments, the loading solution soaks the seeds at room temperature. In some embodiments, the loading solution comprises: (100 - 150) g / L or (0.3 - 0.5) M / L sucrose, (2 - 3) g / L 1 / 2MS, and (150 - 220) g / L or (1.5 - 2.5) M / L glycerol; preferably, the loading solution further comprises (80 - 120) mL / L coconut juice.

[0046] Vitrification: After loading, replace LS with PVS2 and soak the seeds for a certain time. In some embodiments, the soaking time of the seeds can be 0.25 - 2.5 h, such as 0, 0.5 h, 1 h, 0.35 - 1.4 h, 0.5 - 1 h, 0.5 - 1.3 h, 0.8 - 1.2 h, 2 h, 3 h or any value or sub - range therebetween. In some embodiments, the container can be placed on ice for operation. In some embodiments, the plant vitrification solution comprises: (100 - 150) g / L or (0.3 - 0.5) M / L sucrose, (2 - 3) g / L 1 / 2MS, (100 - 200) mL / L glycerol, (100 - 200) mL / L ethylene glycol; preferably, the plant vitrification solution further comprises (100 - 200) mL / L dimethyl sulfoxide.

[0047] Cryopreservation: After vitrification, mix the seeds thoroughly with PVS2, load them into cryotubes, and perform cryopreservation in an environment below - 190°C. Preferably, perform cryopreservation in an environment of (- 196~ - 210)°C. In some embodiments, put the cryotubes into liquid nitrogen and store for a certain time. In some embodiments, the storage time in liquid nitrogen is, for example, 0.5 - 2 h, such as 0.6 - 1.5 h, 0.8 - 1.7 h, 1 - 1.8 h, 1.2 - 1.5 h or any value or sub - range therebetween. Preferably, the storage time in liquid nitrogen is, for example, 1 h.

[0048] Storage: Store the cryopreserved seeds. In some embodiments, the cryopreserved seeds can be stored in an environment below - 20°C for a certain time. For example, the cryopreserved seeds can be stored in liquid nitrogen or at about - 80°C. In some embodiments, the cryopreserved seeds can be stored for at least 100 days.

[0049] Rewarming: After the cryopreservation tube stored with liquid nitrogen is completed, it is placed in a water bath at 40 - 60 °C for rewarming for a certain period of time. In some embodiments, the rewarming time is, for example, 60 - 120 s, such as 65 - 90 s, 70 - 100 s, 80 - 110 s, 85 - 115 s or any value or sub-range therebetween. In some embodiments, the rewarming time is 90 s.

[0050] Unloading: Replace PVS2 with unloading solution UL and soak the seeds for 15 - 30 min. In some embodiments, the unloading treatment time is 15 - 30 min, such as 18 - 25 min, 20 - 28 min, 20 - 25 min, 20 min or any value or sub-range therebetween. In some embodiments, the ambient temperature for soaking the seeds is room temperature. In some embodiments, the unloading solution includes: (360 - 450) g / L sucrose, (2 - 3) g / L 1 / 2MS; preferably, the unloading solution further includes (80 - 120) mL / L coconut milk.

[0051] In some embodiments, the Orchidaceae plants include Phalaenopsis plants, such as but not limited to Kingidium deliciosum, Nothodoritis zhejiangensis, and Phalaenopsis lobbii.

[0052] A cultivation method for Orchidaceae plant seeds or Orchidaceae plant seeds treated by cryopreservation, comprising: sowing Orchidaceae plant seeds on a germination medium. In some embodiments, the germination medium includes: (1 - 5) g / L 1 / 2MS; (10 - 30) g / L sucrose; 5 - 10 g / L carrageenan; and (50 - 150) mL / L coconut milk, or (10 - 40) g / L potato, or (10 - 30) g / L banana, or (10 - 30) g / L corn.

[0053] Further, the seeds are first cultured in the dark for a certain period of time, such as 5 - 15 days, or 7 - 10 days, and then transferred to natural light cycle for cultivation, such as 12 h / d of illumination (illuminated from Monday to Friday, no illumination on Saturday and Sunday), which can make the cultivation effect better.

[0054] In some embodiments, the cryopreservation method for Orchidaceae plant seeds further includes detecting the viability of Orchidaceae plant seeds. The viability test method includes: after unloading is completed, replace UL with TTC (2, 3, 5 - triphenyltetrazolium chloride), mix it with the seeds, place it in a cryopreservation tube, and place it in the dark at room temperature for 18 - 30 h to wait for staining. In some embodiments, the concentration of TTC is 0.5 - 2%. In some embodiments, the concentration of TTC is 1%.

[0055] After staining, use a pipette to aspirate the mixture, take 1 drop of the mixture for slide preparation and observation, count the total number of seeds in the field of view and the number of seeds with red-stained embryos (viable). Calculate the survival rate: Survival rate = (number of viable seeds / total number of seeds) × 100%.

[0056] In some embodiments, the solution ratios involved in the cryopreservation process of Orchidaceae plant seeds can be as follows:

[0057] Loading solution (LS): 137 g / L sucrose + 2.3 g / L 1 / 2MS + 184 g / L glycerol. Or, loading solution: 2.3 g / L 1 / 2MS + 0.4 M / L sucrose + 2 M / L glycerol + 100 mL / L coconut juice.

[0058] 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. Or, plant vitrification solution: 2.3 g / L 1 / 2MS + 300 mL / L glycerol + 150 mL / L ethylene glycol + 0.4 M / L sucrose.

[0059] Unloading solution (UL): 411 g / L sucrose + 2.3 g / L 1 / 2MS. Or, unloading solution: 2.3 g / L 1 / 2MS + 1.2 M / L sucrose + 100 mL / L coconut juice.

[0060] Liquid medium: 2.3 g / L 1 / 2MS powder + 10 g / L sucrose + 100 mL / L fresh coconut juice. When a solid medium is needed, 2.5 g / L carrageenan can be added to the liquid medium.

[0061] Furthermore, 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 stirred quickly and evenly to ensure accurate pH measurement. Put the above solutions (except PVS2) into a horizontal circular autoclave and sterilize for 20 min, and wait until cooled to room temperature. Put PVS2 without dimethyl sulfoxide into the autoclave for sterilization, and then in a laminar flow hood, filter and sterilize dimethyl sulfoxide with a disposable needle filter (PES membrane 0.22 μm Millipore.) and mix it with PVS2 (lacking dimethyl sulfoxide) cooled to room temperature after autoclave sterilization, thus completing the preparation of PVS2. Put the above sterile solutions into a 4°C refrigerator for storage and wait for use.

[0062] Example 1 Cryopreservation of Kingidium deliciosum Seeds

[0063] The solutions involved in this example are as follows:

[0064] Loading solution (LS): 137 g / L sucrose + 2.3 g / L 1 / 2MS + 184 g / L glycerol.

[0065] 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.

[0066] Unloading solution (UL): 411 g / L sucrose + 2.3 g / L 1 / 2MS.

[0067] Selection of experimental materials

[0068] The experimental materials in this experiment were taken from the soilless cultivation greenhouse of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. Plants with good growth vigor were selected for subsequent sampling operations. The experimental material was *Kingidium deliciosum*. The flowering period of *Kingidium deliciosum* is in July. It has 3 - 4 obovate papery leaves, the apex is slightly hooked, and the margin is slightly wavy; it has a paniculate inflorescence, the sepals and petals are white, and there are light purple spotted patterns on the roots; the median sepal is nearly elliptical, and the lateral sepals are obliquely ovate; the lip is 3 - lobed, the base is light yellow, and the apex gradually changes from light purple to white; the stigma is light purple, and the anther cap is white.

[0069] Healthy *Kingidium deliciosum* at the full - flowering stage was selected for cross - pollination. After 100 d, its mature fruits were collected. The mature fruits were disinfected (washed the surface with running water, soaked in 75% alcohol for 15 s, soaked in a diluted 84 solution with 2% available chlorine content for 10 min, washed repeatedly with sterile water, and dried the fruit surface with sterile air), and then cut to obtain a large number of powdery seeds inside the fruit (as Figure 2 shown).

[0070] The vitrification method was used for cryopreservation of *Kingidium deliciosum* seeds. The steps of cryopreservation of *Kingidium deliciosum* seeds by the vitrification method were successively: soaking in the loading solution (LS), soaking in PVS2, plunging into liquid nitrogen (LN), rewarming, and soaking in the unloading solution (UL). In order to detect the viability of the preserved seeds, TTC test was carried out on them.

[0071] In this experiment, by controlling other variables, the seeds were soaked in PVS2 for different durations (0, 0.5, 1, 2, 3 h) for cryopreservation by the vitrification method; by comparing the survival rates after preservation, the most suitable cryopreservation method for *Kingidium deliciosum* seeds was determined. All experimental operations were carried out in a laminar flow hood to ensure a sterile environment. The specific process was as follows:

[0072] Experimental group treatment: Put all the seeds in one sterilized fruit into a 10 mL small beaker, add 5 mL of LS, and soak at room temperature for 30 min. Suck out the LS solution with a pipette, then use a pipettor to aspirate 5 mL of PVS2 on ice and immerse the seeds in PVS2; then place the 10 mL small beaker on ice and soak the seeds for 0, 0.5, 1, 2, and 3 h respectively. After vitrification, mix the seeds well with PVS2, evenly divide them into 3 portions with a pipettor, put them into 3 cryotubes respectively, and plunge into liquid nitrogen at about -196 °C for 1 h. Take out the cryotubes after being stored in liquid nitrogen, put them into a 60 °C water bath for 90 s of rewarming, and put the mixed liquid in the 3 cryotubes into the same 10 mL small beaker. Replace PVS2 with 5 mL of UL and soak the seeds at room temperature for 20 min. After unloading, replace UL with 1 mL of 1% TTC, mix with the seeds, put them into a cryotube, and place them in the dark at room temperature for 24 h for staining. After staining, use a pipettor to aspirate the mixed liquid, take 1 drop of the mixed liquid to make a slide for observation (research-grade upright microscope OLYMPUS BX53), and repeat 3 times. Record it as 3 technical replicates, and each technical replicate has 3 biological replicates. Count the total number of seeds in the field of view under a ×10 magnifying lens (at least 30 seeds in each field of view), and the number of seeds with the embryo stained red (viable). Calculate the survival rate: Survival rate = (number of viable seeds / total number of seeds) × 100%.

[0073] Control (CK) group treatment: Put all the seeds in one sterilized fruit into a cryotube (without the ultra-low temperature preservation step), plunge into liquid nitrogen, after storing for 1 h, conduct TTC test and calculate the survival rate. Both biological replicates and technical replicates are the same as above.

[0074] Figure 3 Results of TTC staining of cryopreserved seeds of Cymbidium erythraeum Lindl. The seeds of Cymbidium erythraeum Lindl. consist of an embryo and a seed coat, without endosperm. Among them, the embryo stained red by TTC is a viable seed; the embryo not stained (gray) or the seed without an embryo in the seed coat is a non-viable seed.

[0075] In this example, the seeds were cryopreserved with 5 different soaking times of PVS2, and 1 control (CK) was set (i.e., directly plunged into liquid nitrogen). Figure 4 The column chart showing the change of the survival rate of the above-mentioned cryopreserved seeds of Cymbidium erythraeum Lindl. with the soaking time of PVS2 is given. From Figure 4 it can be seen that with the increase of the soaking time of PVS2, the seed survival rate shows a trend of first increasing and then decreasing. When the soaking time of PVS2 is 1 h, the survival rate reaches the peak, which is 71.67%. When the soaking time of PVS2 is 0 h, the survival rate is the lowest (13.08%), which is about 10.03% lower than that of the control group.

[0076] In this experiment, Kingidium deliciosum was used as the experimental material. By using the method of controlling variables, the survival rates of seeds after cryopreservation with different PVS2 soaking times were compared to determine the cryopreservation method more suitable for Kingidium deliciosum seeds, providing technical support for protecting the germplasm resources of Kingidium deliciosum. Kingidium deliciosum seeds are suitable for cryopreservation by vitrification method with a PVS2 soaking time of about 1 h, and the survival rate after preservation is 71.67%. By using the method of directly immersing in liquid nitrogen, the survival rate of Kingidium deliciosum seeds is 23.11%. Kingidium deliciosum seeds are only composed of embryo and seed coat, without endosperm and have a low water content. Therefore, after being immersed in liquid nitrogen, the mechanical damage caused by intracellular ice formation is small, so they have a certain activity. Using the preservation method of this example can greatly improve the survival rate of seeds. Therefore, the method of this example is suitable for preserving common orchid seeds, especially the seeds of endangered Phalaenopsis plants.

[0077] Example 2 Cryopreservation of Seeds of Neofinetia falcata by Vitrification

[0078] The plant material in this example is the seeds of Neofinetia falcata, which were collected from the original place in Zhejiang and sent back.

[0079] The preparation methods of the solutions involved in this example include:

[0080] Preparation method of recovery medium: Take 2.3 g / L of 1 / 2MS powder without agar and sucrose, add 10 g / L of sucrose and 100 mL / L of fresh coconut juice. After adjusting the pH to 5.8, add 2.5 g of carrageenan, make up the volume to 1000 mL with distilled water, sterilize in a high-pressure sterilizer at 121 °C for 20 min, pour into plates and seal for later use;

[0081] Preparation method of liquid medium: Take 2.3 g / L of 1 / 2MS powder without agar and sucrose, add 10 g / L of sucrose and 100 mL / L of fresh coconut juice. After adjusting the pH to 5.8, make up the volume to 1000 mL, sterilize in a high-pressure sterilizer at 121 °C for 20 min; cool and keep for later use;

[0082] Preparation method of loading solution: Take 2.3 g / L of 1 / 2MS without agar and sucrose, add 0.4 M / L of sucrose, add 2 M / L of glycerol and 100 mL / L of coconut juice, make up the volume to 1000 mL with distilled water, adjust the pH to 5.8, put it into a high-temperature sterilizer at 121 °C for 20 min, cool and then store in a 4 °C refrigerator for later use;

[0083] Preparation method of PVS2 solution: Take 1 / 2 MS without agar and sucrose at 2.3 g / L, add 300 mL / L glycerol, 150 mL / L ethylene glycol, 0.4 M / L sucrose, make up the volume to 1000 mL with distilled water, adjust the pH to 5.8, put it into a high-temperature sterilizer and sterilize at 121 °C for 20 min. After cooling, add 150 mL of filtered dimethyl sulfoxide (DMSO) and store it in a 4 °C refrigerator for later use (it is recommended to prepare it as needed).

[0084] Preparation method of unloading solution: Take 1 / 2 MS without agar and sucrose at 2.3 g / L, add 1.2 M / L sucrose, 100 mL / L coconut milk, make up the volume to 1000 mL with distilled water, adjust the pH to 5.8, put it into a high-temperature sterilizer and sterilize at 121 °C for 20 min. After cooling, store it in a 4 °C refrigerator for later use.

[0085] In this example, a total of six groups of treatments were set, as shown in Table 1, and each treatment was repeated 5 times. The first group was the blank control group, which was not treated with the loading solution or vitrification, but was directly put into liquid nitrogen for 1 h. After treatment 2-6 were treated with the loading solution, the PVS2 treatment times were 0, 0.5, 1, 2, and 3 h respectively.

[0086] Table 1 Different treatments of cryopreservation of Neofinetia falcata seeds by vitrification

[0087]

[0088] Steps of cryopreservation by vitrification method:

[0089] Under the condition of ensuring sterility throughout the process in a laminar flow hood, take out two fruit pods and surface sterilize them in 75% ethanol for 30 s. Then surface sterilize them with 5% sodium hypochlorite solution for 16 min, rinse them 3 times with sterile water, and roughly blot the water with filter paper. Cut the fruit pods longitudinally with a scalpel and cut them evenly into two parts. Take out the seeds of the two fruit pods, put the seeds of the two different fruit pods into the same cryotube for mixing, divide them into two groups, and record them as two treatments respectively.

[0090] Experimental group treatment (Treatment 2): After the seeds were disinfected and directly knocked into cryotubes, LS (1500 μL) was added, and then thoroughly stirred. The mixture was evenly divided into 5 groups with a pipette gun and placed into 5 cryotubes, 300 μL for each group, and treated for 30 min. The LS solution was sucked out with pipette ①, and then on ice, the seeds were flushed into a cryotube containing PVS2 (1 mL) with a pipette gun. Subsequently, it was immediately placed in liquid nitrogen for 1 h, and thawed in a water bath at 40 °C for 90 s. Then, PVS2 was completely sucked out with pipette ②, and 1 mL of unloading solution was added with a pipette gun to rinse the seeds in pipette ②. Then, the unloading solution was sucked with pipette ③, and the seeds in pipette ③ were rinsed with a pipette gun with liquid medium (a total of 300 μL of liquid medium). It was dispensed with a pipette gun equipped with a 0.5 cm pipette tip with the tip cut off, and the liquid medium (95 μL, mixed with seeds) in the cryotube was transferred into the recovery medium.

[0091] Steps of Treatments 3 to 6 are repeated as those of Treatment 2, except for the different treatment times of PVS2. The specific times are shown in Table 1.

[0092] Control group treatment (Treatment 1): After the seeds were disinfected and directly knocked into cryotubes, they were put into liquid nitrogen for 1 h, thawed in a water bath at 40 °C for 90 s, 1500 μL of liquid medium was injected with a pipette gun, and after thoroughly stirring to mix the seeds, the liquid medium (95 μL, mixed with seeds) in the cryotube was transferred into the recovery medium with a pipette gun equipped with a 0.5 cm pipette tip with the tip cut off.

[0093] Recovery culture and conditions

[0094] The recovery medium used was a 60 mm culture dish. The medium formulation details can be found in the preparation part of Example 1. There were 6 treatments in total, and each treatment had 5 technical replicates. Each replicate was injected into one recovery medium, so there were 30 recovery media in total. The entire experimental operation was carried out in a laminar flow hood to ensure sterility throughout the process.

[0095] All the recovery media were placed in a constant temperature tissue culture room at 25 °C and cultured in the dark for one week. Subsequently, it was transferred to a photoperiod imitating the normal growth environment, that is, the light intensity = 1500 - 2000 lx, light for 12 h / d (from Monday to Friday, no light on Saturday and Sunday), and cultured in a tissue culture room at a temperature of 25 - 28 °C.

[0096] The germination rate was counted 100 days after the recovery culture. Using a stereomicroscope, at 4x magnification, 3 fields of view were taken from each culture dish. Under the condition that the total number of seeds in each field of view exceeded 60, the number of germinated seeds observed was recorded.

[0097] The germination rate of each obtained repetition was statistically analyzed using Microsoft Excel 2022 and IBM SPSS v19.0 (IBM Corp., Armonk, NY, USA) software. Image plotting was completed using OriginPro 2023 software. Among them, the germination rate = the number of germinated seeds / the total number of seeds 100%.

[0098] Figure 5 shows the germination rate of Neofinetia falcata seeds under cryogenic conditions for 100 days according to this embodiment. As Figure 5 shown, as the treatment time of PVS2 increased, the germination rate of Neofinetia falcata seeds showed a trend of first increasing and then decreasing. Under treatment 1 and treatment 2, the germination rates of Neofinetia falcata seeds were the lowest, 44.44% and 41.23% respectively. When the vitrification treatment time increased to 0.5 h, the seed germination rate increased significantly (P < 0.05), and the germination rate was 89.37%. Compared with treatment 1 and 2, the germination rate increased by up to 48.14%. After treatment 3, as the vitrification time increased, the seed germination rate of Neofinetia falcata gradually decreased. After 3 h of vitrification treatment, the seed germination rate was the lowest, 63.52%, which was 25.85% lower than that of treatment 3. It shows that compared with short-term vitrification treatment, the increase of vitrification treatment time can improve the germination rate of Neofinetia falcata seeds, but with the increase of vitrification treatment time, it will also cause damage to the seeds and affect their germination rate.

[0099] Vitrification is a good anti-freezing mechanism. Because the PVS2 solution is very viscous, it will hinder all chemical processes that require molecular diffusion. Its formation will make the cell metabolism very slow and become very stable over time.

[0100] The key to successful cryopreservation by vitrification is to carefully control the dehydration time to prevent damage caused by chemical toxicity or excessive osmotic stress during the PVS2 solution treatment process. Therefore, in order not to affect the germination of seeds after cryopreservation by vitrification, it is necessary to continuously optimize the time of exposure to the PVS2 solution. For many species, direct contact with a high-concentration vitrification solution is toxic. Therefore, samples cryopreserved by vitrification must be pretreated with a medium-concentration cryoprotectant solution to prepare for exposure to the vitrification solution as a transitional acclimation. Among the various time spans tested for PVS2 treatment, as Figure 5 shown, when Neofinetia falcata seeds were treated with PVS2 on ice for 0.5 h, the effect was the best, and the germination rate was as high as 89.37%.

[0101] When using vitrification for cryopreserving seeds of *Nothodoritis zhejiangensis*, treat them with PVS2 solution (1 / 2MS + 30% glycerol + 15% ethylene glycol + 15% dimethyl sulfoxide DMSO + 0.4M sucrose, pH = 5.8) on ice for 0.25 - 3 h, preferably for 0.25 - 2 h, more preferably for 0.25 - 1 h, and most preferably for 0.5 h. For subsequent recovery culture (recovery medium: 1 / 2MS + 10 g / L sucrose + 2.5 g / L carrageenan + 100 mL / L coconut milk, pH = 5.8), first conduct dark culture for one week and then transfer to recovery culture under simulated normal photoperiod, which gives the best effect with a germination rate as high as 89.37%.

[0102] Example 3 Germination Medium for Seeds of *Phalaenopsis lobbii*

[0103] The seeds of *Phalaenopsis lobbii* are from the fruit pods obtained by artificial pollination in the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences.

[0104] For sowing the seeds of *Phalaenopsis lobbii*, this application designed 4 kinds of nutrients as additives to the culture medium. Specifically, different content gradients were set for three additives: potato, banana, and corn, and a coconut milk group was also set to explore the most suitable nutrients and concentration ratios for the germination of *Phalaenopsis lobbii* seeds. A total of 11 groups of treatments were set for the germination medium (as shown in Table 2), and one of them was a blank control group.

[0105] Table 2 Different Treatments of Germination Medium for Seeds of *Phalaenopsis lobbii*

[0106]

[0107] Put the nutrients into a juicer according to the contents in Table 2 to make a puree, and then add it to the culture medium. The culture dishes used in this experiment are 90 mm, and 10 plates are poured for each treatment as 10 replicates.

[0108] Disinfection of Fruit Pods

[0109] In a laminar flow hood, under the condition of ensuring sterility throughout the process, first soak the fruit pods in 75% alcohol for 45 s for surface sterilization. Then surface sterilize with sodium hypochlorite solution (2%) for 10 min, and then rinse 3 times with sterile water and simply blot dry with filter paper.

[0110] Sowing Method and Culture Conditions

[0111] The seeds used in each group of treatments are well-mixed seeds, that is, the seeds used in each treatment are the same. Take the sterilized fruit pods, cut each fruit pod longitudinally with a scalpel, take out all the seeds inside the fruit pod, and beat them in 110 mL of liquid medium for mixing. After mixing, use a pipette with a 0.5 cm cut-off tip to pipette 1000 μL of liquid medium (with mixed seeds) into the prepared Petri dish for Treatment 1. Repeat 10 times to pipette the 10 mL of liquid medium with mixed seeds into the 10 culture media for Treatment 1 respectively. Wrap the sealing film well and label it as Treatment 1.

[0112] Repeat the above steps to complete the aseptic sowing of the remaining 10 groups of treatments.

[0113] Photoperiod culture and observation and recording

[0114] Put all the seeded culture media into a constant-temperature tissue culture room at 25 °C, with a light intensity of 1500 - 2000 lx, a light duration of 12 h / d (from Monday to Friday, no light on Saturday and Sunday), and a culture temperature of 25 - 28 °C. Culture for 60 days and observe and record the germination rate.

[0115] The effects of adding different nutrients on the seed germination rate

[0116] The germination rate results of Rhynchostylis gigantea seeds in different germination media are as Figure 6 shown. As the concentration of the nutrient potato increases gradually from 10 g / L to 30 g / L, the germination rate of Rhynchostylis gigantea seeds also increases accordingly, reaching a maximum of 77.17%. The same is true for the addition of banana as a nutrient. At the corresponding concentration, the highest germination rate of Rhynchostylis gigantea seeds also occurs when 30 g / L of banana is added, reaching 74.83%. However, when corn is added as a nutrient to the culture medium, the germination rate of Rhynchostylis gigantea seeds does not increase with the increase in concentration, but is the highest when 20 g / L of corn is added, at 81.67%. But in Treatment Group 11, the germination rate of the treatment group with 100 mL / L of coconut milk added is the highest, at 87.13%. It shows that the most suitable nutrient for the germination of Rhynchostylis gigantea seeds is coconut milk, and the most suitable culture medium ratio for the germination of Rhynchostylis gigantea seeds is 1 / 2MS + sucrose + 100 mL / L coconut milk + carrageenan, pH = 5.8. The 60-day germination rate is 87.13%.

[0117] In the tissue culture of Phalaenopsis, MS is one of the most common basal media. However, its salt composition and concentration have a great impact on the germination, development and growth of seeds. Therefore, the salt content of the full-strength MS medium is very high. The requirements of seeds for salt content vary with different growth stages. During the germination stage, a lower salt concentration is needed, and too high or too low inorganic salt concentration is not conducive to germination. In this experiment, the basal medium for seed germination is the 1 / 2MS medium with a lower salt concentration, that is, 1 / 2MS + sucrose + 100 mL / L coconut milk is more suitable for the seed germination of Phalaenopsis lobbii, and the seed germination rate is as high as 87.13%. The 1 / 2MS medium has a good promoting effect on the seed germination of Phalaenopsis. Using 1 / 2MS as the basal medium and adding 100 mL / L coconut milk is more suitable for the seed germination of Phalaenopsis lobbii.

[0118] 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 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 disclosure of the present invention.

Claims

1. A method for preserving orchid seeds, comprising: Soak the orchid seeds in the loading solution for 20-40 min; removing the loading solution, and immersing the seeds in a plant vitrification solution to perform a vitrification reaction for 0.5-1 h; Placing the vitrified seeds in an environment below -190°C for ultra-low temperature treatment; as well as The seeds treated with ultra-low temperature are stored.

2. The storage method according to claim 1, further comprising: The seeds are disinfected.

3. The preservation method according to claim 2, wherein the disinfection method comprises: soaking the seeds in alcohol and / or sodium hypochlorite solution; The concentration of the sodium hypochlorite solution is 1.5-3%.

4. The storage method according to claim 1, wherein the loading solution comprises (100-150) g / L sucrose, (2-3) g / L 1 / 2MS, and (150-220) g / L glycerol; or the loading solution comprises (100-150) g / L sucrose, (2-3) g / L 1 / 2MS, (150-220) g / L glycerol, and (80-120) mL / L coconut water.

5. The preservation method according to claim 1, wherein the plant vitrification solution comprises (100-150) g / L sucrose, (2-3) g / L 1 / 2MS, (100-200) mL / L glycerol, and (100-200) mL / L ethylene glycol; or the plant vitrification solution comprises (100-150) g / L sucrose, (2-3) g / L 1 / 2MS, (100-200) mL / L glycerol, and (100-200) mL / L ethylene glycol, and (100-200) mL / L dimethyl sulfoxide. The storage method according to claim 1 , wherein the seeds treated with ultra-low temperature are stored for at least 100 days.

7. The preservation method according to claim 1, wherein the orchid plant is selected from the group consisting of Cymbidium orchidum, Elephant Orchid and Phalaenopsis rosenbergii.

8. A method for rewarming orchid seeds preserved by the preservation method according to any one of claims 1 to 7, comprising: Treating the orchid seeds at a constant temperature of 40-60° C. for 60-120 s; Soaking the seeds in an unloading solution for 15-30 minutes; in, The unloading solution includes: (360-450) g / L sucrose, (2-3) g / L 1 / 2MS, and (80-120) mL / L coconut water.

9. A method for cultivating orchid seeds preserved by the preservation method according to any one of claims 1 to 7 or orchid seeds rewarmed by the method according to claim 8, comprising: The orchid seeds are sown in a germination medium, wherein the germination medium comprises: (1-5) g / L 1 / 2MS; (10-30) g / L sucrose; (50-150) mL / L coconut water, or (10-40) g / L potatoes, or (10-30) g / L bananas, or (10-30) g / L corn; and 5-10 g / L carrageenan.

10. The culture method according to claim 9, wherein the germination medium comprises: 2.3 g / L 1 / 2MS; 20 g / L sucrose; 100 mL / L coconut water, or 30 g / L potatoes, or 30 g / L bananas, or 20 g / L corn; and 7 g / L carrageenan.

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