High-quality anoectochilus formosanus propagation method

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a high-quality anoectochilus formosanus propagation method which comprises the following specific steps: S1, microgravity environment simulation: S1.1, selecting a rotary bioreactor: selecting the rotary bioreactor according to the experimental scale and requirements, so that the rotary bioreactor can generate stable centripetal acceleration to simulate the microgravity environment; according to the method, a unique and optimized growth condition is provided for cell division and proliferation of anoectochilus formosanus by simulating a microgravity environment and utilizing stable centripetal acceleration generated by the rotary bioreactor, and in the environment, cytoskeleton arrangement of anoectochilus formosanus is more ordered, a gene expression mode is optimized, and the growth speed of the anoectochilus formosanus is increased. Therefore, efficient division and proliferation of cells are promoted, the hereditary stability and growth vigor of the seedlings are remarkably improved, and the bred seedlings show higher uniformity and excellence in form, physiological and biochemical characteristics.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a high-quality propagation method for roxburghii roxburghii. Background Art

[0002] Anoectochilus roxburghii, as a rare plant with extremely high medicinal value, occupies an important position in the field of traditional Chinese medicine and health products due to its unique pharmacological components. However, the natural reproduction ability of Anoectochilus roxburghii is limited, its seeds are small and the germination rate is low, which greatly limits its large-scale cultivation and commercial application.

[0003] Traditional methods of propagating Anoectochilus roxburghii mainly rely on seed propagation and tissue culture. However, these methods have many shortcomings. First, the seeds of Anoectochilus roxburghii are small and have low germination ability, which results in a long seed propagation cycle, low germination rate and uneven seedling quality. Secondly, although the traditional tissue culture method can improve the propagation efficiency to a certain extent, it still relies on a large amount of exogenous plant hormones, which not only increases production costs, but may also have an adverse effect on the genetic stability and subsequent growth of the seedlings. In addition, traditional methods also face problems such as long propagation cycle and susceptibility to environmental factors, and it is difficult to meet the high efficiency, high quality and environmental protection requirements of modern agricultural production.

[0004] In order to solve the above problems, it is necessary to optimize the existing golden thread vine breeding method by simulating the microgravity environment in space and using a rotating bioreactor to generate continuous centripetal acceleration, thereby changing the arrangement of the cytoskeleton, affecting gene expression and signal transduction pathways, and thereby promoting the division and proliferation of golden thread vine cells. Summary of the invention

[0005] Technical issues solved

[0006] In view of the above-mentioned shortcomings of the prior art, the present invention provides a high-quality breeding method for Anoectochilus roxburghii, which simulates the microgravity conditions in space and uses a rotating bioreactor to create a continuous centripetal acceleration environment, thereby optimizing the growth environment of Anoectochilus roxburghii cells and promoting the rearrangement of the cytoskeleton and the optimization of gene expression. This method can not only significantly improve the genetic stability and growth potential of Anoectochilus roxburghii seedlings, but also effectively shorten the breeding cycle and reduce production costs. In addition, reducing dependence on exogenous plant hormones is also a highlight of the present invention, which helps to produce safer and healthier Anoectochilus roxburghii seedlings.

[0007] Technical Solution

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] A high-quality propagation method for golden thread lily, the specific steps of the method comprising:

[0010] S1. Microgravity environment simulation:

[0011] S1.1. Select a rotating bioreactor: According to the experimental scale and requirements, select a rotating bioreactor that can generate stable centripetal acceleration to simulate the microgravity environment;

[0012] S1.2, Installation and commissioning: Install the rotating bioreactor on a stable platform and assemble and commission it according to the instructions;

[0013] S1.3. Calibration and verification: Use standard calibration tools to calibrate the rotating bioreactor so that the centripetal acceleration it produces can accurately simulate the required microgravity level;

[0014] S2. Preparation of Anoectochilus roxburghii explants:

[0015] S2.1. Mother plant selection and marking: Select healthy and pest-free plants from the mother plants of Anoectochilus roxburghii as mother plants, and mark them using color marking method for subsequent tracking;

[0016] S2.2. Collection of explants: Use scissors to cut stem tips and stem segments of appropriate length from the mother plant as explants;

[0017] S2.3, surface disinfection: Place the collected explants in a container filled with alcohol and soak for a period of time for preliminary disinfection, then transfer to a sodium hypochlorite solution and continue soaking for a period of time to completely kill surface microorganisms, finally rinse with sterile water and dry;

[0018] S3. Culture medium preparation and inoculation:

[0019] S3.1. Culture medium preparation: Prepare the culture medium according to the growth requirements of Anoectochilus roxburghii and the purpose of the experiment, including basic nutrients, trace elements and plant growth regulators;

[0020] S3.2, high pressure sterilization: Pour the prepared culture medium into the culture bottle and seal it, then put it into the high pressure steam sterilizer for high pressure sterilization to kill the microorganisms therein;

[0021] S3.3, inoculation: inoculate the sterilized explant into the culture medium that has been sterilized by high pressure on a sterile operating table, keep the position and depth of the explant in the culture medium consistent, and gently cover the surface of the culture medium with a layer of sterile gauze to fix the position of the explant;

[0022] S4. Microgravity environment training:

[0023] S4.1. Place in a rotating bioreactor: Place the inoculated culture medium in a rotating bioreactor and fix the position;

[0024] S4.2. Set rotation parameters: Set appropriate rotation speed and direction according to experimental requirements and the performance of the rotation bioreactor to simulate the required microgravity environment;

[0025] S4.3, Environmental control: setting appropriate light intensity, light cycle, temperature and humidity environmental conditions in the rotating bioreactor to promote the division and proliferation of Anoectochilus roxburghii cells;

[0026] S5. Cell division and proliferation monitoring:

[0027] S5.1. Regular sampling: Regular sampling is performed during the culture process to observe the division and proliferation of Anoectochilus roxburghii cells;

[0028] S5.2. Cytoskeleton observation: Use cytoskeleton immunofluorescence double staining technology to observe the arrangement changes of the cytoskeleton. After the sampled cells are fixed, permeabilized and stained, they are observed under a fluorescence microscope and photographed and recorded;

[0029] S5.3. Gene expression and signal transduction detection: Molecular biological methods were used to detect the expression of related genes and changes in signal transduction pathways in Anoectochilus roxburghii cells;

[0030] S6. Subculture and transplanting:

[0031] S6.1. Subculture: When the explants form a large number of clustered buds in a microgravity environment, they are subcultured to expand the scale of reproduction. The clustered buds are cut into small pieces and inoculated into new culture medium and cultured until a sufficient number of seedlings are formed;

[0032] S6.2, seedling hardening treatment: Move the well-growing golden thread vine seedlings from the culture room to the greenhouse for seedling hardening treatment;

[0033] S6.3. Transplantation: When the seedlings grow to the stage where they are ready for transplanting, they are removed from the culture medium and transplanted into the cultivation medium.

[0034] Furthermore, in step S1.1, a rotating bioreactor with a capacity of 100 L is selected according to the experimental scale and requirements so that it can generate a stable centripetal acceleration to simulate a microgravity environment.

[0035] Furthermore, in step S2.2, a stem tip with a length of 0.5-1 cm and a stem segment with a length of 2-3 cm are cut from the mother plant using scissors as explants.

[0036] Furthermore, in step S2.3, the collected explants are placed in a container containing 75% alcohol and soaked for 30-50 seconds for preliminary disinfection, then transferred to a 10% sodium hypochlorite solution and soaked for 5-10 minutes to completely kill surface microorganisms, and finally rinsed with sterile water and dried.

[0037] Furthermore, in step S3.3, the pressure of the high-pressure steam sterilizer is set to 0.11-0.12 MPa, the temperature is 121-123° C., the sterilization time is 15-20 minutes, and the depth of the explant in the culture medium is 0.5-1 cm.

[0038] Furthermore, in step S4.2, the rotation speed is set at 15-40 revolutions per minute according to the experimental requirements and the performance of the rotary bioreactor, and the direction remains constant.

[0039] Furthermore, in step S4.3, the light intensity in the rotating bioreactor is set at 2000-3000 lux, the light cycle is 12-16 hours, the temperature is controlled at 22-26°C, and the humidity is maintained at 60%-70%.

[0040] Furthermore, in step S5.1, samples are taken every 3-5 days to observe the division and proliferation of Anoectochilus roxburghii cells, and the number of samples taken each time is not less than 5 explants.

[0041] Furthermore, in step S5.3, the expression of related genes in the golden thread lotus cells is detected by molecular biological methods, and the related genes include: microtubule protein genes, actin genes, cell cycle protein genes, auxin response genes and gibberellin synthesis and signal transduction related genes.

[0042] Furthermore, in step 6.2, the well-growing golden thread vine seedlings are moved from the culture room to the greenhouse for seedling hardening treatment. Specifically, the humidity in the greenhouse is controlled at 70%-80% for the first 3 days, and then reduced by 5%-10% every day to 40%-60%, the light intensity is increased from 1000 lux to 500-1000 lux every day to 2000-3000 lux, and the ventilation volume is gradually increased to 3-5 times of air change per hour.

[0043] Beneficial Effects

[0044] Compared with the known public technology, the technical solution provided by the present invention has the following advantages:

[0045] Beneficial effects:

[0046] 1. The present invention provides a unique and optimized growth condition for the cell division and proliferation of Anoectochilus roxburghii by simulating a microgravity environment and utilizing the stable centripetal acceleration generated by a rotating bioreactor. In this environment, the cytoskeleton arrangement of Anoectochilus roxburghii is more orderly and the gene expression pattern is optimized, thereby promoting efficient cell division and proliferation, significantly improving the genetic stability and growth potential of the seedlings, and making the bred seedlings show higher uniformity and excellence in morphological, physiological and biochemical characteristics.

[0047] 2. The present invention accelerates the division and proliferation rate of golden thread vine cells by simulating a microgravity environment, thereby significantly shortening the reproduction cycle. At the same time, due to the reduced dependence on exogenous plant hormones and the optimization of culture conditions, the reproduction method of the present invention can greatly reduce production costs. In addition, due to the improvement of seedling quality, subsequent cultivation management also becomes easier and more efficient, further reducing the overall production cost. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the present invention is described clearly and completely. 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.

[0049] The present invention will be further described below in conjunction with the embodiments.

[0050] A high-quality propagation method for Anoectochilus roxburghii, characterized in that the specific steps of the method include:

[0051] S1. Microgravity environment simulation:

[0052] S1.1. Select a rotating bioreactor: According to the experimental scale and requirements, select a rotating bioreactor that can generate stable centripetal acceleration to simulate the microgravity environment;

[0053] S1.2, Installation and commissioning: Install the rotating bioreactor on a stable platform and assemble and commission it according to the instructions;

[0054] S1.3. Calibration and verification: Use standard calibration tools to calibrate the rotating bioreactor so that the centripetal acceleration it produces can accurately simulate the required microgravity level;

[0055] S2. Preparation of Anoectochilus roxburghii explants:

[0056] S2.1. Mother plant selection and marking: Select healthy and pest-free plants from the mother plants of Anoectochilus roxburghii as mother plants, and mark them using color marking method for subsequent tracking;

[0057] S2.2. Collection of explants: Use scissors to cut stem tips and stem segments of appropriate length from the mother plant as explants;

[0058] S2.3, surface disinfection: Place the collected explants in a container filled with alcohol and soak for a period of time for preliminary disinfection, then transfer to a sodium hypochlorite solution and continue soaking for a period of time to completely kill surface microorganisms, finally rinse with sterile water and dry;

[0059] S3. Culture medium preparation and inoculation:

[0060] S3.1. Culture medium preparation: Prepare the culture medium according to the growth requirements of Anoectochilus roxburghii and the purpose of the experiment, including basic nutrients, trace elements and plant growth regulators;

[0061] S3.2, high pressure sterilization: Pour the prepared culture medium into the culture bottle and seal it, then put it into the high pressure steam sterilizer for high pressure sterilization to kill the microorganisms therein;

[0062] S3.3, inoculation: inoculate the sterilized explant into the culture medium that has been sterilized by high pressure on a sterile operating table, keep the position and depth of the explant in the culture medium consistent, and gently cover the surface of the culture medium with a layer of sterile gauze to fix the position of the explant;

[0063] S4. Microgravity environment training:

[0064] S4.1. Place in a rotating bioreactor: Place the inoculated culture medium in a rotating bioreactor and fix the position;

[0065] S4.2. Set rotation parameters: Set appropriate rotation speed and direction according to experimental requirements and the performance of the rotation bioreactor to simulate the required microgravity environment;

[0066] S4.3, Environmental control: setting appropriate light intensity, light cycle, temperature and humidity environmental conditions in the rotating bioreactor to promote the division and proliferation of Anoectochilus roxburghii cells;

[0067] S5. Cell division and proliferation monitoring:

[0068] S5.1. Regular sampling: Regular sampling is performed during the culture process to observe the division and proliferation of Anoectochilus roxburghii cells;

[0069] S5.2. Cytoskeleton observation: Use cytoskeleton immunofluorescence double staining technology to observe the arrangement changes of the cytoskeleton. After the sampled cells are fixed, permeabilized and stained, they are observed under a fluorescence microscope and photographed and recorded;

[0070] S5.3. Gene expression and signal transduction detection: Molecular biological methods were used to detect the expression of related genes and changes in signal transduction pathways in Anoectochilus roxburghii cells;

[0071] S6. Subculture and transplanting:

[0072] S6.1. Subculture: When the explants form a large number of clustered buds in a microgravity environment, they are subcultured to expand the scale of reproduction. The clustered buds are cut into small pieces and inoculated into new culture medium and cultured until a sufficient number of seedlings are formed;

[0073] S6.2, seedling hardening treatment: Move the well-growing golden thread vine seedlings from the culture room to the greenhouse for seedling hardening treatment;

[0074] S6.3. Transplantation: When the seedlings grow to the stage where they are ready for transplanting, they are removed from the culture medium and transplanted into the cultivation medium.

[0075] Embodiment 1

[0076] This embodiment focuses on a high-quality propagation method for Anoectochilus roxburghii. A rotating bioreactor with a capacity of 100 L is selected, installed on a stable platform, assembled and debugged according to the instructions, and healthy and pest-free plants are selected from excellent Anoectochilus roxburghii mother plants as mother plants, marked using a color marking method, and a 0.5 cm long stem tip and a 2 cm long stem segment are cut with a scalpel as explants. The explants are placed in a container filled with 75% alcohol and soaked for 30 seconds for preliminary disinfection, then transferred to a 10% sodium hypochlorite solution and soaked for 5 minutes, and finally sterilized with Rinse with sterile water and dry, prepare culture medium according to the growth requirements of Anoectochilus roxburghii and the purpose of the experiment, pour the prepared culture medium into the culture bottle and seal it, put it into a high-pressure steam sterilizer with a pressure of 0.11MPa and a temperature of 121℃ for sterilization for 15 minutes, inoculate the sterilized explants into the culture medium on a sterile operating table to a depth of 0.5 cm, put the inoculated culture medium into a rotating bioreactor and fix the position, set the rotation speed to 10 revolutions per minute and the direction to be constant to simulate the microgravity environment, and set the light intensity in the reactor to 2000l ux, the light cycle is 12 hours of light, the temperature is controlled at 22℃, and the humidity is maintained at 60%. Samples are taken every 3 days to observe the division and proliferation of Anoectochilus roxburghii cells. The number of samples taken each time is not less than 5 explants. The cytoskeleton immune double fluorescence staining technique is used to observe the changes in cytoskeleton arrangement. The molecular biological methods are used to detect the expression of related genes and changes in signal transduction pathways. When the explants form a large number of clustered buds in a microgravity environment, they are subcultured, the clustered buds are cut into small pieces and inoculated into new culture medium for continued culture. When the Anoectochilus roxburghii cells are grown in a microgravity environment, the cells are cultured for 10 minutes. The Clematis seedlings were moved from the culture room to the greenhouse for seedling hardening. The humidity was controlled at 70% for the first three days, and then decreased by 5% every day to 50%. The light intensity increased from 1000 lux to 2500 lux by 500 lux every day, and the ventilation volume was gradually increased to 3 times per hour. When the seedlings grew to a certain extent and met the conditions for transplanting, they were removed from the culture medium and transplanted into a suitable cultivation medium. Under this rotation speed condition, the cell division rate was relatively slow and the proliferation number was small, but the cell morphology was relatively regular and the gene expression was relatively stable.

[0077] Embodiment 2

[0078] This embodiment further optimizes the high-quality propagation method of Anoectochilus roxburghii. A rotating bioreactor with a capacity of 100 L is selected, installed on a stable platform, assembled and debugged according to the instructions, and healthy and pest-free plants are selected from excellent Anoectochilus roxburghii mother plants as mother plants, marked using a color marking method, and a 0.5 cm long stem tip and a 2 cm long stem segment are cut with a scalpel as explants. The explants are placed in a container filled with 75% alcohol and soaked for 30 seconds for preliminary disinfection, then transferred to a 10% sodium hypochlorite solution and soaked for 5 minutes, and finally sterilized with a sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile sterile Rinse with water and dry, prepare the culture medium according to the growth requirements of Anoectochilus roxburghii and the purpose of the experiment, pour the prepared culture medium into the culture bottle and seal it, put it into a high-pressure steam sterilizer with a pressure of 0.11MPa and a temperature of 121℃ for sterilization for 15 minutes, inoculate the sterilized explants into the culture medium on a sterile operating table to a depth of 0.5 cm, put the inoculated culture medium into a rotating bioreactor and fix the position, set the rotation speed to 15 revolutions per minute and the direction to be constant to simulate the microgravity environment, set the light intensity in the reactor to 2000lux and the light intensity to 121℃. The cycle of light was 12 hours, the temperature was controlled at 22°C, and the humidity was maintained at 60%. Samples were taken every 3 days to observe the division and proliferation of the Anoectochilus roxburghii cells. The number of samples taken each time was not less than 5 explants. The cytoskeleton immunofluorescence staining technique was used to observe the changes in cytoskeleton arrangement. The molecular biological methods were used to detect the expression of related genes and changes in signal transduction pathways. When the explants formed a large number of clustered buds in the microgravity environment, they were subcultured, the clustered buds were cut into small pieces and inoculated into new culture medium for continued cultivation. When the Anoectochilus roxburghii seedlings were removed from the culture room, the cells were cultured for 10 minutes. The seedlings were hardened in the greenhouse. The humidity was controlled at 70% for the first three days, and then decreased by 5% every day to 50%. The light intensity increased from 1000 lux to 2500 lux and increased by 500 lux every day. The ventilation volume was gradually increased to 3 times per hour. When the seedlings grew to a certain extent and were ready for transplanting, they were removed from the culture medium and transplanted into a suitable culture medium. Under this rotation speed condition, the cell division rate was accelerated, the proliferation number increased, the cytoskeleton arrangement was more orderly, the gene expression was more active, and the signal transduction pathway was smoother.

[0079] Embodiment 3

[0080] This embodiment aims to describe in detail a high-quality propagation method for Anoectochilus roxburghii. A rotating bioreactor with a capacity of 100 L is selected, installed on a stable platform, assembled and debugged according to the instructions, and healthy and pest-free plants are selected from excellent Anoectochilus roxburghii mother plants as mother plants, marked using a color marking method, and a 0.5 cm long stem tip and a 2 cm long stem segment are cut with a scalpel as explants. The explants are placed in a container filled with 75% alcohol and soaked for 30 seconds for preliminary disinfection, and then transferred to a 10% sodium hypochlorite solution and soaked for 5 minutes. Finally, rinse with sterile water and dry. Prepare the culture medium according to the growth requirements of Anoectochilus roxburghii and the purpose of the experiment. Pour the prepared culture medium into the culture bottle and seal it. Sterilize it in a high-pressure steam sterilizer with a pressure of 0.11 MPa and a temperature of 121°C for 15 minutes. Inoculate the sterilized explants into the culture medium on a sterile operating table to a depth of 0.5 cm. Put the inoculated culture medium into the rotating bioreactor and fix it in place. Set the rotation speed to 20 revolutions per minute and the direction to be constant to simulate the microgravity environment. Set the light intensity in the reactor to 2000lux, 12-hour light cycle, temperature controlled at 22℃, humidity maintained at 60%, sampling was conducted every 3 days to observe the division and proliferation of Anoectochilus roxburghii cells, and the number of samples taken each time was no less than 5 explants. Cytoskeleton immunofluorescence staining technique was used to observe the changes in cytoskeleton arrangement, and molecular biological methods were used to detect the expression of related genes and changes in signal transduction pathways. When a large number of clustered buds were formed in the explants under microgravity environment, they were subcultured, the clustered buds were cut into small pieces and inoculated into new culture medium for relay culture. Continue to cultivate. When the golden thread vine seedlings are moved from the culture room to the greenhouse for seedling hardening, the humidity is controlled at 70% in the first 3 days, and then reduced by 5% every day to 50%. The light intensity increases from 1000 lux to 2500 lux every day by 500 lux, and the ventilation volume is gradually increased to 3 times of air change per hour. When the seedlings grow to a certain extent and meet the conditions for transplanting, they are taken out of the culture medium and transplanted to a suitable cultivation medium. Under this rotation speed condition, the speed of cell division and proliferation is significantly accelerated, the cluster buds are formed faster, and the cell growth potential is good.

[0081] Embodiment 4

[0082] This embodiment describes in detail a high-quality propagation method for Anoectochilus roxburghii. A rotating bioreactor with a capacity of 100 L is selected, installed on a stable platform, assembled and debugged according to the instructions, and healthy and pest-free plants are selected from excellent Anoectochilus roxburghii mother plants as mother plants, marked using a color marking method, and a 0.5 cm long stem tip and a 2 cm long stem segment are cut with a scalpel as explants. The explants are placed in a container filled with 75% alcohol and soaked for 30 seconds for preliminary disinfection, and then transferred to a 10% sodium hypochlorite solution and soaked for 5 minutes. Finally, rinse with sterile water and dry, prepare the culture medium according to the growth requirements of Anoectochilus roxburghii and the purpose of the experiment, pour the prepared culture medium into the culture bottle and seal it, put it into a high-pressure steam sterilizer with a pressure of 0.11MPa and a temperature of 121℃ for sterilization for 15 minutes, inoculate the sterilized explants into the culture medium on a sterile operating table to a depth of 0.5 cm, put the inoculated culture medium into a rotating bioreactor and fix the position, set the rotation speed to 25 revolutions per minute and the direction to be constant to simulate the microgravity environment, and set the light intensity in the reactor to 2 The explants were placed in a microgravity environment with an ambient temperature of 2000 lux, a 12-hour light cycle, a temperature of 22°C, and a humidity of 60%. Samples were taken every 3 days to observe the division and proliferation of Anoectochilus roxburghii cells. The number of samples taken each time was no less than 5 explants. Cytoskeleton immunofluorescence staining was used to observe changes in cytoskeleton arrangement. Molecular biological methods were used to detect changes in related gene expression and signal transduction pathways. When a large number of clustered buds were formed in the explants under microgravity, they were subcultured, the clustered buds were cut into small pieces and inoculated into new culture medium for further culture. When the golden thread vine seedlings are moved from the culture room to the greenhouse for seedling hardening, the humidity is controlled at 70% for the first 3 days, and then reduced by 5% every day to 50%. The light intensity starts from 1000 lux and increases by 500 lux every day to 2500 lux. The ventilation volume is gradually increased to 3 times per hour. When the seedlings grow to a certain extent and meet the conditions for transplanting, they are taken out of the culture medium and transplanted to a suitable cultivation medium. Under this rotation speed condition, cell division is extremely active and the number of proliferation is large, but some cells will have irregular morphology.

[0083] Embodiment 5

[0084] In this embodiment, a high-quality propagation method of Anoectochilus roxburghii is optimized. A rotating bioreactor with a capacity of 100 L is selected, installed on a stable platform, assembled and debugged according to the instructions, and healthy and pest-free plants are selected from excellent Anoectochilus roxburghii mother plants as mother plants, marked using a color marking method, and a 0.5 cm long stem tip and a 2 cm long stem segment are cut with a scalpel as explants. The explants are placed in a container filled with 75% alcohol and soaked for 30 seconds for preliminary disinfection, and then transferred to a 10% sodium hypochlorite solution and soaked for 5 seconds. The culture medium was prepared according to the growth requirements of Anoectochilus roxburghii and the purpose of the experiment, and the prepared culture medium was poured into the culture bottle and sealed, and sterilized in a high-pressure steam sterilizer with a pressure of 0.11 MPa and a temperature of 121°C for 15 minutes. The sterilized explants were inoculated into the culture medium on a sterile operating table to a depth of 0.5 cm, and the inoculated culture medium was placed in a rotating bioreactor and fixed in position. The rotation speed was set to 30 revolutions per minute and the direction was constant to simulate a microgravity environment. The explants were placed under the environmental conditions of 2000 lux light intensity, 12-hour light cycle, 22°C temperature, and 60% humidity. Samples were taken every 3 days to observe the division and proliferation of Anoectochilus roxburghii cells. The number of samples taken each time was not less than 5 explants. Cytoskeleton immunofluorescence staining technique was used to observe the changes in cytoskeleton arrangement. Molecular biological methods were used to detect the expression of related genes and changes in signal transduction pathways. When a large number of clustered buds were formed in the microgravity environment, they were subcultured, and the clustered buds were cut into small pieces and grafted Plant in a new culture medium and continue to cultivate. When the golden thread vine seedlings are moved from the culture room to the greenhouse for seedling hardening, the humidity is controlled at 70% for the first 3 days, and then reduced by 5% every day to 50%. The light intensity starts from 1000 lux and increases by 500 lux every day to 2500 lux. The ventilation volume is gradually increased to 3 times per hour. When the seedlings grow to a certain extent and are ready for transplanting, they are removed from the culture medium and transplanted to a suitable cultivation medium. Under this rotation speed condition, cells divide and proliferate rapidly, but there will be cell damage.

[0085] Embodiment 6

[0086] This embodiment describes in detail a high-quality propagation method for Anoectochilus roxburghii. A rotating bioreactor with a capacity of 100 L is selected, installed on a stable platform, assembled and debugged according to the instructions, and healthy and pest-free plants are selected from excellent Anoectochilus roxburghii mother plants as mother plants, marked using a color marking method, and a 0.5 cm long stem tip and a 2 cm long stem segment are cut with a scalpel as explants. The explants are placed in a container filled with 75% alcohol and soaked for 30 seconds for preliminary disinfection, then transferred to a 10% sodium hypochlorite solution and soaked for 5 minutes, and finally sterilized with an aqueous solution of 10% sodium hypochlorite. Rinse with bacterial water and dry it, prepare the culture medium according to the growth requirements of Anoectochilus roxburghii and the purpose of the experiment, pour the prepared culture medium into the culture bottle and seal it, put it into a high-pressure steam sterilizer with a pressure of 0.11MPa and a temperature of 121℃ for sterilization for 15 minutes, inoculate the sterilized explants into the culture medium on a sterile operating table to a depth of 0.5 cm, put the inoculated culture medium into a rotating bioreactor and fix the position, set the rotation speed to 35 revolutions per minute and the direction to be constant to simulate the microgravity environment, set the light intensity in the reactor to 2000lux and light intensity to 1000. The lighting cycle was 12 hours, the temperature was controlled at 22°C, and the humidity was maintained at 60%. Samples were taken every 3 days to observe the division and proliferation of the golden thread lotus cells. The number of samples taken each time was not less than 5 explants. The cytoskeleton immunofluorescence staining technique was used to observe the changes in the cytoskeleton arrangement. The molecular biological methods were used to detect the expression of related genes and changes in signal transduction pathways. When the explants formed a large number of clustered buds in the microgravity environment, they were subcultured, the clustered buds were cut into small pieces and inoculated into new culture medium for continued cultivation. When the golden thread lotus seedlings were taken out of the culture room, the cells were cultured for 10 minutes. Move to the greenhouse for seedling hardening. The humidity is controlled at 70% in the first 3 days, and then reduced by 5% every day to 50%. The light intensity starts from 1000 lux and increases by 500 lux every day to 2500 lux. The ventilation volume is gradually increased to 3 times per hour. When the seedlings grow to a certain extent and are ready for transplanting, they are removed from the culture medium and transplanted to a suitable culture medium. Under this rotation speed condition, cell division and proliferation reach a high level in a short time, but the cell stability is poor, and abnormal differentiation and gene expression disorders are prone to occur.

[0087] In summary, different division and proliferation effects of A. roxburghii cells can be achieved by adjusting the rotational speed of the rotary bioreactor. At a lower rotational speed, cell growth is relatively stable but slow. With the increase of the rotational speed, the cell division and proliferation speeds up, but it is also accompanied by risks and unstable factors in terms of cell morphology and gene expression. By adjusting the appropriate rotational speed, the cytoskeleton arrangement of A. roxburghii is more orderly and the gene expression pattern is optimized, which promotes efficient cell division and proliferation, significantly improves the genetic stability and growth potential of the seedlings, and makes the bred seedlings show higher uniformity and excellence in morphological, physiological and biochemical characteristics.

[0088] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A high-quality propagation method for Anoectochilus roxburghii, characterized in that: The specific steps of this method include: S1. Microgravity environment simulation: S1.

1. Select a rotating bioreactor: According to the experimental scale and requirements, select a rotating bioreactor that can generate stable centripetal acceleration to simulate the microgravity environment; S1.2, Installation and commissioning: Install the rotating bioreactor on a stable platform and assemble and commission it according to the instructions; S1.

3. Calibration and verification: Use standard calibration tools to calibrate the rotating bioreactor so that the centripetal acceleration it produces can accurately simulate the required microgravity level; S2. Preparation of Anoectochilus roxburghii explants: S2.

1. Mother plant selection and marking: Select healthy and pest-free plants from the mother plants of Anoectochilus roxburghii as mother plants, and mark them using color marking method for subsequent tracking; S2.

2. Collection of explants: Use scissors to cut stem tips and stem segments of appropriate length from the mother plant as explants; S2.3, surface disinfection: Place the collected explants in a container filled with alcohol and soak for a period of time for preliminary disinfection, then transfer to a sodium hypochlorite solution and continue soaking for a period of time to completely kill surface microorganisms, finally rinse with sterile water and dry; S3. Culture medium preparation and inoculation: S3.

1. Culture medium preparation: Prepare the culture medium according to the growth requirements of Anoectochilus roxburghii and the purpose of the experiment, including basic nutrients, trace elements and plant growth regulators; S3.2, high pressure sterilization: Pour the prepared culture medium into the culture bottle and seal it, then put it into the high pressure steam sterilizer for high pressure sterilization to kill the microorganisms therein; S3.3, inoculation: inoculate the sterilized explant into the culture medium that has been sterilized by high pressure on a sterile operating table, keep the position and depth of the explant in the culture medium consistent, and gently cover the surface of the culture medium with a layer of sterile gauze to fix the position of the explant; S4. Microgravity environment training: S4.

1. Place in a rotating bioreactor: Place the inoculated culture medium in a rotating bioreactor and fix the position; S4.

2. Set rotation parameters: Set the appropriate rotation speed and direction according to the experimental requirements and the performance of the rotation bioreactor to simulate the required microgravity environment; S4.3, Environmental control: Set appropriate light intensity, light cycle, temperature and humidity environmental conditions in the rotating bioreactor to promote the division and proliferation of Anoectochilus roxburghii cells; S5. Cell division and proliferation monitoring: S5.

1. Regular sampling: Regular sampling is performed during the culture process to observe the division and proliferation of Anoectochilus roxburghii cells; S5.

2. Cytoskeleton observation: Use cytoskeleton immunofluorescence double staining technology to observe the arrangement changes of the cytoskeleton. After the sampled cells are fixed, permeabilized and stained, they are observed under a fluorescence microscope and photographed and recorded; S5.

3. Gene expression and signal transduction detection: Molecular biological methods were used to detect the expression of related genes and changes in signal transduction pathways in Anoectochilus roxburghii cells; S6. Subculture and transplanting: S6.

1. Subculture: When the explants form a large number of clustered buds in a microgravity environment, they are subcultured to expand the scale of reproduction. The clustered buds are cut into small pieces and inoculated into new culture medium and cultured until a sufficient number of seedlings are formed; S6.2, seedling hardening treatment: Move the well-growing golden thread vine seedlings from the culture room to the greenhouse for seedling hardening treatment; S6.

3. Transplantation: When the seedlings grow to the stage where they are ready for transplanting, they are removed from the culture medium and transplanted into the cultivation medium.

2. A high-quality propagation method for Anoectochilus roxburghii according to claim 1, characterized in that: In step S1.1, a rotating bioreactor with a capacity of 100 L is selected according to the experimental scale and requirements so that it can generate a stable centripetal acceleration to simulate a microgravity environment.

3. A high-quality propagation method for Anoectochilus roxburghii according to claim 1, characterized in that: In step S2.2, a stem tip with a length of 0.5-1 cm and a stem segment with a length of 2-3 cm are cut from the mother plant using scissors as explants.

4. A high-quality propagation method for Anoectochilus roxburghii according to claim 1, characterized in that: In step S2.3, the collected explants are placed in a container containing 75% alcohol and soaked for 30-50 seconds for preliminary disinfection, then transferred to a 10% sodium hypochlorite solution and soaked for 5-10 minutes to completely kill surface microorganisms, and finally rinsed with sterile water and dried.

5. A high-quality propagation method for Anoectochilus roxburghii according to claim 1, characterized in that: In step S3.3, the pressure of the high-pressure steam sterilizer is set to 0.11-0.12 MPa, the temperature is 121-123° C., the sterilization time is 15-20 minutes, and the depth of the explant in the culture medium is 0.5-1 cm.

6. A high-quality propagation method for Anoectochilus roxburghii according to claim 1, characterized in that: In step S4.2, the rotation speed is set at 15-40 revolutions per minute according to the experimental requirements and the performance of the rotary bioreactor, and the direction remains constant.

7. A high-quality propagation method for Anoectochilus roxburghii according to claim 1, characterized in that: In step S4.3, the light intensity in the rotating bioreactor is set at 2000-3000 lux, the light cycle is 12-16 hours, the temperature is controlled at 22-26° C., and the humidity is maintained at 60%-70%.

8. The high-quality propagation method of Anoectochilus roxburghii according to claim 1, characterized in that: In the step S5.1, samples are taken every 3-5 days to observe the division and proliferation of the Anoectochilus roxburghii cells, and the number of samples taken each time is not less than 5 explants.

9. A high-quality propagation method for Anoectochilus roxburghii according to claim 1, characterized in that: In the step S5.3, the expression of relevant genes in the golden thread lotus cells is detected by molecular biological means, and the relevant genes include: microtubule protein genes, actin genes, cell cycle protein genes, auxin response genes and gibberellin synthesis and signal transduction related genes.

10. A high-quality propagation method for Anoectochilus roxburghii according to claim 1, characterized in that: In the step 6.2, the well-growing golden thread vine seedlings are moved from the culture room to the greenhouse for seedling hardening treatment. Specifically, the humidity in the greenhouse is controlled at 70%-80% for the first 3 days, and then reduced by 5%-10% every day to 40%-60%, the light intensity is increased from 1000 lux to 500-1000 lux every day to 2000-3000 lux, and the ventilation volume is gradually increased to 3-5 times of air change per hour.