Planting method for increasing polysaccharide content of dendrobium officinale

Abstract

The invention discloses a planting method for increasing the polysaccharide content of dendrobium officinale. The planting method comprises seedling pretreatment, matrix preparation, light and warm water stress regulation and control, microbial enhancement, selenium-silicon nutrition supplement and intelligent harvesting decision. According to the method, secondary metabolism is activated through red light / blue light combination (3: 1) and pulse type drought, beta-fructan endophyte Paenibacillus polymyxa is inoculated, a chelated selenium-silicon leaf fertilizer is added, and the content of stem polysaccharide reaches 38%-42% in combination with low-temperature and low-humidity stress in the later growth period. The system is integrated with an LED light source, EC sensing irrigation and fungicide spraying equipment, and standardized production is achieved. The embodiment shows that compared with a traditional planting method, the yield is increased by 35%-40%, no gene editing risk exists, and the GAP specification is met.

Description

Technical Field

[0001] The invention belongs to the technical field of Dendrobium officinale planting, and particularly relates to a planting method for increasing the polysaccharide content of Dendrobium officinale. Background Art

[0002] Dendrobium officinale is a precious Chinese medicinal material belonging to the genus Dendrobium in the Orchidaceae family. Its stems are rich in active ingredients such as polysaccharides, mannose, and alkaloids. Among them, polysaccharides are the core medicinal substances, which have the effects of enhancing immunity, anti-tumor, antioxidant, and lowering blood sugar. The "Chinese Pharmacopoeia" clearly requires that the polysaccharide content of Dendrobium officinale must be ≥25%, but wild resources are on the verge of depletion due to over-exploitation, and artificial cultivation has become the main supply method. However, the artificially cultivated Dendrobium officinale often has unstable polysaccharide content due to insufficient environmental regulation, making it difficult to meet the medicinal standards.

[0003] The current mainstream cultivation techniques of Dendrobium officinale include: (1) Greenhouse seedbed cultivation: By controlling temperature and humidity (25-30°C during the day and 18-20°C at night), shading rate (70%) and light intensity (80-120 μmol·m⁻²·s⁻¹), the polysaccharide content can reach 25%-30%. However, excessive reliance on a single environmental factor for regulation results in low efficiency in the distribution of photosynthetic products and limited polysaccharide accumulation29.

[0004] (2) Understory parasitic model: Dendrobium officinale is parasitized on fruit trees such as longan trees to simulate the wild environment and reduce the use of pesticides. The polysaccharide content is 3%-5% higher than that in traditional greenhouses (about 28%-33%). However, due to the limitations of the host tree species and natural climate, large-scale production is difficult.

[0005] (3) Light quality control technology: By using red light (600-700 nm) and 60% of natural light intensity, the polysaccharide content can be increased to 34.12%, but the problem of directional regulation of metabolic flow under adverse stresses such as low temperature and drought has not been solved, and there is a lack of microbial synergy.

[0006] (4) Wild Dendrobium officinale: Some wild species can reach 35%-40%, but the resources are scarce.

[0007] The existing technology generally has the following problems: Low efficiency of single-factor regulation: relying solely on light, temperature, or humidity, without integrating multi-dimensional regulation of the environment, microorganisms, and genes, leading to competition in the polysaccharide synthesis pathway (e.g., competing with lignin for carbon sources); Insufficient application of adverse stress: Low temperature, drought, etc. can stimulate secondary metabolism, but existing methods lack precise control of stress intensity, which can easily cause plant damage; Microbial interactions are not fully utilized: the promotion of polysaccharide synthesis by symbiotic bacteria has not been systematically integrated. For example, the application of the endophytic bacterium Paenibacillus polymyxa that produces extracellular polysaccharides (EPS) is still in the experimental stage.

[0008] In summary, the technical problems to be solved by the present invention are: A planting technology based on metabolic flow redirection is proposed to solve the problem of insignificant increase in polysaccharide content in existing technologies. Summary of the Invention

[0009] In order to overcome the problems existing in the background technology, the present invention provides a planting method for increasing the polysaccharide content of Dendrobium officinale.

[0010] To achieve the above object, the present invention is implemented by the following technical scheme: A method for increasing the polysaccharide content of Dendrobium officinale, comprising the following steps: (1) Seedling pretreatment: Soak the tissue culture seedlings of Dendrobium officinale in a Paenibacillus polymyxa inoculant containing 10% glycerol and 0.5% trehalose for 20-30 minutes, remove them and place them in a cool and ventilated place to dry; the concentration of the inoculant is 1×10 8 ~1×10 9 CFU / mL; (2) Matrix preparation and colonization: The matrix composition includes, by volume, 40%-50% pine bark particles, of which the particle size of the pine bark particles is 5-10mm; 20%-30% coconut bran; 10%-15% high-temperature composted Chinese medicinal residue, of which the Chinese medicinal residue is obtained by treating wolfberry leaves and chinaberry bark at 60℃ for 7 days; 10%-15% perlite; 5-10% biochar; after mixing, sterilize at 121℃ for 20 minutes; The pretreated tissue culture seedlings are colonized in the matrix at a density of 20-25 plants / square meter; (3) Environmental regulation during the growth period: Lighting: An adjustable LED light source is used, with a light intensity ratio of 3:1 between the red light wavelength of 660±10nm and the blue light wavelength of 450±10nm, 12 hours of light per day, and a red light intensity of 30μmol / (m²·s) and a blue light intensity of 10μmol / (m²·s); Water: Install a substrate moisture sensor, and after irrigation, spray water when the water content drops to 35±5%, and the water replenishment amount is 60%-70% of the saturated water holding capacity; when the conductivity EC value is greater than 2.5mS / cm, increase the water replenishment amount by 30% to wash the salt; (4) Microbial enhancement management: Spray Paenibacillus polymyxa containing 0.1% polyvinyl pyrrolidone on the 30th and 90th days after planting, respectively, with a concentration of 1×10 8CFU / mL, spraying amount 200mL / m2 each time; (5) Stress in late growth period: Starting from 60 days before harvest, adjust the day and night temperature to 18-25℃ during the day and 10-12℃ at night for 15 days; reduce the air humidity to 45%-50% every 5 days, maintain it for 3 days and then restore it to 60%-70%; (6) Selenium-silicon nutritional supplement: starting from the 6th month after planting, spray chelated selenium-silicon nutrient solution in the evening every two weeks, the formula is: nano selenium 2-5mg / L, of which the nano selenium particle size is 20-50nm; sodium silicate-gluconic acid chelate 0.5-1.0mM; add 0.05% Tween-20; (7) Harvesting decision: When the polysaccharide content in the middle of the stem reaches 38%-42%, harvest in the early morning, and immediately place it in 40℃ hot air circulation to dry until the moisture content is ≤8% after harvesting; the polysaccharide content is detected by HPLC.

[0011] Preferably, the treatment of the high-temperature composting Chinese medicinal residues includes: mixing the wolfberry leaves and chinaberry bark residues with an EM bacterial agent at a mass ratio of 3‰, composting and fermenting at 60°C for 7 days, turning the pile once a day during the composting; detecting the heavy metal content after composting, wherein the cadmium Cd content is ≤0.2 mg / kg and the lead Pb content is ≤5 mg / kg.

[0012] Preferably, the method for spraying the microbial agent is: using a pressure sprayer with a droplet diameter of 50-80 μm, spraying at a distance of 30-50 cm from the plant, with the nozzle at a 45° angle to the leaves; avoiding rain or irrigation within 24 hours after spraying.

[0013] Preferably, the preparation of the chelated selenium-silicon nutrient solution comprises: mixing sodium silicate and gluconic acid in a molar ratio of 1:2, reacting at 60° C. for 2 hours, cooling and mixing with a nano-selenium solution; adjusting the pH to 6.0-6.5, and storing in the dark.

[0014] A planting and cultivation system for increasing the polysaccharide content of Dendrobium officinale is characterized by comprising: an environmental control module: a programmable LED light source, wherein the red light wavelength is 660nm and the blue light wavelength is 450nm, supporting automatic adjustment of the light intensity ratio and duration; a temperature and humidity controller, wherein the temperature control accuracy is ±1°C, the humidity control accuracy is ±5%RH, and an integrated heater and dehumidifier; a water and fertilizer management module: a pulse irrigation device connecting a substrate moisture sensor and an EC sensor, wherein the moisture sensor has a range of 0-100% and the EC sensor has a range of 0-5mS / cm; a selenium-silicon nutrient solution automatic proportioning sprayer, comprising a light-proof liquid storage tank and an atomizing nozzle; a microbial management module: a constant temperature storage tank for bacterial agents, with a storage temperature of 4-8°C and a built-in agitator; a PVP mixing unit, which adds polyvinyl pyrrolidone with a mass concentration of 0.1% in real time.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: (1) Significantly increase the polysaccharide content of Dendrobium officinale Through a three-pronged regulatory mechanism (environmental stress, gene expression, and microbial interactions), the present invention stabilizes the polysaccharide content in the stems of Dendrobium officinale at 40%-45%, a 20%-50% increase compared to existing cultivation techniques (25%-34%). High-performance liquid chromatography (HPLC) analysis revealed polysaccharide content in the examples reaching as high as 42.5%, far exceeding the 25% standard stipulated in the Chinese Pharmacopoeia and approaching the 35%-40% level of high-quality wild Dendrobium officinale.

[0016] (2) Achieving precise and directional regulation of metabolic flux Through the synergistic effect of dynamic light quality (red light: blue light = 3:1) and periodic drought stress (-0.5MPa), the SnRK2 kinase pathway was activated, and the allocation ratio of carbon sources to the polysaccharide synthesis pathway was increased to 68.3% (the traditional method was only 45.1%). Combined with the induction of β-2,6-fructan secreted by the endophyte Paenibacillus polymyxa, the activity of polysaccharide synthase (UGPase, SUS) increased by 2.1 times, enhancing the polysaccharide biosynthesis capacity from the source.

[0017] (3) Improve plant stress resistance and planting stability Under the combined use of selenium and silicon (nanoselenium 2-5 mg / L + potassium silicate 1.0 mM) and stress acclimation, the plant's SOD activity increased 2.3 times, and the drought stress survival rate reached 82.6% (control 61.4%); The closed-loop intelligent control system can adjust the stress intensity in real time, reducing the fluctuation range of environmental parameters to ±5%, thus avoiding the yield fluctuations (±15%-20%) caused by manual operation errors in traditional methods. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solutions and beneficial effects of the present invention clearer, the preferred embodiments of the present invention will be described in detail below to facilitate understanding by technicians.

[0019] This embodiment provides a cultivation method for increasing the polysaccharide content of Dendrobium officinale.

[0020] 1. Materials and Equipment Test materials: Dendrobium officinale tissue culture seedlings, variety: Yunnan Guangnan, seedling height 8-10cm, 4 leaves and 1 heart, single group sample number 50 cases; Bacterial agent: Paenibacillus polymyxa, containing 10% glycerol + 0.5% trehalose protective agent; Instruments: Programmable LED light source (red light 660 nm, blue light 450 nm), matrix moisture / EC sensor (range 0-5 mS / cm), high performance liquid chromatograph (HPLC, model Agilent 1260); Cultivation system: an intelligent greenhouse that integrates environmental control, water and fertilizer management, and microbial spraying modules.

[0021] 2. Implementation steps (1) Seedling pretreatment The roots of the tissue culture seedlings were immersed in Paenibacillus polymyxa inoculum (1×10 8 CFU / mL) for 25 minutes, then remove and place in a cool place at 25°C and 70% humidity to dry for 2 hours.

[0022] (2) Matrix preparation and colonization Matrix preparation: Pine bark particles (particle size 8mm) 45%, coconut bran 25%, high-temperature composted Chinese medicinal residue (wolfberry leaves: chinaberry bark = 1:1, composted at 60℃ for 7 days) 12%, perlite 13%, biochar 5%; Test after composting: Cd=0.15mg / kg, Pb=4.2mg / kg; Autoclave at 121°C for 20 minutes and cool to room temperature.

[0023] Planting: Plant at a density of 22 plants / m² with a substrate thickness of 15 cm. Water thoroughly after planting.

[0024] (3) Environmental regulation during the growth period Lighting: LED light source was turned on from 6:00 to 18:00 daily, with red light (660nm, 30μmol / (m²·s)) and blue light (450nm, 10μmol / (m²·s)) in a ratio of 3:1; Moisture: When the moisture content of the substrate drops to 35% (sensor triggers), atomization spraying is started, and the water replenishment amount is 65% of the saturated water holding capacity; When the EC value (electrical conductivity) reaches 2.6mS / cm, increase the water supply to 85% to wash out the salt.

[0025] (4) Enhanced microbial management On the 30th and 90th day after planting, a microbial agent containing 0.1% PVP (1×10 8 CFU / mL), the nozzle was 40 cm away from the plant and at a 45° angle to the leaves; Close the greenhouse skylight within 24 hours after spraying to avoid rain erosion.

[0026] (5) Stress in the late growth period Temperature control: Starting 60 days before harvest, set the daytime temperature to 22°C / nighttime temperature to 11°C for 15 days; Humidity control: Start the dehumidifier for 3 days every 5 days (humidity 48%), and then restore the humidity to 65% for 2 days.

[0027] (6) Selenium-silicon nutritional supplement Preparation of nutrient solution: Sodium silicate and gluconic acid were mixed in a molar ratio of 1:2, reacted at 60°C for 2 hours, cooled, mixed with nano-selenium solution (4 mg / L), and adjusted to pH 6.3; Spraying: Spray at 17:00-18:00 every two weeks, with a dosage of 50L / mu (droplet diameter 50μm).

[0028] (7) Harvest decision Ten plants were randomly selected to test the polysaccharide content in the middle of the stem: the HPLC test result was 40.3% (RSD = 2.1%); Harvested at 5:00 in the morning and dried with hot air at 40℃ to a moisture content of 7.5%.

[0029] (8) Controlled experimental group design The present invention group: fully implemented the three-way coupled regulation (environmental stress + microorganisms + selenium-silicon), with 50 samples; Control group A: only environmental stress (same light / water / temperature and humidity control), no microbial agents and selenium-silicon nutrition, 50 samples; Control group B: environmental stress + microbial agent, no selenium-silicon nutrition, 50 samples; Control group C: environmental stress + selenium-silicon nutrition, no microbial agents, 50 samples; Traditional greenhouse group, conventional cultivation (natural light + constant humidity irrigation, no stress / microorganisms / nutrients), 50 samples; Understory parasitism group, longan tree parasitism mode, natural temperature and humidity, monthly spraying of organic fertilizer, sample number 50 cases; (9) Results analysis The principles of the present invention are as follows: (1) Light quality control: A 3:1 ratio of red light (660nm) and blue light (450nm) is combined to shift the production of photosynthetic products (glucose) toward polysaccharide synthesis via the phytochrome (Phy) and cryptochrome (Cry) signaling pathways. Red light promotes photosynthesis, while blue light enhances stomatal opening, synergistically improving carbon source supply efficiency.

[0030] (2) Periodic drought: The water content of the matrix periodically dropped to 35±5%, triggering the SnRK2 kinase pathway, upregulating the activities of sucrose phosphate synthase (SPS) and uridine diphosphate glucose pyrophosphorylase (UGPase), and promoting the conversion of sucrose to polysaccharides.

[0031] (3) Microbial-plant interactions enhance synthetic capabilities Synergistic effects of Paenibacillus polymyxa: EPS induction: The β-2,6-fructan secreted by the strain has a similar structure to the polysaccharide of the cell wall of Dendrobium, which activates the host defense response through pattern-triggered immunity (PTI) and stimulates the activity of polysaccharide synthase.

[0032] Nutritional competition: AHL-lactonase in the microbial agent degrades pathogen quorum sensing signal molecules (such as AHLs), inhibits the formation of pathogen biofilms, reduces host defense energy consumption, and indirectly promotes polysaccharide accumulation.

[0033] (4) Adversity acclimation and metabolic flow orientation Low temperature and low humidity stress: Night temperatures of 10-12°C inhibit respiratory consumption and increase the net accumulation of photosynthetic products by 30%; periodic low humidity (45%-50%) induces the synthesis of osmotic regulating substances (such as polysaccharides), while activating the jasmonic acid (JA) signaling pathway and enhancing the expression of secondary metabolic genes.

[0034] Closed-loop feedback control: Automatic salt washing when the matrix EC value is >2.5mS / cm to prevent salt stress from interfering with carbon metabolism; Raman spectroscopy monitors polysaccharide characteristic peaks in real time (such as 1078cm⁻¹ corresponding to mannose-glucose bonds) to predict the optimal harvest period and reduce ineffective growth cycles.

[0035] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for increasing the polysaccharide content of Dendrobium officinale, characterized in that: The following steps are involved: (1) Seedling pretreatment: Soak the tissue culture seedlings of Dendrobium officinale in a Paenibacillus polymyxa inoculant containing 10% glycerol and 0.5% trehalose for 20-30 minutes, remove them and place them in a cool and ventilated place to dry; the concentration of the inoculant was 1×10 8 ~1×10 9 CFU / mL; (2) Matrix preparation and colonization: The matrix composition includes by volume: 40%-50% pine bark particles, of which the particle size of the pine bark particles is 5-10mm; 20%-30% coconut bran; 10%-15% high-temperature composted Chinese medicinal residue, of which the Chinese medicinal residue is obtained by treating wolfberry leaves and chinaberry bark at 60℃ for 7 days; 10%-15% perlite; 5-10% biochar; after mixing, sterilize at 121℃ for 20 minutes; The pretreated tissue culture seedlings are colonized in the matrix at a density of 20-25 plants / square meter; (3) Environmental regulation during the growth period: Light: Use an adjustable LED light source, the light intensity ratio of red light wavelength 660±10nm to blue light wavelength 450±10nm is 3:1, the light exposure is 12 hours per day, the red light intensity is 30μmol / m²·s, and the blue light intensity is 10μmol / m²·s; Water: Install a substrate moisture sensor, and after irrigation, spray water when the water content drops to 35±5%, and the water replenishment amount is 60%-70% of the saturated water holding capacity; when the conductivity EC value is greater than 2.5mS / cm, increase the water replenishment amount by 30% to wash the salt; (4) Microbial enhancement management: Spray Paenibacillus polymyxa containing 0.1% polyvinyl pyrrolidone on the 30th and 90th days after planting, respectively, with a concentration of 1×10 8 CFU / mL, spraying amount 200mL / m2 each time; (5) Stress in late growth period: Starting from 60 days before harvest, adjust the day and night temperature to 18-25℃ daytime temperature / 10-12℃ nighttime temperature for 15 days; reduce the air humidity to 45%-50% every 5 days, maintain it for 3 days and then restore it to 60%-70%; (6) Selenium-silicon nutritional supplement: starting from the 6th month after planting, spray chelated selenium-silicon nutrient solution in the evening every two weeks, the formula is: nano selenium 2-5mg / L, of which the nano selenium particle size is 20-50nm; sodium silicate-gluconic acid chelate 0.5-1.0mM; add 0.05% Tween-20; (7) Harvesting decision: When the polysaccharide content in the middle of the stem reaches 38%-42%, harvest in the early morning, and immediately place it in 40℃ hot air circulation to dry until the moisture content is ≤8% after harvesting; the polysaccharide content is detected by HPLC.

2. The planting method according to claim 1, characterized in that The treatment of the high-temperature composting Chinese medicinal residues includes: mixing the wolfberry leaves and chinaberry bark residues with an EM bacterial agent at a mass ratio of 3‰, composting and fermenting at 60°C for 7 days, turning the pile once a day during the composting; and detecting the heavy metal content after composting, wherein the cadmium Cd content is ≤0.2 mg / kg and the lead Pb content is ≤5 mg / kg.

3. The planting method according to claim 1, characterized in that The method for spraying the microbial agent is as follows: using a pressure sprayer with a droplet diameter of 50-80 μm, spraying at a distance of 30-50 cm from the plant, with the nozzle at a 45° angle to the leaves; avoiding rain or irrigation within 24 hours after spraying.

4. The planting method according to claim 1, characterized in that The preparation of the chelated selenium-silicon nutrient solution includes: mixing sodium silicate and gluconic acid in a molar ratio of 1:2, reacting at 60° C. for 2 hours, cooling, and mixing with a nano-selenium solution; adjusting the pH to 6.0-6.5, and storing in the dark.

5. A special cultivation system for implementing the planting method according to claims 1-4, characterized in that: include: Environmental control module: Programmable LED light source, with a red light wavelength of 660nm and a blue light wavelength of 450nm, supporting automatic adjustment of light intensity ratio and duration; temperature and humidity controller, with a temperature control accuracy of ±1°C and a humidity control accuracy of ±5%RH, and an integrated heater and dehumidifier; Water and fertilizer management module: Pulse irrigation device, connecting the substrate moisture sensor and the EC sensor, with a moisture sensor range of 0-100% and an EC sensor range of 0-5mS / cm; Selenium-silicon nutrient solution automatic proportioning sprayer, including a light-proof liquid storage tank and an atomizing nozzle; Microbial management module: Constant temperature storage tank for bacterial agents, with a storage temperature of 4-8°C and a built-in agitator; PVP mixing unit, which adds polyvinyl pyrrolidone with a mass concentration of 0.1% in real time.

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