Planting method for improving content of polysaccharide in dendrobium officinale

Abstract

The application discloses a kind of planting methods for improving the content of Dendrobium candidum polysaccharide, covers seedling pretreatment, substrate preparation, light temperature water stress regulation, microbial reinforcement, selenium-silicon nutrition supplement and intelligent harvesting decision.By red light / blue light combination (3:1) and pulse drought activation secondary metabolism, inoculate beta-fructan endophyte Paenibacillus polymyxa and add chelated selenium-silicon foliar fertilizer, combined with low temperature and low humidity adversity in the later growth stage, the stem polysaccharide content reaches 38%-42%.System integration LED light source, EC sensing irrigation and bacterial agent spraying equipment, realize standardization production.Embodiment shows that the method is 35-40% higher than traditional planting polysaccharide, and there is no gene editing risk, in line with GAP specification.

Description

Technical Field

[0001] This invention belongs to the field of Dendrobium officinale cultivation technology, specifically relating to a cultivation method for increasing the polysaccharide content of Dendrobium officinale. Background Technology

[0002] Dendrobium officinale, a precious traditional Chinese medicine belonging to the genus Dendrobium in the Orchidaceae family, is rich in active ingredients such as polysaccharides, mannose, and alkaloids. Among these, polysaccharides are the core active substances, possessing effects such as enhancing immunity, anti-tumor activity, anti-oxidation, and lowering blood sugar. The Chinese Pharmacopoeia clearly requires that the polysaccharide content of Dendrobium officinale must be ≥25%, but wild resources are nearing depletion due to over-harvesting, making artificial cultivation the main supply method. However, artificially cultivated Dendrobium officinale often suffers from unstable polysaccharide content due to insufficient environmental control, making it difficult to meet medicinal standards.

[0003] Currently, the mainstream cultivation techniques for Dendrobium officinale include:

[0004] (1) Greenhouse seedbed cultivation: By controlling temperature and humidity (25-30℃ during the day and 18-20℃ at night), shading rate (70%) and light intensity (80-120μmol·m⁻²·s⁻¹), polysaccharide content can reach 25%-30%, but it relies too much on the regulation of a single environmental factor, resulting in low efficiency of photosynthetic product distribution and limited polysaccharide accumulation.29

[0005] (2) Understory parasitic mode: Dendrobium officinale is parasitized on fruit trees such as longan trees to simulate the wild environment, reduce the use of pesticides, and the polysaccharide content is 3%-5% higher than that of traditional greenhouses (about 28%-33%). However, due to the host tree species and natural climate, large-scale production is difficult.

[0006] (3) Light quality regulation technology: Using red light (600-700 nm) and 60% natural light intensity, the polysaccharide content can be increased to 34.12%, but the problem of directional regulation of metabolic flux by adverse stresses such as low temperature and drought has not been solved, and there is a lack of microbial synergy.

[0007] (4) Wild Dendrobium officinale: Some wild species can reach 35%-40%, but resources are scarce.

[0008] Existing technologies generally suffer from the following problems:

[0009] Single-factor regulation is inefficient: relying solely on a single factor such as light, temperature, or humidity without integrating multi-dimensional regulation of environment, microorganisms, and genes leads to competition in polysaccharide synthesis pathways (such as competition with lignin for carbon sources).

[0010] Insufficient application of stress management: Low temperature and drought can stimulate secondary metabolism, but existing methods lack precise control of stress intensity, which can easily cause plant damage.

[0011] Microbial interactions are not fully utilized: the promoting effect of symbiotic bacteria on polysaccharide synthesis has not been systematically integrated. For example, the application of the endophytic bacterium Paenibacillus polymyxa, which produces extracellular polysaccharides (EPS), is still in the experimental stage.

[0012] In summary, the technical problem to be solved by this invention is:

[0013] A metabolic flux redirection-based planting technique is proposed to address the problem that existing techniques do not significantly increase polysaccharide content. Summary of the Invention

[0014] In order to overcome the problems existing in the background art, the present invention provides a cultivation method for increasing the polysaccharide content of Dendrobium officinale.

[0015] To achieve the above objectives, the present invention is implemented through the following technical solution: A planting method for increasing the polysaccharide content of Dendrobium officinale, comprising the following steps: (1) Seedling pretreatment: Dendrobium officinale tissue culture seedlings are soaked in Paenibacillus polymyxa inoculant containing 10% glycerol and 0.5% trehalose for 20-30 minutes, then removed and placed in a cool and ventilated place to dry; the inoculant concentration is 1×10 8 ~1×10 9 CFU / mL; (2) Substrate preparation and planting: The substrate composition by volume ratio includes: 40%-50% pine bark particles, of which the particle size of pine bark particles is 5-10mm; 20%-30% coconut coir; 10%-15% high-temperature composted Chinese medicine residue, of which the Chinese medicine residue is obtained by treating wolfberry leaves and bitter neem bark at 60℃ for 7 days; 10%-15% perlite; 5-10% biochar; after mixing, sterilize at 121℃ for 20 minutes; plant the pretreated tissue culture seedlings in the substrate at a density of 20-25 plants / square meter; (3) Environmental control during the growth period: Light: use 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 intensity is 12 hours per day, the red light intensity is 30μmol / (m²·s) and the blue light intensity is 10μmol / (m²·s); Moisture: Install a substrate moisture sensor. After irrigation, when the moisture content drops to 35±5%, perform atomized water replenishment. The replenishment amount is 60%-70% of the saturated water holding capacity. When the conductivity EC value is greater than 2.5mS / cm, increase the replenishment amount by 30% to wash away salt. (4) Microbial enhanced management: On the 30th and 90th day after transplanting, spray Paenibacillus polymyxa inoculant containing 0.1% polyvinylpyrrolidone. The inoculant concentration is 1×10 8CFU / mL, each spraying amount is 200mL / m²; (5) Adverse stress in the later stage of growth: Starting 60 days before harvest, adjust the day and night temperature to 18-25℃ / 10-12℃ 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 nutrient supplementation: Starting from the 6th month after planting, spray chelated selenium-silicon nutrient solution every two weeks in the evening. 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. After harvesting, immediately place it in a 40℃ hot air circulation dryer until the moisture content is ≤8%; the polysaccharide content is detected by HPLC.

[0016] Preferably, the treatment of the medicinal herb residue in the high-temperature composting process includes: mixing wolfberry leaves and neem bark residue with EM bacterial agent at a mass ratio of 3‰, composting at 60℃ for 7 days, turning the pile once a day during the period; and testing the heavy metal content after composting, wherein the cadmium (Cd) content is ≤0.2mg / kg and the lead (Pb) content is ≤5mg / kg.

[0017] Preferably, the method of spraying the microbial agent is as follows: use a pressure sprayer with a droplet diameter of 50-80μm, spray at a distance of 30-50cm from the plant, and make a 45° angle between the nozzle and the leaves; avoid rain or irrigation within 24 hours after spraying.

[0018] Preferably, the preparation of the chelated selenium-silicon nutrient solution includes: mixing sodium silicate and gluconic acid at a molar ratio of 1:2, reacting at 60°C for 2 hours, cooling, and then mixing with a nano-selenium solution; adjusting the pH to 6.0-6.5, and storing in the dark.

[0019] A cultivation system for increasing the polysaccharide content of Dendrobium officinale, 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 light intensity ratio and duration; a temperature and humidity controller, wherein the temperature control accuracy is ±1℃ and the humidity control accuracy is ±5%RH, integrating a heater and a dehumidifier; a water and fertilizer management module: a pulse irrigation device, connected to a substrate moisture sensor and an EC sensor, wherein the moisture sensor range is 0-100% and the EC sensor range is 0-5mS / cm; an automatic selenium-silicon nutrient solution mixing and spraying machine, including a light-proof storage tank and an atomizing nozzle; a microbial management module: a constant temperature storage tank for inoculants, with a storage temperature of 4-8℃ and a built-in stirrer; and a PVP mixing unit, which adds 0.1% polyvinylpyrrolidone by mass in real time.

[0020] Compared with the prior art, the present invention achieves at least the following beneficial effects:

[0021] (1) Significantly increases the polysaccharide content of Dendrobium officinale

[0022] By employing a ternary coupling regulation technology (environmental stress-gene expression-microbe interaction), this invention stabilizes the polysaccharide content in Dendrobium officinale stems at 40%-45%, an increase of 20%-50% compared to existing cultivation techniques (25%-34%). High-performance liquid chromatography (HPLC) analysis showed that the polysaccharide content in the examples reached a maximum of 42.5%, far exceeding the 25% standard stipulated in the Chinese Pharmacopoeia and approaching the level of high-quality wild Dendrobium officinale (35%-40%).

[0023] (2) Achieve precise and targeted regulation of metabolic flux

[0024] By synergistically combining dynamic light quality (red light:blue light = 3:1) with periodic drought stress (-0.5 MPa), the SnRK2 kinase pathway was activated, increasing the allocation of carbon sources to the polysaccharide synthesis pathway to 68.3% (compared to only 45.1% by traditional methods).

[0025] Induced by β-2,6-fructan secreted by the endophytic bacterium Paenibacillus polymyxa, the activity of polysaccharide synthases (UGPase, SUS) increased by 2.1 times, thereby enhancing the ability of polysaccharide biosynthesis from the source.

[0026] (3) Enhance plant stress resistance and planting stability

[0027] Under the combined use of selenium-silicon (nano selenium 2-5 mg / L + potassium silicate 1.0 mM) and stress acclimatization, the SOD activity of the plants increased by 2.3 times, and the survival rate under drought stress reached 82.6% (compared to 61.4% in the control group).

[0028] 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 production fluctuations (±15%-20%) caused by human operation errors in traditional methods. Detailed Implementation

[0029] To make the objectives, 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 those skilled in the art.

[0030] This embodiment provides a cultivation method for increasing the polysaccharide content of Dendrobium officinale.

[0031] 1. Materials and Equipment

[0032] Test materials: Dendrobium officinale tissue culture seedlings, variety: Yunnan Guangnan variety, seedling height 8-10cm, 4 leaves and 1 heart, 50 samples per group;

[0033] Inoculant: Paenibacillus polymyxa, containing 10% glycerol + 0.5% trehalose protectant;

[0034] Instruments: Programmable LED light source (660nm red light, 450nm blue light), matrix moisture / EC sensor (range 0-5mS / cm), high performance liquid chromatograph (HPLC, model Agilent 1260);

[0035] Cultivation system: An intelligent greenhouse integrating environmental control, water and fertilizer management, and microbial spraying modules.

[0036] 2. Implementation Steps

[0037] (1) Seedling pretreatment

[0038] The roots of the tissue culture seedlings were immersed in Paenibacillus polymyxa inoculant (1×10⁻⁶). 8 After soaking in water (CFU / mL) for 25 minutes, remove the contents and air dry in a cool place at 25℃ and 70% humidity for 2 hours.

[0039] (2) Substrate preparation and planting

[0040] Matrix preparation:

[0041] Pine bark granules (8mm diameter) 45%, coconut coir 25%, high-temperature composted Chinese herbal medicine residue (goji berry leaves: neem bark = 1:1, composted at 60℃ for 7 days) 12%, perlite 13%, biochar 5%;

[0042] Post-composting tests: Cd = 0.15 mg / kg, Pb = 4.2 mg / kg;

[0043] Autoclave at 121℃ for 20 minutes, then cool to room temperature.

[0044] Planting: Plant at a density of 22 plants / m², with a substrate thickness of 15cm, and water thoroughly after planting.

[0045] (3) Environmental regulation during the growth period

[0046] Lighting: Turn on the LED light source from 6:00 to 18:00 every day, and irradiate with red light (660nm, 30μmol / (m²·s)) and blue light (450nm, 10μmol / (m²·s)) in a 3:1 ratio;

[0047] Moisture:

[0048] When the substrate moisture content drops to 35% (triggered by the sensor), the atomized spraying is activated, and the water replenishment is 65% of the saturated water holding capacity.

[0049] When the EC value (conductivity) reaches 2.6 mS / cm, increase the water replenishment to 85% for salt washing.

[0050] (4) Enhanced management of microorganisms

[0051] On the 30th and 90th day after transplanting, spray with a microbial agent containing 0.1% PVP (1×10⁻⁶) using a pressure sprayer (droplet diameter 60μm). 8 (CFU / mL), nozzle 40cm from plant, at a 45° angle to leaves;

[0052] Close the greenhouse skylights within 24 hours after spraying to prevent rainwater from washing away the pesticide.

[0053] (5) Adverse stress in the later stages of growth

[0054] Temperature control: Starting 60 days before harvest, set the daytime temperature to 22℃ and the nighttime temperature to 11℃ for 15 consecutive days;

[0055] Humidity control: Run the dehumidifier for 3 days (48% humidity) every 5 days, then restore the humidity to 65% for 2 days.

[0056] (6) Selenium-silicon nutritional supplements

[0057] Nutrient solution preparation: Sodium silicate and gluconic acid are mixed at a molar ratio of 1:2 and reacted at 60°C for 2 hours. After cooling, it is mixed with nano selenium solution (4 mg / L) and the pH is adjusted to 6.3.

[0058] Spraying: Spray every two weeks from 17:00 to 18:00, at a rate of 50L / acre (droplet diameter 50μm).

[0059] (7) Harvesting decision

[0060] Ten plants were randomly selected to test the polysaccharide content in the middle of their stems: the HPLC result was 40.3% (RSD=2.1%).

[0061] Harvested at 5:00 AM and dried with hot air at 40℃ until the moisture content is 7.5%.

[0062] (8) Control experimental group design

[0063] This invention group: Complete implementation of ternary coupled regulation (environmental stress + microorganisms + selenium-silicon), with 50 samples;

[0064] Control group A: Only environmental stress (same light / water / temperature and humidity control), no microbial agents and selenium-silicon nutrition, sample size 50;

[0065] Control group B: Environmental stress + microbial inoculants, no selenium-silicon nutrition, sample size 50 cases;

[0066] Control group C: Environmental stress + selenium-silicon nutrition, no microbial agents, sample size 50 cases;

[0067] Traditional greenhouse group, conventional planting (natural light + constant humidity irrigation, no stress / microorganisms / nutrients), sample size 50;

[0068] Understory parasitism group, longan tree parasitism mode, natural temperature and humidity, monthly application of organic fertilizer, 50 samples;

[0069] (9) Results Analysis

[0070]

[0071] The principle of this invention is as follows:

[0072] (1) Light quality regulation:

[0073] A 3:1 combination of red (660nm) and blue (450nm) light induces the conversion of photosynthetic products (glucose) towards 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.

[0074] (2) Periodic drought:

[0075] The matrix moisture content periodically decreased to 35±5%, triggering the SnRK2 kinase pathway, upregulating the activity of sucrose phosphate synthase (SPS) and uridine diphosphate glucose pyrophosphorylase (UGPase), and promoting the conversion of sucrose to polysaccharides.

[0076] (3) Microbial-plant interaction enhances synthesis capacity

[0077] Synergistic effects of Paenibacillus polymyxa:

[0078] EPS induction: The β-2,6-fructan secreted by the strain has a similar structure to the polysaccharide in the cell wall of Dendrobium. It activates the host defense response through pattern-triggered immunity (PTI) and stimulates the activity of polysaccharide synthase.

[0079] Nutritional competition: AHL-lactonase in the microbial agent degrades pathogenic quorum sensing signaling molecules (such as AHLs), inhibits pathogenic biofilm formation, reduces host defense energy consumption, and indirectly promotes polysaccharide accumulation.

[0080] (4) Adaptation to stress and orientation of metabolic flux

[0081] Low temperature and low humidity stress: Night temperature of 10-12℃ inhibits respiratory consumption and increases the net accumulation of photosynthetic products by 30%; periodic low humidity (45%-50%) induces the synthesis of osmotic regulators (such as polysaccharides), while activating the jasmonic acid (JA) signaling pathway and enhancing the expression of secondary metabolic genes.

[0082] Closed-loop feedback control: Automatic salt washing when the matrix EC value is >2.5mS / cm to avoid salt stress 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.

[0083] 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 intended to limit it. 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 to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A cultivation method for increasing the polysaccharide content of Dendrobium officinale, characterized in that, Includes 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, then remove them and place them in a cool and ventilated place to dry; the concentration of the inoculant is 1×10⁸~1×10⁹. CFU / mL; (2) Substrate preparation and planting: The substrate composition by volume ratio includes: 40%-50% pine bark particles, of which the pine bark particles have a particle size of 5-10mm; 20%-30% coconut coir; 10%-15% high-temperature composted Chinese medicine residue, of which the Chinese medicine residue is obtained by treating wolfberry leaves and bitter neem bark at 60℃ for 7 days; 10%-15% perlite; 5-10% biochar; after mixing, sterilize at 121℃ for 20 minutes; plant the pretreated tissue culture seedlings in the substrate at a density of 20-25 plants / square meter; (3) Environmental control during the growth period: Light: Use adjustable LED light source, red light wavelength 660±10nm and blue light wavelength The light intensity ratio of 450±10nm is 3:1, the daily light exposure is 12 hours, 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, when the water content drops to 35±5%, perform atomized water replenishment, and the replenishment amount is 60%-70% of the saturated water holding capacity; when the conductivity EC value is greater than 2.5mS / cm, increase the replenishment amount by 30% to wash away salt; (4) microbial enhanced management: spray Paenibacilluspolymyxa bacterial agent containing 0.1% polyvinylpyrrolidone on the 30th and 90th days after planting, and the bacterial agent concentration is 1×10⁸ CFU / mL, each spraying amount is 200mL / m²; (5) Adverse stress in the later stage of growth: starting 60 days before harvest, adjust the day and night temperature to 18-25℃ / 10-12℃ 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 nutrient supplementation: starting from the 6th month after planting, spray chelated selenium-silicon nutrient solution every two weeks in the evening. 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 a 40℃ hot air circulation dryer until the moisture content is ≤8%; the polysaccharide content is detected by HPLC.

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

3. The planting method according to claim 1, characterized in that, The method for spraying the microbial agent is as follows: use a pressure sprayer with a droplet diameter of 50-80μm, spray at a distance of 30-50cm from the plant, and make a 45° angle between the nozzle and the leaves; avoid 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 at a molar ratio of 1:2, reacting at 60°C for 2 hours, cooling and then mixing with nano-selenium solution; adjusting the pH to 6.0-6.5 and storing in the dark.

5. A dedicated cultivation system for implementing the planting method according to any one of claims 1-4, characterized in that, include: Environmental control module: Programmable LED light source, with red light wavelength of 660nm and blue light wavelength of 450nm, supporting automatic adjustment of light intensity ratio and duration; temperature and humidity controller, with temperature control accuracy of ±1℃ and humidity control accuracy of ±5%RH, integrating heater and dehumidifier; Water and fertilizer management module: Pulse irrigation device, connected to substrate moisture sensor and EC sensor, with moisture sensor range of 0-100% and EC sensor range of 0-5mS / cm; Selenium-silicon nutrient solution automatic proportioning sprayer, including light-proof storage tank and atomizing nozzle; Microbial management module: Constant temperature storage tank for microbial agents, with storage temperature of 4-8℃ and built-in stirrer; PVP mixing unit, real-time addition of 0.1% polyvinylpyrrolidone by mass concentration.

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