Rhododendron tissue culture rapid propagation method

Abstract

The invention relates to the technical field of plant tissue culture, and discloses an azalea tissue culture and rapid propagation method which comprises the following steps: S1, preparing an azalea explant and a culture medium; s2, preparing a dynamic purification culture device and sterilizing; s3, inoculation and dynamic purification culture: inoculating the stem segments into a proliferation culture medium in a growth area in a sterile environment; starting dynamic purification; s4, carrying out subculture multiplication; s5, rooting and seedling hardening. By arranging the dynamic purification culture device, small molecule metabolites generated in a growth area can be continuously removed in the rhododendron tissue culture process, dependence on traditional antioxidants is not needed, the competitive effect of the antioxidants and metabolite adsorption materials is avoided, the stable culture environment can be maintained for a long time, inhibition of metabolite accumulation on plant cell metabolism is reduced, and the survival rate of the rhododendron is increased. The survival of explants and the normal differentiation and growth of bud clusters are effectively guaranteed, and the stability of the tissue culture process and the overall quality of seedlings are improved.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a method for rapid propagation of rhododendron tissue culture. Background Technology

[0002] Azalea ( Rhododendron spp. Rhododendrons are important ornamental woody flowering plants in the horticultural field, with rich flower colors and high ornamental value, and are widely used in landscaping, home gardening, and other scenarios. With the large-scale development of the rhododendron industry, the demand for high-quality seedlings continues to increase. Tissue culture technology, due to its advantages such as high propagation efficiency and the ability to maintain the excellent characteristics of varieties, has become one of the core technical means for large-scale rhododendron seedling production.

[0003] In current rhododendron tissue culture processes, the industry generally adopts the in vitro explant culture mode. Typically, tender shoots or leaves of plants are selected as explants. After cleaning and disinfection, they are inoculated into culture media based on MS or WPM formulas and cultured in single-chamber culture containers. By controlling the temperature, light intensity and cycle of the culture environment, the explants are induced to differentiate into bud clusters. After subculture, rooting culture and hardening, seedlings that can be transplanted are finally obtained.

[0004] In existing rhododendron tissue culture techniques, small molecule metabolites are produced when explants are damaged or during cell metabolism. These metabolites are difficult to remove effectively within the single-chamber culture container and tend to accumulate in the culture medium. Accumulated metabolites inhibit the normal metabolic activity of rhododendron cells, leading to browning of the explants. They also affect the differentiation efficiency and growth status of shoot clusters, and in severe cases, cause culture failure, thus hindering the large-scale application of rhododendron tissue culture and the improvement of seedling quality. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a rapid propagation method for rhododendron tissue culture, which solves the problems of browning caused by the accumulation of metabolites and the impact on seedling quality during rhododendron tissue culture.

[0006] To achieve the above objectives, the present invention provides a method for rapid propagation of rhododendrons through tissue culture, comprising the following steps: S1. Preparation of rhododendron explants and culture media: Select healthy rhododendron shoots, clean and disinfect them, and cut them into stem segments containing axillary buds; prepare proliferation culture media and rooting culture media suitable for rhododendron tissue culture; S2. Prepare and sterilize the dynamic purification culture device: The device includes a growth zone for culturing explants and a purification zone for processing metabolites. The growth zone and the purification zone are provided with an isolation structure that allows small molecule metabolites to pass through. The device, the proliferation culture medium and the adsorbent material filling the purification zone are sterilized. S3. Inoculation and dynamic purification culture: Under sterile conditions, the stem segments are inoculated into the proliferation culture medium in the growth zone; dynamic purification is initiated, allowing small molecule metabolites produced in the growth zone to enter the purification zone through the isolation structure and be adsorbed by the adsorption material; at the same time, the device is placed in a culture environment with a temperature of 25±2℃, a light intensity of 2000 lux, and a photoperiod of 14h / d. S4. Subculture and Propagation: After the stem segments in the growth zone form bud clusters, the bud clusters are divided and transferred to a new dynamic purification culture device, and the dynamic purification culture steps of S3 are repeated. S5. Rooting and hardening off: After the plants grow to 2-3cm, transfer them to a rooting medium and culture them until they root. After cleaning the roots of the rooted plants, transplant them into the cultivation substrate. Keep the humidity high in the early stage, and then gradually reduce the humidity until the plants adapt to the natural environment to obtain rhododendron tissue culture seedlings.

[0007] Preferably, in step S2, the growth zone and purification zone of the dynamic purification culture device are independent chambers with a double-layer concentric columnar structure; the isolation structure is a hydrophilic modified polyethersulfone membrane with a molecular weight cutoff of 500-1000 Daltons; the inner wall of the growth zone is provided with a cathode, and the outer wall of the container outside the purification zone is provided with an anode. The cathode and anode are connected to a power source and a constant voltage of 1.5-3V is applied to form an electric field pointing from the growth zone to the purification zone.

[0008] Preferably, both the cathode and anode are platinum-iridium alloy wires with a diameter of 0.5 mm, wherein the platinum content is 90% by mass and the iridium content is 10% by mass; the power supply is an adjustable DC power supply with a voltage output range of 0-5V and a current output range of 1-100μA.

[0009] Preferably, the adsorbent material is an amino-functionalized mesoporous silica microsphere or an immobilized polyphenol oxidase microsphere; the amino-functionalized mesoporous silica microsphere has a particle size of 50-100 μm; the immobilized polyphenol oxidase microsphere is prepared by immobilizing polyphenol oxidase on an activated carrier microsphere through a cross-linking method.

[0010] Preferably, the preparation process of the rhododendron explant in step S1 is as follows: select semi-lignified young shoots of the current year, rinse them sequentially with tap water for 30 minutes, wash them with sterile water containing 0.1% (v / v) Tween-20 for 10 minutes, soak them in 75% (v / v) ethanol for 30 seconds, rinse them with sterile water 3 times, treat them with sodium hypochlorite solution containing 0.1% (w / v) available chlorine for 8 minutes, and rinse them with sterile water 5-6 times; the cut stem segments contain 1-2 axillary buds and are 1.5 cm in length.

[0011] Preferably, in step S1, the proliferation medium is a modified Woody Plant Medium with 30 g / L sucrose, 6 g / L agar, 2.0 mg / L zeatin and 0.1 mg / L indolebutyric acid added; the rooting medium is 1 / 2 MS medium with 20 g / L sucrose, 6 g / L agar and 1.0 mg / L indolebutyric acid added; the pH of both media is adjusted to 5.8.

[0012] Preferably, in step S2, sterilization is performed using high-pressure steam sterilization at 121°C for 20 minutes; five stem segments are inoculated into the growth zone of each dynamic purification culture device; and the adsorbent material is mixed with organic-free phosphate buffer to form a suspension before sterilization.

[0013] Preferably, the light source for the culture environment in step S3 is a white LED light source; during subculture in S5, the divided bud clusters contain 2-3 healthy buds, and the structure and sterilization method of the new device are consistent with those of the device in S2.

[0014] Preferably, in step S5, the cultivation substrate is a mixture of peat moss and perlite in a volume ratio of 2:1; the initial high humidity environment has a relative humidity of 85%-90%, which is maintained by a water tray in the seedling box; the humidity is gradually reduced by 5%-10% per day until it reaches 60%-65%, and this is continued for 5-7 days.

[0015] Preferably, the pH of the culture medium is adjusted using a 0.1 mol / L hydrochloric acid solution or a 0.1 mol / L sodium hydroxide solution.

[0016] This invention provides a rapid propagation method for rhododendrons through tissue culture. It has the following beneficial effects: 1. This invention, by setting up a dynamic purification culture device, can continuously remove small molecule metabolites generated in the growth zone during the tissue culture of azaleas. It does not rely on traditional antioxidants, avoids the competition between antioxidants and metabolite adsorption materials, can maintain a stable culture environment for a long time, reduces the inhibition of plant cell metabolism by metabolite accumulation, effectively ensures the survival of explants and the normal differentiation and growth of bud clusters, and improves the stability of the tissue culture process and the overall quality of seedlings.

[0017] 2. This invention optimizes explant treatment and culture medium preparation according to the characteristics of azaleas. It selects semi-lignified young shoots of the current year and adopts a step-by-step sterilization process to reduce cell damage during sterilization. At the same time, it prepares a suitable proliferation and rooting culture medium, controls the sucrose concentration and the ratio of growth regulators to meet the physiological needs of azalea cells, reduces the physiological stress caused by high concentrations of inorganic salts or inappropriate hormones, and promotes robust growth of bud clusters and efficient formation of adventitious roots.

[0018] 3. This invention employs a gradual dehumidification strategy during the seedling hardening stage and uses a mixed substrate of peat moss and perlite. By slowly adjusting the humidity, it helps seedlings gradually adapt to the natural environment, reducing the risk of transpiration water loss caused by direct transplanting. At the same time, the mixed substrate has both water retention and fertilizer retention properties as well as air permeability, preventing root rot caused by water accumulation. This helps seedlings smoothly transition to a natural growth state and ensures normal plant growth after transplanting. Attached Figure Description

[0019] Figure 1 This is a flowchart of the present invention. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] It should be noted that the method described in this invention is applicable to the genus Rhododendron ( Rhododendron spp. Tissue culture of various plants. Those skilled in the art will understand that the following embodiments are merely preferred examples of the present invention and are not intended to limit the scope of protection of the present invention. The scope of protection of the present invention should be determined by the scope defined in the claims. Please see the appendix Figure 1 This invention provides a method for rapid propagation of rhododendrons through tissue culture, specifically including: I. Preparation of Rhododendron Explants and Culture Media 1. Explant preparation: Select healthy rhododendron plants free from pests and diseases, and cut semi-lignified young shoots from the current year as explant materials.

[0022] The technical basis for selecting this type of tender shoot is that semi-lignified tender shoots have strong cell division ability and high differentiation potential, and the degree of lignification of the stem segments is moderate, which can reduce the risk of cell damage during disinfection and reduce the probability of browning in subsequent culture.

[0023] Explant treatment includes the following steps: 1.1 Preliminary cleaning: Rinse the tender shoots under running water (tap water, water pressure 0.1-0.2MPa) for 30 minutes. The purpose is to remove the floating dust, mud and sand and some surface microorganisms attached to the surface of the tender shoots, so as to reduce the pressure of subsequent disinfection.

[0024] 1.2 Surface dewaxing: After rinsing, transfer the tender shoots to a sterile water system containing 0.1% (v / v) Tween-20 and place them on a shaker for 10 minutes of shaking and washing. Tween-20, as a nonionic surfactant, can reduce the interfacial tension between water and the surface of the tender shoots, destroy the waxy layer structure of the tender shoot epidermis, and allow the subsequent disinfectant to fully contact the epidermal cells, thereby improving the disinfection effect. The shaking rate is controlled at 120-150 rpm, which can ensure the uniformity of washing and avoid the tender shoot stems from breaking due to excessive speed.

[0025] 1.3 Ethanol disinfection: After shaking, transfer the tender shoots to a clean bench, pick them up with sterile tweezers and immerse them in 75% (v / v) ethanol for 30 seconds. Ethanol can quickly penetrate the cell membrane of microorganisms, denature proteins, and thus kill bacteria, fungal spores and other microorganisms on the surface of the tender shoots. The reason for limiting it to 30 seconds is that ethanol is also toxic to plant cells. If the soaking is too long, it will cause the epidermal cells of the tender shoots to dehydrate and die, affecting subsequent differentiation.

[0026] 1.4 Sodium hypochlorite disinfection: After ethanol disinfection, immediately rinse the tender shoots three times with sterile water (each rinse lasting no less than 1 minute) to remove residual ethanol; then transfer the tender shoots to a sodium hypochlorite solution containing 0.1% (w / v) available chlorine and stir continuously for 8 minutes. Sodium hypochlorite generates hypochlorous acid in the aqueous solution. Hypochlorous acid has strong oxidizing properties and can penetrate deep into the epidermal crevices to kill microorganisms, compensating for the insufficient depth of ethanol disinfection; stirring ensures that the sodium hypochlorite solution is in full contact with the surface of the tender shoots, avoiding incomplete local disinfection.

[0027] 1.5. Sterile water rinsing: After sodium hypochlorite treatment, rinse the tender shoots with sterile water 5-6 times (each rinse lasting 1-2 minutes) to completely remove residual sodium hypochlorite. If sodium hypochlorite remains, it will slowly release chlorine gas during subsequent culture, inhibiting plant cell metabolism and causing explant yellowing.

[0028] 1.6 Stem segment cutting: Use a sterile scalpel to cut the disinfected tender shoots into stem segments containing 1-2 axillary buds. The length of the stem segments should be controlled to 1.5cm. Retaining 1-2 axillary buds is because axillary buds are the core part of bud differentiation and can be directly used as the differentiation starting point, shortening the culture cycle. The length of 1.5cm can ensure that the stem segments have sufficient nutrient reserves and avoid the inconvenience of inoculation operation and nutrient competition in the culture medium caused by excessive length.

[0029] 2. Culture medium preparation 2.1 Preparation of proliferation culture medium The modified WoodyPlantMedium was used as the base medium. The technical basis for selecting this medium is that its nitrogen, phosphorus and potassium concentrations are suitable for the growth needs of woody plants, and its ammonium nitrate and potassium dihydrogen phosphate contents are lower than those of MS medium, which can avoid physiological stress caused by high concentrations of inorganic salts in acid-loving plants such as azaleas.

[0030] The improved and added components are as follows: Carbon source: Add 30g / L sucrose. Sucrose not only provides energy for plant cells, but also regulates the osmotic pressure of the culture medium (osmotic pressure controlled at 0.4-0.5MPa). This osmotic pressure range matches the osmotic pressure of the rhododendron cell protoplast, preventing cell dehydration or water absorption and rupture.

[0031] Solidifying agent: Add 6 g / L agar. Agar dissolves at 100℃ and solidifies below 40℃, forming a stable solid matrix to support explant growth. The addition of 6 g / L ensures that the medium has moderate hardness, which facilitates explant insertion without affecting root penetration (in the subsequent rooting stage).

[0032] Growth regulators: Add 2.0 mg / L zeatin and 0.1 mg / L indolebutyric acid. Zeatin is a cytokinin that can promote axillary bud germination and bud cluster differentiation. A concentration of 2.0 mg / L can effectively induce bud primordia formation and avoid the result of dense bud clusters and weak growth due to excessive concentration. Indolebutyric acid is an auxin that works synergistically with zeatin to promote cell elongation and improve bud cluster vigor. A concentration of 0.1 mg / L can avoid excessive callus formation caused by excessive auxin.

[0033] pH adjustment: Adjust the pH of the culture medium to 5.8 using 0.1 mol / L hydrochloric acid solution or 0.1 mol / L sodium hydroxide solution. Azalea roots are suitable for growth in a slightly acidic environment. This pH value can ensure the solubility of nutrients (such as trace elements such as iron and manganese) in the culture medium, avoid the precipitation of trace elements caused by excessively high pH, ​​and at the same time maintain the activity of plant cell enzymes (such as cellulase and pectinase) within a suitable range.

[0034] This proliferation culture medium does not contain antioxidants such as ascorbic acid, citric acid, or polyvinylpyrrolidone. The technical basis for this is that although such antioxidants can temporarily inhibit phenolic oxidation, they will compete with the adsorbent material in the subsequent dynamic purification device to bind phenolic substances, reducing purification efficiency. Furthermore, this invention can continuously remove phenolic metabolites through dynamic purification without relying on antioxidants.

[0035] 2.2 Preparation of Rooting Culture Medium Using 1 / 2 MS medium as the basal medium, the nutrient concentration of MS medium is relatively high. Diluting it by 1 / 2 can reduce the stress of inorganic salts on the roots and meet the needs of rhododendron adventitious root induction.

[0036] The following components were added: Carbon source: Adding 20g / L sucrose gradually enhances the photosynthetic capacity of plants during the rooting stage, reducing their demand for carbon sources. 20g / L sucrose can reduce the risk of microbial contamination in the culture medium and avoid excessive root growth caused by excessive carbon sources.

[0037] Solidifying agent: Add 6 g / L agar, with the same function as the proliferation medium.

[0038] Growth regulator: Add 1.0 mg / L indolebutyric acid. Indolebutyric acid can promote the formation of adventitious root primordia. A concentration of 1.0 mg / L can effectively induce root differentiation and avoid rooting delay caused by too low a concentration or root malformation caused by too high a concentration.

[0039] pH adjustment: Same as the proliferation medium, adjust to 5.8.

[0040] II. Prepare and sterilize the dynamic purification culture device. 2.1 Device Structure and Assembly The core of the dynamic purification and cultivation device consists of four parts: the container body, the isolation structure, the electrode assembly, and the adsorption material. The design and principle of each part are as follows: Container body: Made of medical-grade polycarbonate through injection molding into a double-layer concentric columnar structure. The outer column has a diameter of 10-12cm and a height of 15-18cm; the inner column (growth zone) has a diameter of 5-6cm and the same height as the outer layer; a ring-shaped sandwich (purification zone) is formed between the two columns, with a width of 2-3cm. The selection criteria for medical-grade polycarbonate are: the material has a light transmittance of ≥90%, which can ensure the light penetration rate during cultivation and meet the needs of plant photosynthesis; at the same time, it can withstand 121℃ high-pressure steam sterilization, has strong chemical stability, and does not react with culture medium components or metabolites. The purpose of the double-layer concentric columnar structure design is to maximize the effective contact area of ​​the isolation structure, so that the metabolites generated in the growth zone can quickly diffuse to the surface of the isolation structure, thereby improving the purification efficiency.

[0041] Isolation Structure: A hydrophilic modified polyethersulfone membrane is used, with a membrane thickness of 100-120 μm and a molecular weight cutoff of 500-1000 Daltons. The hydrophilic modification enhances the membrane's permeability to water and water-soluble metabolites, preventing metabolite adhesion and blockage caused by hydrophobic membranes. The molecular weight cutoff is determined based on the fact that the main harmful metabolites produced in rhododendron tissue culture are phenolic substances (such as chlorogenic acid and caffeic acid, with a molecular weight of 200-400 Da). This cutoff ensures that phenolic substances can pass freely. However, sucrose (molecular weight 342 Da, hydration radius of about 0.5 nm), EDTA-Fe (chelate structure, hydration radius of about 0.8 nm), and growth regulators (such as zeatin, with a molecular weight exceeding 1000 Da after binding with proteins in the culture medium) in the culture medium cannot pass through the membrane structure due to their molecular size or binding state, thus achieving selective separation of metabolite removal and nutrient retention.

[0042] Installation steps of the isolation structure: Cut the polyethersulfone membrane into a circle that matches the cross-section of the container body (diameter slightly larger than the outer diameter of the inner column). First, immerse the membrane in 75% (v / v) ethanol for 10 minutes to remove residual impurities from the membrane surface. Then rinse three times with sterile water to remove the ethanol. Lay the membrane flat on the step between the inner and outer columns and press it together using the upper and lower flanges (PTFE material). Place a silicone sealing ring (hardness 50-60 Shore A) between the flange and the membrane. Tighten the flange bolts (torque...). ), ensuring that the exchange of matter between the growth zone and the purification zone is achieved only through the membrane, with no leakage. 3. Electrode assembly: Both the cathode and anode are made of platinum-iridium alloy wire (90% platinum by mass and 10% iridium by mass), with a diameter of 0.5mm.

[0043] The platinum-iridium alloy was chosen because: it is chemically inert and does not oxidize or corrode in the slightly acidic environment of the culture medium or during sterilization, and no metal ions are released, thus avoiding toxicity to plant cells; at the same time, it has stable electrical conductivity and can maintain a uniform electric field strength.

[0044] Electrode installation method: A platinum-iridium alloy wire is wound into a ring (5-6cm in diameter) that matches the inner wall of the inner column and serves as the cathode. It is fixed to the inner wall of the inner column using a polytetrafluoroethylene bracket, with a distance of 1-2mm between it and the surface of the isolation structure. Another platinum-iridium alloy wire is wound into a ring (10-12cm in diameter) that matches the outer wall of the outer column and serves as the anode. It is fixed to the outer wall of the outer column, with a distance of 1-2mm between it and the surface of the isolation structure. The electrode leads are led out through the sealed interface (silicone material) at the top of the container body. The interface is sealed with an interference fit to prevent steam from entering during sterilization or contamination during cultivation.

[0045] Adsorption materials: Amino-functionalized mesoporous silica microspheres or immobilized polyphenol oxidase microspheres were selected. The preparation and working principle of the two materials are as follows: Amino-functionalized mesoporous silica microspheres: Spherical mesoporous silica with a particle size of 50-100 μm was placed in toluene solution, and 3-aminopropyltriethoxysilane was added. The mixture was refluxed at 80 °C for 6 hours to graft amino groups onto the surface of the silica microspheres. After the reaction, the microspheres were separated by centrifugation, washed three times with anhydrous ethanol, and vacuum dried at 60 °C for 4 hours.

[0046] The adsorption principle of this material is as follows: amino groups form hydrogen bonds with the phenolic hydroxyl groups of phenolic substances, resulting in strong binding forces (binding constant). It can efficiently adsorb phenolic substances through the isolation structure; the mesoporous structure (pore size 5-10nm) can provide a large specific surface area (≥800m² / g), which can improve the adsorption capacity (each gram of material can adsorb 50-80mg of phenolic substances).

[0047] Immobilized polyphenol oxidase microspheres: Take activated agarose gel microspheres (particle size 50-100 μm), add purified polyphenol oxidase solution (enzyme activity 1000 U / mL), and incubate with shaking at 30℃ for 2 hours; then add glutaraldehyde solution (concentration 2%), and continue incubation for 1 hour to covalently immobilize polyphenol oxidase on the surface of agarose gel microspheres through cross-linking with glutaraldehyde; after immobilization, wash 3 times with phosphate buffer (pH 6.0) to remove unimmobilized enzyme.

[0048] The working principle of this material is as follows: polyphenol oxidase can catalyze the oxidation of phenolic substances into quinone substances, and the quinone substances can further polymerize to form insoluble macromolecules (molecular weight exceeding 10,000 Da), which are precipitated in the purification zone to prevent the secondary diffusion of phenolic substances back to the growth zone; the immobilization treatment can keep the enzyme active (enzyme activity retention rate ≥80%), while preventing the enzyme from dissolving and entering the growth zone and affecting plant cells.

[0049] Filling the adsorbent material: Mix any of the above adsorbent materials with organic-free phosphate buffer (pH 6.0, concentration 0.05 mol / L) at a mass-to-volume ratio of 1:5 to prepare a suspension; inject the suspension into the purification zone, filling it to 80%-90% of the zone's volume, leaving some space to prevent overflow due to volume expansion during sterilization. The role of the phosphate buffer is to maintain pH stability in the purification zone and ensure the activity of the adsorbent material (especially the immobilized enzyme).

[0050] 2.2 Sterilization treatment The assembled dynamic purification culture device (including the adsorption material suspension), the prepared proliferation culture medium and the rooting culture medium were respectively subjected to high-pressure steam sterilization at 121℃, 0.1MPa and 20 minutes.

[0051] This condition can kill heat-resistant microorganisms such as bacterial spores and fungal spores. The 20-minute heat preservation time ensures that the inside of the device and the center of the culture medium reach the sterilization temperature, avoiding incomplete sterilization caused by cold spots. After sterilization, the device and culture medium are transferred to a clean bench and cooled to room temperature (25-28℃) for later use. During the cooling process, the device should be kept away from the outside air to prevent contamination.

[0052] III. Inoculation and Dynamic Purification Culture The specific steps are as follows: 3.1 Vaccination Procedure Inside the clean bench, open the top cover of the dynamic purification culture device, use sterile forceps to pick up the prepared explant stem segments, and vertically insert them into the proliferation culture medium in the growth zone. The insertion depth should be 1 / 3 to 1 / 2 of the stem segment length. The insertion depth should be controlled as follows: too shallow and the explant may fall over; too deep and the base may rot due to lack of oxygen. Inoculate 5 stem segments in each growth zone of the device, with the inoculation positions evenly distributed (1-2 cm apart) to avoid competition for light and nutrients between stem segments that may affect growth. Immediately after inoculation, close the cover and seal the cover with a silicone sealing ring to prevent external microorganisms from entering.

[0053] 3.2 Dynamic Purification Start-up and Parameter Control 3.21. Electric Field Establishment: Connect the cathode and anode leads of the device to an adjustable DC power supply, turn on the power supply, and apply a constant voltage of 1.5-3V. The voltage parameters are determined as follows: below 1.5V, the electric field strength (E=V / d, where d is the distance between the two electrodes, approximately 3-4cm, and the electric field strength is 0.4-0.5V / cm) is insufficient to drive the rapid migration of phenolic ions, resulting in low purification efficiency; above 3V, the electric field strength exceeds 1.0V / cm, which will cause the water molecules in the culture medium to undergo electrolysis (generating...). The generation of bubbles affects the exchange of substances. At the same time, excessively high current (above 100μA) will cause local temperature rise and damage plant cells. The current output range of the power supply is controlled between 1-100μA. Current monitoring can determine whether the adsorbent material is saturated (after adsorption saturation, phenolic substances decrease and the current drops to below 10μA).

[0054] The principle of electric field-assisted purification: In a culture medium at pH 5.8, phenolic substances (Ar-OH) partially dissociate into negatively charged phenoxy ions. Under the influence of an electric field, phenoxy ions are subjected to an electric force pointing towards the anode (F=qE, where q is the ion charge), causing them to migrate directionally to the surface of the isolation structure and enter the purification zone through the membrane. Compared to simple concentration gradient diffusion, the diffusion coefficient of concentration gradient diffusion is... An electric field can increase the migration rate of metabolites by 3-5 times.

[0055] 3.22. Cultivation environment control: Place the device for starting dynamic purification in the light cultivation room and control the cultivation temperature at 25±2℃.

[0056] This temperature range is the optimal temperature for cell division and metabolism in rhododendrons. At 25℃, cytokinin activity is the highest and the bud differentiation rate is the fastest. Temperature fluctuations should be controlled within ±2℃ to avoid cell stress responses (such as decreased enzyme activity and fluctuations in photosynthetic rate) caused by sudden temperature changes.

[0057] The lighting parameters are controlled as follows: light intensity 2000 lux, light cycle 14 hours of light and 10 hours of darkness.

[0058] The light intensity was selected based on the following criteria: Rhododendrons are semi-shade plants, and 2000 lux can meet the light requirements for photosynthesis (the light saturation point is about 2500 lux). This avoids chlorophyll degradation (photo-oxidation) caused by excessive light or insufficient photosynthetic products caused by insufficient light. The 14-hour light cycle simulates the light cycle of the natural growing season, which can promote the accumulation of photosynthetic products and provide energy for bud differentiation.

[0059] The illumination uses a white LED light source with a spectrum covering the visible light range of 400-700nm, which matches the absorption spectrum of plant photosynthesis. It has high light efficiency (≥100lm / W) and low heat generation, thus avoiding the influence of light source heat generation on the cultivation temperature.

[0060] During the cultivation process, observe the condition inside the device daily and record the growth status of the bud clusters (such as bud length and number of leaves) and the status of the adsorption material in the purification zone (such as color changes; after adsorbing phenols, silica microspheres turn light yellow, and immobilized enzyme microspheres turn brown). When the color depth of the adsorption material reaches saturation (silica microspheres are dark yellow, and enzyme microspheres are dark brown), replace the device in time (corresponding to the subculture proliferation step).

[0061] IV. Subgeneration and Proliferation When the explant stem segments in the growth zone have been cultured for 25-30 days and have formed a bud cluster with a height of 3-4 cm and 3-5 healthy buds, subculture propagation is carried out. The basis for choosing this time is that the bud cluster has the ability to grow independently at this time and the survival rate after division is high. If the culture time is too long, the bud cluster will weaken due to nutrient consumption and the adsorption material will be close to saturation, resulting in a decrease in purification efficiency.

[0062] The succession process is as follows: Inside the clean bench, open the cover of the old device and use a sterile scalpel to cut and separate the bud clusters from the base. Each separated bud cluster should retain 2-3 healthy buds. Remove yellowed and thin weak buds to prevent them from consuming nutrients. The cutting tools should be wiped and disinfected with 75% ethanol and disinfected after each cut to prevent cross-contamination.

[0063] Prepare a new dynamic purification culture device, and inoculate the divided bud clusters into the proliferation medium of the new device, with the inoculation density still being 5 bud clusters per device.

[0064] Repeat the aforementioned dynamic purification and culture steps, apply a voltage of 1.5-3V, maintain the conditions of 25±2℃, 2000 lux, and 14h / d light, and culture for 25-30 days to achieve batch proliferation of bud clusters. Each subculture can increase the number of bud clusters by 2-3 times. Through 3-4 subcultures, a large number of robust bud clusters can be obtained.

[0065] V. Rooting and Hardening Off 5.1 Rooting Culture When the subcultured bud clusters grow to 2-3 cm in height and each bud cluster has 2-3 unfolded leaves, they are then cultured for rooting. Bud clusters at this growth stage have the ability to root, and the leaves can perform photosynthesis to provide the energy required for rooting.

[0066] The rooting procedure is as follows: Inside the clean bench, a sterile scalpel is used to cut individual plants from the base of the bud cluster, removing residual culture medium and aging tissue at the base to prevent the aging tissue from rotting and affecting rooting.

[0067] Individual plants are inoculated into a dynamic purification culture device containing rooting medium (the purification zone still needs to be filled with adsorbent material to avoid browning caused by phenolic substances produced during the rooting process). The inoculation density is 8-10 plants per device, with a plant spacing of 0.5-1cm to prevent the roots from tangling together during growth.

[0068] The cultivation environment is controlled as in 3.2 (temperature 25±2℃, light 2000 lux, 14h / d light). The electric field voltage can be reduced to 1.0-1.5V (the amount of phenolic substances produced during the rooting stage is reduced, so high voltage is not required). Cultivate for 15-20 days, and end the rooting culture when 3-5 strong adventitious roots with a length of 1-2cm have formed at the base of the plant.

[0069] 5.2 Seedling hardening The purpose of hardening off seedlings is to gradually adapt tissue culture seedlings from a sterile, high-humidity, and stable environment to the external natural environment, thereby improving the transplant survival rate. The steps are as follows: Adaptation by opening the lid: Transfer the rooting culture device to a greenhouse (temperature 22-28℃, relative humidity 70%-80%), open the lid and leave it for 2-3 days to allow the seedlings to gradually adapt to changes in external humidity and light, avoiding transpiration and water loss caused by direct transplanting.

[0070] Root cleaning: Gently rinse the seedling roots with sterile water to remove any attached agar. If agar remains, it will absorb moisture and mold after transplanting, leading to root diseases. Rinse gently to avoid damaging the young roots.

[0071] Transplanting: After cleaning, transplant the seedlings into the cultivation substrate, which is a mixture of peat moss and perlite in a volume ratio of 2:1. The role of peat moss is to retain water and fertilizer (water holding capacity ≥60%) and provide the organic matter needed for seedling growth; the role of perlite is to increase the aeration of the substrate (porosity ≥40%) and prevent water accumulation in the substrate from causing root rot. The transplanting depth is 1-2cm from the base of the seedling. After transplanting, gently compact the substrate to ensure close contact between the roots and the substrate.

[0072] Humidity control: After transplanting, place the seedlings in a seedling box and maintain a relative humidity of 85%-90% in the early stage. Maintain humidity by placing a water tray in the seedling box with water to a depth of 1-2cm and using water evaporation. Observe the substrate humidity daily. If the substrate surface is dry, spray sterile water with a sprayer to keep the substrate moist but not waterlogged.

[0073] Gradually reduce humidity: Start gradually reducing humidity 3 days after transplanting, decreasing the relative humidity by 5%-10% each day. Specifically, open 1 / 5 of the ventilation opening in the seedling box on the first day, 1 / 3 on the second day, 1 / 2 on the third day, and fully open the seedling box on the fifth day. By the seventh day, the relative humidity should be reduced to 60%-65%. At this time, the seedlings have formed a complete cuticle and transpiration is stable.

[0074] Post-planting management: After the humidity is reduced, transfer the seedlings to the greenhouse for regular management, control the temperature at 22-28℃, and the light intensity at 3000-4000 lux (gradually increase to natural light). Water and fertilize regularly (use NPK compound fertilizer diluted 1000 times once a month). After 30-40 days of cultivation, when the seedlings are growing vigorously, they can be transplanted out of the nursery.

[0075] (vi) Effect verification experiment To verify the effectiveness of the method of this invention in solving the problem of browning caused by metabolite accumulation and affecting seedling quality in rhododendron tissue culture, a comparative experiment was conducted between an experimental group (using the method of this invention) and a control group (using existing traditional tissue culture methods). The experimental design and results are as follows: Experimental materials and grouping: 200 semi-lignified young shoots of the Belgian rhododendron variety from the same batch and with consistent growth status were selected and randomly divided into two groups of 100 shoots each. Experimental group: The specific operation strictly follows the steps (I) to (V) of this implementation method.

[0076] Control group: The existing traditional rhododendron tissue culture method was used, namely single-chamber glass culture bottle (without dynamic purification structure) + modified WPM proliferation medium with 0.1 g / L ascorbic acid added + the same explant disinfection steps as the experimental group + routine hardening-off (direct transplanting to an environment with 70% humidity). The culture environment (temperature 25±2℃, light intensity 2000 lux, photoperiod 14h / d) was completely the same as the experimental group, with the only difference being whether a dynamic purification device was used and whether antioxidants were added to the culture medium.

[0077] The experimental period was 60 days (including 30 days of proliferation culture + 20 days of rooting culture + 10 days of hardening-off). The following indicators were recorded on day 30 of proliferation culture, day 20 of rooting culture, and day 10 of hardening-off. Each group was repeated three times, and the average value was taken: Browning rate: On day 30 of the propagation culture, the proportion of explants or bud clusters that showed browning (browning at the base of the stem segment or the edge of the leaf) was counted. The calculation formula was: number of browned stem segments / total number of inoculated stem segments × 100%.

[0078] Explant survival rate: On day 30 of proliferation culture, the proportion of stem segments that did not turn brown and could differentiate normally to form bud clusters was counted. The calculation formula was: number of surviving stem segments / total number of inoculated stem segments × 100%.

[0079] Proliferation coefficient: On the 30th day of proliferation culture, the average number of robust buds (height ≥ 0.5 cm) differentiated from each surviving stem segment in each group was counted.

[0080] Rooting rate: On the 20th day of rooting culture, the proportion of plants that can form 3 or more adventitious roots with a length of ≥1cm is counted. The calculation formula is: number of rooted plants / total number of transplanted plants × 100%.

[0081] Experimental Results and Analysis (1) Browning rate: The browning rate of the experimental group was 8.2%, while that of the control group was 45.6%. The reason for the difference is that the experimental group continuously removed small molecule metabolites such as phenols from the growth zone through a dynamic purification culture device, without relying on ascorbic acid; the single-chamber culture flask of the control group could not remove metabolites, and ascorbic acid could only temporarily inhibit the oxidation of phenols, which would still accumulate and cause browning as the culture time was extended.

[0082] (2) Explant survival rate: The survival rate of the experimental group was 91.5%, while that of the control group was 54.3%. The difference was due to the fact that the experimental group did not accumulate metabolites that inhibited cell metabolism, and the explants could differentiate normally to form shoot clusters; the control group was browned, which led to the death of a large number of explants and significantly reduced the survival rate.

[0083] (3) Proliferation coefficient: The proliferation coefficient of the experimental group was 4.2, while that of the control group was 2.1. The reason for the difference is that the stable culture environment (no metabolite stress) of the experimental group can promote the healthy differentiation of bud clusters, while the browning and metabolite inhibition of the control group resulted in fewer bud clusters and weaker growth.

[0084] (4) Rooting rate: The rooting rate of the experimental group was 89.7%, while that of the control group was 58.2%. The reason for the difference is that the bud clusters formed in the proliferation stage of the experimental group were robust, and the accumulation of metabolites was avoided through dynamic purification during the rooting culture, which was conducive to the formation of adventitious roots; the proportion of weak buds in the control group was high, and the residual metabolites during the rooting process inhibited root differentiation.

[0085]

[0086] The above results demonstrate that the present invention, through the design of a dynamic purification culture device, can effectively solve the problems of metabolite accumulation leading to browning and affecting seedling quality in existing traditional methods, and significantly improve the stability of rhododendron tissue culture and seedling quality.

[0087] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for rapid propagation of rhododendron tissue culture, characterized by, The method comprises the following steps: S1, preparing rhododendron flower explants and culture medium: selecting healthy tender shoots of rhododendron flower, cutting the shoots into stem segments containing axillary buds after washing and disinfecting; preparing proliferation culture medium and rooting culture medium suitable for rhododendron flower tissue culture; S2, preparing a dynamic purification culture device and sterilizing: the dynamic purification culture device comprises a growth zone for culturing explants and a purification zone for processing metabolites, and an isolation structure allowing small molecule metabolites to pass between the growth zone and the purification zone; sterilizing the device, the proliferation culture medium and the adsorption material filled in the purification zone; S3, inoculation and dynamic purification culture: inoculating the stem segments into the proliferation culture medium in the growth zone in a sterile environment; starting dynamic purification, so that the small molecule metabolites generated in the growth zone enter the purification zone through the isolation structure and are adsorbed by the adsorption material; at the same time, placing the device in a culture environment with a temperature of 25±2℃, a light intensity of 2000 lux and a light cycle of 14h / d; S4, subculture and proliferation: after the stem segments in the growth zone form a bud cluster, dividing the bud cluster and transferring it to a new dynamic purification culture device, repeating the dynamic purification culture step S3; S5, rooting and seedling raising: after the plant grows to 2-3 cm, transferring it to the rooting culture medium for culture until rooting; washing the roots of the rooted plant and transplanting it to a cultivation substrate, maintaining high humidity in the early stage, and gradually reducing the humidity to adapt the plant to the natural environment, to obtain rhododendron tissue culture seedlings.

2. The method for rapid propagation of rhododendrons by tissue culture according to claim 1, characterized in that, The growth zone and the purification zone of the dynamic purification culture device in step S2 are independent chambers with a double-layer concentric columnar structure; the isolation structure is a hydrophilic modified polyether sulfone membrane with a molecular weight cut-off value of 500-1000 daltons; the inner wall of the growth zone is provided with a cathode, and the outer wall of the container outside the purification zone is provided with an anode, the cathode and the anode are connected to a power supply and a constant voltage of 1.5-3V is applied, forming an electric field pointing from the growth zone to the purification zone.

3. The method for rapid propagation of rhododendrons by tissue culture according to claim 2, characterized in that, Both the cathode and the anode are platinum-iridium alloy wires with a diameter of 0.5mm, in which the mass percentage of platinum is 90% and the mass percentage of iridium is 10%; the power supply is an adjustable direct current power supply with a voltage output range of 0-5V and a current output range of 1-100μA.

4. The method for rapid propagation of rhododendrons by tissue culture according to claim 2, characterized in that, The adsorption material is amino-functionalized mesoporous silica gel microspheres or immobilized polyphenol oxidase microspheres; the particle size of the amino-functionalized mesoporous silica gel microspheres is 50-100μm; the immobilized polyphenol oxidase microspheres are prepared by fixing polyphenol oxidase on activated carrier microspheres by cross-linking method.

5. The method for rapid propagation of rhododendrons by tissue culture according to claim 1, characterized in that, The preparation process of the rhododendron flower explants in step S1 is as follows: selecting current-year semi-lignified tender shoots, sequentially washing with tap water for 30 minutes, oscillating washing with sterile water containing 0.1%(v / v) Tween-20 for 10 minutes, soaking in 75%(v / v) ethanol for 30 seconds, washing with sterile water for 3 times, treating with 0.1%(w / v) effective chlorine sodium hypochlorite solution for 8 minutes, and washing with sterile water for 5-6 times; the cut stem segments contain 1-2 axillary buds with a length of 1.5cm.

6. The method for rapid propagation of rhododendrons by tissue culture according to claim 1, characterized in that, The proliferation medium in step S1 is modified Woody Plant Medium, with 30 g / L sucrose, 6 g / L agar, 2.0 mg / L zeatin and 0.1 mg / L indole butyric acid; the rooting medium is 1 / 2MS medium, with 20 g / L sucrose, 6 g / L agar and 1.0 mg / L indole butyric acid; the pH of the two media is adjusted to 5.

8.

7. The method for rapid propagation of rhododendrons by tissue culture according to claim 1, characterized in that, The sterilization in step S2 is high-pressure steam sterilization, with the condition of 121 ℃ for 20 minutes; 5 stem segments are inoculated in the growth area of each dynamic purification culture device; the adsorption material is mixed with organic-free phosphate buffer solution into a suspension before sterilization.

8. The method for rapid propagation of rhododendrons by tissue culture according to claim 1, characterized in that, The illumination of the culture environment in step S3 is white LED light source; in step S5, the divided bud cluster contains 2-3 robust buds, the structure of the new device and the sterilization method are consistent with those in step S2.

9. The method for rapid propagation of rhododendrons by tissue culture according to claim 1, characterized in that, The cultivation substrate in step S5 is peat soil mixed with perlite at a volume ratio of 2:1; the relative humidity of the initial high-humidity environment is 85%-90%, which is maintained by a water tray in the seedling box; the step-by-step humidity reduction mode is to reduce the relative humidity by 5%-10% per day until 60%-65%, which lasts for 5-7 days.

10. The method for rapid propagation of rhododendrons by tissue culture according to claim 6, characterized in that, The pH adjustment of the medium adopts 0.1 mol / L hydrochloric acid solution or 0.1 mol / L sodium hydroxide solution.

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