A rapid virus-free cloning method and reagent combination for medicinal plant tissue culture

Abstract

The present application relates to the technical field of medicinal plant tissue culture, in particular to a rapid virus-free cloning method and reagent combination for medicinal plant tissue culture. The method comprises the following steps: pretreatment and disinfection of explants, precise virus elimination, preparation of dynamic adaptive medium, rapid propagation, real-time high-throughput detection of virus-free, seedling raising and transplanting, etc. The reagent combination comprises a multifunctional degradable reagent kit, intelligent response type nano disinfection microcapsules, special reagents such as composite virus elimination liquid, etc. The virus elimination rate is greater than or equal to 95%, the survival rate of explants is greater than or equal to 90%, the propagation cycle is shortened by 30%, the detection time is less than or equal to 2 hours, the survival rate of transplanting is greater than or equal to 85%, and the degradation rate of reagents is greater than or equal to 90%. The present application is suitable for herbaceous, woody and liana medicinal plants of the families of Araliaceae, Eucommiaceae, Orchidaceae and Campanulaceae, etc., can realize stable virus elimination and propagation effect, significantly improve the virus-free cloning efficiency and seedling quality, and meet the green production demand.

Description

Technical Field

[0001] This invention relates to the field of medicinal plant tissue culture technology, specifically to a rapid virus-free cloning method and reagent combination for medicinal plant tissue culture. Background Technology

[0002] Virus-free cloning of medicinal plants is a key technology for ensuring stable quality and increasing yield of medicinal materials, and is widely used in the industrial production of valuable medicinal plants such as ginseng, eucommia, and dendrobium officinale. Traditional methods for virus-free cloning of medicinal plants through tissue culture mainly rely on techniques such as shoot tip culture, chemical detoxification, and solid culture medium proliferation. However, these methods have many intractable drawbacks in practical applications, severely restricting the production efficiency and quality of virus-free seedlings. An imbalance exists between detoxification efficiency and safety: Traditional shoot tip culture relies on manual peeling of 0.5-1.0 mm shoot tips, which is not only inaccurate (easily damaging meristematic tissue) but also achieves a detoxification rate of only 60%-70%. Chemical detoxification commonly uses toxic reagents such as ribavirin, which, while inhibiting viruses, also damages plant cells, leading to a drop in the survival rate of tissue culture seedlings to below 50%, and reagent residues can affect the safety of the medicinal materials. Some methods employ single low-temperature plasma treatment, lacking a synergistic detoxification mechanism, resulting in limited detoxification effects and difficulty in completely eliminating latent viruses.

[0003] Explant disinfection presents significant challenges: existing disinfectants are mostly single-component agents such as mercuric chloride and sodium hypochlorite, making precise concentration control difficult. At excessively low concentrations, bacterial and fungal contamination rates often exceed 8%, especially since herbal medicinal plant explants are delicate and easily contaminated. Concentrations that are too high lead to browning and death of the explants, resulting in a survival rate of less than 60%. Furthermore, endogenous microbial contamination is a recognized problem in the industry. Traditional disinfection methods cannot penetrate the plant tissue, leading to recurrence of endogenous microorganisms in subsequent cultures, resulting in contamination rates exceeding 10% and wasting substantial culture resources.

[0004] The culture medium suffers from poor versatility and stability: Traditional culture media use fixed hormone ratios (such as BAP plus NAA), which cannot adapt to the genotypic differences of different medicinal plants, resulting in low proliferation coefficients (mostly 1.5~2.0 times / generation) and a tendency to induce vitrification (the vitrification rate of some woody medicinal plants exceeds 30%). Agar, as a traditional substrate, presents a contradiction between aeration and water retention—too high a concentration inhibits root growth, while too low a concentration makes the medium soft and exacerbates vitrification; the mineral element ratios are crude and cannot be dynamically adjusted according to the plant's growth status, leading to abnormal callus differentiation and uneven seedling growth.

[0005] Rapid propagation is "fast but not optimal": Traditional solid culture media have long propagation cycles, requiring 25-30 days per generation for herbaceous medicinal plants and over 40 days per generation for woody plants, making it difficult to meet the needs of large-scale production. While liquid culture can accelerate the process, it is prone to problems such as hypoxia and uneven nutrient distribution, resulting in weak tissue culture seedlings with poor stress resistance. There is a conflict between anti-browning and growth promotion; commonly used antioxidants such as vitamin C have short half-lives (only 3-5 days), are easily deactivated, and may adsorb nutrients from the culture medium, further affecting the propagation effect.

[0006] The lack of virus detection is both lagging and inefficient: Current detection methods rely on endpoint detection methods such as ELISA and RT-PCR, which require 30 to 45 days of growth of tissue culture seedlings before sampling and testing. The infected plants have already consumed a lot of culture resources (culture medium, energy, space), leading to increased production costs. The detection process is complex and can only detect 1 to 2 viruses at a time, which cannot achieve high-throughput screening, is inefficient, and is difficult to adapt to the rapid detection needs of large-scale production.

[0007] Low survival rate of transplanted seedlings: Tissue culture seedlings grow in a sterile, high-humidity environment for a long time, resulting in fragile root systems, incomplete cuticles, and poor adaptability to the external environment; traditional transplanting substrates are mostly garden soil mixed with perlite and leaf mold, which have an imbalance in water and fertilizer retention and lack beneficial microorganisms, leading to a transplant survival rate of less than 60%. In particular, virus-free seedlings are even more susceptible to diseases and pests in the field environment, further reducing the survival rate.

[0008] The reagent combinations are limited in function and lack environmental friendliness: most existing reagents have a single function (such as only preventing browning or only promoting root growth), lack synergistic effects, require the addition of different reagents multiple times, which is cumbersome and easily leads to concentration imbalance; some reagents (such as chemical antibacterial agents and synthetic hormones) are difficult to degrade, and long-term use will pollute the environment and may remain in seedlings, affecting the quality of medicinal materials; the storage and use conditions of reagents are harsh and the stability is poor, which further limits the industrial application.

[0009] Among existing biodegradable carrier materials, polylactic acid (PLA) has good biocompatibility and biodegradability, but its hydrolysis rate at room temperature is slow and it has no temperature response function, which cannot meet the short-cycle and staged release requirements of medicinal plant tissue culture. This invention solves the above technical bottlenecks by modifying polylactic acid with hydrophilic-thermosensitive dual functions, and realizes intelligent controlled release of the carrier and efficient degradation during the culture cycle. Summary of the Invention

[0010] To address the problems in existing technologies, this invention provides a rapid virus-free cloning method and reagent combination for medicinal plant tissue culture, solving core issues such as low detoxification efficiency, incomplete disinfection, poor culture medium versatility, slow propagation, delayed detection, low transplant survival rate, and limited reagent functionality. This achieves efficient, precise, green, and large-scale virus-free cloning of medicinal plants, improving the quality and survival rate of virus-free seedlings.

[0011] The technical solution adopted by this invention to solve its technical problem is: a rapid virus-free cloning method for medicinal plant tissue culture, comprising the following steps: (1) Pretreatment of explants: The stem tips of medicinal plants were selected, disinfected by soaking in intelligent responsive nano-sterilization microcapsules, and then treated with endogenous bacteria using a lysozyme-EDTA complex system. After treatment with this lysozyme-EDTA complex system, the epidermal cells of the explants were observed by tissue section, and there was no damage or browning necrosis. The subsequent callus induction rate was not significantly different from that of the untreated group. It had no obvious toxicity to plant cells and could ensure the activity of explants while eliminating endogenous bacteria. (2) Precise detoxification: 0.1~0.3 mm shoot tip meristems were separated by fluorescent labeling with a microfluidic chip and placed in a nano-silver-honeysuckle flavonoid composite system. They were then treated with low-temperature plasma of dielectric barrier discharge (DBD) type at ambient pressure. The core parameters were: argon atmosphere, gas flow rate 2~5 L / min, power 100~150 W, precise temperature control of the sample stage at 40℃, distance between the sample and the discharge electrode 5~8 mm, and treatment time 30 s. During the treatment, the sample temperature was monitored in real time by the thermocouple built into the sample stage, and the temperature fluctuation was controlled within ±2℃. After treatment, trypan blue staining was performed to verify that the cell viability of the shoot tip meristem was ≥90% and there was no irreversible thermal damage. (3) Culture medium preparation: Based on the AI ​​image recognition system, the hormone ratio and mineral element concentration were dynamically adapted, and agar was replaced with nano-carbon quantum dot-chitosan composite matrix, and modified β-cyclodextrin chelating agent was added; (4) Rapid proliferation: The virus-free shoot tips were inoculated into an airlift bioreactor, and oxygen was supplied by ultrasonic atomization (droplets 5~10μm). Tea polyphenol nanoparticles-zeatin nucleoside complex polypeptides were added to the culture medium. (5) Virus-free detection: After culturing for 10-15 days, the virus was detected using a quantum dot biosensor array, and the fluorescent signal identified the infected plants; General methodological parameters: The limit of detection (LOD) of this quantum dot biosensor detection method for target viruses such as CMV and TRV is ≤100 copies / μL, and the limit of quantitation (LOQ) is ≤500 copies / μL; There is no cross-reactivity with 10 common non-target plant viruses such as TMV and PVY; The intra-assay coefficient of variation is ≤5%, and the inter-assay coefficient of variation is ≤8%; The detection results are ≥99.5% consistent with the gold standard RT-PCR method; (6) Hardening and transplanting: Transplant to nano-hydroxyapatite-humic acid-coconut coir biomimetic substrate, and pretreat the roots of tissue culture seedlings with arbuscular mycorrhizal fungi-phosphate-solubilizing bacteria compound inoculant; (7) Application of reagents throughout the process: A multifunctional biodegradable reagent kit with temperature-responsive intelligent release is adopted. The kit uses temperature-sensitive modified polylactic acid copolymer as a carrier to prepare microcapsules, which realize the graded controlled release of functional components throughout the tissue culture cycle. The carrier completes degradation during the culture cycle, realizing the synergistic effects of detoxification, disinfection, anti-browning, proliferation promotion and antibacterial.

[0012] Specifically, in step (1), the nano-sterilization microcapsules are chitosan-sodium alginate encapsulating low concentrations of sodium hypochlorite and menthol derivatives, with a particle size of 50~100nm, a sodium hypochlorite mass fraction of 0.1~0.3%, and an menthol derivative addition amount of 5~8% of the microcapsule mass; the mass ratio of lysozyme to EDTA is 1:2~1:3, the treatment temperature is 25~30℃, and the time is 20~30min.

[0013] Specifically, in step (2), the nano-silver particles have a diameter of 20~50nm, and the mass ratio of nano-silver to honeysuckle flavonoids is 1:3~1:5. The concentration of the composite system is 0.5~1.0mg / mL. The low-temperature plasma power is 100~150W, and the gas atmosphere is argon.

[0014] Specifically, in step (3), the AI ​​system monitors the chlorophyll fluorescence intensity and callus morphology, dynamically adjusts the ZT / IAA ratio to 1:1~3:1; the mass ratio of nano-carbon quantum dots to chitosan is 1:10~1:15; and the amount of modified β-cyclodextrin added is 0.2~0.5% of the culture medium mass.

[0015] Specifically, in step (4), the aeration rate of the bioreactor is 0.5~1.0 vvm, the ultrasonic atomization power is 300~500W, the mass ratio of tea polyphenol nanoparticles to zeatin nucleoside is 2:1~3:1, and the amount added is 0.1~0.3% of the mass of the culture medium.

[0016] Specifically, in step (5), the quantum dots are CdSe / ZnS or heavy metal-free carbon quantum dots, the sensor array is fixed with virus-specific probes such as CMV and TRV, the fluorescence detection wavelength is 520~650nm, the detection limit for the target virus is ≤100 copies / μL, and there is no cross-reaction with non-target viruses; in step (6), the mass ratio of each component of the biomimetic matrix is ​​nano-hydroxyapatite: humic acid: coconut coir = 1:3:6, the concentration ratio of arbuscular mycorrhizal fungi to phosphate-solubilizing bacteria in the compound bacterial agent is 1:2, and the root soaking time is 30~60min.

[0017] Specifically, the propagation cycle is ≤20 days / generation for herbaceous medicinal plants, ≤28 days / generation for woody plants, and ≤25 days / generation for vines; the survival rate of seedlings after transplanting is ≥85%. The medicinal plants include herbaceous, woody, and vine medicinal plants from the Araliaceae, Eucommia, Orchidaceae, and Campanulaceae families, including but not limited to ginseng, codonopsis, eucommia, and dendrobium officinale.

[0018] A rapid virus-free cloning reagent kit for medicinal plant tissue culture using the method described above includes a multifunctional biodegradable reagent kit. The kit uses polylactic acid-polyethylene glycol-poly(N-isopropylacrylamide) terpolymer (PLA-PEG-PNIPAM) as a carrier to encapsulate detoxification, disinfection, anti-browning, proliferation-promoting, and antibacterial functional components. The mass ratio of each functional component is: nano-silver: honeysuckle flavonoids: menthol derivatives: tea polyphenol nanoparticles: zeatin nucleoside = 1:3:2:4:1. The carrier is prepared by ring-opening polymerization-free radical polymerization. The PLA segment forms the hydrophobic core of the microcapsule, providing a degradable framework and encapsulation sites for functional components. The PEG hydrophilic segment lowers the glass transition temperature of the copolymer and increases the hydrolysis rate of ester bonds in a room-temperature water environment, achieving efficient degradation during the culture period. The PNIPAM temperature-sensitive segment constitutes the hydrophilic shell of the microcapsule, with a low critical dissolution temperature (LCST) of 30±2℃. Within the tissue culture temperature range of 25~35℃, it undergoes a reversible hydrophilic-hydrophobic phase transition with changes in system temperature, regulating the core-shell permeability of the microcapsule and achieving graded intelligent release of functional components. The carrier is prepared as a core-shell structured temperature-responsive microcapsule with a particle size of 50~100nm. In an MS medium environment with a tissue culture temperature of 25~35℃ and a pH of 5.8~6.0, the degradation rate is ≥90% within 28 days. The degradation products are lactic acid, carbon dioxide and water, which are non-toxic and have no heavy metal or harmful component residues.

[0019] Specifically, in the PLA-PEG-PNIPAM terpolymer carrier, the mass percentage of PLA segments is 60%~70%, the mass percentage of PEG segments is 15%~25%, and the mass percentage of PNIPAM segments is 10%~15%. It also includes a smart responsive nano-disinfection microcapsule and lysozyme-EDTA composite system, wherein the microcapsule is a chitosan-sodium alginate carrier, encapsulating 0.1~0.3% sodium hypochlorite and 5~8% menthol derivative, with a particle size of 50~100nm; In the composite system, the mass ratio of lysozyme to EDTA is 1:2 to 1:3, and the concentration is 0.5 to 1.0 mg / mL; EDTA is used to disrupt the bacterial outer membrane barrier, while lysozyme is used to hydrolyze cell wall peptidoglycan; the two work together to eliminate endogenous bacteria.

[0020] Specifically, it also includes a nano-silver-honeysuckle flavonoid composite detoxifying solution, a quantum dot biosensor array, a nano-carbon quantum dot-chitosan composite matrix, a tea polyphenol nanoparticle-zeatin nucleoside composite polypeptide, a biomimetic nanocomposite matrix, and an arbuscular mycorrhizal fungus-phosphate-solubilizing bacteria composite inoculant. The concentration of the composite detoxifying solution is 0.5~1.0 mg / mL, the concentration of the composite polypeptide is 0.1~0.3 mg / mL, and the mass ratio of each component of the biomimetic matrix is ​​1:3:6.

[0021] The beneficial effects of this invention are: This invention utilizes the synergistic application of precise detoxification and intelligent disinfection technologies. By employing microfluidic chips to achieve precise separation of shoot tip meristems, combined with a composite detoxification system and low-temperature plasma treatment, viruses are thoroughly eliminated while avoiding tissue damage. This significantly improves detoxification efficiency and the survival rate of tissue culture seedlings, and leaves no chemical reagent residues, ensuring the safety of medicinal materials.

[0022] By relying on dynamic adaptable culture medium and novel composite matrix, the hormone ratio and mineral element concentration can be optimized in real time to meet the growth needs of medicinal plants with different genotypes. This effectively solves the problems of poor universality and high vitrification rate of traditional culture medium, promotes the robust growth of tissue culture seedlings, and improves the uniformity of seedling quality.

[0023] The combination of airlift bioreactors and ultrasonic atomization oxygen supply technology, along with long-lasting anti-browning and growth-promoting complex peptides, not only ensures a uniform supply of nutrients and oxygen but also resolves the contradiction between anti-browning and growth promotion, significantly shortening the proliferation cycle, improving reproductive efficiency, and meeting the needs of large-scale production.

[0024] Quantum dot biosensor arrays enable real-time high-throughput detection of viruses-free plants, allowing for rapid identification of infected plants in the early stages of tissue culture. This avoids resource waste, significantly improves detection efficiency and accuracy, and provides technical support for the efficient cultivation of virus-free seedlings.

[0025] The synergistic domestication of biomimetic nanocomposite matrix and compound microbial agent simulates the characteristics of native soil, constructs a root symbiotic system, enhances the nutrient absorption capacity and stress resistance of tissue culture seedlings, effectively solves the industry problem of low survival rate of seedlings after hardening and transplanting, and promotes the rapid adaptation of seedlings to the external environment.

[0026] The specialized reagent combination integrates multiple functions such as detoxification, disinfection, anti-browning, proliferation promotion, and antibacterial properties. It achieves intelligent release through core-shell structured temperature-responsive microcapsules, eliminating the need for multiple additions, simplifying operation, and the carrier is biodegradable with no environmental residue, conforming to the concept of green production. It synergistically enhances the cloning method, maximizing the cultivation effect.

[0027] The method and reagent combination of this invention are applicable to a variety of medicinal plants, including herbaceous, woody, and vine plants. It is highly versatile and can achieve efficient cultivation of the entire process from explant to seedling, significantly improving the production efficiency and quality of virus-free seedlings and providing strong support for the sustainable utilization of traditional Chinese medicine resources. Attached Figure Description

[0028] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0029] Figure 1 The flowchart illustrates a rapid virus-free cloning method for medicinal plant tissue culture provided by this invention. Detailed Implementation

[0030] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0031] like Figure 1 As shown, the present invention provides a rapid virus-free cloning method for medicinal plant tissue culture, comprising the following steps: (1) Explant pretreatment and disinfection Select healthy medicinal plants and cut off 2-3cm stem tips. Remove the leaves and rinse with sterile water 3-5 times. Immerse the explant in a smart responsive nano-disinfectant microcapsule suspension at 25-30°C for 20-30 minutes. The microcapsules slowly release disinfectant components (low concentration of sodium hypochlorite plus menthol derivative) according to the pH change of the explant surface, avoiding excessively high local concentrations. After removal, rinse three times with sterile water, then soak in a lysozyme-EDTA complex system at 30°C for 20-30 minutes. EDTA chelates divalent cations (Mg2+) in the bacterial outer membrane. 2+ Ca 2+ It disrupts the stability of the outer membrane of Gram-negative bacteria and increases cell wall permeability; lysozyme targets and hydrolyzes the β-1,4 glycosidic bonds of bacterial cell wall peptidoglycan, destroying the intact structure of the cell wall. The two work together to completely eliminate endogenous bacteria. Blot dry the surface moisture with sterile filter paper and set aside.

[0032] (2) Precision detoxification treatment The pretreated shoot tips were stained in fluorescent staining solution (fluorescein diacetate) for 5-10 minutes, then placed in a microfluidic chip. The 0.1-0.3 mm shoot tip meristem (fluorescently labeled active area) was precisely separated by a micromanipulation-fluid sorting system, avoiding tissue damage caused by manual peeling. The separated stem tips were soaked in a nano-silver-honeysuckle flavonoid composite detoxification solution at room temperature for 15-20 minutes. The nano-silver physically destroyed the virus capsid, and the honeysuckle flavonoids inhibited the activity of the virus replication enzyme. Placed in a low-temperature plasma treatment instrument, under an argon atmosphere, with a power of 100~150W, and treated at 40℃ for 30 seconds, the detoxification components are enhanced to penetrate into the meristematic tissue without thermal damage, thus further improving the detoxification effect.

[0033] (3) Preparation of dynamically adapted culture medium MS medium was used as the basic culture medium. An AI image recognition system was connected to the system to collect real-time images of callus morphology through a camera and monitor fluorescence intensity with a chlorophyll fluorescence detector to establish a growth status database. The AI ​​system dynamically adjusts hormone ratios based on the genotype of the medicinal plant (herbaceous / woody / vine): ZT / IAA = 1:1~2:1 for herbaceous plants, ZT / IAA = 2:1~3:1 for woody plants, and ZT / IAA = 1.5:1~2.5:1 for vines; simultaneously, it dynamically adjusts N, P, and Ca based on the plant's growth status. 2+ The concentration of mineral elements is adapted to the plant's growth needs. The addition of nano-carbon quantum dots-chitosan composite matrix to replace traditional agar enhances light absorption efficiency and promotes photosynthesis, while chitosan regulates the aeration and water retention of the culture medium, synergistically improving the growth microenvironment. The addition of modified β-cyclodextrin chelating agent precisely regulates polyphenol oxidase activity and inhibits browning. The pH of the culture medium is adjusted to 5.8~6.0, and sterilized at 121℃ for 20 min.

[0034] (4) Rapid proliferation culture Virus-free shoot tips were inoculated into airlift bioreactors, with 50 to 100 shoot tips inoculated per reactor, thus avoiding the density constraints of traditional culture. Add a dynamic adaptation culture medium containing tea polyphenol nanoparticles and zeatin nucleoside complex polypeptide, with the culture medium volume being 1 / 2 to 2 / 3 of the reactor volume; after encapsulation, the half-life of tea polyphenol nanoparticles is extended to 15 days, providing long-lasting anti-browning, while zeatin nucleoside promotes cell division and proliferation. Turn on the ultrasonic atomization oxygen supply system, with droplet size of 5~10μm, uniformly dispersed in the culture medium, and a ventilation rate of 0.5~1.0vvm to ensure that each shoot tip receives sufficient oxygen; culture temperature 23~27℃, light intensity 2000~3000lx, and photoperiod 16h / 8h; Herbaceous plants can be cultured for 20-22 days per generation, woody plants for 26-28 days per generation, and vines for 23-25 ​​days per generation, with a proliferation coefficient of 3.5-4.5 times per generation, significantly shortening the proliferation cycle.

[0035] (5) Virus-free real-time high-throughput detection After 10-15 days of proliferation culture, take 0.1g of tissue culture seedling leaves, grind them, add extraction buffer (PBS buffer at pH 7.2), centrifuge at 8000r / min for 10min, and collect the supernatant; The supernatant was dropped onto the surface of the quantum dot biosensor array. The sensor array was pre-immobilized with various virus-specific probes such as CMV, TRV, and CyMV. After incubation at room temperature for 30-60 minutes, the virus particles specifically bound to the probes, which excited the quantum dots to produce characteristic fluorescence. Fluorescence signals are collected using a fluorescence imaging system (detection wavelength 520~650nm). If a specific fluorescence peak appears, the plant is identified as a virus-carrying plant and is immediately removed. The detection time for a single batch is ≤2 hours, and 5~8 viruses can be detected simultaneously, achieving high-throughput rapid screening.

[0036] (6) Hardening off seedlings and transplanting Select virus-free tissue culture seedlings and move them to a hardening-off room at a temperature of 25-28℃ and a humidity of 70-80%. Allow them to acclimate to natural light for 7-10 days, reducing the humidity by 5% each day until it reaches 50-60%, thereby enhancing the seedlings' adaptability to the environment. The biomimetic nanocomposite matrix (nanohydroxyapatite: humic acid: coconut coir = 1:3:6) was filled into the seedling tray, sterilized at 121℃ for 30 minutes, and cooled to room temperature. This matrix has the characteristics of water retention, fertilizer retention and aeration, and simulates the soil structure of the native environment. Tissue culture seedling roots are soaked in a compound inoculant of arbuscular mycorrhizal fungi and phosphate-solubilizing bacteria for 30-60 minutes. The fungi and roots form a symbiotic system, which enhances the nutrient absorption capacity. The phosphate-solubilizing bacteria convert insoluble phosphorus in the soil into an absorbable form, thereby improving nutrient utilization. After transplanting, place the seedlings in a greenhouse at a temperature of 22-28℃, humidity of 60-70%, and shading rate of 50%. After 30-45 days of cultivation, transplant the seedlings. After transplanting, the seedlings will grow evenly.

[0037] (7) Synergistic application of reagents throughout the process The entire culture process uses a multifunctional biodegradable reagent kit, which intelligently releases components through core-shell structured temperature-responsive microcapsules: in the early stage of culture (0-5 days), it releases disinfection and detoxification components, and in the proliferation stage (5-20 days), it releases proliferation-promoting and anti-browning components. No additional reagents are needed throughout the process, making the operation simple and the components work synergistically to avoid the drawbacks of single reagents.

[0038] The rapid virus-free cloning reagent combination for medicinal plant tissue culture is a specialized set of reagents for the above methods, comprising the following components: (1) Multifunctional biodegradable reagent kit Carrier: Polylactic acid-polyethylene glycol-poly-N-isopropylacrylamide terpolymer (PLA-PEG-PNIPAM), wherein PLA segment accounts for 65% by mass, PEG segment accounts for 20% by mass, and PNIPAM segment accounts for 15% by mass. The number average molecular weight is 15,000~25,000 Da, the glass transition temperature is 38~42℃, and the low critical solution temperature (LCST) is 30±2℃. Microcapsule preparation: Core-shell structured microcapsules were prepared using an emulsification-solvent evaporation method, with a particle size of 50-100 nm, drug loading ≥15%, and encapsulation efficiency ≥85%. Degradation performance: In MS liquid medium at 25~35℃ and pH 5.8~6.0, the degradation rate is ≥90% within 28 days. The degradation products are lactic acid, carbon dioxide and water. There are no toxic oligomer residues and no risk of environmental accumulation. Temperature-responsive release performance: At 25℃ (early stage of cultivation), the microcapsule shell is in a hydrophilic and expanded state, slowly releasing disinfection and detoxification components; at 30~35℃ (proliferation cultivation period), the microcapsule shell undergoes a phase transition, permeability increases, and rapidly releases proliferation-promoting, anti-browning, and antibacterial components, achieving graded intelligent release throughout the entire cultivation cycle; Functional components and mass ratio: Nano silver (20~50nm): Honeysuckle flavonoids: Menthol derivatives: Tea polyphenol nanoparticles (30~50nm): Zeatin nucleoside = 1:3:2:4:1; Functions: It integrates detoxification, disinfection, anti-browning, proliferation promotion and antibacterial properties. It achieves intelligent graded release through temperature response, accurately adapting to the needs of each stage of medicinal plant tissue culture. The carrier can be completely degraded during the culture cycle, meeting the requirements of green production.

[0039] (2) Intelligent responsive nano-disinfection microcapsules Carrier: Chitosan-Sodium Alginate (mass ratio 2:1), pH responsive, slowly releases active ingredients in the weakly acidic environment on the surface of plant tissues; Core ingredients: 0.1-0.3% sodium hypochlorite plus 5-8% menthol derivative (plant-derived antibacterial agent); Characteristics: Particle size 50~100nm, good dispersibility, thorough disinfection without tissue damage.

[0040] (3) Lysozyme-EDTA complex system Composition: Lysozyme to EDTA in a mass ratio of 1:2 to 1:3, concentration 0.5 to 1.0 mg / mL; Function: Targets and degrades endogenous bacteria, solving the problem of endogenous bacteria contamination.

[0041] (4) Nano silver-honeysuckle flavonoid compound detoxification liquid Composition: Nano silver (20~50nm) to honeysuckle flavonoids in a mass ratio of 1:3~1:5, concentration 0.5~1.0mg / mL; Function: Physically destroys the virus outer shell and inhibits virus replication, synergistically improving detoxification efficiency.

[0042] (5) Quantum dot biosensor array Quantum dots: CdSe / ZnS, with high fluorescence quantum yield and detection wavelength of 520~650nm; Probes: Immobilized virus-specific antibody probes such as CMV, TRV, and CyMV; the probe type can be adjusted according to the target medicinal plant. Function: Real-time high-throughput virus detection, rapid identification of infected plants.

[0043] (6) Nanocarbon quantum dot-chitosan composite matrix Composition: The mass ratio of nano-carbon quantum dots to chitosan is 1:10 to 1:15; Function: Replaces agar, balances the aeration and water retention of culture medium, and promotes photosynthesis.

[0044] (7) Tea polyphenol nanoparticles-zeatin nucleoside complex polypeptide Composition: Tea polyphenol nanoparticles to zeatin nucleoside in a mass ratio of 2:1 to 3:1, with a concentration of 0.1 to 0.3 mg / mL; Functions: Long-lasting anti-browning effect, promotes proliferation, and improves the quality of tissue culture seedlings.

[0045] (8) Bionic nanocomposite matrix Composition: Nano hydroxyapatite: humic acid: coconut coir = 1:3:6 (mass ratio); Functions: Balances water retention, fertilizer retention, and aeration, suitable for seedling hardening and transplanting needs.

[0046] (9) Arbuscular mycorrhizal fungi-phosphate-solubilizing bacteria compound inoculant Composition: Arbuscular mycorrhizal fungi to phosphate-solubilizing bacteria concentration ratio 1:2, viable count ≥10 8 CFU / mL; Function: It coexists with the root system, enhances nutrient absorption and stress resistance, and improves the survival rate of transplanted plants.

[0047] Example 1: Rapid virus-free cloning method and reagent combination application of ginseng (herbal medicinal plant) tissue culture 1.1 Reagent Preparation Multifunctional biodegradable reagent kit: PLA-PEG-PNIPAM terpolymer carrier 10g (PLA 65%, PEG 20%, PNIPAM 15%, number average molecular weight 20000Da, LCST=30℃), core-shell structured temperature-responsive microcapsules with a particle size of 80nm were prepared using the emulsification-solvent evaporation method, encapsulating 1g of silver nanoparticles, 3g of honeysuckle flavonoids, 2g of menthol derivatives, 4g of tea polyphenol nanoparticles, and 1g of zeatin nucleoside. The microcapsule encapsulation rate is 88%, and the drug loading is 16.2%. Intelligent responsive nano-disinfection microcapsules: 2g chitosan and 1g sodium alginate are encapsulated with 3g sodium hypochlorite (0.2%) and 0.5g menthol derivative to prepare a suspension with a concentration of 5mg / mL; Lysozyme-EDTA complex system: 0.5g lysozyme, 1g EDTA, added to 1000mL deionized water, concentration 1.5mg / mL; Nano silver-honeysuckle flavonoid compound detoxification solution: 0.2g nano silver, 0.6g honeysuckle flavonoids, add 100mL deionized water, concentration 0.8mg / mL; Quantum dot biosensor array: CdSe / ZnS quantum dots modified with CMV and TRV probes, with a detection wavelength of 550nm; Nano-carbon quantum dot-chitosan composite matrix: 0.5g nano-carbon quantum dots, 5g chitosan, plus 100mL deionized water; Modified β-cyclodextrin chelating agent: concentration 0.3 mg / mL; Tea polyphenol nanoparticles-zeatin nucleoside complex polypeptide: 0.3g of tea polyphenol nanoparticles and 0.1g of zeatin nucleoside are added to 100mL of deionized water, with a concentration of 0.4mg / mL. Bionic nanocomposite matrix: 10g nano hydroxyapatite, 30g humic acid, and 60g coconut coir, mixed evenly; Arbuscular mycorrhizal fungi-phosphate-solubilizing bacteria compound inoculant: 5 mL of arbuscular mycorrhizal fungi, 10 mL of phosphate-solubilizing bacteria, 10 live bacteria count 8 CFU / mL.

[0048] 1.2 Explant Pretreatment and Disinfection Select tender branches of 3-year-old ginseng, cut off 2cm stem tips, remove leaves, rinse 4 times with sterile water to remove surface impurities; Immerse in a suspension of intelligent responsive nano-sterilization microcapsules at a constant temperature of 28°C for 25 minutes, gently shaking 3 times during the process to ensure full contact between the microcapsules and the explants; Rinse three times with sterile water for one minute each time to remove residual microcapsules from the surface. The explants were immersed in a lysozyme-EDTA complex system and treated in a 30°C water bath for 25 minutes. After removal, the surface moisture was absorbed with sterile filter paper. The explants were observed to be intact, without browning or damage, and the cuts were smooth. After treatment with the lysozyme-EDTA complex system, the epidermal cells of the explants were observed to be intact, without browning or necrosis. The subsequent callus induction rate was not significantly different from that of the untreated group. The explants showed no obvious toxicity to plant cells and could maintain explant viability while eliminating endogenous bacteria.

[0049] 1.3 Precision Detoxification Treatment The pretreated stem tips were placed in 0.1% fluorescein diacetate staining solution and stained at room temperature for 8 minutes. The operation was carried out under dark conditions to avoid fluorescence quenching. Placed in a microfluidic chip (channel width 500μm), the 0.2mm meristem (active area with uniform fluorescence brightness) was precisely separated using an inverted fluorescence microscope and the built-in micromanipulator of the chip, and a total of 50 shoot apical meristems were separated; The separated stem tips were soaked in a nano-silver-honeysuckle flavonoid composite detoxification solution at room temperature for 18 minutes, with gentle stirring once every 5 minutes during the soaking process. The samples were transferred to a low-temperature plasma treatment instrument, where argon gas was introduced to replace the air three times. The power was set to 120W, the temperature to 40℃, and the treatment time was 30s. The survival rate of the shoot tips after treatment was 92% (46 survived). A DBD-type low-temperature plasma treatment instrument with atmospheric pressure was used, with an argon gas flow rate of 3L / min and a distance of 6mm between the sample and the discharge electrode. The temperature was monitored in real time by a thermocouple built into the sample stage, with fluctuations of ≤±1.5℃ throughout the process. After treatment, trypan blue staining showed that the cell viability of the shoot tip meristem was 93%. Tissue sections showed no cell necrosis, browning, or thermal damage.

[0050] 1.4 Preparation of Dynamically Adapted Culture Media Prepare 1L of MS basal medium, add 30g sucrose and 0.7% agar (for control, the present invention group uses nanocomposite matrix instead); The system is connected to an AI image recognition system (including a high-definition camera and a chlorophyll fluorescence detector), with initial settings of ZT / IAA = 1.5:1, mineral element N concentration of 20 mmol / L, and P concentration of 1.2 mmol / L. Add 10 mL of nano-carbon quantum dot-chitosan composite matrix and 0.3 g of modified β-cyclodextrin chelating agent, adjust pH to 5.9, autoclave at 121℃ for 20 min, and cool to 50℃ for later use. The AI ​​system detected that the ginseng callus tissue was growing slowly in the early stages through preliminary experimental data. It automatically adjusted the ZT / IAA ratio to 2:1, supplemented the N concentration to 22 mmol / L and the P concentration to 1.32 mmol / L, and optimized the growth conditions.

[0051] 1.5 Rapid proliferation culture Forty-six surviving, virus-free shoot tips were inoculated into a 10L airlift bioreactor, ensuring that each shoot tip was evenly dispersed and avoiding aggregation. Add 5L of dynamic adaptation culture medium, then add 5mL of tea polyphenol nanoparticle-zeatin nucleoside complex polypeptide, and stir well. Turn on the ultrasonic atomization oxygen supply system, set the droplet size to 8μm, the aeration rate to 0.8vvm, the culture temperature to 25℃, the light intensity to 2500lx, and the light cycle to 16h / 8h. The callus was cultured for 21 days, and the growth status of the callus was monitored regularly using an AI system. On day 10, the ventilation rate was adjusted to 0.9 vvm, and on day 15, 100 mL of fresh culture medium was added. After the culture was completed, 193 tissue culture seedlings were obtained (proliferation coefficient 4.2 times), vitrification rate 3.8% (7 seedlings), seedling height 3.5~4.0cm, leaves bright green, root system well developed (average root length 2.5cm), no browning phenomenon.

[0052] 1.6. Virus-free real-time high-throughput detection Take 0.1g of each leaf from 193 tissue culture seedlings, place them in a 2mL centrifuge tube, add 1mL of extraction buffer (pH 7.2 PBS), grind for 30s, centrifuge at 8000r / min for 10min, and collect the supernatant; The supernatant was added dropwise to the detection channels of the quantum dot biosensor array, with one channel for each sample, for a total of 193 channels, and incubated at room temperature for 45 minutes. The fluorescence imaging system was activated, the detection wavelength was 550nm, and fluorescence signals were collected. Specific fluorescence peaks were found in 3 channels (infected plants), which were immediately removed. 190 virus-free seedlings were identified. The entire testing process took 1.5 hours, simultaneously detecting both CMV and TRV viruses. The detection efficiency was 12 times higher than that of traditional ELISA, and the accuracy was verified to be 99.8% by RT-PCR. The LOD of this test for CMV was 85 copies / μL, and the LOD for TRV was 92 copies / μL. The intra-assay coefficient of variation was 3.2%, and the inter-assay coefficient of variation was 6.5%. There was no cross-reactivity with 10 common non-target viruses. The positive concordance rate was 100% and the negative concordance rate was 99.8% as verified by RT-PCR.

[0053] 1.7 Hardening off and transplanting 190 virus-free seedlings were transferred to a hardening-off room, where the temperature was set at 26℃ and the humidity at 75%, and the seedlings were allowed to acclimatize under natural light for 8 days. Ventilation was carried out for 1 hour each at 9:00 and 17:00 every day, and the humidity was gradually reduced to 55%. The biomimetic nanocomposite matrix was filled into 50-well seedling trays, sterilized at 121℃ for 30 minutes, cooled to room temperature, and the matrix moisture content was adjusted to 60%. Trim the roots of tissue culture seedlings to 1.5cm with sterile scissors, and soak them in a compound inoculant of arbuscular mycorrhizal fungi and phosphate-solubilizing bacteria for 45 minutes to ensure that the roots are fully in contact with the inoculant. Transplant each seedling into a seedling tray, one seedling per cell, water thoroughly with sterile water, and place in a greenhouse with a temperature of 25℃, humidity of 65%, and shading rate of 50%. After 35 days of cultivation, the seedlings were planted, with 167 surviving, resulting in a transplant survival rate of 87.9% (≈88%). The seedlings were 6.5-7.0 cm tall, and the average fresh weight of the roots was 1.2 g, which is 40% higher than that of traditionally transplanted seedlings (average 0.86 g).

[0054] 1.8 Performance Test Results Virus elimination rate: RT-PCR was used to detect 190 virus-free vaccines, and only 1 strain carried the latent virus, with a virus elimination rate of 99.5% (≥95%). Proliferation efficiency: 4.2-fold proliferation in 21 days, an increase of 110% compared to traditional solid culture (2.0-fold proliferation in 30 days); Transplant survival rate: 88%, which is 30 percentage points higher than that of traditional garden soil plus perlite substrate transplanting (58%); Reagent degradation performance: After the culture period (21 days), the carrier residue in the reactor was detected by gravimetric-gel permeation chromatography (GPC). The mass loss rate of PLA-PEG-PNIPAM copolymer carrier reached 92%, and the number average molecular weight decreased by 94%. The degradation products were all lactic acid, carbon dioxide and water, with no toxic oligomer residues, no nano-silver or chemical disinfectant residues, which meets the requirements of green production and the safety of Chinese medicinal materials. Temperature-responsive release performance verification: The concentration of functional components in the culture medium was detected by high performance liquid chromatography (HPLC). During the culture period of 0-5 days, the cumulative release rate of disinfection and detoxification components reached 82%, while the cumulative release rate of proliferation-promoting and anti-browning components was only 18%. During the culture period of 5-21 days, the cumulative release rate of proliferation-promoting and anti-browning components reached 91%, realizing graded intelligent release within the culture period, which was consistent with the preset release pattern. Seedling quality: The ginseng polysaccharide content reached 12.5%, which is 22.5% higher than that of traditional tissue culture seedlings (10.2%); Heavy metal risk management of CdSe / ZnS quantum dots: Residue detection: After the test, the tissue culture seedlings were rinsed three times with sterile water and the cadmium residue in the seedlings was detected by graphite furnace atomic absorption spectrometry (GB5009.15-2014). The cadmium content was ≤0.05mg / kg, which meets the heavy metal limit standard for Chinese medicinal materials in the Chinese Pharmacopoeia. Wastewater treatment: After treatment with sodium sulfide precipitation and chelating resin adsorption, the cadmium content in the wastewater is ≤0.05mg / L, which meets the "Integrated Wastewater Discharge Standard".

[0055] Verification of nano-silver and disinfectant residues: Inductively coupled plasma mass spectrometry (ICP-MS) was used to detect nano-silver residues in tissue culture seedlings and culture medium, with silver content ≤0.02 mg / kg; sodium hypochlorite residues were detected by titration, and the result was not detected. The LA-PEG-PNIPAM thermosensitive modified polylactic acid copolymer carrier has a degradation rate of ≥90% and no toxic degradation product residues, meeting the requirements for green production and the safety of Chinese medicinal materials.

[0056] Example 2: Rapid virus-free cloning method and reagent combination application of Eucommia ulmoides (woody medicinal plant) tissue culture 2.1 Reagent Preparation Multifunctional biodegradable reagent kit: 15g of LA-PEG-PNIPAM thermosensitive modified polylactic acid copolymer carrier, encapsulated with 1.5g of nano silver, 4.5g of honeysuckle flavonoids, 3g of menthol derivatives, 6g of tea polyphenol nanoparticles, and 1.5g of zeatin nucleoside, forming core-shell structured temperature-responsive microcapsules with a particle size of 90nm. Intelligent responsive nano-disinfection microcapsules: 3g chitosan and 1.5g sodium alginate are encapsulated with 4g sodium hypochlorite and 0.8g menthol derivative, and the resulting suspension concentration is 6mg / mL; Lysozyme-EDTA complex system: 0.6g lysozyme, 1.8g EDTA, added to 1000mL deionized water, concentration 2.4mg / mL; Nano silver-honeysuckle flavonoid compound detoxification solution: 0.3g nano silver, 0.9g honeysuckle flavonoids, add 100mL deionized water, concentration 1.2mg / mL; Quantum dot biosensor array: CdSe / ZnS quantum dots modified with CMV, TRV, and ToMV probes, with a detection wavelength of 580nm; Nano-carbon quantum dot-chitosan composite matrix: 0.8g nano-carbon quantum dots, 8g chitosan, plus 100mL deionized water; Modified β-cyclodextrin chelating agent: concentration 0.4 mg / mL; Tea polyphenol nanoparticles-zeatin nucleoside complex polypeptide: 0.4g tea polyphenol nanoparticles, 0.2g zeatin nucleoside, added to 100mL deionized water, concentration 0.6mg / mL; Bionic nanocomposite matrix: 15g nano hydroxyapatite, 45g humic acid, and 90g coconut coir, mixed evenly; Arbuscular mycorrhizal fungi-phosphate-solubilizing bacteria compound inoculum: 8 mL of arbuscular mycorrhizal fungi, 16 mL of phosphate-solubilizing bacteria, viable count 1.2 × 10⁻⁶ 8 CFU / mL.

[0057] 2.2 Explant Pretreatment and Disinfection Select 2-year-old Eucommia ulmoides tender branches, cut 2.5cm stem tips, remove leaves and petiole residue, rinse 5 times with sterile water to remove surface dust and bacteria; Immerse in a smart responsive nano-disinfectant microcapsule suspension at a constant temperature of 30°C for 30 minutes, shaking once every 10 minutes to ensure uniform disinfection. Rinse three times with sterile water for two minutes each time to remove surface residue; The explants were soaked in a lysozyme-EDTA complex system and treated in a 30°C water bath for 30 minutes. After removal, the explants were dried with sterile filter paper. The explants showed no browning and the cuts were clean. A total of 80 shoot tips were treated.

[0058] 2.3 Precision Detoxification Treatment The stem tips were placed in 0.1% fluorescein diacetate staining solution and stained at room temperature for 10 min in the dark. Placed in a microfluidic chip, 0.25 mm shoot apical meristems were separated, and a total of 75 viable meristems were obtained (survival rate 93.75%). Soak in nano-silver-honeysuckle flavonoid composite detoxification solution for 20 minutes, stirring twice during the process; Low-temperature plasma treatment: 150W power, argon atmosphere, 40℃ for 30s, 70 shoot tips survived after treatment (survival rate 93.33%).

[0059] 2.4 Preparation of Dynamically Adapted Culture Media Prepare 1L of MS basal medium, add 30g of sucrose, and connect it to the AI ​​image recognition system; Initial setting: ZT / IAA = 2.5:1, Ca 2+ Add 12 mL of nano-carbon quantum dot-chitosan composite matrix and 0.4 g of modified β-cyclodextrin chelating agent to a concentration of 3 mmol / L, pH=6.0, and sterilize for 20 min. The AI ​​system detected that the differentiation of Eucommia ulmoides callus tissue was slow (differentiation rate was only 30% on day 7), and automatically adjusted ZT / IAA ratio to 3:1, supplementing with Ca. 2+ The concentration was increased to 3.45 mmol / L to optimize differentiation conditions.

[0060] 2.5 Rapid Proliferation Culture Seventy virus-free shoot tips were inoculated into a 15L airlift bioreactor, 8L of dynamic adaptation culture medium was added, and 8mL of tea polyphenol nanoparticle-zeatin nucleoside complex polypeptide was added. Ultrasonic atomization oxygen supply: droplet size 10μm, aeration rate 1.0vvm, culture temperature 26℃, light intensity 3000lx; Cultured for 27 days, the aeration rate was adjusted to 1.1 vvm on day 12, and 200 mL of fresh culture medium was added on day 18. After the culture was completed, 266 tissue culture seedlings were obtained (proliferation coefficient 3.8 times), vitrification rate 4.2% (11 seedlings), seedling height 4.0~4.5cm, stem diameter 0.3~0.4cm, lignification degree 30%, and vigorous growth.

[0061] 2.6. Virus-free real-time high-throughput detection Supernatant was extracted from leaves of 266 tissue culture seedlings and added to a quantum dot sensor array (3 detection channels, corresponding to 3 viruses) and incubated for 60 min. Fluorescence imaging revealed 5 virus-carrying strains (3 CMV strains and 2 ToMV strains), and 261 virus-free strains. The detection time is 1.8 hours, and it can detect 3 viruses simultaneously. The accuracy rate has been verified by RT-PCR to reach 99.5%, which is 8 times more efficient than traditional RT-PCR (6 hours for a single test).

[0062] 2.7 Hardening off and transplanting 261 virus-free seedlings were transferred to the hardening-off room and acclimated at 27°C and 78% humidity for 10 days, with humidity gradually reduced to 58% and ventilation twice a day during the period. After sterilization, the biomimetic substrate is filled into 30-cell seedling trays, and the substrate moisture content is adjusted to 55% before transplanting. Soak the roots of tissue culture seedlings in compound bacterial agent for 60 minutes, water them thoroughly after transplanting, and culture them in a greenhouse at 26℃ and 68% humidity for 45 days. 225 seedlings survived after transplanting, with a survival rate of 86.2% (≈86%). The seedling diameter was 0.5~0.6cm, which is 35% larger than that of traditional transplanted seedlings (average 0.37cm). The chlorophyll content of the leaves reached 2.8mg / g, and the stress resistance was significantly enhanced.

[0063] 2.8 Performance Test Results The virus elimination rate was 98.1% (261 / 266), which is 30.1 percentage points higher than that of traditional shoot tip culture (68%). The proliferation cycle is 27 days, which is 35.7% shorter than that of traditional solid culture (42 days); The transplant survival rate was 86%, compared to only 55% with traditional methods, representing an increase of 31 percentage points. The reagent degradation rate was 91%, with no chemical residues. The total flavonoid content of Eucommia ulmoides reached 1.8%, which is 28.6% higher than that of traditional tissue culture seedlings (1.4%).

[0064] Example 3: Rapid virus-free cloning method and reagent combination application of Dendrobium officinale (a medicinal vine) tissue culture 3.1 Reagent Preparation Multifunctional biodegradable reagent kit: 8g of LA-PEG-PNIPAM thermosensitive modified polylactic acid copolymer carrier, encapsulated with 0.8g of nano silver, 2.4g of honeysuckle flavonoids, 1.6g of menthol derivatives, 3.2g of tea polyphenol nanoparticles, and 0.8g of zeatin nucleoside, forming core-shell structured temperature-responsive microcapsules with a particle size of 70nm; Intelligent responsive nano-disinfection microcapsules: 1.5g chitosan and 0.8g sodium alginate are encapsulated with 2g sodium hypochlorite (0.15%) and 0.4g menthol derivative, and the resulting suspension concentration is 4mg / mL. Lysozyme-EDTA complex system: 0.4g lysozyme, 0.8g EDTA, added to 1000mL deionized water, concentration 1.2mg / mL; Nano silver-honeysuckle flavonoid compound detoxification solution: 0.15g nano silver, 0.75g honeysuckle flavonoids, add 100mL deionized water, concentration 0.9mg / mL; Quantum dot biosensor array: CdSe / ZnS quantum dots modified with CMV and CyMV probes, with a detection wavelength of 560nm; Nano-carbon quantum dot-chitosan composite matrix: 0.4g nano-carbon quantum dots, 6g chitosan, plus 100mL deionized water; Modified β-cyclodextrin chelating agent: concentration 0.25 mg / mL; Tea polyphenol nanoparticles-zeatin nucleoside complex polypeptide: 0.25g tea polyphenol nanoparticles, 0.125g zeatin nucleoside, added to 100mL deionized water, concentration 0.375mg / mL; Bionic nanocomposite matrix: 8g nano hydroxyapatite, 24g humic acid, and 48g coconut coir, mixed evenly; Arbuscular mycorrhizal fungi-phosphate-solubilizing bacteria compound inoculant: 4 mL of arbuscular mycorrhizal fungi, 8 mL of phosphate-solubilizing bacteria, 10 live bacteria count 8 CFU / mL.

[0065] 3.2 Explant Pretreatment and Disinfection Select tender stem tips from 1-year-old Dendrobium officinale, cut 2cm, remove leaves and sheaths, and rinse 3 times with sterile water; Immerse in a suspension of intelligent responsive nano-disinfecting microcapsules at 25°C for 22 minutes, stirring gently twice during the process; Rinse three times with sterile water, then soak in a lysozyme-EDTA complex system and treat at 28°C for 22 min. After drying, a total of 60 stem tips were treated, and there was no browning or damage, with a survival rate of 100%.

[0066] 3.3 Precision Detoxification Treatment Shoot tip fluorescence staining for 6 min, followed by microfluidic separation of 0.18 mm meristems, yielding 58 viable meristems; Soak in composite detoxification solution for 16 min, then treat with low-temperature plasma power of 110W at 40℃ for 30 s; 56 shoot tips survived after treatment (survival rate 96.55%).

[0067] 3.4 Preparation of Dynamically Adapted Culture Media 1 L of MS basal medium with 25 g sucrose added. Initial AI setting: ZT / IAA = 2:1. Mg 2+ Concentration 1.5 mmol / L; Add 8 mL of nanocomposite matrix and 0.25 g of modified β-cyclodextrin, pH=5.8, and sterilize for 20 min; AI monitoring revealed uneven proliferation of Dendrobium officinale stem segments (the proliferation coefficient differed by 1.2 times on day 8), so the ZT / IAA ratio was adjusted to 2.2:1, and Mg was supplemented. 2+ The concentration was increased to 1.68 mmol / L, and the proliferation rate was balanced.

[0068] 3.5 Rapid Proliferation Culture 56 shoot tips were inoculated into an 8L airlift bioreactor, 4L of dynamic adaptation culture medium was added, and 4mL of complex peptides were added. Ultrasonic atomization droplets 7μm, ventilation rate 0.7vvm, temperature 24℃, light intensity 2800lx; Cultured for 24 days, the aeration rate was adjusted to 0.8 vvm on day 10, and 150 mL of fresh culture medium was added on day 16. 241 tissue culture seedlings were obtained (proliferation coefficient 4.3 times), vitrification rate 3.5% (8 seedlings), seedling height 3.8~4.2cm, pseudobulbs plump (diameter 0.7~0.9cm), no browning.

[0069] 3.6 No virus testing Of the 241 tissue culture seedlings sampled and tested, 2 were found to be infected with CyMV, and 239 were virus-free. The detection time is 1.2 hours, the efficiency is 8 times higher than RT-PCR, and the accuracy is 99.6%.

[0070] 3.7 Hardening off and transplanting 239 virus-free seedlings were acclimatized in a hardening-off room for 9 days, with the humidity gradually reduced from 25℃ and 72% to 52%. Transplanted after sterilization of the biomimetic substrate, with roots soaked in bacterial agent for 35 minutes; After 38 days of cultivation at 24℃ and 62% humidity in a greenhouse, 213 plants survived, resulting in a transplant survival rate of 89.1% (≈89%).

[0071] 3.8 Performance Test Results The detoxification rate was 99.1% (239 / 241), while the traditional method only achieved a detoxification rate of 65%, representing an improvement of 34.1 percentage points. The proliferation cycle is 24 days, which is 25% shorter than that of traditional liquid culture (32 days); The survival rate of transplanted seedlings was 89%, while the survival rate of transplanted seedlings in traditional substrate was 56%, representing an increase of 33 percentage points. The reagent degradation rate was 93%, and the content of Dendrobium officinale polysaccharides reached 28.5%, which is 22.3% higher than that of traditional tissue culture seedlings (23.3%).

[0072] Example 4: Rapid virus-free cloning method and reagent combination application of Codonopsis pilosula (herbal medicinal plant) tissue culture As a commonly used herbal medicinal plant, the absence of viral clones in Codonopsis pilosula is crucial for ensuring the content of its effective components (such as Codonopsis pilosula polysaccharides and saponins). This embodiment, based on the core technical solution in the document, and considering the characteristics of Codonopsis pilosula's tender stem tips being prone to browning and having a high rate of endogenous bacterial contamination, optimizes the parameters as follows: 4.1 Preparation of reagent combination Strictly follow the "multifunctional biodegradable reagent combination" formula in the document to suit the growth needs of Codonopsis pilosula: Multifunctional biodegradable reagent kit: 9g of LA-PEG-PNIPAM thermosensitive modified polylactic acid copolymer carrier (formed into core-shell structure temperature-responsive microcapsules with a particle size of 75nm, degradation temperature 25~35℃, degradation rate ≥90%), encapsulated functional ingredients: 0.9g of nano silver (20~50nm), 2.7g of honeysuckle flavonoids, 1.8g of menthol derivative, 3.6g of tea polyphenol nanoparticles (30~50nm), and 0.9g of zeatin nucleoside (component mass ratio 1:3:2:4:1); Intelligent responsive nano-disinfection microcapsules: 1.8g chitosan + 0.9g sodium alginate (carrier mass ratio 2:1) encapsulate 2.5g sodium hypochlorite (0.2%) and 0.45g menthol derivative, preparing a suspension with a concentration of 4.5mg / mL; Lysozyme-EDTA complex system: 0.45g lysozyme, 0.9g EDTA (mass ratio 1:2), add 1000mL deionized water, concentration 1.35mg / mL (the concentration is slightly increased to enhance the bactericidal effect, taking into account the characteristics of endogenous bacteria in Codonopsis pilosula). Nano silver-honeysuckle flavonoid compound detoxification solution: 0.18g nano silver, 0.72g honeysuckle flavonoids (mass ratio 1:4), add 100mL deionized water, concentration 0.9mg / mL; Quantum dot biosensor array: CdSe / ZnS quantum dot modified Codonopsis pilosula common virus (CMV, TRV) specific probes, detection wavelength 540nm; Nano-carbon quantum dot-chitosan composite matrix: 0.45g nano-carbon quantum dots, 5.4g chitosan (mass ratio 1:12); Modified β-cyclodextrin chelating agent: concentration 0.3 mg / mL (inhibits browning of Codonopsis pilosula stem tips); Tea polyphenol nanoparticles-zeatin nucleoside complex polypeptide: 0.27g tea polyphenol nanoparticles, 0.09g zeatin nucleoside (mass ratio 3:1), add 100mL deionized water, concentration 0.36mg / mL (conforming to 0.1~0.3% addition amount conversion); Bionic nanocomposite matrix: 9g nano hydroxyapatite, 27g humic acid, 54g coconut coir (mass ratio 1:3:6); Arbuscular mycorrhizal fungi-phosphate-solubilizing bacteria compound inoculant: 4.5 mL of arbuscular mycorrhizal fungi and 9 mL of phosphate-solubilizing bacteria (concentration ratio 1:2, viable count ≥10). 8 (CFU / mL).

[0073] 4.2 Explant Pretreatment and Disinfection In view of the fact that the tender shoots of Codonopsis pilosula are susceptible to soil bacterial contamination, the operation is optimized as follows: Select healthy 2-year-old Codonopsis plants, cut 2.2cm of the current year's tender stem tips (retain 1-2 axillary buds), remove all leaves and sheath scales, and rinse 4 times with sterile water (30s each time to remove surface soil residue). Immerse in a suspension of intelligent responsive nano-disinfecting microcapsules, and immerse at a constant temperature of 28℃ with shaking (100r / min) for 28 minutes (the microcapsules slowly release disinfecting components from the weakly acidic environment on the surface of the Codonopsis pilosula stem tip, avoiding local browning). Rinse three times with sterile water (1.5 min each time) to remove residual microcapsules on the surface; Transferred to a lysozyme-EDTA complex system and treated in a 30℃ water bath for 26 min (EDTA disrupts the cell wall of endogenous bacteria in Codonopsis pilosula, and lysozyme targets and degrades bacterial proteins). Sterile filter paper was used to absorb surface moisture. A total of 65 stem tips were treated. The appearance showed no browning, the cuts were smooth, and the initial survival rate was 100%.

[0074] 4.3 Precision Detoxification Treatment Taking advantage of the relatively slender meristem of Codonopsis pilosula stem tip, the separation and detoxification parameters were optimized: The pretreated stem tips were placed in 0.1% fluorescein diacetate staining solution and stained at room temperature in the dark for 7 min (to avoid fluorescence quenching and ensure the identification of active regions). Placed in a microfluidic chip (channel width 400μm, adapted to the thin stem tip of Codonopsis pilosula), the active area with uniform fluorescence was located by an inverted fluorescence microscope, and the 0.22mm stem tip meristem was accurately separated, and a total of 62 active meristems were obtained (separation accuracy 95.4%). The separated stem tips were soaked in nano-silver-honeysuckle flavonoid composite detoxification solution and left to stand at room temperature for 17 minutes (stirring gently once every 6 minutes to ensure full contact with the detoxification solution). The sample was transferred to a low-temperature plasma treatment instrument, and argon gas was introduced to replace the air three times (1 minute each time). The power was set to 130W and the temperature to 40℃, and the treatment lasted for 30 seconds (to avoid thermal damage to the meristematic tissue of Codonopsis pilosula). After treatment, 59 shoot tips survived (survival rate of 95.2%, significantly higher than the 80% of traditional manual stripping).

[0075] 4.4 Preparation of Dynamically Adapted Culture Media Based on the characteristic of slow callus differentiation in Codonopsis pilosula, conditions are dynamically optimized using an AI system: Prepare 1L of MS basal medium and add 28g of sucrose (Codonopsis pilosula prefers a low-sugar environment, so the amount should be slightly less than 30g for ginseng). The system is connected to an AI image recognition system (including a high-definition camera and a chlorophyll fluorescence detector), with an initial ZT / IAA ratio of 1.8:1 (the ratio range for herbaceous plants is 1:1 to 2:1, which is suitable for the propagation requirements of Codonopsis pilosula), and mineral element N concentration of 19 mmol / L and P concentration of 1.1 mmol / L. Add 9 mL of nano-carbon quantum dot-chitosan composite matrix and 0.27 g of modified β-cyclodextrin chelating agent (to enhance anti-browning effect), and adjust pH to 5.85 (the suitable pH range for Codonopsis pilosula is 5.8~6.0). Sterilize at 121℃ with high-pressure steam for 20 minutes, then cool to 50℃ for later use. The AI ​​system detected that the differentiation rate of Codonopsis pilosula callus was only 28% on day 7. It automatically adjusted ZT / IAA to 2.0:1, supplemented N concentration to 21 mmol / L and P concentration to 1.21 mmol / L, and after optimization, the differentiation rate increased to 65% on day 10.

[0076] 4.5 Rapid Proliferation Culture Using an airlift bioreactor to solve the problem of hypoxia in Codonopsis pilosula liquid culture: Fifty-nine virus-free stem tips were inoculated into a 9L airlift bioreactor (inoculation density of 6.6 stem tips / L per reactor to avoid browning of Codonopsis pilosula stem tips). Add 4.5L of dynamic adaptation culture medium, then add 4.5mL of tea polyphenol nanoparticle-zeatin nucleoside complex polypeptide (stir well to ensure consistent concentration). Turn on the ultrasonic atomization oxygen supply system, set the droplet size to 7.5μm (to meet the oxygen requirements of Codonopsis pilosula's thin stem tips), the aeration rate to 0.85vvm, the cultivation temperature to 24℃ (the suitable growth temperature for Codonopsis pilosula is 23~27℃), the light intensity to 2600lx, and the photoperiod to 16h / 8h. During the cultivation process, the AI ​​system monitored the process in real time: on day 9, the ventilation rate was adjusted to 0.9 vvm (due to the accelerated proliferation of Codonopsis pilosula callus tissue, which increases oxygen demand), and on day 15, 120 mL of fresh culture medium was added (to avoid nutrient depletion). After 20 days of cultivation (herb proliferation cycle ≤ 20 days / generation), 236 tissue culture seedlings were obtained, with a proliferation coefficient of 4.0 times (significantly higher than the 1.8 times of traditional solid culture), a vitrification rate of 3.4% (only 8 seedlings), a seedling height of 3.6~4.1cm, a stem diameter of 0.28~0.32cm, and no browning phenomenon (the long-term anti-browning effect of tea polyphenol nanoparticles is significant).

[0077] 4.6 Virus-free real-time high-throughput detection For the main viruses (CMV, TRV) in Codonopsis pilosula, the quantum dot detection technology described in the document is used: Take 0.1g of each leaf from 236 tissue culture seedlings, place them in a 2mL centrifuge tube, add 1mL of extraction buffer (PBS buffer at pH 7.2), grind for 35s, centrifuge at 8000r / min for 10min, and collect the supernatant. The supernatant was added dropwise to the detection channels of the quantum dot biosensor array (one channel per sample, for a total of 236 channels) and incubated at room temperature for 42 min (the binding efficiency of Codonopsis pilosula virus was slightly lower, so the incubation was extended by 5 min). The fluorescence imaging system (detection wavelength 540nm) was activated to collect fluorescence signals. Two channels were found to have specific fluorescence peaks (both of which were CMV-infected plants), and these plants were immediately removed. A total of 234 virus-free vaccine strains were obtained, and the testing time was 1.4 hours (meeting the requirement of ≤2 hours). Two viruses were detected simultaneously, and the accuracy rate was verified by RT-PCR to be 99.7% (traditional ELISA testing requires 48 hours, and the efficiency was improved by 34 times).

[0078] 4.7 Hardening off and transplanting To address the issue of the relatively thin and weak root system of Codonopsis pilosula tissue culture seedlings, the acclimatization and transplanting conditions were optimized: 234 virus-free seedlings were transferred to the hardening-off room, where the temperature was set at 26℃ and the humidity at 75% (the initial humidity for Codonopsis pilosula seedling hardening-off needs to be slightly higher), and natural light was used for acclimatization for 8 days. During the acclimatization period, ventilate for 1.2 hours each at 9:00 and 16:00 every day, and gradually reduce the humidity to 55% (to prevent the Codonopsis pilosula leaves from losing water and wilting). The biomimetic nanocomposite matrix (nano hydroxyapatite: humic acid: coconut coir = 1:3:6) was filled into a 50-well seedling tray, sterilized at 121℃ for 30 minutes, and after cooling, the matrix moisture content was adjusted to 62% (Codonopsis pilosula prefers a moist matrix, slightly higher than 60% for ginseng). Trim the roots of Codonopsis pilosula tissue culture seedlings to 1.6cm using sterile scissors (retaining the main lateral roots), and soak them in a compound inoculant of arbuscular mycorrhizal fungi and phosphate-solubilizing bacteria for 42 minutes (to ensure that the roots are fully in contact with the inoculant and enhance nutrient absorption). Transplant each seedling into a seedling tray (one seedling per hole), water thoroughly with sterile water, and place in a greenhouse with a temperature of 25℃, humidity of 65%, and shading rate of 55% (Codonopsis pilosula is sensitive to strong light, so slightly increase the shading rate). After 38 days of cultivation, the seedlings were transplanted, with 205 surviving, resulting in a transplant survival rate of 87.6% (meeting the requirement of ≥85%, significantly higher than the 56% of traditional garden soil transplanting). The seedlings were 6.8~7.3cm tall after transplanting, and the average fresh weight of the roots was 1.1g (52.8% higher than the 0.72g of traditional transplanted seedlings).

[0079] 4.8 Performance Test Results The effects of virus-free cloning of Codonopsis pilosula are as follows: Virus elimination rate: RT-PCR detection of 234 virus-free seedlings showed that only 1 seedling carried latent TRV virus, with a virus elimination rate of 99.6% (≥95% requirement), which is 33.6 percentage points higher than that of traditional shoot tip culture (66%). Proliferation efficiency: The proliferation cycle is 20 days / generation, which is 28.6% shorter than the traditional solid culture (28 days) (meeting the technical target of "reducing by 30%)", the proliferation coefficient is 4.0 times, and the vitrification rate is 3.4% (the traditional method exceeds 30%). Transplant survival rate: 87.6%, which is 31.6 percentage points higher than that of traditional substrate transplanting (56%). After planting, the seedlings grow uniformly and are free from diseases and pests. Reagent safety: After the culture was completed, the reagent residue in the reactor was tested. The degradation rate of the LA-PEG-PNIPAM thermosensitive modified polylactic acid copolymer carrier was 92.3% (≥90% requirement), and there was no residue of nano silver or sodium hypochlorite. Medicinal material quality: The polysaccharide content of Codonopsis pilosula reaches 16.8% (13.2% for traditional tissue culture seedlings), the saponin content is 0.52% (0.38% for traditional tissue culture seedlings), and the effective components are increased by 27.3%~36.8%, which meets the quality standards of Codonopsis pilosula medicinal materials in the Chinese Pharmacopoeia.

[0080] Comparative Example 1: Traditional shoot tip culture plus chemical detoxification plus solid culture medium plus a single reagent Process: Select ginseng stem tips, manually peel off 0.8 mm stem tips, and soak in 0.1% ribavirin solution for 2 h (chemical detoxification); disinfect explants with 0.1% mercuric chloride for 10 min; use MS solid medium (BAP plus NAA = 1:1), add vitamin C to prevent browning (concentration 0.5 mg / mL), solid culture for 30 days / generation; after 40 days of culture, use ELISA to detect CMV virus; transplant with traditional garden soil plus perlite (mass ratio 2:1) substrate.

[0081] Performance: Virus elimination rate 67.3%, explant disinfection contamination rate 12.5%, survival rate 58%; proliferation coefficient 2.1 times / generation, vitrification rate 32%; vitamin C anti-browning effect lasts only 3 days, with a later browning rate of 25%; detection time 48 hours, one virus detected per batch; transplant survival rate 58%; reagent residue rate 15%, ginseng polysaccharide content 10.2%.

[0082] Defects: Low detoxification efficiency, chemical reagent residue; high disinfection contamination rate, low survival rate; slow proliferation, severe vitrification; poor resistance to browning; delayed detection, low efficiency; poor transplant survival rate, poor seedling quality.

[0083] Comparative Example 2: Ordinary sodium hypochlorite disinfection, fixation culture medium, liquid shake-flask culture, and conventional detection. Process: Eucommia ulmoides stem tips were disinfected with 5% sodium hypochlorite for 15 min; MS medium (ZT / IAA=2:1, fixed ratio); liquid shake flask culture (rotation speed 120 r / min); virus detection was performed by RT-PCR after 35 days of culture; transplanting was carried out with a substrate of leaf mold and perlite (mass ratio 3:1); disinfection reagent, detoxification reagent and anti-browning reagent were added separately, and the operation was carried out step by step.

[0084] Performance: Virus elimination rate 70.5%, explant browning rate 28%, survival rate 62%; proliferation coefficient 2.3 times / generation, proliferation cycle 40 days; 15% of tissue culture seedlings were weak due to hypoxia in liquid culture; detection time 6 hours, two viruses detected in a single batch; transplant survival rate 55%; vitrification rate 28%, total flavonoid content of Eucommia ulmoides 1.4%; reagent addition is cumbersome and concentration imbalance is easy to occur.

[0085] Disadvantages: Sterilization leads to browning; poor compatibility with culture media; hypoxia in liquid culture results in slow proliferation; low detection efficiency; low transplant survival rate; limited reagent functionality and cumbersome operation.

[0086] Control Example 3: Low-temperature plasma detoxification plus traditional agar medium plus ELISA detection plus ordinary matrix Process: Dendrobium officinale stem tips were manually peeled off to 0.6 mm, and detoxified by single low-temperature plasma (50℃, 60s); MS medium (agar as substrate); solid culture for 32 days / generation; ELISA detection was performed after 45 days of culture; transplanting was carried out using traditional humus soil plus coconut coir (mass ratio 2:1) substrate; single-function reagents were used, with no synergistic effect.

[0087] Performance: Detoxification rate 72.1%, stem tip damage rate 35%, survival rate 60%; proliferation coefficient 2.0 times / generation, vitrification rate 29%; detection time 72h, low efficiency; transplant survival rate 56%; single reagent function, requires multiple additions, complex operation; Dendrobium officinale polysaccharide content 23.3%.

[0088] Disadvantages: Limited effectiveness of single-method detoxification, severe damage to shoot tips; poor aeration of culture medium; delayed detection, resulting in resource waste; low transplant survival rate; limited reagent functionality and cumbersome operation.

[0089] Comparative Example 4: A combination of conventional disinfection and fixation hormone-containing culture medium, traditional transplanting, and no special reagents. Process: Ginseng explants were sterilized with 0.1% mercuric chloride for 8 min; MS medium (fixed hormone ratio, no AI adaptation); solid culture for 35 days / generation; no dedicated detoxification reagent, relying solely on shoot tip stripping for detoxification; traditional substrate transplanting; no integrated reagents, multiple reagents were purchased separately and mixed for use.

[0090] Performance: Explant contamination rate 15%, browning rate 30%; proliferation coefficient 1.8 times / generation, vitrification rate 35%; detoxification rate 65%; transplant survival rate 52%; antagonistic effect after reagent mixing, poor anti-browning effect; uneven seedling quality, polysaccharide content 9.8%.

[0091] Defects: The contradiction between disinfection contamination and browning; poor universality of culture media; poor anti-browning effect; slow proliferation; low transplant survival rate; no synergistic effect of reagents, resulting in poor effect.

[0092] This invention addresses the core deficiency of existing virus-free cloning methods for medicinal plants by innovating a complete process encompassing precise detoxification, intelligent disinfection, dynamically adapted culture medium, rapid proliferation, real-time detection, and biomimetic transplanting, along with the synergistic application of a combination of multifunctional biodegradable reagents. Compared to the control, this invention improves the detoxification rate by over 25%, explant survival rate by over 30%, shortens the proliferation cycle by 30%, increases detection efficiency by 10 times, improves transplant survival rate by over 30%, leaves no reagent residue and is biodegradable, and increases the content of effective components in seedlings by over 20%.

[0093] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of protection claimed by the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A rapid, virus-free cloning method for medicinal plant tissue culture, characterized in that, Includes the following steps: (1) Pretreatment of explants: Select the stem tips of medicinal plants, disinfect them by soaking in intelligent responsive nano-disinfection microcapsules, and then treat the endogenous bacteria with a lysozyme-EDTA complex system; (2) Precise detoxification: 0.1~0.3mm shoot tip meristems were separated by fluorescent labeling with microfluidic chip, placed in a nano-silver-honeysuckle flavonoid composite system, and treated with low-temperature plasma of dielectric barrier discharge type at 40℃ for 30s; (3) Culture medium preparation: Based on the AI ​​image recognition system, the hormone ratio and mineral element concentration were dynamically adapted, and agar was replaced with nano-carbon quantum dot-chitosan composite matrix, and modified β-cyclodextrin chelating agent was added; (4) Rapid proliferation: The virus-free shoot tips were inoculated into an airlift bioreactor, and oxygen was supplied by ultrasonic atomization of droplets of 5~10μm. The culture medium was supplemented with tea polyphenol nanoparticles-zeatin nucleoside complex polypeptide. (5) Virus-free detection: After culturing for 10-15 days, the virus was detected by a quantum dot biosensor array, and the fluorescent signal was used to identify infected plants; (6) Hardening and transplanting: Transplant to nano-hydroxyapatite-humic acid-coconut coir biomimetic substrate, and pretreat the roots of tissue culture seedlings with arbuscular mycorrhizal fungi-phosphate-solubilizing bacteria compound inoculant; (7) Application of reagents throughout the process: A multifunctional biodegradable reagent kit with temperature-responsive intelligent release is adopted. The kit uses temperature-sensitive modified polylactic acid copolymer as a carrier to prepare microcapsules, which realize the graded controlled release of functional components throughout the tissue culture cycle. The carrier completes degradation during the culture cycle, realizing the synergistic effects of detoxification, disinfection, anti-browning, proliferation promotion and antibacterial.

2. The rapid virus-free cloning method for medicinal plant tissue culture according to claim 1, characterized in that: In step (1), the nano-sterilization microcapsules are made of chitosan-sodium alginate encapsulating low concentrations of sodium hypochlorite and menthol derivatives, with a particle size of 50~100nm, a sodium hypochlorite mass fraction of 0.1~0.3%, and an menthol derivative addition amount of 5~8% of the microcapsule mass; the mass ratio of lysozyme to EDTA is 1:2~1:3, the treatment temperature is 25~30℃, and the time is 20~30min.

3. The rapid virus-free cloning method for medicinal plant tissue culture according to claim 1, characterized in that: In step (2), the nano-silver particles have a diameter of 20~50nm and a mass ratio of 1:3~1:5 with honeysuckle flavonoids. The concentration of the composite system is 0.5~1.0mg / mL. The low-temperature plasma is a normal pressure dielectric barrier discharge type with a power of 100~150W. The gas atmosphere is argon with a gas flow rate of 2~5L / min. The distance between the sample and the discharge electrode is 5~8mm. The sample stage temperature is controlled at 40℃ and the processing time is 30s.

4. The rapid virus-free cloning method for medicinal plant tissue culture according to claim 1, characterized in that: In step (3), the AI ​​system monitors the chlorophyll fluorescence intensity and callus morphology, dynamically adjusts the ZT / IAA ratio to 1:1~3:1; the mass ratio of nano-carbon quantum dots to chitosan is 1:10~1:15; and the amount of modified β-cyclodextrin added is 0.2~0.5% of the culture medium mass.

5. The rapid virus-free cloning method for medicinal plant tissue culture according to claim 1, characterized in that: In step (4), the aeration rate of the bioreactor is 0.5~1.0 vvm, the ultrasonic atomization power is 300~500W, the mass ratio of tea polyphenol nanoparticles to zeatin nucleoside is 2:1~3:1, and the amount added is 0.1~0.3% of the mass of the culture medium.

6. The rapid virus-free cloning method for medicinal plant tissue culture according to claim 1, characterized in that: In step (5), the quantum dots are CdSe / ZnS, the sensor array is used to fix CMV and TRV virus-specific probes, the fluorescence detection wavelength is 520~650nm, the detection limit for the target virus is ≤100 copies / μL, and there is no cross-reaction with non-target viruses; in step (6), the mass ratio of each component of the biomimetic matrix is ​​nano-hydroxyapatite: humic acid: coconut coir = 1:3:6, the concentration ratio of arbuscular mycorrhizal fungi to phosphate-solubilizing bacteria in the compound bacterial agent is 1:2, and the root soaking time is 30~60min.

7. The rapid virus-free cloning method for medicinal plant tissue culture according to claim 1, characterized in that: The propagation cycle is ≤20 days / generation for herbaceous medicinal plants, ≤28 days / generation for woody plants, and ≤25 days / generation for vines; the survival rate of seedlings after transplanting is ≥85%. The medicinal plants include herbaceous, woody, and vine medicinal plants from the Araliaceae, Eucommia, Orchidaceae, and Campanulaceae families, including but not limited to ginseng, codonopsis, eucommia, and dendrobium officinale.

8. A rapid virus-free cloning reagent combination for medicinal plant tissue culture using the method of any one of claims 1-7, characterized in that, The kit includes a multifunctional biodegradable reagent kit, which uses polylactic acid-polyethylene glycol-polyN-isopropylacrylamide terpolymer as a carrier to encapsulate detoxification, disinfection, anti-browning, proliferation-promoting, and antibacterial functional components. The mass ratio of each functional component is: nano silver: honeysuckle flavonoids: menthol derivatives: tea polyphenol nanoparticles: zeatin nucleoside = 1:3:2:4:

1. The carrier is prepared as a core-shell structured temperature-responsive microcapsule with a particle size of 50~100nm. Its lower critical dissolution temperature is 30±2℃. In an MS medium environment with a tissue culture temperature of 25~35℃ and a pH of 5.8~6.0, the degradation rate is ≥90% within 28 days. The degradation products are lactic acid, carbon dioxide and water, which are non-toxic and have no heavy metal or harmful component residues.

9. The rapid virus-free cloning reagent combination for medicinal plant tissue culture according to claim 8, characterized in that: In the PLA-PEG-PNIPAM terpolymer carrier, the mass percentage of PLA segments is 60%~70%, the mass percentage of PEG segments is 15%~25%, and the mass percentage of PNIPAM segments is 10%~15%. It also includes a smart responsive nano-disinfection microcapsule and lysozyme-EDTA composite system, wherein the microcapsule is a chitosan-sodium alginate carrier, encapsulating 0.1~0.3% sodium hypochlorite and 5~8% menthol derivative, with a particle size of 50~100nm; In the composite system, the mass ratio of lysozyme to EDTA is 1:2 to 1:3, and the concentration is 0.5 to 1.0 mg / mL; EDTA is used to disrupt the bacterial outer membrane barrier, while lysozyme is used to hydrolyze cell wall peptidoglycan; the two work together to eliminate endogenous bacteria.

10. The rapid virus-free cloning reagent combination for medicinal plant tissue culture according to claim 8, characterized in that: It also includes a nano-silver-honeysuckle flavonoid composite detoxifying solution, a quantum dot biosensor array, a nano-carbon quantum dot-chitosan composite matrix, a tea polyphenol nanoparticle-zeatin nucleoside composite polypeptide, a biomimetic nanocomposite matrix, and an arbuscular mycorrhizal fungus-phosphate-solubilizing bacteria composite inoculant. The concentration of the composite detoxifying solution is 0.5~1.0 mg / mL, the concentration of the composite polypeptide is 0.1~0.3 mg / mL, and the mass ratio of each component of the biomimetic matrix is ​​1:3:6.

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