Intelligent tissue culture method for polygonatum cyrtonema

Abstract

The invention discloses an intelligent polygonatum cyrtonema tissue culture method in the field of plant tissue culture, and the method comprises the following steps: collecting terminal buds of polygonatum cyrtonema as explants, carrying out disinfection and anti-oxidation pretreatment, inoculating the terminal buds into a culture medium containing intelligent response hydrogel, and the hydrogel can release a plant growth regulator to induce formation of calluses; after the calluses are transferred to a differential culture medium, hydrogel is triggered to release cytokinin by adjusting the temperature, and adventitious bud differentiation is promoted; after rootless seedlings are inoculated to a rooting culture medium, hydrogel is activated by adjusting the pH value to release growth promoting substances, and adventitious roots are induced to be generated. According to the method, accurate controlled release of the growth regulator is realized by utilizing the response characteristic of the intelligent hydrogel, and the efficiency and the stability of tissue culture are improved.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to an intelligent tissue culture method for Polygonatum multiflorum. Background Technology

[0002] Polygonatum multiflorum, a traditional and precious Chinese medicinal herb, possesses extensive medicinal and health-promoting value. With increasing market demand, its wild resources are becoming increasingly depleted due to over-harvesting, making artificial cultivation the primary means of meeting market needs. However, the traditional propagation method of Polygonatum multiflorum relies mainly on underground tubers. This asexual reproduction method suffers from low propagation coefficients, long cycles, and susceptibility to pests and diseases, severely restricting the large-scale and standardized production of seedlings. While tissue culture technology, as an effective means of rapid propagation, has been successfully applied to various medicinal plants, it still faces many technical bottlenecks in the industrialized seedling production of Polygonatum multiflorum, necessitating the exploration of more efficient and stable cultivation strategies.

[0003] Currently, the tissue culture process of Polygonatum multiflorum still faces many challenges. First, the explant establishment stage is susceptible to contamination and oxidative browning; conventional sterilization methods are often incomplete and easily damage tissue viability, leading to significant fluctuations in the success rate of primary cultures. Second, during critical stages such as callus induction, adventitious bud differentiation, and rooting, plant growth regulators are often applied by directly adding them to the culture medium. This method suffers from uneven hormone distribution in the medium, easy decomposition and inactivation, and an inability to dynamically regulate hormones according to the culture progress, resulting in low morphogenesis efficiency and inconsistent quality of regenerated seedlings. Furthermore, the culture process requires multiple transfers of culture medium, which is not only cumbersome and increases the risk of contamination, but also causes physiological stress to the cultures due to frequent external interventions, affecting their normal growth and development.

[0004] The limitations of existing tissue culture technology ultimately lie in its "static" management of the culture environment, which cannot achieve "dynamic" and precise control that matches the needs of plant growth and development. Therefore, developing a culture system that can intelligently respond to specific environmental signals (such as temperature and pH) during the culture stage and release active substances as needed is of great significance for improving the automation and success rate of Polygonatum odoratum tissue culture. This intelligent method is expected to reduce human intervention, maintain the stability of the culture system, and provide technical support for obtaining a large number of uniform and robust regenerated plants, thereby promoting the high-quality development of the Polygonatum odoratum seed industry. This invention is proposed against this backdrop. Summary of the Invention

[0005] The purpose of this invention is to provide an intelligent tissue culture method for Polygonatum multiflorum, which solves the technical problems of the existing methods for adding plant growth regulators in Polygonatum multiflorum tissue culture, which are crude and cannot accurately respond to the needs of each stage, resulting in a cumbersome culture process and unstable quality of regenerated seedlings.

[0006] The present invention achieves the above objectives through the following technical solutions: A method for intelligent tissue culture of Polygonatum multiflorum includes the following steps: S1. Collect the terminal buds of Polygonatum multiflorum and screen out explants with a polysaccharide content higher than 15%. The explants are treated with 74-76% ethanol, then disinfected with mercuric chloride and rinsed with sterile water. The disinfected explants are pretreated by soaking in an antioxidant solution containing ascorbic acid and citric acid to obtain pretreated explants. S2, 6-benzylaminopurine, thidiazuron, and naphthaleneacetic acid were added to MS medium, along with sucrose and agar; porous Polygonatum polysaccharide-chitosan-montmorillonite composite hydrogel was added to the medium to form a smart response culture system; pretreated explants were inoculated into the smart response culture system and then cultured; during the culture process, the porous Polygonatum polysaccharide-chitosan-montmorillonite composite hydrogel released embedded growth-regulating substances, inducing the explants to form callus tissue; S3, the callus tissue was transferred to differentiation medium and porous Polygonatum polysaccharide-chitosan-montmorillonite composite hydrogel was added; the culture temperature was adjusted to 27-29℃ to trigger the release of cytokinins from the thermosensitive component in the porous Polygonatum polysaccharide-chitosan-montmorillonite composite hydrogel, which promoted the differentiation of callus tissue into adventitious shoots; wherein, the thermosensitive component is hydroxypropyl methylcellulose; S4. Rootless seedlings were inoculated into rooting medium and porous Polygonatum polysaccharide-chitosan-montmorillonite composite hydrogel was added. By adjusting the pH of the medium to 5.7-5.9, the pH response mechanism of the material was activated, releasing naphthaleneacetic acid and sodium nitroprusside, which promoted the formation of adventitious roots.

[0007] According to a preferred embodiment of the present invention, in step S2, the growth regulating substance is composed of 6-benzylaminopurine, thiazolinone, and naphthaleneacetic acid.

[0008] According to a preferred embodiment of the present invention, the preparation steps of the differentiation medium include: using MS (Murashige and Skoog) basal medium as a base, adding 25g to 35g of sucrose per liter of medium as a carbon source and 5g to 7g of agar as a solidifying agent. Before sterilization of the medium, adding 0.5mg / L to 1.5mg / L of 6-benzylaminopurine and 0.05mg / L to 0.15mg / L of thiamethoxam, which work synergistically to initiate cell differentiation. Subsequently, the pH of the medium is adjusted to 5.7 to 5.9, and sterilized at 121°C and pressure for 15 to 20 minutes. When the medium cools to 50°C to 60°C, a pre-prepared porous Polygonatum sibiricum polysaccharide-chitosan-montmorillonite composite hydrogel is added under aseptic conditions. The amount of hydrogel added is 0.04% to 0.06% (w / w) of the total mass of the medium, and it is gently stirred to disperse it evenly. The hydrogel added at this stage contains embedded additional cytokinins, which will be released in response to temperature changes during subsequent culture.

[0009] According to a preferred embodiment of the present invention, the preparation steps of the rooting medium include: using 1 / 2 MS medium (i.e., MS medium with half the concentration of macro- and micro-elements) as a base, adding 15g to 25g of sucrose and 5g to 7g of agar per liter of medium. Before sterilization of the medium, naphthaleneacetic acid (NAA) at a concentration of 0.1mg / L to 0.5mg / L is added as the dominant factor for root induction, and activated carbon at a concentration of 50mg / L to 150mg / L may be selectively added to adsorb harmful metabolites. Subsequently, the initial pH of the medium is adjusted to 5.6 to 5.8, and autoclaved (121°C, 15-20min). After sterilization, under aseptic conditions, a porous Polygonatum polysaccharide-chitosan-montmorillonite composite hydrogel, also accounting for 0.04% to 0.06% (w / w) of the total mass, is added to each liter of medium. This hydrogel mainly encapsulates NAA and the nitric oxide donor sodium nitroprusside at this stage. Before or after inoculation with rootless seedlings, the pH of the culture medium is precisely adjusted to the target range of 5.7 to 5.9 using a sterile acid or alkali solution to activate the pH response mechanism of the hydrogel and initiate the programmed release of growth substances.

[0010] In this invention, the composite hydrogel plays an intelligent responsive role during the tissue culture of Polygonatum cyrtonema, precisely regulating the release behavior of plant growth regulators. During the callus induction stage, the hydrogel is added to the culture medium, and its three-dimensional network structure encapsulates plant growth regulators through physical adsorption and chemical bonding. In the early stages of culture, the hydrogel slowly diffuses and releases the regulators, inducing explant dedifferentiation and callus formation, thanks to the porosity and biodegradability of the network structure. When the callus is transferred to the differentiation medium, the culture temperature rises to a specific range, triggering the hydrogel's thermosensitive mechanism: the hydroxypropyl methylcellulose component undergoes chain segment contraction or expansion above the critical temperature, altering the network pore size and promoting the rapid release of encapsulated cytokinins, accelerating callus differentiation into adventitious shoots; the montmorillonite layered structure regulates the release rate during this process, avoiding a burst release effect. During the rooting stage, after rootless seedlings are inoculated into the culture medium, the pH is adjusted to a slightly acidic condition to activate the pH response mechanism of the hydrogel: the amino groups on the chitosan molecular chain are protonated in the acidic environment, leading to increased electrostatic repulsion and network swelling. Simultaneously, the Schiff base bonds partially hydrolyze under acidic conditions, enhancing pore connectivity and releasing substances such as auxin and sodium nitroprusside, promoting adventitious root formation. Polygonatum polysaccharides, as bioactive components, may synergistically regulate the metabolism of endogenous plant hormones, enhancing stress resistance. The entire culture system achieves intelligent response of the hydrogel through external stimuli (temperature, pH), simulating the natural growth environment of plants and improving the efficiency and stability of tissue culture. This mechanism stems from the multi-component synergistic design of the material, where the organic-inorganic hybrid structure enhances the response precision, and the biocompatibility of natural polysaccharides reduces phytotoxicity, providing a new strategy for the industrialized production of medicinal plants.

[0011] According to a preferred embodiment of the present invention, in step S1, the explant is treated with 74-76% ethanol for 30-40 seconds; and the mercuric chloride is disinfected for 8-10 minutes.

[0012] According to a preferred embodiment of the present invention, in step S2, the culture temperature after inoculation of pretreated explants is 23-25°C, and the light intensity is 40-50 μmol·m⁻². -2 ·s -1 .

[0013] According to a preferred embodiment of the present invention, in step S3, the mass of the porous Polygonatum polysaccharide-chitosan-montmorillonite composite hydrogel added accounts for 0.04-0.06% of the total mass of the substances in the culture medium.

[0014] According to a preferred embodiment of the present invention, in step S4, the height of the rootless seedling is 2-3 cm.

[0015] According to a preferred embodiment of the present invention, the preparation steps of the porous Polygonatum polysaccharide-chitosan-montmorillonite composite hydrogel include: A1. The dried rhizomes of Polygonatum odoratum were extracted by reflux with petroleum ether. The residue was extracted with ethanol and then extracted by reflux with pure water. The extracts were combined and concentrated, ethanol was added to precipitate the residue, and after dialysis, the residue was freeze-dried to obtain crude polysaccharide of Polygonatum odoratum. Subsequently, the crude polysaccharide of Polygonatum odoratum was modified by aldehyde to obtain aldehyde-modified Polygonatum odoratum polysaccharide. A2, natural bentonite was purified and sodium-modified to obtain sodium-modified montmorillonite; aldehyde-modified Polygonatum polysaccharide was mixed with sodium-modified montmorillonite and reacted in phosphate buffer at pH 7.4 to form organically modified montmorillonite; A3. Chitosan is dissolved in acetic acid solution, hydroxypropyl methylcellulose and genipin are added, and the mixture is stirred and reacted at 44-46℃ to form a transparent hydrogel precursor with temperature-responsive properties. A4, aldehyde-modified Polygonatum polysaccharide is mixed with organically modified montmorillonite, added to a transparent hydrogel precursor with temperature-responsive properties, and a plant growth regulator is added. The pH of the system is adjusted to 7.2-7.4, and cross-linking is carried out at 48-52℃. Microcapsules are formed using a microfluidic device. The specific components of the plant growth regulator are: 6-benzylaminopurine, thidiazuron, and naphthaleneacetic acid for the callus induction stage; cytokinin and 6-benzylaminopurine for the adventitious shoot differentiation stage; and naphthaleneacetic acid and sodium nitroprusside for the rooting stage.

[0016] According to a preferred embodiment of the present invention, the step of reflux extraction of dried Polygonatum cyrtonema rhizomes with petroleum ether comprises: taking 100g of dried Polygonatum cyrtonema rhizome powder, placing it in a 2000mL round-bottom flask, and adding 1000mL of petroleum ether (boiling range 60-90℃). Connecting a reflux condenser, and heating under reflux in an 85℃ water bath for 2 hours. After stopping heating, discarding the petroleum ether, and allowing the residue to air dry naturally in a fume hood until no solvent odor remains, obtaining defatted Polygonatum cyrtonema powder.

[0017] According to a preferred embodiment of the present invention, the step of aldehyde modification of crude Polygonatum polysaccharide includes: taking 5g of purified crude Polygonatum polysaccharide and dissolving it in 500mL of deionized water to prepare a 10g / L polysaccharide solution. The solution is placed in an ice-water bath, magnetically stirred, and protected from light. A solution prepared by dissolving 2.14g of sodium periodate (NaIO4) in 100mL of deionized water is slowly added dropwise (the molar ratio of sodium periodate to polysaccharide glycosyl units is approximately 1.2:1). The reaction system temperature is controlled below 10℃, and the pH is adjusted and maintained at 5.0 using 0.1mol / L sodium hydroxide (NaOH) solution. After reacting for 6 hours, 10mL of ethylene glycol is added to terminate the reaction, and stirring continues for 30 minutes. The reaction solution is then transferred to a dialysis bag with a molecular weight cutoff of 3500Da and dialyzed with deionized water for 72 hours, changing the water every 8 hours. Finally, the dialysate is freeze-dried to obtain white flocculent aldehyde-modified Polygonatum polysaccharide.

[0018] According to a preferred embodiment of the present invention, the steps of purifying and sodium-modifying natural bentonite include: taking 100g of natural bentonite ore powder, adding it to 2000mL of deionized water, stirring at high speed (800rpm) for 2 hours, letting it stand for 1 hour, collecting the upper suspension, and discarding the bottom sand and gravel precipitate. This purification process is repeated three times. Adding 500mL of a 1mol / L sodium chloride (NaCl) solution to the purified suspension, and stirring at 60℃ for 4 hours. After the reaction, centrifuging (4000rpm, 10min) to collect the solid, repeatedly washing and centrifuging with deionized water until no white precipitate (AgCl) is produced in the supernatant when tested with 0.1mol / L silver nitrate solution. Drying the obtained sodium-modified montmorillonite in a 60℃ oven for 24 hours, grinding it, and passing it through a 200-mesh sieve for later use.

[0019] According to a preferred embodiment of the present invention, the step of forming microcapsules using a microfluidic device includes: using a glass microfluidic chip with a flow focusing structure; using a hydrogel prepolymer solution containing 2% (w / v) aldehyde-modified Polygonatum odoratum polysaccharide, 1% (w / v) organically modified montmorillonite, and 1.5% (w / v) chitosan (degree of deacetylation ≥95%) as the dispersed phase; and using mineral oil containing 2% (v / v) Span-80 as the continuous phase. The injection flow rate of the dispersed phase is controlled at 10 μL / min and the injection flow rate of the continuous phase is controlled at 500 μL / min using a precision injection pump. Within the chip channels, the dispersed phase is sheared to form droplets with uniform particle size (approximately 150 μm). The droplets flow into a polytetrafluoroethylene (PTFE) collection tube placed in a 50°C constant temperature water bath and remain for 30 minutes to allow the Schiff base reaction to fully crosslink and solidify. Finally, the resulting microcapsule mixture was centrifuged (1000 rpm, 2 min), the upper oil phase was discarded, and the mixture was washed twice with n-hexane and anhydrous ethanol, and finally transferred to phosphate-buffered saline (PBS) at pH 7.4 for storage.

[0020] The preparation steps of the transparent hydrogel precursor with temperature-responsive properties include: Step 1: Chitosan acid dissolution: Accurately weigh 2.0 g of chitosan powder with a degree of deacetylation ≥90% and a viscosity of 200 mPa·s, and place it in a clean container. Measure 100 mL of a 1% (v / v) acetic acid solution, and slowly add the chitosan powder to the acetic acid solution while continuously stirring. Stir at room temperature (approximately 25°C) for at least 2 hours until the chitosan is completely dissolved, forming a clear and transparent viscous solution. Step 2: Addition of temperature-sensitive components and crosslinking agents: Add the following to the chitosan acetic acid solution in sequence: hydroxypropyl methylcellulose (HPMC), with a gelation temperature of approximately 70°C, at an addition amount of 1.5 g; and genipin, a natural crosslinking agent, at an addition amount of 30 mg. Maintain moderate stirring speed (400 rpm) during the addition process to ensure that all components are fully dispersed and that the crosslinking reaction begins. Step 3: Precursor formation via isothermal reaction; place the mixed solution in a isothermal water bath and react continuously for 7 hours at 45℃±1℃ (stirring speed can be reduced to 300rpm to avoid excessive bubble generation). During this process, genipin reacts with the amino groups on the chitosan molecular chains, while HPMC segments entangle with chitosan, ultimately forming a uniform, transparent, and temperature-responsive three-dimensional network structure hydrogel precursor. This precursor exists in a sol state below ambient temperature, making it easy to process and mix; as it approaches or reaches physiological temperature (37℃), its hydrophobicity increases, the network structure shrinks, and its stiffness increases.

[0021] According to a preferred embodiment of the present invention, the specific components of the plant growth regulator are: 6-benzylaminopurine, thidiazuron and naphthaleneacetic acid for the callus induction stage; cytokinin and 6-benzylaminopurine for the adventitious shoot differentiation stage; and naphthaleneacetic acid and sodium nitroprusside for the rooting stage.

[0022] In this invention, the porous Polygonatum polysaccharide-chitosan-montmorillonite composite hydrogel is prepared based on the synergistic effect of multiple chemical reactions to form a three-dimensional network structure with intelligent responsive characteristics. First, crude polysaccharide is obtained from the rhizome of Polygonatum multiflorum through extraction and purification, followed by aldehyde modification to introduce active aldehyde groups, laying the foundation for subsequent Schiff base reactions. Natural bentonite is purified and sodium-modified to obtain layered sodium-modified montmorillonite, which possesses excellent ion exchange capacity and adsorption performance. The aldehyde-modified Polygonatum polysaccharide reacts with sodium-modified montmorillonite in neutral phosphate buffer, forming organically modified montmorillonite through electrostatic interactions and hydrogen bonding, enhancing the biocompatibility and stability of the material. On the other hand, chitosan dissolved in acetic acid solution reacted with hydroxypropyl methylcellulose and genipin under mild heating conditions. Genipin, as a natural crosslinking agent, underwent Michael addition or Schiff base reaction with the amino groups of chitosan, forming a transparent hydrogel precursor with temperature-responsive properties. The addition of hydroxypropyl methylcellulose endowed the polymer network with thermosensitivity, causing it to undergo a phase transition within a specific temperature range. Finally, aldehyde-modified Polygonatum polysaccharide and organically modified montmorillonite were mixed into the hydrogel precursor, and the system was adjusted to weakly alkaline conditions. Crosslinking based on Schiff base reaction was carried out under heating conditions, where the aldehyde groups of aldehyde-modified Polygonatum polysaccharide reacted with the amino groups of chitosan to form imine bonds, constructing a stable three-dimensional network structure. Montmorillonite, as an inorganic filler, was dispersed in the network to improve the mechanical strength and adsorption capacity of the hydrogel. Microcapsules were formed using microfluidic technology to achieve the encapsulation and controlled release of plant growth regulators. The entire preparation process fully utilized the bioactivity of natural polysaccharides, the nano-effect of montmorillonite, and the responsiveness of smart polymers, achieving multifunctional integration of materials.

[0023] According to a preferred embodiment of the present invention, in step A1, the ethanol extraction time is 1-2 hours.

[0024] According to a preferred embodiment of the present invention, in step A2, the reaction time in the phosphate buffer is 6-8 hours.

[0025] According to a preferred embodiment of the present invention, in step A3, the stirring reaction time is 6-8 hours at 44-46°C.

[0026] According to a preferred embodiment of the present invention, in step A4, the mass ratio of organically modified montmorillonite to aldehyde-modified Polygonatum polysaccharide is 1:2.

[0027] The beneficial effects of this invention are as follows: This invention provides an intelligent tissue culture method for Polygonatum multiflorum, which, by introducing intelligent responsive hydrogel materials, achieves precise and programmed controlled release of plant growth regulators during tissue culture, demonstrating significant technical effects and outstanding advantages. Firstly, this method significantly improves the success rate and stability of Polygonatum multiflorum tissue culture. In traditional methods, browning and contamination of explants are the main causes of initial culture failure. This invention, by optimizing the sterilization process and combining it with pretreatment with solutions containing specific antioxidant components, effectively inhibits the oxidation reaction of explant cut surfaces, maximizing tissue viability and laying a good foundation for subsequent culture. More importantly, by pre-screening explants with high polysaccharide content, the superior quality of the starting material is ensured, giving it stronger regeneration potential and improving the efficiency of the entire culture system from the source. During callus induction, adventitious shoot differentiation, and rooting stages, the presence of intelligent hydrogels replaces the traditional frequent addition of hormones, avoiding the physiological impact on the culture caused by fluctuations in hormone concentration or degradation, thus ensuring the smooth progress of the culture process and the consistency of the regenerated seedling population.

[0028] Secondly, the core technical effect of this invention lies in achieving intelligent and on-demand supply of plant growth regulators. This intelligent hydrogel can respond to specific environmental signals and precisely release specific active substances at different stages of the cultivation process. During the callus induction stage, the hydrogel continuously and stably releases basic growth regulators, creating a suitable induction environment. When adventitious shoot differentiation needs to be induced, simply adjusting the cultivation temperature to a specific range triggers the temperature-sensitive components in the hydrogel, releasing high concentrations of cytokinins, effectively promoting callus morphogenesis. Similarly, during the rooting stage, fine-tuning the pH of the culture medium to a slightly acidic condition activates the hydrogel's pH response mechanism, releasing auxin-like substances that promote root growth. This "signal-triggered, precise release" model simulates the natural process of hormone regulation in plants, achieving a leap from a "static" to a "dynamic" cultivation environment, greatly reducing human intervention, labor intensity, and pollution risks, making large-scale, automated seedling cultivation possible.

[0029] Finally, the intelligent hydrogel material used in this invention possesses innovation and multiple synergistic effects. This hydrogel, modified from polysaccharides extracted from Polygonatum cyrtonema as one of the main raw materials, exhibits good biocompatibility, reducing the potential adverse effects of exogenous materials on plant tissues. Its unique three-dimensional network porous structure not only provides an efficient loading space for plant growth regulators but also facilitates the exchange of water, nutrients, and metabolites, creating a more suitable microenvironment for the culture. The introduction of montmorillonite enhances the material's adsorption and structural stability, while natural polymers such as chitosan endow it with degradability and certain antibacterial properties. The entire preparation process is carried out under isothermal conditions via Schiff base reaction, ensuring controllable conditions and resulting in uniform and stable microcapsule structures. The successful development of this intelligent carrier based on natural materials not only provides technical support for this method but also offers new ideas and references for the innovation of tissue culture techniques for other medicinal plants. Detailed Implementation

[0030] The following detailed embodiments are only used to further illustrate this application and should not be construed as limiting the scope of protection of this application. Those skilled in the art can make some non-essential improvements and adjustments to this application based on the above application content.

[0031] The following is information on domestic suppliers of the relevant equipment and materials: The ascorbic acid was purchased from Shijiazhuang Weisheng Pharmaceutical Co., Ltd.

[0032] The citric acid was purchased from Shandong Lemon Biochemical Co., Ltd.

[0033] The MS culture medium was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0034] The 6-benzylaminopurine was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0035] The thiafenone was purchased from Chengdu Grecia Chemical Technology Co., Ltd.

[0036] The naphthaleneacetic acid was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0037] The sucrose was purchased from Guangxi Yangpu Nanhua Sugar Industry Group Co., Ltd.

[0038] The agar was purchased from Qingdao Mingyue Seaweed Group Co., Ltd.

[0039] The dried rhizomes of Polygonatum multiflorum were purchased from Anhui Jiuxianzun Dendrobium Huoshan Co., Ltd.

[0040] The natural bentonite was purchased from Zhejiang Fenghong New Materials Co., Ltd.

[0041] The chitosan was purchased from Jinan Haidebei Marine Biotechnology Co., Ltd.

[0042] The hydroxypropyl methylcellulose was purchased from Huzhou Zhanwang Pharmaceutical Co., Ltd.

[0043] The ginipin was purchased from Wuhan Sewell Biotechnology Co., Ltd.

[0044] Preparation Example 1 This preparation example provides a method for preparing a differentiation medium: the preparation steps include: using MS (Murashige and Skoog) basal medium as a base, adding 25g of sucrose as a carbon source and 5g of agar as a solidifying agent per liter of medium. Before sterilization, 0.5mg / L of 6-benzylaminopurine and 0.05mg / L of thidiazuron are added to the medium; these two work synergistically to initiate cell differentiation. Subsequently, the pH of the medium is adjusted to 5.7 and sterilized at 121°C under pressure for 15 minutes. When the medium cools to 50°C, a pre-prepared porous Polygonatum sibiricum polysaccharide-chitosan-montmorillonite composite hydrogel is added under aseptic conditions. The amount of hydrogel added is 0.04% (w / w) of the total mass of the medium, and it is gently stirred to ensure uniform dispersion. The hydrogel added at this stage embeds additional cytokinins, which will be released in response to temperature changes during subsequent culture.

[0045] Preparation Example 2 This preparation example provides a method for preparing a rooting medium: using 1 / 2 MS medium (i.e., MS medium with half the concentration of macro- and micro-elements) as a base, 25 g of sucrose and 5 g of agar are added per liter of medium. Before sterilization, 0.1 mg / L naphthaleneacetic acid (NAA) is added as the dominant factor for root induction, and 50 mg / L activated carbon is optionally added to adsorb harmful metabolites. Subsequently, the initial pH of the medium is adjusted to 5.6, and autoclaving is performed (121 °C, 15 min). After sterilization, under aseptic conditions, 0.04% (w / w) of porous Polygonatum sibiricum polysaccharide-chitosan-montmorillonite composite hydrogel is added per liter of medium. This hydrogel mainly encapsulates NAA and the nitric oxide donor sodium nitroprusside at this stage. Before or after inoculation with rootless seedlings, the pH of the culture medium is precisely adjusted to the target range of 5.7 using a sterile acid or alkali solution to activate the pH response mechanism of the hydrogel and initiate the programmed release of growth substances.

[0046] Preparation Example 3 This preparation example provides a step-by-step guide for preparing defatted Polygonatum odoratum powder: Take 100g of dried Polygonatum odoratum rhizome powder and place it in a 2000mL round-bottom flask. Add 1000mL of petroleum ether (boiling range 60-90℃). Connect a reflux condenser and heat under reflux in an 85℃ water bath for 2 hours. After stopping heating, discard the petroleum ether and allow the residue to air dry naturally in a fume hood until no solvent odor remains, thus obtaining defatted Polygonatum odoratum powder.

[0047] Preparation Example 4 This preparation example provides a step-by-step guide for preparing aldehyde-modified Polygonatum polysaccharide: 5g of purified crude Polygonatum polysaccharide was dissolved in 500mL of deionized water to prepare a 10g / L polysaccharide solution. The solution was placed in an ice-water bath, magnetically stirred, and protected from light. A solution prepared by dissolving 2.14g of sodium periodate (NaIO4) in 100mL of deionized water was slowly added dropwise (the molar ratio of sodium periodate to polysaccharide glycosyl units was approximately 1.2:1). The reaction temperature was controlled below 10℃, and the pH was adjusted and maintained at 5.0 using 0.1mol / L sodium hydroxide (NaOH) solution. After 6 hours of reaction, 10mL of ethylene glycol was added to terminate the reaction, and stirring was continued for 30 minutes. The reaction solution was then transferred to a dialysis bag with a molecular weight cutoff of 3500Da and dialyzed with deionized water for 72 hours, changing the water every 8 hours. Finally, the dialysate was freeze-dried to obtain white flocculent aldehyde-modified Polygonatum polysaccharide.

[0048] Preparation Example 5 This preparation example provides a step-by-step guide for preparing sodium montmorillonite: Take 100g of natural bentonite powder, add it to 2000mL of deionized water, stir at high speed (800rpm) for 2 hours, let stand for 1 hour, collect the upper suspension, and discard the bottom sediment. This purification process is repeated three times. Add 500mL of 1mol / L sodium chloride (NaCl) solution to the purified suspension, and stir at 60℃ for 4 hours. After the reaction, centrifuge (4000rpm, 10min) to collect the solid, wash repeatedly with deionized water and centrifuge until no white precipitate (AgCl) is formed in the supernatant when tested with 0.1mol / L silver nitrate solution. Dry the obtained sodium montmorillonite in a 60℃ oven for 24 hours, grind it, and pass it through a 200-mesh sieve for later use.

[0049] Preparation Example 6 This preparation example provides a microcapsule preparation procedure using a glass microfluidic chip with a flow focusing structure. A hydrogel prepolymer solution containing 2% (w / v) aldehyde-modified Polygonatum odoratum polysaccharide, 1% (w / v) organically modified montmorillonite, and 1.5% (w / v) chitosan (degree of deacetylation ≥95%) is used as the dispersed phase. Mineral oil containing 2% (v / v) Span-80 is used as the continuous phase. The injection flow rate of the dispersed phase is controlled at 10 μL / min using a precision injection pump, and the injection flow rate of the continuous phase is controlled at 500 μL / min. Within the chip channels, the dispersed phase is sheared to form droplets with uniform particle size (approximately 150 μm). The droplets flow into a polytetrafluoroethylene (PTFE) collection tube placed in a 50°C constant temperature water bath, and the reaction is allowed to proceed for 30 minutes to allow the Schiff base reaction to fully crosslink and solidify. Finally, the resulting microcapsule mixture was centrifuged (1000 rpm, 2 min), the upper oil phase was discarded, and the mixture was washed twice with n-hexane and anhydrous ethanol, and finally transferred to phosphate-buffered saline (PBS) at pH 7.4 for storage.

[0050] Preparation Example 7 This preparation example provides a step-by-step preparation of a transparent hydrogel precursor with temperature-responsive properties, comprising: Step 1: Chitosan acid dissolution: Accurately weigh 2.0 g of chitosan powder with a degree of deacetylation ≥90% and a viscosity of 200 mPa·s, and place it in a clean container. Measure 100 mL of a 1% (v / v) acetic acid solution, and slowly add the chitosan powder to the acetic acid solution while continuously stirring. Stir at room temperature (approximately 25°C) for at least 2 hours until the chitosan is completely dissolved, forming a clear and transparent viscous solution. Step 2: Addition of temperature-sensitive components and crosslinking agents: Add the following to the chitosan acetic acid solution in sequence: hydroxypropyl methylcellulose (HPMC), with a gelation temperature of approximately 70°C, at an addition amount of 1.5 g; and genipin, a natural crosslinking agent, at an addition amount of 30 mg. Maintain moderate stirring speed (400 rpm) during the addition process to ensure that the components are fully dispersed and that the crosslinking reaction begins. Step 3: Precursor formation via isothermal reaction; place the mixed solution in a isothermal water bath and react continuously for 7 hours at 45℃±1℃ (stirring speed can be reduced to 300rpm to avoid excessive bubble generation). During this process, genipin reacts with the amino groups on the chitosan molecular chains, while HPMC segments entangle with chitosan, ultimately forming a uniform, transparent, and temperature-responsive three-dimensional network structure hydrogel precursor. This precursor exists in a sol state below ambient temperature, making it easy to process and mix; as it approaches or reaches physiological temperature (37℃), its hydrophobicity increases, the network structure shrinks, and its stiffness increases.

[0051] Preparation Example 8 This preparation example provides a reaction procedure for preparing a plant growth regulator: The regulator is prepared according to the needs of different culture stages: For the callus induction stage, 2 mg of 6-benzylaminopurine, 0.4 mg of thiamethoxam, and 0.4 mg of naphthaleneacetic acid are dissolved in 1 mL of 75% ethanol and then mixed with 10 mL of pH 7.3 phosphate buffer; for the adventitious shoot differentiation stage, 3.5 mg of 6-benzylaminopurine is dissolved in 1 mL of 75% ethanol and then mixed with 10 mL of pH 7.3 phosphate buffer; for the rooting stage, 2.5 mg of naphthaleneacetic acid and 100 mg of sodium nitroprusside are dissolved in 1 mL of 75% ethanol and then mixed with 10 mL of pH 7.3 phosphate buffer.

[0052] Example 1: A method for intelligent tissue culture of Polygonatum multiflorum, the preparation steps of the porous Polygonatum multiflorum polysaccharide-chitosan-montmorillonite composite hydrogel include: Step A1, take 100g of dried Polygonatum multiflorum rhizome, extract with 200ml of petroleum ether under reflux for 2 hours, extract the residue with 150ml of 80% ethanol for 1.5 hours, then extract with 300ml of pure water under reflux for 3 hours, combine the ethanol and water extracts, concentrate to 50ml, add 4 times the volume of anhydrous ethanol to precipitate, dialyze for 48 hours, freeze dry to obtain about 5g of crude Polygonatum multiflorum polysaccharide; take 3g of this crude polysaccharide for aldehyde modification to obtain 2.8g of aldehyde-modified Polygonatum multiflorum polysaccharide. Step A2: 50g of natural bentonite was purified and sodium-modified to obtain 45g of sodium-modified montmorillonite. 1g of sodium-modified montmorillonite was mixed with 2g of aldehyde-modified Polygonatum odoratum polysaccharide, and 100ml of phosphate buffer solution with a pH of 7.4 was added. The mixture was reacted for 7 hours to form organically modified montmorillonite. Step A3: 2g of chitosan was dissolved in 100ml of 1% (v / v) acetic acid solution, and 1.5g of hydroxypropyl methylcellulose and 30mg of genipin were added. The mixture was stirred at 45℃ for 7 hours to form a transparent hydrogel precursor with temperature-responsive properties. Step A4: 2g of aldehyde-modified Polygonatum odoratum polysaccharide was mixed with 1g of organically modified montmorillonite and added to the above transparent hydrogel precursor. The pH of the system was adjusted to 7.3, and a plant growth regulator was added. The mixture was subjected to a cross-linking reaction at 50℃ for 2 hours. Microcapsules with uniform particle size were formed using a microfluidic device, resulting in a porous Polygonatum odoratum polysaccharide-chitosan-montmorillonite composite hydrogel. The tissue culture steps included: S1, collecting terminal buds of Polygonatum multiflorum and screening explants with a polysaccharide content of 16%; treating the explants with 75% ethanol for 35 seconds, then disinfecting with 0.1% mercuric chloride solution for 9 minutes, and rinsing three times with sterile water; pretreating the disinfected explants by soaking them in an antioxidant solution containing 1 g / L ascorbic acid and 0.5 g / L citric acid for 30 minutes. S2, preparing 1 L of MS medium, adding 0.5 mg 6-benzylaminopurine, 0.1 mg thidiazuron, and 0.1 mg naphthaleneacetic acid, adding 30 g sucrose and 6 g agar; adding 0.5 g of the above-prepared hydrogel to form a smart responsive culture system; inoculating the pretreated explants and incubating at 25 °C and a light intensity of 45 μmol·m⁻². -2 ·s -1Under the specified conditions, callus formation was induced after 30 days of culture. S3, the callus was transferred to differentiation medium, which consisted of 1 L MS medium with 30 g sucrose, 6 g agar, and 0.5 g hydrogel. The culture temperature was adjusted to 28°C and cultured for 25 days to promote callus differentiation and adventitious shoot formation. S4, rootless seedlings with a height of 2.5 cm were inoculated into rooting medium, which consisted of 1 L 1 / 2 MS medium with 20 g sucrose, 6 g agar, and 0.5 g hydrogel. The pH of the medium was adjusted to 5.8 and cultured for 20 days to promote adventitious root formation. In this embodiment, the differentiation medium, rooting medium, defatted Polygonatum odoratum powder, aldehyde-modified Polygonatum odoratum polysaccharide, sodium montmorillonite, microcapsules, temperature-responsive transparent hydrogel precursor, and plant growth regulator were all prepared using the substances obtained in Preparation Examples 1-8.

[0053] Example 2, the specific implementation method is the same as Example 1, except that it is an intelligent tissue culture method for Polygonatum multiflorum. The preparation steps of the porous Polygonatum multiflorum polysaccharide-chitosan-montmorillonite composite hydrogel include: Step A1, take 100g of Polygonatum multiflorum rhizome, extract and purify it to obtain 5g of crude Polygonatum multiflorum polysaccharide; take 3.5g of crude polysaccharide and perform aldehyde modification to obtain 3.2g of aldehyde-modified Polygonatum multiflorum polysaccharide. Step A2, take 45g of sodium montmorillonite; take 1.2g of sodium montmorillonite and mix it with 2.4g of aldehyde-modified Polygonatum multiflorum polysaccharide, and react in 100ml of phosphate buffer at pH 7.4 for 6 hours to form organically modified montmorillonite. Step A3, take 2.2g of chitosan and dissolve it in 100ml of 1% acetic acid solution, add 1.6g of hydroxypropyl methylcellulose and 35mg of genipin, stir and react at 44℃ for 8 hours to form a hydrogel precursor. Step A4: Mix 2.4g of aldehyde-modified Polygonatum polysaccharide with 1.2g of organically modified montmorillonite, add the hydrogel precursor and plant growth regulator, adjust the pH to 7.2, and cross-link at 48℃ for 2 hours to form microcapsule hydrogels. Tissue culture steps include: S1: Treat explants with 74% ethanol (v / v) for 30 seconds, then sterilize with 0.1% mercuric chloride (w / w) for 8 minutes; pretreat in an antioxidant solution containing 1g / L ascorbic acid and 0.5g / L citric acid for 30 minutes. S2: Prepare 1L MS medium, add 0.6mg 6-benzylaminopurine, 0.05mg thidiazuron, and 0.05mg naphthaleneacetic acid, add 25g sucrose and 5g agar; add 0.4g hydrogel. Incubate at 23℃ and 40μmol·m⁻² light intensity. -2 ·s -1Under the specified conditions, the plants were cultured for 30 days. S3: The differentiation medium consisted of 1 L MS medium with 25 g sucrose and 5 g agar, and 0.4 g hydrogel was added; the temperature was adjusted to 27°C and the plants were cultured for 25 days. S4: Rootless seedlings with a height of 2 cm were inoculated into rooting medium consisting of 1 L 1 / 2 MS medium with 15 g sucrose and 5 g agar, and 0.4 g hydrogel was added; the pH was adjusted to 5.7 and the plants were cultured for 20 days. In this example, the differentiation medium, rooting medium, defatted Polygonatum odoratum powder, aldehyde-modified Polygonatum odoratum polysaccharide, sodium montmorillonite, microcapsules, temperature-responsive transparent hydrogel precursor, and plant growth regulator were all derived from the substances prepared in Examples 1-8.

[0054] Example 3, the specific implementation method is the same as Example 1, except that an intelligent tissue culture method for Polygonatum multiflorum is provided. The preparation steps of the porous Polygonatum multiflorum polysaccharide-chitosan-montmorillonite composite hydrogel include: Step A1, take 100g of Polygonatum multiflorum rhizome to obtain 5.2g of crude polysaccharide; take 4g of crude polysaccharide and modify it to obtain 3.7g of aldehyde-modified Polygonatum multiflorum polysaccharide. Step A2, take 1.5g of sodium montmorillonite and mix it with 3g of aldehyde-modified Polygonatum multiflorum polysaccharide, and react it in 100ml of phosphate buffer at pH 7.4 for 8 hours. Step A3, take 2.5g of chitosan and dissolve it in 100ml of 1% acetic acid solution, add 1.7g of hydroxypropyl methylcellulose and 40mg of genipin, and react it at 46℃ for 6 hours. Step A4, mix 3g of aldehyde-modified Polygonatum multiflorum polysaccharide with 1.5g of organically modified montmorillonite, add a plant growth regulator, adjust the pH to 7.4, and crosslink at 52℃ for 2 hours to form microcapsules. The tissue culture procedure included: S1, explants were treated with 76% ethanol for 40 seconds, then sterilized with 0.1% mercuric chloride for 10 minutes; pretreated for 30 minutes in an antioxidant solution containing 1 g / L ascorbic acid and 0.5 g / L citric acid. S2, 1.5 mg 6-benzylaminopurine, 0.15 mg thidiazuron, and 0.15 mg naphthaleneacetic acid were added to 1 L of MS medium, along with 35 g sucrose and 7 g agar; 0.6 g hydrogel was added. The culture was incubated at 22 °C under a light intensity of 50 μmol·m⁻¹. -2 ·s -1 Under the specified conditions, the plants were cultured for 30 days. In S3, the differentiation medium consisted of 1 L MS medium with 35 g sucrose and 7 g agar, and 0.6 g hydrogel was added; the temperature was adjusted to 29°C and the plants were cultured for 25 days. In S4, rootless seedlings with a height of 3 cm were inoculated into rooting medium consisting of 1 L 1 / 2 MS medium with 25 g sucrose and 7 g agar, and 0.6 g hydrogel was added; the pH was adjusted to 5.9 and the plants were cultured for 20 days. The differentiation medium, rooting medium, defatted Polygonatum odoratum powder, aldehyde-modified Polygonatum odoratum polysaccharide, sodium montmorillonite, microcapsules, and the transparent hydrogel precursor and plant growth regulator with temperature-responsive properties in this example were prepared using the substances obtained in Preparation Examples 1-8.

[0055] Comparative Example 1 The specific implementation method is the same as in Example 1, except that the hydrogel preparation steps do not include the addition of hydroxypropyl methylcellulose. In step A3, only 2g of chitosan is dissolved in 100ml of 1% acetic acid solution, 30mg of genipin is added, and the mixture is reacted at 45°C for 7 hours to form a common hydrogel precursor. In step A4, 2g of aldehyde-modified Polygonatum polysaccharide is cross-linked with 1g of organically modified montmorillonite. The tissue culture steps are exactly the same as in Example 1, except that this common hydrogel is added to the differentiation medium in S3, and the mixture is cultured at 28°C. The differentiation medium, rooting medium, defatted Polygonatum polysaccharide powder, aldehyde-modified Polygonatum polysaccharide, sodium montmorillonite, microcapsules, and temperature-responsive transparent hydrogel precursor in this comparative example are the substances obtained in Preparation Examples 1-7.

[0056] Comparative Example 2 The specific implementation method is the same as in Example 1, except that the organic modification of aldehyde-modified Polygonatum polysaccharide and montmorillonite is omitted in the hydrogel preparation step of the Polygonatum multiflorum tissue culture method. In step A4, the hydrogel precursor obtained in step A3 is directly crosslinked at 50°C for 2 hours without adding aldehyde-modified Polygonatum polysaccharide and montmorillonite. The tissue culture steps are exactly the same as in Example 1, except that this hydrogel is added to the rooting medium in S4 and the pH is adjusted to 5.8 for culture. The differentiation medium, rooting medium, defatted Polygonatum multiflorum powder, aldehyde-modified Polygonatum polysaccharide, sodium montmorillonite, microcapsules, temperature-responsive transparent hydrogel precursor, and plant growth regulator in this comparative example are the substances obtained in Preparation Examples 1-8.

[0057] Comparative Example 3 The specific implementation method is the same as in Example 1, except that a traditional tissue culture method for Polygonatum multiflorum does not use smart hydrogels at all. In the tissue culture steps, step S1 is the same as in Example 1. In step S2, 0.5 mg of 6-benzylaminopurine, 0.1 mg of thidiazuron, and 0.1 mg of naphthaleneacetic acid, 30 g of sucrose, and 6 g of agar are directly added to 1 L of MS medium. The differentiation medium in step S3 is 1 L of MS medium with 0.2 mg of 6-benzylaminopurine, 30 g of sucrose, and 6 g of agar added. The rooting medium in step S4 is 1 L of 1 / 2 MS medium with 0.3 mg of naphthaleneacetic acid, 1 mg of sodium nitroprusside, 20 g of sucrose, and 6 g of agar added. The culture conditions are exactly the same as in Example 1. The differentiation medium, rooting medium, defatted Polygonatum multiflorum powder, aldehyde-modified Polygonatum multiflorum polysaccharide, sodium montmorillonite, microcapsules, and the transparent hydrogel precursor and plant growth regulator with temperature-responsive properties in this comparative example are the substances obtained in Preparation Examples 1-8.

[0058] Performance testing The intelligent tissue culture method for Polygonatum multiflorum in Examples 1-3 and Comparative Examples 1-3 was subjected to performance testing according to the following steps: To verify the effectiveness of the intelligent tissue culture method for Polygonatum multiflorum of the present invention, the culture results obtained in Examples 1-3 and Comparative Examples 1-3 were systematically evaluated. Test indicators included callus induction rate, callus browning rate, adventitious bud differentiation rate, average number of differentiated buds per callus piece, rooting rate, average number of roots, average root length, and seedling transplant survival rate. The specific method is as follows: In each treatment group (Examples 1, 2, 3, Comparative Examples 1, 2, 3), three replicates were set up, with 50 explants or callus pieces inoculated in each replicate. The callus induction rate was calculated on day 30 after the primary culture, determining the percentage of explants forming callus to the total number of explants. The callus browning rate was observed and calculated at the same time point, determining the percentage of severely browned callus pieces to the total number of callus pieces. The adventitious bud differentiation rate was calculated on day 25 after differentiation culture. The percentage of callus pieces producing adventitious buds was counted out of the total number of inoculated callus pieces, and the number of adventitious buds produced on each differentiated callus piece was also counted, with the average value calculated. The rooting rate was calculated on day 20 after rooting culture. The percentage of rootless seedlings producing adventitious roots was counted out of the total number of rootless seedlings, and the number and length of roots of all rooted seedlings were measured, with the average value calculated. The seedling transplant survival rate was determined as follows: healthy tissue culture seedlings were selected, the root culture medium was washed off, and they were transplanted into a substrate composed of vermiculite and peat moss. After 30 days of cultivation under greenhouse conditions, the percentage of surviving seedlings was counted. All data were analyzed using one-way ANOVA with SPSS software, and significance was tested using Duncan's new multiple range test.

[0059] Test results: Table 1: Test results of each embodiment and comparative example

[0060] Note: The data in the table is formatted as "mean ± standard deviation". Different letters after the data in the same row indicate that the difference is significant at the p < 0.05 level.

[0061] As shown in Table 1, the performance test results and comparative analysis of Examples 1 to 3 and Comparative Examples 1 to 3 clearly demonstrate that this invention, by introducing intelligent responsive hydrogel technology, effectively solves the core technical problem of the current extensive method of adding plant growth regulators in Polygonatum cyrtonema tissue culture, which fails to accurately respond to the needs of each stage. Specifically, Examples 1 to 3 exhibited extremely high induction rates (all above 96%) and extremely low browning rates (all below 6.5%) during the callus induction stage. This is attributed to the sustained-release effect of the intelligent hydrogel on the growth regulator in the early stage of culture, avoiding the instantaneous impact of high-concentration hormones on explants and creating a stable microenvironment for cell dedifferentiation. In contrast, Comparative Example 3, using the traditional one-time hormone addition method, showed a significantly reduced induction rate of 78.6% and a browning rate as high as 45.7%, directly proving that extensive addition easily leads to excessively high local concentrations, causing tissue toxicity and oxidative browning.

[0062] During the adventitious shoot differentiation stage, the adventitious shoot differentiation rates of Examples 1 to 3 were all above 93%, and each callus tissue produced a large number of shoots (more than 6.5). The key was that when the culture temperature was raised to 27-29℃, the thermosensitive component in the hydrogel responded promptly and released sufficient cytokinin, precisely meeting the differentiation requirements. In contrast, Comparative Example 1, due to the lack of a thermosensitive component in its hydrogel (no hydroxypropyl methylcellulose), could not achieve temperature-triggered release, resulting in a sharp drop in its differentiation rate to 75.4%, with an average of only 3.2 shoots. This clearly demonstrates that the lack of a precise response mechanism leads to insufficient differentiation momentum. Comparative Example 3 also performed poorly at this stage, further confirming that traditional methods cannot synchronize hormone supply with the culture process.

[0063] During the rooting stage, the rooting rates of Examples 1 to 3 all exceeded 95%, with well-developed root systems (more than 5.3 roots, each over 4.0 cm in length), and a transplant survival rate as high as approximately 94%. This was attributed to the activation of the hydrogel's pH response mechanism by adjusting the pH to a slightly acidic level, enabling the efficient release of auxin and nitric oxide donors when needed, strongly promoting root growth and resulting in robust seedlings. In contrast, Comparative Example 2, lacking the pH-responsive enhancement structure constructed from aldehyde-modified Polygonatum polysaccharide and montmorillonite, exhibited significantly lower rooting rates, root numbers, root lengths, and transplant survival rates compared to the examples, indicating that a lack of precise pH-triggered release capability severely weakens the rooting effect. Comparative Example 3 showed the worst rooting indicators, further highlighting the limitations of traditional one-time hormone addition.

[0064] In summary, Examples 1 to 3 utilize smart hydrogels to achieve on-demand and precise release of plant growth regulators during the three key stages of callus induction, adventitious bud differentiation, and rooting. This transforms the traditional static and extensive hormone management model into a dynamic and intelligent regulation model, significantly simplifying the operation process (eliminating the need for multiple transfers and hormone additions) and fundamentally improving the quality consistency and stability of regenerated seedlings, successfully addressing the core defects of existing technologies.

[0065] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for intelligent tissue culture of Polygonatum multiflorum, characterized in that, Includes the following steps: S1. Collect the terminal buds of Polygonatum multiflorum and screen out explants with a polysaccharide content higher than 15%. The explants are treated with 74-76% ethanol, then disinfected with mercuric chloride and rinsed with sterile water. The disinfected explants are pretreated by soaking in an antioxidant solution containing ascorbic acid and citric acid to obtain pretreated explants. S2, 6-benzylaminopurine, thidiazuron, and naphthaleneacetic acid were added to MS medium, along with sucrose and agar; porous Polygonatum polysaccharide-chitosan-montmorillonite composite hydrogel was added to the medium to form a smart response culture system; pretreated explants were inoculated into the smart response culture system and then cultured; during the culture process, the porous Polygonatum polysaccharide-chitosan-montmorillonite composite hydrogel released embedded growth-regulating substances, inducing the explants to form callus tissue; S3, the callus tissue was transferred to differentiation medium and porous Polygonatum polysaccharide-chitosan-montmorillonite composite hydrogel was added; the culture temperature was adjusted to 27-29℃ to trigger the release of cytokinins from the thermosensitive component in the porous Polygonatum polysaccharide-chitosan-montmorillonite composite hydrogel, which promoted the differentiation of callus tissue into adventitious shoots; wherein, the thermosensitive component is hydroxypropyl methylcellulose; S4. Rootless seedlings were inoculated into rooting medium and porous Polygonatum polysaccharide-chitosan-montmorillonite composite hydrogel was added. By adjusting the pH of the medium to 5.7-5.9, the pH response mechanism of the material was activated, releasing naphthaleneacetic acid and sodium nitroprusside, which promoted the formation of adventitious roots.

2. The intelligent tissue culture method for Polygonatum multiflorum according to claim 1, characterized in that, In step S1, the explants are treated with 74-76% ethanol for 30-40 seconds; and the mercuric chloride is disinfected for 8-10 minutes.

3. The intelligent tissue culture method for Polygonatum multiflorum according to claim 1, characterized in that, In step S2, the culture temperature after inoculation of pretreated explants is 23-25℃, and the light intensity is 40-50 μmol·m⁻². -2 ·s -1 .

4. The intelligent tissue culture method for Polygonatum multiflorum according to claim 1, characterized in that, In step S3, the mass of the porous Polygonatum polysaccharide-chitosan-montmorillonite composite hydrogel added accounts for 0.04-0.06% of the total mass of the substances in the culture medium.

5. The intelligent tissue culture method for Polygonatum multiflorum according to claim 1, characterized in that, In step S4, the height of the rootless seedling is 2-3 cm.

6. The intelligent tissue culture method for Polygonatum multiflorum according to any one of claims 1-5, characterized in that, The preparation steps of the porous Polygonatum polysaccharide-chitosan-montmorillonite composite hydrogel include: A1. The dried rhizomes of Polygonatum odoratum were extracted by reflux with petroleum ether. The residue was extracted with ethanol and then extracted by reflux with pure water. The extracts were combined and concentrated, ethanol was added to precipitate the residue, and after dialysis, the residue was freeze-dried to obtain crude polysaccharide of Polygonatum odoratum. Subsequently, the crude polysaccharide of Polygonatum odoratum was modified by aldehyde to obtain aldehyde-modified Polygonatum odoratum polysaccharide. A2, natural bentonite was purified and sodium-modified to obtain sodium-modified montmorillonite; aldehyde-modified Polygonatum polysaccharide was mixed with sodium-modified montmorillonite and reacted in phosphate buffer at pH 7.4 to form organically modified montmorillonite; A3. Chitosan is dissolved in acetic acid solution, hydroxypropyl methylcellulose and genipin are added, and the mixture is stirred and reacted at 44-46℃ to form a transparent hydrogel precursor with temperature-responsive properties. A4, aldehyde-modified Polygonatum polysaccharide is mixed with organically modified montmorillonite, added to a transparent hydrogel precursor with temperature-responsive properties, and a plant growth regulator is added. The pH of the system is adjusted to 7.2-7.4, and cross-linking is carried out at 48-52℃. Microcapsules are formed using a microfluidic device. The specific components of the plant growth regulator are: 6-benzylaminopurine, thidiazuron, and naphthaleneacetic acid for the callus induction stage; cytokinin and 6-benzylaminopurine for the adventitious shoot differentiation stage; and naphthaleneacetic acid and sodium nitroprusside for the rooting stage.

7. The intelligent tissue culture method for Polygonatum multiflorum according to claim 6, characterized in that, In step A1, the ethanol extraction time is 1-2 hours.

8. The intelligent tissue culture method for Polygonatum multiflorum according to claim 6, characterized in that, In step A2, the reaction time in the phosphate buffer is 6-8 hours.

9. The intelligent tissue culture method for Polygonatum multiflorum according to claim 6, characterized in that, In step A3, the stirring reaction time is 6-8 hours at 44-46℃.

10. The intelligent tissue culture method for Polygonatum multiflorum according to claim 6, characterized in that, In step A4, the mass ratio of organically modified montmorillonite to aldehyde-modified Polygonatum polysaccharide is 1:2.

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