A high-efficiency in-vitro regeneration and breeding method for thallus of Herba Zanthoxylum

Abstract

The application provides a high-efficiency regeneration breeding method for Trichosanthes rosthornii stem tips in vitro, and relates to the field of seedling cultivation, and comprises the following steps: S1, material selection: selecting tender dormant bud bodies at the top of underground stems of Trichosanthes rosthornii as inoculation explants, S2, material pretreatment: cleaning the material in S1, S3, explant surface disinfection and stem tip trimming, S4, primary induction and callus culture, S5, bud induction and proliferation culture, and S6, rooting induction culture. The method not only solves the problems such as high contamination rate, weak proliferated seedlings and poor rooting quality in the existing tissue culture technology, but also solves deep recessive problems such as endophyte contamination, silent death, genetic drift of subculture, vascular defects and mycorrhizal loss, so that the adaptability and survival rate of Trichosanthes rosthornii tissue culture seedlings after transplanting are improved.

Description

Technical Field

[0001] This invention relates to the field of seed and seedling cultivation technology, specifically to a high-efficiency in vitro regeneration and propagation method for *Heliotropium indicum* stem tips. Background Technology

[0002] Sunflower ( Zingiber striolatum Diels, also known as wild ginger or Guanyin flower, is a perennial herbaceous plant belonging to the ginger family (Zingiberaceae). It is mainly distributed in Sichuan, Guizhou, Guangxi, Hubei, Hunan, Jiangxi, Guangdong and other provinces. It is a special vegetable that is both food and medicine. It is rich in amino acids, protein, dietary fiber and various trace elements. In traditional medicine, it can be used to promote blood circulation and regulate menstruation, relieve phlegm and cough, and detoxify and reduce swelling.

[0003] Currently, *Gynostemma pentaphyllum* relies on underground stems for asexual reproduction, and coupled with wild harvesting, this has led to germplasm degradation and resource depletion. This not only increases the risk of stem and root rot diseases, but also puts continuous pressure on the survival of wild resources. At the same time, traditional division propagation methods are inefficient and prone to causing the accumulation of pathogens and a decline in seedling quality, severely restricting the sustainable utilization, large-scale cultivation, and industrial development of *Gynostemma pentaphyllum* germplasm resources.

[0004] Currently, there are some reports on tissue culture technology for *Heliotropium indicum*, but existing tissue culture techniques still have the following problems: First, there are obvious problems such as high explant contamination rate, difficulty in balancing disinfection intensity and survival rate, contradiction between proliferation efficiency and seedling quality, poor root development, and insufficient transplant stability; Second, there are deep-seated hidden problems such as latent contamination by endophytic bacteria, starch-induced silent death, phenolic acid-mediated dedifferentiation arrest, epigenetic drift of successors, root and stem vascular connection disorder, root autotoxicity decline, and lack of mycorrhizal symbiosis. Although these deep-seated hidden problems are easily masked in small-batch experiments, they will cause significant efficiency losses and trait degradation in large-scale production. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to provide a high-efficiency in vitro regeneration and propagation method for *Liriope muscari* stem tips. This method not only solves the obvious problems in existing tissue culture techniques, such as high contamination rate, weak seedlings, and poor rooting quality, but also solves the deep-seated hidden problems such as endophytic fungal contamination, silent death, epigenetic drift of successors, vascular defects, and mycorrhizal absence. This establishes an efficient and stable in vitro propagation system and improves the adaptability and survival rate after transplanting.

[0006] The objective of this invention is achieved through the following technical solution: A highly efficient in vitro regeneration and propagation method for *Ligustrum lucidum* stem tips includes: Step S1, Material Selection: Select young dormant buds from the top of the underground stem of *Heliotropium indicum* as explants (preferably select individuals with plump buds, tightly wrapped bracts, and no browning or rot), and trim them to a length of 0.5 ± 0.1 cm. Step S2, Material Pretreatment: Clean the material from step S1; Step S3, Explant Surface Disinfection and Stem Tip Trimming: Turn on the ultra-clean workbench and perform ultraviolet disinfection for 25-35 minutes, and perform gradient surface disinfection on the pretreated explants; after disinfection, transfer the explants to the workbench, and use a scalpel and forceps to peel off the scale-like bracts on the outside of the bud layer by layer, cut off the aging cut at the lower end of the stem segment, and trim it into a stem tip with meristem; Step S4, Primary Induction and Callus Culture: Use sterile filter paper to blot dry the surface moisture of the stem tip with meristem, and vertically inoculate it into a culture bottle containing primary induction medium (inoculate one stem tip with meristem in each bottle, ensuring full contact between the cut and the medium) for primary culture; Step S5, Sprout Induction and Proliferation Culture: After the initial culture is completed, select sterile sprouts that are morphologically complete, vigorous, and free of disease spots (i.e., no browning, no vitrification, no necrotic spots) as the propagation starting material; and transfer the propagation starting material to a culture bottle containing propagation culture medium (one plantlet per bottle) for propagation culture. Step S6, Rooting Induction Culture: Select healthy, sterile seedlings with a height of 3-4 cm and fully expanded leaves from the proliferation culture stage, remove the aging callus and excess leaves at the base, and transfer each seedling to a culture bottle containing rooting induction medium for rooting culture to obtain tissue culture seedlings.

[0007] Based on further optimization of the above scheme, step S2 specifically includes: First, removing the soil and residual leaves from the surface of the explant rhizome and rinsing it initially with running water; then immersing it in a 0.5% (v / v) household detergent (i.e., dishwashing liquid, commercially available brands can be used, with sodium fatty alcohol polyoxyethylene ether sulfate as the main surfactant and an effective content ≥15%) aqueous solution for 8–12 minutes, stirring at 50–80 rpm for 10–20 seconds every 3–5 minutes (using the surfactant components in the dishwashing liquid to wet the surface of the explant, dissolve the surface wax, and remove soil attachments); then rinsing it with tap water, and then transferring it to a 0.1% (w / w) carbendazim (N-(2-benzoimidazole)-methyl carbamate) solution for 8–12 minutes (killing the fungal spores attached to the surface); finally, rinsing it with tap water, sealing the container with gauze, and rinsing it continuously under running water for 8–12 minutes to complete the pretreatment.

[0008] Based on further optimization of the above scheme, step S3, the gradient surface disinfection of the explant specifically includes: First, immersing the sterile container containing the explant in a 75% ethanol solution for 9-11 seconds, shaking the container twice per second during this period; then, removing the explant and rinsing it 1-2 times with sterile distilled water (to initially wash away the ethanol); then, adding the explant to a 2% sodium hypochlorite solution and treating it for 9-11 minutes, shaking the container once per second for 10-15 seconds every 2 minutes during this period; finally, discarding the sodium hypochlorite solution and rinsing repeatedly with sterile distilled water 4-5 times (to thoroughly remove residual disinfectant).

[0009] Based on further optimization of the above scheme, in step S4, the initial culture is specifically as follows: after inoculation, the culture bottle is transferred to a dark culture room and cultured at a constant temperature of 28±0.5℃ and an air humidity of 60%~70%RH for 7~10 days (to induce callus initiation and bud sprouting, and to avoid light exacerbating phenolic oxidation and browning). Then, the culture bottle is transferred to a light culture room and cultured for 18~22 days with a light intensity of 1800~2000Lux and a light cycle of 12h / d. During this period, the contamination, browning, callus occurrence and bud sprouting are observed and recorded regularly.

[0010] Based on further optimization of the above scheme, in step S4, the primary induction medium includes MS medium, sucrose, 6-BA (6-benzylaminopurine), NAA (1-naphthaleneacetic acid), acid-hydrolyzed casein, spermidine, ε-polylysine hydrochloride, trehalose, pectin oligosaccharide (degree of polymerization 3-7), 5-ALA (5-aminolevulinate), fluorine-modified mesoporous silica nanoparticles, disodium pyrroloquinoline quinone, chitosan quaternary ammonium salt-cinnamaldehyde emulsion, and agar, and the pH is adjusted to 5.8.

[0011] Based on further optimization of the above scheme, the concentrations of sucrose, 6-BA, NAA, acid-hydrolyzed casein, spermidine, ε-polylysine hydrochloride, trehalose, pectin oligosaccharide, 5-ALA, fluorine-modified mesoporous silica nanoparticles, disodium pyrroloquinoline quinone, chitosan quaternary ammonium salt-cinnamaldehyde emulsion, and agar in the primary induction culture medium are 29.5–30.5 g / L, 0.9–1.1 mg / L, and 0.9–1.1 mg / L, respectively. / L, 0.9~1.1mg / L, 0.24~0.26g / L, 19.5~20.5mg / L, 0.03~0.05g / L, 0.11~0.13g / L, 0.04~0.06g / L, 4.5~5.5mg / L, 0.02~0.04g / L, 3.8~4.2mg / L, 5~7mL / L, 7.3~7.7g / L.

[0012] By using ε-polylysine hydrochloride as a microbial-derived cationic polypeptide, the permeability of endophytic bacterial cell membranes is disrupted, broadly inhibiting endophytic bacteria such as Pseudomonas, without damaging plant cells. It forms a positive synergy with the cell membrane protection effect of spermidine, maintaining cell structural integrity, reducing bacterial-mediated fungal colonization sites, and lowering the risk of fungal colonization. At the same time, chitosan quaternary ammonium salt-cinnamaldehyde emulsion targets and adsorbs fungal hyphae with positive charge, and penetrates into the intercellular spaces of explant cells with nanoscale particle size. Cinnamaldehyde inhibits the synthesis of ergosterol in fungi, thereby inhibiting latent endophytic fungi such as anthrax and Fusarium. The two work synergistically to achieve full coverage of endophytic bacteria and fungi. Trehalose is used to slow down the rapid hydrolysis of endogenous starch granules in shoot tips, avoiding plasmolysis and autophagy caused by a sudden increase in cell osmotic pressure, while stabilizing the conformation of cell membrane proteins and intracellular enzymes. Pyrroloquinoline quinone disodium salt regulates plastid redox homeostasis, promoting the orderly transformation of starch granules into chloroplasts and preventing silent cell death caused by ROS bursts in the two organelles. Together with spermidine, it forms a multi-layered protection system of "osmotic pressure protection - membrane structure stabilization - organelle function repair." By downregulating polyphenol oxidase gene expression and inhibiting free phenol oxidation and browning through spermidine, pectin oligosaccharides maintain cell wall integrity in the early stages of dedifferentiation, reduce the continuous release of bound phenolic acids, and activate cell cycle pathways as signaling molecules, thus advancing callus initiation. These two components work synergistically to address the problems of delayed browning and dedifferentiation. 5-Aminolevulinate was used to downregulate the expression of endogenous abscisic acid (ABA) synthesis genes, breaking the physiological dormancy of underground buds and narrowing the germination time difference between different buds. Fluorine-modified mesoporous silica nanoparticles, with their nanoscale particle size, mediated the cell wall perforation effect to enter the cell. Through the competitive binding of PP2C phosphatase in the ABA receptor complex via the hydrophobic mesoporous structure of the fluorinated nanoparticles, they indirectly released transcriptional repression of cyclin (CYCD). Due to their hydrophobic properties, they did not adsorb polar hormones and nutrients in the culture medium. At the same time, acid hydrolysis of casein provided a complex organic nitrogen source and growth factors, alleviating the metabolic stress of primary explants transitioning from heterotrophic to autotrophic. All three factors synergistically improved the synchronicity and overall efficiency of primary induction.

[0013] Based on further optimization of the above scheme, the specific steps for preparing pectin oligosaccharides are as follows: Food-grade pectin (esterification degree DE < 30%, galacturonic acid content ≥ 75%) is added to purified water at 50–55°C, and stirred at 200–300 rpm for 30–40 min at a constant temperature. The mass-to-volume ratio of food-grade pectin to purified water is 1.5–2 g: 100 mL, and the pH is adjusted to 4.4–4.6. Then, endogalacturonase (enzyme activity ≥ 30000 U / g) is added, with a mass ratio of endogalacturonase to food-grade pectin powder of 0.5–0.7: 100. The mixture is stirred at 150–200 rpm for 3.5–4.5 h at a constant temperature of 50 ± 0.5°C. Then, the temperature is raised to 95–100°C and held for 9–11 min (to terminate enzyme inactivation). After decolorization with activated carbon and desalination by ion exchange, the solution is filtered through a polyethersulfone (PES) ultrafiltration membrane with a molecular weight cutoff of 3 kDa at a constant temperature of 30–35°C and an operating pressure of 0.25–0.35 MPa. The permeate is collected, followed by filtration through a PES ultrafiltration membrane with a molecular weight cutoff of 0.5 kDa at an operating pressure of 0.3–0.4 MPa. The retentate is collected. Finally, the solution is concentrated under vacuum at 55–60°C and -0.09–-0.095 MPa to a solids content of 20%–25%. Then, it is spray-dried using a centrifugal spray dryer with an inlet air temperature of 145–155°C, an outlet air temperature of 75–80°C, a feed rate of 5–10 mL / min, and a centrifugal atomizer speed of 18000–20000 rpm until the moisture content is ≤5%.

[0014] Based on further optimization of the above scheme, the specific steps for preparing the fluorine-modified mesoporous silica nanoparticles are as follows: Hexadecyltrimethylammonium bromide (CTAB) is dissolved in ultrapure water to prepare a 0.5% (w / w) CTAB solution, and the pH is adjusted to 10–11 using a 2 mol / L sodium hydroxide solution. The solution is then heated in a water bath to 58–62°C. Next, tetraethyl orthosilicate and 1H,1H,2H,2H-perfluorodecyltriethoxysilane are mixed at a mass ratio of 19:1 and added dropwise to the CTAB solution at a rate of 0.5–1 mL / min. The volume ratio of the mixture to the CTAB solution is 1:40. The mixture is stirred at 350–400 rpm while being added dropwise. After the addition is complete, stirring continues for 1 minute. The reaction was carried out for 8–2.2 hours, maintaining a constant temperature of 58–62℃ throughout. After the reaction, the mixture was centrifuged at 8000–10000 rpm for 10–15 minutes, the supernatant was discarded, and the bottom precipitate was collected. Then, anhydrous ethanol was added to the precipitate to the original volume, and the mixture was sonicated at 20 kHz and 95–105 W for 4–6 minutes. The mixture was centrifuged again for 9–11 minutes, the supernatant was discarded, and the washing was repeated 3–4 times. Finally, the washed precipitate was placed in a fume hood to air dry at room temperature, and then transferred to a muffle furnace. The temperature was increased to 545–555℃ at a rate of 1.8–2.2℃ / min, and calcined at this temperature for 4.8–5.2 hours. After naturally cooling to room temperature, fluorine-modified mesoporous silica nanoparticles with a pore size of 2–5 nm were obtained.

[0015] Based on further optimization of the above scheme, the specific steps for preparing the chitosan quaternary ammonium salt-cinnamaldehyde emulsion are as follows: First, dissolve the chitosan quaternary ammonium salt (degree of substitution ≥90%) in ultrapure water, and stir at 200-300 rpm for 20-30 minutes in a constant temperature water bath at 40-45℃ to prepare a 1% (w / w) aqueous solution, which is then pre-cooled to 0-4℃; then, mix cinnamaldehyde and Tween 80 at a mass ratio of 4:1, and pre-cool to 0-4℃ to obtain the oil phase; subsequently, under constant temperature conditions of 0-4℃, add the pre-cooled water... The solution was placed in the jacketed reaction cup of a high-speed shear emulsifier, and the rotation speed was adjusted to 10,000–12,000 rpm. Then, the oil phase was added dropwise to the aqueous solution at a rate of 0.8–1.2 mL / min, with a mass ratio of oil phase to aqueous solution of 1:9. After the addition was completed, the emulsifier was kept at a constant temperature and rotation speed for 9–11 min to obtain a crude emulsion. Finally, the crude emulsion was transferred to a high-pressure homogenizer and homogenized three times at a pressure of 100 MPa. After each homogenization, the emulsion was cooled in an ice bath at 0–4 °C for 4–6 min to obtain the final product.

[0016] Based on further optimization of the above scheme, in step S5, the proliferation culture is specifically as follows: the culture bottle is placed in an environment of 28±0.5℃ and air humidity of 60%~70%RH, and cultured with a light intensity of 2000~2200Lux and a light cycle of 12h / d, and subcultured once every 19~21 days; during subculture, the clustered buds are cut into single plants (the aging callus and weak buds at the base are removed, and the strong single buds are retained for transfer).

[0017] Based on further optimization of the above scheme, in step S5, the proliferation and propagation culture medium includes MS medium, sucrose, 6-BA, NAA, fucoidan (degree of polymerization 2-8), 24-epibrassinolide, monosilicate aqueous solution, azacitidine, methyl jasmonic acid, α-lipoic acid, β-nicotinamide mononucleotide, acarbose, nobiletin and agar, and the pH is adjusted to 5.8.

[0018] Based on further optimization of the above scheme, the concentrations of sucrose, 6-BA, NAA, fucoidan oligosaccharide, 24-epibrassinolide, monosilicate aqueous solution, azacitidine, methyl jasmonic acid, α-lipoic acid, β-nicotinamide mononucleotide, acarbose, nobiletin, and agar are respectively 29.5–30.5 g / L, 1.9–2.1 mg / L, 0.4–0.6 mg / L, 24.5–25.5 mg / L, 0.07–0.09 mg / L, 18–19 mL / L, 0.11–0.13 mg / L, 0.04–0.06 mg / L, 7.5–8.5 mg / L, 2.4–2.6 mg / L, 19–21 mg / L, 0.7–1.3 mg / L, and 6.8–7.2 g / L.

[0019] Azacitidine reduces abnormally elevated DNA methylation levels during subculture, maintains normal expression of key genes for rooting and growth, and addresses epigenetic drift at the nuclear level. β-nicotinamide mononucleotide activates mitochondrial and chloroplast DNA repair enzymes, addressing seedling vigor decline caused by accumulated organelle genome mutations. Together, these two mechanisms form a dual-layer protection of "nuclear epigenetic stability and organelle genome stability," increasing the number of subcultures and rooting rate. This study utilizes alginate oligosaccharides to activate the endogenous cytokinin signaling pathway and upregulate histidine kinase receptor expression, thereby enhancing the sensitivity of seedlings to exogenous 6-BA. This synergistic effect with 6-BA and NAA (exogenous supplementation and endogenous sensitization) significantly slows down hormone desensitization. 24-epibrassin lactone is used to synergistically promote vascular tissue differentiation within the shoot, enhancing the independence and vascular integrity of lateral buds and reducing vascular damage during cutting and grafting. Methyl jasmonate regulates auxin polar transport, inhibiting disordered callus proliferation at the base of clustered shoots and reducing the ineffective consumption of nutrients by callus clusters. Norihesperidin promotes receptor endocytosis and recycling, maintaining cell membrane receptor density and addressing hormone desensitization at its root. Acarbose blocks the extracellular hydrolysis of sucrose by callus, directing nutrients to the shoot tip tissue with high sucrose transporter protein expression. These five synergistic effects achieve stable receptor numbers, enhanced receptor activity, targeted nutrient distribution, and sound vascular structure, effectively improving the rate of robust seedlings. By using α-lipoic acid to enhance the photochemical efficiency of PSII and strengthen the photosynthetic autotrophic capacity of seedlings, and by using monosilicic acid aqueous solution to improve tissue mechanical strength and reduce hormone-induced vitrification edema, the two synergistic effects can significantly reduce the rate of photooxidative burns under strong light after transplanting and significantly shorten the seedling recovery period.

[0020] Based on further optimization of the above scheme, the specific preparation method of monosilicic acid aqueous solution is as follows: First, tetraethyl orthosilicate (TEOS) is added to anhydrous ethanol and stirred at 180–220 rpm for 18–22 min at room temperature to form an organic phase. The mass-to-volume ratio of tetraethyl orthosilicate to anhydrous ethanol is 347 g: 200 mL. Then, the pH of ultrapure water is adjusted to 2.5–3.0 using 37% (v / v) concentrated hydrochloric acid to obtain an acidic aqueous solution. The volume ratio of ultrapure water to anhydrous ethanol is 3:1. The acidic aqueous solution is then added dropwise to the organic phase at a rate of 1–2 mL / min, maintaining a constant temperature of 39–41 °C throughout the process, while stirring at 230–270 rpm. Afterward, the mixture was stirred at a constant temperature for 2.3–2.7 h to obtain a hydrolysate. The hydrolysate was then transferred to a rotary evaporator and distilled under reduced pressure at 54–56 °C and -0.085–-0.095 MPa for 40–60 min (to completely remove ethanol and some water from the system). Sorbitol and citric acid were then added sequentially to the concentrate, and the mixture was stirred at 35–40 °C and 200–250 rpm for 15–20 min. The mass ratio of sorbitol, citric acid, and tetraethyl orthosilicate was 15:5:347. The pH was adjusted to 2.8–3.2 using 0.1 mol / L dilute hydrochloric acid. Finally, the mixture was brought to a final volume with ultrapure water to obtain a monosilicic acid aqueous solution with a SiO2 content of 10 g / L.

[0021] Based on further optimization of the above scheme, in step S6, the rooting culture is specifically as follows: the culture bottle is placed in an environment of 25±1℃ and air humidity of 60%~70%RH, and cultured with a light intensity of 2000~3000Lux and a light cycle of 12h / d for 29~31 days.

[0022] Based on further optimization of the above scheme, in step S6, the rooting induction medium includes MS medium, sucrose, 6-BA, NAA, melatonin, L-rhamnose, chitosan oligosaccharide, strigolactone analog (GR24), hydroxypropyl-β-cyclodextrin, chitosan oligosaccharide (degree of polymerization 4-6), manganese dioxide nanozyme, trichomoniasisin and agar, and the pH is adjusted to 5.8.

[0023] Based on further optimization of the above scheme, the concentrations of sucrose, 6-BA, NAA, melatonin, L-rhamnose, chitosan oligosaccharide, strigolactone analog, hydroxypropyl-β-cyclodextrin, chitosan oligosaccharide, manganese dioxide nanozyme, saliva extract, and agar are 29.5–30.5 g / L, 2.4–2.6 mg / L, 0.9–1.1 mg / L, 0.5–0.7 mg / L, 11.5–12.5 mg / L, 0.04–0.06 g / L, 0.02–0.04 mg / L, 0.11–0.13 g / L, 0.04–0.06 g / L, 3.8–4.2 mg / L, 0.2–0.4 mg / L, and 6.8–7.2 g / L, respectively.

[0024] By using strigolactone analogs to regulate the directional differentiation of vascular tissue, adventitious root vascular cylinders are induced to extend towards the stem vascular bundles, forming continuous xylem and phloem transport channels. This fundamentally solves the problem of pseudoroots (poor vascular bundle connection) and optimizes root architecture, promoting taproot elongation and orderly lateral root development, while reducing disordered fibrous root clustering. Combined with tuftin to activate adenylate cyclase and upregulate intracellular cAMP levels, the entire vascular chain construction of "intraroot vascular differentiation and connection point connection" is achieved through the regulation of auxin polar transport, thereby improving root transport efficiency and reducing transplant wilting rate. Hydroxypropyl-β-cyclodextrin's hydrophobic interior specifically adsorbs hydrophobic autotoxins such as phenolic acids and fatty acids secreted by roots, reducing the concentration of free autotoxins in the culture medium, relieving their inhibition of root tip meristem, maintaining continuous root tip elongation and root hair development, thus avoiding root physiological decline in the later stages of rooting; chitosan oligosaccharide induces the differentiation of more root hairs in the root epidermis, increasing the root absorption area, while simultaneously forming an antibacterial protective film on the root surface; melatonin balances the auxin concentration at the base of different buds, shortening the rooting initiation time difference, significantly improving the uniformity of batch seedlings, while inducing programmed cell death in the root cortex, promoting aerenchyma formation, and alleviating anaerobic root rot after transplanting; L-rhamnose promotes the deposition of cellulose and pectin in the root cortex cell wall, improving the mechanical toughness of fleshy roots; manganese dioxide nanoenzymes with SOD-like and CAT-like dual enzyme activities provide long-lasting catalysis to remove endogenous ROS, maintaining root tip meristem vitality; thus, by utilizing endogenous and exogenous synergistic effects, the problems of autotoxin and premature oxidative aging are solved, increasing the total root length and shortening the transplanting recovery period. By pre-activating the mycorrhizal symbiotic signaling pathway in the root system with chitosan oligosaccharides, the expression of nutrient transport proteins is upregulated, which solves the problem of stunted seedlings due to nutrient deficiency after transplanting from the root, allowing sterile tissue culture seedlings to quickly adapt to the soil symbiotic environment.

[0025] Based on further optimization of the above scheme, the specific steps for preparing the manganese dioxide nanozyme are as follows: First, potassium permanganate is added to ultrapure water and stirred at 300-400 rpm for 15-20 min at room temperature to prepare a 0.05 mol / L potassium permanganate solution. The potassium permanganate solution is then transferred to a constant temperature water bath stirring device and stirred continuously at 250-300 rpm. The water bath is heated and the temperature is stabilized at 78-82℃. Then, a 0.08 mol / L mannitol solution is prepared using ultrapure water, and the mannitol solution is added dropwise to the potassium permanganate solution at a rate of 1-1.5 mL / min, with a volume ratio of mannitol solution to potassium permanganate solution of 1:1. Under constant temperature conditions of 78-82℃, the mannitol solution is added dropwise while stirring. Stir at 300–350 rpm. After the addition is complete, continue stirring for 28–32 minutes. After the reaction is complete, remove the reaction solution from the bath and allow it to cool naturally to room temperature. Select a regenerated cellulose dialysis bag with a molecular weight cutoff of 1 kDa, and first activate it by soaking it in ultrapure water for 9–11 minutes. Then, fill the cooled reaction solution into the dialysis bag and seal both ends tightly (to prevent leakage). Finally, place the dialysis bag in a container filled with ultrapure water, with a volume ratio of dialysis solution to feed solution of not less than 50:1. Dialyze the entire process at 0–4°C, changing the ultrapure water every 6 hours, for a total of 24 hours (to thoroughly remove unreacted potassium permanganate, mannitol, and small molecule salt impurities), and you will obtain manganese dioxide nanozyme (particle size 4–6 nm).

[0026] The following are the technical effects of this invention: This invention constructs a synergistic system of "antibacterial, anti-browning, cell stabilization, dormancy breaking, and differentiation promotion" through primary induction culture medium. On the one hand, a broad-spectrum antibacterial system simultaneously covers endophytic bacteria and latent endophytic fungi, and combined with gradient disinfection technology, it significantly reduces the primary contamination rate under the premise of low cell damage, solving the problems of difficulty in balancing explant disinfection intensity and survival rate, and latent endophytic contamination. On the other hand, through multiple mechanisms of osmotic pressure stabilization, organelle protection, and browning regulation, it blocks cell autophagy and silent death caused by starch decomposition, alleviates phenolic acid-mediated dedifferentiation arrest, and breaks the physiological dormancy of underground buds, narrowing the germination time difference between different buds. Ultimately, the overall system achieves low primary culture contamination rate, low browning rate, high bud germination synchronicity, and fast callus initiation speed, significantly improving the sterile seedling acquisition rate and laying a high-quality starting material for subsequent propagation.

[0027] By using a proliferation culture medium, a multi-pathway synergy of "epistemological stability, hormone sensitization, nutrient orientation, vascular health, and enhanced photosynthesis" is achieved. This involves sensitizing endogenous hormone signals and maintaining receptor homeostasis to delay hormone desensitization during subculture. Combined with auxin polarity regulation to inhibit ineffective callus growth at the base, nutrient supply is directed to the shoot tip meristem, ensuring a high proliferation rate while reducing ineffective nutrient consumption and increasing the proportion of robust shoots. Simultaneously, by addressing both nuclear genome epigenetic modification and organelle genome repair, epigenetic drift and seedling vigor decline after multiple subcultures are suppressed. Furthermore, the photosynthetic capacity and tissue mechanical strength of the shoots are simultaneously enhanced, reducing the risk of vitrification and shortening the subsequent transplanting recovery period. Ultimately, this achieves stable proliferation efficiency and a high rate of robust seedlings after multiple subcultures, with intact seedling morphology, sound vascular structure, and strong physiological activity, solving the problems of difficulty in balancing proliferation efficiency and seedling quality, and epigenetic drift during subculture, inherent in traditional tissue culture.

[0028] By using a rooting induction medium, a regulatory system of "vascular connectivity, configuration optimization, anti-aging and survival maintenance, and symbiotic pre-adaptation" was constructed. First, it directionally regulates vascular tissue differentiation, connecting the xylem and phloem transport channels between the rootstock and stem, addressing the root-rooting problem of "apparent rooting but poor transport" at its source, while simultaneously optimizing root configuration and promoting the elongation of the taproot and the orderly development of lateral roots. Second, through the adsorption of autotoxic substances and the removal of endogenous reactive oxygen species, it alleviates root autotoxicity decline and premature oxidative aging in the later stages of rooting, maintaining the continuous meristematic capacity of the root tip and normal root hair development. Third, it pre-activates mycorrhizal symbiotic signaling pathways and upregulates the expression of nutrient transport proteins, allowing aseptic tissue culture seedlings to adapt to the soil symbiotic environment in advance, solving the problem of nutrient deficiency and stunted seedlings after transplanting. Ultimately, it achieves high root uniformity, sound root function, strong fleshy root toughness, low wilting rate after transplanting, short recovery period, and significantly improved survival rate and environmental adaptability of tissue culture seedlings, solving problems such as poor root development and insufficient transplanting stability in traditional tissue culture.

[0029] The method of this invention significantly improves the induction rate and proliferation coefficient of buds, and the buds are robust, tender green, and free from deformities. At the same time, it obtains robust root systems and effectively improves the quality of tissue culture seedlings, providing a high-quality seedling foundation for subsequent hardening and transplanting, which is conducive to the standardized mass production of Hedysarum heterotropoides seedlings, the improvement of genetic traits, and the protection of biodiversity. Attached Figure Description

[0030] Figure 1 This is a schematic diagram illustrating the induction of callus formation from *Heliotropium indicum* explants in an embodiment of the present invention.

[0031] Figure 2 This is a schematic diagram illustrating the effect of inducing and propagating the explant shoots of *Liriope muscari* in an embodiment of the present invention.

[0032] Figure 3 This is a schematic diagram of the rooting culture of *Liriope muscari* explants in an embodiment of the present invention.

[0033] Figure 4 This is a schematic diagram of tissue culture seedlings of *Liriope muscari* in an embodiment of the present invention. Detailed Implementation

[0034] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0035] Example 1: A highly efficient in vitro regeneration and propagation method for *Ligustrum lucidum* stem tips includes: Step S1, Material Selection: Select young dormant buds from the top of the underground stem of *Heliotropium indicum* as explants (preferably select individuals with plump buds, tightly wrapped bracts, and no browning or rot), and trim them to a length of 0.5 ± 0.1 cm. Step S2, Material Pretreatment: The material from Step S1 is cleaned, specifically including: First, removing the soil and residual leaves from the surface of the explant rhizome and rinsing it with running water; then immersing it in a 0.5% (v / v) household detergent (i.e., dishwashing liquid, commercially available brands can be used, with sodium fatty alcohol polyoxyethylene ether sulfate as the main surfactant and an effective content ≥15%) aqueous solution for 8 minutes, stirring at 50 rpm for 20 seconds every 3 minutes (using the surfactant components in the dishwashing liquid to wet the surface of the explant, dissolve the surface wax, and remove soil attachments); then rinsing it with tap water, and then transferring it to a 0.1% (w / w) carbendazim (N-(2-benzoimidazole)-methyl carbamate) solution for 8 minutes (to kill the fungal spores attached to the surface); finally, rinsing it with tap water, sealing the container with gauze, and rinsing it continuously under running water for 8 minutes to complete the pretreatment.

[0036] Step S3, Explant Surface Disinfection and Shoot Tip Trimming: Turn on the laminar flow hood and perform ultraviolet disinfection for 25 minutes. Then, perform gradient surface disinfection on the pretreated explants, specifically including: First, immerse the sterile container containing the explants in a 75% ethanol solution for 9 seconds, shaking the container twice per second during this period; then, remove the explants and rinse them once with sterile distilled water (to initially wash away the ethanol); then, add the explants to a 2% sodium hypochlorite solution and treat for 9 minutes, shaking the container once per second for 10 seconds every 2 minutes during this period; finally, discard the sodium hypochlorite solution and rinse repeatedly with sterile distilled water four times (to thoroughly remove residual disinfectant).

[0037] After disinfection, the explants were moved to the workbench, and the scale-like bracts on the outside of the buds were peeled off layer by layer with a scalpel and forceps. The aging cut at the lower end of the stem segment was removed and the stem tip was trimmed with meristem.

[0038] Step S4, Primary Induction and Callus Culture: Use sterile filter paper to absorb the surface moisture of the shoot tip with meristem and vertically inoculate it into a culture bottle containing primary induction medium (inoculate one shoot tip with meristem in each bottle, ensuring full contact between the cut and the medium) for primary culture. Specifically, after inoculation, transfer the culture bottle to a dark culture room and culture at 28±0.5℃ and 60%RH for 10 days (to induce callus initiation and bud sprouting, and to avoid light-induced phenolic oxidation and browning). Then transfer the culture bottle to a light culture room and culture for 22 days with a light intensity of 1800 Lux and a light cycle of 12h / d. During this period, regularly observe and record the contamination, browning, callus occurrence, and bud sprouting.

[0039] The primary induction medium included MS medium, sucrose, 6-BA (6-benzylaminopurine), NAA (1-naphthaleneacetic acid), acid-hydrolyzed casein, spermidine, ε-polylysine hydrochloride, trehalose, pectin oligosaccharides (degree of polymerization 3-7), 5-ALA (5-aminolevulinate), fluorine-modified mesoporous silica nanoparticles, disodium pyrroloquinoline quinone, chitosan quaternary ammonium salt-cinnamaldehyde emulsion, and agar, with the pH adjusted to 5.8. The concentrations of sucrose, 6-BA, NAA, acid-hydrolyzed casein, spermidine, ε-polylysine hydrochloride, trehalose, pectin oligosaccharide, 5-ALA, fluorine-modified mesoporous silica nanoparticles, disodium pyrroloquinoline quinone, chitosan quaternary ammonium salt-cinnamaldehyde emulsion, and agar were 29.5 g / L, 0.9 mg / L, 0.24 g / L, 19.5 mg / L, 0.03 g / L, 0.11 g / L, 0.04 g / L, 4.5 mg / L, 0.02 g / L, 3.8 mg / L, 5 mL / L, and 7.3 g / L, respectively.

[0040] The specific steps for preparing fluorine-modified mesoporous silica nanoparticles are as follows: Hexadecyltrimethylammonium bromide (CTAB) is dissolved in ultrapure water to prepare a 0.5% (w / w) CTAB solution. The pH is adjusted to 10.5 using a 2 mol / L sodium hydroxide solution, and the solution is heated to 60°C in a water bath. Then, tetraethyl orthosilicate and 1H,1H,2H,2H-perfluorodecyltriethoxysilane are mixed at a mass ratio of 19:1 and added dropwise to the CTAB solution at a rate of 0.7 mL / min. The volume ratio of the mixed solution to the CTAB solution is 1:40. The mixture is stirred at 375 rpm while being added dropwise until the addition is complete. Afterward, stirring was continued for 2 hours, maintaining a constant temperature of 60℃ throughout the process. After the reaction was completed, the mixture was centrifuged at 9000 rpm for 12.5 min, the supernatant was discarded, and the bottom precipitate was collected. Then, anhydrous ethanol was added to the precipitate to the original volume, and the mixture was sonicated at 20 kHz and 100 W for 5 min. The mixture was centrifuged again for 10 min, the supernatant was discarded, and the washing was repeated 3 times. Finally, the washed precipitate was placed in a fume hood to air dry at room temperature, and then transferred to a muffle furnace. The temperature was increased to 550℃ at a rate of 2℃ / min, and calcined at a constant temperature for 5 hours. After naturally cooling to room temperature, fluorine-modified mesoporous silica nanoparticles with a pore size of 2-5 nm were obtained.

[0041] The specific steps for preparing chitosan quaternary ammonium salt-cinnamaldehyde emulsion are as follows: First, dissolve chitosan quaternary ammonium salt (substitution degree ≥90%) in ultrapure water and stir at 250 rpm for 25 min in a 42℃ constant temperature water bath to prepare a 1% (w / w) aqueous solution, which is then pre-cooled to 2℃. Next, mix cinnamaldehyde and Tween 80 at a mass ratio of 4:1 and pre-cool to 2℃ to obtain the oil phase. Then, under constant temperature of 2℃, place the pre-cooled aqueous solution in the jacketed reaction cup of a high-speed shear emulsifier and adjust the speed to 11000 rpm. Then, add the oil phase dropwise to the aqueous solution at a rate of 1 mL / min, with a mass ratio of oil phase to aqueous solution of 1:9. After the addition is completed, emulsify at constant temperature and speed for 10 min to obtain a crude emulsion. Finally, transfer the crude emulsion to a high-pressure homogenizer and homogenize it three times under a pressure of 100 MPa. After each homogenization, cool it in an ice bath at 2℃ for 5 min to obtain the final product.

[0042] Step S5, Bud Induction and Proliferation Culture: After the initial culture is completed, select sterile buds that are morphologically complete, vigorous, and free of disease spots (i.e., no browning, no vitrification, no necrotic spots) as the starting material for propagation. Transfer the starting material to culture bottles containing propagation culture medium (one plantlet per bottle) for propagation culture. Specifically, place the culture bottles in an environment of 28±0.5℃ and 60%RH, with a light intensity of 2000Lux and a photoperiod of 12h / d. Subculture every 21 days. During subculture, cut the clustered buds into single plants (remove aging callus and weak buds from the base, and retain strong single buds for transfer).

[0043] The proliferation and propagation medium includes MS medium, sucrose, 6-BA, NAA, fucoidan (degree of polymerization 2-8), 24-epibrassin lactone, monosilicate aqueous solution, azacitidine, methyl jasmonic acid, α-lipoic acid, β-nicotinamide mononucleotide, acarbose, nobiletin and agar, and the pH is adjusted to 5.8. The concentrations of sucrose, 6-BA, NAA, fucoidan oligosaccharide, 24-epibrassinolide, monosilicate aqueous solution, azacitidine, methyl jasmonic acid, α-lipoic acid, β-nicotinamide mononucleotide, acarbose, nobiletin, and agar were 29.5 g / L, 1.9 mg / L, 0.4 mg / L, 24.5 mg / L, 0.07 mg / L, 18 mL / L, 0.11 mg / L, 0.04 mg / L, 7.5 mg / L, 2.4 mg / L, 19 mg / L, 0.7 mg / L, and 6.8 g / L, respectively.

[0044] Step S6, Rooting Induction Culture: Select robust, 3-4cm tall, and fully expanded sterile seedlings from the proliferation culture stage. Remove aging callus and excess leaves from the base. Transfer each seedling individually to a culture bottle containing rooting induction medium for rooting culture. Specifically, place the culture bottle in an environment of 25±1℃ and 60%RH, with a light intensity of 2000Lux and a photoperiod of 12h / d for 31 days.

[0045] The rooting induction medium included MS medium, sucrose, 6-BA, NAA, melatonin, L-rhamnose, chitosan oligosaccharide, strigolactone analog (GR24), hydroxypropyl-β-cyclodextrin, chitosan oligosaccharide (degree of polymerization 4-6), manganese dioxide nanozyme, trichoderma, and agar, with the pH adjusted to 5.8. The concentrations of sucrose, 6-BA, NAA, melatonin, L-rhamnose, chitosan oligosaccharide, strigolactone analog, hydroxypropyl-β-cyclodextrin, chitosan oligosaccharide, manganese dioxide nanozyme, trichoderma, and agar were 29.5 g / L, 2.4 mg / L, 0.9 mg / L, 0.5 mg / L, 11.5 mg / L, 0.04 g / L, 0.02 mg / L, 0.11 g / L, 0.04 g / L, 3.8 mg / L, 0.2 mg / L, and 6.8 g / L, respectively.

[0046] The specific steps for preparing manganese dioxide nanozymes are as follows: First, potassium permanganate is added to ultrapure water and stirred at 350 rpm for 17 min at room temperature to prepare a 0.05 mol / L potassium permanganate solution. The potassium permanganate solution is then transferred to a constant temperature water bath stirring device and stirred continuously at 275 rpm. The water bath is heated and the temperature is stabilized at 80℃. Next, a 0.08 mol / L mannitol solution is prepared using ultrapure water, and the mannitol solution is added dropwise to the potassium permanganate solution at a rate of 1.2 mL / min, with a volume ratio of mannitol solution to potassium permanganate solution of 1:1. Under constant temperature of 80℃, the solution is added dropwise while stirring at 325 rpm. After the addition is complete, stir continuously for 30 minutes. After the reaction is complete, remove the reaction solution from the bath and allow it to cool naturally to room temperature. Select a regenerated cellulose dialysis bag with a molecular weight cutoff of 1 kDa, first soak it in ultrapure water for 10 minutes to activate it, then fill the cooled reaction solution into the dialysis bag and seal both ends (to prevent leakage). Finally, place the dialysis bag in a container filled with ultrapure water, with the volume ratio of the dialysis solution to the feed solution not less than 50:1. Dialyze the entire process at 2°C and replace the ultrapure water every 6 hours, for a total of 24 hours (to thoroughly remove unreacted potassium permanganate, mannitol, and small molecule salt impurities), and you will get manganese dioxide nanozyme (particle size 4-6 nm).

[0047] Example 2: A highly efficient in vitro regeneration and propagation method for *Ligustrum lucidum* stem tips includes: Step S1, Material Selection: Select young dormant buds from the top of the underground stem of *Heliotropium indicum* as explants (preferably select individuals with plump buds, tightly wrapped bracts, and no browning or rot), and trim them to a length of 0.5 ± 0.1 cm. Step S2, Material Pretreatment: The material from Step S1 is cleaned, specifically including: First, removing the soil and residual leaves from the surface of the explant rhizome and rinsing it with running water; then immersing it in a 0.5% (v / v) household detergent (i.e., dishwashing liquid, commercially available brands can be used, with sodium fatty alcohol polyoxyethylene ether sulfate as the main surfactant and an effective content ≥15%) aqueous solution for 10 minutes, stirring at 65 rpm for 15 seconds every 4 minutes (using the surfactant components in the dishwashing liquid to wet the surface of the explant, dissolve the surface wax, and remove soil attachments); then rinsing it with tap water, and then transferring it to a 0.1% (w / w) carbendazim (N-(2-benzoimidazole)-methyl carbamate) solution for 10 minutes (killing the fungal spores attached to the surface); finally, rinsing it with tap water, sealing the container with gauze, and rinsing it continuously under running water for 10 minutes to complete the pretreatment.

[0048] Step S3, Explant Surface Disinfection and Shoot Tip Trimming: Turn on the laminar flow hood and perform ultraviolet disinfection for 30 minutes. Then, perform gradient surface disinfection on the pretreated explants, specifically including: First, immerse the sterile container containing the explants in a 75% ethanol solution for 10 seconds, shaking the container twice per second during this period; then, remove the explants and rinse them once with sterile distilled water (to initially remove ethanol); then, add the explants to a 2% sodium hypochlorite solution and treat for 10 minutes, shaking the container once per second for 12 seconds every 2 minutes during this period; finally, discard the sodium hypochlorite solution and rinse repeatedly with sterile distilled water four times (to thoroughly remove residual disinfectant).

[0049] After disinfection, the explants were moved to the workbench, and the scale-like bracts on the outside of the buds were peeled off layer by layer with a scalpel and forceps. The aging cut at the lower end of the stem segment was removed and the stem tip was trimmed with meristem.

[0050] Step S4, Primary Induction and Callus Culture: Use sterile filter paper to absorb the surface moisture of the shoot tip with meristem and vertically inoculate it into a culture bottle containing primary induction medium (inoculate one shoot tip with meristem in each bottle, ensuring full contact between the cut and the medium) for primary culture. Specifically, after inoculation, transfer the culture bottle to a dark culture room and culture at 28±0.5℃ and 65%RH for 8.5 days (to induce callus initiation and bud sprouting, and to avoid light-induced phenolic oxidation and browning). Then transfer the culture bottle to a light culture room and culture for 20 days with a light intensity of 1900 Lux and a light cycle of 12h / d. During this period, regularly observe and record the contamination, browning, callus occurrence, and bud sprouting.

[0051] The primary induction medium included MS medium, sucrose, 6-BA (6-benzylaminopurine), NAA (1-naphthaleneacetic acid), acid-hydrolyzed casein, spermidine, ε-polylysine hydrochloride, trehalose, pectin oligosaccharides (degree of polymerization 3-7), 5-ALA (5-aminolevulinate), fluorine-modified mesoporous silica nanoparticles, disodium pyrroloquinoline quinone, chitosan quaternary ammonium salt-cinnamaldehyde emulsion, and agar, with the pH adjusted to 5.8. The concentrations of sucrose, 6-BA, NAA, acid-hydrolyzed casein, spermidine, ε-polylysine hydrochloride, trehalose, pectin oligosaccharide, 5-ALA, fluorine-modified mesoporous silica nanoparticles, disodium pyrroloquinoline quinone, chitosan quaternary ammonium salt-cinnamaldehyde emulsion, and agar were 30 g / L, 1 mg / L, 1 mg / L, 0.25 g / L, 20 mg / L, 0.04 g / L, 0.12 g / L, 0.05 g / L, 5 mg / L, 0.03 g / L, 4 mg / L, 6 mL / L, and 7.5 g / L, respectively.

[0052] The preparation steps for fluorine-modified mesoporous silica nanoparticles were the same as in Example 1. The preparation steps for the chitosan quaternary ammonium salt-cinnamaldehyde emulsion were the same as in Example 1.

[0053] Step S5, Bud Induction and Proliferation Culture: After the initial culture is completed, select sterile buds that are morphologically complete, vigorous, and free of disease spots (i.e., no browning, no vitrification, no necrotic spots) as the starting material for propagation. Transfer the starting material to culture bottles containing propagation culture medium (one plantlet per bottle) for propagation culture. Specifically, place the culture bottles in an environment of 28±0.5℃ and 65%RH, with a light intensity of 2100Lux and a photoperiod of 12h / d. Subculture every 20 days. During subculture, cut the clustered buds into single plants (remove aging callus and weak buds from the base, and retain strong single buds for transfer).

[0054] The proliferation and propagation medium includes MS medium, sucrose, 6-BA, NAA, fucoidan (degree of polymerization 2-8), 24-epibrassin lactone, monosilicate aqueous solution, azacitidine, methyl jasmonic acid, α-lipoic acid, β-nicotinamide mononucleotide, acarbose, nobiletin and agar, and the pH is adjusted to 5.8. The concentrations of sucrose, 6-BA, NAA, fucoidan oligosaccharide, 24-epibrassinolide, monosilicate aqueous solution, azacitidine, methyl jasmonic acid, α-lipoic acid, β-nicotinamide mononucleotide, acarbose, nobiletin, and agar were 30 g / L, 2 mg / L, 0.5 mg / L, 25 mg / L, 0.08 mg / L, 18.5 mL / L, 0.12 mg / L, 0.05 mg / L, 8 mg / L, 2.5 mg / L, 20 mg / L, 1 mg / L, and 7 g / L, respectively.

[0055] Step S6, Rooting Induction Culture: Select robust, 3-4cm tall, and fully expanded sterile seedlings from the proliferation culture stage. Remove aging callus and excess leaves from the base. Transfer each seedling individually to a culture bottle containing rooting induction medium for rooting culture. Specifically, place the culture bottle in an environment of 25±1℃ and 65%RH, with a light intensity of 2500Lux and a photoperiod of 12h / d for 30 days.

[0056] The rooting induction medium included MS medium, sucrose, 6-BA, NAA, melatonin, L-rhamnose, chitosan oligosaccharide, strigolactone analog (GR24), hydroxypropyl-β-cyclodextrin, chitosan oligosaccharide (degree of polymerization 4-6), manganese dioxide nanozyme, trichoderma, and agar, with the pH adjusted to 5.8. The concentrations of sucrose, 6-BA, NAA, melatonin, L-rhamnose, chitosan oligosaccharide, strigolactone analog, hydroxypropyl-β-cyclodextrin, chitosan oligosaccharide, manganese dioxide nanozyme, trichoderma, and agar were 30 g / L, 2.5 mg / L, 1 mg / L, 0.6 mg / L, 12 mg / L, 0.05 g / L, 0.03 mg / L, 0.12 g / L, 0.05 g / L, 4 mg / L, 0.3 mg / L, and 7 g / L, respectively.

[0057] The preparation steps for manganese dioxide nanozymes are the same as those in Example 1.

[0058] Example 3: A highly efficient in vitro regeneration and propagation method for *Ligustrum lucidum* stem tips includes: Step S1, Material Selection: Select young dormant buds from the top of the underground stem of *Heliotropium indicum* as explants (preferably select individuals with plump buds, tightly wrapped bracts, and no browning or rot), and trim them to a length of 0.5 ± 0.1 cm. Step S2, Material Pretreatment: The material from Step S1 is cleaned, specifically including: First, removing the soil and residual leaves from the surface of the explant rhizome and rinsing it with running water; then immersing it in a 0.5% (v / v) household detergent (i.e., dishwashing liquid, commercially available brands can be used, with sodium fatty alcohol polyoxyethylene ether sulfate as the main surfactant and an effective content ≥15%) aqueous solution for 12 minutes, stirring at 80 rpm for 10 seconds every 5 minutes (using the surfactant components in the dishwashing liquid to wet the surface of the explant, dissolve the surface wax, and remove soil attachments); then rinsing it with tap water, and then transferring it to a 0.1% (w / w) carbendazim (N-(2-benzoimidazole)-methyl carbamate) solution for 12 minutes (killing the fungal spores attached to the surface); finally, rinsing it with tap water, sealing the container with gauze, and rinsing it continuously under running water for 12 minutes to complete the pretreatment.

[0059] Step S3, Explant Surface Disinfection and Shoot Tip Trimming: Turn on the laminar flow hood and perform ultraviolet disinfection for 35 minutes. Then, perform gradient surface disinfection on the pretreated explants, specifically including: First, immerse the sterile container containing the explants in a 75% ethanol solution for 11 seconds, shaking the container twice per second during this period; then, remove the explants and rinse them twice with sterile distilled water (to initially remove ethanol); next, add the explants to a 2% sodium hypochlorite solution and treat for 11 minutes, shaking the container once per second for 15 seconds every 2 minutes during this period; finally, discard the sodium hypochlorite solution and rinse repeatedly five times with sterile distilled water (to thoroughly remove residual disinfectant).

[0060] After disinfection, the explants were moved to the workbench, and the scale-like bracts on the outside of the buds were peeled off layer by layer with a scalpel and forceps. The aging cut at the lower end of the stem segment was removed and the stem tip was trimmed with meristem.

[0061] Step S4, Primary Induction and Callus Culture: Use sterile filter paper to absorb the surface moisture of the shoot tip with meristem and vertically inoculate it into a culture bottle containing primary induction medium (inoculate one shoot tip with meristem in each bottle, ensuring full contact between the cut and the medium) for primary culture. Specifically, after inoculation, transfer the culture bottle to a dark culture room and culture at 28±0.5℃ and 70%RH for 7 days (to induce callus initiation and bud sprouting, and to avoid light-induced phenolic oxidation and browning). Then transfer the culture bottle to a light culture room and culture for 18 days with a light intensity of 2000 Lux and a light cycle of 12h / d. During this period, regularly observe and record contamination, browning, callus occurrence, and bud sprouting.

[0062] The primary induction medium included MS medium, sucrose, 6-BA (6-benzylaminopurine), NAA (1-naphthaleneacetic acid), acid-hydrolyzed casein, spermidine, ε-polylysine hydrochloride, trehalose, pectin oligosaccharides (degree of polymerization 3-7), 5-ALA (5-aminolevulinate), fluorine-modified mesoporous silica nanoparticles, disodium pyrroloquinoline quinone, chitosan quaternary ammonium salt-cinnamaldehyde emulsion, and agar, with the pH adjusted to 5.8. The concentrations of sucrose, 6-BA, NAA, acid-hydrolyzed casein, spermidine, ε-polylysine hydrochloride, trehalose, pectin oligosaccharide, 5-ALA, fluorine-modified mesoporous silica nanoparticles, disodium pyrroloquinoline quinone, chitosan quaternary ammonium salt-cinnamaldehyde emulsion, and agar were 30.5 g / L, 1.1 mg / L, 1.1 mg / L, 0.26 g / L, 20.5 mg / L, 0.05 g / L, 0.13 g / L, 0.06 g / L, 5.5 mg / L, 0.04 g / L, 4.2 mg / L, 7 mL / L, and 7.7 g / L, respectively.

[0063] The preparation steps for fluorine-modified mesoporous silica nanoparticles were the same as in Example 1. The preparation steps for the chitosan quaternary ammonium salt-cinnamaldehyde emulsion were the same as in Example 1.

[0064] Step S5, Bud Induction and Proliferation Culture: After the initial culture is completed, select sterile buds that are morphologically complete, vigorous, and free of disease spots (i.e., no browning, no vitrification, no necrotic spots) as the starting material for propagation. Transfer the starting material to culture bottles containing propagation culture medium (one plantlet per bottle) for propagation culture. Specifically, place the culture bottles in an environment of 28±0.5℃ and 70%RH, with a light intensity of 2200Lux and a photoperiod of 12h / d. Subculture every 19 days. During subculture, cut the clustered buds into single plants (remove aging callus and weak buds from the base, and retain strong single buds for transfer).

[0065] The proliferation and propagation medium includes MS medium, sucrose, 6-BA, NAA, fucoidan (degree of polymerization 2-8), 24-epibrassin lactone, monosilicate aqueous solution, azacitidine, methyl jasmonic acid, α-lipoic acid, β-nicotinamide mononucleotide, acarbose, nobiletin and agar, and the pH is adjusted to 5.8. The concentrations of sucrose, 6-BA, NAA, fucoidan oligosaccharide, 24-epibrassinolone, monosilicate aqueous solution, azacitidine, methyl jasmonic acid, α-lipoic acid, β-nicotinamide mononucleotide, acarbose, nobiletin, and agar were 30.5 g / L, 2.1 mg / L, 0.6 mg / L, 25.5 mg / L, 0.09 mg / L, 19 mL / L, 0.13 mg / L, 0.06 mg / L, 8.5 mg / L, 2.6 mg / L, 21 mg / L, 1.3 mg / L, and 7.2 g / L, respectively.

[0066] Step S6, Rooting Induction Culture: Select robust, 3-4cm tall, and fully expanded sterile seedlings from the proliferation culture stage. Remove aging callus and excess leaves from the base. Transfer each seedling individually to a culture bottle containing rooting induction medium for rooting culture. Specifically, place the culture bottle in an environment of 25±1℃ and 70%RH, with a light intensity of 3000Lux and a photoperiod of 12h / d for 29 days.

[0067] The rooting induction medium included MS medium, sucrose, 6-BA, NAA, melatonin, L-rhamnose, chitosan oligosaccharide, strigolactone analog (GR24), hydroxypropyl-β-cyclodextrin, chitosan oligosaccharide (degree of polymerization 4-6), manganese dioxide nanozyme, trichoderma, and agar, with the pH adjusted to 5.8. The concentrations of sucrose, 6-BA, NAA, melatonin, L-rhamnose, chitosan oligosaccharide, strigolactone analog, hydroxypropyl-β-cyclodextrin, chitosan oligosaccharide, manganese dioxide nanozyme, trichoderma, and agar were 30.5 g / L, 2.6 mg / L, 1.1 mg / L, 0.7 mg / L, 12.5 mg / L, 0.06 g / L, 0.04 mg / L, 0.13 g / L, 0.06 g / L, 4.2 mg / L, 0.4 mg / L, and 7.2 g / L, respectively.

[0068] The preparation steps for manganese dioxide nanozymes are the same as those in Example 1.

[0069] Example 4: As another preferred embodiment of the present invention, based on any one of Examples 1 to 3, the pectin oligosaccharide preparation steps are as follows: food-grade pectin (degree of esterification DE < 30%, galacturonic acid content ≥ 75%) is added to purified water at 52°C, and stirred at 250 rpm for 35 min at a constant temperature. The mass-to-volume ratio of food-grade pectin to purified water is 1.75 g: 100 mL, and the pH is adjusted to 4.5. Then, endogalacturonase (enzyme activity ≥ 30000 U / g) is added, and the mass ratio of endogalacturonase to food-grade pectin powder is 0.6: 100. The mixture is stirred at 175 rpm for 4 h at a constant temperature of 50 ± 0.5°C. Then, the temperature is raised to 97°C and kept at that temperature for 1 hour. The enzyme was deactivated at 0 min (to terminate enzyme inactivation), and then decolorized with activated carbon and desalted by ion exchange. The solution was then filtered through a polyethersulfone (PES) ultrafiltration membrane with a molecular weight cutoff of 3 kDa at an operating pressure of 0.3 MPa at 32°C. The permeate was collected, followed by filtration through a PES ultrafiltration membrane with a molecular weight cutoff of 0.5 kDa at an operating pressure of 0.35 MPa, and the retentate was collected. Finally, the solution was vacuum concentrated at 57°C and -0.09 MPa to a solids content of 22%. It was then spray-dried using a centrifugal spray dryer with an inlet air temperature of 150°C, an outlet air temperature of 77°C, a feed rate of 7.5 mL / min, and a centrifugal atomizer speed of 19000 rpm until the moisture content was ≤5%.

[0070] The activated carbon decolorization process is as follows: 767 pharmaceutical-grade activated carbon (200-300 mesh) is used. The system pH is maintained at 4.2 at 62℃. Activated carbon is added to the enzyme-inactivated mixture at a mass-to-volume ratio of 10 g:1 L, and the mixture is stirred at 200 rpm for 40 min. Afterward, the activated carbon is removed using a Buchner funnel, followed by fine filtration using a 0.45 μm microporous membrane to obtain a clear enzymatic hydrolysate. The ion exchange desalination process is as follows: a cation-anion column series process is used. The cation column uses 001×7 strong acid styrene-based cation exchange resin with a height-to-diameter ratio of 4:1, and the anion column uses D301 macroporous weakly basic styrene-based anion exchange resin with a height-to-diameter ratio of 4:1. The volume of the feed solution flowing through per hour is 1.2 times the resin packing volume. Ion exchange desalination is carried out at room temperature (25±1℃).

[0071] The specific preparation method of monosilicic acid aqueous solution is as follows: First, tetraethyl orthosilicate (TEOS) is added to anhydrous ethanol and stirred at 200 rpm for 20 min at room temperature to form an organic phase. The mass-to-volume ratio of tetraethyl orthosilicate to anhydrous ethanol is 347 g: 200 mL. Then, the pH of ultrapure water is adjusted to 2.7 using 37% (v / v) concentrated hydrochloric acid to obtain an acidic aqueous solution. The volume ratio of ultrapure water to anhydrous ethanol is 3:1. The acidic aqueous solution is added dropwise to the organic phase at a rate of 1.5 mL / min, maintaining a constant temperature of 40°C throughout the process, while stirring at 250 rpm. After the addition was complete, the mixture was stirred at a constant temperature for 2.5 hours to obtain the hydrolysate. Then, the hydrolysate was transferred to a rotary evaporator and distilled under reduced pressure at 55℃ and -0.09MPa for 50 minutes (to completely remove the ethanol and some water from the system). Sorbitol and citric acid were then added to the concentrate in sequence, and the mixture was stirred at 225 rpm for 17 minutes at 37℃. The mass ratio of sorbitol, citric acid and tetraethyl orthosilicate was 15:5:347. The pH was adjusted to 3 with 0.1 mol / L dilute hydrochloric acid. Finally, the volume was brought to a final volume with ultrapure water to obtain a monosilicic acid aqueous solution with a SiO2 content of 10 g / L.

[0072] Example 5: As another preferred embodiment of the present invention, based on any one of Examples 1 to 3, after rooting culture, seedling hardening and transplanting acclimatization are also carried out, specifically including: moving the culture bottle to a room temperature diffused light environment, opening the bottle cap to harden the seedlings for 3 to 5 days (preferably 4 days, gradually adapting to external humidity and light), during which a small amount of sterile water can be added to keep the culture medium moist to avoid rapid water loss and wilting; then, taking out the tissue culture seedlings and washing the culture medium attached to the roots with clean water (to avoid culture medium residue causing substrate mold and root rot); then, transplanting them into a mixed substrate of peat, perlite and vermiculite in a mass ratio of 2:1:1, watering thoroughly after planting, covering with a moisturizing film to maintain air humidity above 80% RH, and gradually removing the film for ventilation after one week; finally, shading 50% for the first two weeks after transplanting (to avoid direct strong sunlight), and then gradually increasing the light, and managing water and fertilizer as usual.

[0073] Comparative Example 1: A method for in vitro cultivation of *Ligustrum lucidum* stem tips includes: Step S1, Material Selection: Same as Step S1 in Example 2.

[0074] Step S2, material pretreatment: consistent with step S2 in Example 2.

[0075] Step S3, Explant surface disinfection and stem tip trimming: Same as step S3 in Example 2.

[0076] Step S4, Primary Induction and Callus Culture: Use sterile filter paper to absorb the surface moisture of the stem tip with meristem and vertically inoculate it into a culture bottle containing primary induction medium (inoculate one stem tip with meristem in each bottle to ensure full contact between the cut and the culture medium) for primary culture. The primary culture method is the same as step S4 in Example 2.

[0077] The primary induction medium included MS medium, sucrose, 6-BA (6-benzylaminopurine), NAA (1-naphthaleneacetic acid), acid-hydrolyzed casein, spermidine, ε-polylysine hydrochloride, trehalose, pectin oligosaccharides (degree of polymerization 3-7), 5-ALA (5-aminolevulinate), silica nanoparticles, disodium pyrroloquinoline quinone, chitosan quaternary ammonium salt-cinnamaldehyde emulsion, and agar, with the pH adjusted to 5.8. The concentrations of sucrose, 6-BA, NAA, acid-hydrolyzed casein, spermidine, ε-polylysine hydrochloride, trehalose, pectin oligosaccharide, 5-ALA, fluorine-modified mesoporous silica nanoparticles, disodium pyrroloquinoline quinone, chitosan quaternary ammonium salt-cinnamaldehyde emulsion, and agar were 30 g / L, 1 mg / L, 1 mg / L, 0.25 g / L, 20 mg / L, 0.04 g / L, 0.12 g / L, 0.05 g / L, 5 mg / L, 0.03 g / L, 4 mg / L, 6 mL / L, and 7.5 g / L, respectively.

[0078] Step S5, bud induction and propagation culture: consistent with step S2 in Example 2.

[0079] Step S6, Rooting Induction Culture: Same as step S6 in Example 2.

[0080] Comparative Example 2: A method for in vitro cultivation of *Ligustrum lucidum* stem tips includes: Step S1, Material Selection: Same as Step S1 in Example 2.

[0081] Step S2, material pretreatment: consistent with step S2 in Example 2.

[0082] Step S3, Explant surface disinfection and stem tip trimming: Same as step S3 in Example 2.

[0083] Step S4, Primary Induction and Callus Culture: Use sterile filter paper to absorb the surface moisture of the stem tip with meristem and vertically inoculate it into a culture bottle containing primary induction medium (inoculate one stem tip with meristem in each bottle to ensure full contact between the cut and the culture medium) for primary culture. The primary culture method is the same as step S4 in Example 2.

[0084] The primary induction medium included MS medium, sucrose, 6-BA (6-benzylaminopurine), NAA (1-naphthaleneacetic acid), acid-hydrolyzed casein, spermidine, ε-polylysine hydrochloride, trehalose, pectin oligosaccharides (degree of polymerization 3-7), 5-ALA (5-aminolevulinate), fluorine-modified mesoporous silica nanoparticles, chitosan quaternary ammonium salt-cinnamaldehyde emulsion, and agar, with the pH adjusted to 5.8. The concentrations of sucrose, 6-BA, NAA, acid-hydrolyzed casein, spermidine, ε-polylysine hydrochloride, trehalose, pectin oligosaccharide, 5-ALA, fluorine-modified mesoporous silica nanoparticles, chitosan quaternary ammonium salt-cinnamaldehyde emulsion, and agar were 30 g / L, 1 mg / L, 1 mg / L, 0.25 g / L, 20 mg / L, 0.04 g / L, 0.12 g / L, 0.05 g / L, 5 mg / L, 0.03 g / L, 6 mL / L, and 7.5 g / L, respectively.

[0085] Step S5, bud induction and propagation culture: consistent with step S2 in Example 2.

[0086] Step S6, Rooting Induction Culture: Same as step S6 in Example 2.

[0087] Comparative Example 3: A method for in vitro cultivation of *Ligustrum lucidum* stem tips includes: Step S1, Material Selection: Same as Step S1 in Example 2.

[0088] Step S2, material pretreatment: consistent with step S2 in Example 2.

[0089] Step S3, Explant surface disinfection and stem tip trimming: Same as step S3 in Example 2.

[0090] Step S4, Primary Induction and Callus Culture: Use sterile filter paper to absorb the surface moisture of the stem tip with meristem and vertically inoculate it into a culture bottle containing primary induction medium (inoculate one stem tip with meristem in each bottle to ensure full contact between the cut and the culture medium) for primary culture. The primary culture method is the same as step S4 in Example 2.

[0091] The primary induction medium included MS medium, sucrose, 6-BA (6-benzylaminopurine), NAA (1-naphthaleneacetic acid), acid-hydrolyzed casein, spermidine, ε-polylysine hydrochloride, trehalose, pectin oligosaccharides (degree of polymerization 3-7), 5-ALA (5-aminolevulinate), fluorine-modified mesoporous silica nanoparticles, disodium pyrroloquinoline quinone, cinnamaldehyde, and agar, with the pH adjusted to 5.8. The concentrations of sucrose, 6-BA, NAA, acid-hydrolyzed casein, spermidine, ε-polylysine hydrochloride, trehalose, pectin oligosaccharide, 5-ALA, fluorinated mesoporous silica nanoparticles, disodium pyrroloquinoline quinone, cinnamaldehyde, and agar were 30 g / L, 1 mg / L, 1 mg / L, 0.25 g / L, 20 mg / L, 0.04 g / L, 0.12 g / L, 0.05 g / L, 5 mg / L, 0.03 g / L, 4 mg / L, 0.06 g / L, and 7.5 g / L, respectively.

[0092] Step S5, bud induction and propagation culture: consistent with step S2 in Example 2.

[0093] Step S6, Rooting Induction Culture: Same as step S6 in Example 2.

[0094] Comparative Example 4: A method for in vitro cultivation of *Ligustrum lucidum* stem tips includes: Step S1, Material Selection: Same as Step S1 in Example 2.

[0095] Step S2, material pretreatment: consistent with step S2 in Example 2.

[0096] Step S3, Explant surface disinfection and stem tip trimming: Same as step S3 in Example 2.

[0097] Step S4, primary induction and callus culture: consistent with step S4 in Example 2.

[0098] Step S5, bud induction and proliferation culture: After the initial culture is completed, select sterile buds that are morphologically complete, vigorous, and free of disease spots (i.e., no browning, no vitrification, no necrotic spots) as the starting material for proliferation; and transfer the starting material for proliferation to a culture bottle containing proliferation culture medium (one bud per bottle) for proliferation culture. The proliferation culture method is the same as step S5 in Example 2.

[0099] The propagation medium included MS medium, sucrose, 6-BA, NAA, fucoidan oligosaccharides (degree of polymerization 2–8), 24-epibrassinolone, azacitidine, methyl jasmonic acid, α-lipoic acid, β-nicotinamide mononucleotide, acarbose, norihesperidin, and agar, with the pH adjusted to 5.8. The concentrations of sucrose, 6-BA, NAA, fucoidan oligosaccharides, 24-epibrassinolone, azacitidine, methyl jasmonic acid, α-lipoic acid, β-nicotinamide mononucleotide, acarbose, norihesperidin, and agar were 30 g / L, 2 mg / L, 0.5 mg / L, 25 mg / L, 0.08 mg / L, 0.12 mg / L, 0.05 mg / L, 8 mg / L, 2.5 mg / L, 20 mg / L, 1 mg / L, and 7 g / L, respectively.

[0100] Step S6, Rooting Induction Culture: Same as step S6 in Example 2.

[0101] Comparative Example 5: A method for in vitro cultivation of *Ligustrum lucidum* stem tips includes: Step S1, Material Selection: Same as Step S1 in Example 2.

[0102] Step S2, material pretreatment: consistent with step S2 in Example 2.

[0103] Step S3, Explant surface disinfection and stem tip trimming: Same as step S3 in Example 2.

[0104] Step S4, primary induction and callus culture: consistent with step S4 in Example 2.

[0105] Step S5, bud induction and proliferation culture: After the initial culture is completed, select sterile buds that are morphologically complete, vigorous, and free of disease spots (i.e., no browning, no vitrification, no necrotic spots) as the starting material for proliferation; and transfer the starting material for proliferation to a culture bottle containing proliferation culture medium (one bud per bottle) for proliferation culture. The proliferation culture method is the same as step S5 in Example 2.

[0106] The propagation medium included MS medium, sucrose, 6-BA, NAA, fucoidan oligosaccharides (degree of polymerization 2-8), 24-epibrassinolide, aqueous monosilicate, azacitidine, methyl jasmonic acid, β-nicotinamide mononucleotide, acarbose, norihesperidin, and agar, with the pH adjusted to 5.8. The concentrations of sucrose, 6-BA, NAA, fucoidan oligosaccharides, 24-epibrassinolide, aqueous monosilicate, azacitidine, methyl jasmonic acid, β-nicotinamide mononucleotide, acarbose, norihesperidin, and agar were 30 g / L, 2 mg / L, 0.5 mg / L, 25 mg / L, 0.08 mg / L, 18.5 mL / L, 0.12 mg / L, 0.05 mg / L, 2.5 mg / L, 20 mg / L, 1 mg / L, and 7 g / L, respectively.

[0107] Step S6, Rooting Induction Culture: Same as step S6 in Example 2.

[0108] Comparative Example 6: A method for in vitro cultivation of *Ligustrum lucidum* stem tips includes: Step S1, Material Selection: Same as Step S1 in Example 2.

[0109] Step S2, material pretreatment: consistent with step S2 in Example 2.

[0110] Step S3, Explant surface disinfection and stem tip trimming: Same as step S3 in Example 2.

[0111] Step S4, primary induction and callus culture: consistent with step S4 in Example 2.

[0112] Step S5, bud induction and proliferation culture: After the initial culture is completed, select sterile buds that are morphologically complete, vigorous, and free of disease spots (i.e., no browning, no vitrification, no necrotic spots) as the starting material for proliferation; and transfer the starting material for proliferation to a culture bottle containing proliferation culture medium (one bud per bottle) for proliferation culture. The proliferation culture method is the same as step S5 in Example 2.

[0113] The propagation medium included MS medium, sucrose, 6-BA, NAA, fucoidan oligosaccharides (degree of polymerization 2-8), 24-epibrassinolide, monosilicic acid aqueous solution, azacitidine, methyl jasmonic acid, α-lipoic acid, acarbose, nobiletin, and agar, with the pH adjusted to 5.8. The concentrations of sucrose, 6-BA, NAA, fucoidan oligosaccharides, 24-epibrassinolide, monosilicic acid aqueous solution, azacitidine, methyl jasmonic acid, α-lipoic acid, acarbose, nobiletin, and agar were 30 g / L, 2 mg / L, 0.5 mg / L, 25 mg / L, 0.08 mg / L, 18.5 mL / L, 0.12 mg / L, 0.05 mg / L, 8 mg / L, 20 mg / L, 1 mg / L, and 7 g / L, respectively.

[0114] Step S6, Rooting Induction Culture: Same as step S6 in Example 2.

[0115] Comparative Example 7: A method for in vitro cultivation of *Ligustrum lucidum* stem tips includes: Step S1, Material Selection: Same as Step S1 in Example 2.

[0116] Step S2, material pretreatment: consistent with step S2 in Example 2.

[0117] Step S3, Explant surface disinfection and stem tip trimming: Same as step S3 in Example 2.

[0118] Step S4, primary induction and callus culture: consistent with step S4 in Example 2.

[0119] Step S5, bud induction and propagation culture: consistent with step S5 in Example 2.

[0120] Step S6, Rooting Induction Culture: Select healthy, sterile seedlings with a height of 3-4 cm and fully expanded leaves from the proliferation culture stage, remove the aging callus and excess leaves at the base, and transfer each seedling to a culture bottle containing rooting induction culture medium for rooting culture. The rooting culture method is the same as step S6 in Example 2.

[0121] The rooting induction medium included MS medium, sucrose, 6-BA, NAA, melatonin, chitosan oligosaccharide, strigolactone analog (GR24), hydroxypropyl-β-cyclodextrin, chitosan oligosaccharide (degree of polymerization 4-6), manganese dioxide nanozyme, trichoderma, and agar, with the pH adjusted to 5.8. The concentrations of sucrose, 6-BA, NAA, melatonin, chitosan oligosaccharide, strigolactone analog, hydroxypropyl-β-cyclodextrin, chitosan oligosaccharide, manganese dioxide nanozyme, trichoderma, and agar were 30 g / L, 2.5 mg / L, 1 mg / L, 0.6 mg / L, 0.05 g / L, 0.03 mg / L, 0.12 g / L, 0.05 g / L, 4 mg / L, 0.3 mg / L, and 7 g / L, respectively.

[0122] Comparative Example 8: A method for in vitro cultivation of *Ligustrum lucidum* stem tips includes: Step S1, Material Selection: Same as Step S1 in Example 2.

[0123] Step S2, material pretreatment: consistent with step S2 in Example 2.

[0124] Step S3, Explant surface disinfection and stem tip trimming: Same as step S3 in Example 2.

[0125] Step S4, primary induction and callus culture: consistent with step S4 in Example 2.

[0126] Step S5, bud induction and propagation culture: consistent with step S5 in Example 2.

[0127] Step S6, Rooting Induction Culture: Select healthy, sterile seedlings with a height of 3-4 cm and fully expanded leaves from the proliferation culture stage, remove the aging callus and excess leaves at the base, and transfer each seedling to a culture bottle containing rooting induction culture medium for rooting culture. The rooting culture method is the same as step S6 in Example 2.

[0128] The rooting induction medium included MS medium, sucrose, 6-BA, NAA, melatonin, L-rhamnose, chitosan oligosaccharide, strigolactone analog (GR24), hydroxypropyl-β-cyclodextrin, chitosan oligosaccharide (degree of polymerization 4-6), trichoderma, and agar, with the pH adjusted to 5.8. The concentrations of sucrose, 6-BA, NAA, melatonin, L-rhamnose, chitosan oligosaccharide, strigolactone analog, hydroxypropyl-β-cyclodextrin, chitosan oligosaccharide, trichoderma, and agar were 30 g / L, 2.5 mg / L, 1 mg / L, 0.6 mg / L, 12 mg / L, 0.05 g / L, 0.03 mg / L, 0.12 g / L, 0.05 g / L, 0.3 mg / L, and 7 g / L, respectively.

[0129] Comparative Example 9: A method for in vitro cultivation of *Ligustrum lucidum* stem tips includes: Step S1, Material Selection: Same as Step S1 in Example 2.

[0130] Step S2, material pretreatment: consistent with step S2 in Example 2.

[0131] Step S3, Explant surface disinfection and stem tip trimming: Same as step S3 in Example 2.

[0132] Step S4, primary induction and callus culture: consistent with step S4 in Example 2.

[0133] Step S5, bud induction and propagation culture: consistent with step S5 in Example 2.

[0134] Step S6, Rooting Induction Culture: Select healthy, sterile seedlings with a height of 3-4 cm and fully expanded leaves from the proliferation culture stage, remove the aging callus and excess leaves at the base, and transfer each seedling to a culture bottle containing rooting induction culture medium for rooting culture. The rooting culture method is the same as step S6 in Example 2.

[0135] The rooting induction medium included MS medium, sucrose, 6-BA, NAA, melatonin, L-rhamnose, chitosan oligosaccharide, strigolactone analog (GR24), hydroxypropyl-β-cyclodextrin, chitosan oligosaccharide (degree of polymerization 4-6), manganese dioxide nanozyme, and agar, with the pH adjusted to 5.8. The concentrations of sucrose, 6-BA, NAA, melatonin, L-rhamnose, chitosan oligosaccharide, strigolactone analog, hydroxypropyl-β-cyclodextrin, chitosan oligosaccharide, manganese dioxide nanozyme, and agar were 30 g / L, 2.5 mg / L, 1 mg / L, 0.6 mg / L, 12 mg / L, 0.05 g / L, 0.03 mg / L, 0.12 g / L, 0.05 g / L, 4 mg / L, and 7 g / L, respectively.

[0136] The explant contamination rate, callus induction rate, shoot proliferation coefficient, proportion of healthy and effective shoots, upper limit of stable subculturing, root induction rate, root conduction efficiency (relative value), rooting rate, and transplant survival rate of the embryos cultured in Examples 1 to 3 and Comparative Examples 1 to 9 were tested respectively; among which: Explant contamination rate = (number of contaminated embryos / number of inoculated explants) × 100%; Callus induction rate = (number of explants that form healthy callus and have bud sprouting / total number of inoculated explants) × 100%; effective induction is indicated by dense, light green callus, plump and expansive buds, and no browning or rotting. Bud proliferation coefficient = total number of new buds at the end of the cultivation cycle / initial number of buds at the time of inoculation; The percentage of healthy and effective buds = (number of healthy buds meeting the transfer criteria / total number of buds) × 100%; The upper limit of stable subculture is the maximum number of generations that can be continuously transferred under the premise that the proliferation coefficient, effective shoot ratio, and rooting rate are not lower than the qualified threshold during continuous subculture (which reflects the long-term stability of the tissue culture system). Root induction rate = (Number of rooted tissue culture seedlings / Total number of inoculated rooted seedlings) × 100%; Root transport efficiency = (dye ascent rate of tissue culture seedlings / dye ascent rate of wild divisions of *Heliotropium indicum*) × 100%; Specific test method: Take healthy tissue culture seedlings that have been rooted for 30 days, wash off the culture medium at the base, absorb the surface moisture, and then completely immerse the roots in a 0.5% acidic red ink solution and let them stand under light for 2 hours; After that, take them out and rinse the surface with clean water, longitudinally cut the root-stem junction, and measure the height at which the red dye is transported from the roots to the stem segment; Simultaneously, use wild divisions of *Heliotropium indicum* of the same age as a control to calculate the relative transport efficiency. Rooting rate = (Number of plants with at least 3 healthy roots after cultivation / Total number of seedlings before rooting cultivation) × 100%; Transplant survival rate = (number of plants that survived and sprouted new leaves 20 days after transplanting / total number of plants with good roots selected before transplanting) × 100%; all transplanting was carried out in accordance with the method described in Example 5; The final test results are shown in the table below (all results are averages from multiple tests):

[0137] As shown in the table above, the *Heliotropium indicum* shoot tip in vitro regeneration system of this invention achieves efficient propagation throughout the entire process of "low contamination, high induction, strong proliferation, stable subculture, superior rooting, and high survival" by combining specific culture media in the three stages of primary generation, proliferation, and rooting. This effectively reduces the explant contamination rate, increases the callus induction rate, enhances the shoot proliferation coefficient, improves the rooting rate and transplanting seedling rate, and ensures the robustness and survival stability of the final plants.

Claims

1. A method for high-efficiency regeneration and propagation of *Litsea cubeba* stem tips in vitro, characterized in that: include: Step S1, Material Selection: Select young dormant buds from the top of the underground stem of *Heliotropium indicum* as explants and trim them to a length of 0.5 ± 0.1 cm. Step S2, Material Pretreatment: Clean the material from step S1; Step S3, Explant Surface Disinfection and Stem Tip Trimming: Turn on the ultra-clean workbench and perform ultraviolet disinfection for 25-35 minutes, and perform gradient surface disinfection on the pretreated explants; after disinfection, transfer the explants to the workbench, and use a scalpel and forceps to peel off the scale-like bracts on the outside of the bud layer by layer, cut off the aging cut at the lower end of the stem segment, and trim it into a stem tip with meristem; Step S4, Primary Induction and Callus Culture: Use sterile filter paper to absorb the surface moisture of the shoot tip with meristem, and vertically inoculate it into a culture flask containing primary induction medium for primary culture; Step S5, bud induction and proliferation culture: After the initial culture is completed, select morphologically complete, vigorous, and disease-free sterile buds as the proliferation starting material; and transfer the proliferation starting material to a culture bottle containing proliferation culture medium for proliferation culture. Step S6, Rooting Induction Culture: Select healthy, sterile seedlings with a height of 3-4 cm and fully expanded leaves from the proliferation culture stage, remove the aging callus and excess leaves at the base, and transfer each seedling to a culture bottle containing rooting induction medium for rooting culture to obtain tissue culture seedlings.

2. The method for high-efficiency regeneration and propagation of *Litsea cubeba* stem tips in vitro according to claim 1, characterized in that: Step S2 specifically includes: First, removing the soil and residual leaves from the surface of the explant rhizome and rinsing it initially with running water; then immersing it in a 0.5% (v / v) household detergent solution for 8–12 minutes, stirring at 50–80 rpm for 10–20 seconds every 3–5 minutes; then rinsing it with tap water and transferring it to a 0.1% (w / w) carbendazim solution for 8–12 minutes; finally, rinsing it with tap water, sealing the container with gauze, and rinsing it continuously under running water for 8–12 minutes to complete the pretreatment.

3. A method for high-efficiency in vitro regeneration and propagation of *Litsea cubeba* stem tips according to claim 1 or 2, characterized in that: In step S3, the gradient surface disinfection of the explants specifically includes: first, immersing the sterile container containing the explants in a 75% ethanol solution for 9–11 seconds, shaking the container twice per second during this period; then, removing the explants and rinsing them 1–2 times with sterile distilled water; next, adding the explants to a 2% sodium hypochlorite solution and treating them for 9–11 minutes, shaking the container once per second for 10–15 seconds every 2 minutes during this period; finally, discarding the sodium hypochlorite solution and rinsing the container repeatedly with sterile distilled water 4–5 times.

4. A method for high-efficiency in vitro regeneration and propagation of *Litsea cubeba* stem tips according to claim 2 or 3, characterized in that: In step S4, the initial culture is as follows: after inoculation, the culture bottle is transferred to a dark culture room and cultured at a constant temperature of 28±0.5℃ and an air humidity of 60%~70%RH for 7~10 days. Then, the culture bottle is transferred to a light culture room and cultured at a light intensity of 1800~2000Lux and a light cycle of 12h / d for 18~22 days. During this period, the contamination, browning, callus occurrence and bud sprouting are observed and recorded regularly.

5. A method for high-efficiency in vitro regeneration and propagation of *Litsea cubeba* stem tips according to claim 3 or 4, characterized in that: In step S4, the primary induction medium includes MS medium, sucrose, 6-BA, NAA, acid-hydrolyzed casein, spermidine, ε-polylysine hydrochloride, trehalose, pectin oligosaccharide, 5-ALA, fluorine-modified mesoporous silica nanoparticles, disodium pyrroloquinoline quinone, chitosan quaternary ammonium salt-cinnamaldehyde emulsion, and agar, and the pH is adjusted to 5.

8.

6. A method for high-efficiency in vitro regeneration and propagation of *Litsea cubeba* stem tips according to claim 3 or 5, characterized in that: The concentrations of sucrose, 6-BA, NAA, acid-hydrolyzed casein, spermidine, ε-polylysine hydrochloride, trehalose, pectin oligosaccharide, 5-ALA, fluorinated mesoporous silica nanoparticles, disodium pyrroloquinoline quinone, chitosan quaternary ammonium salt-cinnamaldehyde emulsion, and agar in the primary induction medium were 29.5–30.5 g / L, 0.9–1.1 mg / L, 0.9–1.1 mg / L, 0.24–0.26 g / L, 19.5–20.5 mg / L, 0.03–0.05 g / L, 0.11–0.13 g / L, 0.04–0.06 g / L, 4.5–5.5 mg / L, 0.02–0.04 g / L, 3.8–4.2 mg / L, 5–7 mL / L, and 7.3–7.7 g / L, respectively.

7. The method for high-efficiency regeneration and propagation of *Litsea cubeba* stem tips in vitro according to claim 6, characterized in that: The specific steps for preparing the pectin oligosaccharide are as follows: Food-grade pectin is added to purified water at 50–55°C and stirred at 200–300 rpm for 30–40 minutes at a constant temperature. The mass-to-volume ratio of food-grade pectin to purified water is 1.5–2 g:100 mL, and the pH is adjusted to 4.4–4.

6. Then, endogalacturonase is added, with a mass ratio of endogalacturonase to food-grade pectin powder of 0.5–0.7:

100. The mixture is stirred at 150–200 rpm for 3.5–4.5 hours at a constant temperature of 50±0.5°C. Next, the temperature is raised to 95–100°C and held for 9–11 minutes, followed by activated carbon decolorization and ion exchange desalting. The mixture is then cooled to 30–35°C. At a constant temperature of ℃, the solution is first filtered through a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 3 kDa at an operating pressure of 0.25–0.35 MPa, and the permeate is collected. Then, it is filtered through a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 0.5 kDa at an operating pressure of 0.3–0.4 MPa, and the retentate is collected. Finally, it is vacuum concentrated at 55–60℃ and -0.09–-0.095 MPa until the solid content is 20%–25%. Then, it is spray-dried using a centrifugal spray dryer with an inlet air temperature of 145–155℃, an outlet air temperature of 75–80℃, a feed rate of 5–10 mL / min, and a centrifugal atomizer speed of 18000–20000 rpm until the moisture content is ≤5%.

8. The method for high-efficiency regeneration and propagation of *Litsea cubeba* stem tips in vitro according to claim 6, characterized in that: The specific steps for preparing the fluorine-modified mesoporous silica nanoparticles are as follows: Hexadecyltrimethylammonium bromide is dissolved in ultrapure water to prepare a 0.5% (w / w) CTAB solution, and the pH is adjusted to 10–11 using a 2 mol / L sodium hydroxide solution. The solution is then heated in a water bath to 58–62°C. Next, tetraethyl orthosilicate and 1H,1H,2H,2H-perfluorodecyltriethoxysilane are mixed at a mass ratio of 19:1 and added dropwise to the CTAB solution at a rate of 0.5–1 mL / min. The volume ratio of the mixture to the CTAB solution is 1:

40. The mixture is stirred at 350–400 rpm while being added dropwise. After the addition is complete, stirring continues for 1 minute. The reaction was carried out for 8–2.2 hours, maintaining a constant temperature of 58–62°C throughout. After the reaction, the mixture was centrifuged at 8000–10000 rpm for 10–15 minutes, the supernatant was discarded, and the bottom precipitate was collected. Then, anhydrous ethanol was added to the precipitate to the original volume, and the mixture was sonicated at 20 kHz and 95–105 W for 4–6 minutes. The mixture was centrifuged again for 9–11 minutes, the supernatant was discarded, and the washing was repeated 3–4 times. Finally, the washed precipitate was placed in a fume hood to air dry at room temperature, and then transferred to a muffle furnace. The temperature was increased to 545–555°C at a rate of 1.8–2.2°C / min, and calcined at this temperature for 4.8–5.2 hours. After naturally cooling to room temperature, the precipitate was obtained.

9. The method for high-efficiency regeneration and propagation of *Litsea cubeba* stem tips in vitro according to claim 6, characterized in that: The specific steps for preparing the chitosan quaternary ammonium salt-cinnamaldehyde emulsion are as follows: First, dissolve the chitosan quaternary ammonium salt in ultrapure water and stir at 200-300 rpm for 20-30 minutes in a constant temperature water bath at 40-45℃ to prepare a 1% (w / w) aqueous solution, which is then pre-cooled to 0-4℃. Next, mix cinnamaldehyde and Tween 80 at a mass ratio of 4:1 and pre-cool to 0-4℃ to obtain the oil phase. Then, under constant temperature conditions of 0-4℃, place the pre-cooled aqueous solution in a high-speed shear emulsifier. The oil phase was added dropwise to the aqueous solution at a rate of 0.8–1.2 mL / min in a jacketed reaction vessel, and the rotation speed was adjusted to 10,000–12,000 rpm. The mass ratio of oil phase to aqueous solution was 1:

9. After the addition was completed, the mixture was emulsified at a constant temperature and rotation speed for 9–11 min to obtain a crude emulsion. Finally, the crude emulsion was transferred to a high-pressure homogenizer and homogenized three times at a pressure of 100 MPa. After each homogenization, the mixture was cooled in an ice bath at 0–4 °C for 4–6 min to obtain the final product.

Images