Tissue culture medium and tissue culture method for culturing ginseng stem cells

Abstract

The invention provides a tissue culture medium and a tissue culture method for culturing ginseng stem cells, and belongs to the field of plant biology. The tissue culture medium comprises a germination medium, a growth medium, an induction medium and a proliferation medium; wherein the germination culture medium takes MS or 1 / 2 MS as a basic culture medium, and further comprises GA3, TDZ, melatonin, chitosan oligosaccharide, cane sugar, activated carbon and plant gel; the growth culture medium takes B5 as a basic culture medium, and further comprises 6-BA, NAA, cane sugar, trehalose, calcium nitrate and plant gel; the induction culture medium takes B5 or MS as a basic culture medium, and further comprises 2, 4-D, KT, jasmonic acid methyl ester, melatonin, functionalized graphene oxide, acid hydrolyzed casein, cane sugar and plant gel. Therefore, on the basis of reducing the culture period of the ginseng stem cells, the active ingredient content of the ginseng stem cells is increased.

Description

Technical Field

[0001] The present application relates to the field of plant biotechnology, and in particular to a tissue culture medium and a tissue culture method for culturing ginseng stem cells. Background Art

[0002] Ginseng, a traditional Chinese medicinal herb, faces extinction due to overharvesting of wild resources and a long growth cycle (4-6 years). Artificial cultivation is limited by land resources, pests and diseases, and unstable levels of active ingredients (such as ginsenosides). Tissue culture technology, which rapidly expands resources through stem cell cultivation, has become a key path to preserving the germplasm gene pool and promoting the sustainable development of Traditional Chinese Medicine.

[0003] Early ginseng tissue culture technology required inducing callus tissue to redifferentiate into adventitious roots or stem cells, which had a long cycle and low efficiency. At the same time, conventional culture media could not effectively activate the synthesis pathways of active ingredients such as ginsenosides, resulting in a decrease in the content of active ingredients in the product and unstable efficacy. Early technologies used high concentrations of auxins (such as 2,4-D) and mitogens (such as KT) to accelerate cell division and shorten the culture cycle, but excessive proliferation would inhibit secondary metabolic pathways (such as ginsenoside synthesis). Secondary metabolism usually requires cells to be in a slow growth or stress state to be activated. For example, although high concentrations of 2,4-D in traditional culture media can promote rapid callus formation, they will inhibit the activity of terpenoid synthases (such as HMGR), resulting in saponin content of only 1% to 5% of natural ginseng. Therefore, how to increase the content of active ingredients in ginseng stem cells while reducing the culture cycle is a technical problem that urgently needs to be solved. Summary of the Invention

[0004] The present application provides a tissue culture medium and a tissue culture method for culturing ginseng stem cells to solve the following technical problem: how to increase the content of active ingredients in ginseng stem cells while reducing the culture cycle of ginseng stem cells.

[0005] In a first aspect, the present application provides a tissue culture medium for culturing ginseng stem cells, comprising a germination medium, a growth medium, an induction medium, and a proliferation medium; wherein,

[0006] The germination medium is based on MS or 1 / 2MS and further includes 3.0-4.0 mg / L GA3, 0.1-0.5 mg / L TDZ, 0.05-0.1 mg / L melatonin, 0.1-0.3 g / L chitosan oligosaccharide, 30 g / L sucrose, 1.0-1.5 g / L activated carbon, and 6.0-6.5 g / L plant gel;

[0007] The growth medium is based on B5 as a basic medium and further includes 1.5-2.0 mg / L of 6-BA, 0.4-0.5 mg / L of NAA, 20 g / L of sucrose, 10 g / L of trehalose, 600 mg / L of calcium nitrate, and 6.0-6.5 g / L of plant gel;

[0008] The induction culture medium is based on B5 or MS as a basic culture medium and further includes 1.5-2.5 mg / L of 2,4-D, 0.5 mg / L of KT, 0.2-0.5 g / L of methyl jasmonate, 0.1-0.3 mg / L of melatonin, 0.5-1.0 mg / L of functionalized graphene oxide, 1.0-1.2 g / L of acid hydrolyzed casein, 30 g / L of sucrose, and 6.0-6.5 g / L of plant gel;

[0009] The proliferation culture medium is based on B5 as a basic culture medium and further includes 2.5-2.8 mg / L of 2,4-D, 0.6-0.7 mg / L of KT, 0.1-0.3 mg / L of brassinolide, 0.2-1.5 g / L of reduced glutathione, 0.05-0.1 g / L of selenomethionine, 0.1-0.3 mg / L of chitosan nanoparticles, 0.1-0.5 g / L of humic acid, 0.1-0.5 g / L of sodium alginate-gelatin hydrogel, 0.5-1.0 g / L of Ag-TiO2 nanoparticle-loaded activated carbon, 10 g / L of sucrose, and 20 g / L of trehalose.

[0010] Optionally, the NH4 + With NO3 — The molar ratio is 1:5.

[0011] Optionally, the specific surface area of ​​the functionalized graphene oxide is 500 to 800 m 2 / g, surface modified with carboxyl groups.

[0012] Optionally, in the sodium alginate-gelatin hydrogel, the mass ratio of sodium alginate to gelatin is 3:1 to 5:1.

[0013] Optionally, in the Ag-TiO2 nanoparticle-loaded activated carbon, the mass of the Ag-TiO2 nanoparticles is 1 to 5% of the mass of the activated carbon.

[0014] Optionally, the preparation method of the Ag-TiO2 nanoparticle-loaded activated carbon includes:

[0015] Mixing metatitanic acid with activated carbon, and then performing a hydrothermal reaction to generate TiO2 nanoparticles and load them on the surface of the activated carbon to obtain TiO2-loaded activated carbon; the hydrothermal reaction temperature is 180°C and the time is 12 hours;

[0016] The TiO2-loaded activated carbon was immersed in silver nitrate solution and then irradiated with UVA light source to make Ag + The Ag-TiO2 nanoparticles are photoreduced to Ag nanoparticles on the surface to obtain the Ag-TiO2 nanoparticles loaded activated carbon.

[0017] In a second aspect, the present application provides a method for tissue culture of ginseng stem cells, using any of the tissue culture medium described in the first aspect, comprising the following steps:

[0018] sterilizing the vernalized ginseng seeds and inoculating them into the germination medium to perform germination culture to obtain germinated seeds;

[0019] transferring the germinated seeds to the growth medium to carry out seedling growth culture to obtain sterile seedlings;

[0020] cutting stem segments of the sterile seedlings and inoculating them into an induction medium for induction culture, and gradually reducing the 2,4-D concentration in the induction medium during the induction culture to obtain callus tissue;

[0021] The callus tissue is transferred to a proliferation culture medium for proliferation culture to obtain ginseng stem cells.

[0022] Optionally, a completely dark environment is used during the germination stage, a CO2 concentration of 800 ppm is introduced, the temperature is 25±0.5°C, and the culture time is 15 to 20 days;

[0023] During the seedling growth stage, a light-dark cycle of 14 hours / 10 hours, a red light to blue light ratio of 7:3, a temperature of 25±0.5° C., and a culture time of 40 to 44 days were adopted.

[0024] Optionally, the induction stage is divided into 15 days of dark culture and 15 days of light / dark cycle of 12 / 12 hours, and the far-red light intensity is increased in the latter stage, the temperature is 24±1° C., and the culture time is 30 days;

[0025] During the proliferation stage, a light / dark cycle of 16 hours and 8 hours was used, wherein pulsed light supplementation was performed every 2 hours with an interval of 10 minutes, the temperature was 26±1° C., and the culture time was 62 to 68 days.

[0026] Optionally, the length of the stem segment is 1 to 2 cm.

[0027] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0028] The present application provides a tissue culture medium for culturing ginseng stem cells. By adjusting the hormone ratio in stages (such as GA3+TDZ in the germination stage and high concentration 2,4-D+KT in the proliferation stage), full process control from dormancy breaking to efficient stem cell amplification is achieved to avoid abnormal differentiation. Methyl jasmonate activates the synthesis pathway of ginsenosides (such as Rg3, Rh2), and combines far-infrared light to enhance light signals, significantly increasing the content of active ingredients. At the same time, melatonin, reduced glutathione, trehalose and other multi-level protections remove ROS, extend cell life and maintain genomic stability. In addition, functionalized graphene oxide enhances nutrient adsorption and signal transduction, and Ag-TiO2 nanoparticles load activated carbon antibacterial-photocatalytic synergy to improve culture efficiency. Thus, on the basis of reducing the culture cycle of ginseng stem cells, the content of active ingredients in ginseng stem cells is increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0030] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0031] Figure 1 A schematic diagram of a process for culturing ginseng stem cells provided in an embodiment of the present application; DETAILED DESCRIPTION

[0032] To make the purpose, technical solutions, and advantages of the embodiments of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings in the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0033] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0034] In a first aspect, the present application provides a tissue culture medium for culturing ginseng stem cells, comprising a germination medium, a growth medium, an induction medium, and a proliferation medium; wherein,

[0035] The germination medium is based on MS or 1 / 2MS and further includes 3.0-4.0 mg / L GA3, 0.1-0.5 mg / L TDZ, 0.05-0.1 mg / L melatonin, 0.1-0.3 g / L chitosan oligosaccharide, 30 g / L sucrose, 1.0-1.5 g / L activated carbon, and 6.0-6.5 g / L plant gel;

[0036] The growth medium is based on B5 as a basic medium and further includes 1.5-2.0 mg / L of 6-BA, 0.4-0.5 mg / L of NAA, 20 g / L of sucrose, 10 g / L of trehalose, 600 mg / L of calcium nitrate, and 6.0-6.5 g / L of plant gel;

[0037] The induction culture medium is based on B5 or MS as a basic culture medium and further includes 1.5-2.5 mg / L of 2,4-D, 0.5 mg / L of KT, 0.2-0.5 g / L of methyl jasmonate, 0.1-0.3 mg / L of melatonin, 0.5-1.0 mg / L of functionalized graphene oxide, 1.0-1.2 g / L of acid hydrolyzed casein, 30 g / L of sucrose, and 6.0-6.5 g / L of plant gel;

[0038] The proliferation culture medium is based on B5 as a basic culture medium and further includes 2.5-2.8 mg / L of 2,4-D, 0.6-0.7 mg / L of KT, 0.1-0.3 mg / L of brassinolide, 0.2-1.5 g / L of reduced glutathione, 0.05-0.1 g / L of selenomethionine, 0.1-0.3 mg / L of chitosan nanoparticles, 0.1-0.5 g / L of humic acid, 0.1-0.5 g / L of sodium alginate-gelatin hydrogel, 0.5-1.0 g / L of Ag-TiO2 nanoparticle-loaded activated carbon, 10 g / L of sucrose, and 20 g / L of trehalose.

[0039] It should be noted that MS medium is the most commonly used basal medium in plant tissue culture. Characterized by a high inorganic salt concentration and a balanced ion ratio, it is suitable for rapid proliferation and callus induction. 1 / 2MS medium is a diluted version of MS medium, primarily reducing the concentration of macronutrients (by half), while other components are the same as MS. B5 medium, developed by Gamborg et al., features low ammonium and high nitrate levels and is suitable for woody plants and the induction of secondary metabolites.

[0040] The germination medium is used to break dormancy, activate embryo germination, and reduce the risk of oxidative damage and contamination; the growth medium is used to balance organ differentiation (buds and roots) to support the morphological construction and nutrient reserves of seedlings; the induction medium is used to induce callus formation, activate secondary metabolism (such as saponin synthesis), and maintain pluripotency. The proliferation medium is used to efficiently amplify stem cells, maintain genomic stability, and simulate the natural microenvironment to enhance cell activity. At the same time, this application rationally designs the components of the culture medium at each stage, and the specific functions of each component are as follows:

[0041] (1) Germination medium

[0042] GA3 (3.0-4.0 mg / L): Gibberellic acid GA3 breaks seed dormancy, activates hydrolases such as α-amylase, and promotes explant embryo germination.

[0043] TDZ (0.1-0.5 mg / L): The cytokinin analog TDZ induces cell division, forms callus tissue, and reduces the risk of browning.

[0044] Melatonin (0.05-0.1mg / L): scavenges reactive oxygen species (ROS), inhibits oxidative damage to explants, and improves survival rate.

[0045] Chitosan oligosaccharide (0.1-0.3g / L): activates plant immune genes (such as PR protein), inhibits fungal contamination, and promotes cell wall relaxation.

[0046] Activated carbon (1.0-1.5g / L): adsorbs phenolic toxins, regulates osmotic pressure, and maintains pH stability of the culture medium.

[0047] GA3+TDZ: Synergistically activate metabolism and cell division, accelerating the embryo to break out of dormancy.

[0048] Melatonin + chitosan oligosaccharide: dual antioxidant and antibacterial protection, reducing the initial contamination rate of explants.

[0049] (2) Growth medium

[0050] 6-BA (1.5-2.0 mg / L) and NAA (0.4-0.5 mg / L): 6-BA promotes bud differentiation, NAA induces root development, and the hormone ratio (3:1) balances organogenesis.

[0051] Trehalose (10g / L): protects cell membrane integrity, prevents dehydration damage, and assists sucrose as a carbon source.

[0052] Calcium nitrate (600 mg / L): provides Ca 2+ Strengthen cell walls, NO3 — Promote nitrogen metabolism and enhance disease resistance.

[0053] 6-BA / NAA ratio (3:1): can precisely regulate bud and root differentiation and avoid excessive callus proliferation.

[0054] Trehalose + calcium nitrate: Combining osmotic protection and nutritional fortification to support the healthy growth of seedlings.

[0055] (3) Induction medium

[0056] 2,4-D (1.5-2.5 mg / L) and KT (0.5 mg / L): 2,4-D maintains the undifferentiated state, while KT promotes cell proliferation and simulates injury signals to induce callus formation.

[0057] Methyl jasmonate (0.2-0.5g / L): activates secondary metabolic pathways (such as ginsenoside synthesis) and enhances adaptability to adversity.

[0058] Functionalized graphene oxide (0.5-1.0 mg / L): Nanocarrier enhances nutrient adsorption and transport, regulates cell membrane potential and promotes signal transduction.

[0059] 2,4-D+methyl jasmonate: maintains the undifferentiated state while activating secondary metabolism and enhancing the accumulation of active ingredients.

[0060] Graphene oxide + acid hydrolyzed casein: Nanomaterials enhance nutrient delivery, and casein provides amino acids to support protein synthesis.

[0061] (4) Proliferation culture medium

[0062] 2,4-D (2.5-2.8 mg / L) and KT (0.6-0.7 mg / L): High concentrations of 2,4-D inhibit differentiation, while KT accelerates division, achieving large-scale expansion of stem cells.

[0063] Reduced glutathione (0.2-1.5g / L): neutralizes ROS, repairs DNA / protein oxidative damage, and maintains genomic stability.

[0064] Sodium alginate-gelatin hydrogel (0.1-0.5g / L): Three-dimensional scaffold simulates the natural cell microenvironment and promotes cell adhesion and proliferation.

[0065] Glutathione + selenomethionine: A dual antioxidant system that prolongs cell life and reduces apoptosis during the expansion process.

[0066] Chitosan nanoparticles + Ag-TiO2 nanoparticles: slow-release nutrition and antibacterial synergy, inhibit contamination and improve amplification efficiency.

[0067] It can be seen that this application has the following significant advantages by rationally designing the components of the culture medium at each stage: (1) Precise stage-by-stage regulation: Through the hormone gradient (such as GA3→6-BA→2,4-D), step-by-step regulation from germination to proliferation is achieved to avoid abnormal differentiation. (2) Multi-level antioxidant system: melatonin, glutathione, trehalose and other synergistic protection significantly improve cell survival rate and functional activity. (3) Nanomaterial enhancement: The application of functionalized graphene oxide and Ag-TiO2 nanoparticles loaded with activated carbon breaks through the efficiency bottleneck of traditional culture medium. (4) Bionic microenvironment construction: Sodium alginate-gelatin hydrogel simulates the extracellular matrix to support stem cell attachment and directional differentiation.

[0068] In some embodiments, the pH value of the germination medium is 5.75-5.85, the pH value of the growth medium is 5.75-5.85, the pH value of the induction medium is 5.75-5.85, and the pH value of the proliferation medium is 5.75-5.85.

[0069] The pH value of the defined culture medium is 5.75-5.85. This slightly acidic environment, close to the optimal pH range for ginseng cell protoplasts, activates hydrolytic enzymes (such as cellulase and pectinase), promotes cell wall relaxation, and enhances nutrient absorption. Simultaneously, a stable pH reduces the accumulation of metabolites such as lactic acid, prevents cell acidification damage, and maintains mitochondrial function. Furthermore, the acidic environment protects the activity of hormones such as GA3 and TDZ, prolonging their action time.

[0070] In some embodiments, the NH4 + With NO3 — The molar ratio is 1:5.

[0071] NH4 in defined growth medium + With NO3 — The molar ratio is 1:5, high proportion of NO3 — (5 times that of NH4 + ) as the main nitrogen source to promote protein synthesis; a small amount of NH4 + Supplement amino groups to avoid ammonium poisoning. At the same time, NO3 — As an osmotically active substance, with Ca 2+ (calcium nitrate) synergistically maintains cell membrane stability. In addition, NO3 — Reductase activity is regulated by the ratio, promoting nitrogen metabolism efficiency and supporting rapid growth of seedlings.

[0072] In some embodiments, the specific surface area of ​​the functionalized graphene oxide is 500 to 800 m 2 / g, surface modified with carboxyl groups.

[0073] The specific surface area of ​​functionalized graphene oxide is limited to 500-800 m 2 / g, and its surface is modified with carboxyl groups. Its large surface area enhances nutrient loading capacity and allows for the sustained release of hormones (such as 2,4-D) into the cellular microenvironment. Simultaneously, the carboxyl groups (-COOH) enhance the hydrophilicity of graphene oxide, preventing nanoparticle aggregation and ensuring uniform distribution in the culture medium. Furthermore, the surface charge regulates cell membrane potential, promotes calcium influx, and accelerates differentiation signaling.

[0074] In some embodiments, in the sodium alginate-gelatin hydrogel, the mass ratio of sodium alginate to gelatin is 3:1 to 5:1.

[0075] The mass ratio of sodium alginate to gelatin hydrogel is limited to 3:1 to 5:1. A high ratio of sodium alginate (3:1) forms a dense network, providing mechanical support and mimicking the rigidity of the extracellular matrix (ECM). Meanwhile, gelatin contains RGD peptides, which enhance stem cell adhesion and promote integrin signaling. Furthermore, the optimized ratio ensures the gel remains stable at 37°C, facilitating subsequent cell harvesting.

[0076] In some embodiments, in the Ag-TiO2 nanoparticle-loaded activated carbon, the mass of the Ag-TiO2 nanoparticles is 1 to 5% of the mass of the activated carbon.

[0077] Ag nanoparticles release Ag + , destroying pathogen cell membranes and reducing contamination risks. Simultaneously, TiO2 generates reactive oxygen species (ROS) under light, synergizing with Ag to enhance the bactericidal effect while simultaneously degrading metabolic toxins. Furthermore, the high surface area of ​​activated carbon adsorbs harmful substances (such as phenols), while the sustained release of nanoparticles prolongs the antibacterial cycle.

[0078] In some embodiments, the method for preparing the Ag-TiO2 nanoparticle-loaded activated carbon comprises:

[0079] Mixing metatitanic acid with activated carbon, and then performing a hydrothermal reaction to generate TiO2 nanoparticles and load them on the surface of the activated carbon to obtain TiO2-loaded activated carbon; the hydrothermal reaction temperature is 180°C and the time is 12 hours;

[0080] The TiO2-loaded activated carbon was immersed in silver nitrate solution and then irradiated with UVA light source to make Ag + The Ag-TiO2 nanoparticles are photoreduced to Ag nanoparticles on the surface to obtain the Ag-TiO2 nanoparticles loaded activated carbon.

[0081] Figure 1 This is a schematic diagram of a process for culturing ginseng stem cells according to an embodiment of the present application.

[0082] Second, as Figure 1As shown, the present application provides a tissue culture method for ginseng stem cells, using any tissue culture medium described in the first aspect, comprising the following steps:

[0083] S1, disinfecting the vernalized ginseng seeds and inoculating them into the germination medium to perform germination culture to obtain germinated seeds;

[0084] S2, transferring the germinated seeds to the growth medium for seedling growth and culture to obtain sterile seedlings;

[0085] S3, cutting stem segments of the sterile seedlings and inoculating them into an induction medium for induction culture, and gradually reducing the 2,4-D concentration in the induction medium during the induction culture to obtain callus tissue;

[0086] S4. Transferring the callus tissue to a proliferation culture medium for proliferation culture to obtain ginseng stem cells.

[0087] In some embodiments, a completely dark environment is used during the germination stage, a CO2 concentration of 800 ppm is introduced, the temperature is 25±0.5°C, and the culture time is 15 to 20 days;

[0088] During the seedling growth stage, a light / dark cycle of 14 / 10 hours was used, wherein the ratio of red light to blue light was 7:3, the temperature was 25±0.5°C, and the culture time was 40 to 44 days;

[0089] The induction stage is divided into the first 15 days of dark culture and the last 15 days of 12 / 12 hour light and dark cycle, and the far-red light intensity is increased in the last stage, the temperature is 24±1°C, and the culture time is 30 days;

[0090] During the proliferation stage, a light / dark cycle of 16 hours and 8 hours was used, wherein pulsed light supplementation was performed every 2 hours with an interval of 10 minutes, the temperature was 26±1° C., and the culture time was 62 to 68 days.

[0091] This application simulates the natural growth rhythm of ginseng by precisely controlling environmental factors such as light, temperature, air, and hormones, and at the same time combines the biological characteristics of plant stem cells (such as pluripotency and self-renewal ability) to achieve fully controllable cultivation from seeds to stem cells.

[0092] During the germination and cultivation stage, ginseng seeds are usually kept in dark conditions covered with soil when germinating in a natural environment. A completely dark environment can simulate the natural germination process, avoid light interference with the internal hormones of the seeds (such as gibberellins), promote the radicle to break through the seed coat, and reduce the damage to the embryo caused by photooxidation. The CO2 concentration is limited to 800ppm. High concentrations of CO2 can enhance the efficiency of seed respiratory metabolism, accelerate the decomposition of storage substances (such as starch and fat) in the seeds, provide energy for germination, and inhibit the risk of contamination by certain fungi. The temperature is limited to 25±0.5℃, which is slightly higher than the conventional ginseng seed germination temperature (15-20℃), but combined with the physiological state of the seeds after vernalization treatment, it can accelerate enzyme activity, shorten the germination cycle, and ensure the uniformity of germination. The culture time is limited to 15-20 days. This period of time ensures that the seeds complete the entire process of imbibition, enzyme activation and radicle breaking through the seed coat, avoiding premature transfer that leads to fragile seedlings.

[0093] During the seedling growth stage, the light and dark cycle is limited to 14 / 10 hours (red light: blue light = 7:3). Red light (wavelength 630-660nm) promotes stem elongation and chlorophyll synthesis, and blue light (wavelength 450-470nm) enhances stomatal opening and photosynthetic efficiency. The 7:3 ratio can balance the morphological construction of seedlings and the accumulation of photosynthetic products. The 14-hour light cycle simulates the long-day conditions of ginseng's natural growing season and promotes the healthy growth of seedlings. The temperature is limited to 25±0.5℃, maintaining the same temperature control as the germination stage, reducing the stress of environmental fluctuations on seedlings, and at the same time complying with the optimal growth temperature range of ginseng seedlings (the upper limit of 18-22℃), accelerating biomass accumulation. The cultivation time is limited to 40-44 days to ensure that the seedlings form 3-4 true leaves and a strong root system, providing sufficient nutrient reserves for subsequent stem segment collection.

[0094] During the induction phase, the first 15 days of culture are in darkness. Dark conditions inhibit phytochrome activity, reduce differentiation signals, and promote dedifferentiation of explant (stem) cells, leading to callus formation. This also reduces light-induced oxidative stress. For the next 15 days, a 12 / 12 hour light / dark cycle (with far-red light enhancement) is applied. Far-red light (730 nm) activates the Pr form of phytochrome, promoting callus differentiation and laying the structural foundation for subsequent proliferation. Adjusting the photoperiod gradually introduces light, simulating the circadian rhythm of the natural environment and promoting the transition of cellular metabolism from dedifferentiation to redifferentiation. The concentration of 2,4-D, a synthetic auxin analog, is gradually reduced. Initial high concentrations induce rapid cell division and callus formation; gradually reducing the concentration later reduces hormone dependence, promotes cellular homeostasis, and avoids tissue deformities caused by excessive proliferation. The temperature is maintained at 24 ± 1°C, slightly lower than that during the seedling stage, slowing metabolic rates, prolonging the callus formation cycle, and improving cell uniformity.

[0095] During the proliferation culture stage, the light-dark cycle is 16 / 8 hours (pulsed lighting), and the long photoperiod simulates the long-day conditions in summer, promotes cytokinin synthesis, and accelerates stem cell proliferation. Pulsed lighting (10 minutes of light every 2 hours) activates photoperiod-related genes (such as LHY and TOC1) through short-period light signal fluctuations, enhances cell cycle synchronization, and reduces the accumulation of metabolic byproducts. The temperature is limited to 26±1°C. Higher temperatures (close to the upper limit of heat tolerance of mature ginseng plants) can accelerate enzymatic reactions, shorten the cell cycle, and avoid heat stress damage through precise temperature control. The culture time is 62 to 68 days. This cycle ensures that stem cells complete multiple rounds of proliferation and accumulate active ingredients such as rare ginsenosides (such as Rg3 and Rh2), meeting the dual requirements of industrial production for biomass and effective ingredients.

[0096] In some embodiments, the stem segment is 1 to 2 cm in length.

[0097] The stem segment length is limited to 1-2 cm, which ensures that the explant contains sufficient meristematic tissue (such as shoot apical meristematic cells) while avoiding the risk of nutrient competition or incision infection due to excessive length.

[0098] In summary, this application achieves efficient and stable culture of ginseng stem cells by precisely controlling culture medium components (such as hormones and nanomaterials), optimizing environmental parameters (light, temperature, pH), and constructing a biomimetic microenvironment. Its core advantages are:

[0099] (1) Multi-stage precision control system: By adjusting the hormone ratio in stages (such as GA3+TDZ in the germination stage and high concentration 2,4-D+KT in the proliferation stage), the whole process from breaking dormancy to efficient expansion of stem cells is controlled to avoid abnormal differentiation. At the same time, combined with the light cycle (such as red light: blue light = 7:3, pulsed light supplement) and temperature gradient (25±0.5℃→26±1℃), the natural growth rhythm of ginseng is simulated to optimize the synchronization of cell metabolism and proliferation. In addition, dynamic culture medium optimization, such as the gradient reduction of 2,4-D concentration in the induction culture medium, reduces hormone dependence and improves callus uniformity.

[0100] (2) Efficient accumulation of active ingredients and activation of secondary metabolism: Methyl jasmonate (0.2-0.5 g / L) activates the synthesis pathway of ginsenosides (such as Rg3 and Rh2), and combined with far-infrared light to enhance the light signal, significantly increases the content of active ingredients. At the same time, melatonin, reduced glutathione, trehalose and other multi-level protections can remove ROS, prolong cell life and maintain genome stability. In addition, functionalized graphene oxide (specific surface area 500-800 m 2 / g) enhances nutrient adsorption and signal transduction, and Ag-TiO2 nanoparticles loaded with activated carbon have antibacterial and photocatalytic synergy to improve culture efficiency.

[0101] (3) Pollution control and resource sustainability: Chitosan oligosaccharides inhibit fungal contamination, Ag-TiO2 nanoparticles destroy pathogen cell membranes, and activated carbon adsorbs toxins, reducing the contamination rate. At the same time, stem cell culture replaces traditional cultivation to alleviate pressure on wild ginseng resources and preserve germplasm genetic diversity. In addition, tissue culture technology does not require large-scale arable land, reduces pesticide use, and reduces labor costs, meeting the needs of ecological sustainable development.

[0102] (4) Industrial application potential: Sodium alginate-gelatin hydrogel (mass ratio 3:1-5:1) simulates the extracellular matrix, supports stem cell attachment and directional differentiation, and is suitable for large-scale production. At the same time, the Box-Behnken design was used to optimize the culture conditions (such as pH 5.75-5.85, NH4 + / NO3 - The ratio is 1:5), improving product consistency and meeting the needs of pharmaceutical / cosmetic raw materials. In addition, the proliferation medium combines pulsed light supplementation with high concentrations of 2,4-D to increase stem cell expansion efficiency by three times and shorten the biomass accumulation period to 62-68 days.

[0103] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods in the following examples where specific conditions are not specified are generally measured according to industry standards. If there are no corresponding industry standards, then the methods are carried out according to general international standards, conventional conditions, or the conditions recommended by the manufacturer.

[0104] Example 1

[0105] This embodiment provides a tissue culture medium for culturing ginseng stem cells, including a germination medium, a growth medium, an induction medium, and a proliferation medium; wherein,

[0106] The germination medium is based on MS and further includes 3.5 mg / L of GA3 (CAS No. 77-06-5), 0.3 mg / L of TDZ (CAS No. 51707-55-2), 0.08 mg / L of melatonin (CAS No. 73-31-4), 0.2 g / L of chitosan oligosaccharide (CAS No. 148411-57-8), 30 g / L of sucrose (CAS No. 57-50-1), 1.2 g / L of activated carbon (CAS No. 64365-11-3), and 6.1 g / L of phytogel (CAS No. 71010-52-1).

[0107] The growth medium is based on B5 as a basal medium and further includes 1.8 mg / L of 6-BA (CAS No. 1214-39-7), 0.45 mg / L of NAA (CAS No. 86-87-3), 20 g / L of sucrose (CAS No. 57-50-1), 10 g / L of trehalose (CAS No. 99-20-7), 600 mg / L of calcium nitrate, and 6.2 g / L of phytol (CAS No. 71010-52-1).

[0108] The induction medium is based on B5 or MS as a basic medium, and further includes 1.5-2.5 mg / L of 2,4-D (CAS No. 94-75-7), 0.5 mg / L of KT (CAS No. 525-79-1), 0.3 g / L of methyl jasmonate (CAS No. 39924-52-2), 0.2 mg / L of melatonin, 0.8 mg / L of functionalized graphene oxide, 1.1 g / L of acid hydrolyzed casein (CAS No. 9000-71-9), 30 g / L of sucrose, and 6.2 g / L of plant gel;

[0109] The specific surface area of ​​the functionalized graphene oxide is 600 m 2 / g, surface modified with carboxyl groups.

[0110] The method for preparing functionalized graphene oxide includes dispersing graphene oxide (CAS number 012111-13-4) in nitric acid, heating to 70°C, and continuing the reaction for 12 hours. Carboxyl groups are formed by breaking carbon chains at the edges and defects of the graphene oxide. After the reaction, the graphene oxide is centrifuged and washed until neutral, thereby obtaining functionalized graphene oxide.

[0111] The proliferation culture medium is based on B5 as a basic culture medium and further includes 2.5-2.8 mg / L of 2,4-D, 0.65 mg / L of KT, 0.2 mg / L of brassinolide (CAS No. 78821-43-9), 1.0 g / L of reduced glutathione (CAS No. 70-18-8), 0.08 g / L of selenomethionine (CAS No. 3211-76-5), 0.2 mg / L of chitosan nanoparticles (CAS No. 9012-76-4), and 0.2 mg / L of glutathione. L, humic acid (CAS number 1415-93-6) 0.3g / L, sodium alginate-gelatin hydrogel (CAS number of sodium alginate is 9005-38-3, CAS number of gelatin is 9000-70-8) 0.3g / L, Ag-TiO2 nanoparticles loaded activated carbon 0.8g / L, sucrose 10g / L, trehalose (CAS number 525-79-1) 20g / L.

[0112] Wherein, in the sodium alginate-gelatin hydrogel, the mass ratio of sodium alginate to gelatin is 4:1.

[0113] In the Ag-TiO2 nanoparticle-loaded activated carbon, the mass of the Ag-TiO2 nanoparticles is 3% of the mass of the activated carbon.

[0114] The preparation method of Ag-TiO2 nanoparticle-loaded activated carbon comprises: mixing metatitanic acid with activated carbon, and then performing a hydrothermal reaction to generate TiO2 nanoparticles and load them on the surface of the activated carbon to obtain TiO2-loaded activated carbon; the temperature of the hydrothermal reaction is 180°C and the time is 12 hours; immersing the TiO2-loaded activated carbon in a silver nitrate solution, and then irradiating it with a UVA light source to make the Ag + The Ag-TiO2 nanoparticles are photoreduced to Ag nanoparticles on the surface to obtain the Ag-TiO2 nanoparticles loaded activated carbon.

[0115] Based on the above tissue culture medium for culturing ginseng stem cells, this embodiment also provides a tissue culture method for culturing ginseng stem cells, comprising the following steps:

[0116] S11, disinfecting the vernalized ginseng seeds and inoculating them into the germination medium to perform germination culture to obtain germinated seeds;

[0117] S21, transferring the germinated seeds to the growth medium for seedling growth and culture to obtain sterile seedlings;

[0118] S31, cutting stem segments of the sterile seedlings and inoculating them into an induction medium for induction culture, and gradually reducing the 2,4-D concentration in the induction medium during the induction culture to obtain callus tissue;

[0119] S41. Transferring the callus tissue to a proliferation culture medium for proliferation culture to obtain ginseng stem cells.

[0120] Wherein, during the germination stage, a completely dark environment is adopted, a CO2 concentration of 800 ppm is introduced, a temperature of 25±0.5°C, and a culture time of 15 to 20 days;

[0121] During the seedling growth stage, a light / dark cycle of 14 / 10 hours was used, wherein the ratio of red light to blue light was 7:3, the temperature was 25±0.5°C, and the culture time was 40 to 44 days;

[0122] The induction stage is divided into 15 days of dark culture and 15 days of 12 / 12 hour light-dark cycle, and the far-red light intensity is increased in the latter stage, the temperature is 24±1°C, and the culture time is 30 days; during the first 10 days of culture, the 2,4-D concentration is 2.5 mg / L, from the 10th to the 20th day of culture, the 2,4-D concentration is 2 mg / L, and during the last 10 days of culture, the 2,4-D concentration is 1.5 mg / L;

[0123] During the proliferation stage, a light / dark cycle of 16 hours and 8 hours was used, wherein pulsed light supplementation was performed every 2 hours with an interval of 10 minutes, the temperature was 26±1° C., and the culture time was 62 to 68 days.

[0124] The length of the stem segment is 1.5 cm.

[0125] Example 2

[0126] This embodiment provides a tissue culture medium for culturing ginseng stem cells, including a germination medium, a growth medium, an induction medium, and a proliferation medium; wherein,

[0127] The germination medium is based on MS and further includes 3.0 mg / L of GA3 (CAS No. 77-06-5), 0.5 mg / L of TDZ (CAS No. 51707-55-2), 0.05 mg / L of melatonin (CAS No. 73-31-4), 0.3 g / L of chitosan oligosaccharide (CAS No. 148411-57-8), 30 g / L of sucrose (CAS No. 57-50-1), 1.0 g / L of activated carbon (CAS No. 64365-11-3), and 6.5 g / L of phytogel (CAS No. 71010-52-1).

[0128] The growth medium is based on B5 as a basal medium and further includes 1.5 mg / L of 6-BA (CAS No. 1214-39-7), 0.4 mg / L of NAA (CAS No. 86-87-3), 20 g / L of sucrose (CAS No. 57-50-1), 10 g / L of trehalose (CAS No. 99-20-7), 600 mg / L of calcium nitrate, and 6.0 g / L of phytol (CAS No. 71010-52-1).

[0129] The induction medium is based on B5 or MS as a basic medium, and further includes 1.5-2.5 mg / L of 2,4-D (CAS No. 94-75-7), 0.5 mg / L of KT (CAS No. 525-79-1), 0.2 g / L of methyl jasmonate (CAS No. 39924-52-2), 0.3 mg / L of melatonin, 0.5 mg / L of functionalized graphene oxide, 1.2 g / L of acid hydrolyzed casein (CAS No. 9000-71-9), 30 g / L of sucrose, and 6.0 g / L of plant gel;

[0130] The specific surface area of ​​the functionalized graphene oxide is 800 m 2 / g, surface modified with carboxyl groups.

[0131] The method for preparing functionalized graphene oxide includes dispersing graphene oxide (CAS number 012111-13-4) in nitric acid, heating to 70°C, and continuing the reaction for 12 hours. Carboxyl groups are formed by breaking carbon chains at the edges and defects of the graphene oxide. After the reaction, the graphene oxide is centrifuged and washed until neutral, thereby obtaining functionalized graphene oxide.

[0132] The proliferation culture medium is based on B5 as the basic culture medium and also includes 2,4-D 2.8 mg / L, KT 0.6 mg / L, brassinolide (CAS No. 78821-43-9) 0.3 mg / L, reduced glutathione (CAS No. 70-18-8) 0.2 g / L, selenomethionine (CAS No. 3211-76-5) 0.1 g / L, chitosan nanoparticles (CAS No. 9012-76-4) 0.3 mg / L, humic acid (CAS No. 1415-93-6) 0.1 g / L, sodium alginate-gelatin hydrogel (CAS No. 9005-38-3 of sodium alginate, CAS No. 9000-70-8 of gelatin) 0.1 g / L, Ag-TiO2 nanoparticles loaded with activated carbon 1.0 g / L, sucrose 10 g / L, trehalose (CAS No. 525-79-1) 20 g / L.

[0133] Wherein, in the sodium alginate-gelatin hydrogel, the mass ratio of sodium alginate to gelatin is 3:1.

[0134] In the Ag-TiO2 nanoparticle-loaded activated carbon, the mass of the Ag-TiO2 nanoparticles is 1% of the mass of the activated carbon.

[0135] The preparation method of Ag-TiO2 nanoparticle-loaded activated carbon comprises: mixing metatitanic acid with activated carbon, and then performing a hydrothermal reaction to generate TiO2 nanoparticles and load them on the surface of the activated carbon to obtain TiO2-loaded activated carbon; the temperature of the hydrothermal reaction is 180°C and the time is 12 hours; immersing the TiO2-loaded activated carbon in a silver nitrate solution, and then irradiating it with a UVA light source to make the Ag + The Ag-TiO2 nanoparticles are photoreduced to Ag nanoparticles on the surface to obtain the Ag-TiO2 nanoparticles loaded activated carbon.

[0136] Based on the above tissue culture medium for culturing ginseng stem cells, this embodiment also provides a tissue culture method for culturing ginseng stem cells, comprising the following steps:

[0137] S11, disinfecting the vernalized ginseng seeds and inoculating them into the germination medium to perform germination culture to obtain germinated seeds;

[0138] S21, transferring the germinated seeds to the growth medium for seedling growth and culture to obtain sterile seedlings;

[0139] S31, cutting stem segments of the sterile seedlings and inoculating them into an induction medium for induction culture, and gradually reducing the 2,4-D concentration in the induction medium during the induction culture to obtain callus tissue;

[0140] S41. Transferring the callus tissue to a proliferation culture medium for proliferation culture to obtain ginseng stem cells.

[0141] Wherein, during the germination stage, a completely dark environment is adopted, a CO2 concentration of 800 ppm is introduced, a temperature of 25±0.5°C, and a culture time of 15 to 20 days;

[0142] During the seedling growth stage, a light / dark cycle of 14 / 10 hours was used, wherein the ratio of red light to blue light was 7:3, the temperature was 25±0.5°C, and the culture time was 40 to 44 days;

[0143] The induction stage is divided into 15 days of dark culture and 15 days of 12 / 12 hour light-dark cycle, and the far-red light intensity is increased in the latter stage, the temperature is 24±1°C, and the culture time is 30 days; during the first 10 days of culture, the 2,4-D concentration is 2.5 mg / L, from the 10th to the 20th day of culture, the 2,4-D concentration is 2 mg / L, and during the last 10 days of culture, the 2,4-D concentration is 1.5 mg / L;

[0144] During the proliferation stage, a light / dark cycle of 16 hours and 8 hours was used, wherein pulsed light supplementation was performed every 2 hours with an interval of 10 minutes, the temperature was 26±1° C., and the culture time was 62 to 68 days.

[0145] The length of the stem segment is 1 cm.

[0146] Example 3

[0147] This embodiment provides a tissue culture medium for culturing ginseng stem cells, including a germination medium, a growth medium, an induction medium, and a proliferation medium; wherein,

[0148] The germination medium is based on MS and further includes 4.0 mg / L of GA3 (CAS No. 77-06-5), 0.1 mg / L of TDZ (CAS No. 51707-55-2), 0.1 mg / L of melatonin (CAS No. 73-31-4), 0.1 g / L of chitosan oligosaccharide (CAS No. 148411-57-8), 30 g / L of sucrose (CAS No. 57-50-1), 1.5 g / L of activated carbon (CAS No. 64365-11-3), and 6.5 g / L of phytogel (CAS No. 71010-52-1).

[0149] The growth medium is based on B5 as a basal medium and further includes 1.5 mg / L of 6-BA (CAS No. 1214-39-7), 0.5 mg / L of NAA (CAS No. 86-87-3), 20 g / L of sucrose (CAS No. 57-50-1), 10 g / L of trehalose (CAS No. 99-20-7), 600 mg / L of calcium nitrate, and 6.2 g / L of phytol (CAS No. 71010-52-1).

[0150] The induction medium is based on B5 or MS as a basic medium, and further includes 1.5-2.5 mg / L of 2,4-D (CAS No. 94-75-7), 0.5 mg / L of KT (CAS No. 525-79-1), 0.2 g / L of methyl jasmonate (CAS No. 39924-52-2), 0.1 mg / L of melatonin, 0.5 mg / L of functionalized graphene oxide, 1.0 g / L of acid hydrolyzed casein (CAS No. 9000-71-9), 30 g / L of sucrose, and 6.5 g / L of plant gel;

[0151] The specific surface area of ​​the functionalized graphene oxide is 500 m 2 / g, surface modified with carboxyl groups.

[0152] The method for preparing functionalized graphene oxide includes dispersing graphene oxide (CAS number 012111-13-4) in nitric acid, heating to 70°C, and continuing the reaction for 12 hours. Carboxyl groups are formed by breaking carbon chains at the edges and defects of the graphene oxide. After the reaction, the graphene oxide is centrifuged and washed until neutral, thereby obtaining functionalized graphene oxide.

[0153] The proliferation culture medium is based on B5 as the basic culture medium and also includes 2,4-D 2.8 mg / L, KT 0.6 mg / L, brassinolide (CAS No. 78821-43-9) 0.2 mg / L, reduced glutathione (CAS No. 70-18-8) 1.5 g / L, selenomethionine (CAS No. 3211-76-5) 0.1 g / L, chitosan nanoparticles (CAS No. 9012-76-4) 0.3 mg / L, humic acid (CAS No. 1415-93-6) 0.1 g / L, sodium alginate-gelatin hydrogel (CAS No. 9005-38-3 of sodium alginate, CAS No. 9000-70-8 of gelatin) 0.1 g / L, Ag-TiO2 nanoparticles loaded with activated carbon 1.0 g / L, sucrose 10 g / L, trehalose (CAS No. 525-79-1) 20 g / L.

[0154] Wherein, in the sodium alginate-gelatin hydrogel, the mass ratio of sodium alginate to gelatin is 5:1.

[0155] In the Ag-TiO2 nanoparticle-loaded activated carbon, the mass of the Ag-TiO2 nanoparticles is 5% of the mass of the activated carbon.

[0156] The preparation method of Ag-TiO2 nanoparticle-loaded activated carbon comprises: mixing metatitanic acid with activated carbon, and then performing a hydrothermal reaction to generate TiO2 nanoparticles and load them on the surface of the activated carbon to obtain TiO2-loaded activated carbon; the temperature of the hydrothermal reaction is 180°C and the time is 12 hours; immersing the TiO2-loaded activated carbon in a silver nitrate solution, and then irradiating it with a UVA light source to make the Ag + The Ag-TiO2 nanoparticles are photoreduced to Ag nanoparticles on the surface to obtain the Ag-TiO2 nanoparticles loaded activated carbon.

[0157] Based on the above tissue culture medium for culturing ginseng stem cells, this embodiment also provides a tissue culture method for culturing ginseng stem cells, comprising the following steps:

[0158] S11, disinfecting the vernalized ginseng seeds and inoculating them into the germination medium to perform germination culture to obtain germinated seeds;

[0159] S21, transferring the germinated seeds to the growth medium for seedling growth and culture to obtain sterile seedlings;

[0160] S31, cutting stem segments of the sterile seedlings and inoculating them into an induction medium for induction culture, and gradually reducing the 2,4-D concentration in the induction medium during the induction culture to obtain callus tissue;

[0161] S41. Transferring the callus tissue to a proliferation culture medium for proliferation culture to obtain ginseng stem cells.

[0162] Wherein, during the germination stage, a completely dark environment is adopted, a CO2 concentration of 800 ppm is introduced, a temperature of 25±0.5°C, and a culture time of 15 to 20 days;

[0163] During the seedling growth stage, a light / dark cycle of 14 / 10 hours was used, wherein the ratio of red light to blue light was 7:3, the temperature was 25±0.5°C, and the culture time was 40 to 44 days;

[0164] The induction stage is divided into 15 days of dark culture and 15 days of 12 / 12 hour light-dark cycle, and the far-red light intensity is increased in the latter stage, the temperature is 24±1°C, and the culture time is 30 days; during the first 10 days of culture, the 2,4-D concentration is 2.5 mg / L, from the 10th to the 20th day of culture, the 2,4-D concentration is 2 mg / L, and during the last 10 days of culture, the 2,4-D concentration is 1.5 mg / L;

[0165] During the proliferation stage, a light / dark cycle of 16 hours and 8 hours was used, wherein pulsed light supplementation was performed every 2 hours with an interval of 10 minutes, the temperature was 26±1° C., and the culture time was 62 to 68 days.

[0166] The length of the stem segment is 2 cm.

[0167] Comparative Example 1

[0168] This comparative example is modified as follows based on Example 1:

[0169] No chitosan oligosaccharide was included in the germination medium.

[0170] Comparative Example 2

[0171] This comparative example is modified as follows based on Example 1:

[0172] Trehalose was not included in the growth medium.

[0173] Comparative Example 3

[0174] This comparative example is modified as follows based on Example 1:

[0175] Methyl jasmonate was not included in the induction medium.

[0176] Comparative Example 4

[0177] This comparative example is modified as follows based on Example 1:

[0178] No functionalized graphene oxide was included in the induction medium.

[0179] Comparative Example 5

[0180] This comparative example is modified as follows based on Example 1:

[0181] The proliferation medium does not contain sodium alginate-gelatin hydrogel.

[0182] Comparative Example 6

[0183] This comparative example is modified as follows based on Example 1:

[0184] No Ag-TiO2 nanoparticles were included in the proliferation medium.

[0185] The tissue culture methods of ginseng stem cells in Examples 1 to 3 and Comparative Examples 1 to 6 were used to measure callus induction rate, browning rate, ginsenoside content, cell proliferation efficiency, contamination rate, and callus induction rate. The results are shown in Table 1. The specific measurement methods are as follows:

[0186] Callus induction rate: The percentage of callus-forming explants was counted, and effectiveness was determined by morphological observation (amorphous, white, or pale yellow cell clusters). Induction rate = (number of explants forming callus / total number of explants) × 100%.

[0187] Browning rate: Observe the browning of the explant surface and count the number of browning samples. Browning rate = (number of browning explants / total number of explants) × 100%.

[0188] Ginsenoside content: Quantitative analysis was performed using high performance liquid chromatography (HPLC) by comparing the retention time and peak area of ​​standards (such as ginsenosides Rg3 and Rh2).

[0189] Cell proliferation efficiency: CCK-8 method was used to detect cell metabolic activity (OD value).

[0190] Contamination rate: Count the proportion of samples with bacterial plaques or turbidity in the culture medium.

[0191] Table 1 Performance test results of ginseng stem cell tissue culture method

[0192]

[0193] As shown in Table 1, Examples 1 to 33 achieved efficient callus induction (95% to 98%) and highly active saponin synthesis (170 to 190 mg / g) by functionalizing key components such as graphene oxide, methyl jasmonate, and three-dimensional hydrogel.

[0194] In Comparative Example 1, there is no chitosan oligosaccharide. The lack of chitosan oligosaccharide leads to a decrease in antibacterial ability, aggravated oxidative damage to the explants, and accumulation of phenolic toxins causing browning. At the same time, the risk of fungal contamination increases significantly after the lack of chitosan oligosaccharide.

[0195] Comparative Example 2 lacks trehalose, which leads to osmotic pressure imbalance and cell dehydration damage. At the same time, insufficient osmotic protection impairs cell membrane integrity and reduces metabolic activity.

[0196] Comparative Example 3 does not contain methyl jasmonate. The lack of methyl jasmonate results in the inability to activate secondary metabolic pathways (such as saponin synthesis) and a decrease in HMGR enzyme activity.

[0197] Comparative Example 4 does not contain functionalized graphene oxide. The absence of graphene oxide prevents the sustained release of hormone (2,4-D), leading to unstable cell division signals. Furthermore, nutrient absorption efficiency is reduced, and cell synchronization is decreased.

[0198] Comparative Example 5 lacks the sodium alginate-gelatin hydrogel, lacking the three-dimensional scaffold support, resulting in decreased cell adhesion and restricted proliferation. Furthermore, the absence of the hydrogel leads to insufficient activation of the integrin signaling pathway and increased differentiation tendency.

[0199] Comparative Example 6 does not contain Ag-TiO2 nanoparticles. The lack of Ag-TiO2 leads to a decrease in antibacterial ability and an increase in the risk of bacterial / fungal contamination.

[0200] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a hard limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numbers within the range. For example, the description of a range from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0201] In addition, in the description of this application, the terms "including", "comprising", etc. mean "including but not limited to". In this document, relational terms such as "first" and "second" are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations.

[0202] The foregoing is merely a list of specific embodiments of the present application, intended to enable those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the broadest scope consistent with the principles and novel features of the present application.

Claims

1. A tissue culture medium for culturing ginseng stem cells, characterized in that: It includes germination medium, growth medium, induction medium and proliferation medium; wherein, The germination medium is based on MS or 1 / 2MS and further includes 3.0-4.0 mg / L GA3, 0.1-0.5 mg / L TDZ, 0.05-0.1 mg / L melatonin, 0.1-0.3 g / L chitosan oligosaccharide, 30 g / L sucrose, 1.0-1.5 g / L activated carbon, and 6.0-6.5 g / L plant gel; The growth medium is based on B5 as a basic medium and further includes 1.5-2.0 mg / L of 6-BA, 0.4-0.5 mg / L of NAA, 20 g / L of sucrose, 10 g / L of trehalose, 600 mg / L of calcium nitrate, and 6.0-6.5 g / L of plant gel; The induction culture medium is based on B5 or MS as a basic culture medium and further includes 1.5-2.5 mg / L of 2,4-D, 0.5 mg / L of KT, 0.2-0.5 g / L of methyl jasmonate, 0.1-0.3 mg / L of melatonin, 0.5-1.0 mg / L of functionalized graphene oxide, 1.0-1.2 g / L of acid hydrolyzed casein, 30 g / L of sucrose, and 6.0-6.5 g / L of plant gel; The proliferation culture medium is based on B5 as a basic culture medium and further includes 2.5-2.8 mg / L of 2,4-D, 0.6-0.7 mg / L of KT, 0.1-0.3 mg / L of brassinolide, 0.2-1.5 g / L of reduced glutathione, 0.05-0.1 g / L of selenomethionine, 0.1-0.3 mg / L of chitosan nanoparticles, 0.1-0.5 g / L of humic acid, 0.1-0.5 g / L of sodium alginate-gelatin hydrogel, 0.5-1.0 g / L of Ag-TiO2 nanoparticle-loaded activated carbon, 10 g / L of sucrose, and 20 g / L of trehalose.

2. The tissue culture medium for culturing ginseng stem cells according to claim 1, wherein NH4 in the growth medium + With NO3 — The molar ratio is 1:

5.

3. The tissue culture medium for culturing ginseng stem cells according to claim 1, wherein The specific surface area of ​​the functionalized graphene oxide is 500 to 800 m 2 / g, surface modified with carboxyl groups.

4. The tissue culture medium for culturing ginseng stem cells according to claim 1, wherein In the sodium alginate-gelatin hydrogel, the mass ratio of sodium alginate to gelatin is 3:1 to 5:

1.

5. The tissue culture medium for culturing ginseng stem cells according to claim 1, wherein In the Ag-TiO2 nanoparticle-loaded activated carbon, the mass of the Ag-TiO2 nanoparticles is 1-5% of the mass of the activated carbon.

6. The tissue culture medium for culturing ginseng stem cells according to claim 5, characterized in that: The preparation method of the Ag-TiO2 nanoparticle-loaded activated carbon comprises: Mixing metatitanic acid with activated carbon, and then performing a hydrothermal reaction to generate TiO2 nanoparticles and load them on the surface of the activated carbon to obtain TiO2-loaded activated carbon; the hydrothermal reaction temperature is 180°C and the time is 12 hours; The TiO2-loaded activated carbon was immersed in silver nitrate solution and then irradiated with UVA light source to make Ag + The Ag-TiO2 nanoparticles are photoreduced to Ag nanoparticles on the surface to obtain the Ag-TiO2 nanoparticles loaded activated carbon.

7. A tissue culture method for ginseng stem cells, characterized in that: Using the tissue culture medium according to any one of claims 1 to 6, comprising the following steps: sterilizing the vernalized ginseng seeds and inoculating them into the germination medium to perform germination culture to obtain germinated seeds; transferring the germinated seeds to the growth medium to carry out seedling growth culture to obtain sterile seedlings; cutting stem segments of the sterile seedlings and inoculating them into an induction medium for induction culture, and gradually reducing the 2,4-D concentration in the induction medium during the induction culture to obtain callus tissue; The callus tissue is transferred to a proliferation culture medium for proliferation culture to obtain ginseng stem cells.

8. The tissue culture method of ginseng stem cells according to claim 7, characterized in that: During the germination stage, a completely dark environment is used, a CO2 concentration of 800 ppm is introduced, the temperature is 25±0.5°C, and the incubation time is 15 to 20 days; During the seedling growth stage, a light-dark cycle of 14 hours / 10 hours, a red light to blue light ratio of 7:3, a temperature of 25±0.5° C., and a culture time of 40 to 44 days were adopted.

9. The tissue culture method of ginseng stem cells according to claim 7, characterized in that: The induction stage is divided into the first 15 days of dark culture and the last 15 days of 12 / 12 hour light and dark cycle, and the far-red light intensity is increased in the last stage, the temperature is 24±1°C, and the culture time is 30 days; During the proliferation stage, a light / dark cycle of 16 hours and 8 hours was used, wherein pulsed light supplementation was performed every 2 hours with an interval of 10 minutes, the temperature was 26±1° C., and the culture time was 62 to 68 days. The tissue culture method of ginseng stem cells according to claim 7, characterized in that: The length of the stem segment is 1 to 2 cm.

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