Method for obtaining a medicinal plant stem cell clone and a high-yield cell line

Abstract

The present application relates to the technical field of biology, in particular to a method for obtaining a medicinal plant stem cell clone and a high-yield cell line, which adopts the establishment of a suspension cell line, the acquisition of single cells, cell clone culture and the screening of clone cells to obtain a cell line with high yield of specific active substances.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a method for obtaining a medicinal plant stem cell clone and a high-yield cell line. BACKGROUND

[0002] Medicinal plants are an important source of natural medicines, health products and cosmetic raw materials. Efficient extraction of their active ingredients has always been a research hotspot in the field of biological medicine. Among them, plant stem cells have the characteristics of unlimited proliferation and stable synthesis of specific active substances, and have become an ideal material for large-scale production of medicinal ingredients. Obtaining a high-yield cell line with consistent genetic background through single cell cloning technology not only solves the problems of shortage of wild medicinal plant resources and quality fluctuations, but also provides a stable cell source for the industrialized production of active ingredients, which has important academic value and application prospect.

[0003] Currently, plant single cell cloning technology mainly includes key links such as single cell separation, culture proliferation and clone screening, but the existing technology still has many limitations in practical application. In the aspect of single cell clone culture, the traditional method mostly adopts single-layer solid culture or single liquid culture: in single-layer solid culture, the nutrient distribution of the culture medium is uneven, and the single cell is difficult to proliferate due to nutrient deficiency or physical compression, and the clone formation rate is low; although liquid culture can improve the nutrient supply, single cells are easy to aggregate into groups, and the lack of physical support leads to difficulty in tracking cell division, making it difficult to accurately observe the clone formation process. In addition, the existing liquid supplementing method mostly uses direct supplementation of the basic culture medium, without considering the cell autocrine substances (such as growth factors and signal molecules) required for single cell proliferation, resulting in slow cell proliferation, long clone formation period and low efficiency.

[0004] Therefore, it is a key technical requirement in the field of plant stem cell cloning to develop an integrated technology for specific medicinal plants, which integrates efficient suspension line establishment, high-purity single cell separation, rapid clone culture and accurate screening, solves the problems of long period, low efficiency and blind screening in the existing method, and becomes a key technical requirement to be solved. SUMMARY

[0005] The purpose of the present application is to provide a method for obtaining a medicinal plant stem cell clone and a high-yield cell line with short period and high efficiency.

[0006] In order to achieve the above-mentioned application purpose, the technical scheme adopted by the present application is as follows:

[0007] A method for obtaining a medicinal plant stem cell clone and a high-yield cell line, characterized in that it comprises the following steps:

[0008] (1) Establishment of suspension cell lines: Select the cambium of medicinal plant stem segments, inoculate them into species-specific solid culture medium to induce stem cells, culture for 3-4 weeks, then transfer them to the corresponding liquid culture medium in shake flasks for culture, change the culture medium every 2-3 weeks, and obtain stable suspension cell lines after 2-3 passages;

[0009] (2) Single cell acquisition: The stable suspension cell line from step (1) was sieved step by step, the filtered cell slurry was collected, the supernatant was removed by centrifugation and the cells were collected; the cells were resuspended in liquid culture medium and diluted to a cell concentration of 3-6×10⁻⁶. 5 The inoculum density was 3-6 × 10⁶ cells / ml, and after mixing with the culture medium, the final inoculum density was achieved. 4 pcs / ml;

[0010] (3) Cell clone culture: Layered culture is used. The culture medium is mixed with the cell dilution mixture obtained in step (2). The bottom of the culture dish is marked with a grid to locate the cells. Cell division is observed. During the process, the medium is replenished and it is observed whether small cell clusters visible to the naked eye are formed.

[0011] (4) Screening of cloned cells: Select the cell clusters obtained in step (3) and seed them into 12-well cell culture plates. By comparing growth status, color and texture, growth rate and content of specific active substances, high-yield and stable cell lines are screened.

[0012] Furthermore, the medicinal plants mentioned are licorice, sandalwood, or yew.

[0013] Furthermore, the formulation of the licorice-specific solid culture medium is: MS + 0.5 mg / L NAA + 0.5 mg / L 6-BA + 0.5 mg / L 2,4-D + 30 g / L sucrose + 0.4% plant gel.

[0014] Furthermore, the formulation of the sandalwood-specific solid culture medium is: MS + 0.5 mg / L LDZ + 30 g / L sucrose + 0.4% plant gel.

[0015] Furthermore, the formula for the yew-specific solid culture medium is: B5 + 0.5 mg / L NAA + 1 g / L cypermethrin + 0.1% CH + 30 g / L sucrose + 0.4% plant gel.

[0016] Furthermore, the liquid culture medium is a formulation for removing plant gel from a solid culture medium of the corresponding species.

[0017] Furthermore, the culture medium for the layered culture in step (3) is a double-layer culture medium, wherein the bottom layer is a solid culture medium with a plant gel concentration of 0.5%, and the top layer is a solid culture medium with a plant gel concentration of 0.2% and the cell dilution mixture obtained in step (2).

[0018] Furthermore, the culture medium for the layered culture in step (3) is a double-layer culture medium, wherein the bottom layer is a solid culture medium with a plant gel concentration of 0.5%, and the top layer is a mixture of liquid culture medium with a plant gel concentration and cell dilution obtained in step (2).

[0019] Furthermore, in step (3), the replenishment of fluid refers to mixing the supernatant of the cell exponential growth phase with the liquid culture medium in a ratio of 1:1 to 1:1.5.

[0020] Furthermore, in step (2), the cells are filtered through a 200-mesh sieve to remove large cell clusters before being passed through 70μm and 40μm sieves to obtain a single-cell suspension.

[0021] The "specific active substances" mentioned in this application are determined based on the different medicinal plant cells cultivated, and are the active substances with medicinal value that the high-yield cell lines ultimately want to obtain; for example, in licorice, the specific active substances obtained are glycyrrhizin and glycyrrhizin flavonoids; in yew, the specific active substance obtained is paclitaxel. For different medicinal plants, those skilled in the art should be able to know the active substances that need to be obtained.

[0022] This invention optimizes the entire process of establishing species-specific suspension cell lines, isolating high-purity single cells, performing stratified synergistic culture, and screening phenotypes and bioactive substances. Compared with existing technologies, it achieves significant technical advantages, as detailed below:

[0023] 1. Species-specific solid and liquid culture medium formulations for licorice, sandalwood, and yew are precisely matched to the hormone requirements of stem cells of different medicinal plants, which shortens the establishment cycle of stable suspension cell lines, increases the stem cell induction rate, and stabilizes cell activity, providing high-quality starting materials for subsequent single-cell cloning.

[0024] 2. A step-by-step sieving and centrifugation process is employed to effectively remove large cell clusters and impurities, thereby improving single-cell purity; simultaneously, precise density adjustment (3-6×10⁻⁶) is used. 4 (cells / ml) to avoid cell overcrowding or sparseness, single cell viability retention rate ≥95%, significantly reducing mixed cell clusters in subsequent clonal culture and ensuring genetic consistency of clonal population;

[0025] 3. Layered synergistic culture technology solves the problems of uneven nutrition and difficulty in tracking in traditional monolayer culture by using a structural design of "bottom layer support + upper layer nutrition" and targeted replenishment of "exponential growth phase supernatant + culture medium" (utilizing cell auto-secreted growth factors);

[0026] 4. By linking the phenotypic screening of cell clusters’ “growth status, color, texture, and growth rate” with the precise detection of “specific active substance content”, the blindness of relying solely on phenotypic screening in the past is avoided. Attached Figure Description

[0027] Figure 1 Licorice suspension cell line;

[0028] Figure 2 Photos of licorice suspension cell lines before and after sieving;

[0029] Figure 3 : Licorice cell density adjusted photo;

[0030] Figure 4 Licorice cell cloning;

[0031] Figure 5 Spectrum of glycyrrhizic acid standard;

[0032] Figure 6 : Glycyrrhizic acid sample spectrum;

[0033] Figure 7 : Spectrum of licorice standard;

[0034] Figure 8 Licorice cell atlas;

[0035] Figure 9 : Taxus chinensis suspension cell line;

[0036] Figure 10 : Yew cell cloning;

[0037] Figure 11 Sandalwood suspension cell line;

[0038] Figure 12 Sandalwood cell cloning. Detailed Implementation

[0039] The present invention will be further described below through specific embodiments, but the scope of protection of this application is not limited to the embodiments.

[0040] Plant sources used in the examples:

[0041] Licorice: The licorice was collected from Ningxia and was kindly provided by the South China Botanical Garden.

[0042] Sandalwood: The sandalwood (Santalumalbum) was a gift from the South China Botanical Garden, Chinese Academy of Sciences.

[0043] Yew: Southern yew (Taxus wallichiana) purchased from the Lianshui Yew Plantation in Huai'an, Jiangsu.

[0044] Example 1: Licorice single-cell cloning and acquisition of high-yield cell lines

[0045] 1. Material preparation

[0046] Medicinal plant material: annual stem segments of licorice (Glycyrrhiza glabra).

[0047] Reagents and culture media: MS medium, NAA (naphthaleneacetic acid), 6-BA (6-benzylaminopurine), 2,4-D (2,4-dichlorophenoxyacetic acid), sucrose, plant gel (Sigma); high performance liquid chromatograph (Agilent 1260), cell counting chamber, 50 mL centrifuge tubes, 60 mm culture dishes.

[0048] 2. Establishment of suspension cell lines

[0049] (1) Take licorice stem segments, disinfect them with 75% ethanol for 30 seconds, rinse them with sterile water 3 times, soak them in 10% sodium hypochlorite solution for 12 minutes, and rinse them with sterile water 5 times; peel off the cambium tissue of the stem segments (about 0.5cm × 0.5cm) under a stereomicroscope.

[0050] (2) The cambium tissue was inoculated into licorice-specific solid culture medium: MS + 0.5 mg / L NAA + 0.5 mg / L 6-BA + 0.5 mg / L 2,4-D + 30 g / L sucrose + 0.4% plant gel, and the pH was adjusted to 5.8. It was then placed in a 25°C, light-proof incubator and cultured. After 3 weeks, white, loose stem cell clusters were visible.

[0051] (3) Transfer the stem cell clusters to liquid culture medium (same formula as the solid culture medium above, except for the removal of plant gel), and culture them on a shaker (120 rpm, 25°C, protected from light). Change the liquid culture medium every 2 weeks. After two passages, a stable suspension cell line is obtained. See [link to relevant documentation]. Figure 1 Licorice suspension cell line. (Uniform single cells and small cell clusters are visible under microscopic examination)

[0052] 3. Single-cell acquisition

[0053] (1) Take 10 mL of a stable suspension cell line and filter it sequentially through a 200-mesh sieve (to remove large cell clusters), a 70 μm sieve, and a 40 μm sieve. Collect the filtrate into a 50 mL centrifuge tube; see Figure 2 Photos of licorice cell lines before and after screening.

[0054] (2) Centrifuge at 4000 rpm for 10 min, discard the supernatant, add 5 mL of licorice liquid culture medium to resuspend the cells, take 10 μL of the cell suspension and count the cells under a microscope using a cell counting chamber, and adjust the cell concentration to 5 × 10⁻⁶. 5 per mL.

[0055] (3) Mix cells and liquid culture medium at a ratio of 1:9 to achieve a final inoculation density of 5 × 10⁻⁶ cells / ml. 4Quantity / mL, for later use. See Figure 3 Adjustment of licorice cell density.

[0056] 4. Cell clonal culture (solid-state + solid-state bilayer culture), see Figure 4 Licorice cell cloning.

[0057] (1) Prepare licorice solid culture medium with 0.5% plant gel (the formula is the same as the solid culture medium in step 2, with a plant gel concentration of 0.5%) and licorice solid culture medium with 0.2% plant gel (plant gel concentration of 0.2%), and sterilize at 121℃ for 20 min.

[0058] (2) Cool 0.5% gel medium to 65°C, mix it with the supernatant of licorice cell index growth phase at a ratio of 1:1, and quickly pour it into 60 mm culture dishes (10 mL per dish), and cool it to room temperature to solidify (bottom layer).

[0059] (3) Cool the 0.2% gel culture medium to 40°C, mix the cell suspension and culture medium at a ratio of 1:9, take 5 mL and pour it on top of the bottom layer, control the thickness of the top layer to about 1 mm, and let it cool and solidify.

[0060] (4) Mark the cell positions by drawing a grid on the bottom of the culture dish, place it in a 25°C dark incubator, observe the cell division every 3 days, and add 2mL of "exponential growth phase supernatant + liquid culture medium (1:1)" mixture on the 10th day.

[0061] 5. Cloning screening and detection of active substances

[0062] (1) After 21 days of culture, cell clusters with a diameter of 0.5-1 mm were observed (clonal formation rate of 65%). Ten cell clusters with regular shape and vigorous growth were selected and seeded into 12-well plates (each well containing 2 mL of licorice liquid culture medium). The cells were cultured in a shaker at 25°C. After 2 weeks of culture, the cells were transferred to 250 ml shake flasks for propagation.

[0063] (2) After 4 weeks, the three fastest-growing cell lines were selected, and their bioactive substance content was tested:

[0064] Glycyrrhizic acid was determined by high performance liquid chromatography (General Rule 0512).

[0065] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the stationary phase; acetonitrile as mobile phase A and 0.05% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; the detection wavelength was 237 nm. The theoretical plate number, calculated based on the glycyrrhizin peak, should be no less than 5000.

[0066]

[0067] Preparation of reference solution: Take appropriate amounts of glycyrrhizin reference standard and ammonium glycyrrhizate reference standard, accurately weigh them, and add 70% ethanol to prepare solutions containing 20 μg of glycyrrhizin and 0.2 mg of ammonium glycyrrhizate per 1 ml, respectively. (Weight of glycyrrhizic acid = weight of ammonium glycyrrhizate / 1.0207).

[0068] Preparation of the test solution: Weigh approximately 0.2 g of the powder (passed through a No. 3 sieve) accurately, place it in a stoppered conical flask, accurately add 100 ml of 70% ethanol, seal tightly, weigh, sonicate (power 250 W, frequency 40 kHz) for 30 minutes, cool, weigh again, replenish the lost weight with 70% ethanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0069] Assay: Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine the results. See below for details. Figure 5 Glycyrrhizic acid standard chromatogram and Figure 6 Glycyrrhizic acid sample spectrum.

[0070] For the determination of glycyrrhizin in licorice, referring to the Chinese Pharmacopoeia and the research methods of Hu Xinhua et al., HPLC was used.

[0071] Chromatographic column: Shimadzu C18 column (250mm×4.6mm, 5μm, HSS); mobile phase: acetonitrile (A)-0.05% phosphoric acid (B) aqueous solution; gradient elution (elution program shown in the table below); flow rate: 0.8mL / min; detection wavelength: 280nm; column temperature: 30℃; injection volume: 10μL.

[0072]

[0073] See diagram Figure 7 and Figure 8 .

[0074] Licorice flavonoid detection:

[0075] (1) Sample preparation

[0076] Licorice callus or suspension cultured cells were dried at 65°C, ground into powder using a mortar and pestle, and then passed through a 50-mesh sieve. 0.1 g of the sieved powder was accurately weighed, and 1 mL of 90% ethanol was added. The mixture was extracted using an ultrasonic extractor at 60°C for 30 min. After ultrasonic extraction, the mixture was centrifuged at 10,000 rpm for 10 min at room temperature. The supernatant was collected and diluted to 1 mL with 90% ethanol for analysis.

[0077] (2) Sample determination

[0078] Take 60 μL of the test solution (blank is 90% ethanol), add 15 μL of 5% sodium nitrite solution, let stand for 6 min, add 15 μL of 10% aluminum nitrate solution, let stand for 6 min, then add 120 μL of 1mol / L sodium hydroxide solution, and finally add 90 μL of 90% ethanol. Shake well and let stand for 15 min. Measure the absorbance at 502 nm using an ELISA reader.

[0079] (3) Preparation of standard curve

[0080] Prepare a 0.2 mg / mL rutin standard solution. Pipette 1.00 mL, 2.00 mL, 3.00 mL, 4.00 mL, and 5.00 mL of the rutin standard solution into 5 mL volumetric flasks, and dilute to the mark with 90% ethanol. Perform the determination as described in step 1, and plot the standard curve.

[0081] Example 2: Single-cell cloning of Taxus chinensis and acquisition of high-yield cell lines

[0082] 1. Materials and reagents:

[0083] One-year-old stem segments of Taxus wallichiana; B5 medium, NAA, thiamethoxam, casein hydrolysate (CH), sucrose, plant gel; high performance liquid chromatography (equipped with a Unitary C18 column).

[0084] 2. Establishment of suspension cell lines, see [link to documentation]. Figure 9 Photograph of yew tree suspension cells.

[0085] (1) The stem segments were disinfected in the same way as in Example 1. The cambium tissue was peeled off and inoculated into a solid culture medium of Taxus chinensis: B5 + 0.5 mg / L NAA + 1 g / L cypermethrin + 0.1% CH + 30 g / L sucrose + 0.4% plant gel. The cells were cultured at 25°C in the dark for 4 weeks to obtain stem cell clusters.

[0086] (2) Transfer to liquid culture medium (same as above, but remove plant gel), culture on a shaker (110 rpm), passage once every 3 weeks, and obtain a stable suspension cell line after 3 passages.

[0087] 3. Single-cell acquisition and clonal culture (solid + liquid bilayer culture), see [link / reference]. Figure 10 Taxus cell cloning.

[0088] (1) Cell screening, centrifugation, and density adjustment were the same as in Example 1, with a final seeding density of 4 × 10⁻⁶. 4 per mL.

[0089] (2) Cool 0.5% gel medium (B5 formula) to 60°C, mix with yew cell supernatant at a ratio of 1:1.5, pour plate to solidify (bottom layer); mix cell suspension with liquid medium (1:9), take 2 mL and spread it on the bottom layer surface, and culture at 25°C in the dark.

[0090] (3) During the culture period, 1 mL of “supernatant + liquid culture medium (1:1.5)” mixture was added every 5 days, and cell clusters were observed after 45 days.

[0091] 4. Paclitaxel detection

[0092] The active substance detected in yew cells is paclitaxel, and the detection method is as follows:

[0093] (1) Reference solution

[0094] Accurately weigh 9.99 mg of paclitaxel reference standard and place it in a 25 mL brown volumetric flask. Dissolve and dilute to the mark with methanol to obtain the reference solution. Accurately measure 0.1, 0.2, 0.5, 1.0, 1.5, and 2.0 mL of the reference solution and place them in 10 mL volumetric flasks. Dilute to the mark with methanol and shake well to prepare reference solutions with concentrations of 3.99, 7.98, 19.95, 39.90, 59.85, and 79.80 mg / L, respectively. Store at 4°C.

[0095] (2) Test solution

[0096] Accurately pipette 5.0 mL of cell suspension into a 150 mL Erlenmeyer flask, add 15 mL of methanol:acetic acid (95:5), and sonicate for 30 min. After extraction, cool to room temperature, make up for weight loss, centrifuge at high speed for 3 min, and filter the supernatant through a 0.45 μm microporous membrane. Store at 4 °C.

[0097] (3) Preparation of standard curve

[0098] Accurately pipette 20 μL of paclitaxel reference solutions of different concentrations under item "(1)", determine the peak area according to chromatographic conditions, inject in parallel three times, and take the average value. Perform linear regression with the concentration (mg / L) of each paclitaxel reference standard as the abscissa (X) and the chromatographic peak area of ​​paclitaxel as the ordinate (Y), and obtain the linear equation Y=19.309X+27.518, r2=0.9995. The results show that paclitaxel has a good linear relationship with peak area in the concentration range of 3.99~79.80 mg / L.

[0099] (4) Chromatographic conditions

[0100] Chromatographic column: Unitary C18 column (150mm × 4.6mm, 5μm).

[0101] Mobile phase: (A) water-(B) acetonitrile, gradient elution program is shown in the table below, flow rate: 1.0 mL / min, injection volume: 20 μL, column temperature: 30℃, detection wavelength: 227 nm.

[0102]

[0103] Example 3: Single-cell cloning of sandalwood and acquisition of high-yield cell lines

[0104] 1. Materials and Reagents

[0105] Sandalwood (Santalum album) stem segments; MS medium, TDZ (thiabendazole), diethyl ether, anhydrous sodium sulfate; gas chromatography-mass spectrometry (Agilent 7890A-5975C).

[0106] 2. Establishment of suspension cell lines, see... Figure 11 Sandalwood suspension cell line.

[0107] (1) After disinfection, the stem segments were inoculated into sandalwood solid culture medium: MS + 0.5 mg / LTDZ + 30 g / L sucrose + 0.4% plant gel, and cultured at 28℃ in the dark for 3 weeks to obtain pale yellow stem cell clusters.

[0108] (2) Transfer to liquid culture medium (remove plant gel), culture on a shaker (120 rpm), and obtain a stable suspension after two subcultures.

[0109] 3. Single-cell cloning and screening

[0110] (1) Prepare sandalwood solid culture medium with 0.5% plant gel (the formula is the same as the solid culture medium in step 2, with a plant gel concentration of 0.5%) and sandalwood solid culture medium with 0.2% plant gel (plant gel concentration of 0.2%), and sterilize at 121℃ for 20 min.

[0111] (2) Cool 0.5% gel medium to 65°C, mix it with the supernatant of sandalwood cell exponential growth phase at a ratio of 1:1, and quickly pour it into 60 mm culture dishes (10 mL per dish), and cool it to room temperature to solidify (bottom layer).

[0112] (3) Cool the 0.2% gel culture medium to 40°C, mix the cell suspension and culture medium at a ratio of 1:9, take 5 mL and pour it on top of the bottom layer, control the thickness of the top layer to about 1 mm, and let it cool and solidify.

[0113] (4) Mark cell positions by drawing a tic-tac-toe grid on the bottom of the culture dish, place it in a 25°C incubator in the dark, and observe cell division every 3 days. On the 10th day, add 2 mL of a mixture of "exponential growth phase supernatant + liquid culture medium (1:1)". See Figure 12 Sandalwood cell cloning.

[0114] 4. Clone screening and detection of active substances

[0115] (1) After 21 days of culture, cell clusters with a diameter of 0.5-1 mm were observed. Ten cell clusters with regular shape and vigorous growth were selected and inoculated into 12-well plates (each well containing 2 mL of sandalwood liquid culture medium). The cells were cultured in a shaker at 25°C. After 2 weeks of culture, the cells were transferred to 250 ml shake flasks for propagation.

[0116] (2) After 4 weeks, the three fastest-growing cell lines were selected and their active substance content was tested.

[0117] The active substances detected in sandalwood cells were α-santalol and β-santalol, and the detection method is as follows:

[0118] 1. Extraction method: Cold ether extraction of sandalwood volatile oil.

[0119] Weigh approximately 20g of each sample powder (passed through a 20-mesh sieve) accurately and place it in a 250mL Erlenmeyer flask. Add approximately 200mL of anhydrous diethyl ether and soak at room temperature for 3 days. Filter the solution. Soak the residue again for 2 days using the same method. Filter the solution and combine the two filtrates. Allow the solution to evaporate naturally at room temperature to a final volume of 10mL. Add 1.5g of anhydrous sodium sulfate and dehydrate the solution at room temperature for 2-3 days. Filter the solution and allow it to evaporate naturally at room temperature to a final volume of 1mL. Transfer the solution to a small bottle for later use.

[0120] 2. Programmable heating method:

[0121] An Agilent HP-5 flexible quartz capillary column (30 m × 0.32 mm, 0.25 μm) was used. The injection port temperature was 270 °C, with a temperature program of (column temperature 90 °C, increased to 160 °C at 5 °C / min, then to 165 °C at 0.5 °C / min, then to 180 °C at 1 °C / min, and finally to 230 °C at 10 °C / min). The vaporization chamber temperature was 250 °C. The carrier gas was high-purity helium (99.999% purity). The column inlet pressure was 52.6 kPa, the carrier gas flow rate was 1.0 mL / min, the injection volume was 1 μL, and the split ratio was 20:1. The ion source was an EI source with an ion source temperature of 230 °C, a quadrupole temperature of 150 °C, an electron energy of 70 eV, an emission current of 34.6 μA, an interface temperature of 280 °C, a solvent delay of 3 min, and a mass range of 10–550 amu.

Claims

1. A method for cloning medicinal plant stem cells and obtaining high-yield cell lines, characterized in that, The medicinal plant is licorice, and the method includes the following steps: (1) Establishment of suspension cell lines: Select medicinal plant stem segments, peel off their cambium, inoculate them into species-specific solid culture medium to induce stem cells, culture for 3-4 weeks, then transfer them to the corresponding liquid culture medium in shake flasks for culture, change the culture medium every 2-3 weeks, and obtain stable suspension cell lines after 2-3 passages; The specific solid culture medium is: MS + 0.5 mg / L NAA + 0.5 mg / L 6-BA + 0.5 mg / L 2,4-D + 30 g / L sucrose + 0.4% plant gel; The liquid culture medium is a formulation for removing plant gel from a solid culture medium of the corresponding species; (2) Single cell acquisition: The stable suspension cell line from step (1) was filtered through a 200-mesh sieve, a 70-μm sieve, and a 40-μm sieve to remove large cell clusters. The filtrate was collected, and the supernatant was removed by centrifugation before collecting the cells. The cells were then resuspended in liquid culture medium and diluted to a cell concentration of 3–6 × 10⁻⁶. 5 Inoculation density: cells / mL, then mixed with liquid culture medium to achieve a final inoculation density of 3–6 × 10⁻⁶. 4 cells / mL; (3) Cell clone culture: A double-layer culture is adopted, wherein the bottom layer is a solid culture medium of the corresponding species with a plant gel concentration of 0.5%, and the upper layer is a mixture of the solid culture medium of the corresponding species with a plant gel concentration of 0.2% and the cell dilution mixture obtained in step (2); after the mixture is added to the culture dish, a grid is drawn on the bottom of the dish for positioning, and culture is carried out under the dark. During the period, replenishment solution is added every 10 days. The replenishment solution is made by mixing the supernatant of the cell exponential growth phase with the corresponding liquid culture medium at a volume ratio of 1:1-1:1.5 until small cell clusters visible to the naked eye are formed. (4) Screening of cloned cells: The cell clusters obtained in step (3) are first seeded into 12-well cell culture plates, and then further seeded into shake flasks for propagation and culture. During the propagation process, high-yield and stable cell lines are screened by comparing growth status, color and texture, growth rate and content of specific active substances. The specific active substances mentioned above are glycyrrhizin and glycyrrhizin flavonoids from licorice.

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