Culture method of hormone autotrophic ginseng cell line and ginseng cell line

Abstract

The invention belongs to the technical field of plant cell engineering, and particularly relates to a culture method of a hormone autotrophic ginseng cell line and the ginseng cell line. The culture method comprises the following steps: peeling, cleaning and disinfecting a ginseng embryo, slicing, inoculating to an MS solid culture medium, and inducing to obtain an embryogenic cell line; transferring to an MS liquid culture medium for suspension culture to obtain a stable suspension cell line; carrying out nursing culture after treatment with a composite mutagenic agent; and transferring the growing cell cluster to a hormone-free MS solid culture medium for multiple subculture, and screening out a cell line independent of exogenous auxin. The cell line can proliferate without exogenous hormones, the residual risk is eliminated, the cost is reduced, and the cell line is suitable for the fields of food and medicine and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of plant cell engineering technology, specifically relating to a method for culturing a hormone-autotrophic ginseng cell line and the ginseng cell line itself. Background Technology

[0002] Ginseng, a traditional and precious Chinese medicinal herb, enjoys high market demand. However, traditional field cultivation methods face challenges such as long planting cycles, stringent environmental requirements, and difficulties in continuous cropping, resulting in high production costs. To overcome these limitations, ginseng tissue and cell culture technology has emerged. This technology boasts significant advantages, including short production cycles, no seasonal or geographical limitations, ease of large-scale and standardized production, and no pesticide pollution, demonstrating broad application prospects.

[0003] However, existing plant cell culture technologies, including ginseng cell culture, generally rely on adding exogenous plant hormones, such as auxins and cytokinins, to the culture medium to induce and maintain cell proliferation and growth. While this method effectively promotes cell growth, it also introduces several insurmountable drawbacks. First, the use of exogenous hormones significantly increases production costs. Second, and more critically, the harvested cell products are prone to containing exogenous hormone residues. This not only increases the complexity and cost of subsequent separation and purification processes but also potentially threatens the safety of the final product, making it difficult to meet the high safety standards required by the food, health product, and pharmaceutical industries.

[0004] like:

[0005] Chinese Patent Application No. 202110425119.3 discloses a method for isolating and culturing ginseng stem cells using a bioreactor, which involves adding 3 mg / L of 2,4-dichlorophenoxyacetic acid, 2 mg / L of gibberellin, and 1 mg / L of kinetin to the culture medium.

[0006] Chinese Patent Application No. 201710435874.3 discloses a method for preparing ginseng stem cells, which describes a culture medium for ginseng stem cells containing 0.5 mg / L naphthaleneacetic acid and 4.0 mg / L indolebutyric acid.

[0007] Chinese Patent Application No. 202411570691.9 discloses a tissue culture medium and a tissue culture method for culturing ginseng stem cells. It describes a culture medium for ginseng stem cells in which kinetin and 2,4-dichlorophenoxyacetic acid are added.

[0008] The existing technology described above induces ginseng cell growth by adding auxin or cytokinin to the culture medium. However, cells cultured in this way are prone to exceeding hormone residue limits after harvest, affecting the safety of subsequent products. Developing a cell line that can be cultured industrially without relying on exogenous plant hormones would be of great significance for reducing production costs and improving product safety.

[0009] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the Invention

[0010] The purpose of this application is to provide a method for preparing a hormone-autotrophic ginseng cell line, so as to solve the problems of existing ginseng cell culture relying on exogenous hormones, which leads to the safety hazards of hormone residues in the product and high production costs.

[0011] To achieve the above objectives, the present invention provides a method for culturing a hormone-autotrophic ginseng cell line, the method comprising the following steps:

[0012] (1) The ginseng embryos were peeled, cleaned, and disinfected. After being sliced, they were inoculated onto a solid culture medium for induction and proliferation to obtain embryogenic cell lines.

[0013] (2) The embryonic cell line was transferred to a liquid culture medium for suspension culture to obtain a suspension cell line;

[0014] (3) The suspension cell line was treated with mutagenesis and then cultured under supervision;

[0015] (4) After the care culture is completed, the cell clusters growing on the care culture medium are transferred to hormone-free solid culture medium for multiple subcultures to screen out cell lines that can grow without the help of exogenous growth hormone.

[0016] Preferably, in step (1), the method of peeling, cleaning and disinfecting the ginseng embryo is as follows: peel the mature ginseng embryo and wash it with running water for 10-20 minutes, then disinfect it with 75-80 vol% ethanol for 30-40 seconds; disinfect it with 0.5-0.8 wt% sodium hypochlorite for 5-10 minutes, wash it with 0.5% silver nitrate 2-3 times, each time for 5-8 minutes, and finally wash it with sterile water 2-3 times, each time for 1-2 minutes.

[0017] Preferably, in step (1), the method of inoculating the slicing onto the solid culture medium is as follows: the sterilized ginseng embryo slices are inoculated onto MS solid culture medium, and 2,4-dichlorophenoxyacetic acid and kinetin are added simultaneously. Wherein:

[0018] MS medium is a known culture medium. The composition of MS medium (mg / L) is as follows: potassium nitrate 1900, ammonium nitrate 1650, calcium chloride dihydrate 440, magnesium sulfate 370, potassium dihydrogen phosphate 170, manganese sulfate 22.3, boric acid 6.2, zinc sulfate 8.6, potassium iodide 0.83, copper sulfate 0.025, sodium molybdate 0.25, cobalt chloride 0.025, disodium EDTA 74.6, ferrous sulfate 55.6, inositol 200, ammonium sulfate hydrochloride 0.2, glycine 4, pyridoxine hydrochloride 1.0.

[0019] Preferably, the concentration of 2,4-dichlorophenoxyacetic acid added is 2-4 mg / L, and the concentration of kinetin added is 0.1-0.2 mg / L.

[0020] Preferably, in step (1), the method of inducing proliferation is as follows: cultured in the dark at 22-24℃ for 20-30 days to obtain an embryonic cell line.

[0021] Preferably, in step (2), the embryonic cell line is transferred to MS liquid medium containing 2,4-dichlorophenoxyacetic acid and cultured for 30-35 days under shaking conditions of 22-24℃ and 120-140r / min. The cells are subcultured twice to obtain a stable proliferating cell line.

[0022] Preferably, in step (3), the suspended cell line is treated with 0.1-0.2 wt% hydroxylamine hydrochloride, 20-25 mg / L 5'-bromouracil, and 0-0.15 wt% ethyl methanesulfonate for 1-1.5 h.

[0023] Preferably, in step (3), the nurturing culture method is as follows: the mutagenized cells are evenly spread on sterile filter paper of the nurturing culture medium and cultured in the dark at 22-24℃ for 30-35 days.

[0024] Based on the same technical concept, another aspect of the present invention is to provide a ginseng cell line obtained by a method for culturing a hormone-autotrophic ginseng cell line.

[0025] The beneficial effects of this invention are as follows:

[0026] This invention, through the construction of a "mutation-recovery-hormone-free screening" technical system, successfully obtained a hormone-autotrophic ginseng cell line, fundamentally solving the problems of high cost, safety risks, and complex production processes caused by the dependence on exogenous hormones in existing ginseng cell culture technologies. Specific beneficial effects are as follows:

[0027] 1. Solve the problem of hormone residues and improve product safety.

[0028] In existing technologies, ginseng cell culture relies on exogenous auxins, which can lead to residual exogenous hormones in the harvested cell products, increasing the difficulty of subsequent purification and threatening food and medicinal safety. The hormone-autotrophic cell line screened in this invention can proliferate stably without the addition of any exogenous plant hormones, eliminating the risk of hormone residues at the source and making it more suitable for fields with stringent safety requirements, such as food, health products, and pharmaceuticals.

[0029] 2. Reduce production costs and simplify production processes.

[0030] Exogenous plant hormones are expensive, and their concentration control requires precise operation, increasing culture costs and process complexity. The cell line of this invention does not require exogenous hormones, thus eliminating hormone procurement costs and simplifying culture medium preparation and hormone concentration control. This reduces manpower and equipment investment in the production process, making it more conducive to the standardized, automated, and industrialized production of ginseng cells.

[0031] 3. Break through traditional cultivation limitations and improve production efficiency.

[0032] Traditional ginseng field cultivation suffers from problems such as long cycles (several years), geographical / seasonal limitations, and difficulties in continuous cropping. This invention, based on cell culture technology and combined with the autotrophic properties of hormones, enables rapid proliferation in laboratories or factories (single culture cycle 30-35 days), unaffected by the natural environment. This significantly shortens the production cycle, increases yield per unit time, and provides a new path to resolving the supply and demand imbalance in the ginseng market.

[0033] 4. The technical methods are reliable and the results are reproducible.

[0034] This invention induces cellular genetic variations through chemical or physical mutagenesis, combined with nurturing culture (or liquid regeneration culture) to aid in the recovery of damaged cells, and finally screens target cell lines using hormone-free culture medium. The technical pathway is clear and feasible. Molecular biological analysis (transcriptome sequencing, quantitative real-time PCR) confirms that the hormone autotrophic trait of the target cell line originates from the activation of endogenous auxin signaling pathways (upregulation of genes such as AUX1, AUX / IAA, GH3, and SAUR), mechanistically validating the scientific validity of the method and the stability of the results. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 This is a real photo of a cell cluster under care and culture after mutagenesis.

[0037] Figure 2 This is a comparison of the growth of the mutant cell line g37 and the control cell line on hormone-free culture medium.

[0038] Figure 3 This is a diagram showing the results of differentially expressed genes.

[0039] Figure 4 This is a graph showing the results of KEGG enrichment analysis (biological processes).

[0040] Figure 5 This is a graph showing the results of the GO enrichment analysis (Mut_vs_Control_G).

[0041] Figure 6 This is a graph showing the results of KEGG enrichment analysis.

[0042] Figure 7 This is a diagram of the auxin signaling pathway.

[0043] Figure 8 This is a graph showing the expression levels of the AUX1 gene in the control cell line (WT) and the mutant cell line (g37).

[0044] Figure 9 This is a graph showing the expression levels of the AUX / IAA-1 gene in the control cell line (WT) and the mutant cell line (g37).

[0045] Figure 10 This is a graph showing the expression levels of the GH3-1 gene in the control cell line (WT) and the mutant cell line (g37).

[0046] Figure 11 This is a graph showing the expression levels of the GH3-2 gene in the control cell line (WT) and the mutant cell line (g37).

[0047] Figure 12 This is a graph showing the expression levels of the SAUR-3 gene in the control cell line (WT) and the mutant cell line (g37).

[0048] Figure 13 This is a graph showing the expression levels of the SAUR-6 gene in the control cell line (WT) and the mutant cell line (g37). Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0050] Example 1

[0051] This embodiment provides a method for culturing hormone-autotrophic ginseng cell lines. This method combines chemical mutagenesis, nurture culture recovery, and hormone-free screening to successfully screen and prepare the target cell line from cells derived from ginseng embryos.

[0052] The method specifically includes the following steps:

[0053] Step (1): Select plump, mature ginseng seeds and, under aseptic conditions, peel off the complete embryos as explants. To ensure sterility, the embryos undergo a series of strict surface disinfection treatments: rinse the peeled embryos under running water for about 10 minutes to remove surface impurities, then immerse them in a 75% ethanol solution for rapid disinfection for 30 seconds, and then transfer them to a 0.5% sodium hypochlorite solution for 5 minutes. To neutralize and wash away residual sodium hypochlorite, wash twice with a 0.5% silver nitrate solution, each time for 5 minutes. Finally, rinse twice with sterile water for 1 minute each time to thoroughly remove disinfectant residue.

[0054] After sterilization, the cleaned embryos were sliced ​​into thin slices on a sterile operating table and evenly inoculated onto pre-prepared solid culture media (approximately 100 slices per media). In this embodiment, the basal medium was MS medium. To induce the formation of embryogenic cell masses, 2.0 mg / L of 2,4-dichlorophenoxyacetic acid and 0.1 mg / L of kinetin were added to the MS solid culture medium. 2,4-dichlorophenoxyacetic acid, as a highly efficient synthetic auxin, effectively promotes cell dedifferentiation and proliferation, while kinetin, as a cytokinin, works synergistically with auxin to maintain cell division capacity. The inoculated culture dishes were placed in a 22°C incubator for dark culture to inhibit differentiation and promote callus growth.

[0055] After approximately 25 days of culture, numerous pale yellow, loosely textured embryogenic cell clusters were induced on the embryo sections. Rapidly growing and well-morphologically sound fragments were selected and transferred to liquid culture medium for suspension culture. This liquid culture medium was also MS-based, with the hormone formulation adjusted to include only 2.0 mg / L of 2,4-dichlorophenoxyacetic acid to promote rapid cell proliferation in a liquid environment. The Erlenmeyer flasks containing the cells and liquid culture medium were placed on a shaker and cultured at 22°C and 120 rpm. This ensured uniform cell dispersion, adequate oxygen and nutrients, and prevented cell aggregation. Subculture was performed every 30 days. After two consecutive subcultures, a stable, highly viable suspension cell line was obtained. This cell line served as the control cell line WT in subsequent experiments, without any mutagenesis treatment.

[0056] Step (2): Mutagenesis treatment of the cell line is a key step in generating genetic variations and obtaining potential hormone autotrophic mutants. Specifically, WT suspension cells in good condition during the logarithmic growth phase are collected by static incubation or low-speed centrifugation. Then, a compound chemical mutagen is added to the culture medium. In this embodiment, to improve the mutagenesis efficiency and the breadth of the mutation spectrum, a compound treatment scheme consisting of three chemical mutagens with different mechanisms of action—hydroxylamine hydrochloride, 5'-bromouracil, and ethyl methanesulfonate—is used, with final concentrations of 0.1%, 20 mg / L, and 0%, respectively. Hydroxylamine hydrochloride mainly acts on cytosine in DNA; 5'-bromouracil, as an analog of thymine, can be mistakenly incorporated into DNA replication; and ethyl methanesulfonate, as an alkylating agent, can alkylate guanine in DNA. The cells are mixed evenly with the culture medium containing the above compound mutagen and treated with shaking at 22 degrees Celsius and 120 rpm for 1.5 hours. The treatment time and concentration are designed to ensure sufficient mutagenesis efficiency while minimizing cell lethality.

[0057] In fact, there are several options for the ratio and processing time of hydroxylamine hydrochloride, 5'-bromouracil, and ethyl methanesulfonate, as shown in Table 1. This embodiment selects the first option.

[0058] Table 1

[0059]

[0060] Step (3): Perform restorative culture on the mutagenized cells. Given the strong toxicity and damage of mutagenesis to cells, direct selective culture would lead to the death of a large number of cells. Therefore, a recovery phase is needed to allow damaged cells to repair damage and restore physiological function and division capacity. In one embodiment of this application, caregiver culture is used as a preferred restorative culture method.

[0061] The core of foster culture lies in utilizing the growth factors and signaling molecules secreted by healthy homologous cells (i.e., feeder cells) to support the survival and growth of damaged cells. The culture medium preparation method is as follows: In a sterile culture dish 1, first pour a layer of solid culture medium containing standard MS nutrients but without exogenous hormones as a basal nutrient layer; after it solidifies, take a small amount of the unmutated control cell line WT and evenly spread it on the surface of the solid culture medium to form a thin feeder cell layer, which simulates the microenvironment of cells in tissues; then, cover the feeder cell layer with a sterile filter paper that has been autoclaved. The sterile filter paper is designed to allow nutrients and signaling molecules secreted by the feeder cells to permeate upwards, while physically separating the feeder cells from the mutagenic cells to be recovered, ensuring the accuracy of subsequent screening. Specifically... Figure 1 As shown, the cell clusters were cultured after mutagenesis, and each cluster of cells represents a cell line.

[0062] After the mutagenesis treatment, the Erlenmeyer flasks containing the mutagenic cells were allowed to stand for a short time. Most of the supernatant containing the mutagen was carefully discarded. The remaining concentrated cell suspension was then evenly spread onto sterile filter paper containing pre-prepared nucleation medium. The culture dish was sealed and placed in a dark incubator at 22 degrees Celsius for nucleation. After 30 days of recovery culture, many mutagenic cells were observed to re-proliferate on the sterile filter paper, forming visible, pale yellow or light milky white cell clusters.

[0063] Step (4): Screening culture on exogenous hormone-free medium. This step is the core of applying selection pressure to isolate mutant cell lines with true hormone autotrophic capacity from a large number of cells that have recovered growth. Specifically, using sterile forceps, rapidly growing, large, normally colored, and fluffy cell clusters are carefully selected from the sterile filter paper of the nurturing medium. Each selected cell cluster is treated as an independent clone and inoculated onto fresh MS solid medium without any exogenous plant hormones. For strict comparison, the unmutated control cell line WT is also inoculated into identical hormone-free MS solid medium in clusters of similar size. All inoculated culture dishes are incubated at 22 degrees Celsius in the dark, and the growth of each cell line is observed and recorded regularly over the next 30 days.

[0064] Step (5): Obtain the target cell line. Significant differences can be observed after 30 days of selection culture. Please refer to... Figure 2 This figure shows a comparison of the screening results for hormone-autotrophic cell lines. As shown, the control cell line WT essentially stopped growing on the hormone-free medium, with no significant increase in cell mass volume compared to inoculation, and its color gradually turned brown. Some cells showed signs of death, indicating that its growth and survival were heavily dependent on exogenous hormones. In stark contrast, one of the selected mutagenic cell lines, named g37, exhibited vigorous proliferation on the same hormone-free medium. Its cell mass volume increased significantly, its color remained a bright light milky white, and its morphology was full and fluffy. These results indicate that the g37 cell line has acquired the trait of being able to proliferate stably on its own without dependence on exogenous hormones, thus becoming a hormone-autotrophic cell line. Through continuous subculturing of the g37 cell line, its hormone-autotrophic trait can be stably inherited, thus successfully preparing a hormone-autotrophic ginseng cell line.

[0065] To reveal the intrinsic mechanism by which the g37 cell line acquires hormone autotrophic traits at the molecular level and to provide a scientific basis for the beneficial effects of this application, this embodiment further conducted a series of molecular biological analyses.

[0066] Transcriptome sequencing was performed first. Specifically, total RNA was extracted from control cell lines and the mutagenized cell line g37, which had been cultured on hormone-free medium for 30 days, with three replicates per group. RNA extraction was conducted using a commercially available RNA extraction kit. RNA quality control was performed by the sequencing company, and only RNA that passed quality control was used for transcriptome sequencing. The transcriptome analysis workflow followed standard procedures.

[0067] The sequencing data undergoes a series of bioinformatics analyses. First, the raw sequencing data is quality controlled to remove low-quality and adapter sequences, resulting in high-quality clean sequences. Then, these clean sequences are aligned with the ginseng reference genome, and the expression level of each gene is calculated based on the alignment results. Differentially expressed genes are identified by comparing the expression levels of each gene in the g37 and WT cell lines.

[0068] Differentially expressed gene results as follows Figure 3 As shown, the results indicate that the mutagenic cell line g37 showed significantly upregulated expression of 1093 genes and significantly downregulated expression of 1253 genes compared to the control cell line, which confirms that mutagenesis treatment does indeed have a wide-ranging effect on cells at the transcriptional level.

[0069] Further enrichment analysis of GO and KEGG was performed. Figure 4 , Figure 5 The results showed that the differentially expressed genes between the mutant cell line g37 and the control cell line were mainly enriched in: DNA-binding transcription factor activity, extracellular regions, oxidoreductase reactions, defense responses, stress responses, transcription factor activity, auxin-activated signaling pathways, DNA-template transcription, and sequence-specific DNA binding.

[0070] KEGG enrichment analysis results are as follows: Figure 6 As shown, the differentially expressed genes between the mutant cell line g37 and the control cell line are enriched in: phenylpropane biosynthesis; unsaturated fatty acid biosynthesis; plant-pathogen interaction; diterpenoid biosynthesis; and plant hormone signal transduction. Because auxin is crucial for plant cell growth and division, the auxin signaling pathway is specifically listed below.

[0071] Figure 7This is a diagram of the auxin signaling pathway, with red boxes indicating upregulated expression levels. The results show that the mutagenic cell line g37 has genes with significantly upregulated expression levels in the auxin signaling pathway compared to the control cell line. More specifically, the diagram highlights differentially expressed genes in the auxin signaling transduction pathway. In this pathway diagram, genes at several key nodes show significant upregulated expression in the g37 cell line and are highlighted. These genes include the inward transporter AUX1, responsible for transporting auxin into the cell, and downstream early response gene families in the auxin signaling pathway, such as the GH3 family and the SAUR family.

[0072] This embodiment further validates the auxin signaling pathway-related genes using real-time quantitative PCR, specifically as follows:

[0073] Step 1: Use an RNA extraction kit to extract total RNA from a cell sample of approximately 0.7 grams, and then use a reverse transcription kit to synthesize cDNA.

[0074] The reverse transcription reaction system consisted of 20 μL of total RNA, 1 μL of EVO M-MLV RTase Enzyme Mix, 1 μL of Primer Mix, 4 μL of 5×RTase Reaction Buffer Mix, and 12 μL of RNase Free Water. Reaction conditions: 37℃ for 15 min; 85℃ for 5 s; 4℃ for 10 min.

[0075] Step 2: The reaction system for quantitative real-time PCR (10 μL) includes 5 μL of 2×Realab Green PCR FastMixture; 1 μL of Primer Mix; 2 μL of cDNA; and 2 μL of RNase-free water. The quantitative real-time PCR reaction program is: 95℃ for 2 min, (95℃ for 10 s, 60℃ for 30 s) × 45 cycles, followed by 95℃ for 1 min.

[0076] Step 3: RT-qPCR was used to detect differentially expressed gene levels in the control cell line (WT) and the mutant cell line (g37). RT-qPCR data were plotted using GraphPad software, and the results are shown below. Figures 8-13The results of RT-qPCR showed that, compared with the control cell line WT, the relative expression levels of genes such as auxin introporter AUX1, early response genes AUX / IAA-1, GH3-1, GH3-2, SAUR-3, and SAUR-6 were significantly increased in the mutagenic cell line g37. This result is highly consistent with the transcriptome sequencing data, thus strongly confirming that the molecular basis for hormone autotrophy in the g37 cell line lies in the systematic activation of its endogenous auxin signaling pathway, leading to enhanced cell response to extremely low concentrations of endogenous auxin or the synthesis of more auxin, thereby freeing it from dependence on exogenous hormones.

[0077] Example 2

[0078] This embodiment provides a variation of the method for preparing hormone-autotrophic ginseng cell lines, aiming to demonstrate that, in addition to chemical mutagenesis, physical mutagenesis can also achieve the technical objectives of this application. The overall process of this embodiment is similar to that of Embodiment 1, with the main difference being the specific method of mutagenesis treatment in step (2).

[0079] Step (1): Establish a ginseng suspension cell line. The operation of this step is exactly the same as in Example 1.

[0080] Step (2) involves mutagenesis of the cell line. As an optional implementation, this embodiment employs a physical mutagenesis method, specifically ultraviolet (UV) irradiation. Suspension cells in the logarithmic growth phase are taken, and a portion of the culture medium on the surface of the cell cluster is absorbed using a sterile filter paper to make the cells semi-dry. Approximately 2 grams of wet-weight cells are weighed and evenly spread on the bottom of a sterile culture dish to form a thin layer. This ensures that all cells receive a uniform UV dose. The culture dish with the lid open is placed in a preheated UV crosslinker, and the irradiation wavelength is set to 254 nm, with a total irradiation dose of 15 joules / m². After irradiation, to avoid the influence of photorepair on the mutagenesis effect, fresh, hormone-free MS liquid culture medium should be immediately added to the culture dish under light-protected conditions. The cells are gently washed off the bottom of the dish and resuspended using a pipette.

[0081] Step (3) involves restorative culture of the mutated cells. This step is exactly the same as in Example 1, using the same caretaker culture method.

[0082] Steps (4) and (5) involve hormone-free screening and obtaining the target cell line. These two steps are performed exactly the same as in Example 1. Well-grown cell clusters are selected from the filter paper used for nurture culture and seeded onto MS solid medium free of exogenous hormones for screening. After a period of culture, some of the mutated cell lines (e.g., g-uv-1) exhibit vigorous growth, while the control cell line WT shows growth stagnation.

[0083] It is understood that this embodiment demonstrates that the "mutation-recovery-screening" technical path proposed in this application has universality. The mutagenesis step can be either chemical mutagenesis or physical mutagenesis, such as ultraviolet irradiation or gamma ray irradiation or other physical methods that can induce gene mutations.

[0084] In summary, the method for preparing a hormone-autotrophic ginseng cell line provided in this application creatively combines three core steps: "mutation treatment," "restorative culture," and "exogenous hormone-free screening culture." This method can stably and efficiently screen novel cell lines from various ginseng tissue sources that can proliferate stably without exogenous hormones. This method not only fundamentally solves the potential safety hazards of hormone residues in products from traditional ginseng cell culture techniques and significantly reduces production costs, but its scientific mechanism has also been strongly confirmed at the molecular biology level, possessing extremely high industrial application value.

[0085] Conclusion

[0086] This invention induces embryogenic cell lines from ginseng embryos, transfers them to liquid culture medium to obtain stable suspension cell lines, treats these suspension cells with a mutagen and performs nucleated culture, and finally transfers them to hormone-free solid culture medium for multiple subcultures, thereby screening out cell lines that can grow without the dependence of exogenous auxin. Subsequent transcriptome sequencing combined with quantitative real-time PCR analysis confirmed enhanced auxin signaling pathways in this cell line. Differentially expressed genes AUX1 encodes an auxin inward transporter, AUX / IAA encodes members of the Aux / IAA protein family involved in auxin signaling, GH3 encodes an auxin-responsive protein, and SAUR encodes an auxin-responsive protein. Based on the signal pathway diagram obtained from KEGG enrichment analysis, the possible mechanism by which the mutant cell line g37 can grow on auxin-free medium is that the expression of AUX1, a protein that mediates the inward transport of auxin, is upregulated in g37 cells, leading to more auxin entering the nucleus and regulating the transcription of genes. Ultimately, this regulates the transcription of auxin-responsive genes such as AUX / IAA, GH3, and SAUR, thereby allowing the proliferation of the mutant cell line g37 to be independent of exogenously added auxin. Therefore, the mechanism by which the growth of this cell line is independent of exogenous auxin can be explained at the gene level.

[0087] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for culturing a hormone-autotrophic ginseng cell line, characterized in that, The cultivation method includes the following steps: (1) The ginseng embryos were peeled, cleaned, and disinfected. After being sliced, they were inoculated onto a solid culture medium for induction and proliferation to obtain embryogenic cell lines. (2) The embryonic cell line was transferred to a liquid culture medium for suspension culture to obtain a suspension cell line; (3) The suspension cell line was treated with mutagenesis and then cultured under supervision; (4) After the care culture is completed, the cell clusters growing on the care culture medium are transferred to hormone-free solid culture medium for multiple subcultures to screen out cell lines that can grow without the help of exogenous growth hormone.

2. The method for culturing the hormone-autotrophic ginseng cell line according to claim 1, characterized in that, In step (1), the method of peeling, cleaning and disinfecting the ginseng embryo is as follows: peel the mature ginseng embryo and wash it with running water for 10-20 minutes, then disinfect it with 75-80 vol% ethanol for 30-40 seconds; disinfect it with 0.5-0.8 wt% sodium hypochlorite for 5-10 minutes, wash it with 0.5% silver nitrate 2-3 times, each time for 5-8 minutes, and finally wash it with sterile water 2-3 times, each time for 1-2 minutes.

3. The method for culturing the hormone-autotrophic ginseng cell line according to claim 1, characterized in that, In step (1), the method of inoculating the slicing onto the solid culture medium is as follows: the sterilized ginseng embryo slices are inoculated onto MS solid culture medium, and 2,4-dichlorophenoxyacetic acid and kinetin are added at the same time.

4. The method for culturing the hormone-autotrophic ginseng cell line according to claim 3, characterized in that, The concentration of 2,4-dichlorophenoxyacetic acid added is 2-4 mg / L, and the concentration of kinetin added is 0.1-0.2 mg / L.

5. The method for culturing the hormone-autotrophic ginseng cell line according to claim 1, characterized in that, In step (1), the method of inducing proliferation is as follows: cultured in the dark at 22-24℃ for 20-30 days to obtain embryonic cell lines.

6. The method for culturing the hormone-autotrophic ginseng cell line according to claim 1, characterized in that, In step (2), the embryonic cell line is transferred to MS liquid medium containing 2,4-dichlorophenoxyacetic acid and cultured for 30-35 days under shaking conditions of 22-24℃ and 120-140r / min. The cell line is subcultured twice to obtain a stable proliferating cell line.

7. The method for culturing the hormone-autotrophic ginseng cell line according to claim 1, characterized in that, In step (3), the suspension cell line is treated with 0.1-0.2 wt% hydroxylamine hydrochloride, 20-25 mg / L 5'-bromouracil and 0-0.15 wt% ethyl methanesulfonate for 1-1.5 h.

8. The method for culturing the hormone-autotrophic ginseng cell line according to claim 1, characterized in that, In step (3), the nucleation method is as follows: the mutagenized cells are evenly spread on sterile filter paper of nucleation medium and cultured in the dark at 22-24℃ for 30-35 days.

9. The ginseng cell line obtained by the culture method of the hormone-autotrophic ginseng cell line according to any one of claims 1-8.

Images