Construction method of pseudo-ginseng cell line

Abstract

The invention relates to the technical field of fermentation of medicinal material production, and discloses a construction method of a pseudo-ginseng cell line, which comprises the following steps: obtaining a target organ of a pseudo-ginseng plant; inducing calluses through a solid culture medium by adopting a target organ; and culturing and evaluating the callus in an oscillation fermentation system to obtain the working cell strain and the adaptive liquid culture medium thereof. According to the technical scheme provided by the invention, cells from different tissue parts of the same or different plants can form cell strain lines with different yields and different product components under selective and optimized culture conditions; the cell strain line can generate different drug properties and different economic values. In the dimensions, compared with traditional planting type pseudo-ginseng, the pseudo-ginseng planting method has the advantages of richness of germplasm and diversity of product application. When the obtained working cell strain is applied to a liquid fermentation method of pseudo-ginseng cells, compared with a traditional pseudo-ginseng planting method, pseudo-ginseng biomass which is richer in medicine property and higher in quality is more efficiently produced.

Description

Technical Field

[0001] This invention relates to the field of fermentation technology in medicinal material production, and in particular to a method for constructing a Panax notoginseng cell line. Background Technology

[0002] Rare medicinal plant resources refer to plant species that have significant medicinal value but are naturally distributed in limited quantities, scarce, or endangered. These plants play an irreplaceable role in traditional medicine and modern drug development, but their survival faces severe challenges due to over-harvesting, habitat destruction, and other factors. Therefore, the production technology of rare medicinal materials is a crucial link in the pharmaceutical and health industries. However, current medicinal material cultivation technology faces multiple technical bottlenecks, limiting industrial upgrading. The following are the main difficulties and specific manifestations of the medicinal material industry:

[0003] (1) Backward germplasm resources and breeding technology. A lack of high-quality germplasm: The germplasm of traditional Chinese medicinal herbs has severely degenerated (e.g., the yield of Rehmannia glutinosa in Henan has decreased by 30% compared to 20 years ago); high-purity seedlings rely on imports (e.g., 90% of saffron bulbs are imported from Iran and Spain); hybridization breeding technology is weak; most medicinal herbs still use traditional breeding methods; and the approval cycle for new varieties is as long as 10 years or more (compared to the 3-5 year breeding cycle for flowers in the Netherlands). Lack of seed and seedling standards: Only 15% of commonly used Chinese medicinal herbs nationwide (e.g., ginseng and astragalus) have national or industry seedling standards; local varieties are mixed (e.g., there is a mixed planting of "Mawei Gui" and "Zhujie Gui" in Gansu).

[0004] (2) Extensive planting methods and difficulty in implementing standardization. Traditional experience dominates, and scientific data is lacking: More than 60% of small farmers rely on the experience of their ancestors to determine the sowing amount and fertilization timing, resulting in yield differences of 2-3 times in the same production area (e.g., the yield of Panax notoginseng in Wenshan, Yunnan, ranges from 200 kg to 600 kg per mu). The implementation rate of GAP (Good Agricultural Practices) is less than 10%, and some provisions are out of touch with reality (e.g., requiring a 10-year rotation cycle for medicinal herbs with continuous cropping obstacles, which is far beyond the affordability of farmers). There is no solution to the problem of continuous cropping obstacles: Continuous cropping of medicinal herbs such as Rehmannia glutinosa, Atractylodes macrocephala, and Panax notoginseng leads to the outbreak of soil-borne diseases, reducing yield by more than 50%, and existing solutions (such as soil fumigation) cost as much as 3,000 yuan per mu and pollute the environment.

[0005] (3) Shortcomings in green pest and disease control technologies. High dependence on chemical pesticides: Highly toxic organochlorine pesticides (such as pentachloronitrobenzene) are still widely used in ginseng cultivation, while similar production areas in South Korea have fully promoted biological pesticides (such as Bacillus preparations). In the EU's MRLs (Maximum Residue Limits) standards, 38 commonly used Chinese medicinal materials have been found to have exceeded the limits for pesticides (such as the 2023 case in Germany where wolfberries were detained due to excessive levels of acetamiprid). Difficulty in promoting biological control technologies: The application cost of natural enemy insects (such as predatory mites to control spider mites) is twice that of chemical pesticides, resulting in low acceptance among farmers. Poor stability of microbial agents (such as the field survival rate of Bacillus subtilis is less than 30%).

[0006] (4) Insufficient application of mechanization and intelligent technology. Lack of machinery suitable for specific crops: The mechanical damage rate during harvesting of root and rhizome medicinal herbs (such as Salvia miltiorrhiza and Angelica dahurica) exceeds 20%, compared to less than 5% for potatoes harvested in the Netherlands. The mechanization rate in mountainous medicinal herb producing areas (such as the Coptis chinensis planting area in Chongqing) is less than 15%. Obstacles to the implementation of digital technology: The cost of a single set of IoT monitoring equipment exceeds 20,000 yuan, which is difficult for small farmers to afford. The accuracy rate of AI pest and disease identification systems is only 70% (e.g., the misjudgment rate of leaf edge scorching reaches 25%), far lower than that of field crops (e.g., the disease identification rate of rice is 95%).

[0007] Panax notoginseng is a rare medicinal herb. As an important Chinese medicinal herb, Panax notoginseng has a huge market, involving multiple links such as planting, processing, circulation and end consumption. The national Panax notoginseng circulation market size is about RMB 15 billion to 20 billion (including medicinal material markets, pharmaceutical company procurement, e-commerce and other channels).

[0008] The cultivation of Panax notoginseng inevitably encounters the aforementioned industry challenges, particularly in terms of germplasm decline, continuous cropping obstacles, excessive pesticide residues in products, and low land use efficiency. These issues have become major obstacles to the production of Panax notoginseng raw materials. Therefore, a new method should be developed that can partially or completely solve the problems of Panax notoginseng cultivation.

[0009] Currently, a new method for obtaining Panax notoginseng raw materials—the fermentation method of Panax notoginseng cells—can be used. This method involves obtaining tissues from the same or different Panax notoginseng plants, inducing them into callus tissue, and then introducing the callus tissue into a sealed fermentation tank for aerobic fermentation, which can efficiently obtain high-quality Panax notoginseng cell biomass.

[0010] Obtaining high-quality Panax notoginseng biomass involves not only obtaining cell lines, but also obtaining a group of cell lines with different medicinal properties and fermentation performances, thereby enabling the fermentation process and fermentation products to exhibit diversity and higher economic value. This method for constructing Panax notoginseng cell lines perfectly solves the traditional problems in Panax notoginseng breeding, including:

[0011] (1) Long growth cycle and high self-incompatibility. Long growth cycle: Panax notoginseng typically takes more than 3 years from sowing to flowering and seed setting. Completing a full breeding cycle of "sowing-harvesting seeds-sowing the next generation" takes at least 3-4 years. If multiple generations of selection and trait stabilization are required, the breeding of a new variety may take more than ten or even several decades. This greatly slows down the breeding process and increases time and economic costs. High self-incompatibility: Panax notoginseng is a typical cross-pollinated plant, and self-pollination is almost fruitless or has a very low fruit set rate. This means that in the breeding process, it is impossible to quickly homogenize superior genes and fix superior traits through self-pollination. Breeders must carry out artificial hybridization, but the hybrid offspring will produce huge trait segregation, requiring a large population and a long generation to select and stabilize the target line.

[0012] (2) Conflicts between highly complex traits and breeding objectives. There are diverse breeding objectives for Panax notoginseng, and some of these objectives are inherently conflicting. Multi-objective breeding: An ideal Panax notoginseng variety needs to simultaneously possess: high yield – large root biomass; high quality – high content of major medicinal components (saponins, etc.); strong disease resistance – especially against major diseases such as root rot, black spot, and circular spot; strong stress resistance – drought tolerance, waterlogging tolerance, cold tolerance, etc.; trade-offs between traits – these excellent traits often have a "give-and-take" relationship. For example, high-yielding varieties may have weaker disease resistance; varieties with high saponin content may have slower growth rates. How to break these unfavorable genetic linkages and integrate multiple excellent genes into the same variety is a huge technical challenge.

[0013] (3) Scarcity of germplasm resources and narrow genetic base. Depletion of wild resources: Wild populations of Panax notoginseng are extremely rare, nearly endangered, resulting in very limited access to superior wild genetic resources for breeders. Wild resources typically contain strong genes for disease resistance and stress tolerance. Low genetic diversity in cultivated populations: For a long time, Panax notoginseng cultivation has relied mainly on traditional seed propagation. After years of artificial selection, the genetic background has become relatively narrow, with insufficient sources of superior genes, limiting breeding potential. This is akin to "inbreeding," making it difficult to produce breakthrough variations.

[0014] In summary, a method for constructing a highly efficient Panax notoginseng cell line adapted to plant cell fermentation has significant practical implications. This invention relates to a method for constructing a Panax notoginseng cell line. Summary of the Invention

[0015] This invention proposes a method for constructing Panax notoginseng cell lines to solve the technical problems of monotonous Panax notoginseng cell lines and poor fermentation effect in the prior art.

[0016] To address the aforementioned technical problems, this invention proposes a method for constructing a Panax notoginseng cell line, comprising:

[0017] Obtain the target organs from Panax notoginseng plants;

[0018] Callus tissue was induced from the target organ using a solid culture medium.

[0019] The callus tissue was cultured in a shaking fermentation system to obtain working cell lines and liquid culture medium.

[0020] Optionally, the target organ includes at least one of the following: root, stem, leaf, flower, fruit, and seed.

[0021] Optionally, the solid culture medium includes a basal culture medium, a carbon source, plant growth hormones, and cytokinins.

[0022] Optionally, the basal culture medium includes B5 solid culture medium or MS solid culture medium.

[0023] Optionally, the basal culture medium includes at least one of MS20, MS2, NTM52, B5.6, B5.7, B5.7h&h, B5.12, and IND30.

[0024] Optionally, the step of inducing callus tissue from the target organ using a solid culture medium includes:

[0025] The target organ is cultured in the solid culture medium at 23-27°C for 10-25 days to induce the callus tissue.

[0026] Optionally, obtaining the target organ of the Panax notoginseng plant includes:

[0027] Select Panax notoginseng seedlings;

[0028] The Panax notoginseng seedlings were first cleaned;

[0029] Cut the Panax notoginseng seedlings into seedling segments;

[0030] Transfer the seedling segments to the workbench;

[0031] The seedling segments were soaked in an ethanol solution;

[0032] Discard the ethanol solution;

[0033] The seedling segments were then subjected to a second cleaning.

[0034] The seedling segments were soaked in a mercuric chloride solution;

[0035] Discard the mercuric chloride solution;

[0036] The seedling segments were then subjected to a third cleaning.

[0037] The target organ is obtained by absorbing the moisture from the seedling segment.

[0038] Optionally, the concentration of the ethanol solution is 70-80%.

[0039] Optionally, the concentration of the mercuric chloride solution is 0.08-0.12%.

[0040] Optionally, the second washing of the seedling segment includes:

[0041] The seedling segments were then washed a second time with sterile water.

[0042] The third cleaning of the seedling segment includes:

[0043] The seedling segments were then washed a third time with sterile water.

[0044] Compared with the prior art, the method for constructing Panax notoginseng cell lines of the present invention has the following advantages:

[0045] (1) This invention can form working cell lines of Panax notoginseng with rich medicinal properties, and ensure that the germplasm is properly preserved and disseminated in a controlled manner. This technology can rapidly and economically form cell lines, including cell lines formed from tissues of wild Panax notoginseng plants from different habitats such as Yunnan and Guangxi, and cell lines from different organs of the same Panax notoginseng plant, such as roots, stems, leaves, flowers, fruits, and seeds, to establish a library. This not only ensures the stability of the germplasm, but also has rich medicinal diversity, meeting the raw material needs of a wider range of health products. This technology can form hundreds or even thousands of cell lines with different fermentation performance, product characteristics and other biological traits within 0.5-1 year, which is hundreds of times faster than the traditional Panax notoginseng breeding rate.

[0046] (2) This technology can economically and effectively produce Panax notoginseng cell culture, partially replacing traditional Panax notoginseng cultivation; the yield per mu of this technology is 100 times higher than that of cultivated Panax notoginseng, saving about 99% or more of the cultivated area.

[0047] (3) The product quality is more stable, safe, and pollution-free. This technology can allow cells to accumulate about 11% total saponins of Panax notoginseng within 14 days, and contains rare components such as NotR1, which is rarely found in cultivated medicinal materials.

[0048] In summary, the method for constructing Panax notoginseng cell lines of the present invention enables the formation of cell lines with different yields and compositions from cells of the same plant or different tissues of different plants under selective and optimized culture conditions. These cell lines with different yields can produce different medicinal properties and different economic values. In these dimensions, it offers greater germplasm richness and product application diversity than traditionally cultivated Panax notoginseng. When the working cell lines obtained from the method for constructing Panax notoginseng cell lines of the present invention are applied to the liquid fermentation method of Panax notoginseng cell lines, the fermentation effect of Panax notoginseng cells can be improved. Attached Figure Description

[0049] One or more embodiments are illustrated by way of example with reference to the accompanying drawings. These illustrations do not constitute a limitation on the embodiments. Elements having the same reference numerals in the drawings are denoted as similar elements. Unless otherwise stated, the figures in the drawings are not to be limited by scale.

[0050] Figure 1 This is a flowchart of the method for constructing a Panax notoginseng cell line and the liquid fermentation method in one embodiment of the present invention;

[0051] Figure 2 This is a graph showing experimental data for saponin testing in one embodiment of the present invention. Detailed Implementation

[0052] To facilitate understanding of the present invention, the present invention will be described in more detail below with reference to the accompanying drawings and specific embodiments.

[0053] An embodiment of the present invention provides a method for constructing a Panax notoginseng cell line, comprising:

[0054] A1. Obtain the target organ from the Panax notoginseng plant;

[0055] A2. Callus tissue was induced from the target organ using a solid culture medium.

[0056] A3. The callus tissue is cultured in a shaking fermentation system to obtain working cell lines and liquid culture medium.

[0057] Plant cell suspension culture or fermentation technology is a synthetic biology technique developed by utilizing the natural instincts of plant cells, such as their "dryness" and "totipotency," combined with fermentation engineering, cell engineering, enzyme engineering, and genetic engineering. Plant cell fermentation technology can improve the above-mentioned production pain points from multiple dimensions.

[0058] First, plant cell fermentation technology can optimize germplasm resources and standardize seeds, thereby ensuring the stable biological activity of fermentation products. Selected new cell lines of medicinal plants can guarantee stable product yields and composition profiles, overcoming the dependence of authentic medicinal materials on comprehensive geographical resources such as latitude, longitude, hydrology, climate, and weather.

[0059] Secondly, plant cell fermentation technology can solve the problems of extensive planting methods and difficulty in standardization in traditional Chinese medicine cultivation. Plant cell fermentation technology uses stable cell line preservation techniques to ensure stable metabolic synthesis of the cell lines, a closed fermentation environment, and stable and controllable fermentation conditions, thus ensuring stable and controllable separation, extraction, and purification conditions. Therefore, it is highly conducive to achieving standardized production.

[0060] Third, plant cell fermentation technology can solve the pollution problems caused by pesticides and fertilizers in the traditional Chinese medicine planting process. The plant cell fermentation process is a closed, sterile, pure culture process that naturally avoids contact with pesticide and fertilizer residues, resulting in pollution-free production, and the product naturally does not contain endotoxins.

[0061] Fourth, plant cell fermentation technology inherently possesses the potential to integrate with and continuously iterate upon mechanized and intelligent technologies. By combining mechanized and intelligent technologies, plant cell fermentation technology leverages the genetic potential, reaction rate, and product quality of cell lines, achieving yields that surpass those of traditional Chinese medicine production by 3 to 5 geometric progressions. Furthermore, this technology significantly reduces the need for manual labor, thus representing intelligent manufacturing.

[0062] In terms of germplasm resource assurance, this technology has obvious advantages: (1) Germplasm stability. Since it is asexual reproduction, once the cell line is determined, the stability of the variety and the high efficiency of rejuvenation can be guaranteed under long-term operation. Traditional Panax notoginseng seeds have a short lifespan, surviving only for 1 week at room temperature and less than 1 year at low temperature. However, plant cell fermentation technology uses liquid nitrogen low-temperature refrigeration, which can guarantee preservation for several years or even 10 years; (2) Germplasm resource diversity: This technology can quickly and economically form cell lines, including cell lines formed from wild Panax notoginseng from different habitats such as Yunnan and Guangxi, and can also establish cell lines from different organs of different Panax notoginseng plants, such as roots, stems, leaves, flowers, fruits, and seeds, to establish a library, which can not only ensure germplasm stability, but also have rich diversity to meet the raw material needs of a wider range of health products; (3) Breeding efficiency. The rejuvenation of Panax notoginseng cell lines can be completed in 30-45 days (basically within 2-3 fermentation cycles), while planting Panax notoginseng requires 5-10 years. This is because Panax notoginseng takes 3-5 years to flower and bear fruit; (4) Quality standardization. Because the definition and evaluation standards of traditional Chinese medicine are difficult to standardize within the industry, it is challenging to achieve uniformity in terms of molecular identity, geographical environment, and fingerprint profiles of medicinal components. Plant cell fermentation, however, can control the basic characteristics through two indicators: the active component profile and fermentation performance characteristics, thus possessing standardized evaluation criteria and component stability.

[0063] In terms of production efficiency, the annual yield per mu (a Chinese unit of area, approximately 0.067 hectares) of plant cell fermentation technology surpasses that of Panax notoginseng by several geometric progressions. Based on the data from this experiment, a 100-mu (a Chinese unit of area, approximately 6.67 hectares) plant cell fermentation plant can produce 259.2 tons of Panax notoginseng cell dry matter annually (=12 tanks * 60 tons * 24 batches * 15 kg / ton), while planting 100 mu of Panax notoginseng can produce only 3 tons annually (100 mu * 1 / 5 * 150 kg / mu), resulting in a yield per mu ratio of 86.4 and saving approximately 99.2% of cultivated land.

[0064] In terms of product quality, Panax notoginseng cell fermentation technology has two typical advantages over cultivated Panax notoginseng: (1) Advantage in quality. Panax notoginseng cell fermentation technology uses GMP-level fermentation technology, with no chemical fertilizers or pesticides used throughout the process, naturally eliminating the residue of pollutants. In contrast, Panax notoginseng cultivation faces the challenges of fertilizer application and pest control. In particular, to resist the typical continuous cropping obstacle of Panax notoginseng, the land must either be left fallow for 5-10 years or high-input agricultural processes such as high-concentration soil disinfection and process pest control must be implemented. Therefore, the quality of Panax notoginseng products often faces problems such as pollution residues; (2) Advantage in function. Based on the components required by the human body, this technology can achieve its goals by utilizing controllable culture medium components, types and concentrations of stimulants, fermentation strategies, etc. Moreover, the rich cell lines have a wide range of medicinal properties, which can be applied to the treatment or conditioning needs of different indications or sub-health conditions; (3) Full plant utilization. Panax notoginseng cell lines that have undergone liquid fermentation have the characteristics of thin cell walls and low levels of difficult-to-utilize components such as cellulose, which are easily digested by human or animal digestive enzyme systems, making them suitable for direct human consumption and highly bioavailable. The traditional consumption of Panax notoginseng inevitably involves consuming the indigestible parts, such as the tuberous roots, stems, and leaves with varying degrees of fibrosis and lignification.

[0065] In terms of its advantage in integrating with future technologies for innovation, Panax notoginseng's plant cell technology, being a product of interdisciplinary integration, naturally absorbs and innovates upon the excellent achievements of advanced manufacturing technologies such as synthetic biology, fermentation engineering, and smart science. For example, Panax notoginseng cell fermentation technology can incorporate single-cell sequencing and gene editing from synthetic biology to form cell lines with extremely high yields; it can also incorporate the latest achievements of smart science in fermentation engineering, combining "dry" prediction with "wet" detection to continuously tap into yield potential.

[0066] Therefore, developing Panax notoginseng cell fermentation technology can solve the inefficiency problems in the Panax notoginseng planting industry, such as the "joint liability barrier" and competition for land with grain crops. It can also address the pollution risks associated with fertilizers and pesticides during the planting process, and ensure the richness of product efficacy and quality safety. This provides an optimal solution for human land health, environmental safety, food safety, and life health, possessing significant practical significance and commercial value. Furthermore, as a common plant cell fermentation technology, Panax notoginseng cell fermentation technology can also provide valuable insights for the development of cell fermentation technologies for other rare medicinal plants, possessing extremely high academic value.

[0067] In summary, the method for constructing the Panax notoginseng cell line in this embodiment has the following advantages:

[0068] (1) To form working cell lines of Panax notoginseng with rich medicinal properties and ensure that the germplasm is properly preserved and disseminated in a controlled manner. This technology can quickly and economically form cell lines, including cell lines formed from wild Panax notoginseng from different habitats such as Yunnan and Guangxi, and can also establish cell banks from different organs of different Panax notoginseng plants such as roots, stems, leaves, flowers, fruits and seeds. This can not only ensure the stability of germplasm, but also have rich medicinal diversity, and meet the raw material needs of a wider range of health products;

[0069] (2) This technology can economically and effectively produce Panax notoginseng cell culture, partially replacing traditional Panax notoginseng cultivation; the yield per mu of this technology is 80 times higher than that of cultivated Panax notoginseng, saving about 99% or more of the cultivated area.

[0070] (3) The product quality is more stable, safe, and pollution-free. This technology can allow cells to accumulate about 11% total saponins of Panax notoginseng within 14 days, and contains rare components such as NotR1, which is rarely found in cultivated medicinal materials.

[0071] In one embodiment, the target organ includes at least one of the following: root, stem, leaf, flower, fruit, and seed.

[0072] In one embodiment, the solid culture medium includes a basal culture medium, a carbon source, plant growth hormones, and cytokinins.

[0073] In one embodiment, as shown in Table 1, the basal culture medium includes at least one of MS20, MS2, NTM52, B5.6, B5.7, B5.7h&h, B5.12, and IND30.

[0074] The culture medium formulations adjusted based on B5 solid medium or MS solid medium are shown in Table 1 below:

[0075] Table 1. Composition of the culture medium used to culture Panax notoginseng cell lines

[0076]

[0077] The embodiments of the present invention also propose a liquid fermentation method based on the above-described method for constructing Panax notoginseng cell lines, comprising:

[0078] B1. Obtain working cell lines;

[0079] B2. Obtain the liquid culture medium;

[0080] B3. Inoculate the working cell line into the liquid culture medium to obtain a seed culture;

[0081] B4. Inoculate the seed liquid into a fermenter for fermentation culture to obtain fermentation broth;

[0082] B5. Dehydrate the fermentation broth to obtain a concentrated slurry;

[0083] B6. Prepare the target product from the slurry.

[0084] Steps B1 and B2 can be combined into step A3. That is, working cell lines and liquid culture medium can be obtained through step A3.

[0085] In one embodiment, obtaining the working cell line includes:

[0086] A4. Dehydrate the mixture of the working cell line and the liquid culture medium;

[0087] A5. Immerse the dehydrated mixture in a cryoprotectant solution and freeze for storage;

[0088] A6. Revive the cryopreserved mixture to obtain the working cell line.

[0089] In one embodiment, the cryoprotectant is liquid nitrogen.

[0090] In one embodiment, the process of preparing the concentrated slurry into the target product includes:

[0091] The concentrated slurry is dried to obtain the target product as a dried product.

[0092] In one embodiment, the process of preparing the concentrated slurry into the target product includes:

[0093] Food preservatives are added to the concentrated slurry to obtain the target product, which is made from fresh ingredients.

[0094] In one embodiment, the food preservative includes tea polyphenols, potassium sorbate, and e-polylysine.

[0095] In one embodiment, after adding a food preservative to the concentrated slurry to obtain the fresh target product, the process further includes:

[0096] The fresh target product is sealed and stored at 8-15℃ for more than one year.

[0097] Screening and shake-flask yield evaluation of Panax notoginseng cell lines

[0098] 1.1 Materials

[0099] Panax notoginseng seedlings were randomly purchased by Langoufei Aiteng Biomedical Co., Ltd. in Wenshan, Yunnan Province, planted in nutrient soil, and used as explant material after two weeks of growth.

[0100] 1.2 Culture medium

[0101] MS20 was used as the basal medium. Meanwhile, to create diverse cell lines and compare fermentation yields, NTM52, IND30, and B5.6 were also used to evaluate the fermentation yields of different cell lines. Their formulations are shown in Table 1.

[0102] 1.3 Disinfection of explants

[0103] First, rinse the Panax notoginseng seedlings with running water and cut them into appropriate sizes. Then, place them in a clean bench, add 75% ethanol (by volume) and soak for 30 seconds. After pouring out the ethanol, rinse three times with sterile water. Next, pour in 0.1% mercuric chloride solution for 5 minutes to disinfect. After pouring out the mercuric chloride solution, rinse five times with sterile water. Finally, use sterile filter paper to absorb the moisture.

[0104] 1.4 Explant inoculation and callus induction

[0105] Place the sterilized taproots, lateral roots, stem segments, leaves, flower buds, fruits, and seeds into petri dishes. Use sterile scissors and a scalpel to cut each organ into small pieces of (1-5) mm × (1-5) mm. After inoculation, place the dishes in an incubator for cultivation, controlling the temperature at 23-27℃ in complete darkness, and regularly observe the changes in the explants and the growth of callus tissue. If contamination occurs, promptly remove the contaminated petri dishes.

[0106] Each callus formed on the plate is then divided into 9 equal parts and placed on an MS20 plate to form new callus. This process is repeated 2-3 times until the resulting callus can be cultured in liquid.

[0107] 1.5 Fermentation Yield Evaluation

[0108] After 2-3 divisions and subcultures of the callus tissue, select vigorous and loosely structured callus tissues. Inoculate individual callus tissue clusters at a rate of 60-120 g / L (fresh weight) into 300 mL Erlenmeyer flasks containing 100 mL of sterile liquid culture medium. Incubate on a shaker at 25°C and 100 rpm. Add 50 μM methyl jasmonate and 100 μM silver thiosulfate on days 0, 7, and 14. Measure relevant parameters every 7 days. Details are as follows.

[0109] Determination of five specific saponins: Cell clusters were obtained by centrifuging 10-20 mL of cell fermentation broth. The cell clusters were extracted twice with a 1:10 weight ratio of 40% ethanol solution, and the extracts were combined to determine the content of the five saponins in the solution. HPLC was used for detection. The five saponins are ginsenoside Rd (CAS 52705-93-8), Rb1 (CAS 41753-43-9), Rg1 (CAS22427-39-0), Re (CAS 52286-59-6), and notoginsenoside R1 (Not R1, CAS 80418-24-2).

[0110] 1.6 Experimental Results

[0111] from Figure 2 It can be seen that after callus induction and callus liquid fermentation, the ginsenoside composition (proportion of 5 saponins) and ginsenoside concentration (proportion of fresh weight) of tissues from the same plant are very different.

[0112] like Figure 2 The figures show the yield of ginsenosides in suspension cultures generated from primary callus. The concentration and composition of total ginsenosides are also shown. The first bar graph for each cell line shows the ginsenoside yield under uninduced conditions. Subsequent bar graphs show the ginsenoside yield after induction with 50 μM MJS and 100 μM SLTS for 7, 14, and 21 days. The origin of the cell lines is labeled R (root), S (stem), L (leaf), and P (petiole). MJS stands for methyl jasmonate, and SLTS stands for silver thiosulfate.

[0113] Therefore, it can be concluded that cells from different tissues of the same plant, under selective and optimized culture conditions, can form cell lines with different yields and compositions. These cell lines with different yields can produce different medicinal properties and different economic values. In these dimensions, it has greater germplasm richness and product application diversity than traditionally cultivated Panax notoginseng.

[0114] In summary, the working cell lines obtained by the method for constructing Panax notoginseng cell lines in this embodiment can effectively construct cell lines rich in medicinal properties when applied to the liquid fermentation method of Panax notoginseng cells. This results in a wide range of applications. Furthermore, the fermentation technology is highly efficient, produces high-quality products, maintains stable cell performance and yield, and is environmentally friendly, energy-saving, water-saving, and land-saving. It is a technology worthy of key support and should be protected by patents.

Claims

1. A method for constructing a Panax notoginseng cell line, characterized in that, include: Obtain the target organs from Panax notoginseng plants; Callus tissue was induced from the target organ using a solid culture medium. The callus tissue was cultured in a shaking fermentation system to obtain working cell lines and liquid culture medium.

2. The method for constructing the Panax notoginseng cell line according to claim 1, characterized in that, The target organ includes at least one of the following: root, stem, leaf, flower, fruit, and seed.

3. The method for constructing the Panax notoginseng cell line according to claim 1, characterized in that, The solid culture medium includes a basal culture medium, a carbon source, plant growth hormones, and cytokinins.

4. The method for constructing the Panax notoginseng cell line according to claim 3, characterized in that, The basal culture medium includes B5 solid culture medium or MS solid culture medium.

5. The method for constructing the Panax notoginseng cell line according to claim 4, characterized in that, The basal culture medium includes at least one of MS20, MS2, NTM52, B5.6, B5.7, B5.7h&h, B5.12, and IND30.

6. The method for constructing the Panax notoginseng cell line according to claim 3, characterized in that, The step involves inducing callus tissue from the target organ using a solid culture medium, including: The target organ was cultured in the basal culture medium at 23-27°C for 10-25 days to induce the callus tissue.

7. The method for constructing the Panax notoginseng cell line according to claim 1, characterized in that, The target organs obtained from the Panax notoginseng plant include: Select Panax notoginseng seedlings; The Panax notoginseng seedlings were first cleaned; Cut the Panax notoginseng seedlings into seedling segments; Transfer the seedling segments to the workbench; The seedling segments were soaked in an ethanol solution; Discard the ethanol solution; The seedling segments were then subjected to a second cleaning. The seedling segments were soaked in a mercuric chloride solution; Discard the mercuric chloride solution; The seedling segments were then subjected to a third cleaning. The target organ is obtained by absorbing the moisture from the seedling segment.

8. The method for constructing the Panax notoginseng cell line according to claim 7, characterized in that, The concentration of the ethanol solution is 70-80%.

9. The method for constructing the Panax notoginseng cell line according to claim 7, characterized in that, The concentration of the mercuric chloride solution is 0.08-0.12%.

10. The method for constructing the Panax notoginseng cell line according to claim 7, characterized in that, The second cleaning of the seedling segment includes: The seedling segments were then washed a second time with sterile water. The third cleaning of the seedling segment includes: The seedling segments were then washed a third time with sterile water.

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