Method for culturing suspension cells of saussurea saluenensis

Abstract

The embodiment of the specification provides a culture method for suspension cells of saussurea saluenensis, which comprises the following steps: selecting seeds of saussurea saluenensis, inoculating the seeds into a solid culture medium containing hormone, and inducing to form a primary callus, the hormone comprising naphthylacetic acid and kinetin; adjusting the concentration of hormones in the solid culture medium, and screening out target calluses; and inoculating the suspension cells obtained based on the target callus into an optimized liquid MS culture medium for culturing to obtain the target saussurea saluenensis suspension cells. According to the process, by optimizing induction of calluses, screening an efficient cell line, establishing a suspension cell culture system and optimizing culture medium components, the content of total flavonoids in a saussurea saluenensis cell culture is remarkably increased, and the industrial production standard is met.

Description

Technical Field

[0001] This specification relates to the technical field of plant cell culture, and particularly to a method for suspension culture of Saussurea salwinensis Anthony cells. Background Art

[0002] Plants of the genus Saussurea in the Compositae family have attracted much attention due to their rich medicinal value. In particular, Saussurea involucrata, the total flavonoid compounds contained in it have been widely proven to have significant pharmacological effects, including anti-inflammatory, antioxidant, and anti-tumor effects, etc. These bioactive components have given Saussurea involucrata a long application history in traditional medicine, and with the continuous deepening of modern medical research, its medicinal value has been further confirmed and expanded. With the rapid development of biotechnology, plant cell culture technology has become an effective way to replace traditional extraction methods. This technology can efficiently produce the required plant secondary metabolites by precisely controlling the culture conditions in the laboratory or standardized production environment.

[0003] Saussurea salwinensis Anthony, as a closely related species of Saussurea involucrata in the genus Saussurea, is mainly distributed in the high-altitude areas of Yunnan and Tibet in China, growing in special environments such as hillside shrubs, meadows, and scree slopes at an altitude of 3500 - 4450 meters. The growth rate of Saussurea salwinensis Anthony is slow, and its distribution area is limited. Coupled with the relatively lack of scientific research, these factors have restricted its large-scale promotion in commercial applications.

[0004] Therefore, it is desired to provide a process for inducing, screening, and large-scale culturing of Saussurea salwinensis Anthony suspension cells to obtain total flavonoid components similar to Saussurea salwinensis Anthony efficiently and at low cost. Summary of the Invention

[0005] One or more embodiments of this specification provide a method for culturing Saussurea salwinensis Anthony suspension cells, including the following steps: selecting the seeds of Saussurea salwinensis Anthony, inoculating them into a solid medium containing hormones to induce the formation of primary callus, the hormones including naphthaleneacetic acid and kinetin; adjusting the concentration of the hormones in the solid medium to screen out the target callus; inoculating the suspension cells obtained based on the target callus into an optimized liquid MS medium for culture to obtain the target Saussurea salwinensis Anthony suspension cells.

[0006] In some embodiments, the solid medium further includes sucrose. Before the adjustment, the concentration of naphthaleneacetic acid in the solid medium is 0.5 - 2 mg / L, the concentration of kinetin is 0.5 - 1 mg / L, and the mass concentration of sucrose is 1% - 5%.

[0007] In some embodiments, after the adjustment, the concentration of naphthylacetic acid in the solid culture medium is 1.8 - 2.2 mg / L, and the concentration of kinetin is 0.8 - 1.2 mg / L.

[0008] In some embodiments, the target callus is the cell line with the highest total flavonoid content of secondary metabolites.

[0009] In some embodiments, the concentration of ammonium nitrate in the optimized liquid MS medium is 800–850 mg / L, the concentration of phenylalanine is 0.4–0.6 mg / L, the concentration of cinnamic acid is 0.4–0.6 mg / L, and the concentration of sodium acetate is 0.4–0.6 mg / L.

[0010] In some embodiments, the culture conditions in the optimized liquid MS medium include an oscillation speed of 120 ± 10 rpm, a temperature of 25 ± 2 °C, a light cycle: 15 - 17 hours of light followed by 7 - 9 hours of darkness, and a light intensity of 1000 - 1500 lux.

[0011] In some embodiments, the method further includes subjecting the suspension cells obtained from the target callus to at least one of ultraviolet treatment, methyl jasmonate treatment, and 4 °C cold treatment to further induce the accumulation of secondary metabolites in the cells.

[0012] In some embodiments, the duration of ultraviolet irradiation is 47 - 49 hours or 71 - 73 hours, the duration of 4 °C cold treatment is 71 - 73 hours, the concentration of methyl jasmonate is 95 - 105 μM, and the treatment time of methyl jasmonate is 23 - 25 hours or 47 - 49 hours.

[0013] One or more embodiments of this specification provide a Saussurea salwinensis suspension cell culture. The suspension cell culture is prepared by the above method. The content of secondary metabolites in the dried product of the suspension cell culture is: chlorogenic acid 0.44% - 1.58%, syringin 1.62% - 8.59%, 1,5-dicaffeoylquinic acid 0.26% - 5.67%.

[0014] In some embodiments, the expression of WUS, WOX8, PAL, 4CL, and C4H genes in the suspension cell culture is upregulated. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] This specification will be further described by way of exemplary embodiments, which will be described in detail through the drawings.

[0016] Figure 1 It is the formation process of Saussurea salwinensis callus and 3 different cell lines shown in some embodiments of this specification;

[0017] Figure 2 is the transcriptome of Saussurea salwinensis seedlings, solid-cultured callus, and suspension cultures shown in some embodiments of this specification;

[0018] Figure 3 is the comparison of the expression levels of key genes for differentiation and flavonoid synthesis in Saussurea salwinensis seedlings, callus, and suspension cells shown in some embodiments of this specification;

[0019] Figure 4 is the content of chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid in multiple batches of Saussurea japonica suspension cell cultures shown in some embodiments of this specification;

[0020] Figure 5 is the HPLC fingerprint of Saussurea salwinensis suspension cell culture shown in some embodiments of this specification. Detailed implementation manners

[0021] To more clearly illustrate the technical solutions of the embodiments of this specification, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some examples or embodiments of this specification. For those of ordinary skill in the art, without creative efforts, this specification can also be applied to other similar scenarios based on these drawings.

[0022] As shown in this specification and the claims, unless the context clearly indicates otherwise, words such as "a", "an", "one", and / or "the" are not specifically singular and may also include plural. Generally speaking, the terms "including" and "comprising" only indicate the inclusion of the clearly identified steps and elements, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements.

[0023] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure pertains.

[0024] The embodiments of this specification propose an induction, screening, and large-scale culture technology for Saussurea salwinensis suspension cells. This technology realizes the efficient and low-cost acquisition of Saussurea salwinensis suspension cells containing high-purity total flavonoids by precisely screening the optimal hormone ratio, optimizing the culture medium formula, adjusting the culture conditions, and improving the extraction and purification process.

[0025] The embodiments of this specification provide a method for culturing Saussurea salwinensis suspension cells, including the following steps: Step (1), select the seeds of Saussurea salwinensis and inoculate them into a solid medium containing hormones to induce the formation of primary callus. The hormones include naphthaleneacetic acid (NAA) and kinetin (KT).

[0026] A solid medium refers to a medium used to induce the formation of primary callus from explants. The solid medium includes conventional MS medium, hormones, sucrose, etc.

[0027] The MS medium can include macronutrients, micronutrients, iron salts, organic components, carbon sources, and solidifying agents. In some embodiments, the pH of the MS medium is 5 - 6.

[0028] In some embodiments, the MS medium can include ammonium nitrate (1650 mg / L), potassium nitrate (1900 mg / L), calcium chloride (440 mg / L), magnesium sulfate (370 mg / L), and potassium dihydrogen phosphate (170 mg / L); the micronutrients include potassium iodide (0.83 mg / L), boric acid (6.2 mg / L), manganese sulfate (22.3 mg / L), zinc sulfate (8.6 mg / L), sodium molybdate (0.25 mg / L), copper sulfate (0.025 mg / L), and cobalt chloride (0.025 mg / L); the iron salt is ferrous sulfate (27.8 mg / L) and disodium ethylenediaminetetraacetate (37.3 mg / L); the organic components include inositol (100 mg / L), nicotinic acid (0.5 mg / L), pyridoxine hydrochloride (0.5 mg / L), thiamine hydrochloride (0.1 mg / L), and glycine (2.0 mg / L); the carbon source is sucrose (30 g / L); the solidifying agent is agar (6 - 10 g / L). In some embodiments, the MS medium does not include a solidifying agent.

[0029] According to the requirements of callus induction, specific hormones or hormone - like substances, such as naphthaleneacetic acid and kinetin, etc., can be added to the solid medium.

[0030] In some embodiments, the hormones of the solid medium are selected from at least one of 2,4 - dichlorophenoxyacetic acid (2,4 - D), NAA, KT, and 6 - benzylaminopurine (6 - BA). For the selection of hormones or hormone - like substances, this specification does not make specific limitations.

[0031] In some embodiments, the solid medium also includes sucrose. Before adjustment, the concentration of NAA is 0.1 - 3 mg / L, the concentration of KT is 0.1 - 1.5 mg / L, and the mass concentration of sucrose is 0.1% - 6%.

[0032] In some embodiments, the concentration of NAA in the solid medium is 0.5 - 2 mg / L, the concentration of KT is 0.5 - 1 mg / L, and the mass concentration of sucrose is 1% - 5%.

[0033] In some embodiments, the concentration of NAA in the solid medium is 0.5 mg / L, 1 mg / L, 1.5 mg / L or 2 mg / L, the concentration of KT is 0.5 mg / L, 0.7 mg / L, 0.9 mg / L or 1 mg / L, and the mass concentration of sucrose is 1%, 2%, 3%, 4% or 5%.

[0034] In some embodiments, the concentration of NAA in the solid medium is 2.0 mg / L, the concentration of KT is 0.5 mg / L, and the mass concentration of sucrose is 3%.

[0035] The primary callus refers to the precursor of the target callus.

[0036] In some embodiments, the target callus is the cell line with the highest total flavonoid content of secondary metabolites. In some embodiments, the target callus can be yellow callus, green callus and purple callus (named according to color), and the total flavonoid contents in their dried products (dried at 50 °C for 12 hours) are 4.16%, 9.05% or 7.33% respectively.

[0037] In some embodiments, the target callus is green callus, and the total flavonoid content in its dried product (dried at 50 °C for 12 hours) can be 9.05%.

[0038] Step (2): Adjust the concentration of hormones in the solid medium to screen out the target callus.

[0039] In order to obtain a primary callus with loose structure, it is necessary to adjust the concentration of hormones such as NAA and other components in the solid medium to achieve the rapid growth of the primary callus of Saussurea salwinensis.

[0040] In some embodiments, after adjustment, the concentration of NAA in the solid medium is 1.5 - 2.5 mg / L, and the concentration of KT is 0.5 - 1.5 mg / L.

[0041] In some embodiments, after adjustment, the concentration of NAA in the solid medium is 1.8 - 2.2 mg / L, and the concentration of KT is 0.8 - 1.2 mg / L.

[0042] In some embodiments, after adjustment, the concentration of NAA in the solid medium is 1.8 mg / L, 1.9 mg / L, 2.0 mg / L, 2.1 mg / L or 2.2 mg / L, and the concentration of KT is 0.8 mg / L, 0.9 mg / L, 1 mg / L, 1.1 mg / L or 1.2 mg / L.

[0043] In some embodiments, after adjustment, the concentration of NAA in the solid medium is 2 mg / L, and the concentration of KT is 1 mg / L.

[0044] Step (3): Inoculate the suspension cells obtained from the target callus into an optimized liquid MS medium for culture to obtain the target Saussurea salwinensis suspension cells.

[0045] The process of obtaining suspension cells from the target callus may include: After subculturing the callus, filter out large cell clusters through a sterile filter screen to obtain suspension cells with a uniform state.

[0046] The target Saussurea salwinensis suspension cells refer to the Saussurea salwinensis suspension cells with the highest flavonoid content obtained by culturing the target callus.

[0047] The optimized liquid MS medium refers to a synthetic medium used for culturing the Saussurea salwinensis suspension cells with the highest flavonoid content. In some embodiments, the optimized liquid MS medium includes ammonium nitrate, phenylalanine, cinnamic acid, sodium acetate, NAA, KT, and sucrose, etc. The present specification does not specifically limit the components of the optimized liquid MS medium.

[0048] In some embodiments, the optimized liquid MS medium further includes adjusting the concentration of ammonium nitrate in the solid medium to about one-half of the original. For example, the original concentration of ammonium nitrate in the medium for screening the target callus is 1400 - 1800 mg / L, and the concentration of ammonium nitrate in the optimized liquid MS medium can be 700 - 900 mg / L.

[0049] In some embodiments, the concentration of ammonium nitrate in the optimized liquid MS medium is 750–900 mg / L, the concentration of phenylalanine is 0.2–0.8 mg / L, the concentration of cinnamic acid is 0.2–0.8 mg / L, and the concentration of sodium acetate is 0.2–0.8 mg / L.

[0050] In some embodiments, the concentration of ammonium nitrate in the optimized liquid MS medium is 800–850 mg / L, the concentration of phenylalanine is 0.4–0.6 mg / L, the concentration of cinnamic acid is 0.4–0.6 mg / L, and the concentration of sodium acetate is 0.4–0.6 mg / L.

[0051] In some embodiments, the concentration of ammonium nitrate in the optimized liquid MS medium is 825 mg / L, the concentration of phenylalanine is 0.5 mg / L, the concentration of cinnamic acid is 0.5 mg / L, and the concentration of sodium acetate is 0.5 mg / L. For example, the optimized liquid MS medium includes the following concentration components: 825 mg / L of ammonium nitrate, 0.5 mg / L of phenylalanine, 0.5 mg / L of cinnamic acid, 0.5 mg / L of sodium acetate, 2 mg / L of NAA, 1 mg / L of KT, and sucrose with a mass concentration of 3%.

[0052] In the embodiments of this specification, by adjusting the components in the solid-liquid MS medium, such as the contents of ammonium nitrate, phenylalanine, cinnamic acid, sodium acetate, NAA, KT, and sucrose, etc., the flavonoid content in the target callus of Saussurea salwinensis can be significantly increased.

[0053] In some embodiments, the culture conditions in the optimized liquid MS medium include an oscillation speed of 120 ± 15 rpm, a temperature of 25 ± 3 °C, a light cycle of 13 - 19 hours of light followed by 5 - 10 hours of darkness, and a light intensity of 900 - 1600 lux.

[0054] In some embodiments, the culture conditions in the optimized liquid MS medium include an oscillation speed of 120 ± 10 rpm, a temperature of 25 ± 2 °C, a light cycle of 15 - 17 hours of light followed by 7 - 9 hours of darkness, and a light intensity of 1000 - 1500 lux.

[0055] In some embodiments, the culture conditions in the optimized liquid MS medium include an oscillation speed of 120 rpm, a temperature of 25 °C, a light cycle of 6 hours of light followed by 8 hours of darkness, and a light intensity of 1000 lux.

[0056] In some embodiments, the Saussurea salwinensis suspension cell culture method further includes treating the Saussurea salwinensis suspension cells by at least one of ultraviolet treatment, methyl jasmonate (MeJA) treatment, and 4 °C cold treatment to further induce the accumulation of cell secondary metabolites.

[0057] In some embodiments, the ultraviolet irradiation duration is 40 - 55 hours or 70 - 75 hours; the 4 °C cold treatment time is 45 - 75 hours; the concentration of MeJA is 90 - 210 μM, and the MeJA treatment time is 20 - 30 hours or 45 - 50 hours.

[0058] In some embodiments, the ultraviolet irradiation duration is 47 - 49 hours or 71 - 73 hours; the 4 °C cold treatment time is 71 - 73 hours; the concentration of MeJA is 95 - 105 μM, and the MeJA treatment time is 23 - 25 hours or 47 - 49 hours.

[0059] In some embodiments, the ultraviolet irradiation duration is 47 hours, 48 hours, 49 hours, 71 hours, 72 hours, or 73 hours; the 4 °C cold treatment time is 71 hours, 72 hours, or 73 hours; the concentration of MeJA is 95 μM, 97 μM, 99 μM, 100 μM, 101 μM, 102 μM, 103 μM, or 105 μM, and the MeJA treatment time is 23 hours, 24 hours, 25 hours, 47 hours, 48 hours, or 49 hours.

[0060] In some embodiments, the duration of ultraviolet irradiation is 72 hours; the duration of cold treatment at 4°C is 72 hours; the concentration of MeJA is 100 μM, and the treatment time of MeJA is 48 hours.

[0061] In some embodiments, the flavonoid content of the target Saussurea salwinensis suspension cells obtained by treatment can be 18.9%, 22%, 23.5% or 23.6%. For example, the flavonoid content of the target Saussurea salwinensis suspension cells obtained by cold treatment at 4°C for 72 hours is 23.6%. Another example, the flavonoid content of the target Saussurea salwinensis suspension cells obtained by treatment with MeJA for 48 hours is 23.5%.

[0062] In the embodiments of this specification, by treating the target callus, such as ultraviolet treatment, methyl jasmonate (MeJA) treatment, cold treatment at 4°C, salicylic acid (SA) treatment, etc., the synthesis efficiency of secondary metabolites is further improved, and thus the flavonoid content in Saussurea salwinensis suspension cells can be significantly increased.

[0063] The main active ingredients of the Saussurea involucrata culture include chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid, etc. Given that Saussurea salwinensis and Saussurea involucrata are closely related species belonging to the genus Saussurea in the Compositae family, the two show significant similarities in the components of secondary metabolites.

[0064] The embodiments of this specification also provide a Saussurea salwinensis suspension cell culture, which is prepared by the above method. The content of secondary metabolites in the dried product of this suspension cell culture is: chlorogenic acid 0.44% - 1.58%, syringin 1.62% - 8.59%, 1,5-dicaffeoylquinic acid 0.26% - 5.67%.

[0065] In some embodiments, the expression of genes such as WUS, WOX8, PAL, 4CL, and C4H in the suspension cell culture is up-regulated.

[0066] As used herein, "expression level" and "gene expression level" refer to the relative expression level of a gene.

[0067] The WUS gene (WUSCHEL) plays a key role in the differentiation of plant stem cells. The protein encoded by it is a transcription factor, which has a decisive influence on the maintenance and differentiation of plant stem cells. ARF7 and ARF19 are two important auxin response factors. Auxin induces the transcription of DPa through ARF7 and ARF19 and plays an important role in the maintenance of distal stem cells at the root tip.

[0068] In some embodiments, taking the seedlings of Saussurea salwinensis as a control, the expression levels of WUS in the target callus and the target Saussurea salwinensis suspension cells are up-regulated by 118 times and 1.6 times respectively.

[0069] The WOX8 gene plays a key role in plant (such as Arabidopsis thaliana) development and is involved in multiple important processes such as stem cell maintenance, embryo development, lateral root formation, and response to environmental stress.

[0070] In some embodiments, using the seedlings of Saussurea salwinensis as a control, the expression levels of WOX8 in the target callus and the target Saussurea salwinensis suspension cells were upregulated by 4.1-fold and 4.5-fold, respectively.

[0071] Phenylalanine ammonia-lyase (PAL) is a key enzyme in the phenylpropanoid metabolic pathway of plants. It catalyzes the conversion of phenylalanine to cinnamic acid and then participates in the synthesis of various secondary metabolites, including flavonoids. The PAL gene family contains multiple members (such as PAL1, PAL4, and PAL5) and plays an important role in plant growth and development and response to environmental stress.

[0072] In some embodiments, using the seedlings of Saussurea salwinensis as a control, the PAL1 gene in the target callus and the target Saussurea salwinensis suspension cells was upregulated by 9.3-fold and 2.4-fold, respectively; PAL4 was upregulated by 1.6-fold and 12.2-fold; PAL5 was upregulated by 2.6-fold and 52.8-fold.

[0073] 4-Coumarate:CoA ligase (4CL) plays an important role in flavonoid synthesis and is the first dedicated enzyme in the flavonoid synthesis pathway, controlling the carbon flow from the phenylpropanoid pathway derived from phenylalanine into the synthesis of flavonoids.

[0074] In some embodiments, using the seedlings of Saussurea salwinensis as a control, 4CL in the target callus and the target Saussurea salwinensis suspension cells was upregulated by 1.9-fold and 4.5-fold, respectively.

[0075] Cinnamic acid 4-hydroxylase (C4H) is one of the key enzymes in the flavonoid metabolic pathway and belongs to the cytochrome P450 family (CYP73A). Using NADPH-cytochrome P450 reductase as an electron donor, it catalyzes the hydroxylation of cinnamic acid to form 4-coumaric acid, which is an essential step in the synthesis of flavonoids.

[0076] In some embodiments, using the seedlings of Saussurea salwinensis as a control, C4H in the target callus and the target Saussurea salwinensis suspension cells was upregulated by 5-fold and 169-fold, respectively.

[0077] In the embodiments of this specification, by optimizing the induction of callus, screening highly efficient cell lines, establishing a suspension cell culture system, and optimizing the components of the culture medium, the total flavonoid content in the cell cultures of Saussurea salwinensis has been significantly increased, meeting the standards for industrial production. Combining the main active components of the snow lotus cultures and the expression of related genes, a highly efficient and stable suspension cell culture method for Saussurea salwinensis has been determined. This not only facilitates the sustainable utilization of Saussurea salwinensis resources and reduces interference with the natural environment, but also conforms to the concepts of sustainable development and green chemistry. The cell cultures prepared by this method have a high total flavonoid content and can supply high-quality natural product raw materials for industries such as medicine, health products, and cosmetics, strongly promoting the innovative development of related industries.

[0078] The following examples are some more specific descriptions related to some of the above examples. Some of the content in these examples can also be replaced or combined with the corresponding content in other examples to form new examples. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from regular biochemical reagent companies unless otherwise specified. In the following examples, all quantitative tests are set up with three repeated experiments, and the results are averaged. It should be understood that the following examples are for better explaining the present invention and are not intended to limit the present invention. Example Example 1: Induction of Callus of Saussurea salwinensis

[0079] Mature seeds were isolated from the flower heads of mature plants of Saussurea salwinensis, soaked in a 70% ethanol solution for 30 seconds, and then rinsed with sterile water. Subsequently, the seeds were placed in a 10% sodium hypochlorite solution for 15 minutes of disinfection treatment, and rinsed 5 times with sterile water to remove the residual disinfectant.

[0080] The disinfected seeds were inoculated onto a sterile MS medium, maintaining a constant temperature (25°C) and suitable light conditions (about 16 hours of light per day) to promote the germination process of the seeds.

[0081] After the seeds germinated and formed seedlings, parts such as hypocotyls and cotyledons were selected as explants for plant tissue culture.

[0082] The explants were inoculated into an MS medium containing an appropriate hormone ratio, where the NAA concentration was 3.0 mg / L, the KT concentration was 0.5 mg / L, and 3% sucrose and 1% agar were added. Before using the medium, it was sterilized at 121°C under high temperature and high pressure for 20 minutes to effectively induce the formation of primary callus.

[0083] The inoculated explants were placed under sterile conditions, maintained at a constant temperature of 25 °C, cultured under a light intensity of 1000 Lux, with a photoperiod set to 16 hours of light and 8 hours of darkness, and the growth status of the primary callus was observed regularly. Experimental Example 2: Screening of High-efficiency Cell Lines of Saussurea salwinensis

[0084] After obtaining a sufficient quantity of primary callus of Saussurea salwinensis, referring to the callus induction experience of Saussurea involucrata and Saussurea medusa in the genus Saussurea of the Asteraceae family, with MS salts as the basal medium, the screening work of the optimal hormone ratio combination was carried out.

[0085] Taking the browning tendency, texture softness and growth rate of the callus as evaluation indicators, a hormone combination with bright-colored callus masses, loose texture and rapid growth was screened out. Table 1: Effects of Different Hormone Combinations on the Growth Status of Primary Callus of Saussurea salwinensis

[0086] After screening, this example determined that adding 2 mg / L NAA and 1 mg / L KT, as well as 3% sucrose to the MS medium was the optimal growth condition for primary callus. Based on this condition, the primary callus was subcultured.

[0087] Setting 14 days as a subculture cycle, and continuously subculturing more than 20 times, cell lines with stable growth of yellow, green and purple Saussurea salwinensis were successfully screened out, namely the target callus. Figure 1 It is the formation process of Saussurea salwinensis callus shown in some embodiments of this specification (A: Seed germination; B: Callus induction; C: Formation of primary callus) and 3 different cell lines (D: Yellow cell line; E: Green cell line; F: Purple cell line).

[0088] The total flavonoid contents of wild plants of Saussurea salwinensis and the above three cell lines were determined by ultraviolet-visible spectrophotometry at a wavelength of 260 nm.

[0089] The measurement results showed that the total flavonoid contents in the dried products (dried at 50 °C for 12 hours) of wild plants of Saussurea salwinensis, yellow cell line, green cell line and purple cell line were 0.47%, 4.16%, 9.05% and 7.33% respectively. Therefore, the green cell line was determined as the high-efficiency cell line. Example 3: Establishment and Culture Process Optimization of Suspension Cell Line of Saussurea salwinensis

[0090] The target callus cultured in solid state was inoculated into an MS medium containing 2 mg / L NAA, 1 mg / L KT and 3% sucrose, and cultured with shaking at a rate of 120 rpm, maintaining the temperature at 25 °C, the light cycle was set at 16 hours of light / 8 hours of darkness, and the light intensity was 1000 lux.

[0091] Taking 14 days as a subculture cycle, after subculturing 3 times, the cells were filtered through an 80-mesh sterile filter to remove large cell aggregates, thereby obtaining a suspension cell line with uniform state.

[0092] With an inoculation amount of 40 g / L, using 2 mg / L of NAA and 1 mg / L of KT as the hormone concentration in the medium, the cells were transferred to synthetic media with different nutrient ratios and precursor substances (the basic medium was MS, see Table 2 for details), in order to screen out the synthetic medium with the best growth state of the cell culture and the highest flavonoid content.

[0093] According to the experimental results, the PRK-16 synthetic medium was finally determined as the best growth medium. For the subsequent growth medium of Saussurea salwinensis cells, PRK-16 was used, that is, in the MS medium, the concentration of ammonium nitrate NH 4 NO 3 was adjusted to 825 mg / L, other nutrients remained unchanged, the hormone ratio was 2 mg / L of NAA and 1 mg / L of KT, and the sucrose concentration was 3%.

[0094] Furthermore, in this example, the induction conditions that can significantly promote the increase in total flavonoid content were screened out, and the specific operation steps are shown in Table 3. Through comparative analysis, it was found that after treatment with ultraviolet (UV) irradiation for 48 hours (Prk-16-B), 72 hours (Prk-16-C), and cold treatment at 4 °C for 72 hours (Prk-16-F), as well as treatment with 100 μM of methyl jasmonate (MeJA) for 24 hours (Prk-16-J), 48 hours (Prk-16-K), the total flavonoid content produced by the suspension cells of Saussurea salwinensis could reach 3 g / L.

[0095] Based on the actual industrial production, the Prk-16-K medium and process combination were selected as the subsequent large-scale bioreactor production process. Table 2: Effects of different nutrient ratios and precursor substances on the growth and flavonoid content of Saussurea salwinensis suspension cell cultures Table 3: Comparison of the growth and flavonoid content of Saussurea salwinensis suspension cells under different induction conditions Example 4: Study on Gene Expression Characteristics of Suspension Cell Cultures of Saussurea salwinensis

[0096] Given that there is currently no genomic reference information for Saussurea salwinensis, this example aims to analyze the gene expression characteristics of Saussurea salwinensis seedlings, callus, and suspension cells through transcriptome sequencing technology to reveal the molecular mechanisms underlying its growth, development, and high production of flavonoid secondary metabolites. The specific steps are as follows:

[0097] Collect the seedlings, callus, and suspension cell cultures of Saussurea salwinensis, and extract total RNA from the above materials to ensure the quality and integrity of the RNA.

[0098] Use a nanodrop to detect the concentration and purity of the RNA, ensuring that the A260 / A280 ratio is between 1.8 - 2.0 and the A260 / A230 ratio is greater than 2.0.

[0099] Construct a cDNA library using the extracted total RNA, operating with the TruSeq RNA Sample Preparation Kit, including steps such as RNA fragmentation, cDNA synthesis, end repair, and adapter ligation.

[0100] Perform sequencing using the Illumina HiSeq high-throughput sequencing platform to generate a large number of short read sequences.

[0101] For the raw image data of the sequencing results, use the software Bcl2fastq (v2.20.0.422) for image base calling and perform preliminary quality analysis (during the sequencing process, the built-in software of Illumina determines the retention or discard of each read based on the quality of the first 25 bases of each sequencing fragment, i.e., read), obtaining the raw sequencing data (PassFilter Data), and the results are stored in the FASTQ file format.

[0102] Use the assembly software Trinity (v2.2.0) to de novo assemble the sample data, and perform further sequence splicing and redundancy removal on the assembly results through sequence clustering to obtain long non-redundant Unigene sequences.

[0103] Transcriptome data analysis is as Figure 2 shown. Principal component analysis (PCA) shows( Figure 2In A), the transcriptome data of the three treatment groups (FMJ-plant-1 to FMJ-plant-3, FMJ-callus-1 to FMJ-callus-3, FMJ-cell-1 to FMJ-cell-3) were highly aggregated within their respective groups. Among them, FMJ-plant represents the seedlings of Saussurea salwinensis, FMJ-callus represents the target callus on the solid medium, and FMJ-cell represents the target suspension cells of Saussurea salwinensis. The abscissa PC1 is used to show the distribution of samples in the PC1 direction. If the samples are significantly separated in the PC1 direction, it indicates that PC1 can effectively distinguish these samples. PC1 (29.7%) means that the first principal component explains 29.7% of the variance in the dataset. The ordinate PC2 represents the second principal component, which is the direction with the second largest variance in the data. PC2 (15.5%) means that the second principal component explains 15.5% of the variance. Pearson correlation analysis ( Figure 2 in B) shows that the correlation between samples within the same group is higher than that between samples from different groups (the larger the value, the higher the correlation). In the volcano plot (as shown in Figure 2 C - E), the abscissa: log2(Fold Change) represents the logarithm of the fold change in the expression of genes or molecules (Fold Change), reflecting the amplitude of gene or molecule expression changes under different conditions (such as treatment group vs control group).

[0104] log2(Fold Change) = 0: indicates no change in expression level.

[0105] log2(Fold Change)>0: indicates upregulation of expression level (positive correlation).

[0106] log2(Fold Change)<0: indicates downregulation of expression level (negative correlation).

[0107] The ordinate: -log10(qvalue) represents the negative logarithm of statistical significance, reflecting the statistical significance of gene or molecule expression changes.

[0108] Significantly upregulated genes: located on the right side of the figure (log2(Fold Change)>0) and with a higher ordinate (-log10(q - value) is larger).

[0109] Significantly downregulated genes: located on the left side of the figure (log2(Fold Change)<0) and with a higher ordinate (-log10(q - value) is larger).

[0110] Genes with no significant changes: Located in the middle region of the figure (log2(Fold Change) close to 0) or with a lower ordinate (-log10(q-value) is smaller). As Figure 2 shown in C, compared with the FMJ-plant group, 30,499 transcripts were up-regulated and 33,352 transcripts were down-regulated in the FMJ-callus group; as Figure 2 shown in D, compared with the FMJ-plant group, 46,118 transcripts were up-regulated and 50,024 transcripts were down-regulated in the FMJ-cell group; as Figure 2 shown in E, compared with the FMJ-callus, 51,946 transcripts were up-regulated and 54,078 transcripts were down-regulated in the FMJ-cell group. These results indicate that there are significant differences at the transcriptional level among the seedlings, target callus on solid medium, and target Saussurea kingii suspension cells in liquid medium of Saussurea kingii.

[0111] Comparison of the expression levels of key genes in stem cell differentiation and flavonoid synthesis: Based on the RNA-seq differential gene expression data, this example compared the expression levels of key genes regulating stem cell differentiation and flavonoid synthesis in the seedlings, callus, and green suspension cell lines of Saussurea kingii. The results are as Figure 3 shown. plant represents the seedlings of Saussurea kingii, callus represents the target callus on solid medium, and cell represents the target Saussurea kingii suspension cells. The WUS gene (WUSCHEL) plays a key role in plant stem cell differentiation. The protein encoded by it is a transcription factor and has a decisive influence on the maintenance and differentiation of plant stem cells. ARF7 (Auxin Response Factor 7) and ARF19 (Auxin Response Factor 19) are two important auxin response factors. Auxin induces the transcription of the downstream target gene DPa through ARF7 and ARF19 and plays an important role in the maintenance of distal stem cells at the root tip. The WOX6 (WUSCHEL-related homeobox gene 6) and WOX8 (WUSCHEL-related homeobox gene 8) genes play a key role in the development of Arabidopsis thaliana and are involved in multiple important processes such as stem cell maintenance, embryonic development, lateral root formation, and response to environmental stress. Taking the seedlings of Saussurea kingii as the control, the expression levels of WUS in the solid-cultured callus and liquid-cultured suspension cells were up-regulated by 118 times and 1.6 times respectively; ARF7 was significantly down-regulated by 0.4 times and 0.07 times; there was no significant difference in the expression level of ARF19; the expression levels of WOX6 were up-regulated by 7.2 times and 5.8 times respectively; the expression of WOX8 was up-regulated by 4.1 times and 4.5 times respectively.

[0112] Phenylalanine ammonia-lyase (PAL) is a key enzyme in the phenylpropanoid metabolic pathway of plants. It catalyzes the conversion of phenylalanine to cinnamic acid and then participates in the synthesis of various secondary metabolites, including flavonoids. The PAL gene family contains multiple members (such as PAL1, PAL4, and PAL5), which play important roles in plant growth, development, and response to environmental stresses. Taking the seedlings of Saussurea salwinensis as a control, the PAL1 gene in the solid-cultured callus and liquid-cultured suspension cells was upregulated by 9.3-fold and 2.4-fold, respectively; PAL4 was upregulated by 1.6-fold and 12.2-fold; PAL5 was upregulated by 2.6-fold and 52.8-fold. In flavonoid synthesis, 4-coumarate-CoA ligase (4CL) is particularly important. It is the first dedicated enzyme in the flavonoid synthesis pathway, controlling the carbon flow from the phenylpropanoid pathway derived from phenylalanine into the synthesis of flavonoids. Taking the seedlings of Saussurea salwinensis as a control, 4CL in the solid-cultured callus and liquid-cultured suspension cells was upregulated by 1.9-fold and 4.5-fold, respectively. Cinnamic acid 4-hydroxylase (C4H) is one of the key enzymes in the flavonoid metabolic pathway and belongs to the cytochrome P450 family (CYP73A). Using NADPH-cytochrome P450 reductase as an electron donor, it catalyzes the hydroxylation of cinnamic acid to form 4-coumaric acid, which is an essential step in the synthesis of flavonoids. Taking the seedlings of Saussurea salwinensis as a control, C4H in the solid-cultured callus and liquid-cultured suspension cells was upregulated by 5-fold and 169-fold, respectively. Example 5: Fingerprint and Analysis of Index Compound Contents of Suspension Cell Cultures of Saussurea salwinensis

[0113] Based on the research on the chemical constituents of Saussurea involucrata medicinal materials and cell cultures, the three active substances, chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid, were used as characteristic active components in Saussurea involucrata cultures. Saussurea salwinensis and Saussurea involucrata are closely related species in the genus Saussurea of the Compositae family, and their secondary metabolite components are similar. In this example, three characteristic active components, chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid, in the cultures were selected as characteristic peaks to establish the fingerprint of the suspension cell cultures of Saussurea salwinensis.

[0114] Detection was carried out using high-performance liquid chromatography (HPLC). Mobile phase gradient elution analysis was adopted to compare the separation of different mobile phases at various gradient slopes and optimize the separation effect of the three characteristic peaks. Under the optimized chromatographic conditions, the three characteristic peaks were all well separated, meeting the requirements of various methodological validations.

[0115] Using the fingerprint determination method, the contents of chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid in 30 batches of suspension cell cultures of Saussurea salwinensis were determined. The results are as Figure 4As shown, the content range of chlorogenic acid is 0.44 - 1.58%, the content range of syringin is 1.62 - 8.59%, and the content range of 1,5-dicaffeoylquinic acid is 0.26 - 5.67%. The contents of these three active ingredients fluctuate with factors such as cultivation, harvesting, and storage conditions.

[0116] Extract the characteristic chromatogram data of 3 batches of Saussurea salwinensis suspension cell cultures and wild Saussurea salwinensis, import them into the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition), obtain the standard fingerprint spectrum, and calculate the similarity. The results are as Figure 5 shown. Figure 5 In which A is the wild Saussurea salwinensis plant, Figure 5 and B in it is the Saussurea salwinensis cell culture in the examples of this specification. The similarity of the 3 batches of Saussurea salwinensis cell cultures (Saussurea salwinensis suspension cell cultures) and wild Saussurea salwinensis is greater than 0.89, indicating that the cell cultures and wild plants have good consistency in chemical components.

[0117] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only an example and does not constitute a limitation to this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification, so such modifications, improvements, and corrections still belong to the spirit and scope of the exemplary embodiments of this specification.

[0118] Meanwhile, this specification uses specific terms to describe the embodiments of this specification. Such as "one embodiment", "an embodiment", and / or "some embodiments" mean a certain feature, structure, or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that the "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more at different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0119] In addition, unless clearly stated in the claims, the order of the processing elements and sequences described in this specification, the use of numbers, letters, or other names, is not used to limit the order of the processes and methods in this specification. Although some currently considered useful invention embodiments are discussed through various examples in the above disclosure, it should be understood that such details only serve the purpose of illustration. The appended claims are not limited to the disclosed embodiments. On the contrary, the claims are intended to cover all modifications and equivalent combinations that conform to the essence and scope of the embodiments of this specification.

[0120] Similarly, it should be noted that, in order to simplify the presentation disclosed in this specification and thus assist in the understanding of one or more embodiments of the invention, in the foregoing description of the embodiments of this specification, multiple features are sometimes grouped into one embodiment, drawing, or description thereof. However, this method of disclosure does not mean that the features required by the subject matter of this specification are more than those mentioned in the claims. In fact, the features of the embodiments are fewer than all the features of the individual embodiments disclosed above.

[0121] In some embodiments, numbers are used to describe components and the quantity of attributes. It should be understood that such numbers used for the description of embodiments are, in some examples, modified by the modifiers "about", "approximate", or "substantially". Unless otherwise stated, "about", "approximate", or "substantially" indicate that the said numbers allow a variation of ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, and such approximate values may vary according to the characteristics required by individual embodiments. In some embodiments, the numerical parameters should consider the specified significant digits and adopt the method of retaining general digits. Although the numerical ranges and parameters used in some embodiments of this specification to confirm the breadth of their scope are approximate values, in specific embodiments, the setting of such numerical values is as precise as possible within the feasible range.

[0122] For each patent, patent application, patent application publication, and other materials cited in this specification, such as articles, books, specifications, publications, documents, etc., their entire contents are hereby incorporated into this specification by reference. Except for the application history documents that are inconsistent with or conflict with the content of this specification, and except for the documents that limit the broadest scope of the claims of this specification (currently or subsequently appended to this specification). It should be noted that if there are inconsistencies or conflicts between the descriptions, definitions, and / or uses of terms in the supplementary materials of this specification and the content described in this specification, the descriptions, definitions, and / or uses of terms in this specification shall prevail.

[0123] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, by way of example and not limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.

Claims

1. A method for culturing suspension cells of Saussurea nujiangensis, characterized in that: The following steps are involved: The seeds of Nujiang Saussurea ovata are selected and inoculated into a solid culture medium containing hormones to induce the formation of primary callus tissue, wherein the hormones include naphthylacetic acid and kinetin; adjusting the concentration of the hormone in the solid culture medium to screen out target callus tissue; The suspension cells obtained based on the target callus tissue are inoculated into an optimized liquid MS medium for culture to obtain the target Saussurea nujiangensis suspension cells.

2. The method according to claim 1, characterized in that The solid culture medium also includes sucrose. Before the adjustment, the concentration of naphthaleneacetic acid in the solid culture medium is 0.5-2 mg / L, the concentration of kinetin is 0.5-1 mg / L, and the mass concentration of sucrose is 1%-5%.

3. The method according to claim 1, characterized in that After the adjustment, the concentration of the naphthylacetic acid in the solid culture medium is 1.8-2.2 mg / L, and the concentration of the kinetin is 0.8-1.2 mg / L.

4. The culture method according to claim 1, characterized in that The target callus tissue is a cell line with the highest content of total flavonoids in secondary metabolites.

5. The culture method according to claim 1, characterized in that The optimized liquid MS culture medium has an ammonium nitrate concentration of 800-850 mg / L, a phenylalanine concentration of 0.4-0.6 mg / L, a cinnamic acid concentration of 0.4-0.6 mg / L, and a sodium acetate concentration of 0.4-0.6 mg / L.

6. The culture method according to claim 1, characterized in that The culture conditions in the optimized liquid MS medium include a shaking speed of 120±10 rpm, a temperature of 25±2° C., a light cycle of 15-17 hours of light followed by 7-9 hours of darkness, and a light intensity of 1000-1500 lux.

7. The culture method according to claim 1, characterized in that: The method further comprises subjecting the suspension cells obtained based on the target callus tissue to at least one of ultraviolet light treatment, methyl jasmonate treatment, and 4° C. cold treatment to further induce accumulation of cell secondary metabolites.

8. The culture method according to claim 7, characterized in that: The duration of ultraviolet irradiation is 47-49 hours or 71-73 hours, the duration of cold treatment at 4°C is 71-73 hours, the concentration of methyl jasmonate is 95-105 μM, and the duration of methyl jasmonate treatment is 23-25 ​​hours or 47-49 hours.

9. A suspension cell culture of Saussurea nujiangensis, characterized in that: The cell culture is prepared by the method of claims 1 to 8, and the secondary metabolite content in the dried product of the cell culture is: chlorogenic acid 0.44%-1.58%, syringin 1.62%-8.59%, and 1,5-dicaffeoylquinic acid 0.26%-5.67%.

10. The suspension cell culture according to claim 9, characterized in that The expression of WUS, WOX8, PAL, 4CL, and C4H genes in the suspension cell culture is upregulated.

Images