Method for producing adventitious roots of traditional Chinese medicinal materials for producing saponin
By monitoring the expression of key genes in the indefinite root production medium of Chinese medicinal materials and adding inducers to collect the indefinite roots with high saponin content, the problems of low yield and unstable content in traditional cultivation methods were solved, and efficient and stable saponin production was achieved.
Patent Information
- Application Number
- CN202510095776.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-20
- Publication Date
- 2025-05-06
AI Technical Summary
Traditional Chinese herbal medicine uncertain root planting methods have problems such as high cost, low yield and unstable saponin content, and existing tissue culture technologies are difficult to achieve fine monitoring and efficient production.
By performing subculture in the indefinite root production medium, and regularly monitoring the expression of WOX5 or PIN3 genes, adding inducers according to the changes in the expression level, monitoring the expression of key enzyme genes for saponin synthesis, and finally collecting indefinite roots with higher saponin content.
It improves the saponin yield and content of uncertain roots of traditional Chinese medicinal materials, enhances production efficiency, and solves the problems of low yield and unstable content in traditional methods.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of tissue culture of Chinese medicinal materials, and in particular to a method for producing adventitious roots of Chinese medicinal materials producing saponins. Background Art
[0002] Saponin is a triterpenoid glycoside compound, which can be divided into protopanaxadiol saponin (PPD-type saponin), protopanaxatriol saponin (PPT-type saponin) and oleanane-type. Saponin can reduce DNA damage, reduce the host's susceptibility to mutation, increase immune monitoring and cell apoptosis, and has high medicinal value such as anti-cancer, antioxidant, anti-inflammatory, vasodilation, anti-allergy, and anti-diabetes. In addition, saponin can effectively improve the efficacy of traditional chemotherapy drugs and prevent damage to normal tissues of the human body.
[0003] Saponins are mainly found in terrestrial higher plants, especially in many Chinese medicinal materials, such as ginseng, Panax notoginseng, Panax japonicus, and American ginseng. A large amount of saponins can be synthesized in the adventitious roots of these Chinese medicinal materials. However, traditional planting methods have problems such as high cost, low adventitious root yield, and unstable saponin content. In recent years, researchers have developed a ginseng adventitious root culture process based on tissue culture technology, but it still faces many challenges, such as the lack of sophisticated monitoring technology, differences in adventitious root growth status between different batches, and differences in saponin content.
[0004] Therefore, it is necessary to provide a method for producing adventitious roots of saponin-producing Chinese medicinal materials to improve production efficiency and saponin yield. Summary of the invention
[0005] One or more embodiments of the present specification provide a method for producing adventitious roots of Chinese medicinal materials that produce saponins. The adventitious root production method comprises: subculturing the induced adventitious roots of Chinese medicinal materials in an adventitious root production medium, and regularly monitoring the expression level of the key gene WOX5 for stem cell differentiation or the key gene PIN3 for lateral root development; when the expression level of the WOX5 decreases to a first preset value or the expression level of the PIN3 increases to a second preset value, adding an inducer; and after adding the inducer, regularly monitoring the expression level of the gene encoding the key enzyme for saponin synthesis, and collecting the adventitious roots according to the change in the expression level of the key enzyme for saponin synthesis.
[0006] In some embodiments, the adventitious root production medium comprises a modified MS medium, which comprises components in the following concentrations: 112.5 mg / L NH4NO3, 1900 mg / L KNO3, 60 mg / L KH2PO4, 370 mg / L MgSO4·7H2O, 440 mg / L CaCl2·2H2O, 6.2 mg / L H3BO3, 8.6 mg / L ZnSO4·7H2O, 0.025 mg / L CuSO4·5H2O, 13.9 mg / L FeSO4·7H2O, 18.7 mg / L Na2·EDTA, 0.1 mg / L thiamine hydrochloride and 0.5 mg / L vitamin B6.
[0007] In some embodiments, the adventitious root production medium further comprises a growth regulator and sucrose, wherein the growth regulator comprises 2-20 mg / L indolebutyric acid and 0.1-1 mg / L kinetin, and the concentration of the sucrose is 40 g / L.
[0008] In some embodiments, the first preset value is between 30%-70% of the initially monitored WOX5 expression level, and the second preset value is between 150%-250% of the initially monitored PIN3 expression level, wherein the initial monitoring is started at 21 days of culture.
[0009] In some embodiments, according to the change in the expression level of the key enzyme gene for saponin synthesis, collecting the adventitious roots includes: taking the time of adding the inducer as the starting time, and collecting the adventitious roots when the expression level of the key enzyme gene for saponin synthesis first increases and then recovers to near the expression level of the key enzyme gene for saponin synthesis monitored at the starting time.
[0010] In some embodiments, the key enzyme genes for saponin synthesis include at least one of HGMR1, FPS1, DDS1 and UGT71A27.
[0011] In some embodiments, the inducer comprises methyl jasmonate, and the concentration of the inducer is 50-200 μM.
[0012] In some embodiments, real-time fluorescence quantitative PCR is used to monitor the expression level.
[0013] In some embodiments, the Chinese medicinal material includes at least one of ginseng, American ginseng, Panax japonicus and Panax notoginseng.
[0014] One or more embodiments of the present specification further provide a saponin, wherein the saponin is extracted from the adventitious roots of a Chinese medicinal material produced by the above-mentioned method for producing adventitious roots of a Chinese medicinal material producing saponins. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] This specification will be further described in the form of exemplary embodiments, which will be described in detail by the accompanying drawings. These embodiments are not restrictive, and in these embodiments, the same number represents the same structure, wherein:
[0016] Figure 1 is an exemplary flow chart of a method for producing adventitious roots of saponin-producing Chinese medicinal materials according to some embodiments of this specification;
[0017] Figure 2A-2B It is a schematic diagram of the shake flask culture of ginseng adventitious roots according to Example 3 of this specification;
[0018] Figure 3A-3B It is a schematic diagram of barrel culture of ginseng adventitious roots according to Example 4 of this specification;
[0019] Figure 4A and Figure 4B They are bar graphs showing the changes in the expression levels of the WOX5 gene and the PIN3 gene during the adventitious root subculture process according to some embodiments of the present specification;
[0020] Figure 5A-5D They are bar graphs of the expression changes of HGMR1, FPS1, DDS1 and UGT71A27 genes during the adventitious root subculture process according to some embodiments of the present specification. DETAILED DESCRIPTION
[0021] In order to more clearly illustrate the technical solutions of the embodiments of this specification, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some examples or embodiments of this specification. For ordinary technicians in this field, this specification can also be applied to other similar scenarios based on these drawings without creative work. Unless it is obvious from the language environment or otherwise explained, the same reference numerals in the figures represent the same structure or operation.
[0022] As shown in this specification and claims, unless the context clearly indicates an exception, the words "a", "an", "an" and / or "the" do not refer to the singular and may also include the plural. Generally speaking, the terms "comprise" and "include" only indicate the inclusion of the steps and elements that have been clearly identified, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements.
[0023] Figure 1 is an exemplary flow chart of the method for producing adventitious roots of Chinese medicinal materials producing saponins according to some embodiments of this specification. Figure 1 As shown, process 100 includes the following steps:
[0024] Step S1, subculturing the induced adventitious roots of the Chinese medicinal materials in an adventitious root production medium, and regularly monitoring the expression level of the key gene WOX5 for stem cell differentiation or the key gene PIN3 for lateral root development.
[0025] In some embodiments, the Chinese medicinal material may include at least one of ginseng, American ginseng, Panax japonicus and Panax notoginseng.
[0026] In some embodiments, the adventitious roots of Chinese medicinal materials can be obtained by culturing in an induction medium. For example, inducing adventitious roots of Chinese medicinal materials by induction medium may include the following steps: taking the roots of Chinese medicinal materials as explants, culturing in a callus induction medium, and obtaining callus tissue of Chinese medicinal materials; culturing the callus tissue of Chinese medicinal materials in an adventitious root induction medium to obtain adventitious roots of Chinese medicinal materials. Callus induction medium is used to induce explants to generate callus tissue, and its components include conventional MS medium, hormones (such as gibberellins), sucrose and agar, etc.; according to the needs of callus induction, specific hormones such as gibberellins can be added to the callus induction medium. Adventitious root induction medium is used to induce callus tissue to generate adventitious roots, and its components include conventional MS medium, hormones, sucrose and agar, etc.; according to the needs of adventitious root induction, specific hormones such as naphthaleneacetic acid, 6-benzylpurine, etc. can be added to the adventitious root induction medium.
[0027] After obtaining adventitious roots through callus culture, the culture medium can be replaced and subculture can be performed. Subculture can include continuous multi-generation culture to obtain a large number of adventitious roots. In some embodiments, the adventitious roots of the induced Chinese medicinal materials can be subcultured in an adventitious root production medium.
[0028] In some embodiments, a plant cell fermentation culture device can be used for subculture, and the culture reactor can be a culture barrel reactor. The material of the culture barrel reactor can be a high temperature resistant transparent material that can be directly sterilized.
[0029] Adventitious root production medium refers to a medium for culturing adventitious roots of Chinese medicinal materials with high saponin content. In some embodiments, the adventitious root production medium may include a modified MS medium. The modified MS medium refers to a medium whose ingredients have been adjusted based on the MS medium. In some embodiments, the modified MS medium may include components in the following concentrations: 112.5 mg / L NH4NO3, 1900 mg / L KNO3, 60 mg / L KH2PO4, 370 mg / L MgSO4·7H2O, 440 mg / L CaCl2·2H2O, 6.2 mg / LH3BO3, 8.6 mg / L ZnSO4·7H2O, 0.025 mg / L CuSO4·5H2O, 13.9 mg / L FeSO4·7H2O, 18.7 mg / L Na2·EDTA, 0.1 mg / L thiamine hydrochloride, and 0.5 mg / L vitamin B6.
[0030] In the examples of this specification, by adjusting the components of the MS culture medium, such as reducing the contents of ammonium salts, phosphates, iron salts, unnecessary trace elements and organic elements, the saponin content in the adventitious roots of Chinese medicinal materials can be significantly increased.
[0031] In some embodiments, the adventitious root production medium may further include a growth regulator and sucrose, the growth regulator may include 2-20 mg / L indolebutyric acid and 0.1-1 mg / L kinetin, and the concentration of sucrose is 40 g / L. In some embodiments, the concentration of indolebutyric acid may be 2 mg / L, 8 mg / L, 14 mg / L, or 20 mg / L, etc. In some embodiments, the concentration of kinetin may be 0.1 mg / L, 0.3 mg / L, 0.5 mg / L, 0.7 mg / L, or 1 mg / L, etc.
[0032] Adventitious root growth and saponin synthesis are carried out in stages. No or a small amount of saponin is synthesized during the adventitious root growth period. After the adventitious root growth is completed, saponin synthesis begins in large quantities. WOX5, a key gene for adventitious root stem cell differentiation, and PIN3, a key gene for lateral root development, are two important genes that regulate adventitious root growth and differentiation. During the adventitious root culture process, when the expression of the WOX5 gene decreases or the expression of the PIN3 gene increases, the adventitious root is basically developed and saponin synthesis begins.
[0033] In some embodiments, during the adventitious root subculture process, the expression levels of the WOX5 gene and / or the PIN3 gene are regularly monitored, so that the development of the adventitious roots and the synthesis of saponins can be visually observed.
[0034] Regular monitoring refers to monitoring the gene expression of Chinese medicinal materials according to a certain cycle. The cycle can be determined according to actual conditions or experience. For example, monitoring once a day, monitoring once every two days, etc. In some embodiments, real-time fluorescence quantitative PCR can be used to monitor the gene expression regularly.
[0035] As used herein, "expression amount" and "gene expression amount" refer to the relative expression amount of a gene. The expression amount of the WOX5 gene and / or the PIN3 gene refers to the percentage of the absolute expression amount of the WOX5 gene and / or the PIN3 gene relative to the absolute expression amount of the WOX5 gene and / or the PIN3 gene during initial monitoring. The absolute expression amount can be determined by real-time fluorescence quantitative PCR.
[0036] Step S2, when the expression level of WOX5 decreases to a first preset value or the expression level of PIN3 increases to a second preset value, adding an inducer.
[0037] The first preset value refers to the expression amount of the WOX5 gene used to determine the time point at which the inducer is added. In some embodiments, the first preset value can be 30%-70% of the expression amount of WOX5 initially monitored. In some embodiments, the first preset value can be 30% of the expression amount of WOX5 initially monitored. In some embodiments, the first preset value can be 50% of the expression amount of WOX5 initially monitored. In some embodiments, the first preset value can be 70% of the expression amount of WOX5 initially monitored.
[0038] The second preset value refers to the expression of the PIN3 gene at the time point when the inducing agent is added. In some embodiments, the second preset value can be 150%-250% of the expression of the PIN3 initially monitored. In some embodiments, the second preset value can be 150% of the expression of the PIN3 initially monitored. In some embodiments, the second preset value can be 200% of the expression of the PIN3 initially monitored. In some embodiments, the second preset value can be 250% of the expression of the PIN3 initially monitored.
[0039] Initial monitoring refers to the initial monitoring of the expression of genes in adventitious roots during the subculture process. The average growth period of adventitious roots is 30-45 days from the start of subculture. Initial monitoring is performed in the early stages of the growth period of adventitious roots, which can more comprehensively monitor the dynamic expression of genes in adventitious roots. In some embodiments, initial monitoring can be performed starting from the 21st day of subculture.
[0040] Inducer refers to an agent used to induce saponin synthesis. In some embodiments, the inducer can be methyl jasmonate. In some embodiments, the concentration of the inducer can be 50 to 200 μM. In some embodiments, the concentration of the inducer can be 50 μM. In some embodiments, the concentration of the inducer can be 100 μM. In some embodiments, the concentration of the inducer can be 150 μM. In some embodiments, the concentration of the inducer can be 200 μM.
[0041] In the examples of this specification, by regularly monitoring the changes in the expression levels of WOX5, a key gene for adventitious root stem cell differentiation, and PIN3, a key gene for lateral root development, it is possible to determine whether the adventitious root growth period is completed and whether the saponin synthesis period has begun, thereby determining the optimal time for adding the inducer, and utilizing the inducer to increase the amount of saponin synthesis in the adventitious roots during the saponin synthesis stage.
[0042] Step S3, after adding the inducer, regularly monitoring the expression level of the gene encoding the key enzyme for saponin synthesis, and collecting adventitious roots according to the change in the expression level of the key enzyme for saponin synthesis.
[0043] For instructions on regular monitoring, please refer to the relevant description above. In some embodiments, the time of regular monitoring in step S3 may be different from the time of regular monitoring in step S2, for example, after adding the inducer, the expression of the gene encoding the key enzyme of saponin synthesis is monitored once every 12 hours, once every 2 days, and once every 5 days.
[0044] The key enzyme gene for saponin synthesis is the key gene regulating saponin synthesis. During the saponin synthesis process, the expression level of the key enzyme gene for saponin synthesis gradually increases, reaches the highest expression level, and then gradually decreases and returns to the lowest expression level, indicating that saponin synthesis is basically completed. At this time, collecting adventitious roots can maximize the acquisition of adventitious roots with high saponin content. By monitoring the changes in the expression level of the key enzyme gene for saponin synthesis, it is possible to determine whether saponin synthesis is completed, thereby determining the best time to collect adventitious roots.
[0045] In some embodiments, based on the change in the expression level of the key enzyme gene for saponin synthesis, collecting adventitious roots may include: taking the time of adding the inducer as the starting time, and collecting the adventitious roots when the expression level of the key enzyme gene for saponin synthesis first increases and then recovers to near the expression level of the key enzyme gene for saponin synthesis monitored at the starting time.
[0046] The vicinity of the expression of the saponin synthesis key enzyme gene monitored at the start time can include the range of + / -20% of the expression of the saponin synthesis key enzyme gene monitored at the start time. For example, 80%, 100% or 120% of the expression of the saponin synthesis key enzyme gene monitored at the start time can be recovered.
[0047] The expression level of the key enzyme gene for saponin synthesis is a relative expression level, that is, the absolute expression level of the key enzyme gene for saponin synthesis is the percentage of the absolute expression level of the key enzyme gene for saponin synthesis monitored at the start time. The absolute expression level can be determined by real-time fluorescence quantitative PCR.
[0048] In some embodiments, the key enzyme genes for saponin synthesis may include at least one or more of HGMR1, FPS1, DDS1 and UGT71A27. Taking HGMR1, FPS1, DDS1 and UGT71A27 as examples, the period of collecting adventitious roots is described: when the expression level of HGMR1 increases from 1 times to about 5.5 times, and then returns to about 1 times; when the expression level of FPS1 increases from 1 times to about 9.5 times, and then returns to about 1 times; when the expression level of DDS1 increases from 1 times to about 8.7 times, and then returns to about 1 times; or when the expression level of UGT71A27 increases from 1 times to about 4.5 times, and then returns to about 1 times, adventitious roots are collected. In some embodiments, after the inducing agent is added, the expression level of one gene or the expression levels of multiple genes in HGMR1, FPS1, DDS1 and UGT71A27 can be regularly monitored.
[0049] In the embodiments of this specification, by regularly monitoring the expression levels of one or more key enzyme genes for saponin synthesis, the expression changes of the key enzyme genes for saponin synthesis can be observed in real time, thereby determining whether saponin synthesis is completed, determining the optimal time to collect adventitious roots of Chinese medicinal materials, and obtaining adventitious roots with higher saponin content to the greatest extent possible.
[0050] One or more embodiments of the present disclosure further provide a saponin, which is extracted from the adventitious roots of Chinese medicinal materials produced by the above-mentioned method for producing adventitious roots of Chinese medicinal materials producing saponins.
[0051] Saponin is one of the important active ingredients in traditional Chinese medicine, and has certain pharmacological effects in maintaining blood circulation, improving myocardial ischemia, anti-arrhythmia, anti-shock, sedation, improving intelligence, anti-aging, anti-oxidation, anti-cell proliferation and anti-tumor. There are many types of saponin, and the common saponin components are mainly ginsenoside Rg1, Rb1, Rb2, Rc, Rd, Re and notoginseng saponin R1.
[0052] Saponins in adventitious roots of Chinese medicinal materials can be extracted by a variety of methods. For example, alcohol extraction method, such as using methanol, ethanol, etc. as solvents to extract saponins; adsorption column chromatography, such as using silica gel, alumina, etc. as adsorbents to extract saponins; high-performance liquid chromatography, such as using methanol-water, acetonitrile-water, etc. as eluents, using high-performance liquid chromatography to extract saponins, etc.
[0053] The saponin content in the adventitious roots of Chinese medicinal materials can be detected by a variety of methods, such as high performance liquid chromatography (HPLC), ultraviolet and visible spectrum (UV-vis), etc.
[0054] In the embodiments of the present specification, by optimizing the composition of the adventitious root production medium, the cost of the medium materials is reduced and the saponin content in the adventitious roots is increased; in the adventitious root production process, by real-time monitoring of the changes in the expression levels of key genes, the addition time of the inducer and the collection time of the adventitious roots are determined, so that the saponin content in the produced adventitious roots is greatly improved; combined with the regular monitoring of the adventitious root production medium and gene expression levels, an efficient and stable method for producing adventitious roots of saponin-producing Chinese medicinal materials is determined, and this method can be applied to the adventitious root production of various medicinal plants such as ginseng, American ginseng, japonicus, Panax notoginseng, etc., and has broad application prospects.
[0055] The following is a detailed description of the present invention in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, several adjustments and improvements can be made without departing from the concept of the present invention. These all fall within the scope of protection of the present invention. Example Example 1: Preparation of culture medium
[0056] 1. Prepare ginseng callus induction medium: add 0.2mg / L-0.5mg / L gibberellins (GA), 20g / L-30g / L sucrose and 0.6g / L-0.8g / L agar to the conventional MS medium. The composition of the conventional MS medium is shown in Table 1.
[0057] 2. Prepare ginseng adventitious root induction medium: add 0.2mg / L-0.5mg / L 6-benzylaminopurine (6-BA), 2mg / L-4mg / L naphthaleneacetic acid (NAA), 0.6g / L-0.8g / L agar and 20g / L-40g / L sucrose to the conventional MS medium.
[0058] 3. Prepare ginseng adventitious root production medium: Add 40g / L sucrose, 2mg / L-20mg / L indole-3-butyric acid (IBA), and 0.1mg / L-1mg / L KT to the modified MS medium. The composition of the modified MS medium is shown in Table 1. Table 1 Comparison of the components of conventional MS medium and improved MS medium Example 2: Inducing adventitious roots of ginseng
[0059] (1) Taking wild ginseng roots as explants, soaking them in 75% alcohol for 10-30 seconds, taking out the explants, disinfecting them with 0.1% mercuric chloride solution for 8-12 minutes, and then repeatedly rinsing them with sterile water for 4-5 times, placing them on sterile filter paper to absorb the moisture on the surface of the explants, removing the epidermis on the surface of the explants after disinfection, and inoculating them on ginseng callus induction medium to obtain ginseng callus;
[0060] (2) taking the ginseng callus tissue obtained in step (1), inoculating it into a ginseng adventitious root induction medium, and transferring it once every 30-40 days to obtain a large number of ginseng cells;
[0061] (3) Collect the ginseng cells in step (2), inoculate them into human adventitious root induction medium, culture them in the dark at 22-27° C., transfer the cell tissue once every 30-40 days, and induce ginseng adventitious roots. Example 3: Shake flask culture of ginseng adventitious roots and induction of ginsenosides
[0062] (1) The ginseng adventitious roots obtained in Example 2 were inoculated into the ginseng adventitious root production medium at a ratio of 3 g / L. A total of 8 bottles with a capacity of 650 mL were used, each with 250 mL of medium. The culture was carried out at a speed of 100-120 rpm / min under dark conditions at 25°C. Figure 2A As shown;
[0063] (2) Generally, after 21-35 days of cultivation, a large number of new adventitious roots will sprout around the original roots, such as Figure 2B From the 21st day, samples were taken every 24 hours, and the expression of pgWOX5, a key gene for ginseng stem cell differentiation, and pgPIN3, a key gene for lateral root development, was monitored using real-time fluorescence quantitative PCR (qPCR). The primers for detecting gene expression are shown in Table 2.
[0064] (3) Taking the gene expression level on the 21st day as a reference, when the expression level of pgWOX5 decreased to about 50% and the expression level of pgPIN3 increased to about 200%, 50 μM methyl jasmonate (MeJA) was added for induction;
[0065] (4) Sampling was performed every 24 hours, and the expression changes of the key enzyme genes encoding ginsenoside synthesis, pgHGMR1, pgFPS1, pgDDS1, and pgUGT71A27, were monitored by qPCR technology. The primers for detecting gene expression are shown in Table 2. Taking the gene expression level when MeJA was added as a reference, when the expression level of pgHGMR1 increased from 1 times to about 5.5 times, and then recovered to about 1 times; the expression level of pgFPS1 increased from 1 times to about 9.5 times, and then recovered to about 1 times; the expression level of pgDDS1 increased from 1 times to about 8.7 times, and then recovered to about 1 times; the expression level of pgUGT71A27 increased from 1 times to about 4.5 times, and then recovered to about 1 times, adventitious roots were collected;
[0066] (5) The collected adventitious roots were rinsed with sterile water and then dried in an oven at 50°C for 8-10 hours. Table 2 qPCR primers used to detect key genes for stem cell differentiation, lateral root development, and ginsenoside synthesis
[0067] The adventitious roots of ginseng produced by the above steps have a biomass accumulation of 5 g / L based on dry weight; as shown in Table 3, the total saponin content measured by HPLC method can reach an average of 7.88%, the total saponin content measured by UV method can reach an average of more than 14.16%, the ginsenoside Rg1 content can reach an average of 0.093%, the saponin Re content can reach an average of 0.81%, the saponin Rg2 content can reach an average of 0.04%, the saponin Rb1 content can reach an average of 2.69%, the saponin Rc content can reach an average of 1.49%, the saponin Rb2 content can reach an average of 1.34%, and the saponin Rd content can reach an average of 1.62%. In addition, the ginseng polysaccharide content reaches more than 10%. Table 3 Statistics of saponin content in adventitious roots of ginseng cultured in shake flasks (percentage) Comparative Example 1
[0068] Comparative Example 1 is compared with Example 3. The difference from Example 3 is that the ginseng adventitious root production medium in Comparative Example 1 is based on conventional MS medium with the addition of sucrose 40g / L, 2mg / L-20mg / L indolebutyric acid (Indole-3-butyric acid, IBA), 0.1mg / L-1mg / L KT and 0.5mg / L vitamin B6 (PyridoxineHCl), and includes 3 repetitions, and the remaining steps and conditions are the same as in Example 3.
[0069] As shown in Table 4, the ginseng adventitious roots produced by Comparative Example 1 had an average total saponin content of only 3.083% as measured by HPLC, and an average total saponin content of only more than 6.383% as measured by UV method. The average content of ginsenoside Rg1 was 0.073%, the average content of saponin Re was 0.308%, the average content of saponin Rg2 was 0.017%, the average content of saponin Rb1 was 1.096%, the average content of saponin Rc was 0.626%, the average content of saponin Rb2 was 0.629%, and the average content of saponin Rd was 0.335%. Table 4 Statistics of saponin content in ginseng adventitious roots cultured in conventional MS medium shake flasks (percentage)
[0070] By comparing Example 3 with Comparative Example 1, it was found that, compared with conventional MS culture medium, ginseng adventitious roots cultured in improved MS culture medium can produce higher content of ginsenosides. Example 4: Barrel culture of ginseng adventitious roots and obtaining high-content ginsenosides
[0071] (1) The ginseng adventitious roots obtained in Example 2 were inoculated into the ginseng adventitious root production medium at a ratio of 5-10 g / L fresh weight and cultured in a culture barrel. Figure 3A As shown, the culture barrel includes a hollow cylindrical conical culture tank body, the inoculation port and the air inlet are located at the conical end of the culture tank body, the inoculation port is equipped with a sealing cover, the air inlet is equipped with an air distributor and is tilted, and the rotary air intake can meet the dissolved oxygen demand of the culture medium at a low ventilation volume, the exhaust port is located at the cylindrical end of the culture tank body, and the volume of the culture barrel is 2-100L; it is produced in batches, with a total of 3 batches, and one of the culture barrels in each batch is used as a monitoring barrel to monitor the changes in gene expression, and the rest are used as production barrels;
[0072] (2) After 21 days of cultivation, a large number of new adventitious roots will sprout around the original roots, such as Figure 3B From the 21st day, samples were taken every 24 hours to monitor the expression of pgWOX5 and pgPIN3 genes using qPCR. The primers for detecting gene expression are shown in Table 2;
[0073] (3) Taking the gene expression level on day 21 as a reference, when the expression level of pgWOX5 decreased to about 50% and the expression level of pgPIN3 increased to about 200%, MeJA at a concentration of 200 μM was added to induce the synthesis of ginsenosides;
[0074] (4) Sampling was performed every 24 hours, and the expression changes of the key enzyme genes encoding ginsenoside synthesis, pgHGMR1, pgFPS1, pgDDS1, and pgUGT71A27, were monitored by qPCR technology. The primers for detecting gene expression are shown in Table 2. Taking the gene expression level when MeJA was added as a reference, when the expression level of pgHGMR1 increased from 1 times to about 5.5 times, and then recovered to about 1 times; the expression level of pgFPS1 increased from 1 times to about 9.5 times, and then recovered to about 1 times; the expression level of pgDDS1 increased from 1 times to about 8.7 times, and then recovered to about 1 times; the expression level of pgUGT71A27 increased from 1 times to about 4.5 times, and then recovered to about 1 times, adventitious roots were collected;
[0075] (5) The collected adventitious roots were rinsed with sterile water and then dried in an oven at 50°C for 8-10 hours.
[0076] In step (2), the expression levels of the pgWOX5 gene and the pgPIN3 gene change over time as shown in Figure 4A and Figure 4B In step (4), the expression levels of ginsenoside synthesis key enzyme genes pgHGMR1, pgFPS1, pgDDS1 and pgUGT71A27 changed over time as shown in Figure 5A-5D shown.
[0077] The adventitious roots of ginseng produced by the above steps can accumulate biomass up to 6.14 g / L in terms of dry weight; as shown in Table 5, the total saponin content measured by HPLC method can reach an average of 9.39%, the total saponin content measured by UV method can reach an average of 17.07%, the saponin Rg1 content can reach an average of 0.02%, the saponin Re content can reach an average of 0.32%, the saponin Rg2 content can reach an average of 0.19%, the saponin Rb1 content can reach an average of 2.92%, the saponin Rc content can reach an average of 1.66%, the saponin Rb2 content can reach an average of 1.53%, and the saponin Rd content can reach an average of 2.75%. In addition, the ginseng polysaccharide content reaches more than 10%. Table 5 Statistics of saponin content in barrel-cultured ginseng adventitious roots (percentage) Example 5: Barrel culture of American ginseng adventitious roots and obtaining high-content saponins
[0078] In Example 5, the ginseng adventitious roots of Example 4 were replaced with American ginseng adventitious roots, including 3 repetitions, and the other conditions remained unchanged.
[0079] The adventitious roots of American ginseng obtained by culture in Example 5 have a biomass accumulation of up to 7.25 g / L based on dry weight; as shown in Table 6, the total saponin content measured by HPLC method can reach an average of 7.64%, the total saponin content measured by UV method can reach more than 16.63%, the saponin Rg1 content can reach an average of 0.04%, the saponin Re content can reach an average of 0.42%, the saponin Rg2 content can reach an average of 0.08%, the Rb1 content can reach an average of 2.53%, the saponin Rc content can reach an average of 1.50%, the saponin Rb2 content can reach an average of 1.28%, and the saponin Rd content can reach an average of 1.78%. Table 6 Statistics of saponin content in barrel-cultured adventitious roots of American ginseng (percentage)
[0080] It can be seen from Example 5 that the method for producing adventitious roots of saponin-producing Chinese medicinal materials provided in the examples of this specification is applied to American ginseng, and the saponin content in the adventitious roots of American ginseng produced can still reach a relatively high level. It can be seen that the method provided in this specification can be applied to a variety of medicinal plants, and adventitious roots with a high saponin content can be obtained, which has a wide range of application value.
[0081] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only for example and does not constitute a limitation of 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.
[0082] At the same time, this specification uses specific words to describe the embodiments of this specification. For example, "one embodiment", "an embodiment", and / or "some embodiments" refer to a certain feature, structure or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more in 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.
[0083] In addition, unless explicitly stated in the claims, the order of the processing elements and sequences described in this specification, the use of alphanumeric characters, or the use of other names are not intended to limit the order of the processes and methods of this specification. Although the above disclosure discusses some invention embodiments that are currently considered useful through various examples, it should be understood that such details are only for illustrative purposes, and the attached claims are not limited to the disclosed embodiments. On the contrary, the claims are intended to cover all modifications and equivalent combinations that are consistent with the essence and scope of the embodiments of this specification.
[0084] Similarly, it should be noted that in order to simplify the description disclosed in this specification and thus help understand one or more embodiments of the invention, in the above description of the embodiments of this specification, multiple features are sometimes combined into one embodiment, figure or description thereof. However, this disclosure method does not mean that the features required by the subject matter of this specification are more than the features mentioned in the claims. In fact, the features of the embodiments are less than all the features of the single embodiment disclosed above.
[0085] In some embodiments, numbers describing the number of components and attributes are used. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise specified, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may change according to the required features of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the setting of such numerical values is as accurate as possible within the feasible range.
[0086] Each patent, patent application, patent application publication, and other materials, such as articles, books, specifications, publications, documents, etc., cited in this specification are hereby incorporated by reference in their entirety. Except for application history documents that are inconsistent with or conflicting with the contents of this specification, documents that limit the broadest scope of the claims of this specification (currently or later attached to this specification) are also excluded. It should be noted that if the descriptions, definitions, and / or use of terms in the materials attached to this specification are inconsistent or conflicting with the contents described in this specification, the descriptions, definitions, and / or use of terms in this specification shall prevail.
[0087] 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, as an example and not a 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 producing adventitious roots of saponin-producing Chinese medicinal materials, characterized in that: The method comprises: The induced adventitious roots of the Chinese medicinal materials are subcultured in an adventitious root production medium, and the expression levels of the key gene WOX5 for stem cell differentiation or the key gene PIN3 for lateral root development are regularly monitored; When the expression level of WOX5 decreases to a first preset value or the expression level of PIN3 increases to a second preset value, adding an inducer; and After adding the inducer, the expression level of the gene encoding the key enzyme for saponin synthesis is regularly monitored, and the adventitious roots are collected according to the change in the expression level of the key enzyme for saponin synthesis.
2. The method according to claim 1, characterized in that The adventitious root production medium comprises a modified MS medium, wherein the modified MS medium comprises the following components at the following concentrations: 112.5mg / L NH4NO3, 1900mg / L KNO3, 60mg / L KH2PO4, 370mg / LMgSO4·7H2O, 440mg / LCaCl2·2H2O, 6.2mg / L H3BO3, 8.6mg / L ZnSO4·7H2O, 0.025mg / L CuSO4·5H2O, 13.9mg / LFeSO4·7H2O, 18.7mg / L Na2·EDTA, 0.1mg / L thiamine hydrochloride and 0.5mg / L vitamin B6.
3. The method according to claim 2, characterized in that The adventitious root production medium also includes a growth regulator and sucrose. The growth regulator includes 2-20 mg / L indolebutyric acid and 0.1-1 mg / L kinetin. The concentration of the sucrose is 40 g / L.
4. The method according to claim 1, characterized in that: The first preset value is between 30% and 70% of the initially monitored expression level of WOX5, and the second preset value is between 150% and 250% of the initially monitored expression level of PIN3, wherein the initial monitoring is started at 21 days of culture.
5. The method according to claim 1, characterized in that: According to the change in the expression amount of the key enzyme gene for saponin synthesis, collecting the adventitious roots comprises: Taking the time of adding the inducer as the starting time, when the expression level of the key enzyme gene for saponin synthesis first increases and then recovers to near the expression level of the key enzyme gene for saponin synthesis monitored at the starting time, the adventitious roots are collected.
6. The method according to claim 1, characterized in that The key enzyme genes for saponin synthesis include at least one of HGMR1, FPS1, DDS1 and UGT71A27.
7. The method according to any one of claims 1 to 6, characterized in that The inducer includes methyl jasmonate, and the concentration of the inducer is 50-200 μM.
8. The method according to any one of claims 1 to 6, characterized in that Real-time fluorescence quantitative PCR was used to monitor the expression level.
9. The method according to claim 1, characterized in that: The Chinese medicinal material includes at least one of ginseng, American ginseng, panax notoginseng and rhizoma notoginseng.
10. A saponin extracted from the adventitious roots of a Chinese medicinal material produced by the method for producing adventitious roots of a Chinese medicinal material producing saponins according to any one of claims 1 to 9.