Method for establishing sphallerocarpus gracilis tender stem tissue culture system

Abstract

The invention relates to a method for establishing a sphallerocarpus gracilis tissue culture system, which comprises the following steps: taking a sphallerocarpus gracilis tender stem, sterilizing to obtain a sterile sphallerocarpus gracilis explant, and the sphallerocarpus gracilis tender stem is a fresh and tender stem of an umbelliferae sphallerocarpus gracilis plant. And carrying out induction culture, multiplication culture and rooting culture on the sterile sphallerocarpus gracilis explant to obtain a tissue culture plant. The explant sterilization method has the beneficial effects that on one hand, the explant sterilization method is lowest in pollution rate and highest in survival rate; on the other hand, the quality of the callus induced by taking the stem as the explant is the best, the induction rate and the differentiation rate are the highest, and the overall growth condition is better. On the other hand, the invention provides a bacteriostatic composition which combines the bearing capacity and the bacteriostatic effect of the sphallerocarpus gracilis.

Description

Technical Field

[0001] The present invention relates to the field of plant culture, and particularly to a method for establishing a tissue culture system for the tender stems of Sphallerocarpus gracilis. Background Art

[0002] Sphallerocarpus gracilis is a single-species plant of the genus Sphallerocarpus in the family Umbelliferae. It is known as "little ginseng" in Compendium of Materia Medica, and is also listed as a tonic medicine in the traditional Chinese medicine classic Jingzhu Materia Medica. Its fleshy roots have the effects of invigorating qi and nourishing blood, relieving pain, nourishing yin and strengthening yang, dredging meridians and collaterals, and strengthening the stomach and soothe the liver. It is especially distributed in Qilian Mountains and Yanshan Mountains (within Zhangye City, Gansu Province). It is very rare for Sphallerocarpus gracilis to form a community advantage in a large area in Shandan County, Zhangye City. The Sphallerocarpus gracilis produced in Shandan County, Zhangye City, Gansu Province within the Qilian Mountains is called Shandan Sphallerocarpus gracilis, which is a natural and original ecological treasure with extremely high nutritional, economic and medicinal values. Tissue culture methods for plants in the family Umbelliferae have been reported. For example, Chinese Patent CN1918872A discloses a tissue culture and propagation method for Notopterygium incisum, and Zhang Jiaqing et al. published "Research Progress on Tissue Culture of Medicinal Plants in the Family Umbelliferae" which discloses tissue culture methods for several plants in the family Umbelliferae. However, due to different plant genera and species, the direct application of tissue culture to Sphallerocarpus gracilis has unsatisfactory effects. Summary of the Invention

[0003] Therefore, the present invention provides a method for establishing a tissue culture system for the tender stems of Sphallerocarpus gracilis, which includes the following steps:

[0004] Taking the tender stems of Sphallerocarpus gracilis and obtaining sterile Sphallerocarpus gracilis explants through sterilization treatment, wherein the tender stems of Sphallerocarpus gracilis are the fresh stems of the plant Sphallerocarpus gracilis of the genus Sphallerocarpus in the family Umbelliferae;

[0005] Subjecting the sterile Sphallerocarpus gracilis explants to induction culture, proliferation culture and rooting culture to obtain tissue culture plants.

[0006] For a further tissue culture method of Sphallerocarpus gracilis, the step of taking the tender stems of Sphallerocarpus gracilis and obtaining sterile Sphallerocarpus gracilis explants through sterilization treatment includes the following steps:

[0007] S1 The step of rinsing in HgCl2 solution;

[0008] S2 The step of soaking in alcohol;

[0009] S3 The step of soaking in saturated bleaching powder solution; and, after any of the steps S1 to S3, the step S4 of rinsing with running water or sterile water.

[0010] Further method for tissue culture of Codonopsis lanceolata Benth. et Hook.f., in step S1, the concentration of the HgCl₂ solution is 0.01 - 1%, preferably 0.1%; the rinsing time is 1 - 10 min, preferably 5 min; in step S4, running water is used for rinsing for 10 - 30 min, preferably 10 min.

[0011] Further method for tissue culture of Codonopsis lanceolata Benth. et Hook.f., in step S2, the concentration of the alcohol is 50 - 100% and the soaking time is 10 - 120 s, preferably the concentration of the alcohol is 75% and the soaking time is 30 s; in step S4, sterile water is used for rinsing 1 - 5 times, preferably 3 times.

[0012] Further method for tissue culture of Codonopsis lanceolata Benth. et Hook.f., in step S2, soak in saturated bleaching powder solution for 1 - 30 min, preferably soak for 10 min; in step S4, sterile water is used for rinsing 1 - 5 times, preferably 3 times.

[0013] Further method for tissue culture of Codonopsis lanceolata Benth. et Hook.f., for induction culture, an induction callus medium is used; for proliferation culture, a cluster bud medium is used; for rooting culture, a rooting medium is used; the pH of the induction callus medium, the cluster bud medium and the rooting medium is 5.0 - 7.0 and is supplemented with 1 - 10% sucrose and 0.1 - 5% agar. Preferably, the pH of the induction callus medium, the cluster bud medium and the rooting medium is 5.8 - 6.0 and is supplemented with 3% sucrose and 0.6% agar.

[0014] Further method for tissue culture of Codonopsis lanceolata Benth. et Hook.f., the induction callus medium includes: B5 medium, NAA with a final concentration of 0.1 - 5 mg / L and KT with a final concentration of 0.01 - 5 mg / L; preferably, the induction callus medium includes: B5 medium, NAA with a final concentration of 0.2 mg / L and KT with a final concentration of 0.1 mg / L.

[0015] Further method for tissue culture of Codonopsis lanceolata Benth. et Hook.f., the cluster bud medium includes: B5 medium, NAA with a final concentration of 0.1 - 5 mg / L and 6 - BA with a final concentration of 1 - 10 mg / L; preferably, the cluster bud medium includes: B5 medium, NAA with a final concentration of 0.2 mg / L and 6 - BA with a final concentration of 3.0 mg / L.

[0016] Further method for tissue culture of Codonopsis lanceolata Benth. et Hook.f., the rooting medium includes: 1 / 2MS, NAA with a final concentration of 1 - 10 mg / L; preferably, the rooting medium includes: 1 / 2MS, NAA with a final concentration of 0.5 mg / L.

[0017] The beneficial effects of the present invention are as follows. On the one hand, the explant sterilization method of the present invention has the lowest contamination rate and the highest survival rate. On the other hand, when using the stem as the explant, the quality of the induced callus is the best, the induction rate and differentiation rate are the highest, and the overall growth condition is better. On the further hand, the present invention provides an antibacterial composition that takes into account the tolerance of Radix Adenophorae seu Glehniae and the antibacterial effect. Brief Description of the Drawings

[0018] Figure 1 The tender stem of Radix Adenophorae seu Glehniae used in the embodiments of the present invention;

[0019] Figure 2 Effect of different disinfection times on the contamination rate;

[0020] Figure 3 Effect of different disinfection times on the browning rate;

[0021] Figure 4 Effect of different disinfection times on the survival rate. Detailed Embodiments

[0022] Term Explanation:

[0023] An explant refers to a fragment of an organ, tissue, or cell, etc. cut from a living plant for in vitro culture in plant tissue culture.

[0024] Tissue culture refers to a technique of separating the required tissues, organs, or cells, protoplasts, etc. from a plant body, and through aseptic operation, inoculating them on a culture medium containing various nutrients and plant hormones under aseptic conditions for culture to obtain a regenerated complete plant or produce other products with economic value.

[0025] Subculture refers to the process of transferring callus or culture from the original medium to a fresh medium to continue its growth and reproduction.

[0026] Huangshen, a single-species plant of the genus Sphallerocarpus (Sphallerocarpus gracills) in the Apiaceae family (Xue Hongyan, 2011), also known as Tibetan medicine Sphallerocarpus gracilis, Huangfeng, and small-leaf mountain carrot (in Wanping, Hebei) (Pi Li, 2015). It is golden throughout. It is sporadically distributed in the northwest, northeast, and north China in our country, especially in the Qilian Mountains and the Yanzhi Mountains (within Zhangye City, Gansu Province). It is very rare for such a large area to form a community dominance in Shandan County, Zhangye City (Jia Huixian, 2001). Under the background of ecological civilization construction, studying Huangshen has important ecological and economic significance for soil and water conservation, curbing grassland desertification, and maintaining biodiversity in the Qilian Mountains. According to the Compendium of Materia Medica, Huangshen has the effects of invigorating qi and nourishing blood, tonifying the liver and kidney, and dredging the channels and collaterals. Its fleshy roots are rich in 16 kinds of amino acids essential for the human body and are known as the "little ginseng" (Ma Tingting, 2014). Research shows that Huangshen also has functions such as antioxidant, anti-tumor, enhancing immunity, delaying fatigue, and antibacterial. Huangshen (Tibetan medicine Sphallerocarpus gracilis) has been widely used as the primary medicine in the "Five Roots Powder" and "Tangfan Brain Tonifying Liquid" all the time. Huangshen is also listed as a tonic medicine in the traditional Chinese medicine classic "Jingzhu Materia Medica". As a natural green health food with rich nutrition, high economic and medicinal value, rich in protein, polysaccharides, carotenoids, vitamins, reducing sugars, and trace elements (Pi Li, 2015), Huangshen has become a special product of Gansu and a designated product of the Western Development Trade Fair, with ecological, economic, and social benefits, and has great application potential and development prospects in the fields of food, medicine, chemical industry, etc. The Qilian Mountains National Nature Reserve, 76.4% of whose area is within Zhangye City, is an important ecological security barrier in the west of our country.

[0027] Clustered Bud (or Clonal Bud) refers to the dense bud clusters induced from explants (such as shoot tips, leaves, etc.) under suitable conditions in plant tissue culture. These buds have a multicellular structure, bipolarity, and complete organ primordia and can directly develop into small plants.

[0028] Callus refers to the newly formed tissue on the wound surface after local trauma stimulation of the original plant body. It is composed of living parenchyma cells and can originate from living cells of various tissues in any organ of the plant body.

[0029] NAA (Naphthaleneacetic Acid) is naphthaleneacetic acid, an artificially synthesized plant growth regulator belonging to the auxin hormone category.

[0030] KT (Kinetin) is kinetin, an artificially synthesized cytokinin with the chemical name 6-furfurylamino-purine. It belongs to the plant growth regulator and has physiological functions similar to natural cytokinins.

[0031] 6-BA, namely 6-benzylaminopurine, is a synthetic cytokinin plant hormone and is widely used in the field of plant growth regulation.

[0032] MS culture medium was designed by plant physiologists Murashige and Skoog in 1962 for tobacco cell culture and has now become one of the most commonly used basic culture media in plant tissue culture.

[0033] In some embodiments, the present invention provides a method for establishing a tissue culture system for the tender stems of Codonopsis pilosula var. modesta, comprising the following steps:

[0034] Take the tender stems of Codonopsis pilosula var. modesta and obtain sterile explants of Codonopsis pilosula var. modesta after sterilization treatment. The tender stems of Codonopsis pilosula var. modesta are the fresh stems of the plant Sphallerocarpus gracilis belonging to the genus Sphallerocarpus in the family Umbelliferae.

[0035] Cultivate the sterile explants of Codonopsis pilosula var. modesta through induction culture, proliferation culture and rooting culture to obtain tissue culture plants.

[0036] In some further embodiments of the tissue culture method for Codonopsis pilosula var. modesta, the step of taking the tender stems of Codonopsis pilosula var. modesta and obtaining sterile explants of Codonopsis pilosula var. modesta after sterilization treatment comprises the following steps:

[0037] Step S1: Rinse in HgCl2 solution;

[0038] Step S2: Soak in alcohol;

[0039] Step S3: Soak in saturated bleaching powder solution; and, after any of steps S1 to S3, step S4: Rinse with running water or sterile water.

[0040] In some further embodiments of the tissue culture method for Codonopsis pilosula var. modesta, in step S1, the concentration of the HgCl2 solution is 0.01 - 1%, preferably 0.1%; the rinsing time is 1 - 10 min, preferably 5 min; in step S4, it is to rinse with running water for 1 - 30 min, preferably 10 min.

[0041] In some further embodiments of the tissue culture method for Codonopsis pilosula var. modesta, in step S2, the concentration of the alcohol is 50 - 100% and the soaking time is 10 - 120 s, preferably the concentration of the alcohol is 75% and the soaking time is 30 s; in step S4, it is to rinse with sterile water 1 - 5 times, preferably 3 times.

[0042] In some further embodiments of the Codonopsis pilosula Franch. Nannf. tissue culture method, in the step S2, soak in a saturated bleaching powder solution for 1 to 30 minutes, preferably soak for 10 minutes; the preparation method of the saturated bleaching powder solution is: prepare a saturated aqueous solution of bleaching powder; in the step S4, rinse with sterile water 1 to 5 times, preferably 3 times.

[0043] In some further embodiments of the Codonopsis pilosula Franch. Nannf. tissue culture method, the induction culture uses an induction callus medium, the proliferation culture uses a cluster bud medium, and the rooting culture uses a rooting medium; the pH of the induction callus medium, the cluster bud medium, and the rooting medium is 5.0 to 7.0 and is supplemented with 1 to 10% sucrose and 0.1 to 5% agar. Preferably, the pH of the induction callus medium, the cluster bud medium, and the rooting medium is 5.8 to 6.0 and is supplemented with 3% sucrose and 0.6% agar.

[0044] In some further embodiments of the Codonopsis pilosula Franch. Nannf. tissue culture method, the induction callus medium includes: B5 medium, NAA with a final concentration of 0.1 to 5 mg / L, and KT with a final concentration of 0.01 to 5 mg / L; preferably, the induction callus medium includes: B5 medium, NAA with a final concentration of 0.2 mg / L, and KT with a final concentration of 0.1 mg / L.

[0045] In some further embodiments of the Codonopsis pilosula Franch. Nannf. tissue culture method, the cluster bud medium includes: B5 medium, NAA with a final concentration of 0.1 to 5 mg / L, and 6-BA with a final concentration of 1 to 10 mg / L; preferably, the cluster bud medium includes: B5 medium, NAA with a final concentration of 0.2 mg / L, and 6-BA with a final concentration of 3.0 mg / L.

[0046] In some further embodiments of the Codonopsis pilosula Franch. Nannf. tissue culture method, the rooting medium includes: 1 / 2MS, NAA with a final concentration of 1 to 10 mg / L; preferably, the rooting medium includes: 1 / 2MS, NAA with a final concentration of 0.5 mg / L.

[0047] Example 1 Method for establishing a Codonopsis pilosula Franch. Nannf. tender stem tissue culture system

[0048] Materials: Fresh tender stems of Codonopsis pilosula Franch. Nannf. from Shandan County, Gansu Province (collected from the Shandan Military Horse Farm in Gansu Province in May), as Figure 1 shown.

[0049] Method:

[0050] (1) The tender stems of Codonopsis lanceolata picked in May were sterilized to obtain sterile explants. The explant sterilization method: After shaking and rinsing with a small amount of 0.1% HgCl2 for 5 min, rinsing under running water for 10 min, then putting it into 75% alcohol for 30 s, rinsing with sterile water 3 times, transferring it into the supernatant of saturated bleaching powder, sterilizing for 10 min, and rinsing with sterile water 3 times.

[0051] (2) The sterile explants were subjected to induction culture, proliferation culture and rooting culture to obtain tissue culture plants, where: The induction culture used an induction callus medium: B5 medium + 0.2 mg / L NAA + 0.1 mg / L KT; The proliferation culture used a cluster bud medium: B5 medium + 0.2 mg / L NAA + 3.0 mg / L 6 - BA; The rooting culture used a rooting medium: 1 / 2MS + NAA 0.5 mg / L; All the above media were supplemented with 3% sucrose, 0.6% agar, and pH 5.8 - 6.0.

[0052] The same culture conditions for induction culture, proliferation culture and rooting culture were: temperature 20 - 22 °C, light time 12 h / d. The light intensity for induction culture and proliferation culture was 2000 - 2500 lx, and the light intensity for rooting culture (rooting stage) was 3000 lx.

[0053] Method for establishing a tissue culture system of the tender stems of Codonopsis lanceolata in Example 2

[0054] Different from Example 1, after the induction culture, proliferation culture and rooting culture, it further includes a subculture step, and the subculture step is as follows:

[0055] Before the subculture medium for subculture cooled and solidified, cefuroxime axetil with a final concentration of 30 - 60 mg / L was added, where: The subculture medium: B5 medium + 0.5 mg / L 6 - BA + 0.2 mg / L IBA.

[0056] Method for establishing a tissue culture system of the tender stems of Codonopsis lanceolata in Example 3

[0057] Different from Example 2, the subculture step is specifically:

[0058] Before the subculture medium for subculture cooled and solidified, cefuroxime axetil with a final concentration of 30 - 60 mg / L and magnesium trisilicate (Mg2Si3O8) with a final concentration of 60 - 90 mg / L were added, and stirred for 3 - 5 min.

[0059] Test Example 1 Comparative test of different disinfection conditions

[0060] Test method: According to the method of Example 1, the conditions different from those in the example are shown in Table 1. Each treatment is repeated in 3 groups, with 20 bottles per group, that is, 60 bottles are inoculated for each treatment in the test group. They are placed on the culture rack and cultured at 25°C with a 12-hour light per day. The emergence time and growth of callus are observed from the 1st to the 25th day, and the contamination rate is counted.

[0061] Contamination rate (%) = number of contaminated / total number of inoculations × 100%;

[0062] Browning rate (%) = number of browned / total number of inoculations except for contaminated × 100%;

[0063] Induction rate (%) = number of callus induced / total number of inoculations except for contaminated × 100%.

[0064] Callus morphology: "Light yellow and compact" indicates good quality of callus; "Dark yellow and loose" indicates better quality of callus; "Brownish and loose" indicates poor quality of callus.

[0065] Growth condition: "+" indicates general, "++" indicates better, "+++" indicates good, "++++" indicates the best.

[0066] Test results:

[0067] Table 1 Test results of disinfection conditions

[0068]

[0069] The results are as Figures 2 to 4 shown. After summarizing the sterilization effect of 0.1% HgCl2 and other time data, it is as follows: The induction rates at 5 minutes and 7 minutes are the highest, reaching 100%, but the browning rate at 7 minutes is 2.5%, which is higher than that at 5 minutes, and the emergence time of callus is later than that at 5 minutes. Comparing the sterilization effects of HgCl2 5 minutes + 75% alcohol for 30 seconds, 60 seconds, and 90 seconds, the induction rates of HgCl2 5 minutes + 75% alcohol for 30 seconds and 60 seconds both reach 100%, the induction rate at 90 seconds is 96.76%, but the browning rates at 60 seconds and 90 seconds reach 2.5% and 3.1% respectively, and the emergence time of callus at 60 seconds and 90 seconds is later than that at 30 seconds. Based on the above results, the sterilization effect of HgCl2 5 minutes + 75% alcohol for 30 seconds is the best.

[0070] Test Example 2 Comparative test of different hormone conditions

[0071] Test method: The preparation methods of each sample are basically the same as those in Example 1, and the different conditions are shown in Table 2 and Table 3.

[0072] Table 2 Effects of different hormone ratios on callus induction of young stems of Huang Shen

[0073]

[0074]

[0075] Note: + represents slow callus growth; ++ represents faster callus growth; +++ represents fast and vigorous callus growth.

[0076] Table 3 Effects of Different Hormone Combinations on the Proliferation of Clustered Shoots from the Tender Stems of Codonopsis pilosula var. modesta

[0077]

[0078] Note: + represents slow differentiation growth; ++ represents faster differentiation growth; +++ represents fast and vigorous differentiation growth.

[0079] The results showed that treatment JB5 (B5 + 0.2 mg / L NAA + 0.1 mg / L KT) was an ideal medium for promoting the differentiation growth of callus from Codonopsis pilosula var. modesta (see Table 2); treatment SG4 (B5 + 0.2 mg / L NAA + 3.0 mg / L 6-BA) was an ideal medium for promoting the proliferation of clustered shoots from Codonopsis pilosula var. modesta (see Table 3).

[0080] Test Example 3 Test on the Effects of Antibiotics on the Growth and Contamination Degree of Test-Tube Seedlings of Codonopsis pilosula var. modesta

[0081] Test method: The preparation methods of each sample were basically the same as those in Example 2, except that: before the medium cooled and solidified, antibiotic stock solutions were added at the designed concentrations on the ultra-clean bench, shaken evenly, and marked. For each concentration of each antibiotic, 50 bottles were treated, and 150 bottles were used as the blank control without adding antibiotics.

[0082] After induction culture, proliferation culture, and rooting culture, if there was no bacterial contamination on the surface of the medium or only slight contamination, the young buds of Codonopsis pilosula var. modesta were rinsed 5 times with sterile water and inoculated on the subculture medium containing different concentrations of ampicillin sodium, cefuroxime axetil, and streptomycin sulfate. After 60 days, the contamination rate, proliferation coefficient, plant height, and number of roots were investigated. The antibiotic concentrations were set at 0, 30, 60, and 90 mg / L respectively.

[0083] Table 4 Effects of Different Types and Mass Concentrations of Antibiotics on the Growth and Contamination Degree of Test-Tube Seedlings of Codonopsis pilosula var. modesta

[0084]

[0085] Note: Proliferation coefficient = (number of proliferated plants + number of inoculated plants) / number of inoculated plants. Severe contamination: ++++; Serious contamination: +++; Moderate contamination: ++; Slight contamination: +; No contamination: -

[0086] +; Moderate contamination: ++; Slight contamination: +; No contamination: -

[0087] The results in Table 4 show that when 30 - 60 mg / L of cefuroxime axetil is added to the solid medium for subculture, the contaminated medium shows a sterile state, the test-tube plantlets grow normally, with the highest multiplication coefficient, the highest average plant height, and the most average root numbers.

[0088] Test Example 4: Experiment on the Effect of Antibiotics on the Growth and Contamination Degree of the Test-Tube Plantlets of Huangcan

[0089] Test method: The preparation methods of each sample are basically the same as those in Example 3, except that:

[0090] After induction culture, proliferation culture, and rooting culture, if there is a phenomenon of moderate bacterial contamination on the surface of the medium, take the young buds of Huangcan, rinse them 5 times with sterile water, and inoculate them on the subculture medium containing different concentrations of cefuroxime axetil and magnesium trisilicate. After 60 days, investigate the contamination rate, multiplication coefficient, plant height, and root numbers. The concentrations of cefuroxime axetil (Cef) and magnesium trisilicate (Mag) are set at 60 mg / L and 30 mg / L, 60 mg / L and 60 mg / L, 60 mg / L and 90 mg / L, 60 mg / L and 120 mg / L respectively.

[0091] Table 6: Effect of Different Types and Mass Concentrations of Antibiotics on the Growth and Contamination Degree of the Test-Tube Plantlets of Huangcan

[0092]

[0093] Note: Multiplication coefficient = (number of proliferations + number of inoculations) / number of inoculations. Relatively serious contamination: ++++; serious contamination: ++

[0094] +; moderate contamination: ++; slight contamination: +; no contamination: -

[0095] The results in Table 6 show that when 80 - 90 mg / L of cefuroxime axetil and 60 - 90 mg / L of magnesium trisilicate are added to the solid medium for subculture, even if there is a phenomenon of moderate bacterial contamination on the surface of the primary medium, the subculture medium can be kept in a sterile state during subculture, the test-tube plantlets grow normally, with the highest multiplication coefficient, the highest average plant height, and the most average root numbers.

[0096] Result analysis: Inhibiting the contamination of miscellaneous bacteria is the key to tissue culture. The contamination situation varies depending on the genotype of plant materials and the sampling parts. Regarding the application of antibiotics, different plant materials have different sensitivities to different antibiotics, and when using them, the tolerance of plant materials and the antibacterial effect should be considered. Selecting the appropriate types and concentrations of antibacterial and screening antibiotics is crucial for the success of tissue culture. Different species and different genotypes of the same species of plant materials have very different tolerances to antibiotics, and high concentrations are not easy to be removed from plant materials. Thus, it can be seen that the addition of magnesium trisilicate improves the tolerance of Huangcan to cefuroxime axetil and can be used as a tolerance agent.

[0097] Test Example 5 Inhibiting Contamination by Miscellaneous Bacteria Positive Control Test

[0098] Therefore, the inventors tested a large number of methods for inhibiting contamination by miscellaneous bacteria reported in the prior art, but the effects were not satisfactory. The following is illustrated with Comparative Examples 1 to 4:

[0099] Comparative Example 1

[0100] Different from Example 2, the conditions for inhibiting contamination by miscellaneous bacteria of Chinese Patent CN201910589650 (a tissue culture method of Kirengeshoma palmata) were adopted, specifically: using 200 mg / L of cefotaxime and 100 mg / L of timentin.

[0101] Test results: When the maximum concentration in the tissue culture of C. ginseng was 90 mg / L, the color of the seedling leaves became lighter. When using 200 mg / L of cefotaxime, the C. ginseng materials had been killed, so it could not be used for the tissue culture of C. ginseng.

[0102] Comparative Example 2

[0103] Different from Example 2, the conditions for inhibiting contamination by miscellaneous bacteria of Chinese Patent CN202311042060 (a method for a tissue culture regeneration system of Cyperus esculentus) were adopted, specifically: using 300 - 500 mg / L of cefotaxime.

[0104] Test results: Basically the same as Comparative Example 2. When the maximum concentration in the tissue culture of C. ginseng was 90 mg / L, the color of the seedling leaves became lighter. When using 300 mg / L of cefotaxime, the C. ginseng materials had been killed, so it could not be used for the tissue culture of C. ginseng either.

[0105] Comparative Example 3

[0106] Different from Example 1, the conditions for inhibiting contamination by miscellaneous bacteria of Chinese Patent CN202410398039 (a rapid propagation method for tissue culture of chrysanthemum cut flowers without many lateral branches) were adopted, specifically: using 1000 mg / L of cefotaxime.

[0107] Test results: When using 1000 mg / L of cefotaxime, the C. ginseng materials had been killed. Due to different antibiotic usage methods and a concentration difference of more than ten times, it could not be used for the tissue culture of C. ginseng.

[0108] Comparative Example 4

[0109] Different from Example 1, the conditions for inhibiting contamination by miscellaneous bacteria of Chinese Patent CN202411934470 (a method for rapid propagation of tissue culture of Ficus carica male germplasm materials) were adopted, specifically: using 200 mg / L of cefotaxime sodium.

[0110] Test results: When using 200 mg / L cefotaxime sodium, the materials of Codonopsis lanceolata have been killed. Codonopsis lanceolata belongs to perennial herbaceous plants. Woody plants have a greater tolerance to antibiotics than herbaceous plants and cannot be used for the tissue culture of Codonopsis lanceolata, which is consistent with the conclusion of the test results.

[0111] The above-described embodiments are merely described as the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for establishing a tissue culture system of the tender stems of Codonopsis pilosula var. modesta, characterized in that, It includes the following steps: Take the tender stems of Sphallerocarpus gracilis, a plant of the genus Sphallerocarpus in the family Umbelliferae, and obtain sterile explants of Sphallerocarpus gracilis after sterilization treatment. The tender stems of Sphallerocarpus gracilis are the fresh and tender stems of Sphallerocarpus gracilis. Subject the sterile explants of Sphallerocarpus gracilis to induction culture, proliferation culture, and rooting culture to obtain tissue culture plants.

2. The method for tissue culture of Huangshen as claimed in claim 1, characterized in that, The step of taking the tender stems of Sphallerocarpus gracilis and obtaining sterile explants of Sphallerocarpus gracilis after sterilization treatment includes the following steps: S1: Rinsing step in HgCl2 solution; S2: Soaking step in alcohol; S3: Soaking step in saturated bleaching powder solution; and, after any of the steps S1 to S3, S4: Rinsing step with running water or sterile water.

3. The method for tissue culture of Ophiopogon japonicus as claimed in claim 1, wherein, In the step S1, the concentration of the HgCl2 solution is 0.01 - 1%, preferably 0.1%; the rinsing time is 1 - 10 min, preferably 5 min; in the step S4, it is to rinse with running water for 1 - 30 min, preferably 10 min.

4. The tissue culture method of Codonopsis pilosula Franch. according to claim 1, characterized in that In the step S2, the concentration of the alcohol is 50 - 100%, the soaking time is 10 - 120 s, preferably the concentration of the alcohol is 75% and the soaking time is 30 s; in the step S4, it is to rinse with sterile water for 1 - 5 times, preferably 3 times.

5. The method for tissue culture of Huangcan as claimed in claim 1, characterized in that, In the step S2, soak in saturated bleaching powder solution for 1 - 30 min, preferably soak for 10 min; in the step S4, it is to rinse with sterile water for 1 - 5 times, preferably 3 times.

6. The method for tissue culture of Radix Astragali seu Hedysari according to claim 1, wherein, For the induction culture, an induction callus medium is used; for the proliferation culture, a cluster bud medium is used; for the rooting culture, a rooting medium is used; the pH of the induction callus medium, the cluster bud medium, and the rooting medium is 5.0 - 7.0 and contains 1 - 10% sucrose and 0.1 - 5% agar. Preferably, the pH of the induction callus medium, the cluster bud medium, and the rooting medium is 5.8 - 6.0 and contains 3% sucrose and 0.6% agar.

7. The method for tissue culture of yellow ginseng according to claim 6, wherein, The induction callus medium includes: B5 medium, NAA with a final concentration of 0.1 - 5 mg / L, and KT with a final concentration of 0.01 - 5 mg / L; preferably, the induction callus medium includes: B5 medium, NAA with a final concentration of 0.2 mg / L, and KT with a final concentration of 0.1 mg / L.

8. The tissue culture method of Radix Astragali seu Hedysari according to claim 6, wherein, The cluster bud medium includes: B5 medium, NAA with a final concentration of 0.1 - 5 mg / L, and 6 - BA with a final concentration of 1 - 10 mg / L; preferably, the cluster bud medium includes: B5 medium, NAA with a final concentration of 0.2 mg / L, and 6 - BA with a final concentration of 3.0 mg / L.

9. The tissue culture method of Radix Astragali seu Hedysari according to claim 6, characterized in that, The rooting medium includes: 1 / 2MS, NAA with a final concentration of 1 - 10 mg / L; preferably, the rooting medium includes: 1 / 2MS, NAA with a final concentration of 0.5 mg / L.

10. The method for tissue culture of Radix Codonopsis Pilosulae as claimed in claim 1, wherein, The culture conditions for the induction culture, the proliferation culture and the rooting culture include: temperature 18 - 25 °C, light intensity 1800 - 2800 lx, light duration 6 - 24 h / d. Among them, the light intensity is increased to 2900 - 3500 lx during the rooting stage; preferably, the culture conditions for the induction culture, the proliferation culture and the rooting culture include: temperature 20 - 22 °C, light intensity 2000 - 2500 lx, light duration 12 h / d. Among them, the light intensity is increased to 3000 lx during the rooting stage.

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