Culture medium composition for saussurea involucrate tissue culture and application thereof

Abstract

The invention provides a culture medium composition for saussurea involucrate tissue culture and application thereof, and belongs to the field of plant tissue culture. On the basis of the MS culture medium, the culture medium composition also comprises the following components: 9 to 11 g / L of fructose, 9 to 11 g / L of glucose, 9 to 11 g / L of sucrose, 5 to 7 g / L of agar, 0.5 to 1.5 mg / L of 2, 4-dichlorphenoxyacetic acid, 0.05 to 0.1 mg / L of naphthylacetic acid, 0.05 to 0.2 mg / L of N-phenyl-N '-1, 2, 3-thiadiazole-5-urea, 400 to 600 mg / L of lactoalbumin hydrolysate, 0.2 to 0.4 g / L of activated carbon and 10 to 25 mg / L of methyl jasmonate. By integrating hormone, functional additives (lactoalbumin hydrolysate and activated carbon) and a nano material (silver / copper doped nano zinc oxide), a multi-level synergistic effect is formed, so that the yield of flavonoid compounds is improved on the basis of improving the callus induction rate of saussurea involucrata.

Description

Technical Field

[0001] This application relates to the technical field of plant tissue culture, and particularly relates to a culture medium composition for tissue culture of Saussurea involucrata and its application. Background Art

[0002] Saussurea involucrata is a rare medicinal plant growing in the extreme environment of high altitude (2500 - 4000 meters above sea level), and is known as the "king of herbs in the high mountains". Its roots, stems and leaves are rich in active ingredients such as flavonoids, terpenoids, and polysaccharides, and have significant pharmacological effects such as anti - inflammation, anti - oxidation, and anti - tumor. In particular, it is used in traditional medicine to treat rheumatism, altitude sickness, and immune system diseases. However, due to its long growth cycle (it takes 5 - 7 years to bloom), low natural reproduction rate, combined with over - exploitation and habitat degradation, the wild resources of Saussurea involucrata are on the verge of exhaustion and are listed as a national second - level protected plant. In this context, tissue culture technology has become the key breakthrough for its resource protection and sustainable utilization.

[0003] In the prior art, the dedifferentiation induction rate of conventional culture media (such as MS, B5) for Saussurea involucrata explants (such as leaves, stem segments) is less than 40%, and they are prone to browning and death. At the same time, the synthesis amount of medicinal ingredients such as flavonoids in in vitro culture is only 10% - 30% of that of wild plants, which is difficult to meet the medicinal demand. In addition, high - altitude plants are prone to oxidative stress (ROS accumulation) in in vitro culture, resulting in a decline in metabolic activity and even apoptosis. Therefore, how to improve the yield of flavonoid compounds on the basis of increasing the induction rate of Saussurea involucrata callus is a technical problem that urgently needs to be solved at present. Summary of the Invention

[0004] This application provides a culture medium composition for tissue culture of Saussurea involucrata and its application, so as to solve the following technical problem: how to improve the yield of flavonoid compounds on the basis of increasing the induction rate of Saussurea involucrata callus.

[0005] In the first aspect, this application provides a culture medium composition for tissue culture of Saussurea involucrata. On the basis of the MS culture medium, the culture medium composition further includes: fructose 9 - 11 g / L, glucose 9 - 11 g / L, sucrose 9 - 11 g / L, agar 5 - 7 g / L, 2,4 - dichlorophenoxyacetic acid 0.5 - 1.5 mg / L, naphthylacetic acid 0.05 - 0.1 mg / L, N - phenyl - N'-1,2,3 - thiadiazole - 5 - urea 0.05 - 0.2 mg / L, hydrolyzed milk protein 400 - 600 mg / L, activated carbon 0.2 - 0.4 g / L, methyl jasmonate 10 - 25 mg / L.

[0006] Optionally, the culture medium composition further includes: phenylalanine 40 - 60 mg / L, tyrosine 20 - 40 mg / L.

[0007] Optionally, the culture medium composition further comprises: 6 - 8 mg / L of salicylic acid and 80 - 120 mg / L of chitosan.

[0008] Optionally, the culture medium composition further comprises: 10 - 20 mg / L of amino-functionalized silver / copper-doped magnesium zinc composite nano-zinc oxide.

[0009] Optionally, the preparation method of the amino-functionalized silver / copper-doped magnesium zinc composite nano-zinc oxide comprises:

[0010] Mix zinc acetate, magnesium acetate, silver nitrate, copper nitrate and a solvent, and then adjust the pH to a set pH value to obtain a mixed solution;

[0011] Perform a hydrothermal reaction on the mixed solution, and then perform separation and purification to obtain magnesium zinc composite nano-zinc oxide;

[0012] Mix the magnesium zinc composite nano-zinc oxide with 3-aminopropyltrimethoxysilane and a solvent to perform amino-functionalization modification on the magnesium zinc composite nano-zinc oxide to obtain the amino-functionalized silver / copper-doped magnesium zinc composite nano-zinc oxide.

[0013] Optionally, the mass ratio of the zinc acetate to the magnesium acetate is (8 - 12):1.

[0014] Optionally, the mass concentration of the silver nitrate in the mixed solution is 0.5 - 2%;

[0015] The mass concentration of the copper nitrate in the mixed solution is 0.5 - 2%.

[0016] Optionally, the reaction temperature of the hydrothermal reaction is 150 - 180 °C, and the reaction time is 8 - 12 h.

[0017] In a second aspect, the present application provides an application of a culture medium composition for tissue culture of Saussurea involucrata, and the culture medium composition is used for callus induction or callus subculture of Saussurea involucrata.

[0018] Optionally, during the callus induction period, the culture conditions are dark culture for 7 days and then changed to red light, the light illumination time is 12 h / d, the light intensity is 2200 lx, and the temperature is 23 - 25 °C;

[0019] During the callus subculture period, the culture conditions are alternating irradiation with red light and blue light, the red light illumination time is 12 h / d, the blue light illumination time is 2 h / d, the light intensity is 2200 lx, and the temperature is 25 °C during the day and 18 °C at night.

[0020] The above technical solutions provided by the embodiments of the present application have the following advantages compared with the prior art:

[0021] The present application provides a culture medium composition for tissue culture of Saussurea involucrata Kar. et Kir., which forms a multi-level synergistic effect by integrating hormones (2,4-D, NAA, TDZ), functional additives (hydrolyzed milk protein, activated carbon) and nanomaterials (silver / copper-doped zinc oxide nanoparticles). At the same time, the combination of 2,4-D and TDZ optimizes the balance of dedifferentiation and bud regeneration, reduces the generation of abnormal buds, while hydrolyzed milk protein provides amino acid nutrition, significantly improving the survival rate of callus and the induction rate of regenerated buds. In addition, methyl jasmonate synergistically stimulates the synthesis of flavonoids (such as quercetin, syringin) with phenylalanine / tyrosine, and nanomaterials further strengthen the metabolic pathway by photocatalytically immobilizing key enzymes (CHS, PAL), resulting in significantly higher levels of secondary metabolites than traditional culture methods. Thus, on the basis of improving the induction rate of Saussurea involucrata Kar. et Kir. callus, the yield of flavonoids is increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings herein are incorporated into the specification and form a part of the specification, showing embodiments consistent with the present application, and are used together with the specification to explain the principles of the present application.

[0023] To more clearly illustrate the technical solutions in the embodiments of the present application or in the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

[0024] Figure 1 It is a schematic flow chart of the preparation method of amino-functionalized silver / copper-doped magnesium zinc composite zinc oxide nanoparticles provided by the embodiments of the present application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present application with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative efforts fall within the scope of protection of the present application.

[0026] Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments and equipment used in the present application can be obtained through the market or can be prepared by existing methods.

[0027] In a first aspect, the present application provides a culture medium composition for tissue culture of Saussurea involucrata, and the culture medium composition further includes, on the basis of MS medium: 9-11 g / L of fructose, 9-11 g / L of glucose, 9-11 g / L of sucrose, 5-7 g / L of agar, 0.5-1.5 mg / L of 2,4-dichlorophenoxyacetic acid, 0.05-0.1 mg / L of naphthaleneacetic acid, 0.05-0.2 mg / L of N-phenyl-N'-1,2,3-thiadiazol-5-urea, 400-600 mg / L of hydrolyzed milk protein, 0.2-0.4 g / L of activated carbon, and 10-25 mg / L of methyl jasmonate.

[0028] It should be noted that the MS medium is a high-salt concentration formulation that provides comprehensive mineral nutrition and organic components, and is suitable for cell dedifferentiation, callus induction, organ differentiation, and secondary metabolite synthesis of most plants.

[0029] In some embodiments, the culture medium composition further includes: 40-60 mg / L of phenylalanine and 20-40 mg / L of tyrosine.

[0030] In some embodiments, the culture medium composition further includes: 6-8 mg / L of salicylic acid and 80-120 mg / L of chitosan.

[0031] In some embodiments, the culture medium composition further includes: 10-20 mg / L of amino-functionalized silver / copper-doped magnesium zinc composite nanozinc oxide.

[0032] The culture medium composition provided by the present application realizes the efficient induction of Saussurea involucrata callus and the directional enrichment of secondary metabolites by integrating hormones, functional additives, and nano-catalysis technology, and has both resource protection and medicinal development value. Specifically, the functions of each component are as follows:

[0033] MS medium: It can provide comprehensive mineral nutrition (such as nitrogen, phosphorus, potassium) and vitamins, and support cell dedifferentiation, proliferation, and secondary metabolism.

[0034] Composite carbon source (9-11 g / L of fructose, glucose, and sucrose each): In the induction stage, fructose and glucose provide high osmotic pressure to support the rapid growth of callus; at the same time, in the subculture stage, it promotes root differentiation and the accumulation of secondary metabolites.

[0035] 2,4-dichlorophenoxyacetic acid (2,4-D): Induce the dedifferentiation of explants to form callus. High concentrations are suitable for initial induction and gradually decrease later to inhibit excessive proliferation.

[0036] Naphthaleneacetic acid (NAA): Low concentrations promote the compactness of callus, reduce browning, and synergistically support bud differentiation and root elongation with TDZ.

[0037] N-phenyl-N'-1,2,3-thiadiazole-5-urea (TDZ): Promotes bud regeneration by activating the cytokinin signaling pathway and reduces the formation of deformed buds.

[0038] Hydrolyzed milk protein: Provides amino acids and peptide nutrients, maintains cell viability, and enhances the induction rate of regenerated buds.

[0039] Activated carbon: Adsorbs phenolic oxidation products, reduces browning, and improves the survival rate of callus.

[0040] Methyl jasmonate: Can induce the synthesis of flavonoids (such as quercetin and syringin) and increase the content of secondary metabolites.

[0041] Phenylalanine (40 - 60 mg / L) and tyrosine (20 - 40 mg / L): As precursors for the synthesis of flavonoids and alkaloids, directly promote the production of secondary metabolites.

[0042] Salicylic acid (6 - 8 mg / L) and chitosan (80 - 120 mg / L): Salicylic acid enhances the stress resistance of callus (such as cold tolerance); chitosan enhances metabolic activity by inducing the expression of disease-resistant related genes.

[0043] Amino-functionalized silver / copper-doped magnesium zinc composite nano-zinc oxide: Silver / copper doping significantly enhances the photocatalytic activity, generates free radicals (·OH, ·O 2 - ) under light irradiation, catalyzes the conversion of flavonoid precursors (such as p-coumaric acid) into active flavonoid components (chalcones, naringenin) with an efficiency increase of 50% - 80%. Magnesium doping enhances the material stability, reduces photocorrosion, and prolongs the service life. At the same time, by regulating the redox state, activates the key enzymes for flavonoid synthesis (such as PAL, 4CL, CHS), and accelerates the metabolic flux. Amino-functionalization immobilizes enzyme molecules (such as CHS), making the flavonoid yield reach 3 times that of wild plants. In addition, it can scavenge reactive oxygen species (ROS), protect cells from oxidative damage, inhibit lipid peroxidation and DNA breakage. Magnesium doping enhances the cell membrane stability and reduces ion leakage; copper ions bind to flavonoids, reducing heavy metal toxicity.

[0044] Meanwhile, the present application rationally designs each component of the culture medium, and the components of the Saussurea involucrata culture medium composition have a synergistic effect, as follows:

[0045] Hormonal synergistic regulation of callus development: 2,4-D dominates dedifferentiation, NAA promotes callus compaction, and TDZ activates bud regeneration. The three synergistically achieve a dynamic balance of "dedifferentiation - proliferation - regeneration" under a concentration gradient.

[0046] Carbon source and hormone stage adaptation: During the induction stage, fructose and glucose (high osmotic pressure) accelerate callus growth; during the subculture stage, sucrose (low osmotic pressure) promotes root differentiation and flavonoid accumulation, and synergistically with methyl jasmonate, increases the content of secondary metabolites to 3 times that of wild plants.

[0047] Nanocatalysis and enhancement of secondary metabolism: Through amino-functionalized zinc oxide nanoparticles + methyl jasmonate + precursors (phenylalanine / tyrosine), the nanomaterials generate free radicals (·OH, ·O 2 - ) and catalyze the conversion of precursors (such as p-coumaric acid) into flavonoids; amino-functionalization immobilizes CHS enzyme, methyl jasmonate induces PAL enzyme activity, and phenylalanine / tyrosine provides substrates, and the three work together to improve the flavonoid synthesis efficiency.

[0048] Synergy of stress resistance and metabolic protection: Through salicylic acid + chitosan + zinc oxide nanoparticles, salicylic acid regulates cold resistance, chitosan induces the expression of disease-resistant genes, and the nanomaterials scavenge ROS and stabilize cell membranes, jointly reducing oxidative stress damage and enhancing callus survival rate.

[0049] Synergy of nutrition and function: Through hydrolyzed milk protein + activated carbon, hydrolyzed milk protein provides amino acids to support cell viability, activated carbon adsorbs phenolic toxins, and synergistically with hormones and nanomaterials, maintains metabolic homeostasis and enhances the induction rate of regenerated buds.

[0050] Therefore, by using the gradient combination of "2,4-D (high concentration for dedifferentiation induction) + NAA (low concentration for promoting compactification) + TDZ (precisely activating bud regeneration)", the limitations of traditional single hormones are broken through, and efficient regeneration of callus is achieved; free radicals are generated by photocatalysis to accelerate the conversion of flavonoid precursors, and at the same time, key enzymes (such as CHS, PAL) are immobilized to activate the phenylpropanoid metabolic pathway and improve metabolism; the combination of salicylic acid and chitosan is used to regulate the expression of disease-resistant genes, and the nanomaterials scavenge ROS and stabilize cell membranes to provide a low-stress environment for secondary metabolism.

[0051] Figure 1 Schematic flow chart of the preparation method of amino-functionalized silver / copper-doped magnesium zinc composite zinc oxide provided in the embodiment of the present application.

[0052] In some embodiments, as Figure 1 shown, the preparation method of the amino-functionalized silver / copper-doped magnesium zinc composite zinc oxide includes:

[0053] S1. Mix zinc acetate, magnesium acetate, silver nitrate, copper nitrate and a solvent, and then adjust the pH to a set pH value to obtain a mixed solution;

[0054] S2. Perform a hydrothermal reaction on the mixed solution, and then perform separation and purification to obtain magnesium zinc composite zinc oxide;

[0055] S3. Mix the magnesium-zinc composite nano-zinc oxide with 3-aminopropyltrimethoxysilane and a solvent to perform amino-functionalization modification on the magnesium-zinc composite nano-zinc oxide, thereby obtaining the amino-functionalized silver / copper-doped magnesium-zinc composite nano-zinc oxide.

[0056] In some embodiments, the mass ratio of zinc acetate to magnesium acetate is (8 - 12):1.

[0057] In some embodiments, the mass concentration of silver nitrate in the mixed solution is 0.5 - 2%;

[0058] The mass concentration of copper nitrate in the mixed solution is 0.5 - 2%.

[0059] In some embodiments, the reaction temperature of the hydrothermal reaction is 150 - 180 °C, and the reaction time is 8 - 12 h.

[0060] This application can successfully prepare amino-functionalized silver / copper-doped magnesium-zinc composite nano-zinc oxide through precursor mixing, hydrothermal reaction, and silanization modification.

[0061] In a second aspect, this application provides an application of a culture medium composition for tissue culture of Saussurea involucrata, and the culture medium composition is used for callus induction or callus subculture of Saussurea involucrata.

[0062] In some embodiments, during the callus induction period, the culture conditions are dark culture for 7 days followed by red light, with a light duration of 12 h / d, a light intensity of 2200 lx, and a temperature of 23 - 25 °C;

[0063] During the callus subculture period, the culture conditions are alternating irradiation with red and blue light, with a red light duration of 12 h / d and a blue light duration of 2 h / d, a light intensity of 2200 lx, a daytime temperature of 25 °C, and a nighttime temperature of 18 °C.

[0064] Callus induction period (dark culture for 7 days → red light 12 h / d, 23 - 25 °C): Dark culture can reduce light inhibition, and red light promotes cell division; constant temperature maintains metabolic homeostasis.

[0065] Subculture period (alternating red and blue light, 25 °C / 18 °C day / night): Red light promotes flavonoid synthesis, and blue light regulates cell differentiation; the temperature difference between day and night simulates the native environment and stimulates the accumulation of secondary metabolites.

[0066] Therefore, the culture medium composition for tissue culture of Saussurea involucrata and its application provided by this application have the following advantages:

[0067] (1) Multi-component synergistic effect for efficient induction and metabolic regulation: By integrating hormones (2,4-D, NAA, TDZ), functional additives (hydrolyzed casein, activated carbon), and nanomaterials (Ag / Cu-doped nano-zinc oxide), a multi-level synergistic effect was formed. Meanwhile, the combination of 2,4-D and TDZ optimized the balance between dedifferentiation and bud regeneration, reducing the generation of abnormal buds. At the same time, hydrolyzed casein provided amino acid nutrition, significantly improving the survival rate of callus and the induction rate of regenerated buds. In addition, methyl jasmonate and phenylalanine / tyrosine synergistically stimulated the synthesis of flavonoids (such as quercetin, syringin), and the nanomaterials fixed key enzymes (CHS, PAL) through photocatalysis, further strengthening the metabolic pathway and making the content of secondary metabolites significantly higher than that of traditional culture methods.

[0068] (2) Innovative technology integration to enhance stress resistance and stability: Salicylic acid enhances low-temperature tolerance, chitosan activates the expression of disease-resistant genes, and nano-zinc oxide scavenges reactive oxygen species (ROS). The three work together to reduce oxidative stress damage, especially suitable for simulated cultivation in high-altitude low-temperature environments. At the same time, activated carbon adsorbs phenolic toxins, hydrolyzed casein provides continuous nutrition, combined with day-night temperature difference (25°C during the day / 18°C at night) and alternating red and blue light irradiation, simulating the native habitat of Saussurea involucrata, promoting cell differentiation and metabolite accumulation.

[0069] (3) Dual values of resource protection and medicinal development: Through efficient tissue culture technology, the dependence on endangered wild Saussurea involucrata is reduced, alleviating ecological pressure and meeting the requirements of sustainable resource utilization. At the same time, the contents of key active substances such as flavonoids, polyphenols, and 1,5-dicaffeoylquinic acid are significantly increased, providing high-purity raw materials for medicinal development such as anti-inflammatory, anti-osteoporosis, and anti-radiation.

[0070] (4) Standardized production process and industrialization potential: Dark culture followed by red light induction for cell division, and alternating red and blue light optimization of metabolism during the subculture stage, combined with constant temperature control, achieve high controllability and repeatability of culture conditions. At the same time, amino-functionalized nano-zinc oxide prepared by hydrothermal reaction and silanization modification has both catalytic activity and biocompatibility, providing technical support for industrial production.

[0071] (5) Wide application extensibility: This culture medium is not only suitable for callus induction and subculture, but also can be extended to cluster bud differentiation, rooting, and plant regeneration by adjusting hormone concentrations and light conditions, laying a foundation for the rapid propagation of Saussurea involucrata and the industrialization of cell cultures.

[0072] In summary, through the integration of multidisciplinary technologies, this application solves the problems of low content of secondary metabolites and high browning rate of callus in traditional culture, while taking into account ecological protection and high-value medicinal development, and has significant scientific research and commercial value.

[0073] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions noted in the following embodiments, they are generally determined according to industry standards. If there is no corresponding industry standard, they are carried out according to general international standards, conventional conditions, or the conditions recommended by the manufacturer.

[0074] Example 1

[0075] This example provides a culture medium composition for tissue culture of Saussurea involucrata, and the culture medium composition further includes, on the basis of MS medium: fructose 10 g / L, glucose 10 g / L, sucrose 10 g / L, agar 6 g / L, 2,4-D 1.0 mg / L, NAA 0.075 mg / L, TDZ 0.125 mg / L, hydrolyzed milk protein 500 mg / L, activated carbon 0.3 g / L, methyl jasmonate 17.5 mg / L.

[0076] The seeds of Saussurea involucrata are disinfected with 75% alcohol for 30 s, then disinfected with 0.1% mercuric chloride solution for 10 min, rinsed 3 times with sterile water, and after drying the moisture with sterilized filter paper, they are inoculated onto the culture medium. The culture medium composition: MS + 3.0% sucrose + 0.6% agar powder. After 50 days of seed germination, seedlings with leaves are grown, and the leaves are cut into explants 0.5 cm long and placed flat on the callus induction medium. The callus is subcultured on the subculture medium (the same as the above callus induction medium) for 15 days. Both callus induction and callus subculture are carried out using the above culture medium composition. When in the callus induction period, the culture conditions are dark culture for 7 days and then changed to red light, the light time is 12 h / d, the light intensity is 2200 lx, and the temperature is 23 - 25°C; when in the callus subculture period, the culture conditions are alternating irradiation with red light and blue light, the red light illumination time is 12 h / d, the blue light illumination time is 2 h / d, the light intensity is 2200 lx, the temperature is 25°C during the day and 18°C at night.

[0077] Example 2

[0078] On the basis of what is disclosed in Example 1, the following modifications are made in this example:

[0079] The culture medium composition further includes: phenylalanine 50 mg / L, tyrosine 30 mg / L.

[0080] Example 3

[0081] On the basis of what is disclosed in Example 2, the following modifications are made in this example:

[0082] The culture medium composition further includes: salicylic acid 7 mg / L, chitosan 100 mg / L.

[0083] Example 4

[0084] On the basis of what is disclosed in Example 3, the following modifications are made in this example:

[0085] The culture medium composition further includes: 15 mg / L of amino-functionalized silver / copper-doped magnesium zinc composite nano-zinc oxide.

[0086] Among them, the preparation method of the amino-functionalized silver / copper-doped magnesium zinc composite nano-zinc oxide includes the following steps:

[0087] S11. Dissolve zinc acetate, magnesium acetate, silver nitrate, and copper nitrate in a mixed solvent of water and ethanol in proportion, and then adjust the pH to 9 with sodium hydroxide solution to obtain a mixed solution; the mass ratio of zinc acetate to magnesium acetate is 10:1, the mass concentration of silver nitrate in the mixed solution is 1%; the mass concentration of copper nitrate in the mixed solution is 1%;

[0088] S21. Transfer the mixed solution to a high-pressure reaction kettle, carry out a hydrothermal reaction at 160 °C for 10 h. After the reaction is completed, centrifuge to separate the precipitate, and wash it repeatedly with deionized water and ethanol to obtain magnesium zinc composite nano-zinc oxide;

[0089] S31. Mix the magnesium zinc composite nano-zinc oxide with 3-aminopropyltrimethoxysilane in ethanol, stir at room temperature for 5 hours, realize surface amino-functionalization through a silane coupling reaction, then centrifuge to remove the unreacted silane reagent, wash and dry again to obtain amino-functionalized silver / copper-doped magnesium zinc composite nano-zinc oxide.

[0090] Comparative Example 1

[0091] On the basis of what is disclosed in Example 4, the following modifications are made in this comparative example:

[0092] Do not add TDZ to the culture medium composition.

[0093] Comparative Example 2

[0094] On the basis of what is disclosed in Example 4, the following modifications are made in this comparative example:

[0095] Do not add hydrolyzed milk protein to the culture medium composition.

[0096] Comparative Example 3

[0097] On the basis of what is disclosed in Example 4, the following modifications are made in this comparative example:

[0098] Do not add activated carbon to the culture medium composition.

[0099] Comparative Example 4

[0100] On the basis of what is disclosed in Example 4, the following modifications are made in this comparative example:

[0101] Methyl jasmonate is not added to the culture medium composition.

[0102] The calli of Saussurea involucrata Kar. et Kir. obtained in Examples 1 to 4 and Comparative Examples 1 to 4 were subjected to performance measurement, and the results are shown in Table 1. The measurement methods are as follows:

[0103] Dry weight of callus: The callus cultured for 30 days was taken and weighed after drying to a constant weight at 105 °C.

[0104] Browning rate: The percentage of brown or black callus blocks in the total number was counted.

[0105] Bud regeneration rate: The proportion of callus with differentiated green bud points was counted.

[0106] Total flavonoid content: Determined by ultraviolet spectrophotometry (using rutin as the standard product and measuring at a wavelength of 510 nm).

[0107] Anti-inflammatory activity: Mouse ear swelling experiment, and the ear swelling inhibition rate was measured after intragastric administration of the Saussurea involucrata Kar. et Kir. extract.

[0108] Table 1 Performance of the calli of Saussurea involucrata Kar. et Kir. obtained in Examples 1 to 4 and Comparative Examples 1 to 4

[0109]

[0110] In Table 1, the absence of TDZ in Comparative Example 1 led to the blockage of the cytokinin signaling pathway, and the bud differentiation ability was severely limited. In Comparative Example 2, the lack of amino acids and peptide nutrients significantly decreased cell viability. In Comparative Example 3, the absence of activated carbon led to insufficient adsorption of phenolic toxins and aggravated oxidative stress. In Comparative Example 4, methyl jasmonate is a core inducer for the synthesis of secondary metabolites, and its absence closed the flavonoid synthesis pathway.

[0111] The various embodiments of the present application may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be construed as a rigid limitation on the scope of the present application; therefore, it should be considered that the description of the range has specifically disclosed all possible sub-ranges and the individual values within the range. For example, it should be considered that the description of the range from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within the range, such as 1, 2, 3, 4, 5, and 6, which applies regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any cited number (fraction or integer) within the indicated range.

[0112] In addition, in the description of the specification of the present application, terms such as "include" and "comprise" mean "including but not limited to". In this text, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations.

[0113] The above are only specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features claimed herein.

Claims

1. A culture medium composition for tissue culture of Saussurea involucrata, characterized in that: The culture medium composition further comprises, on the basis of MS culture medium: 9-11 g / L fructose, 9-11 g / L glucose, 9-11 g / L sucrose, 5-7 g / L agar, 0.5-1.5 mg / L 2,4-dichlorophenoxyacetic acid, 0.05-0.1 mg / L naphthylacetic acid, 0.05-0.2 mg / L N-phenyl-N'-1,2,3-thiadiazole-5-urea, 400-600 mg / L hydrolyzed milk protein, 0.2-0.4 g / L activated carbon, and 10-25 mg / L methyl jasmonate.

2. The culture medium composition according to claim 1, characterized in that The culture medium composition also includes: 40-60 mg / L phenylalanine and 20-40 mg / L tyrosine.

3. The culture medium composition according to claim 2, characterized in that The culture medium composition also includes: 6-8 mg / L salicylic acid and 80-120 mg / L chitosan.

4. The culture medium composition according to claim 3, characterized in that The culture medium composition also includes: 10-20 mg / L of amino-functionalized silver / copper-doped magnesium-zinc composite nano zinc oxide.

5. The culture medium composition according to claim 4, characterized in that The preparation method of the amino-functionalized silver / copper-doped magnesium-zinc composite nano zinc oxide comprises: Mixing zinc acetate, magnesium acetate, silver nitrate, copper nitrate and a solvent, and then adjusting the pH to a set pH value to obtain a mixed solution; The mixed solution is subjected to a hydrothermal reaction, and then separated and purified to obtain magnesium-zinc composite nano zinc oxide; The magnesium-zinc composite nano-zinc oxide is mixed with 3-aminopropyltrimethoxysilane and a solvent to carry out amino-functional modification on the magnesium-zinc composite nano-zinc oxide to obtain the amino-functionalized silver / copper-doped magnesium-zinc composite nano-zinc oxide.

6. The culture medium composition according to claim 5, characterized in that The mass ratio of the zinc acetate to the magnesium acetate is (8-12):

1.

7. The culture medium composition according to claim 5, characterized in that The mass concentration of the silver nitrate in the mixed solution is 0.5-2%; The mass concentration of the copper nitrate in the mixed solution is 0.5-2%.

8. The culture medium composition according to claim 5, characterized in that The reaction temperature of the hydrothermal reaction is 150-180° C., and the reaction time is 8-12 hours.

9. An application of a culture medium composition for tissue culture of Saussurea involucrata, characterized in that: The culture medium composition is used for callus induction or callus subculture of Saussurea involucrata.

10. The use according to claim 9, characterized in that: During the callus induction period, the culture conditions were dark culture for 7 days and then switched to red light, with a light duration of 12 h / d, a light intensity of 2200 lx, and a temperature of 23-25°C; During the callus subculture period, the culture conditions were alternating red light and blue light, with the red light illumination time being 12h / d, the blue light illumination time being 2h / d, the light intensity being 2200lx, and the temperature being 25℃ during the day and 18℃ at night.

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