Method for culturing ginseng adventitious roots and application

Abstract

The invention discloses a method for culturing ginseng adventitious roots and application. Relates to the technical field of tissue culture. Comprising the following steps: preparing a tissue culture medium; cutting the disinfected ginseng sections, inoculating the ginseng sections into a tissue culture medium, and culturing until ginseng adventitious roots are obtained; and subculturing the ginseng adventitious roots: inoculating the ginseng adventitious roots to a subculture medium for culture. The method optimizes induction and subculture processes and induction and subculture medium components, improves the content of secondary metabolites such as flavone, and has commercial and medical prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of tissue culture, more particularly to a method for culturing ginseng adventitious roots and application. BACKGROUND

[0002] Ginseng adventitious roots are non-typical root systems growing at the base of ginseng stems or creeping stems, which have the functions of absorbing nutrients and fixing plants, and are also one of the synthesis sites of active ingredients such as ginsenosides. Such root systems exist universally in wild or artificial cultivation, and their morphology and function are influenced by factors such as environment and growth stage, and are regulated by plant hormones and the like. Ginseng adventitious roots are rich in active substances such as ginsenosides and polysaccharides, and have high medicinal or market value.

[0003] In the prior art, the research on ginseng adventitious roots includes optimization of culture medium composition or culture method, in addition, some research focuses on the application of ginseng adventitious root shape, fresh weight or extract, and based on the previous research, there is high research value in further improving secondary metabolites through subculture.

[0004] Therefore, whether a method for culturing ginseng adventitious roots and application can be provided is a problem to be solved by those skilled in the art. SUMMARY

[0005] Therefore, the present application provides a method for culturing ginseng adventitious roots and application. Based on the previous research, the culture method and culture medium are optimized to improve the content of secondary metabolites in ginseng adventitious roots, which has high application value.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: A method for culturing ginseng adventitious roots, comprising the following steps: (1) preparing a tissue culture medium; (2) cutting the sterilized ginseng segments and inoculating them into the tissue culture medium to obtain ginseng adventitious roots; (3) subculturing the ginseng adventitious roots: inoculating the ginseng adventitious roots into a subculture medium for culture; the culture conditions are as follows: temperature 23-25℃, shaking speed 80rpm, ventilation volume 70mL / min, dark culture for 8-12d; temperature 20-23℃, shaking speed 90rpm, ventilation volume 100mL / min, dark culture for 8-12d; temperature 23-25℃, shaking speed 80rpm, ventilation volume 90mL / min, dark culture for 8-12d.

[0007] The beneficial effect is that in subculture, the present application appropriately changes the temperature, speed and ventilation volume to affect the related enzyme activity, metabolism and other processes, and guide the cells to generate more secondary metabolites, which is rarely seen in existing research.

[0008] Preferably, the raw material of the step (1) tissue culture medium is: MS-based medium, 20-30 g / L sucrose, 5-10 g / L glucose, 2-3 mg / L IBA, 0.2-0.3 mg / L NAA, 0.05-0.1 mg / L KT, and 1-2 mg / L AgNO3.

[0009] Preferably, the step (2) culture is: dark culture in a shaking table.

[0010] Preferably, the dark culture in a shaking table is: dark culture in a shaking table at 22-25°C for 30-35 days to grow the ginseng adventitious roots, and the inoculation amount is 5-10 g of the adventitious roots per 100 mL of the ginseng adventitious root culture medium.

[0011] Preferably, the step (3) subculture medium is: 1 / 2MS-based medium, 20-30 g / L sucrose, 10-15 g / L glucose, 1-2 mg / L NAA, 1-1.5 mg / L IBA, and 0.1-0.2 mg / L GA3.

[0012] The application also provides the ginseng adventitious roots obtained by any of the methods.

[0013] The application also provides the application of any of the methods or the above-mentioned ginseng adventitious roots in production, processing, and pharmacy.

[0014] Preferably, the content of the secondary metabolites in the ginseng adventitious roots is increased.

[0015] The beneficial effect is that, in the ginseng adventitious roots, not only the content of total ginsenosides needs to be increased in the early induction, but also the content of other secondary metabolites, such as flavonoids, phenols, and polysaccharides, needs to be increased through further subculture, so as to meet the application requirements in the later stage, such as medicinal application.

[0016] According to the above technical solution, compared with the prior art, the application discloses a method and application for culturing ginseng adventitious roots, and the technical effects are: the induction and subculture process and the composition of the induction and subculture medium are optimized, the content of secondary metabolites, such as flavonoids, is increased, and the method has commercial and medical prospects. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0018] The application discloses a method for culturing ginseng adventitious roots and application The materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified. The experimental steps not mentioned are conventional experimental steps, which will not be described here.

[0019] For example, the conventional detection method is as follows: Preparation of a standard curve: during the determination of the total phenol content, gallic acid is used as a standard, different concentrations of the standard solution are respectively taken into test tubes and diluted to 2 mL, Folin phenol reagent is added into each test tube, Na2CO3 solution is added later, after reaction under the condition of avoiding light, the absorbance is determined by using an ultraviolet spectrophotometer, and a standard curve is drawn.

[0020] Determination of the total phenol content: the harvested adventitious roots are precisely weighed in a conical flask, 80% methanol is added, water bath is carried out at 80 DEG C, after cooling, the filtrate is filtered, diluted to 25 mL, 1 mL of the filtrate is taken into a test tube, 1 mL of 80% methanol is added, and then the operation is carried out according to the standard curve.

[0021] Total phenol content (mg·g 1 ) = CVD / m In the formula, C is the total phenol concentration of the sample solution to be measured (mg·mL 1 ), V is the volume of the sample solution to be measured (mL), D is the dilution multiple, and m is the sample mass (g).

[0022] Determination of the total flavone content Preparation of a standard curve: during the determination of the total flavone content, rutin is used as a standard, different concentrations of the standard solution are respectively taken into test tubes and diluted to 2 mL, 0.3 mL of 5% NaNO2 is added into each test tube, after shaking and reaction for 6 min, 0.3 mL of 10% Al (NO3) 3 is added, after shaking and standing for 6 min, NaOH is added, the OD value is determined by using an ultraviolet spectrophotometer at a wavelength of 510 nm, and a standard curve is drawn. Determination of the total flavone content: the harvested adventitious roots are precisely weighed, respectively placed in 50 mL conical flasks, and 70% ethanol solution is added into the conical flasks, water bath heating is carried out in a water bath kettle at 60 DEG C, after cooling, filtration is carried out, and then diluted to 25 mL, 1 mL is taken into a test tube, and 75% ethanol solution is added. The operation is carried out according to the standard curve. The total flavone content calculation formula is as follows:

[0023] Total flavone content (mg·g 1 ) = CVD / m ​Wherein, C is the total flavonoids concentration in the sample solution to be tested (mg·mL 1 ), V is the volume of the sample solution to be tested (mL), D is the dilution factor, and m is the rhizome mass (g).

[0024] Determination of polysaccharide content: The rhizome was crushed into powder, and then washed by 90% ethanol at 25°C. The precipitate was collected, and 80% ethanol was added to remove the inclusions. Distilled water was added and ultrasonic extraction was performed. After filtration, the filtrate was collected. An equal volume of distilled water was added to the residue and ultrasonic extraction was repeated three times. The above filtrates were combined and diluted to 100 mL to obtain the sample solution to be tested. The absorbance of glucose was determined by the phenol-concentrated sulfuric acid method, and the polysaccharide content was calculated.

[0025] Polysaccharide content / (mg·g 1 )=(CDf)·W 1 In the formula, C is the glucose concentration in the sample solution / (g·L 1 ); D is the dilution factor of the sample solution; f is the conversion factor; and W is the mass of the sample / g.

[0026] Example 1 A method for culturing ginseng adventitious roots, comprising the following steps: (1) preparing a tissue culture medium; (2) cutting the sterilized ginseng segments and inoculating them into the tissue culture medium to obtain ginseng adventitious roots; (3) subculturing the ginseng adventitious roots: inoculating the ginseng adventitious roots into a subculture medium for culture; the culture conditions are as follows: temperature 23°C, shaking speed 80 rpm, air flow 70 mL / min, dark culture for 8 days; temperature 20°C, shaking speed 90 rpm, air flow 100 mL / min, dark culture for 12 days; temperature 23°C, shaking speed 80 rpm, air flow 90 mL / min, dark culture for 8 days.

[0027] To further optimize the technical solution: the raw materials of the tissue culture medium in step (1) are as follows: MS as the basic medium, 21 g / L sucrose, 5 g / L glucose, 2 mg / L IBA, 0.25 mg / L NAA, 0.05 mg / L KT, and 1 mg / L AgNO3.

[0028] To further optimize the technical solution: in step (2), the shaking speed is dark culture.

[0029] To further optimize the technical solution: the shaker dark culture: 22℃ under shaker dark culture 30d grow out of ginseng adventitious roots, inoculation amount is 5g of adventitious roots per 100mL of ginseng adventitious roots medium.

[0030] To further optimize the technical solution: step (3) subculture medium: 1 / 2MS is the basic medium, 20g / L sucrose, 10g / L glucose, 1mg / L NAA, 1mg / L IBA, 0.1mg / L GA3.

[0031] Example 2 A method for culturing ginseng adventitious roots, comprising the following steps: (1) preparing tissue culture medium; (2) cutting the sterilized ginseng segments and inoculating into the tissue culture medium, and culturing to obtain ginseng adventitious roots; (3) subculturing the ginseng adventitious roots: inoculating the ginseng adventitious roots into the subculture medium for culture; the culture conditions are: 24℃, shaker speed 80rpm, ventilation 70mL / min, dark culture for 10d; 22℃, shaker speed 90rpm, ventilation 100mL / min, dark culture for 10d; 24℃, shaker speed 80rpm, ventilation 90mL / min, dark culture for 11d.

[0032] To further optimize the technical solution: the raw materials of step (1) tissue culture medium are: MS as the basic medium, 27g / L sucrose, 7g / L glucose, 2.3mg / L IBA, 0.2mg / L NAA, 0.08mg / L KT, 1.3mg / L AgNO3.

[0033] To further optimize the technical solution: step (2) culture: shaker dark culture.

[0034] To further optimize the technical solution: shaker dark culture: 24℃ under shaker dark culture 32d grow out of ginseng adventitious roots, inoculation amount is 9g of adventitious roots per 100mL of ginseng adventitious roots medium.

[0035] To further optimize the technical solution: step (3) subculture medium: 1 / 2MS is the basic medium, 25g / L sucrose, 12g / L glucose, 1.5mg / L NAA, 1.3mg / L IBA, 0.16mg / L GA3.

[0036] Example 3 A method for culturing ginseng adventitious roots, comprising the following steps: (1) preparing tissue culture medium; (2) cutting the sterilized ginseng segments and inoculating into the tissue culture medium, and culturing to obtain ginseng adventitious roots; (3) subculture of the ginseng adventitious roots: the ginseng adventitious roots are inoculated into the subculture medium for culture; the culture conditions are as follows: temperature 25℃, shaking speed 80rpm, ventilation volume 70mL / min, dark culture for 12d; temperature 20-23℃, shaking speed 90rpm, ventilation volume 100mL / min, dark culture for 8d; temperature 23-25℃, shaking speed 80rpm, ventilation volume 90mL / min, dark culture for 12d.

[0037] For further optimization of the technical scheme: the raw materials of the tissue culture medium in step (1) are as follows: MS as the basic medium, 28g / L sucrose, 9g / L glucose, 2.5mg / L IBA, 0.3mg / L NAA, 0.1mg / L KT, and 2mg / L AgNO3.

[0038] For further optimization of the technical scheme: the shaking speed for dark culture in step (2) is 80rpm.

[0039] For further optimization of the technical scheme: the shaking speed for dark culture is 80rpm at 25℃ for 35d to grow the ginseng adventitious roots, and the inoculation amount is 10g of the adventitious roots per 100mL of the ginseng adventitious root culture medium.

[0040] For further optimization of the technical scheme: the subculture medium in step (3) is 1 / 2MS as the basic medium, 30g / L sucrose, 15g / L glucose, 2mg / L NAA, 1.5mg / L IBA, and 0.2mg / L GA3.

[0041] Verification of technical effects: Experimental example: the 3-year-old fresh ginseng roots are prepared into the ginseng adventitious roots by the method of Example 2.

[0042] Comparative example 1: the 3-year-old fresh ginseng roots are cut into 2cm sections, washed with sterile water for 3 times after conventional disinfection, and placed in the ginseng adventitious root culture medium for dark culture at 24℃ for 32d to obtain the ginseng adventitious roots, and the inoculation amount is 10g per 100mL of the ginseng adventitious root culture medium, and the ginseng adventitious root culture medium is MS+34g / L sucrose+1.5mg / L IBA+0.15mg / L NAA+0.1mg / L KT.

[0043] Subculture: the ginseng adventitious roots are inoculated into the subculture medium for culture; the culture conditions are as follows: temperature 24℃, shaking speed 90rpm, ventilation volume 80mL / min, and dark culture for 31d.

[0044] Subculture medium: 1 / 2MS as the basic medium, 45g / L sucrose, 2mg / L NAA, and 1.5mg / L IBA.

[0045] The flavone and phenol contents in the ginseng adventitious roots of the experimental example and Comparative Example 1 were determined by using the existing conventional detection method.

[0046] The experimental results show (three parallels, the results are the average values) that the flavone content of the experimental example is about 47 mg / g, and that of Comparative Example 1 is about 36 mg / g. The phenol content of the experimental example is about 51 mg / g, and that of Comparative Example 1 is about 42 mg / g.

[0047] The polysaccharide content of the experimental example is about 217 mg / g, and that of Comparative Example 1 is about 174 mg / g.

[0048] The experimental example improves the contents of the secondary metabolites represented by flavone and phenol.

[0049] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be mutually referred to.

[0050] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Accordingly, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for culturing ginseng adventitious roots, characterized by, The method comprises the following steps: (1) preparing a tissue culture medium; (2) cutting the sterilized ginseng stem and inoculating into the tissue culture medium to culture to obtain ginseng adventitious roots; (3) subculturing the ginseng adventitious roots: inoculating the ginseng adventitious roots into a subculture medium to culture; the culture conditions are as follows: temperature 23-25℃, shaking speed 80rpm, ventilation volume 70mL / min, dark culture for 8-12d; temperature 20-23℃, shaking speed 90rpm, ventilation volume 100mL / min, dark culture for 8-12d; temperature 23-25℃, shaking speed 80rpm, ventilation volume 90mL / min, dark culture for 8-12d.

2. The method of claim 1, wherein, The raw materials of the tissue culture medium in step (1) are as follows: MS as a basic medium, 20-30g / L sucrose, 5-10g / L glucose, 2-3mg / L IBA, 0.2-0.3mg / L NAA, 0.05-0.1mg / L KT and 1-2mg / L AgNO3.

3. The method of claim 2, wherein, The culture in step (2) is shaking culture in the dark.

4. The method of claim 3, wherein, The shaking culture in the dark is as follows: shaking culture in the dark at 22-25℃ for 30-35d to grow ginseng adventitious roots, and the inoculation amount is 5-10g of the adventitious roots per 100mL of the ginseng adventitious root culture medium.

5. The method of claim 4, wherein, The subculture medium in step (3) is 1 / 2MS as a basic medium, 20-30g / L sucrose, 10-15g / L glucose, 1-2mg / L NAA, 1-1.5mg / L IBA and 0.1-0.2mg / L GA3.

6. The ginseng adventitious roots obtained by the method in any one of claims 1-5.

7. The use of the method in any one of claims 1-5 or the ginseng adventitious roots in claim 6 in production, processing or medicine.

8. Use according to claim 7, wherein Increasing the content of secondary metabolites in ginseng adventitious roots.