Culture medium for ginseng callus culture and callus culture method

Abstract

The invention provides a culture medium and a culture method for ginseng callus culture, and belongs to the technical field of plant tissue culture. The invention provides a culture medium for ginseng callus culture, B5 is used as a basic culture medium, and the culture medium also comprises DuXiexian, TDZ or KT, 2, 4-D, carbon nanotubes, sucrose and agar. The pH (Potential of Hydrogen) value of the callus induction culture medium is 5.75 to 5.85. The result of the embodiment shows that the culture medium provided by the invention can obviously improve the induction rate of the ginseng callus. Furthermore, a large number of calluses can be obtained in a short time through multiplication culture of the calluses obtained through culture of the callus induction culture medium, the obtained calluses can be directly used for synthesizing ginsenoside or used as a raw material for synthesizing ginsenoside, and then the yield of ginsenoside can be increased.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to a culture medium for culturing ginseng callus and a culture method for the callus. Background Art

[0002] At present, the method for biosynthesis of ginsenosides is to wash, disinfect and cut the fresh roots of perennial ginseng and then induce callus in a solid culture medium containing a certain concentration of growth regulators. Afterwards, the induced callus is induced and differentiated into adventitious roots, and the adventitious roots are used to synthesize ginsenosides. With the development of biotechnology, by transferring the plasmid of Agrobacterium rhizogenes into ginseng hairy roots, this type of root can grow rapidly in a culture medium without plant hormones and increase the yield of ginsenosides in the hairy roots. This method can produce a large number of hairy roots, but the disadvantage is that the content of saponins in the hairy roots is low (Rc content is about 1.0%), the cost of extraction is high, and the yield cannot be greatly increased.

[0003] Ginseng callus can carry out cell growth and metabolic activities under certain conditions, synthesize and accumulate ginsenosides. However, ginseng callus often has the problem of low induction rate, which limits the application of callus in the preparation and extraction of ginsenosides. Summary of the invention

[0004] In view of the defects in the prior art, the object of the present invention is to provide a culture medium for ginseng callus culture, which can greatly improve the induction rate of callus and obtain a large amount of callus in a short period of time.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] The invention provides a culture medium for culturing ginseng callus tissue, the culture medium comprising an induction culture medium, the composition of the induction culture medium comprising: taking B5 as a basic culture medium, and further comprising: 0.5-1.0 mg / L of tolbutamide, 2.0-3.0 mg / L of 2,4-D, 0.5-1.5 mg / L of carbon nanotubes, 20-30 g / L of sucrose and 6.5-7.0 g / L of agar;

[0007] Alternatively, TDZ 0.5-1.0 mg / L, 2,4-D 2.0-3.0 mg / L, carbon nanotubes 0.5-1.5 mg / L, sucrose 20-30 g / L, and agar 6.5-7.0 g / L;

[0008] Alternatively, KT 0.5-1.0 mg / L, 2,4-D 2.0-3.0 mg / L, carbon nanotubes 0.5-1.5 mg / L, sucrose 20-30 g / L, and agar 6.5-7.0 g / L;

[0009] The pH value of the induction medium is 5.75-5.85.

[0010] Preferably, the culture medium comprises a proliferation culture medium, and the composition of the proliferation culture medium comprises: B5 or MS as a basic culture medium, and further comprises: 0.3-1.0 mg / L of toxin ammonium, 2.5-3.5 mg / L of 2,4-D, 0.5-1.0 mg / L of acid hydrolyzed casein, 0.5-1.5 mg / L of carbon nanotubes, 20-30 g / L of sucrose and 6.5-7.0 g / L of agar;

[0011] Alternatively, TDZ 0.3-1.0 mg / L, 2,4-D 2.5-3.5 mg / L, acid hydrolyzed casein 0.5-1.0 mg / L, carbon nanotubes 0.5-1.5 mg / L, sucrose 20-30 g / L and agar 6.5-7.0 g / L;

[0012] Alternatively, KT 0.3-1.0 mg / L, 2,4-D 2.5-3.5 mg / L, acid hydrolyzed casein 0.5-1.0 mg / L, carbon nanotubes 0.5-1.5 mg / L, sucrose 20-30 g / L and agar 6.5-7.0 g / L;

[0013] The pH value of the proliferation culture medium is 5.75-5.85.

[0014] Preferably, the culture medium includes an anti-browning proliferation medium, and the composition of the anti-browning proliferation medium includes: on the basis of the proliferation medium components of the culture medium described in the above technical solution, 1.0-2.0 g / L of glutathione, 1.0-2.0 g / L of vitamin C and 0-1.5 g / L of activated carbon are added.

[0015] The present invention provides application of the culture medium described in the above technical solution in rapid propagation of ginseng.

[0016] The present invention provides a method for culturing ginseng callus, comprising the following steps:

[0017] The sterilized ginseng inflorescence axis is induced and cultured on the culture medium described in the above technical solution.

[0018] Preferably, the induction culture temperature is 24-26°C; the induction culture time is 30-35 days.

[0019] Preferably, the culture method further comprises proliferation culture; the proliferation culture comprises:

[0020] The ginseng callus is cultured on the proliferation medium in the culture medium described in the above technical solution or the anti-browning proliferation medium in the culture medium described in the above technical solution.

[0021] Preferably, the temperature of the proliferation culture is 24-26°C; the time of the proliferation culture is 50-60 days; the proliferation culture includes light culture; the light intensity of the light culture is 2000-3000 lx; ​​the illumination time of the light culture is 12-16 h / d.

[0022] The present invention provides application of the culture medium described in the above technical solution or the culture method described in the above technical solution in preparing ginsenosides.

[0023] Preferably, ginsenosides are extracted and separated from callus tissue cultured using the culture medium or the culture method.

[0024] Beneficial Effects of the Invention

[0025] The invention provides a culture medium for culturing ginseng callus tissue, the culture medium comprising an induction culture medium; the composition of the induction culture medium comprises: taking B5 as a basic culture medium, and further comprising: 0.5-1.0 mg / L of tolbutamide, 2.0-3.0 mg / L of 2,4-D, 0.5-1.5 mg / L of carbon nanotubes, 20-30 g / L of sucrose and 6.5-7.0 g / L of agar; or, 0.5-1.0 mg / L of TDZ, 2.0-3.0 mg / L of 2,4-D, 0.5-1.5 mg / L of carbon nanotubes, 20-30 g / L of sucrose and 6.5-7.0 g / L of agar; or, 0.5-1.0 mg / L, 2,4-D 2.0-3.0 mg / L, carbon nanotubes 0.5-1.5 mg / L, sucrose 20-30 g / L and agar 6.5-7.0 g / L; the pH value of the induction medium is 5.75-5.85. In the callus induction medium provided by the present invention, the toxin, TDZ or KT has the function of promoting cell division, and synergizing with the auxin 2,4-D to promote cell growth. The 2,4-D can dedifferentiate differentiated tissues and cells, and promote cell growth, which is beneficial to the induction of callus; the carbon nanotubes can promote the absorption of water and nutrients by cells, and thus promote the growth of callus. The results of the examples show that the induction medium provided by the present invention can significantly improve the induction rate of ginseng callus.

[0026] Furthermore, the callus tissue obtained by culturing with the induction medium can be proliferated to obtain a large amount of callus tissue in a short period of time, and the obtained callus tissue can be directly used to synthesize ginsenosides or as a raw material for synthesizing ginsenosides, thereby facilitating the increase of the yield of ginsenosides. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0028] Figure 1 This is a diagram of callus induction of young ginseng inflorescence axis;

[0029] Figure 2 This is a diagram of the proliferation of callus tissue of young ginseng inflorescence axis;

[0030] Figure 3 This is a graph showing the proliferation rate of callus tissue of young ginseng inflorescence axis in Example 4. DETAILED DESCRIPTION

[0031] The invention provides a culture medium for culturing ginseng callus tissue, which comprises an induction culture medium.

[0032] As an optional embodiment of the present invention, the induction culture medium uses B5 as the basic culture medium, and is further supplemented with: 0.5-1.0 mg / L of toluidine, 2.0-3.0 mg / L of 2,4-D, 0.5-1.5 mg / L of carbon nanotubes, 20-30 g / L of sucrose and 6.5-7.0 g / L of agar; the pH value of the induction culture medium is 5.75-5.85.

[0033] As another optional embodiment of the present invention, the induction medium uses B5 as the basic medium, and is further supplemented with TDZ 0.5-1.0 mg / L, 2,4-D 2.0-3.0 mg / L, carbon nanotubes 0.5-1.5 mg / L, sucrose 20-30 g / L and agar 6.5-7.0 g / L; the pH value of the induction medium is 5.75-5.85.

[0034] As another optional embodiment of the present invention, the induction medium uses B5 as the basic medium, and is further supplemented with KT 0.5-1.0 mg / L, 2,4-D 2.0-3.0 mg / L, carbon nanotubes 0.5-1.5 mg / L, sucrose 20-30 g / L and agar 6.5-7.0 g / L; the pH value of the induction medium is 5.75-5.85.

[0035] In the present invention, the induction culture medium uses B5 as the basic culture medium. The B5 culture medium can meet the requirements of macroelements and trace elements required in the ginseng callus induction process, and is beneficial to promoting the callus induction process.

[0036] In the present invention, the induction medium includes 0.5-1.0 mg / L of toluidine. As an optional embodiment of the present invention, the mass concentration of toluidine can be 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L or 1.0 mg / L. In the present invention, the induction medium includes 0.5-1.0 mg / L of TDZ. As an optional embodiment of the present invention, the mass concentration of TDZ can be 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L or 1.0 mg / L. In the present invention, the induction medium includes 0.5-1.0 mg / L of KT. As an optional embodiment of the present invention, the mass concentration of KT can be 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L or 1.0 mg / L. In the present invention, the induction medium may include any one of toxaphene, TDZ and KT.

[0037] In the present invention, the induction medium includes 2,4-D 2.0-3.0 mg / L. As an optional embodiment of the present invention, the mass concentration of 2,4-D can be 2.0 mg / L, 2.1 mg / L, 2.2 mg / L, 2.3 mg / L, 2.4 mg / L, 2.5 mg / L, 2.6 mg / L, 2.7 mg / L, 2.8 mg / L, 2.9 mg / L or 3.0 mg / L.

[0038] In the present invention, the induction medium includes 0.5-1.5 mg / L of carbon nanotubes. As an optional embodiment of the present invention, the mass concentration of the carbon nanotubes can be 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1.0 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L or 1.5 mg / L. In the present invention, the carbon nanotubes can include multi-walled carbon nanotubes.

[0039] In the present invention, the induction medium includes 20 to 30 g / L of sucrose. As an optional embodiment of the present invention, the mass concentration of the sucrose can be 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L or 30 g / L.

[0040] In the present invention, the induction medium includes 6.5-7.0 g / L of agar. As an optional embodiment of the present invention, the mass concentration of the agar can be 6.5 g / L, 6.6 g / L, 6.7 g / L, 6.8 g / L, 6.9 g / L or 7.0 g / L.

[0041] In the present invention, the pH of the induction medium may be 5.75-5.85, or 5.8.

[0042] As an optional embodiment of the present invention, after adding the above components to B5, the induction medium may also be added with other components that can be used for induction medium, or may not be added separately.

[0043] In the present invention, the induction culture medium is used for the induction culture of ginseng callus, which can greatly improve the induction rate of callus.

[0044] In the present invention, the culture medium includes a proliferation culture medium, and the composition of the proliferation culture medium includes: B5 or MS as a basic culture medium, and also includes: 0.3-1.0 mg / L of toxin, 2.5-3.5 mg / L of 2,4-D, 0.5-1.0 mg / L of acid-hydrolyzed casein, 0.5-1.5 mg / L of carbon nanotubes, 20-30 g / L of sucrose and 6.5-7.0 g / L of agar; or, 0.3-1.0 mg / L of TDZ, 2.5-3.5 mg / L of 2,4-D, 0.5-1.0 mg / L of acid-hydrolyzed casein, 0.5-1.5 mg / L of carbon nanotubes, 20-30 g / L of sucrose and 6.5-7.0 g / L of agar; or, 0.3-1.0 mg / L of KT, 2,4-D 2.5-3.5 mg / L, 0.5-1.0 mg / L acid hydrolyzed casein, 0.5-1.5 mg / L carbon nanotubes, 20-30 g / L sucrose and 6.5-7.0 g / L agar; the pH value of the proliferation culture medium is 5.75-5.85.

[0045] As an optional embodiment of the present invention, the proliferation culture medium uses B5 or MS as the basic culture medium, and is further supplemented with: 0.3-1.0 mg / L of toxin-containing ampicillin, 2.5-3.5 mg / L of 2,4-D, 0.5-1.0 mg / L of acid-hydrolyzed casein, 0.5-1.5 mg / L of carbon nanotubes, 20-30 g / L of sucrose and 6.5-7.0 g / L of agar; the pH value of the proliferation culture medium is 5.75-5.85.

[0046] As another optional embodiment of the present invention, the proliferation culture medium uses B5 or MS as the basic culture medium, and is further supplemented with: TDZ 0.3-1.0 mg / L, 2,4-D 2.5-3.5 mg / L, acid hydrolyzed casein 0.5-1.0 mg / L, carbon nanotubes 0.5-1.5 mg / L, sucrose 20-30 g / L and agar 6.5-7.0 g / L; the pH value of the proliferation culture medium is 5.75-5.85.

[0047] As another optional embodiment of the present invention, the proliferation culture medium uses B5 or MS as the basic culture medium, and is further supplemented with: KT 0.3-1.0 mg / L, 2,4-D 2.5-3.5 mg / L, acid hydrolyzed casein 0.5-1.0 mg / L, carbon nanotubes 0.5-1.5 mg / L, sucrose 20-30 g / L and agar 6.5-7.0 g / L; the pH value of the proliferation culture medium is 5.75-5.85.

[0048] In the present invention, the proliferation medium uses B5 or MS as the basic medium, preferably B5 as the basic medium. In the process of callus proliferation of the present invention, B5 or MS is used as the basic medium to better promote callus proliferation and increase the proliferation coefficient of callus.

[0049] In the present invention, the proliferation medium includes 0.3-1.0 mg / L of toluidine. As an optional embodiment of the present invention, the mass concentration of toluidine can be 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L or 1.0 mg / L. In the present invention, the proliferation medium includes TDZ 0.3-1.0 mg / L. As an optional embodiment of the present invention, the mass concentration of TDZ can be 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L or 1.0 mg / L. In the present invention, the proliferation medium includes KT 0.3-1.0 mg / L. As an optional embodiment of the present invention, the mass concentration of KT can be 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L or 1.0 mg / L. In the present invention, the proliferation medium can include any one of toxaphene, TDZ and KT.

[0050] In the present invention, the proliferation medium includes 2,4-D 2.5-3.5 mg / L. As an optional embodiment of the present invention, the mass concentration of 2,4-D can be 2.5 mg / L, 2.6 mg / L, 2.7 mg / L, 2.8 mg / L, 2.9 mg / L, 3.0 mg / L, 3.1 mg / L, 3.2 mg / L, 3.3 mg / L, 3.4 mg / L or 3.5 mg / L.

[0051] In the present invention, the proliferation medium includes 0.5-1.0 mg / L of acid hydrolyzed casein. As an optional embodiment of the present invention, the mass concentration of the acid hydrolyzed casein can be 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L or 1.0 mg / L.

[0052] In the present invention, the proliferation medium includes 0.5-1.5 mg / L of carbon nanotubes. As an optional embodiment of the present invention, the mass concentration of the carbon nanotubes can be 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 1.0 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L or 1.5 mg / L. In the present invention, the carbon nanotubes can include multi-walled carbon nanotubes.

[0053] In the present invention, the proliferation medium includes 20 to 30 g / L of sucrose. As an optional embodiment of the present invention, the mass concentration of the sucrose can be 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L or 30 g / L.

[0054] In the present invention, the proliferation medium includes 6.5-7.0 g / L of agar. As an optional embodiment of the present invention, the mass concentration of the agar can be 6.5 g / L, 6.6 g / L, 6.7 g / L, 6.8 g / L, 6.0 g / L or 7.0 g / L.

[0055] In the present invention, the pH of the proliferation medium may be 5.75-5.85, or 5.8.

[0056] As an optional embodiment of the present invention, after adding the above components to B5 or MS, the proliferation medium may also be added with other components that can be used in proliferation medium, or may not be added separately.

[0057] In the proliferation culture medium provided by the present invention, the acid hydrolyzed casein provides an organic nitrogen source required for cell division, thereby facilitating the proliferation of ginseng callus; the chloranthate, TDZ or KT has the function of promoting cell division, and synergizing with auxin 2,4-D to promote cell growth; the 2,4-D can dedifferentiate differentiated tissues and cells, while promoting cell growth; the carbon nanotubes promote the absorption of water and nutrients. The proliferation culture medium provided by the present invention can significantly increase the proliferation coefficient of callus.

[0058] In the present invention, the culture medium also includes an anti-browning proliferation culture medium, and the composition of the anti-browning proliferation culture medium includes the proliferation culture medium described in the above technical solution, and further added with 1.0-2.0 g / L of glutathione, 1.0-2.0 g / L of vitamin C and 0-1.5 g / L of activated carbon.

[0059] In the present invention, the anti-browning proliferation medium includes 1.0 to 2.0 g / L of glutathione. As an optional embodiment of the present invention, the mass concentration of glutathione can be 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L or 2.0 g / L.

[0060] In the present invention, the anti-browning proliferation medium includes 1.0-2.0 g / L of vitamin C. As an optional embodiment of the present invention, the mass concentration of the vitamin C can be 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L or 2.0 g / L.

[0061] In the present invention, the anti-browning proliferation medium includes 0 to 1.5 g / L of activated carbon. As an optional embodiment of the present invention, the mass concentration of the activated carbon can be 0, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L or 1.5 g / L.

[0062] In the present invention, the anti-browning proliferation medium uses B5 or MS as the basic medium, preferably B5 as the basic medium. In the process of callus proliferation of the present invention, B5 or MS is used as the basic medium to better promote callus proliferation and increase the proliferation coefficient of callus.

[0063] In the present invention, the anti-browning proliferation medium includes 0.3-1.0 mg / L of toluidine. As an optional embodiment of the present invention, the mass concentration of toluidine can be 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L or 1.0 mg / L. In the present invention, the anti-browning proliferation medium includes TDZ 0.3-1.0 mg / L. As an optional embodiment of the present invention, the mass concentration of TDZ can be 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L or 1.0 mg / L. In the present invention, the anti-browning proliferation medium includes KT 0.3-1.0 mg / L. As an optional embodiment of the present invention, the mass concentration of KT can be 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L or 1.0 mg / L. In the present invention, the anti-browning proliferation medium can include any one of toxaphene, TDZ and KT.

[0064] In the present invention, the anti-browning proliferation medium includes 2.5-3.5 mg / L of 2,4-D. As an optional embodiment of the present invention, the mass concentration of 2,4-D can be 2.5 mg / L, 2.6 mg / L, 2.7 mg / L, 2.8 mg / L, 2.9 mg / L, 3.0 mg / L, 3.1 mg / L, 3.2 mg / L, 3.3 mg / L, 3.4 mg / L or 3.5 mg / L.

[0065] In the present invention, the anti-browning proliferation medium includes 0.5-1.0 mg / L of acid hydrolyzed casein. As an optional embodiment of the present invention, the mass concentration of the acid hydrolyzed casein can be 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L or 1.0 mg / L.

[0066] In the present invention, the anti-browning proliferation medium includes 0.5-1.5 mg / L of carbon nanotubes. As an optional embodiment of the present invention, the mass concentration of the carbon nanotubes can be 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 1.0 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L or 1.5 mg / L. In the present invention, the carbon nanotubes can include multi-walled carbon nanotubes.

[0067] In the present invention, the anti-browning proliferation medium includes 20-30 g / L of sucrose. As an optional embodiment of the present invention, the mass concentration of the sucrose can be 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L or 30 g / L.

[0068] In the present invention, the anti-browning proliferation medium includes 6.5-7.0 g / L of agar. As an optional embodiment of the present invention, the mass concentration of the agar can be 6.5 g / L, 6.6 g / L, 6.7 g / L, 6.8 g / L, 6.0 g / L or 7.0 g / L.

[0069] In the present invention, the pH of the anti-browning proliferation medium may be 5.75-5.85, or 5.8.

[0070] In the anti-browning proliferation medium provided by the present invention, the glutathione and vitamin C are mainly used as antioxidants to prevent the browning of callus tissue; the activated carbon is mainly used as an adsorbent to adsorb some browning components produced during the anti-oxidation process. The present invention shows through the results of the examples that the anti-browning proliferation medium provided by the present invention can significantly reduce the browning rate of callus tissue, and also help to improve the proliferation coefficient of callus tissue.

[0071] The present invention provides an application of the culture medium described in the above technical solution in the rapid propagation of ginseng. The culture medium described in the above technical solution of the present invention can greatly improve the induction rate of ginseng callus, improve the proliferation coefficient of callus, and is conducive to obtaining a large amount of ginseng callus in a short period of time, thereby facilitating the rapid propagation of ginseng.

[0072] The present invention provides a method for culturing ginseng callus, comprising the following steps:

[0073] The sterilized ginseng inflorescence axis is induced and cultured on the culture medium described in the above technical solution.

[0074] The present invention has no special limitation on the source of the ginseng inflorescence axis, and any conventional ginseng inflorescence axis in the art can be used. In the present invention, the inflorescence axis can be a young and tender inflorescence axis. As an optional embodiment of the present invention, the inflorescence axis can be taken from a ginseng umbel that is about to open. After obtaining the ginseng inflorescence axis, the present invention preferably sterilizes the ginseng inflorescence axis. The length of the ginseng inflorescence axis used for sterilization in the present invention can be 3.0 to 3.5 cm, or 3.0 cm, 3.1 cm, 3.2 cm, 3.3 cm, 3.4 cm or 3.5 cm. The present invention has no special limitation on the sterilization method, and any conventional sterilization method in the art can be used. As an optional embodiment of the present invention, the sterilization method includes: washing the ginseng inflorescence axis and then disinfecting it. The present invention has no special limitation on the washing method, and any conventional washing method in the art can be used. As an optional embodiment of the present invention, the washing method includes: washing the ginseng inflorescence axis in deionized water containing liquid detergent, and then rinsing with sterile water; the washing is accompanied by shaking; the rotation speed of the shaking is 150rpm; the shaking time is 30 to 40min, or 30min, 32min, 34min, 35min, 37min, 39min or 40min; the rinsing time can be 40 to 60min, or 40min, 45min, 50min, 55min or 60min. As an optional embodiment of the present invention, the disinfection includes a first disinfection and a second disinfection; the first disinfection can be disinfected with alcohol having a volume fraction of 75%; the first disinfection time can be 30 to 45s, or 30s, 35s, 40s or 45s. After the first disinfection is completed, the obtained ginseng inflorescence axis is preferably rinsed; the number of rinses can be 3 times; the rinse can be performed with sterile water. After the flushing is completed, the present invention can perform a second disinfection; the second disinfection can be performed using a sodium hypochlorite solution with an effective chlorine content of 2.0%; the second disinfection time can be 15 to 18 minutes, or 15 minutes, 16 minutes, 17 minutes or 18 minutes. After the second disinfection is completed, the obtained ginseng inflorescence axis is preferably rinsed; the number of times of the rinse can be 4 times; the rinse can be performed using sterile water. After the rinse is completed, the present invention obtains a sterilized ginseng inflorescence axis.

[0075] After obtaining the sterilized ginseng inflorescence axis, the present invention induces and cultures the ginseng inflorescence axis on the induction medium described in the above technical solution. After obtaining the sterilized ginseng inflorescence axis, the present invention preferably cuts off about 0.5 cm from both ends of the ginseng inflorescence axis to keep the length of the ginseng inflorescence axis at 2.0 to 2.5 cm, or 2.0 cm, 2.1 cm, 2.2 cm, 2.3 cm, 2.4 cm or 2.5 cm. After obtaining the ginseng inflorescence axis of 2.0 to 2.5 cm, the present invention inoculates the ginseng inflorescence axis onto the induction medium for induction culture. When inoculating, the present invention preferably places the ginseng inflorescence axis horizontally on the induction medium. In the present invention, the temperature of the induction culture is 24-26°C, or 24°C, 25°C or 26°C; the time of the induction culture is 30-35 days, or 30 days, 31 days, 32 days, 33 days, 34 days or 35 days; the induction culture can be carried out in the dark or in the light. If the induction culture of the present invention is carried out in the light, the intensity of the light can be 2000-3000 lx, or 2000 lx, 2500 lx or 3000 lx; ​​the light time can be 12-16 h / d, or 12, 13, 14, 15 or 16 h / d.

[0076] The present invention adopts the culture method described in the technical solution to make the ginseng callus induction rate as high as 70%.

[0077] In the present invention, the culture method further comprises proliferation culture; the proliferation culture comprises:

[0078] The ginseng callus is cultured on the proliferation medium described in the above technical solution or the anti-browning proliferation medium described in the above technical solution.

[0079] In the present invention, the temperature of the proliferation culture is 24-26°C, or 24°C, 25°C or 26°C; the time of the proliferation culture is 50-60d, or 50d, 51d, 52d, 53d, 54d, 55d, 56d, 57d, 58d, 59d or 60d; the proliferation culture includes light culture; the light intensity of the light culture can be 2000-3000lx, or 2000lx, 2500lx or 3000lx; the illumination time of the light culture can be 12h / d.

[0080] In the process of proliferation culture of the present invention, the callus tissue can be subcultured at 30 to 35 days of proliferation culture. In the process of proliferation culture of the present invention, proliferation culture can be carried out in the proliferation culture medium of the above technical solution first; and proliferation culture can be carried out in the anti-browning proliferation culture after culturing to a certain stage.

[0081] The present invention provides application of the culture medium described in the above technical solution or the culture method described in the above technical solution in preparing ginsenosides.

[0082] In the present invention, the method for preparing ginsenosides comprises:

[0083] Ginsenosides are extracted and separated from callus tissue obtained by culture using the induction culture medium, the proliferation culture medium or the anti-browning culture medium, or callus tissue obtained by culture using the culture method.

[0084] The present invention can directly extract and separate ginsenosides through the callus tissue; the callus tissue can also be converted into stem cells for liquid culture for the extraction and separation of ginsenosides.

[0085] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0086] The ginseng seedlings used in the examples of the present invention to obtain explants were purchased from Fusong County, Jilin Province.

[0087] Example 1

[0088] A method for inducing ginseng callus, comprising the following steps:

[0089] (1) Obtaining and disinfecting explants: During the flowering period of ginseng, in mid-May, cut off 3.0 cm of the young inflorescence axis of the ginseng umbel that is about to open, put it in a glass bottle, add 10 times the volume of deionized water and 2 drops of liquid detergent, shake and wash at 150rpm for 30 minutes, and then rinse with running water for 40 minutes. Then transfer the cleaned inflorescence axis to the clean bench, disinfect it with 75% alcohol for 40 seconds, rinse it with sterile water 3 times; then disinfect it with sodium hypochlorite solution with an effective chlorine content of 2.0% for 15 minutes, rinse it with sterile water 4 times, and set it aside.

[0090] (2) Callus induction: Place the sterilized young inflorescence axis on an inoculation plate, cut off about 0.5 cm at each end, leaving 2.0 cm, and place it horizontally on a callus induction medium for induction culture. Figure 1As shown. The number of explants inoculated in this treatment was 30. The composition of the callus induction medium is: the basic medium is B5, and 6.5g / L agar, 20g / L sucrose, 0.5mg / L TDZ, 2.0mg / L 2,4-D and 1.0mg / L multi-walled carbon nanotubes are added. The pH value of the induction medium is 5.8. The conditions for induction culture are: dark culture, and the culture temperature is 25±1℃. After 15 days of induction culture, the explants sprouted, the surface was convex, and a layer of light yellow-green frosty callus was formed; the culture continued, and the callus continued to increase.

[0091] Example 2

[0092] A method for inducing ginseng callus, the steps are the same as (1) and (2) in Example 1, the only difference being that the composition of the callus induction medium is: the basic medium is B5, and 6.5 g / L agar, 20 g / L sucrose, 0.5 mg / L TDZ, 2.0 mg / L 2,4-D and 1.5 mg / L multi-walled carbon nanotubes are added.

[0093] Example 3

[0094] A method for inducing ginseng callus, the steps are the same as (1) and (2) in Example 1, the only difference being that the composition of the callus induction medium is: the basic medium is B5, and 6.5 g / L agar, 20 g / L sucrose, 0.5 mg / L TDZ, 2.0 mg / L 2,4-D and 0.5 mg / L multi-walled carbon nanotubes are added.

[0095] Comparative Example 1

[0096] A method for inducing ginseng callus, the steps are the same as (1) and (2) in Example 1, the only difference being that the composition of the callus induction medium is: the basic medium is B5, and 6.5 g / L agar, 20 g / L sucrose, 0.5 mg / L TDZ and 2.0 mg / L 2,4-D are added, and no multi-walled carbon nanotubes are contained.

[0097] Comparative Example 2

[0098] A method for inducing ginseng callus, the steps are the same as those of Example 1, the only difference being that the callus induction medium is composed of a basic medium of B5, and is further supplemented with 6.5 g / L agar, 20 g / L sucrose, 0.5 mg / L TDZ, 2.0 mg / L 2,4-D and 2.0 mg / L multi-walled carbon nanotubes.

[0099] Application Example 1

[0100] The callus induction culture was carried out for 35 days, and the induction results of the callus were statistically analyzed. The results are shown in Table 1.

[0101] Table 1 Callus induction results of Examples 1 to 3 and Comparative Examples 1 to 2

[0102] Examples Serial number Multi-walled carbon nanotube concentration (mg / L) Number of explants inoculated Callus induction rate (%) Comparative Example 1 1 0 30 60.00 Example 3 2 0.5 30 67.67 Example 1 3 1.0 30 70.00 Example 2 4 1.5 30 70.00 Comparative Example 2 5 2.0 20 56.67

[0103] As shown in Table 1, when the concentration of multi-walled carbon nanotubes in the callus induction medium is between 0.5 and 1.5 mg / L, the callus induction rate is relatively high. The callus obtained from different induction mediums is all light yellow-green.

[0104] Comparative Example 3

[0105] A method for inducing ginseng callus, the steps are the same as (1) and (2) in Example 1, the only difference being that the callus induction medium uses 1 / 2MS medium as the basic medium.

[0106] Comparative Example 4

[0107] A method for inducing ginseng callus, the steps are the same as (1) and (2) in Example 1, the only difference being that the callus induction medium uses MS medium as the basic medium.

[0108] Comparative Example 5

[0109] A method for inducing ginseng callus, the steps are the same as (1) and (2) in Example 1, the only difference being that the callus induction medium uses N6 medium as the basic medium.

[0110] Comparative Example 6

[0111] A method for inducing ginseng callus, the steps are the same as (1) and (2) in Example 1, the only difference being that the callus induction medium uses WPM medium as the basic medium.

[0112] Application Example 2

[0113] Example 1 and Comparative Examples 3-6 were induced and cultured for 35 days, and the induction results of callus tissue were statistically analyzed. The results are shown in Table 2.

[0114] Table 2 Results of callus induction culture of Example 1 and Comparative Examples 3 to 6

[0115] Examples Serial number Basic medium Number of explants inoculated Callus induction rate Comparative Example 3 1 1 / 2MS 30 56.67 Comparative Example 4 2 MS 30 60.00 Example 1 3 B5 30 70.00 Comparative Example 5 4 N6 30 50.00 Comparative Example 6 5 WPM 20 43.33

[0116] As shown in Table 2, callus induction medium with B5 as the basic medium has more advantages in improving induction than other culture media.

[0117] Comparative Example 7

[0118] A method for inducing ginseng callus, the steps are the same as (1) and (2) in Example 1, the only difference being that the composition of the callus induction medium is: the basic medium is B5, and 6.5 g / L agar, 20 g / L sucrose, 1.5 mg / L 2,4-D and 1.0 mg / L multi-walled carbon nanotubes are added, and TDZ is not contained.

[0119] Comparative Example 8

[0120] A method for inducing ginseng callus, the steps are the same as (1) and (2) in Example 1, the only difference being that the composition of the callus induction medium is: the basic medium is B5, and 6.5 g / L agar, 20 g / L sucrose, 2.0 mg / L 2,4-D and 1.0 mg / L multi-walled carbon nanotubes are added, and TDZ is not contained.

[0121] Comparative Example 9

[0122] A method for inducing ginseng callus, the steps are the same as (1) and (2) in Example 1, the only difference being that the composition of the callus induction medium is: the basic medium is B5, and 6.5 g / L agar, 20 g / L sucrose, 2.5 mg / L 2,4-D and 1.0 mg / L multi-walled carbon nanotubes are added, and TDZ is not contained.

[0123] Comparative Example 10

[0124] A method for inducing ginseng callus, the steps are the same as (1) and (2) in Example 1, the only difference being that the composition of the callus induction medium is: the basic medium is B5, and 6.5 g / L agar, 20 g / L sucrose, 3.0 mg / L 2,4-D and 1.0 mg / L multi-walled carbon nanotubes are added, and TDZ is not contained.

[0125] Comparative Example 11

[0126] A method for inducing ginseng callus, the steps are the same as (1) and (2) in Example 1, the only difference being that the composition of the callus induction medium is: the basic medium is B5, and 6.5 g / L agar, 20 g / L sucrose, 3.5 mg / L 2,4-D and 1.0 mg / L multi-walled carbon nanotubes are added, and TDZ is not contained.

[0127] Application Example 3

[0128] Comparative Examples 7 to 11 were induced and cultured for 35 days, and the induction results of callus tissue were statistically analyzed. The results are shown in Table 3.

[0129] Table 3 Comparative Examples 7 to 11 Callus Induction Culture Results

[0130] Examples Serial number 2,4-D concentration (mg / L) Number of explants inoculated Callus induction rate (%) Comparative Example 7 1 1.5 30 36.67 Comparative Example 8 2 2.0 30 50.00 Comparative Example 9 3 2.5 30 53.33 Comparative Example 10 4 3.0 30 53.33 Comparative Example 11 5 3.5 30 40.00

[0131] As shown in Table 3, not adding TDZ has a significant adverse effect on the callus induction results and greatly reduces the callus induction rate.

[0132] Comparative Example 12

[0133] A method for inducing ginseng callus, the steps are the same as (1) and (2) in Example 1, the only difference being that the composition of the callus induction medium is: the basic medium is B5, and 6.5 g / L agar, 20 g / L sucrose, 0.25 mg / L TDZ and 1.5 mg / L multi-walled carbon nanotubes are added, and 2,4-D is not contained.

[0134] Comparative Example 13

[0135] A method for inducing ginseng callus, the steps are the same as (1) and (2) in Example 1, the only difference being that the composition of the callus induction medium is: the basic medium is B5, and 6.5 g / L agar, 20 g / L sucrose, 0.5 mg / L TDZ and 1.5 mg / L multi-walled carbon nanotubes are added, and 2,4-D is not contained.

[0136] Comparative Example 14

[0137] A method for inducing ginseng callus, the steps are the same as (1) and (2) in Example 1, the only difference being that the composition of the callus induction medium is: the basic medium is B5, and 6.5 g / L agar, 20 g / L sucrose, 0.75 mg / L TDZ and 1.5 mg / L multi-walled carbon nanotubes are added, and 2,4-D is not contained.

[0138] Comparative Example 15

[0139] A method for inducing ginseng callus, the steps are the same as (1) and (2) in Example 1, the only difference being that the composition of the callus induction medium is: the basic medium is B5, and 6.5 g / L agar, 20 g / L sucrose, 1.0 mg / L TDZ and 1.5 mg / L multi-walled carbon nanotubes are added, and 2,4-D is not contained.

[0140] Comparative Example 16

[0141] A method for inducing ginseng callus, the steps are the same as (1) and (2) in Example 1, the only difference being that the composition of the callus induction medium is: the basic medium is B5, and 6.5 g / L agar, 20 g / L sucrose, 1.25 mg / L TDZ and 1.5 mg / L multi-walled carbon nanotubes are added, and 2,4-D is not contained.

[0142] Application Example 4

[0143] Comparative Examples 12 to 16 were induced and cultured for 35 days, and the induction results of callus tissue were statistically analyzed. The results are shown in Table 4.

[0144] Table 4 Comparative Examples 12 to 16 Callus Induction Culture Results

[0145] Examples Serial number TDZ concentration (mg / L) Number of explants inoculated Callus induction rate (%) Comparative Example 12 1 0.25 30 13.33 Comparative Example 13 2 0.5 30 36.67 Comparative Example 14 3 0.75 30 40.00 Comparative Example 15 4 1.0 30 40.00 Comparative Example 16 5 1.25 20 26.67

[0146] As shown in Table 4, not adding 2,4-D has a significant adverse effect on the callus induction results and greatly reduces the callus induction rate.

[0147] Example 4

[0148] A callus proliferation culture method, the steps are as follows:

[0149] After 35 days of induction culture of callus induction culture method of Example 1, callus with pale yellow-green color, good granularity and similar growth status was selected for proliferation culture, and the inflorescence axis and callus that had begun to proliferate were transferred to the proliferation culture medium for proliferation culture. Figure 2 As shown. 30 pieces of callus tissue were inoculated in this treatment, and the basic culture medium of the proliferation medium was MS, supplemented with sucrose 30g / L, agar 6.5g / L, TDZ 0.5mg / L, 2,4-D 2.5mg / L, acid hydrolyzed casein 0.5mg / L and multi-walled carbon nanotubes 0.5mg / L. The pH value of the proliferation medium was 5.8. The proliferation culture temperature was 25±1℃, the light intensity was 2000lx, and the lighting time was 12h / d. After 30 days of proliferation culture, the same formula was used for subculture. After a total of 55 days of proliferation culture, the callus tissue proliferated and completely covered the inflorescence axis. The proliferation rate of the callus tissue of the young inflorescence axis of ginseng is shown as follows Figure 3 shown. Figure 3 The left picture in the middle shows the callus tissue during the proliferation culture on June 26; Figure 3 The right picture in the figure shows the callus tissue during the proliferation culture on June 19. Figure 3 It can be seen that the proliferation rate of callus tissue between June 19 and June 26.

[0150] Example 5

[0151] A callus proliferation culture method, the steps are the same as those of Example 4, except that the basic culture medium of the proliferation culture medium is B5.

[0152] Comparative Example 17

[0153] A callus proliferation culture method, the steps are the same as those of Example 4, except that the basic culture medium of the proliferation culture medium is 1 / 2MS.

[0154] Comparative Example 18

[0155] A callus proliferation culture method, the steps are the same as those of Example 4, except that the basic culture medium of the proliferation culture medium is N6.

[0156] Comparative Example 19

[0157] A callus proliferation culture method, the steps are the same as those of Example 4, the difference being that the basic culture medium of the proliferation culture medium is WPM.

[0158] Application Example 5

[0159] The proliferation coefficients of the callus tissues of Examples 4 to 5 and Comparative Examples 17 to 19 after proliferation culture for 30 days were statistically analyzed by weighing. The results are shown in Table 5.

[0160] Table 5 Proliferation culture results of Examples 4 to 5 and Comparative Examples 17 to 19

[0161] Examples Serial number Callus tissue Basic medium Multiplication coefficient / fold Example 4 1 30 MS 2.67 Comparative Example 17 2 30 1 / 2MS 2.55 Example 5 3 30 B5 2.98 Comparative Example 18 4 30 N6 2.03 Comparative Example 19 5 30 WPM 2.35

[0162] It can be seen from Table 5 that when the proliferation culture medium uses MS and B5 as the basic culture medium for proliferation culture, the proliferation coefficient of callus tissue is higher, which is significantly better than the culture effect using other basic culture media.

[0163] Comparative Example 20

[0164] A callus tissue proliferation culture method, the steps are the same as those of Example 4, the only difference is that the composition of the proliferation culture medium is different, the composition of the proliferation culture medium is: the basic culture medium is MS, sucrose 30g / L, agar 6.5g / L, TDZ 0mg / L, 2,4-D 2.5mg / L, acid hydrolyzed casein 0.5mg / L and multi-walled carbon nanotubes 0.5mg / L are added.

[0165] Comparative Example 21

[0166] A callus proliferation culture method, the steps are the same as those of Example 4, the only difference is that the composition of the proliferation culture medium is different, the composition of the proliferation culture medium is: the basic culture medium is MS, sucrose 30g / L, agar 6.5g / L, TDZ 0.5mg / L, 2,4-D 0mg / L, acid hydrolyzed casein 0.5mg / L and multi-walled carbon nanotubes 0.5mg / L are added.

[0167] Comparative Example 22

[0168] A callus proliferation culture method, the steps are the same as those of Example 4, the only difference is that the composition of the proliferation culture medium is different, the composition of the proliferation culture medium is: the basic culture medium is MS, sucrose 30g / L, agar 6.5g / L, TDZ 0.5mg / L, 2,4-D 2.5mg / L, acid hydrolyzed casein 0.5mg / L and multi-walled carbon nanotubes 0mg / L are added.

[0169] Comparative Example 23

[0170] A callus proliferation culture method, the steps are the same as those of Example 4, the only difference is that the composition of the proliferation culture medium is different, the composition of the proliferation culture medium is: the basic culture medium is MS, sucrose 30g / L, agar 6.5g / L, TDZ 0.5mg / L, 2,4-D 2.5mg / L, acid hydrolyzed casein 0mg / L and multi-walled carbon nanotubes 0.5mg / L are added.

[0171] Application Example 6

[0172] The proliferation coefficients of the callus tissues of Comparative Examples 20 to 23 that were cultured for 35 days were statistically analyzed by weighing. The results are shown in Table 6.

[0173] Table 6 Comparative Examples 20 to 23 Proliferation Culture Results

[0174]

[0175] As shown in Table 6, omitting any one of TDZ, 2,4-D, multi-walled carbon nanotubes and acid-hydrolyzed casein will significantly reduce the proliferation coefficient of callus tissue.

[0176] Example 6

[0177] A method for preventing browning during callus proliferation, comprising the following steps:

[0178] After 60 days of proliferation culture using the method of callus proliferation culture in Example 4, callus with light yellow-green color, good granularity and similar growth state was selected for proliferation culture, and the callus was transferred to the anti-browning proliferation medium for continued proliferation culture. 30 callus were inoculated in this treatment, and the composition of the anti-browning proliferation medium was specifically as follows: MS was used as the basic medium, sucrose 30g / L, agar 6.5g / L, glutathione 1.0g / L, vitamin C 1.0g / L, activated carbon 0.5g / L, TDZ 0.3mg / L, 2,4-D 2.5mg / L, acid hydrolyzed casein 0.5mg / L and multi-walled carbon nanotubes 0.5mg / L, and the pH value of the anti-browning proliferation medium was 5.8. The conditions of proliferation culture were the same as those in Example 4.

[0179] The pale yellow-green callus obtained by proliferation culture can be transformed into stem cells, cultured in liquid and proliferated, and then used for the extraction and separation of ginsenosides, or differentiated and cultured to induce the production of clustered buds for the production of sterile ginseng seedlings.

[0180] Example 7

[0181] A method for preventing browning during callus proliferation, the steps are the same as those of Example 6, the only difference being that the composition of the anti-browning proliferation medium is: MS as the basic medium, supplemented with 1.5 g / L glutathione, 1.5 g / L vitamin C, 1.0 g / L activated carbon, 0.3 mg / L TDZ, 2.5 mg / L 2,4-D, 0.5 mg / L acid hydrolyzed casein and 0.5 mg / L multi-walled carbon nanotubes.

[0182] Example 8

[0183] A method for preventing browning during callus proliferation, the steps are the same as those of Example 6, the only difference being that the composition of the anti-browning proliferation medium is: MS as the basic medium, supplemented with 1.5 g / L glutathione, 1.5 g / L vitamin C, 0 g / L activated carbon, 0.3 mg / L TDZ, 2.5 mg / L 2,4-D, 0.5 mg / L acid hydrolyzed casein and 0.5 mg / L multi-walled carbon nanotubes.

[0184] Comparative Example 24

[0185] A method for preventing browning during callus proliferation, the steps are the same as those of Example 6, the only difference being that the composition of the anti-browning proliferation medium is: MS as a basic medium, supplemented with 0 g / L glutathione, 1.5 g / L vitamin C, 1.0 g / L activated carbon, 0.3 mg / L TDZ, 2.5 mg / L 2,4-D, 0.5 mg / L acid hydrolyzed casein and 0.5 mg / L multi-walled carbon nanotubes.

[0186] Comparative Example 25

[0187] A method for preventing browning during callus proliferation, the steps are the same as those of Example 6, the only difference being that the composition of the anti-browning proliferation medium is: MS as the basic medium, supplemented with 1.5 g / L glutathione, 0 g / L vitamin C, 1.0 g / L activated carbon, 0.3 mg / L TDZ, 2.5 mg / L 2,4-D, 0.5 mg / L acid hydrolyzed casein and 0.5 mg / L multi-walled carbon nanotubes.

[0188] Application Example 7

[0189] The browning rate and proliferation coefficient of the callus tissues of Examples 6 to 8 and Comparative Examples 24 to 25 cultured for 35 days were statistically analyzed. The results are shown in Table 7.

[0190] Table 7 Cultivation results of Examples 6 to 8 and Comparative Examples 24 to 25

[0191] Examples Serial number Callus tissue Glutathione Vc Activated carbon Browning rate / % Multiplication coefficient / fold Comparative Example 24 1 30 0 1.5 1.0 60.00 0.95 Comparative Example 25 2 30 1.5 0 1.0 53.33 0.90 Example 8 3 30 1.5 1.5 0 23.33 2.90 Example 7 4 30 1.5 1.5 1.0 20.00 2.98 Example 6 5 30 1.0 1.0 0.5 20.00 2.95

[0192] It can be seen from Table 7 that adding glutathione and Vc and / or activated carbon to the proliferation medium at the same time can significantly reduce the browning rate during the proliferation culture process and is beneficial to improving the proliferation coefficient.

[0193] Example 9

[0194] A method for inducing ginseng callus, comprising the following steps:

[0195] (1) Obtaining and disinfecting explants: During the flowering period of ginseng, in late May, cut off 3.5 cm of the young inflorescence axis of the ginseng umbel that is about to open, put it in a glass bottle, add 10 times the volume of deionized water and 2 drops of liquid detergent, shake and wash at 150rpm for 35 minutes, and then rinse with running water for 50 minutes. Then transfer the cleaned inflorescence axis to the clean bench, disinfect it with 75% alcohol for 35 seconds, rinse it with sterile water three times; then disinfect it with sodium hypochlorite solution with an effective chlorine content of 2.0% for 17 minutes, rinse it with sterile water four times, and set it aside.

[0196] (2) Induction of callus: Place the sterilized young inflorescence axis on an inoculation plate, cut off about 0.5 cm at each end, retain 2.5 cm, and place it horizontally on a callus induction medium for induction culture. The number of explants inoculated in this treatment is 30. The composition of the callus induction medium is: the basic medium is B5, supplemented with 6.5 g / L agar, 20 g / L sucrose, 0.5 mg / L toluidine, 2.0 mg / L 2,4-D and 1.0 mg / L multi-walled carbon nanotubes. The pH value of the callus induction medium is 5.8. The induction culture is carried out in the dark, and the culture temperature is 25±1℃. After 15 days of induction culture, the explants sprouted, the surface was convex, and a layer of light yellow-green frosty callus was formed; the culture continued, and the callus continued to increase.

[0197] After 35 days of induction culture, the callus induction rate was statistically analyzed, and it was found that the callus induction rate was 70% when the method was used for callus induction.

[0198] Example 10

[0199] A callus proliferation culture method, the steps are as follows:

[0200] Among the callus tissues obtained by inducing and culturing for 35 days using the callus induction and culture method of Example 9, callus tissues with light yellow-green color, good granularity and similar growth state are selected for proliferation culture, specifically: the inflorescence axis and the callus tissue that has begun to proliferate are transferred to the proliferation culture medium for proliferation culture. 30 pieces of callus tissues were inoculated in this treatment, and the basic culture medium of the proliferation culture medium was MS, with 30g / L sucrose, 6.5g / L agar, 0.5mg / L toluidine, 2,4-D 2.5mg / L, 0.5mg / L acid hydrolyzed casein and 0.5mg / L multi-walled carbon nanotubes. The pH value of the proliferation culture medium was 5.8. The proliferation culture temperature was 25±1°C, the light intensity was 2000lx, and the illumination time was 12h / d. When the proliferation culture was 30d, the same formula was used for subculture. After 55d of proliferation culture, the callus tissue proliferated and completely covered the inflorescence axis.

[0201] After 35 days of proliferation culture, the proliferation coefficient of callus tissue was statistically analyzed, and it was found that when the method was used for proliferation culture of callus tissue, the proliferation coefficient of callus tissue was 2.95 times.

[0202] Embodiment 11

[0203] A method for preventing browning during callus proliferation, comprising the following steps:

[0204] After 60 days of proliferation culture using the method of callus proliferation culture in Example 10, callus with light yellow-green color, good granularity and similar growth state was selected for proliferation culture, and the callus was transferred to the anti-browning proliferation medium for continued proliferation culture. 30 callus were inoculated in this treatment, and the composition of the anti-browning proliferation medium was: MS as the basic medium, supplemented with sucrose 30g / L, agar 6.5g / L, glutathione 1.0g / L, vitamin C 1.0g / L, activated carbon 0.5g / L, toxoplasmosis 0.3mg / L, 2,4-D 2.5mg / L, acid hydrolyzed casein 0.5mg / L and multi-walled carbon nanotubes 0.5mg / L, and the pH value of the anti-browning proliferation medium was 5.8. The conditions of proliferation culture were the same as those in Example 10, specifically: proliferation culture temperature 25±1°C, light intensity 2000lx, and light time 12h / d. The pale yellow-green callus obtained by proliferation culture can be used for the extraction and separation of ginsenosides, or can be converted into stem cells for liquid culture and further used for the extraction and separation of ginsenosides.

[0205] The browning rate and proliferation coefficient of the callus cultured for 30 days in Example 11 were statistically analyzed, and the browning rate was 20% and the proliferation coefficient was 2.98 times.

[0206] Example 12

[0207] A method for inducing ginseng callus, comprising the following steps:

[0208] (1) Obtaining and disinfecting explants: During the flowering period of ginseng, in early May, cut off 3.0 cm of the young inflorescence axis of the ginseng umbel that is about to open, put it in a glass bottle, add 10 times the volume of deionized water and 2 drops of liquid detergent, shake and wash at 150rpm for 40 minutes, and then rinse with running water for 60 minutes. Then transfer the cleaned inflorescence axis to the clean bench, disinfect it with 75% alcohol for 45 seconds, rinse it with sterile water three times; then disinfect it with sodium hypochlorite solution with an effective chlorine content of 2.0% for 18 minutes, rinse it with sterile water four times, and set it aside.

[0209] (2) Callus induction: Place the sterilized young inflorescence axis on an inoculation plate, cut off about 0.5 cm at each end, retain 2.0 cm, and place it horizontally on a callus induction medium for induction culture. The number of explants inoculated in this treatment is 30. The composition of the callus induction medium is: the basic medium is B5, supplemented with 6.5 g / L agar, 20 g / L sucrose, 1.0 mg / L KT, 3.0 mg / L 2,4-D and 1.0 mg / L multi-walled carbon nanotubes. The pH value of the callus induction medium is 5.8. The induction culture is carried out in the dark, and the culture temperature is 25±1℃. After 15 days of induction culture, the explants sprouted, the surface was convex, and a layer of light yellow-green frosty callus was formed; the culture continued, and the callus continued to increase.

[0210] After 30 days of induction culture, the callus induction rate was statistically analyzed, and it was found that when the method was used for callus induction, the callus induction rate was 70%.

[0211] Example 13

[0212] A callus proliferation culture method, the steps are as follows:

[0213] Among the calli obtained by inducing and culturing for 35 days using the callus induction and culture method of Example 12, calli with light yellow-green color, good granularity and similar growth state are selected for proliferation culture, and the inflorescence axis and the callus that have begun to proliferate are transferred to the proliferation culture medium for proliferation culture. 30 calli were inoculated in this treatment, and the basic culture medium of the proliferation culture medium was MS, with 30g / L sucrose, 6.5g / L agar, 1.0mg / L KT, 3.0mg / L 2,4-D, 1.0mg / L acid hydrolyzed casein and 1.0mg / L multi-walled carbon nanotubes. The pH value of the proliferation culture medium was 5.8. The proliferation culture temperature was 25±1°C, the light intensity was 2000lx, and the illumination time was 12h / d. When the proliferation culture was 30d, the same formula was used for subculture. After 55d of proliferation culture, the callus proliferated and completely covered the inflorescence axis.

[0214] Embodiment 14

[0215] A method for preventing browning during callus proliferation, comprising the following steps:

[0216] After 60 days of proliferation culture using the method of callus proliferation culture in Example 13, callus with light yellow-green color, good granularity and similar growth state was selected for proliferation culture, and the callus was transferred to the anti-browning proliferation medium for continued proliferation culture. 30 callus were inoculated in this treatment, and the composition of the anti-browning proliferation medium was: MS as the basic medium, supplemented with sucrose 30g / L, agar 6.5g / L, glutathione 1.5g / L, vitamin C 1.5g / L, activated carbon 1.0g / L, KT0.5mg / L, 2,4-D 2.5mg / L, acid hydrolyzed casein 1.0mg / L and multi-walled carbon nanotubes 1.0mg / L, and the pH value of the anti-browning proliferation medium was 5.8. The conditions of proliferation culture were the same as those in Example 10. The light yellow-green callus obtained by proliferation culture can be used for the extraction and separation of ginsenosides, or converted into stem cells for liquid culture, and then used for the extraction and separation of ginsenosides.

[0217] The browning rate and proliferation coefficient of the callus cultured for 30 days in Example 14 were statistically analyzed, and the browning rate was 23.33% and the proliferation coefficient was 2.90.

[0218] Comparative Example 26

[0219] A method for inducing ginseng callus tissue, the steps are the same as those of Example 1, the only difference being that the petiole of ginseng leaflets is used as the explant instead of the ginseng inflorescence axis as the explant in Example 1.

[0220] The induction rate of ginseng callus tissue was finally 0, and no callus tissue was induced.

[0221] In summary, the culture medium and culture method for callus tissue culture provided by the present invention can obtain a large amount of callus tissue in a short period of time. The obtained callus tissue can be used for tissue culture and rapid propagation of ginseng, or can be directly used to synthesize ginsenosides or as a raw material for synthesizing ginsenosides, thereby helping to increase the yield of ginsenosides.

[0222] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A culture medium for ginseng callus culture, characterized in that: The culture medium includes an induction culture medium, and the composition of the induction culture medium includes: B5 as a basic culture medium, and also includes: 0.5-1.0 mg / L of tolbutamide, 2.0-3.0 mg / L of 2,4-D, 0.5-1.5 mg / L of carbon nanotubes, 20-30 g / L of sucrose and 6.5-7.0 g / L of agar; Alternatively, TDZ 0.5-1.0 mg / L, 2,4-D 2.0-3.0 mg / L, carbon nanotubes 0.5-1.5 mg / L, sucrose 20-30 g / L, and agar 6.5-7.0 g / L; Alternatively, KT 0.5-1.0 mg / L, 2,4-D 2.0-3.0 mg / L, carbon nanotubes 0.5-1.5 mg / L, sucrose 20-30 g / L, and agar 6.5-7.0 g / L; The pH value of the induction medium is 5.75-5.

85.

2. The culture medium according to claim 1, characterized in that The culture medium comprises a proliferation culture medium, and the composition of the proliferation culture medium comprises: taking B5 or MS as a basic culture medium, and further comprising: 0.3-1.0 mg / L of tolbutamide, 2.5-3.5 mg / L of 2,4-D, 0.5-1.0 mg / L of acid hydrolyzed casein, 0.5-1.5 mg / L of carbon nanotubes, 20-30 g / L of sucrose and 6.5-7.0 g / L of agar; Alternatively, TDZ 0.3-1.0 mg / L, 2,4-D 2.5-3.5 mg / L, acid hydrolyzed casein 0.5-1.0 mg / L, carbon nanotubes 0.5-1.5 mg / L, sucrose 20-30 g / L and agar 6.5-7.0 g / L; Alternatively, KT 0.3-1.0 mg / L, 2,4-D 2.5-3.5 mg / L, acid hydrolyzed casein 0.5-1.0 mg / L, carbon nanotubes 0.5-1.5 mg / L, sucrose 20-30 g / L and agar 6.5-7.0 g / L; The pH value of the proliferation culture medium is 5.75-5.

85.

3. The culture medium according to claim 1, characterized in that The culture medium includes an anti-browning proliferation culture medium, and the composition of the anti-browning proliferation culture medium includes: on the basis of the proliferation culture medium components of the culture medium according to claim 2, 1.0-2.0 g / L of glutathione, 1.0-2.0 g / L of vitamin C and 0-1.5 g / L of activated carbon are added.

4. Use of the culture medium according to any one of claims 1 to 3 in the rapid propagation of ginseng.

5. A method for culturing ginseng callus, characterized in that: The following steps are involved: The sterilized ginseng inflorescence axis is induced and cultured on the culture medium according to claim 1.

6. The culture method according to claim 5, characterized in that: The induction culture temperature is 24-26° C.; the induction culture time is 30-35 days.

7. The culture method according to claim 5, characterized in that: The culture method further comprises proliferation culture; the proliferation culture comprises: The ginseng callus is cultured on the proliferation medium in the culture medium of claim 2 or the anti-browning proliferation medium in the culture medium of claim 3.

8. The culture method according to claim 7, characterized in that: The temperature of the proliferation culture is 24-26° C.; the time of the proliferation culture is 50-60 days; the proliferation culture includes light culture; the light intensity of the light culture is 2000-3000 lx; ​​the light time of the light culture is 12-16 h / d.

9. Use of the culture medium according to any one of claims 1 to 3 or the culture method according to any one of claims 5 to 8 in the preparation of ginsenosides.

10. The use according to claim 9, characterized in that: Ginsenosides are extracted and separated from the callus tissue cultured using the culture medium or the culture method.

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