A culture medium for ginseng callus culture and a culture method for callus

Abstract

This invention provides a culture medium and method for culturing ginseng callus, belonging to the field of plant tissue culture technology. The culture medium for ginseng callus culture uses B5 as the basic medium and further includes: thiamethoxam, TDZ or KT, 2,4-D, carbon nanotubes, sucrose, and agar; the pH of the callus induction medium is 5.75-5.85. Example results show that the culture medium provided by this invention can significantly improve the induction rate of ginseng callus. Furthermore, the callus obtained from the callus induction medium can be proliferated to obtain a large amount of callus in a short period. The obtained callus can be directly used to synthesize ginsenosides or as a raw material for ginsenoside synthesis, thereby improving the yield of ginsenosides.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a culture medium for ginseng callus culture and a method for culturing callus. Background Technology

[0002] Currently, the biosynthesis of ginsenosides involves washing, disinfecting, and cutting fresh roots of perennial ginseng, then inducing callus formation in a solid culture medium containing a certain concentration of growth regulators. The induced callus is then induced and differentiated into adventitious roots, which are then used to synthesize ginsenosides. With the development of biotechnology, by transferring plasmids of Agrobacterium rhizogenes into ginseng hairy roots, this type of root can grow rapidly in a hormone-free culture medium, increasing the yield of ginsenosides in the hairy roots. This method can produce a large number of hairy roots, but its disadvantages include a low ginsenoside content (Rc content approximately 1.0%), high extraction costs, and an inability to significantly increase yield.

[0003] Under certain conditions, ginseng callus tissue can undergo cell growth and metabolic activities, synthesizing and accumulating ginsenosides. However, ginseng callus tissue often suffers from low induction rates, which limits its application in the preparation and extraction of ginsenosides. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the purpose of this invention is to provide a culture medium for culturing ginseng callus, which can significantly improve the induction rate of callus and obtain a large amount of callus in a short period of time.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] This invention provides a culture medium for ginseng callus culture, the culture medium including an induction medium, the composition of which includes: B5 as the basic medium, and further includes: 0.5-1.0 mg / L of Picloram, 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 to 5.85.

[0010] Preferably, the culture medium includes a proliferation medium, the composition of which includes: B5 or MS as the basic medium, and further includes: 0.3-1.0 mg / L of chlorhexidine, 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 medium is 5.75 to 5.85.

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

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

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

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

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

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

[0020] Ginseng callus tissue was cultured and proliferated on the proliferation medium or the anti-browning proliferation medium described in the above technical solution.

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

[0022] This invention provides the application of the culture medium or culture method described in the above technical solution in the preparation of ginsenosides.

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

[0024] Beneficial effects of the present invention

[0025] This invention provides a culture medium for ginseng callus culture, the culture medium comprising an induction medium; the composition of the induction medium includes: B5 as the basic medium, further comprising: 0.5-1.0 mg / L of chlorhexidine, 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, KT The induction medium contains 0.5–1.0 mg / L of ginseng, 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 of the induction medium is 5.75–5.85. In the callus induction medium provided by this invention, the ginseng, TDZ, or KT promotes cell division and synergistically promotes cell growth with auxin 2,4-D. The 2,4-D can dedifferentiate differentiated tissues and cells while promoting cell growth, thus facilitating callus induction; the carbon nanotubes promote the absorption of water and nutrients by cells, thereby promoting callus growth. The results of the examples show that the induction medium provided by this invention can significantly improve the induction rate of ginseng callus.

[0026] Furthermore, the callus tissue obtained from the induction culture medium can be proliferated to obtain a large amount of callus tissue in a short period of time. The obtained callus tissue can be directly used to synthesize ginsenosides or as a raw material for the synthesis of ginsenosides, thereby helping to increase the yield of ginsenosides. Attached Figure Description

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0028] Figure 1 Diagram showing callus induction in young ginseng inflorescence axis;

[0029] Figure 2 A diagram illustrating the proliferation of callus tissue from a young ginseng inflorescence axis;

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

[0031] The present invention provides a culture medium for culturing ginseng callus, the culture medium comprising an induction medium.

[0032] As an optional embodiment of the present invention, the induction medium uses B5 as the basic medium and further adds: 0.5-1.0 mg / L of chlortetracycline, 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 medium is 5.75-5.85.

[0033] In 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] In 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 this invention, the induction medium uses B5 as the basic medium. The B5 medium can meet the requirements of macro- and micro-elements required in the induction of ginseng callus, which is beneficial to promoting the induction of callus.

[0036] In this invention, the induction medium includes 0.5–1.0 mg / L of chlorhexidine. As an optional embodiment of this invention, the mass concentration of chlorhexidine 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 this invention, the induction medium includes 0.5–1.0 mg / L of diethyldimethylamine (TDZ). As an optional embodiment of this 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 this invention, the induction medium includes 0.5–1.0 mg / L of hydroxylamine (KT). As an optional embodiment of this 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 this invention, the induction culture medium may include any one of cyproconazole, TDZ, and KT.

[0037] In this invention, the induction medium contains 2,4-D at a concentration of 2.0–3.0 mg / L. As an optional embodiment of this 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 this invention, the induction culture medium comprises 0.5–1.5 mg / L of carbon nanotubes. As an optional embodiment of this 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 this invention, the carbon nanotubes may include multi-walled carbon nanotubes.

[0039] In this invention, the induction culture medium contains 20-30 g / L of sucrose. As an optional embodiment of this 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 this invention, the induction culture medium comprises 6.5–7.0 g / L of agar. As an optional embodiment of this 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 this invention, the pH of the induction culture medium can be 5.75-5.85 or 5.8.

[0042] As an optional embodiment of the present invention, in addition to the above components, other components that can be used as induction medium may also be added to the induction medium based on B5, or no additional components may be added.

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

[0044] In this invention, the culture medium includes a proliferation medium, the composition of which includes: using B5 or MS as the basal medium, and further comprising: 0.3–1.0 mg / L of chlorhexidine, 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, and 0.5–1.5 mg / L of 2,4-D. The culture medium contains 2.5–3.5 mg / L of acid-hydrolyzed casein, 0.5–1.0 mg / L of carbon nanotubes, 20–30 g / L of sucrose, and 6.5–7.0 g / L of agar; the pH of the culture medium is 5.75–5.85.

[0045] As an optional embodiment of the present invention, the proliferation medium uses B5 or MS as the basic medium and further adds: 0.3-1.0 mg / L of chlorhexidine, 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 medium is 5.75-5.85.

[0046] In another optional embodiment of the present invention, the proliferation medium uses B5 or MS as the basic medium and further adds: 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 medium is 5.75-5.85.

[0047] In another optional embodiment of the present invention, the proliferation medium uses B5 or MS as the basic medium and further adds: 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 medium is 5.75-5.85.

[0048] In this invention, the proliferation medium uses B5 or MS as the basal medium, preferably B5. During callus proliferation, using B5 or MS as the basal medium can better promote callus proliferation and increase the callus proliferation coefficient.

[0049] In this invention, the proliferation medium contains 0.3–1.0 mg / L of atrazine. As an optional embodiment of this invention, the mass concentration of atrazine 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 this invention, the proliferation medium contains 0.3–1.0 mg / L of TDZ. As an optional embodiment of this 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 this invention, the proliferation medium contains 0.3–1.0 mg / L of KT. 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 culture medium may include any one of thiamethoxam, TDZ, and KT.

[0050] In this invention, the proliferation medium contains 2,4-D at a concentration of 2.5–3.5 mg / L. As an optional embodiment of this 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 this invention, the proliferation medium comprises 0.5–1.0 mg / L of acid-hydrolyzed casein. As an optional embodiment of this 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 this invention, the proliferation culture medium comprises 0.5–1.5 mg / L of carbon nanotubes. As an optional embodiment of this 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 this invention, the carbon nanotubes may include multi-walled carbon nanotubes.

[0053] In this invention, the proliferation medium comprises 20-30 g / L of sucrose. As an optional embodiment of this 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 this invention, the proliferation medium comprises 6.5–7.0 g / L of agar. As an optional embodiment of this 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 this invention, the pH of the proliferation medium can be 5.75-5.85 or 5.8.

[0056] As an optional embodiment of the present invention, after adding the above components to the B5 or MS culture medium, other components that can be used in the culture medium may also be added, or no additional components may be added.

[0057] The proliferation medium provided by this invention provides an organic nitrogen source for cell division, thereby promoting the proliferation of ginseng callus tissue. The oxytocin, TDZ, or KT promotes cell division and synergistically promotes cell growth with auxin 2,4-D. 2,4-D can dedifferentiate differentiated tissues and cells while simultaneously promoting cell growth. The carbon nanotubes promote the absorption of water and nutrients. The proliferation medium provided by this invention can significantly increase the proliferation coefficient of callus tissue.

[0058] In this invention, the culture medium further includes an anti-browning proliferation culture medium, which is composed of the proliferation culture medium described in the above technical solution, and further contains 1.0-2.0 g / L glutathione, 1.0-2.0 g / L vitamin C and 0-1.5 g / L activated carbon.

[0059] In this invention, the anti-browning proliferation culture medium includes 1.0–2.0 g / L of glutathione. As an optional embodiment of this 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 this invention, the anti-browning proliferation culture medium includes 1.0–2.0 g / L of vitamin C. As an optional embodiment of this invention, the mass concentration of 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 this invention, the anti-browning proliferation culture medium includes 0-1.5 g / L of activated carbon. As an optional embodiment of this 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 this invention, the anti-browning proliferation medium uses B5 or MS as the basal medium, preferably B5. During callus proliferation, using B5 or MS as the basal medium can better promote callus proliferation and increase the callus proliferation coefficient.

[0063] In this invention, the anti-browning proliferation medium includes 0.3–1.0 mg / L of atrazine. As an optional embodiment of this invention, the mass concentration of atrazine 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 this invention, the anti-browning proliferation medium includes 0.3–1.0 mg / L of TDZ. As an optional embodiment of this 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 this invention, the anti-browning proliferation medium includes 0.3–1.0 mg / L of KT. 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 culture medium may include any one of atrazine, TDZ, and KT.

[0064] In this invention, the anti-browning proliferation medium contains 2,4-D at a concentration of 2.5–3.5 mg / L. As an optional embodiment of this 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 this invention, the anti-browning proliferation medium includes 0.5–1.0 mg / L of acid-hydrolyzed casein. As an optional embodiment of this 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 this invention, the anti-browning proliferation culture medium includes 0.5–1.5 mg / L of carbon nanotubes. As an optional embodiment of this 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 this invention, the carbon nanotubes may include multi-walled carbon nanotubes.

[0067] In this invention, the anti-browning proliferation culture medium comprises 20-30 g / L of sucrose. As an optional embodiment of this 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 this invention, the anti-browning proliferation culture medium comprises 6.5–7.0 g / L of agar. As an optional embodiment of this 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 this invention, the pH of the anti-browning proliferation medium can be 5.75-5.85 or 5.8.

[0070] In the anti-browning proliferation culture medium provided by this invention, glutathione and vitamin C mainly act as antioxidants to prevent callus browning; activated carbon mainly acts as an adsorbent to adsorb some browning components generated during the antioxidant process. The results of the embodiments of this invention show that the anti-browning proliferation culture medium provided by this invention can significantly reduce the browning rate of callus tissue and also help to increase the proliferation coefficient of callus tissue.

[0071] This invention provides the application of the culture medium described in the above-mentioned technical solution in the rapid propagation of ginseng. Using the culture medium described in the above-mentioned technical solution of this invention can significantly improve the induction rate and proliferation coefficient of ginseng callus, which is beneficial for obtaining a large amount of ginseng callus in a short period of time, thereby facilitating the rapid propagation of ginseng.

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

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

[0074] This invention does not specifically limit the source of the ginseng inflorescence axis; any conventional ginseng inflorescence axis in the art can be used. In this invention, the inflorescence axis can be a young inflorescence axis. As an optional embodiment of this invention, the inflorescence axis can be taken from a ginseng umbel that is about to open. After obtaining the ginseng inflorescence axis, this invention preferably sterilizes the ginseng inflorescence axis. The length of the ginseng inflorescence axis used for sterilization in this invention can be 3.0–3.5 cm, or 3.0 cm, 3.1 cm, 3.2 cm, 3.3 cm, 3.4 cm, or 3.5 cm. This invention does not specifically limit the sterilization method; any conventional sterilization method in the art can be used. As an optional embodiment of this invention, the sterilization method includes: washing the ginseng inflorescence axis and then disinfecting it. This invention does not specifically limit the washing method; 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, followed by rinsing with sterile water; the washing is accompanied by agitation; the agitation speed is 150 rpm; the agitation time is 30-40 min, or 30 min, 32 min, 34 min, 35 min, 37 min, 39 min, or 40 min; the rinsing time is 40-60 min, or 40 min, 45 min, 50 min, 55 min, or 60 min. As an optional embodiment of the present invention, the disinfection includes a first disinfection and a second disinfection; the first disinfection can be performed using 75% alcohol by volume; the first disinfection time can be 30-45 s, or 30 s, 35 s, 40 s, or 45 s. After the first disinfection is completed, the obtained ginseng inflorescence axis is preferably rinsed; the rinsing can be performed three times; the rinsing can be performed using sterile water. After rinsing, the present invention can perform a second disinfection; the second disinfection can be carried out using a sodium hypochlorite solution with an effective chlorine content of 2.0%; the second disinfection time can be 15-18 minutes, or 15 minutes, 16 minutes, 17 minutes, or 18 minutes. After the second disinfection, the obtained ginseng inflorescence axis is preferably rinsed; the rinsing can be performed 4 times; the rinsing can be performed using sterile water. After rinsing, the present invention obtains sterilized ginseng inflorescence axis.

[0075] After obtaining the sterilized ginseng inflorescence axis, the present invention induces its culture on the induction medium described in the above-mentioned technical solution. After obtaining the sterilized ginseng inflorescence axis, the present invention preferably removes approximately 0.5 cm from both ends of the ginseng inflorescence axis, maintaining its length at 2.0–2.5 cm, but it can also be 2.0 cm, 2.1 cm, 2.2 cm, 2.3 cm, 2.4 cm, or 2.5 cm. After obtaining the 2.0–2.5 cm ginseng inflorescence axis, the present invention inoculates the ginseng inflorescence axis onto the induction medium for induction culture. During inoculation, the present invention preferably places the ginseng inflorescence axis horizontally on the induction medium. In this invention, the induction culture temperature is 24–26°C, or it can be 24°C, 25°C, or 26°C; the induction culture time is 30–35 days, or it can be 30 days, 31 days, 32 days, 33 days, 34 days, or 35 days; the induction culture can be carried out under dark conditions or under light conditions. If the induction culture is carried out under light conditions, the light intensity can be 2000–3000 lx, or it can be 2000 lx, 2500 lx, or 3000 lx; ​​the light exposure time can be 12–16 h / d, or it can be 12, 13, 14, 15, or 16 h / d.

[0076] The present invention employs the cultivation method described in the above technical solution, which can achieve a ginseng callus induction rate of up to 70%.

[0077] In this invention, the cultivation method further includes proliferation culture; the proliferation culture includes:

[0078] Ginseng callus tissue was cultured and proliferated on the proliferation culture medium or the anti-browning proliferation culture medium described in the above technical solution.

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

[0080] In the proliferation culture process described in this invention, the callus tissue can be subcultured after 30-35 days of proliferation culture. Alternatively, the proliferation culture process can be carried out first in the proliferation culture medium described in the above technical solution; after reaching a certain stage, proliferation culture can then be carried out on an anti-browning proliferation culture medium.

[0081] This invention provides the application of the culture medium or culture method described in the above technical solution in the preparation of ginsenosides.

[0082] In this invention, the method for preparing ginsenosides includes:

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

[0084] The present invention can directly extract and separate ginsenosides from the callus tissue; or the callus tissue can be converted into stem cells and cultured in liquid for the extraction and separation of ginsenosides.

[0085] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0086] The ginseng seedlings used in this embodiment of the 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) Acquisition and disinfection of explants: During the flowering period of ginseng, in mid-May, 3.0 cm of the tender inflorescence axis of the ginseng umbel that is about to open was cut, placed in a glass bottle, 10 times the volume of deionized water and 2 drops of liquid detergent were added, and the mixture was shaken and washed at 150 rpm for 30 min, followed by rinsing with running water for 40 min. Then the cleaned inflorescence axis was transferred to a clean bench, disinfected with 75% alcohol for 40 s, and rinsed 3 times with sterile water; then disinfected with sodium hypochlorite solution with an effective chlorine content of 2.0% for 15 min, and rinsed 4 times with sterile water for later use.

[0090] (2) Callus induction: Sterilized young inflorescence axes are placed on an inoculation tray, with approximately 0.5 cm removed from each end, leaving 2.0 cm. The tray is then placed horizontally on callus induction medium for induction culture. Figure 1As shown. The number of explants inoculated in this treatment was 30. The callus induction medium consisted of: basic medium B5, supplemented with 6.5 g / L agar, 20 g / L sucrose, 0.5 mg / L TDZ, 2.0 mg / L 2,4-D, and 1.0 mg / L multi-walled carbon nanotubes. The pH of the induction medium was 5.8. The induction culture conditions were: dark culture at a temperature of 25 ± 1℃. After 15 days of induction culture, the explants began to sprout, their surfaces bulged, and a layer of pale yellowish-green, frosty callus tissue formed; with continued culture, the callus tissue 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 is that the composition of the callus induction culture medium is: the basic culture 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 is that the composition of the callus induction culture medium is: the basic culture 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 is that the composition of the callus induction culture medium is: the basic culture medium is B5, and it also contains 6.5 g / L agar, 20 g / L sucrose, 0.5 mg / L TDZ and 2.0 mg / L 2,4-D, and does not contain multi-walled carbon nanotubes.

[0097] Comparative Example 2

[0098] A method for inducing ginseng callus, the steps are the same as in Example 1, the only difference being: the composition of the callus induction culture medium is: the basic culture medium is B5, and it also contains 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] Callus was induced and cultured for 35 days, and the induction results were statistically analyzed. The results are shown in Table 1.

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

[0102] Example 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] Table 1 shows that the callus induction rate was higher when the concentration of multi-walled carbon nanotubes in the callus induction medium was between 0.5 and 1.5 mg / L. The callus tissue obtained from different induction media was all pale 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 is based on 1 / 2MS 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 is MS medium as the base 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 is based on N6 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 is based on WPM medium.

[0112] Application Example 2

[0113] Examples 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 in Examples 1 and Comparative Examples 3-6

[0115] Example Serial Number Basic culture 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, B5 is the basic culture medium for callus induction, and it has a greater advantage in improving induction compared to 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 is that the composition of the callus induction culture medium is: the basic culture medium is B5, and it also contains 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, and does not contain TDZ.

[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 is that the composition of the callus induction culture medium is: the basic culture medium is B5, and it also contains 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, and does not contain TDZ.

[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 is that the composition of the callus induction culture medium is: the basic culture medium is B5, and it also contains 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, and does not contain TDZ.

[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 is that the composition of the callus induction culture medium is: the basic culture medium is B5, and it also contains 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, and does not contain TDZ.

[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 is that the composition of the callus induction culture medium is: the basic culture medium is B5, and it also contains 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, and does not contain TDZ.

[0127] Application Example 3

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

[0129] Table 3. Results of callus induction culture for comparative examples 7–11

[0130] Example 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, the absence of TDZ has a significant adverse effect on callus induction results, and will greatly reduce 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 is that the composition of the callus induction culture medium is: the basic culture medium is B5, and it also contains 6.5 g / L agar, 20 g / L sucrose, 0.25 mg / L TDZ and 1.5 mg / L multi-walled carbon nanotubes, and does not contain 2,4-D.

[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 is that the composition of the callus induction culture medium is: the basic culture medium is B5, and it also contains 6.5 g / L agar, 20 g / L sucrose, 0.5 mg / L TDZ and 1.5 mg / L multi-walled carbon nanotubes, and does not contain 2,4-D.

[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 is that the composition of the callus induction culture medium is: the basic culture medium is B5, and it also contains 6.5 g / L agar, 20 g / L sucrose, 0.75 mg / L TDZ and 1.5 mg / L multi-walled carbon nanotubes, and does not contain 2,4-D.

[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 is that the composition of the callus induction culture medium is: the basic culture medium is B5, and it also contains 6.5 g / L agar, 20 g / L sucrose, 1.0 mg / L TDZ and 1.5 mg / L multi-walled carbon nanotubes, and does not contain 2,4-D.

[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 is that the composition of the callus induction culture medium is: the basic culture medium is B5, and it also contains 6.5 g / L agar, 20 g / L sucrose, 1.25 mg / L TDZ and 1.5 mg / L multi-walled carbon nanotubes, and does not contain 2,4-D.

[0142] Application Example 4

[0143] Comparative examples 12–16 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. Results of callus induction culture of Comparative Examples 12–16

[0145] Example 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, the absence of 2,4-D has a significant adverse effect on callus induction results, and will greatly reduce the callus induction rate.

[0147] Example 4

[0148] A method for callus proliferation culture, comprising the following steps:

[0149] After inducing and culturing the callus tissue using the method described in Example 1 for 35 days, callus tissues that were pale yellowish-green, had good granularity, and were in a similar growth state were selected for proliferation culture. The inflorescence axis and the callus tissues that had begun to proliferate were transferred together to a proliferation medium for further proliferation culture. Figure 2 As shown. Thirty callus tissue samples were inoculated using MS medium supplemented with 30 g / L sucrose, 6.5 g / L agar, 0.5 mg / L TDZ, 2,4-D 2.5 mg / L, acid-hydrolyzed casein 0.5 mg / L, and multi-walled carbon nanotubes 0.5 mg / L. The pH of the proliferation medium was 5.8. The proliferation culture temperature was 25 ± 1℃, the light intensity was 2000 lx, and the photoperiod was 12 h / d. After 30 days of proliferation culture, the same formulation was used for subculturing. After a total of 55 days of proliferation culture, the callus tissue proliferated and completely covered the inflorescence axis. The proliferation rate of ginseng young inflorescence axis callus tissue is shown in the figure. Figure 3 As shown. Figure 3 The left image shows callus tissue during the proliferation culture process on June 26th; Figure 3 The right image shows callus tissue during its proliferation culture process on June 19th. Figure 3 It can be seen that the proliferation rate of callus tissue was between June 19 and June 26.

[0150] Example 5

[0151] A method for callus proliferation culture, the steps are the same as in Example 4, the difference being that the basic culture medium for the proliferation medium is B5.

[0152] Comparative Example 17

[0153] A method for callus proliferation culture, the steps are the same as in Example 4, the difference being that the basic culture medium for proliferation culture is 1 / 2 MS.

[0154] Comparative Example 18

[0155] A method for callus proliferation culture, the steps are the same as in Example 4, the difference being that the basic culture medium for the proliferation medium is N6.

[0156] Comparative Example 19

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

[0158] Application Example 5

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

[0160] Table 5. Results of proliferation culture in Examples 4-5 and Comparative Examples 17-19

[0161] Example Serial Number Callus tissue (block) Basic culture medium Proliferation coefficient / times 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] As shown in Table 5, when MS and B5 were used as the basic culture media for proliferation culture, the proliferation coefficient of the callus tissue was higher, which was significantly better than the culture effect using other basic culture media.

[0163] Comparative Example 20

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

[0165] Comparative Example 21

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

[0167] Comparative Example 22

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

[0169] Comparative Example 23

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

[0171] Application Example 6

[0172] The proliferation coefficients of callus tissues cultured for 35 days in comparative examples 20–23 were statistically analyzed by weighing, and the results are shown in Table 6.

[0173] Table 6. Results of proliferation culture of Comparative Examples 20–23

[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 culture, comprising the following steps:

[0178] After 60 days of callus proliferation culture using the method described in Example 4, pale yellow-green callus with good granularity and similar growth status was selected for further proliferation culture. The callus was then transferred to an anti-browning proliferation medium for continued proliferation culture. Thirty callus pieces were inoculated for this treatment. The anti-browning proliferation medium consisted of MS as the basal medium, supplemented with 30 g / L sucrose, 6.5 g / L agar, 1.0 g / L glutathione, 1.0 g / L vitamin C, 0.5 g / L activated charcoal, 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. The pH of the anti-browning proliferation medium was 5.8. The proliferation culture conditions were the same as in Example 4.

[0179] The pale yellow-green callus tissue obtained from proliferation culture can be transformed into stem cells, which can then be cultured in liquid and proliferated for the extraction and separation of ginsenosides, or differentiated and cultured to induce the production of clustered shoots 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 in Example 6, the only difference being: the composition of the anti-browning proliferation medium is: MS as the basic medium, supplemented with glutathione 1.5 g / L, vitamin C 1.5 g / L, activated carbon 1.0 g / L, TDZ 0.3 mg / L, 2,4-D 2.5 mg / L, acid-hydrolyzed casein 0.5 mg / L and multi-walled carbon nanotubes 0.5 mg / L.

[0182] Example 8

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

[0184] Comparative Example 24

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

[0188] Application Example 7

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

[0190] Table 7. Culture results of Examples 6-8 and Comparative Examples 24-25

[0191] Example Serial Number Callus tissue (block) Glutathione Vc Activated carbon Browning rate / % Proliferation coefficient / times 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] As shown in Table 7, the simultaneous addition of glutathione, vitamin C, and / or activated carbon to the proliferation medium significantly reduces the browning rate during the proliferation process and also helps to increase the proliferation coefficient.

[0193] Example 9

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

[0195] (1) Acquisition and disinfection of explants: During the flowering period of ginseng, in late May, 3.5 cm of tender inflorescence axis of ginseng umbels that are about to open were cut, placed in a glass bottle, and 10 times the volume of deionized water and 2 drops of liquid detergent were added. The mixture was shaken and washed at 150 rpm for 35 min, and then rinsed with running water for 50 min. The cleaned inflorescence axis was then transferred to a clean bench and disinfected with 75% alcohol for 35 s, and rinsed 3 times with sterile water; then disinfected with sodium hypochlorite solution with an effective chlorine content of 2.0% for 17 min, and rinsed 4 times with sterile water for later use.

[0196] (2) Callus induction: Sterilized young inflorescence axes were placed on inoculation trays, with approximately 0.5 cm removed from each end, leaving 2.5 cm. These were then placed horizontally on callus induction medium for induction culture. The number of explants inoculated was 30. The callus induction medium consisted of B5 basal medium supplemented with 6.5 g / L agar, 20 g / L sucrose, 0.5 mg / L thiamethoxam, 2.0 mg / L 2,4-D, and 1.0 mg / L multi-walled carbon nanotubes. The pH of the callus induction medium was 5.8. Induction culture was conducted in the dark at a temperature of 25 ± 1℃. After 15 days of induction culture, the explants began to sprout, their surfaces bulged, and a layer of pale yellow-green, frost-like callus formed. Continued culture resulted in a continuous increase in callus formation.

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

[0198] Example 10

[0199] A method for callus proliferation culture, comprising the following steps:

[0200] From the callus tissue induced and cultured for 35 days using the method described in Example 9, callus tissues that were pale yellowish-green, had good granularity, and similar growth status were selected for proliferation culture. Specifically, the inflorescence axis and the callus tissue that had begun to proliferate were transferred together to a proliferation medium for proliferation culture. Thirty callus tissues were inoculated for this treatment. The basic medium for proliferation culture was MS, supplemented with 30 g / L sucrose, 6.5 g / L agar, 0.5 mg / L thiamethoxam, 2,4-D 2.5 mg / L, acid-hydrolyzed casein 0.5 mg / L, and multi-walled carbon nanotubes 0.5 mg / L. The pH of the proliferation medium was 5.8. The proliferation culture temperature was 25 ± 1 °C, the light intensity was 2000 lx, and the photoperiod was 12 h / d. After 30 days of proliferation culture, the same formulation was used for subculturing. After 55 days of proliferation culture, the callus tissue proliferated and completely covered the inflorescence axis.

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

[0202] Example 11

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

[0204] After 60 days of callus proliferation culture using the method described in Example 10, pale yellow-green callus with good granularity and similar growth status was selected for further proliferation culture. The callus was then transferred to an anti-browning proliferation medium for continued proliferation culture. Thirty callus pieces were inoculated for this treatment. The anti-browning proliferation medium consisted of MS as the basal medium, supplemented with 30 g / L sucrose, 6.5 g / L agar, 1.0 g / L glutathione, 1.0 g / L vitamin C, 0.5 g / L activated charcoal, 0.3 mg / L chlorhexidine, 2.5 mg / L 2,4-D, 0.5 mg / L acid-hydrolyzed casein, and 0.5 mg / L multi-walled carbon nanotubes. The pH of the anti-browning proliferation medium was 5.8. The proliferation culture conditions were the same as in Example 10, specifically: a proliferation culture temperature of 25 ± 1 °C, a light intensity of 2000 lx, and a photoperiod of 12 h / d. The pale yellow-green callus tissue obtained from proliferation culture can be used for the extraction and separation of ginsenosides, or it can be transformed into stem cells, cultured in liquid, and then 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. 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) Acquisition and disinfection of explants: During the flowering period of ginseng, in early May, 3.0 cm of tender umbel-shaped inflorescence stalks of ginseng that are about to open were cut and placed in a glass bottle. Ten times the volume of deionized water and 2 drops of liquid detergent were added, and the plants were shaken and washed at 150 rpm for 40 min, followed by rinsing with running water for 60 min. The cleaned inflorescence stalks were then transferred to a clean bench, disinfected with 75% alcohol for 45 s, and rinsed 3 times with sterile water; then disinfected with sodium hypochlorite solution with an effective chlorine content of 2.0% for 18 min, and rinsed 4 times with sterile water for later use.

[0209] (2) Callus induction: Sterilized young inflorescence axes were placed on inoculation trays, with approximately 0.5 cm removed from each end, leaving 2.0 cm. These were then placed horizontally on callus induction medium for induction culture. The number of explants inoculated for this treatment was 30. The callus induction medium consisted of B5 basal medium 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 of the callus induction medium was 5.8. Induction culture was conducted in the dark at a temperature of 25 ± 1℃. After 15 days of induction culture, the explants began to sprout, their surfaces bulged, and a layer of pale yellowish-green, frost-like callus formed. With continued culture, the amount of callus increased.

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

[0211] Example 13

[0212] A method for callus proliferation culture, comprising the following steps:

[0213] From the callus tissue induced and cultured for 35 days using the method described in Example 12, callus tissues that were pale yellowish-green, had good granularity, and were in a similar growth state were selected for proliferation culture. The inflorescence axis and the callus tissues that had begun to proliferate were transferred together to a proliferation medium for further proliferation culture. Thirty callus tissues were inoculated for this treatment. The basic medium for proliferation culture was MS, supplemented with 30 g / L sucrose, 6.5 g / L agar, 1.0 mg / L KT, 3.0 mg / L 2,4-D, 1.0 mg / L acid-hydrolyzed casein, and 1.0 mg / L multi-walled carbon nanotubes. The pH of the proliferation medium was 5.8. The proliferation culture temperature was 25 ± 1 °C, the light intensity was 2000 lx, and the photoperiod was 12 h / d. After 30 days of proliferation culture, the same formulation was used for subculturing. After 55 days of proliferation culture, the callus tissues proliferated and completely covered the inflorescence axis.

[0214] Example 14

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

[0216] After 60 days of proliferation culture using the callus proliferation culture method of Example 13, pale yellow-green callus tissues with good granularity and similar growth status were selected for further proliferation culture. The callus tissues were then transferred to an anti-browning proliferation medium for continued proliferation culture. Thirty callus tissues were inoculated for this treatment. The anti-browning proliferation medium consisted of MS basal medium supplemented with 30 g / L sucrose, 6.5 g / L agar, 1.5 g / L glutathione, 1.5 g / L vitamin C, 1.0 g / L activated charcoal, 0.5 mg / L KT, 2.5 mg / L 2,4-D, 1.0 mg / L acid-hydrolyzed casein, and 1.0 mg / L multi-walled carbon nanotubes. The pH of the anti-browning proliferation medium was 5.8. The proliferation culture conditions were the same as in Example 10. The pale yellow-green callus tissues obtained from the proliferation culture can be used for the extraction and separation of ginsenosides, or they can be 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. The browning rate was 23.33% and the proliferation coefficient was 2.90.

[0218] Comparative Example 26

[0219] A method for inducing ginseng callus, with the same steps as in Example 1, except that the petioles of ginseng leaflets are used as explants instead of the ginseng inflorescence axis used in Example 1.

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

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

[0222] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A culture medium for culturing ginseng callus, characterized in that, The culture medium includes an induction medium, which is composed of B5 as the basic medium and also contains: 0.5-1.0 mg / L of chlorhexidine, 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, using B5 as the basic culture medium, the following are also added: 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, using B5 as the basic culture medium, the following additional components were added: 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; The explants used for ginseng callus induction are ginseng inflorescence axes; The carbon nanotubes are multi-walled carbon nanotubes.

2. The culture medium according to claim 1, characterized in that, The culture medium includes a proliferation medium, which is composed of B5 or MS as the basic medium, and further supplemented with: 0.3-1.0 mg / L of chlorhexidine, 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, using B5 or MS as the basal medium, the following are also added: 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, using B5 or MS as the basal medium, the following are also added: 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 medium is 5.75 to 5.

85.

3. The culture medium according to claim 1, characterized in that, The culture medium includes an anti-browning proliferation medium, which is composed of: glutathione 1.0-2.0 g / L, vitamin C 1.0-2.0 g / L and activated charcoal 0-1.5 g / L, based on the proliferation medium components of the culture medium described in claim 2.

4. The application 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, Includes the following steps: The sterilized ginseng inflorescence axis was induced and cultured on the culture medium described in claim 1.

6. The cultivation 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 cultivation method according to claim 5, characterized in that, The cultivation method further includes proliferation culture; the proliferation culture includes: The ginseng callus tissue was cultured and proliferated on the proliferation medium in claim 2 or the anti-browning proliferation medium in claim 3.

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

9. The 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 application according to claim 9, characterized in that, Ginsenosides were extracted and separated from the callus tissue obtained by the culture medium or the culture method described above.

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