Culture method capable of improving physiological activity of calluses and adventitious roots of ginseng

Abstract

The invention relates to a culture method capable of improving physiological activity of ginseng callus and adventitious roots, which comprises the following steps: constructing a nutrition system and an environment system, and placing an induction material in the nutrition system and the environment system for cultivation. The growth regulator is a combination of 2, 4-dichlorphenoxyacetic acid, naphthylacetic acid and 6-benzyladenine, the use amount of cane sugar is increased, and the induction rate of the ginseng stem callus can reach 90.1.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, in particular to a culture method capable of improving the physiological activity of ginseng callus and adventitious roots. Background Art

[0002] In recent years, with the increasing application of natural raw materials in the field of cosmetics, plant-derived active ingredients have become the core driving force of industrial innovation. According to the "Catalogue of Used Cosmetic Raw Materials (2021 Edition)", more than 3,400 plant raw materials have been included in the cosmetics system, highlighting their important position as efficient and safe raw materials. Although traditional extraction processes (such as solvent extraction, supercritical separation, etc.) can obtain plant active substances, they are highly dependent on wild or cultivated resources and face bottlenecks such as long planting cycles, scarce resources, and poor ingredient stability. Taking ginseng as an example, its roots are rich in high-value active substances such as saponins and β-sitosterol, and have significant potential in the field of skin care. However, the traditional culture model has defects such as high bacterial infection rate, low efficiency of adventitious root induction, and insufficient accumulation of physiologically active substances, which seriously restrict industrial applications.

[0003] The rise of plant cell culture technology has provided a new path to solve the above problems. Currently, there are at least the following technical problems in ginseng callus and adventitious root culture:

[0004] Optimization of culture medium components: Existing technologies lack a systematic design of the nutrient system. In particular, the regulatory mechanisms of the inorganic and organic ratios, growth regulator combinations, and concentrations on the synthesis of active ingredients remain unclear.

[0005] Insufficient adaptability of environmental parameters: There is a lack of standardized coordinated control solutions for key environmental factors such as temperature, humidity, and pH;

[0006] Metabolic efficiency bottleneck: The single supply mode of carbon sources such as sucrose in traditional methods makes it difficult to effectively improve the production efficiency of secondary metabolites.

[0007] Therefore, there is an urgent need to develop a cultivation method that integrates the coordinated regulation of nutrition and environment systems. Summary of the Invention

[0008] (1) Technical issues to be resolved

[0009] The technical problem to be solved by the present invention is to provide a culture method capable of improving the physiological activity of ginseng callus and adventitious roots.

[0010] (2) Technical solution

[0011] In order to solve the above problems, the present invention provides a culture method for improving the physiological activity of ginseng callus and adventitious roots, which at least includes constructing a nutrition system and an environmental system;

[0012] The nutrient system includes a basic solvent, inorganic matter, organic matter, a growth regulator, and a coagulant;

[0013] Environmental systems include temperature, humidity, and pH;

[0014] The induction material is placed in the nutrition system and the environmental system for cultivation.

[0015] The organic matter at least includes: thiamine hydrochloride, pyridoxine hydrochloride, niacin, inositol, glycine and sucrose.

[0016] Wherein, the inorganic substances include at least: KNO3, NH4NO3, KH2PO4, MgSO4·7H2O, CaCl2·2H2O, MnSO4·4H2O, H3BO3, KI, ZnSO4·7H2O, Na2MoO4·2H2O, CuSO4·5H2O, CoCl2·6H2O, Na2EDTA, FeSO4·7H2O.

[0017] Wherein, the growth regulator includes at least one or more of 2,4-dichlorophenoxyacetic acid, naphthylacetic acid, 6-benzyladenine and kinetin.

[0018] Furthermore, the growth regulators are 2,4-dichlorophenoxyacetic acid, naphthylacetic acid and 6-benzyladenine.

[0019] Furthermore, the mass ratio of 2,4-dichlorophenoxyacetic acid, naphthylacetic acid and 6-benzyladenine in the growth regulator is 4:3:4.

[0020] Wherein, sucrose is additionally added to the growth regulator.

[0021] Wherein, the nutritional system includes

[0022] Inorganic substances: 1900 mg / L KNO3, 165 mg / L NH4NO3, 170 mg / L KH2PO4, 370 mg / L MgSO4·7H2O, 440 mg / L CaCl2·2H2O, 22.3 mg / L MnSO4·4H2O, 6.2 mg / L H3BO3, 0.83 mg / L KI, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.25 mg / L CuSO4·5H2O, 0.25 mg / L CoCl2·6H2O, 37.3 mg / L Na2EDTA, 27.8 mg / L FeSO4·7H2O;

[0023] Organic matter: 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L niacin, 100 mg / L inositol, 2 mg / L glycine, and 30,000 mg / L sucrose;

[0024] Growth regulators: 0.05-5 mg / L 2,4-dichlorophenoxyacetic acid, 0.05-5 mg / L naphthaleneacetic acid, 0.05-10 mg / L 6-benzyladenine, 0.05-5 mg / L kinetin;

[0025] Coagulant: 10000 mg / L agar or plant gel;

[0026] The environmental system includes: temperature of 23-28°C, humidity of 65-80%, and pH of 5.8;

[0027] The induction material is placed in the nutrition system and the environmental system for cultivation.

[0028] Wherein, the nutritional system includes

[0029] Inorganic substances: 1900 mg / L KNO3, 165 mg / L NH4NO3, 170 mg / L KH2PO4, 370 mg / L MgSO4·7H2O, 440 mg / L CaCl2·2H2O, 22.3 mg / L MnSO4·4H2O, 6.2 mg / L H3BO3, 0.83 mg / L KI, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.25 mg / L CuSO4·5H2O, 0.25 mg / L CoCl2·6H2O, 37.3 mg / L Na2EDTA, 27.8 mg / L FeSO4·7H2O;

[0030] Organic matter: 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L niacin, 100 mg / L inositol, 2 mg / L glycine, and 30,000 mg / L sucrose;

[0031] Growth regulators: 2.0 mg / L 2,4-dichlorophenoxyacetic acid, 1.5 mg / L naphthaleneacetic acid, 2.0 mg / L 6-benzyladenine;

[0032] An additional 35,000 mg / L of sucrose was added to the growth regulator;

[0033] Coagulant: 10000 mg / L agar or plant gel;

[0034] The environmental system includes: temperature of 23-28°C, humidity of 65-80%, and pH of 5.8;

[0035] The induction material is placed in the nutrition system and the environmental system for cultivation.

[0036] (3) Beneficial effects

[0037] The above technical solution of the present invention has the following advantages: the culture method provided by the present invention and the synergistic effect of growth regulators can achieve an induction rate of ginseng stem callus of up to 90.1%. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The drawings of the present invention are provided for illustrative purposes only, and the proportions and quantities of the components in the drawings may not necessarily be consistent with the actual product.

[0039] Figure 1 This is a comparison photo of the adventitious root growth results in the embodiment of the present invention;

[0040] Figure 2 These are comparison photos of callus amplification results in the examples of the present invention. DETAILED DESCRIPTION

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0042] The present invention provides a method for increasing the physiological activity of ginseng callus and adventitious roots, which comprises at least:

[0043] Construct nutrition system and environmental system;

[0044] The nutrient system includes a basic solvent, inorganic matter, organic matter, a growth regulator, and a coagulant;

[0045] The base solvent may be water.

[0046] The organic matter at least includes: thiamine hydrochloride, pyridoxine hydrochloride, niacin, inositol, glycine and sucrose.

[0047] Inorganic substances include at least: KNO3, NH4NO3, KH2PO4, MgSO4·7H2O, CaCl2·2H2O, MnSO4·4H2O, H3BO3, KI, ZnSO4·7H2O, Na2MoO4·2H2O, CuSO4·5H2O, CoCl2·6H2O, Na2EDTA, FeSO4·7H2O.

[0048] The growth regulator includes at least one or more of 2,4-dichlorophenoxyacetic acid, naphthylacetic acid, 6-benzyladenine and kinetin.

[0049] The solidifying agent is agar or vegetable gel;

[0050] Environmental systems include temperature, humidity, and pH;

[0051] The induction material is placed in the nutrition system and the environmental system for cultivation.

[0052] Preferably, the growth regulator is 2,4-dichlorophenoxyacetic acid, naphthylacetic acid and 6-benzyladenine.

[0053] More preferably, the mass ratio of 2,4-dichlorophenoxyacetic acid, naphthylacetic acid and 6-benzyladenine in the growth regulator is 4:3:4.

[0054] Still more preferably, sucrose is additionally added to the growth regulator.

[0055] In one embodiment, the present invention provides a method for cultivating ginseng callus and adventitious roots to improve their physiological activity, wherein the nutritional system comprises:

[0056] Inorganic substances: 1900 mg / L KNO3, 165 mg / L NH4NO3, 170 mg / L KH2PO4, 370 mg / L MgSO4·7H2O, 440 mg / L CaCl2·2H2O, 22.3 mg / L MnSO4·4H2O, 6.2 mg / L H3BO3, 0.83 mg / L KI, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.25 mg / L CuSO4·5H2O, 0.25 mg / L CoCl2·6H2O, 37.3 mg / L Na2EDTA, 27.8 mg / L FeSO4·7H2O;

[0057] Organic matter: 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L niacin, 100 mg / L inositol, 2 mg / L glycine, and 30,000 mg / L sucrose;

[0058] Growth regulators: 0.05-5 mg / L 2,4-dichlorophenoxyacetic acid, 0.05-5 mg / L naphthaleneacetic acid, 0.05-10 mg / L 6-benzyladenine, 0.05-5 mg / L kinetin;

[0059] Coagulant: 10000 mg / L agar or plant gel;

[0060] The environmental system includes: temperature of 23-28°C, humidity of 65-80%, and pH of 5.8;

[0061] The induction material is placed in the nutrition system and the environmental system for cultivation.

[0062] In the present invention, the ginseng roots and tender stems used as induction materials can be treated as follows: first, they are cleaned with tap water, rinsed under running water for 1-2 hours, then washed in a 70% ethanol solution for 30s-60s, then soaked in a 5%-15% NaClO bleach, shaken at a speed of 70-130 rpm for 5-25 minutes, and then rinsed with sterile distilled water 4-5 times under a sterile environment.

[0063] The ginseng rhizomes are peeled to retain a tissue tuber with a length of 0.5-1 cm, and the tissue is cut and inoculated into the nutrition system and the environmental system for cultivation. The ginseng callus tissue is induced after culturing for 3-6 weeks.

[0064] In another embodiment, the present invention provides a culture method for improving the physiological activity of ginseng callus and adventitious roots, wherein the nutrition system comprises:

[0065] Basic solvent: 1L water;

[0066] Inorganic substances: 1900 mg / L KNO3, 165 mg / L NH4NO3, 170 mg / L KH2PO4, 370 mg / L MgSO4·7H2O, 440 mg / L CaCl2·2H2O, 22.3 mg / L MnSO4·4H2O, 6.2 mg / L H3BO3, 0.83 mg / L KI, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.25 mg / L CuSO4·5H2O, 0.25 mg / L CoCl2·6H2O, 37.3 mg / L Na2EDTA, 27.8 mg / L FeSO4·7H2O;

[0067] Organic matter: 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L niacin, 100 mg / L inositol, 2 mg / L glycine, and 30,000 mg / L sucrose;

[0068] Growth regulators: 2.0 mg / L 2,4-dichlorophenoxyacetic acid, 1.5 mg / L naphthaleneacetic acid, 2.0 mg / L 6-benzyladenine;

[0069] An additional 35,000 mg / L of sucrose was added to the growth regulator;

[0070] Coagulant: 10000 mg / L agar or plant gel;

[0071] The environmental system includes: temperature of 23-28°C, humidity of 65-80%, and pH of 5.8;

[0072] The ginseng roots and tender stems used as induction materials can be treated as follows: first wash them with tap water, rinse them under running water for 1-2 hours, then wash them in 70% ethanol solution for 30s-60s, then soak them in 5%-15% NaClO bleach, shake them at a speed of 70-130rpm for 5-25min, and then rinse them with sterilized distilled water 4-5 times under a sterile environment.

[0073] The ginseng rhizomes are peeled to retain 0.5-1 cm long tissue tubers, and the tissues are cut and inoculated into the nutrition system and the environmental system for cultivation to induce ginseng callus tissue. Figure 1 As shown in the figure, the growth of adventitious roots can be clearly observed. Figure 2 As shown, callus expansion can be clearly observed.

[0074] In this example, the regulatory effects of a combination of growth regulators on the callus and adventitious root induction rate of ginseng were tested.

[0075] The ginseng roots used as induction materials are first cleaned with tap water and rinsed under running water for 1-2 hours. The roots are then rinsed in a 70% ethanol solution for 30-60 seconds. The roots are then immersed in a 5%-15% NaClO bleach solution, shaken at 70-130 rpm for 5-25 minutes, and then rinsed 4-5 times with sterile distilled water under a sterile environment. The ginseng roots are peeled, retaining a 0.5-1 cm length of tissue tuber. The roots are then cultured in the nutrient and environmental systems disclosed herein.

[0076] The other factors remained unchanged, and only the ratio of growth regulators 2,4-dichlorophenoxyacetic acid, naphthylacetic acid, and 6-benzyladenine was used as a variable.

[0077] Experimental results list:

[0078]

[0079] Among them, 2.0 mg / L 2,4-dichlorophenoxyacetic acid, 1.5 mg / L naphthylacetic acid, and 2.0 mg / L 6-benzyladenine were the best combination, with an induction rate of 90.1%.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that not every embodiment contains only one independent technical solution. In the absence of a conflict of solutions, the various technical features mentioned in each embodiment can be combined in any manner to form other implementation methods that can be understood by those skilled in the art.

[0081] In addition, without departing from the scope of the present invention, the technical solutions described in the aforementioned embodiments may be modified, or some of the technical features thereof may be replaced by equivalents, without causing the essence of the corresponding technical solutions to depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for increasing the physiological activity of ginseng callus and adventitious roots, characterized in that: At least: Construct nutrition system and environmental system; The nutrient system includes a basic solvent, inorganic matter, organic matter, a growth regulator, and a coagulant; Environmental systems include temperature, humidity, and pH; The induction material is placed in the nutrition system and the environmental system for cultivation.

2. A method for increasing the physiological activity of ginseng callus and adventitious roots according to claim 1, characterized in that: The organic matter at least includes: thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid, inositol, glycine and sucrose.

3. The method for increasing the physiological activity of ginseng callus and adventitious roots according to claim 1, wherein: The inorganic substances include at least: KNO3, NH4NO3, KH2PO4, MgSO4·7H2O, CaCl2·2H2O, MnSO4·4H2O, H3BO3, KI, ZnSO4·7H2O, Na2MoO4·2H2O, CuSO4·5H2O, CoCl2·6H2O, Na2EDTA, and FeSO4·7H2O.

4. A method for increasing the physiological activity of ginseng callus and adventitious roots according to claim 1, characterized in that: The growth regulator comprises at least one or more of 2,4-dichlorophenoxyacetic acid, naphthylacetic acid, 6-benzyladenine and kinetin.

5. A method for increasing the physiological activity of ginseng callus and adventitious roots according to claim 4, characterized in that: The growth regulators are 2,4-dichlorophenoxyacetic acid, naphthylacetic acid and 6-benzyladenine.

6. A method for increasing the physiological activity of ginseng callus and adventitious roots according to claim 5, characterized in that: The mass ratio of 2,4-dichlorophenoxyacetic acid, naphthylacetic acid and 6-benzyladenine in the growth regulator is 4:3:

4.

7. A method for increasing the physiological activity of ginseng callus and adventitious roots according to claim 6, characterized in that: Sucrose is additionally added to the growth regulator.

8. The method for increasing the physiological activity of ginseng callus and adventitious roots according to claim 1, wherein: The nutritional system comprises: Inorganic substances: 1900 mg / L KNO3, 165 mg / L NH4NO3, 170 mg / L KH2PO4, 370 mg / L MgSO4·7H2O, 440 mg / L CaCl2·2H2O, 22.3 mg / L MnSO4·4H2O, 6.2 mg / L H3BO3, 0.83 mg / L KI, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.25 mg / L CuSO4·5H2O, 0.25 mg / L CoCl2·6H2O, 37.3 mg / L Na2EDTA, 27.8 mg / L FeSO4·7H2O; Organic matter: 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L niacin, 100 mg / L inositol, 2 mg / L glycine, and 30,000 mg / L sucrose; Growth regulators: 0.05-5 mg / L 2,4-dichlorophenoxyacetic acid, 0.05-5 mg / L naphthaleneacetic acid, 0.05-10 mg / L 6-benzyladenine, 0.05-5 mg / L kinetin; Coagulant: 10000 mg / L agar or plant gel; The environmental system includes: temperature of 23-28°C, humidity of 65-80%, and pH of 5.8; The induction material is placed in the nutrition system and the environmental system for cultivation.

9. The method for cultivating ginseng callus and adventitious roots for improving physiological activity according to claim 1, wherein: The nutritional system comprises: Inorganic substances: 1900 mg / L KNO3, 165 mg / L NH4NO3, 170 mg / L KH2PO4, 370 mg / L MgSO4·7H2O, 440 mg / L CaCl2·2H2O, 22.3 mg / L MnSO4·4H2O, 6.2 mg / L H3BO3, 0.83 mg / L KI, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.25 mg / L CuSO4·5H2O, 0.25 mg / L CoCl2·6H2O, 37.3 mg / L Na2EDTA, 27.8 mg / L FeSO4·7H2O; Organic matter: 0.1 mg / L thiamine hydrochloride, 0.5 mg / L pyridoxine hydrochloride, 0.5 mg / L niacin, 100 mg / L inositol, 2 mg / L glycine, and 30,000 mg / L sucrose; Growth regulators: 2.0 mg / L 2,4-dichlorophenoxyacetic acid, 1.5 mg / L naphthaleneacetic acid, 2.0 mg / L 6-benzyladenine; An additional 35,000 mg / L of sucrose was added to the growth regulator; Coagulant: 10000 mg / L agar or plant gel; The environmental system includes: temperature of 23-28°C, humidity of 65-80%, and pH of 5.8; The induction material is placed in the nutrition system and the environmental system for cultivation.

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