Tissue culture medium and tissue culture method for culturing ginseng stem cells

Abstract

The invention belongs to the technical field of plant biology, and particularly relates to a tissue culture medium and a tissue culture method for culturing ginseng stem cells. The invention provides a tissue culture medium and a tissue culture method for culturing ginseng stem cells. The tissue culture medium comprises a germination medium, a growth medium, an induction medium and a proliferation medium. Under the combined action of NAA, 2, 4-D, 6-BA, KT, gibberellin GA3, acid hydrolyzed casein, carbon nanotubes, activated carbon, reduced glutathione and vitamin C, the tissue culture medium provided by the invention is beneficial to induction and proliferation of calluses and further conversion of the calluses into stem cells, so that browning of the calluses is effectively prevented. Results of the embodiment show that the tissue culture medium can induce ginseng seeds to germinate in a short time, the browning rate is 10%, the browning rate is low, a large number of ginseng stem cells can be rapidly obtained, and a foundation is laid for application of the ginseng stem cells.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology, and particularly relates to a tissue culture medium and a tissue culture method for culturing ginseng stem cells. Background Art

[0002] Ginseng (Panaxginseng) is a perennial herbaceous plant of the genus Panax in the Araliaceae family. Ginseng can reach a height of 60 cm; the main root is fusiform; the leaves are palmately compound, 3 to 6 in whorls at the top of the stem; the umbel is solitary at the top of the stem, the inflorescence is about 1.5 cm in diameter, with 30 to 50 small flowers; the pedicel is usually longer than the leaves, the pedicel is 15 to 30 cm long, with longitudinal stripes; the flowers are light yellow-green. The fruit is oblate, bright red, 6 to 7 mm in diameter; the seeds are kidney-shaped and milky white.

[0003] Ginseng is distributed in eastern Liaoning, eastern Jilin and eastern Heilongjiang in my country. It is generally grown in deciduous broad-leaved forests or coniferous broad-leaved mixed forests at an altitude of several hundred meters. Ginseng prefers sandy loam with loose texture, good ventilation, good drainage and fertile nutrients; Ginseng likes shade, and a cool and humid climate is beneficial to its growth; it tolerates low temperatures, avoids strong direct sunlight, and prefers diffused weak light.

[0004] The fleshy root of ginseng is a tonic medicine, suitable for regulating blood pressure, restoring heart function, neurasthenia and physical weakness, and also has the effects of expectoration, stomachic, diuretic and excitement. The stems, leaves, flowers, fruits and processing by-products of ginseng are all raw materials for light industry. Ginseng cultivation is an important project for farmers' diversified operations and can provide a large amount of taxes and profits to the country.

[0005] Stem cells are a type of multipotent cells with the ability to self-replicate. Under certain conditions, they can differentiate into multiple functional cells. According to the developmental stage of stem cells, they are divided into embryonic stem cells and adult stem cells. According to the developmental potential of stem cells, they are divided into totipotent stem cells, pluripotent stem cells and unipotent stem cells. Stem cells are a type of immature, underdifferentiated cells that have the potential to regenerate various tissues, organs and the human body. They are called "universal cells" in the medical field. In the prior art, ginseng roots are usually used as explants, and dedifferentiation is difficult. Summary of the invention

[0006] The invention provides a tissue culture medium and a tissue culture method for culturing ginseng stem cells. The tissue culture medium of the invention can be used to obtain ginseng stem cells by tissue culture with ginseng seeds as explants.

[0007] In order to solve the above technical problems, the present invention proposes the following technical solutions:

[0008] The present invention provides a tissue culture medium for culturing ginseng stem cells, the tissue culture medium comprising a germination medium, a growth medium, an induction medium and a proliferation medium;

[0009] The germination medium uses MS or 1 / 2MS as a basic medium, and also includes 2.0-4.0 mg / L GA3, 0.5-2.0 g / L activated carbon, 30 g / L sucrose and 6.5-7.0 g / L agar;

[0010] The growth medium uses B5 as the basic medium and also includes 1.0-3.0 mg / L 6-BA, 0.3-0.5 mg / L NAA, 30 g / L sucrose and 6.5-7.0 g / L agar;

[0011] The induction culture medium uses B5 or MS as a basic culture medium, and also includes 0.2-0.5 mg / L KT, 1.5-2.5 mg / L 2,4-D, 0.5-1.0 g / L acid hydrolyzed casein, 1.0-2.0 mg / L carbon nanotubes, 30 g / L sucrose and 6.5-7.0 g / L agar;

[0012] The proliferation culture medium uses B5 as a basic culture medium and also includes 0.5-0.8 mg / L KT, 2.0-3.0 mg / L 2,4-D, 1.0-2.0 mg / L carbon nanotubes, 0.5-2.0 g / L acid hydrolyzed casein, 0.2-1.5 g / L reduced glutathione, 0.5-1.0 g / L vitamin C, 0.5-2.0 g / L activated carbon, 30 g / L sucrose and 6.5-7.0 g / L agar.

[0013] Preferably, the pH value of the germination medium is 5.75-5.85, the pH value of the growth medium is 5.75-5.85, the pH value of the induction medium is 5.75-5.85, and the pH value of the proliferation medium is 5.75-5.85.

[0014] The present invention provides a tissue culture method for ginseng stem cells, the tissue culture method adopts the tissue culture medium described in the above technical solution, and comprises the following steps:

[0015] Inoculating ginseng seeds into a germination medium for germination culture to obtain germinated seeds; the ginseng seeds are seeds that have been subjected to vernalization treatment;

[0016] The germinated seeds are transferred to a growth medium for seedling growth culture to obtain sterile seedlings with a height of 5.0 to 8.0 cm;

[0017] Cutting the stem segments of the sterile seedlings and transferring them to an induction medium for induction culture to obtain callus tissue;

[0018] The callus tissue is transferred to a proliferation culture medium for proliferation culture to obtain ginseng stem cells.

[0019] Preferably, the ginseng seeds are peeled seeds and contain embryo and endosperm.

[0020] Preferably, the ginseng seeds are cleaned and disinfected before inoculation, and the disinfection includes first disinfecting with 75% alcohol for 30 to 45 seconds, and rinsing with sterile water for 2 to 3 times; then disinfecting with sodium hypochlorite solution with an effective chlorine content of 2.0% to 3.0% for 6 to 9 minutes, and rinsing with sterile water for 4 to 5 times.

[0021] Preferably, the temperatures of the germination culture, seedling growth culture, induction culture and proliferation culture are all 25±1°C.

[0022] Preferably, the germination culture is dark culture; the seedling growth culture and proliferation culture are both carried out under light-dark alternation conditions; and the induction culture can be carried out under light-dark alternation conditions or in darkness.

[0023] Preferably, the time of the light culture with alternating light and dark is 12 to 16 h / d; the intensity of the light culture is 800 to 1200 lx; ​​and the time of the dark culture with alternating light and dark is 8 to 12 h / d.

[0024] Preferably, the germination culture time is 15 to 32 days; the seedling growth culture time is 35 to 60 days; the induction culture time is 28 to 45 days; and the proliferation culture time is 60 to 80 days.

[0025] Preferably, the length of the stem segment is 1.5 to 2.5 cm.

[0026] Beneficial effects of the present invention: The present invention provides a tissue culture medium for culturing ginseng stem cells, wherein the tissue culture medium comprises a germination medium, a growth medium, an induction medium and a proliferation medium.

[0027] In the tissue culture medium provided by the present invention, the main function of gibberellin (GA3) is to promote cell elongation and embryoid growth. Activated carbon can simulate the dark conditions for seed germination, and can also effectively reduce the oxidation reaction and pollution of growth regulators, ensure the purity of the culture environment, improve the success rate of culture, and reduce the browning rate. Naphthaleneacetic acid, as a plant growth regulator, promotes cell division, promotes growth and enhances resistance; 6-benzylaminopurine is a synthetic cytokinin plant growth regulator, which promotes cell division and induces bud production in tissue culture medium; 2,4-dichlorophenoxyacetic acid (2,4-D) is a plant growth regulator that stimulates plant growth. 2,4-D, KT, carbon nanotubes and acid hydrolyzed casein can promote the formation and proliferation of callus. Reduced glutathione and vitamin C can reduce the browning rate of callus.

[0028] The combined action of basic culture medium, NAA, 2,4-D, 6-BA, KT, gibberellin GA3, acid hydrolyzed casein, carbon nanotubes, activated carbon, reduced glutathione and vitamin C is conducive to inducing callus tissue, proliferation, and further transformation into stem cells, effectively preventing callus tissue from browning. The results of the embodiment show that the tissue culture medium of the present invention can induce ginseng seeds to germinate in a short time, obtain ginseng stem cells, and reduce the browning rate.

[0029] The present invention also provides a tissue culture method for ginseng stem cells. According to the tissue culture method of the present invention, seeds treated with low temperature and GA3 can germinate after two weeks, and the germination rate of the seeds can reach 70%. The young stem segments of the obtained seedlings can be used for inducing callus groups, and the induction rate of callus tissue reaches 90%. The browning rate of callus tissue is low, and the proliferation coefficient of callus tissue in solid culture medium is about 5 times, so that a large number of ginseng stem cells can be quickly obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the morphological diagram of the seeds of Example 1 after germination for 25 days;

[0031] Figure 2 This is the morphological diagram of the seedlings grown and cultured for 28 days in Example 1;

[0032] Figure 3 The morphological diagram of callus induction culture of Example 1, wherein the left side is the morphological diagram of callus induction culture 7 days, the right side marked 6.19 is the morphological diagram of callus induction culture 21 days, and the right side marked 6.26 is the morphological diagram of callus induction culture 28 days;

[0033] Figure 4 This is a morphological diagram of callus induction culture for 25 days in Example 1;

[0034] Figure 5This is a morphological picture of the callus cells (i.e. stem cells) of Example 1 under a microscope. DETAILED DESCRIPTION

[0035] The invention provides a tissue culture medium for culturing ginseng stem cells. The tissue culture medium comprises a germination medium, a growth medium, an induction medium and a proliferation medium.

[0036] The germination culture medium of the present invention uses MS or 1 / 2MS as a basic culture medium and further comprises 2.0-4.0 mg / L GA3, 0.5-2.0 g / L activated carbon, 30 g / L sucrose and 6.5-7.0 g / L agar.

[0037] As an optional embodiment, the concentration of GA3 in the germination medium provided by the present invention can be 2.0-4.0 mg / L; in a specific embodiment of the present invention, the concentration of GA3 in the germination medium can be 2.0, 2.5, 3.0, 3.5 or 4.0 mg / L. The concentration of activated carbon in the germination medium can be 0.5-2.0 g / L; in a specific embodiment of the present invention, the concentration of activated carbon in the germination medium can be 0.5, 1.0, 1.5 or 2.0 g / L. The concentration of agar in the germination medium can be 6.5-7.0 g / L, and in a specific embodiment of the present invention, the concentration of agar in the germination medium can be 6.5, 6.7, 6.9 or 7.0 g / L. In the present invention, GA3 at an appropriate concentration can promote the germination of ginseng seeds, increase the germination rate, and shorten the germination process; activated carbon simulates the soil germination environment of seeds and promotes the germination of ginseng seeds. In the present invention, the pH value of the germination medium may be 5.75-5.85, or 5.80.

[0038] The growth medium provided by the invention uses B5 as a basic medium and further comprises 1.0-3.0 mg / L 6-BA, 0.3-0.5 mg / L NAA, 30 g / L sucrose and 6.5-7.0 g / L agar.

[0039] As an optional embodiment, the concentration of 6-BA in the growth medium provided by the present invention can be 1.0-3.0 mg / L; in a specific embodiment of the present invention, the concentration of 6-BA in the growth medium can be 1.0, 1.5, 2.0, 2.5 or 3.0 mg / L. The concentration of NAA in the growth medium can be 0.3-0.5 mg / L; in a specific embodiment of the present invention, the concentration of NAA in the growth medium can be 0.3, 0.35, 0.4, 0.45 or 0.5 mg / L. The concentration of agar in the growth medium can be 6.5-7.0 g / L, and in a specific embodiment of the present invention, the concentration of agar in the growth medium can be 6.5, 6.7, 6.9 or 7.0 g / L. In the present invention, 6-BA and NAA can promote the growth of ginseng buds at appropriate concentrations to obtain sterile seedlings. In the present invention, the pH value of the growth medium can be 5.75-5.85, or 5.80.

[0040] The induction culture medium provided by the invention uses B5 or MS as a basic culture medium and further comprises 0.2-0.5 mg / L KT, 1.5-2.5 mg / L 2,4-D, 0.5-1.0 g / L acid hydrolyzed casein, 1.0-2.0 mg / L carbon nanotubes, 30 g / L sucrose and 6.5-7.0 g / L agar.

[0041] As an optional embodiment, the concentration of KT in the induction medium provided by the present invention can be 0.2-0.5 mg / L, or 0.3-0.4 mg / L; in a specific embodiment of the present invention, the concentration of KT in the induction medium can be 0.2, 0.3, 0.4 or 0.5 mg / L. The concentration of 2,4-D in the induction medium can be 1.5-2.5 mg / L, or 1.8-2.3 mg / L; in a specific embodiment of the present invention, the concentration of 2,4-D in the induction medium can be 1.5, 1.7, 1.9, 2.3 or 2.5 mg / L. The concentration of acid-hydrolyzed casein in the induction medium can be 0.5-1.0 g / L, or 0.6-0.9 g / L; in a specific embodiment of the present invention, the concentration of acid-hydrolyzed casein in the induction medium can be 0.5, 0.7, 0.8, 0.9 or 1.0 g / L. The concentration of carbon nanotubes in the induction medium can be 1.0-2.0 mg / L, or 1.2-1.8 mg / L; in a specific embodiment of the present invention, the carbon nanotubes in the induction medium can be multi-walled carbon nanotubes, and the concentration of multi-walled carbon nanotubes can be 1.0, 1.2, 1.4, 1.6, 1.8 or 2.0 mg / L. The concentration of agar in the induction medium can be 6.5-7.0 g / L, or 6.6-6.8 g / L; in a specific embodiment of the present invention, the concentration of agar in the induction medium can be 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0 g / L. In the present invention, KT, 2,4-D, acid hydrolyzed casein and carbon nanotubes can promote the formation of ginseng callus at an appropriate concentration ratio. In the present invention, the pH value of the induction medium can be 5.75-5.85, or 5.80.

[0042] The proliferation culture medium provided by the present invention uses B5 as a basic culture medium and also includes 0.5-0.8 mg / L KT, 2.0-3.0 mg / L 2,4-D, 1.0-2.0 mg / L carbon nanotubes, 0.5-2.0 g / L acid hydrolyzed casein, 0.2-1.5 g / L reduced glutathione, 0.5-1.0 g / L vitamin C, 0.5-2.0 g / L activated carbon, 30 g / L sucrose and 6.5-7.0 g / L agar.

[0043] As an optional embodiment, the concentration of KT in the proliferation medium provided by the present invention can be 0.5-0.8 mg / L, or 0.6-0.7 mg / L; in a specific embodiment of the present invention, the concentration of KT in the proliferation medium can be 0.5, 0.6, 0.7 or 0.8 mg / L. The concentration of 2,4-D in the proliferation medium can be 2.0-3.0 mg / L, or 2.3-2.8 mg / L; in a specific embodiment of the present invention, the concentration of 2,4-D in the proliferation medium can be 2.0, 2.2, 2.5, 2.7 or 3.0 mg / L. The concentration of acid-hydrolyzed casein in the proliferation medium can be 0.5-2.0 g / L, or 1.0-1.8 g / L; in a specific embodiment of the present invention, the concentration of acid-hydrolyzed casein in the proliferation medium can be 0.5, 0.7, 1.0, 1.2, 1.5, 1.8 or 2.0 g / L. The carbon nanotubes in the proliferation medium may be multi-walled carbon nanotubes, and the concentration of the multi-walled carbon nanotubes may be 1.0-2.0 mg / L, or 1.2-1.8 mg / L; in a specific embodiment of the present invention, the concentration of the multi-walled carbon nanotubes in the proliferation medium may be 1.0, 1.2, 1.4, 1.6, 1.8 or 2.0 mg / L. The concentration of reduced glutathione in the proliferation medium may be 0.2-1.5 g / L, or 0.8-1.3 g / L; in a specific embodiment of the present invention, the concentration of reduced glutathione in the proliferation medium may be 0.5, 0.7, 0.9, 1.1, 1.3 or 1.5 mg / L. The concentration of vitamin C in the proliferation medium may be 0.5-1.0 g / L, or 0.5-0.8 g / L; in a specific embodiment of the present invention, the concentration of vitamin C in the proliferation medium may be 0.5, 0.6, 0.8 or 1.0 g / L. The concentration of activated carbon in the proliferation medium can be 0.5-2.0 g / L, or 1.0-1.8 g / L; in a specific embodiment of the present invention, the concentration of activated carbon in the proliferation medium can be 0.5, 0.7, 0.9, 1.1, 1.3, 1.5 or 1.8 g / L. The concentration of agar in the proliferation medium can be 6.5-7.0 g / L, or 6.6-6.8 g / L; in a specific embodiment of the present invention, the concentration of agar in the proliferation medium can be 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0 g / L. Reduced glutathione and vitamin C are antioxidant substances. The present invention can promote the proliferation of ginseng callus tissue under appropriate concentration ratios of KT, 2,4-D, acid hydrolyzed casein, carbon nanotubes, reduced glutathione, vitamin C, and activated carbon, and further transform them into stem cells, reduce the browning rate of callus tissue, and once browning occurs, biochar can also absorb the undesirable components produced in browning. In the present invention, the pH value of the proliferation medium may be 5.75-5.85, or 5.80.

[0044] The germination medium, growth medium, induction medium and proliferation medium provided by the present invention all contain sucrose, which can be a carbon source required for plant growth, promote bud germination, callus generation and proliferation. The present invention promotes ginseng seed germination and seedling growth under the joint action of 2,4-D, GA3, 6-BA, NAA, KT, carbon nanotubes, acid hydrolyzed casein, reduced glutathione, vitamin C, activated carbon and sucrose, and induces the generation and proliferation of callus. The tissue culture medium provided by the present invention has a good application effect, and a large number of stem cells can be obtained at the same time for the production of various products. The present invention does not specifically limit the sources of the B5 medium, MS medium, 1 / 2MS, GA3, 2,4-D, 6-BA, NAA, KT, carbon nanotubes, acid hydrolyzed casein, reduced glutathione, vitamin C, activated carbon, sucrose and agar, and conventional commercially available products can be used. In the embodiment of the present invention, the acid hydrolyzed casein used is purchased from Gao Jiaoyan (Beijing) Technology Co., Ltd.

[0045] The present invention provides a tissue culture method for ginseng stem cells, the tissue culture method adopts the tissue culture medium described in the above technical solution, and comprises the following steps:

[0046] placing ginseng seeds in a germination medium for germination culture to obtain germinated seeds; the ginseng seeds are seeds that have been subjected to vernalization treatment;

[0047] The germinated seeds are transferred to a growth medium for seedling growth culture to obtain sterile seedlings with a height of 5.0 to 8.0 cm;

[0048] cutting stem segments of the sterile seedlings and transferring them to an induction medium for induction culture to obtain callus tissue;

[0049] The callus tissue is transferred to a proliferation culture medium for proliferation culture to obtain ginseng stem cells.

[0050] The present invention places the ginseng seeds into a germination medium for germination culture, and the ginseng seeds are seeds after vernalization treatment. As an embodiment, the ginseng seeds of the present invention can be red berries in the ginseng seed harvesting period. The vernalization treatment method can be wet sand storage. The temperature of the wet sand storage can be 15°C, and the time of the wet sand storage can be 70 to 90 days. The selection of the sand storage time and method of the present invention is to break the dormancy of the ginseng seeds and shorten its germination time. After the vernalization treatment, the present invention also includes peeling the seeds. The present invention does not specifically limit the peeling method, and conventional methods can be used. In a specific embodiment of the present invention, the ginseng seeds are cut along the longitudinal diameter of the seeds, the peel is removed, and peeled seeds are obtained; the peeled seeds contain embryos and endosperms.

[0051] As an embodiment, after obtaining the peeled seeds, the present invention may further include: cleaning and disinfecting the peeled seeds; the cleaning preferably includes deionized water cleaning and running water rinsing in sequence; the present invention has no special restrictions on the cleaning method, and it is sufficient to ensure that the cleaning is clean. As an implementable embodiment, the deionized water cleaning time can be 20 to 30 minutes, or 25 minutes; liquid detergent can be added during the deionized water cleaning process; the present invention has no special restrictions on the liquid detergent, and it is sufficient to ensure that the cleaning is clean. The liquid detergent of the present invention can be detergent and / or laundry detergent. The present invention preferably first uses liquid detergent to shake and wash for 20 to 30 minutes, rinses with running water for 30 to 60 minutes, and then moves to a clean bench; disinfects with 75% alcohol for 30 to 45 seconds, rinses with sterile water 2 to 3 times, and then disinfects with sodium hypochlorite solution with an effective chlorine content of 2.0% to 3.0% for 6 to 9 minutes, and then rinses with sterile water 4 to 5 times. The present invention preferably washes with sterile water 2 to 3 times, more preferably 3 times; the disinfection is preferably carried out with a sodium hypochlorite solution having an effective chlorine content of 2% to 3%, more preferably with a sodium hypochlorite solution having an effective chlorine content of about 3%, and the disinfection time of the present invention is preferably 6 to 9 minutes, more preferably 8 minutes; and finally preferably washes with sterile water 4 to 5 times, more preferably 4 times. In a specific embodiment of the present invention, the blade is first rinsed with 75% ethanol for 45 seconds, and rinsed with sterile water 3 times; then soaked in a sodium hypochlorite solution having an effective chlorine content of 2% for 7 minutes, and rinsed with sterile water 4 times.

[0052] After the peeled seeds of the present invention are cleaned and disinfected, the peeled seeds can be inoculated into a germination medium for germination culture to obtain germinated seeds. As an embodiment, the inoculation method of the present invention is preferably horizontally placed on the induction medium.

[0053] As an embodiment, the temperature of the germination culture can be 24-26°C, or 25°C; the time of the germination culture can be 15-32 days, or 20-25 days. The germination culture can be dark culture. The time of the induction culture of the present invention is calculated from the time when the peeled seeds are inoculated on the germination medium until the buds germinate to 1.5-2.0 cm. Dark culture helps to promote seed germination.

[0054] After the seeds germinate, the present invention transfers the germinated seeds to a growth medium for seedling growth culture to obtain sterile seedlings with a height of 5.0 to 8.0 cm. As an embodiment, the temperature of the seedling growth culture can be 24 to 26°C, or 25°C; the time of the seedling growth culture can be 35 to 60 days, or 45 to 55 days. The seedling growth culture of the present invention can be carried out under alternating light and dark conditions, and the illumination time can be 12 to 16 h / d, or 14 to 16 h / d. The dark culture time of the present invention is 8 to 12 h / d, or 8 to 10 h / d. The intensity of the light culture of the present invention can be 800 to 1200 lx, or 1200 lx.

[0055] After obtaining the sterile seedlings, the present invention cuts the stem segments of the sterile seedlings and transfers them to an induction culture medium for induction culture to obtain callus tissue.

[0056] As an embodiment, the length of the stem segment of the present invention can be 2.0-2.5 cm. The temperature of the induction culture can be 24-26°C, or 25°C; the time of the induction culture can be 28-45 days, or 30-40 days. The induction culture can be carried out under light-dark alternation conditions, or under dark conditions. When carried out under light-dark alternation conditions, the time of the light-dark alternation illumination culture of the present invention can be 12-16 h / d, or 14-16 h / d; the intensity of the illumination culture can be 800-1200 lx, or 900-1100 lx; ​​the time of the light-dark alternation dark culture can be 8-12 h / d, or 8-10 h / d. The present invention can transfer the stem segment to the induction culture medium for callus induction culture until a callus mass with a diameter of more than 1.0 cm is obtained. The callus induction rate of the young stem segment of the present invention is high, and the material is convenient to obtain, and is not restricted by season.

[0057] A callus mass with a diameter of more than 1.0 cm is obtained. The present invention can transfer the callus mass to a proliferation culture medium for proliferation culture to obtain ginseng stem cells.

[0058] As an embodiment, the temperature of the proliferation culture can be 24-26°C, or 25°C; the time of the proliferation culture can be 60-80 days, or 70-80 days. The proliferation culture can be carried out under light-dark alternation conditions. When carried out under light-dark alternation conditions, the time of the light-dark alternation illumination culture can be 12-16h / d, or 14-16h / d; the intensity of the illumination culture can be 800-1200lx, or 900-1100lx; the time of the light-dark alternation dark culture can be 8-12h / d, or 8-10h / d. As an embodiment, after 35-40 days of the proliferation culture, the present invention can transfer the callus tissue to a new proliferation culture medium for proliferation culture, and the composition of the proliferation culture medium remains unchanged. The number of transfers can be 1, and the transfer can provide more nutrients for the proliferation of the callus tissue and prevent the callus tissue from browning.

[0059] The proliferation culture time of the present invention is calculated from the transfer of the generated callus mass (i.e., the embryo that has generated callus tissue) to the proliferation culture medium until the callus mass with a diameter of 1.5 to 2.5 cm is obtained. The callus mass with a diameter of 1.5 to 2.5 cm of the present invention is the obtained ginseng stem cell.

[0060] In the prior art, ginseng roots are usually used as explants, which makes dedifferentiation difficult. The innovation of the present invention is to use ginseng seeds as explants, combined with growth regulators, to induce and proliferate callus tissue, thereby laying the foundation for the large-scale cultivation of ginseng stem cells.

[0061] 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.

[0062] Example 1

[0063] 1. Seed acquisition: In mid-July 2023, when the ginseng berry peel turns red, pick the inflorescence, bring it back to the laboratory, remove the peel, put the seeds in wet sand, maintain the temperature at (15±1)℃, take the seeds out of the wet sand after 95 days, clean them and set aside.

[0064] 2. Disinfection of explants: Cut the cleaned seeds along the longitudinal diameter, remove the seed coat, and obtain skinless seeds, which contain embryos and endosperms. Put the peeled seeds into a glass bottle, add 30 times the volume of deionized water and 3 drops of liquid detergent, wash with 150rpm vibration for 25 minutes, and then rinse with running water for 60 minutes. Then transfer the cleaned peeled seeds to the clean bench, disinfect with 75% volume alcohol for 45 seconds, and then rinse with sterile water 3 times; then disinfect with sodium hypochlorite solution with an effective chlorine content of 2.0% for 7.0 minutes, and then rinse with sterile water 4 times for standby use.

[0065] 3. Seed germination: The peeled seeds disinfected in step 2 are horizontally inoculated onto the seed germination medium for germination culture. The composition of the germination medium is: 1 / 2MS medium as the basic tissue culture medium, and only 3.0 mg / L GA3, 2.0 mg / L activated carbon, 30 g / L sucrose and 6.5 g / L agar are added. The pH value is 5.8. Seed germination is cultured in the dark. The first day of horizontal inoculation of the embryo into the germination medium is the first day of germination culture. After 7 days of germination culture, the embryo begins to germinate. After 20 days of germination culture, the seeds germinate into buds about 1.5 cm long. The morphology of seeds at 25 days of germination culture is shown in Figure 1 .

[0066] 4. Seedling growth and culture: After 32 days of seed germination culture in step 3, transfer the germinated seeds to the seedling growth medium for seedling growth culture. The composition of the seedling growth medium is: B5 tissue culture medium as the basic medium, and only 2.0 mg / L 6-BA, 0.5 mg / L NAA, 30 g / L sucrose and 6.5 g / L agar are added; the pH value is 5.8. The seedling growth culture is a light and dark alternating culture with a light intensity of 1200 lx, a light time of 16 h / d, and a dark culture time of 8 h / d. The morphology of the seedlings after 28 days of growth culture is shown in Figure 2 After 35 days of cultivation, the diameter of the main root of the seedling increased to 0.2-0.3 cm, the fleshy root was about 3.0 cm long, and the height of the seedling grew to about 5.0 cm-6.0 cm.

[0067] 5. Induction of callus: Cut the ginseng seedling stems with a height of about 6.0 cm in step 3 into stem segments with a length of about 2.0 cm, and place them horizontally on the callus induction medium for callus induction culture. The induction medium is composed of B5 as the basic medium, and only KT 0.3 mg / L, 2,4-D 2.0 mg / L, acid hydrolyzed casein 1.0 g / L, multi-walled carbon nanotubes 1.0 mg / L, sucrose 30 g / L and 6.5 g / L agar are added; the pH value is 5.8, and the proliferation culture is light-dark alternating culture. The light culture time of light-dark alternating culture is 12h / d, the dark culture time is 12h / d, and the light intensity of light culture is 1200lx; after 21 days of culture, it can be observed that light yellow-green callus differentiates from the green stem segment; continue to culture, the callus begins to proliferate in small quantities, and when the yellow-green cells are densely distributed on the stem segment. Figure 3 The morphological diagram of callus induction culture of Example 1, wherein the left side is the morphological diagram of callus induction culture 7d, the right side marked 6.19 is the morphological diagram of callus induction culture 21d, the right side marked 6.26 is the morphological diagram of callus induction culture 28d, and the morphological diagram of callus induction culture 25d is shown in FIG. Figure 4 The morphology of the callus obtained after 21 days of culture under the microscope is shown in Figure 5 .

[0068] 6. Callus proliferation: After 35 days of induction culture, the stem segments densely covered with callus tissue cells in step 5 were transferred to callus proliferation medium for proliferation culture. The proliferation medium composition is: B5 as the basic medium, and only KT 0.5mg / L, 2,4-D 2.5mg / L, multi-walled carbon nanotubes 1.0mg / L, acid hydrolyzed casein 1.0g / L, 0.2g / L reduced glutathione, 0.5g / L vitamin C, 1.0g / L activated carbon, 30g / L sucrose and 6.8g / L agar are added; pH value is 5.8.

[0069] After 35 days of proliferation culture, the callus tissue was transferred to a new proliferation medium and continued to be cultured for 30 to 40 days. The composition of the proliferation medium remained unchanged. To prevent the callus tissue from browning, antioxidant substances glutathione and vitamin C were added to the proliferation medium. The ginseng callus tissue obtained after proliferation culture is ginseng stem cells, see Figure 5 .

[0070] Example 2

[0071] Only steps 1 to 3 of Example 1 were performed, with the only difference being that the germination medium was changed. The composition of the germination medium was: MS medium as the basic tissue culture medium, and only 3.0 mg / L GA3, 2.0 mg / L activated carbon, 30 g / L sucrose and 6.5 g / L agar were added, and the pH value was 5.8.

[0072] Comparative Example 1

[0073] Only steps 1 to 3 of Example 1 were performed, with the only difference being that the germination medium was changed. The composition of the germination medium was: B5 medium as the basic tissue culture medium, and only 3.0 mg / L GA3, 2.0 mg / L activated carbon, 30 g / L sucrose and 6.5 g / L agar were added, and the pH value was 5.8.

[0074] Comparative Example 2

[0075] Only steps 1 to 3 of Example 1 were performed, with the only difference being that the germination medium was changed. The composition of the germination medium was: the germination medium was composed of: N6 medium as the basic tissue culture medium, and only 3.0 mg / L GA3, 2.0 mg / L activated carbon, 30 g / L sucrose and 6.5 g / L agar were added, and the pH value was 5.8.

[0076] Comparative Example 3

[0077] Only steps 1 to 3 of Example 1 were performed, with the only difference being that the germination medium was changed. The composition of the germination medium was: the germination medium was composed of: WPM medium as the basic tissue culture medium, and only 3.0 mg / L GA3, 2.0 mg / L activated carbon, 30 g / L sucrose and 6.5 g / L agar were added, and the pH value was 5.8.

[0078] The germination rates of ginseng seeds in Examples 1 to 2 and Comparative Examples 1 to 3 were measured, and the results are shown in Table 1. As can be seen from Table 1, the germination rate of ginseng seeds in the germination medium of the present invention is high.

[0079] Table 1 Effects of different basic culture media on ginseng seed germination rate

[0080]

[0081]

[0082] Example 3

[0083] Only steps 1 to 5 of Example 1 were performed. The only difference was that the induction medium was changed. The composition of the induction medium was: MS was used as the basic medium, and only KT 0.3 mg / L, 2,4-D 2.0 mg / L, acid hydrolyzed casein 1.0 g / L, multi-walled carbon nanotubes 1.0 mg / L, sucrose 30 g / L and agar 6.5 g / L were added; the pH value was 5.8.

[0084] Comparative Example 4

[0085] Only steps 1 to 5 of Example 1 were performed. The only difference was that the induction medium was changed. The composition of the induction medium was: 1 / 2MS was used as the basic medium, and only KT 0.3 mg / L, 2,4-D 2.0 mg / L, acid hydrolyzed casein 1.0 g / L, multi-walled carbon nanotubes 1.0 mg / L, sucrose 30 g / L and agar 6.5 g / L were added; the pH value was 5.8.

[0086] Comparative Example 5

[0087] Only steps 1 to 5 of Example 1 were performed. The only difference was that the induction medium was changed. The composition of the induction medium was: N6 was used as the basic medium, and only KT 0.3 mg / L, 2,4-D 2.0 mg / L, acid hydrolyzed casein 1.0 g / L, multi-walled carbon nanotubes 1.0 mg / L, sucrose 30 g / L and agar 6.5 g / L were added; the pH value was 5.8.

[0088] Comparative Example 6

[0089] Only steps 1 to 5 of Example 1 were performed. The only difference was that the induction medium was changed. The composition of the induction medium was: WPM was used as the basic medium, and only KT 0.3 mg / L, 2,4-D 2.0 mg / L, acid hydrolyzed casein 1.0 g / L, multi-walled carbon nanotubes 1.0 mg / L, sucrose 30 g / L and agar 6.5 g / L were added; the pH value was 5.8.

[0090] 30 days after the ginseng seedling stem segments were transferred, the ginseng callus induction rates of Examples 1 and 3 and Comparative Examples 4 to 6 were measured. The results are shown in Table 2.

[0091] Callus induction rate (%) = number of stem segments induced with callus / number of inoculated stem segments × 100

[0092] As shown in Table 2, the induction medium of the present invention has a high induction rate of ginseng callus.

[0093] Table 2 Effects of different basic culture media on callus induction rate of ginseng stem segments

[0094]

[0095]

[0096] Examples 4 to 14 and Comparative Examples 7 to 24 all only performed steps 1 to 5 of Example 1. The only difference from Example 1 was that the induction medium was changed. The composition of the induction medium was: B5 was used as the basic medium, and only KT, 2,4-D, 1.0 g / L acid hydrolyzed casein, 1.0 mg / L multi-walled carbon nanotubes, 30 g / L sucrose and 6.5 g / L agar were added; the pH value was 5.8. The concentrations of KT and 2,4-D in the induction medium of Examples 4 to 14 and Comparative Examples 7 to 24 are shown in Table 3.

[0097] 30 days after the ginseng seedling stem segments were transferred, the callus induction rates of the stem segments of Examples 4 to 14 and Comparative Examples 7 to 24 were calculated. The results are shown in Table 3.

[0098] As shown in Table 3, the induction rate of ginseng callus using the induction medium of the present invention is high.

[0099] Table 3 Effects of KT and 2,4-D concentrations on callus induction rate of young ginseng stem segments

[0100]

[0101]

[0102] Comparative Examples 25 to 36 only performed steps 1 to 5 of Example 1. The only difference between them and Example 1 was that the induction medium was changed. The composition of the induction medium was: B5 was used as the basic medium, and only cytokinin, auxin, 1.0 g / L acid hydrolyzed casein, 1.0 mg / L multi-walled carbon nanotubes, 30 g / L sucrose and 6.5 g / L agar were added; the pH value was 5.8. The types and concentrations of cytokinin and auxin in the induction medium used in Comparative Examples 25 to 36 are shown in Table 4.

[0103] 30 days after the ginseng seedling stem segments were transferred, the callus induction rates of the stem segments of comparative examples 25 to 36 were calculated. The results are shown in Table 4.

[0104] As shown in Table 4, the induction medium of the present invention has a high induction rate of ginseng callus.

[0105] Table 4 Effects of different hormones on callus induction of ginseng stem segments

[0106]

[0107]

[0108] Examples 15 to 19 and Comparative Examples 37 to 54 all only performed steps 1 to 6 of Example 1. The only difference from Example 1 is that the proliferation medium is changed. The composition of the proliferation medium is: B5 is used as the basic medium, and 0.5 mg / L KT, 2.5 mg / L 2,4-D, acid hydrolyzed casein, multi-walled carbon nanotubes, 0.2 g / L reduced glutathione, 0.5 g / L vitamin C, 1.0 g / L activated carbon, 30 g / L sucrose and 6.8 g / L agar are added. The concentrations of acid hydrolyzed casein and multi-walled carbon nanotubes in the proliferation medium of Examples 15 to 25 and Comparative Examples 37 to 54 are shown in Table 5.

[0109] 30 days after the ginseng seedling stem segments were transferred, the proliferation coefficients of Example 1, Examples 15 to 25 and Comparative Examples 37 to 54 were calculated by weighing method. The results are shown in Table 5.

[0110] Proliferation coefficient (fold) = weight of (tissue culture bottle + culture medium + callus tissue) 30 days after transfer / weight of (tissue culture bottle + culture medium + callus tissue) at the time of inoculation.

[0111] As shown in Table 5, the proliferation coefficient of ginseng callus in the proliferation medium of the present invention is high.

[0112] Table 5 Effects of acid hydrolyzed casein and multi-walled carbon nanotubes on callus proliferation in proliferation medium

[0113]

[0114]

[0115] Comparative Examples 55 to 58 only performed steps 1 to 6 of Example 1. The only difference from Example 1 is that the proliferation medium is changed. The composition of the proliferation medium is: B5 is used as the basic medium, and 0.5 mg / L KT, 2.5 mg / L 2,4-D, 1.0 g / L acid hydrolyzed casein, 1.0 mg / L multi-walled carbon nanotubes, reduced glutathione, vitamin C, activated carbon, 30 g / L sucrose and 6.8 g / L agar are added. The concentrations of reduced glutathione, vitamin C and activated carbon in the proliferation medium of Comparative Examples 55 to 58 are shown in Table 6. According to Table 6, the browning rate of the tissue culture medium provided by the present invention is low.

[0116] Table 6 Effects of antioxidant substances on the browning rate of ginseng callus

[0117]

[0118]

[0119] In summary, the technical solution provided by the present invention has a high callus induction rate, a high proliferation coefficient, and a low browning rate. The tissue culture medium and tissue culture method for inducing the production of ginseng stem cells provided by the present invention can obtain a large number of ginseng stem cells and shorten the culture cycle.

[0120] 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 tissue culture medium for culturing ginseng stem cells, characterized in that: The tissue culture medium includes germination medium, growth medium, induction medium and proliferation medium; The germination medium uses MS or 1 / 2MS as a basic medium, and also includes 2.0-4.0 mg / L GA3, 0.5-2.0 g / L activated carbon, 30 g / L sucrose and 6.5-7.0 g / L agar; The growth medium uses B5 as the basic medium and also includes 1.0-3.0 mg / L 6-BA, 0.3-0.5 mg / L NAA, 30 g / L sucrose and 6.5-7.0 g / L agar; The induction culture medium uses B5 or MS as a basic culture medium, and also includes 0.2-0.5 mg / L KT, 1.5-2.5 mg / L 2,4-D, 0.5-1.0 g / L acid hydrolyzed casein, 1.0-2.0 mg / L carbon nanotubes, 30 g / L sucrose and 6.5-7.0 g / L agar; The proliferation culture medium uses B5 as a basic culture medium and also includes 0.5-0.8 mg / L KT, 2.0-3.0 mg / L 2,4-D, 1.0-2.0 mg / L carbon nanotubes, 0.5-2.0 g / L acid hydrolyzed casein, 0.2-1.5 g / L reduced glutathione, 0.5-1.0 g / L vitamin C, 0.5-2.0 g / L activated carbon, 30 g / L sucrose and 6.5-7.0 g / L agar.

2. The tissue culture medium according to claim 1, characterized in that The pH value of the germination medium is 5.75-5.85, the pH value of the growth medium is 5.75-5.85, the pH value of the induction medium is 5.75-5.85, and the pH value of the proliferation medium is 5.75-5.

85.

3. A method for tissue culture of ginseng stem cells, characterized in that: The tissue culture method adopts the tissue culture medium according to claim 1 or 2, comprising the following steps: Inoculating ginseng seeds into a germination medium for germination culture to obtain germinated seeds; the ginseng seeds are seeds that have been subjected to vernalization treatment; The germinated seeds are transferred to a growth medium for seedling growth culture to obtain sterile seedlings with a height of 5.0 to 8.0 cm; Cutting the stem segments of the sterile seedlings and transferring them to an induction medium for induction culture to obtain callus tissue; The callus tissue is transferred to a proliferation culture medium for proliferation culture to obtain ginseng stem cells.

4. The tissue culture method according to claim 3, characterized in that: The ginseng seeds are peeled seeds and contain embryo and endosperm.

5. The tissue culture method according to claim 3, characterized in that: The ginseng seeds are also cleaned and disinfected before inoculation. The disinfection includes first disinfecting with 75% alcohol for 30-45 seconds and rinsing with sterile water for 2-3 times; then disinfecting with sodium hypochlorite solution with an effective chlorine content of 2.0%-3.0% for 6-9 minutes and rinsing with sterile water for 4-5 times.

6. The tissue culture method according to claim 3, characterized in that: The temperatures of the germination culture, seedling growth culture, induction culture and proliferation culture are all 25±1°C.

7. The tissue culture method according to claim 3, characterized in that: The germination culture is dark culture; the seedling growth culture and proliferation culture are both carried out under light-dark alternation conditions; and the induction culture can be carried out under light-dark alternation conditions or in darkness.

8. The tissue culture method according to claim 6, characterized in that: The time of the light culture with alternating light and dark is 12 to 16 h / d; the intensity of the light culture is 800 to 1200 lx; ​​the time of the dark culture with alternating light and dark is 8 to 12 h / d.

9. The tissue culture method according to claim 7, characterized in that: The time of the germination culture is 15 to 32 days; the time of the seedling growth culture is 35 to 60 days; the time of the induction culture is 28 to 45 days; and the time of the proliferation culture is 60 to 80 days.

10. The tissue culture method according to claim 3, characterized in that: The length of the stem segment is 1.5 to 2.5 cm.

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