Patent Application
20250194484
The present disclosure belongs to the technical field of plant tissue culture, and in particular relates to a method for separating and culturing ginseng stem cells.
Ginseng (Panax ginseng C. A. Mey.) is a plant of the genus Panax of Araliaceae, distributed in China, Japan, and Korea, and its rhizome is a precious Chinese medicinal material, known as the “king of herbs”. Ginseng tastes sweet and slightly bitter, and is slightly warm. It has the effects of reinforcing vital energy, restoring the pulse for relieving desertion, tonifying the spleen and benefiting the lung, promoting fluid production and nourishing blood, calming the nerves and improving the intelligence. It is mainly used for verging on desertion due to weak health, reducing appetite due to spleen deficiency, asthma and cough due to lung deficiency, thirst due to body fluid deficiency, insomnia due to palpitation, etc. Ginsenoside is the main active ingredient of ginseng, and has the effects of resisting fatigue, delaying aging, regulating the central nervous system, improving the immunity of the body, improving the blood supply insufficiency of heart and cerebral vessels, and inhibiting the production of tumor cells. In recent years, ginseng has been widely used in various cosmetics, health care products and drinks, and the market prospect of ginseng is very broad.
At present, wild ginseng resources are almost extinct due to excessive digging, environmental destruction, etc., and field cultivation is the main source of ginseng. However, ginseng grows slowly has a long planting period, and requires strict environmental conditions. Its quality is easily affected by climate, cultivation conditions, diseases and insect pests. Its cultivation techniques are complex, and there are also problems such as excessive pesticide residues and old ginseng soil. These problems greatly limit the development prospects of artificial cultivation of ginseng. The supply of ginseng cultivated in the field is difficult to meet the market demand. Ginseng tissue culture technology has a short cycle, is not limited by seasons, and is prone to large-scale industrial production, which has a great development prospect.
At present, adventitious roots are generally induced by ginseng callus, which needs to induce callus first and then induce adventitious roots, which requires a long experimental cycle, and has complex operation steps and high pollution risk. In addition, there is also a problem that the content of ginsenoside in cultivated ginseng is low, which is difficult to meet the needs of clinical applications. For example, Chinese patent application No. 201410698528.0 discloses a method for inducing the proliferation of ginseng adventitious roots, which comprises the following steps: cutting ginseng tissue culture seedlings into small tissue pieces, inoculating the small tissue pieces into a solid induction medium to induce formation of adventitious roots, cutting the adventitious roots into small adventitious root pieces, and inoculating the small adventitious root pieces into a liquid proliferation medium for proliferation culture of adventitious roots; wherein the solid induction medium and the liquid proliferation medium use a 1/2 MS (—N) medium as a minimal medium, and contains 1-10 mg/L of indolebutyric acid. In the above solution, the tissue culture seedlings of 28-32 days old are cut into small pieces to directly induce adventitious roots, the tissue culture seedlings are young and tender, and have strong differentiation ability. In fact, it takes at least 28-32 days to obtain tissue culture seedlings through seed germination or explant culture, and explant culture still needs callus induction. Thus, the cycle is not actually shortened.
Ginseng stem cells are undifferentiated cells with infinite division capacity. At present, ginseng related pharmaceutical active ingredients can be produced by isolating and culturing ginseng stem cells. At present, in the prior art, when ginseng stem cells are isolated, differentiated tissues and organs are used as explants, and are induced into callus cells with differentiation capacity through culture in a three-dimensional culture stage (a dedifferentiation process). But the cells are intrinsically derived from differentiated somatic cells, and therefore have limited division capacity, and low stress resistance. In industrial production, cell line degeneration is prone to occur, and the division capacity is weak.
Root tips of ginseng adventitious roots have meristems, and strong division capacity. Therefore, if a method for culturing ginseng adventitious roots from mature ginseng, and then further culturing ginseng stem cells with shorter time and simpler steps can be explored, a large number of high-performance ginseng stem cells can be obtained in a short time, which has broad significance.
In view of this, the present disclosure is specifically proposed.
The technical problem to be solved by the present disclosure is to overcome the deficiencies in the prior art and provide a method for separating and culturing ginseng stem cells. In the present disclosure, first, adventitious roots are induced from various parts of mature ginseng by a one-step method, and then stem cells are directly dissected and isolated from root tips of the prepared adventitious roots for culture to obtain ginseng stem cells. The steps are more simple, convenient and time-saving.
In order to solve the above technical problem, a basic idea of the technical solution adopted by the present disclosure is as follows:
The root tips of the ginseng adventitious roots have more meristems. According to the method for separating and culturing ginseng stem cells of the present disclosure, the meristems can be directly dissected and isolated from the root tips of the ginseng adventitious roots without screening in an osmotic environment, and an osmotic agent, which can save steps, shorten the time, and save the cost.
The isolated ginseng stem cells of the present disclosure are subsequently subjected to dark culture in the stem cell induction medium, the stem cell subculture medium, and the stem cell liquid medium with suitable composition and ratios. The growth speed of ginseng stem cells is fast, and ginseng stem cells can be rapidly obtained.
The present disclosure uses the one-step method for culturing the ginseng adventitious roots from mature ginseng without an intermediate step of callus induction, and the adventitious roots can be directly induced from the mature ginseng in one step, thereby simplifying the induction steps and shortening the induction time.
In a further solution, in the step (3), the stem cell induction medium includes 2-4 mg/L of gibberellin, 0.6-1 mg/L of kinetin, 2-4 mg/L of indoleacetic acid, 15-75 mg/L of ascorbic acid, 50-150 mg/L of citric acid, 20-60 g/L of sucrose, 1-6 g/L of Phytagel, 1-2.4 g/L of a 1/2 MS medium, and 1-2.5 g/L of a B5 medium.
As one preferred embodiment, the stem cell induction medium includes 3 mg/L of gibberellin, 0.8 mg/L of kinetin, 2.5 mg/L of indoleacetic acid, 50 mg/L of ascorbic acid, 100 mg/L of citric acid, 40 g/L of sucrose, 3 g/L of Phytagel, 1.8 g/L of the 1/2 MS medium, and 1.5 g/L of the B5 medium.
The stem cell induction medium of the present disclosure contains indoleacetic acid, gibberellin and kinetin, which can induce stem cell growth. And ascorbic acid and citric acid contained in the stem cell induction medium of the present disclosure can produce synergistic antioxidant effects. The ingredients of the induction medium act synergistically to enable stem cells to rapidly grow in the initial stage.
In a further solution, in the step (4), the stem cell subculture medium includes 2-4 mg/L of 2,4-dichlorophenoxyacetic acid, 1-3 mg/L of gibberellin, 0.8-1.2 mg/L of kinetin, 20-60 g/L of sucrose, 1-6 g/L of Phytagel, 1-2.4 g/L of a 1/2 MS medium, and 0.6-1.4 g/L of a WPM medium.
As one preferred embodiment, the stem cell subculture medium includes 3 mg/L of 2,4-dichlorophenoxyacetic acid, 2 mg/L of gibberellin, 1 mg/L of kinetin, 35 g/L of sucrose, 3 g/L of Phytagel, 1.8 g/L of the 1/2 MS medium, and 0.8 g/L of the WPM medium.
The stem cell subculture medium of the present disclosure contains 2,4-Dichlorophenoxyacetic acid, gibberellin and kinetin, greatly increasing the growth rate of stem cells.
In a further solution, in the steps (3) and (4), the dark culture is performed at 20-25° C. until the inoculated stem cells grow a large number of cell masses.
In a further solution, in the step (5), the stem cell liquid medium includes 2-4 mg/L of 2,4-dichlorophenoxyacetic acid, 1-3 mg/L of gibberellin, 0.8-1.2 mg/L of kinetin, 20-60 g/L of sucrose, 1-2.4 g/L of a 1/2 MS medium and 0.6-1.4 g/L of a WPM medium.
As one preferred embodiment, the stem cell liquid medium includes 3 mg/L of 2,4-dichlorophenoxyacetic acid, 2 mg/L of gibberellin, 1 mg/L of kinetin, 35 g/L of sucrose, 1.8 g/L of the 1/2 MS medium, and 0.8 g/L of the WPM medium.
Further, in the step (5), the dark culture is performed at 20-25° C. at 100-150 rpm for passage once every 3-5 weeks.
In a further solution, the pH of the stem cell induction medium, the stem cell subculture medium and the stem cell liquid medium of the present disclosure is 5.6-6.0.
In a further solution, the pH of the stem cell induction medium, the pH of the stem cell subculture medium, and the pH of the stem cell liquid medium of the present disclosure are adjusted by using NaOH or KOH to facilitate stem cell growth.
In a further solution, in the step (2), the root tips of the ginseng adventitious roots cultured in (1) are taken and observed under a microscope, the stem cell area is determined based on the characteristics of the stem cells, and a root tip stem cell area is obtained by micromanipulation of dissecting and cutting.
The root tips are observed under the microscope, and irregular parts of cell shape and arrangement are growing points, i.e., a meristem cell aggregation area, which contains multiple vacuoles in the cells. Micromanipulation using the scalpel for cutting is performed to obtain the root tip stem cell area.
In a further solution, in the step (1), a method for culturing the ginseng adventitious roots by the one-step method according to the present disclosure specifically includes: washing and disinfecting mature ginseng, cutting the disinfected ginseng into slices, and inoculating the slices into an induction medium to induce adventitious roots of ginseng; re-inoculating the obtained adventitious roots of ginseng into an induction medium for subculture and propagation; and then cutting the obtained adventitious roots of ginseng into segments, and inoculating the segments of the adventitious root of ginseng into a liquid medium for culture to obtain adventitious roots.
At present, adventitious roots are generally induced by ginseng callus, which needs to in induce callus first and then induce adventitious roots, which requires a long experimental cycle, and has complex operation steps and high pollution risk. In addition, there is also a problem that the content of ginsenoside in cultivated ginseng is low, which is difficult to meet the needs of clinical applications.
In view of the long age, high maturity and difficulty in differentiation of mature ginseng, there is no report that adventitious roots can be directly induced from mature ginseng. After a large number of experiments, the present disclosure unexpectedly found that adventitious roots can be directly induced from mature ginseng slices in a specific induction medium, so that the adventitious roots can be directly induced from the mature ginseng in one step without an intermediate step of callus induction, thereby simplifying the induction steps and shortening the induction time.
In a further solution, the induction medium includes 1-6 mg/L of naphthylacetic acid, 0.2-1 mg/L of gibberellin, 0.1-0.6 mg/L of kinetin, 0.075-1.5 g/L of citric acid, 0.03-1 g/L of ascorbic acid, 20-60 g/L of sucrose, 1-6 g/L of Phytagel, 1-4 g/L of a B5 medium, and 1-2.4 g/L of a WPM medium.
The induction medium in the above solution enables direct induction of adventitious roots from the parts of the mature ginseng without the intermediate step of callus induction, thereby simplifying the induction steps and shortening the induction time.
In this solution, naphthylacetic acid is a plant growth regulator and gibberellin is a plant hormone, which can both promote adventitious root formation. Kinetin is a cytokinin that can promote division of cells. Citric acid and ascorbic acid can produce synergistic antioxidant effects, prevent browning of mature ginseng tissue in vitro, and facilitate direct induction of adventitious roots from the mature ginseng tissue in vitro. The ingredients in the induction medium act synergistically, eventually achieving direct induction of adventitious roots from the parts of the mature ginseng without the intermediate step of callus induction.
In a further solution, the induction medium includes 4 mg/L of naphthylacetic acid, 0.6 mg/L of gibberellin, 0.4 mg/L of kinetin, 0.1 g/L of citric acid, 0.05 g/L of ascorbic acid, 30 g/L of sucrose, 3 g/L of Phytagel, 1.55 g/L of a B5 medium and 1.21 g/L of a WPM medium.
The induction medium in the above ratio of ingredients has the best effect on the induction of adventitious roots from mature ginseng. The number of the produced adventitious roots is large and the quality is good, which is conducive to next propagation, and increases the content of active ingredients in adventitious roots.
In a further solution, the liquid medium uses a B5 medium, a WPM medium or a 1/2 MS medium as a basic medium, and contains 4 mg/L of indolebutyric acid, 0.1 g/L of citric acid, 0.05 g/L of ascorbic acid, 30 g/L of sucrose.
In the present disclosure, when further liquid culture is carried out on the induced adventitious roots, a conventional medium, such as a 1/2 MS medium, can be adopted, which is consistent with the commonly used adventitious root medium, indicating that the adventitious roots induced by the one-step method of the present disclosure, and adventitious roots obtained by other two-step processes have no difference, and both can be cultured by using the conventional medium, and meanwhile, the induction time is shortened and the pollution risk is reduced.
In a further solution, the slices are thin slices having a width of 0.5-0.7 cm, a length of 0.5-0.7 cm, and a thickness of 0.2-0.5 mm.
In a further solution, the mature ginseng has an age of 3 years or more;
The centennial ginseng is very rare in nature, and has a high edible and medicinal value, and can nourish the five internal organs, soothe the nerves, calm the soul, stop palpitation, eliminate evil spirits, improve eyesight, and make happy and improve intelligence. And its value is much greater than that of planted ginseng with a short age. The present disclosure does not require the intermediate step of callus induction, adventitious roots can be directly induced from centennial ginseng slices in one step, which can not only simplify the induction steps, and shorten the induction time, but also obtain unique functional components in the female parent centennial ginseng, thereby obtaining adventitious roots with better nutritional values.
In a further solution, taproots, or rhizomes, or adventitious roots on rhizomes, or lateral roots, or fibrous roots of the mature ginseng are washed and disinfected, sliced and inoculated into an induction medium to induce ginseng adventitious roots.
In a further solution, the ginseng is selected from wild ginseng, transplant wild ginseng, ginseng under forest, and garden ginseng; and
As one specific preferred embodiment, in the step (1), culturing the adventitious roots by the one-step method specifically includes the following steps:
In a further solution, the pH of the induction medium or the liquid medium of the present disclosure is 5.6-6.0.
In a further solution, the pH of the induction medium and the pH of the liquid medium of the present disclosure are adjusted by using NaOH or KOH to facilitate adventitious root growth.
It should be noted that in the present disclosure, the WPM medium, the B5 medium, the 1/2 MS medium, and the like are known in the art.
Ingredients are as follows: 950 mg/L of potassium nitrate, 825 mg/L of ammonium nitrate, 220 mg/L of calcium chloride dihydrate, 185 mg/L of magnesium sulfate, 85 mg/L of potassium dihydrogen phosphate, 11.15 mg/L of manganese sulfate, 4.3 mg/L of zinc sulfate, 3.1 mg/L of boric acid, 0.415 mg/L of potassium iodide, 0.125 mg/L of sodium molybdate, 0.0125 mg/L of copper sulfate, 0.0125 mg/L of cobalt chloride, 37.3 mg/L of disodium ethylenediamine tetraacetate, 27.8 mg/L of ferrous sulfate, 100 mg/L of inositol, 2 mg/L of glycine, 0.5 mg/L of hydrochloric acid, 0.5 mg/L of pyridoxine hydrochloride, and 0.1 mg/L of thiamine hydrochloride.
Ingredients are as follows: 2500 mg/L of potassium nitrate KNO3, 250 mg/L of MgSO4·7H2O, 150 mg/L of CaCI2·2H2O, 134 mg/L of (NH4)2SO4, 150 mg/L of NaH2PO4H2O, 0.075 mg/L of KI, 3.0 mg/L of H3BO3, 10 mg/L of MnSO4·4H2O, 2.0 mg/L of ZnSO4·7H2O, 0.25 mg/L of NazMoO4·2H2O, 0.025 mg/L of CoCI2: 6H2O, 0.025 mg/L of CuSO4·5H2O, 37.3 mg/L of Naz-EDTA, 27.8 mg/L of FeSO4·7H2O, 100 mg/L of inositol, 1.0 mg/L of nicotinic acid, 1.0 mg/L of pyridoxine hydrochloride, and 10 mg/L of thiamine hydrochloride.
Ingredients are as follows: 400 mg/L of ammonium nitrate, 556 mg/L of calcium nitrate tetrahydrate, 990 mg/L of potassium sulfate, 72 mg/L of anhydrous calcium chloride, 170 mg/L of potassium dihydrogen phosphate, 0.25 mg/L of sodium molybdate dihydrate, 180 mg/L of anhydrous magnesium sulfate, 22.4 mg/L of manganese sulfate monohydrate, 8.6 mg/L of zinc sulfate heptahydrate, 0.25 mg/L of copper sulfate pentahydrate, 27.8 mg/L of ferrous sulfate heptahydrate, 37.3 mg/L of disodium ethylenediamine tetraacetate, 100 mg/L of inositol, 1 mg/L of vitamin B1, 0.5 mg/L of nicotinic acid, 0.5 mg/L of vitamin B6, and 2 mg/L of glycine, with a pH of 5.2.
After the above technical solution is adopted, compared with the prior art, the present disclosure has the following beneficial effects:
Specific embodiments of the present discolsure will be further described below in detail in combination with the accompanying drawings.
The drawings serving as one part of the present disclosure are intended to provide a further understanding for the present disclosure. Schematic embodiments of the present disclosure and the descriptions thereof are intended to explain the present disclosure, rather than an improper limitation of the present disclosure. Obviously, the drawings described below are merely some embodiments. On the premise of not paying inventive labor, those of ordinary skill in the art can further obtain other drawings according to these drawings. In the drawings:
To make the objectives, technical solutions, and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments will be clearly and completely described below. The following embodiments are used to describe the present disclosure but not to limit the scope of the present disclosure.
It should be noted that a preparation method or a detection method, which is not specifically defined in the present disclosure, can be performed by using a method that can be used to achieve its purpose in the prior art of the field.
Rhizomes and adventitious roots on rhizomes were removed from 20-year-old wild ginseng, leaving the taproots. The taproots were washed and sterilized, cut into slices having a width of 0.6 cm, a length of 0.7 cm, and a thickness of 0.3 mm, and inoculated into an induction medium for dark culture at 22±1° C. for 4-5 weeks to induce wild ginseng adventitious roots; wherein the induction medium includes 4 mg/L of naphthylacetic acid, 0.6 mg/L of gibberellin, 0.4 mg/L of kinetin, 0.1 g/L of citric acid, 0.05 g/L of ascorbic acid, 30 g/L of sucrose, 3 g/L of Phytagel, 1.55 g/L of a B5 medium and 1.21 g/L of a WPM medium, with a pH of 5.8;
the wild ginseng adventitious roots obtained in the step (1) were inoculated into the same induction medium as that in the step (1) for dark culture under the same conditions for 4-5 weeks; and
the wild ginseng adventitious roots obtained in the step (2) were cut into tissue of about 1 cm in length, and inoculated into a liquid medium for culture on a shaker at 22±1° C. at 130 rpm for 3-4 weeks to obtain adventitious roots; wherein the liquid medium contains 4 mg/L of indolebutyric acid, 0.1 g/L of citric acid, 0.05 g/L of ascorbic acid, a 1/2 MS medium and 30 g/L of sucrose, with a pH of 5.8.
In this embodiment, the adventitious roots produced on the induction medium in the step (1) are shown in
Rhizomes and adventitious roots on rhizomes of 10-year-old wild ginseng were removed, leaving the taproots. The taproots were washed and sterilized, cut into slices having a width of 0.5 cm, a length of 0.6 cm, and a thickness of 0.4 mm, and inoculated into an induction medium for dark culture at 22±1° C. for 4-5 weeks to induce wild ginseng adventitious roots; wherein the induction medium includes 4 mg/L of naphthylacetic acid, 0.6 mg/L of gibberellin, 0.4 mg/L of kinetin, 0.1 g/L of citric acid, 0.05 g/L of ascorbic acid, 30 g/L of sucrose, 3 g/L of Phytagel, 1.55 g/L of a B5 medium and 1.21 g/L of a WPM medium, with a pH of 5.8;
the wild ginseng adventitious roots obtained in the step (1) were inoculated into the same induction medium as that in the step (1) for dark culture under the same conditions for 4-5 weeks; and
the wild ginseng adventitious roots obtained in the step (2) were cut into tissue of about 1.5 cm in length, and inoculated into a liquid medium for culture on a shaker at 22±1° C. at 120 rpm for 3-4 weeks to obtain adventitious roots; wherein the liquid medium contains 4 mg/L of indolebutyric acid, 0.1 g/L of citric acid, 0.05 g/L of ascorbic acid, a 1/2 MS medium and 30 g/L of sucrose, with a pH of 5.8.
Similar to the results of Embodiment 1, adventitious roots can be induced in one step in the step (1) of this embodiment.
Rhizomes of 6-year-old garden ginseng were washed and sterilized, cut into slices having a width of 0.5 cm, a length of 0.6 cm, and a thickness of 0.3 mm, and inoculated into an induction medium for dark culture at 22±1° C. for 4-5 weeks to induce adventitious roots; wherein the induction medium includes 6 mg/L of naphthylacetic acid, 0.2 mg/L of gibberellin, 0.4 mg/L of kinetin, 1.2 g/L of citric acid, 0.1 g/L of ascorbic acid, 20 g/L of sucrose, 5 g/L of Phytagel, 4 g/L of a B5 medium and 1.8 g/L of a WPM medium, with a pH of 5.6;
the adventitious roots obtained in the step (1) were inoculated into the same induction medium as that in the step (1) for dark culture under the same conditions for 4-5 weeks; and
the adventitious roots obtained in the step (2) were cut into tissue of about 1 cm in length, and inoculated into a liquid medium for culture on a shaker at 22±1° C. at 110 rpm for 3-4 weeks to obtain adventitious roots; wherein the liquid medium contains 4 mg/L of indolebutyric acid, 0.1 g/L of citric acid, 0.05 g/L of ascorbic acid, a 1/2 MS medium and 30 g/L of sucrose, with a pH of 5.6.
Similar to the results of Embodiment 1, adventitious roots can be induced in one step in the step (1) of this embodiment.
Adventitious roots on rhizomes of 15-year-old ginseng under forest were washed and sterilized, cut into slices having a width of 0.7 cm, a length of 0.7 cm, and a thickness of 0.5 mm, and inoculated into an induction medium for dark culture at 22±1° C. for 4-5 weeks to induce adventitious roots; wherein the induction medium includes 5 mg/L of naphthylacetic acid, 1 mg/L of gibberellin, 0.1 mg/L of kinetin, 0.075 g/L of citric acid, 0.03 g/L of ascorbic acid, 40 g/L of sucrose, 4 g/L of Phytagel, 2 g/L of a B5 medium and 1 g/L of a WPM medium, with a pH of 6.0;
the adventitious roots obtained in the step (1) were inoculated into the same induction medium as that in the step (1) for dark culture under the same conditions for 4-5 weeks; and
the adventitious roots obtained in the step (2) were cut into tissue of about 2 cm in length, and inoculated into a liquid medium for culture on a shaker at 22±1° C. at 130 rpm for 3-4 weeks to obtain adventitious roots; wherein the liquid medium contains 4 mg/L of indolebutyric acid, 0.1 g/L of citric acid, 0.05 g/L of ascorbic acid, a WPM medium and 30 g/L of sucrose, with a pH of 6.0.
Similar to the results of Embodiment 1, adventitious roots can be induced in one step in the step (1) of this embodiment.
Taproots of 20-year-old transplant wild ginseng were washed and sterilized, cut into slices having a width of 0.5 cm, a length of 0.6 cm, and a thickness of 0.4 mm, and inoculated into an induction medium for dark culture at 22±1° C. for 4-5 weeks to induce wild ginseng adventitious roots; wherein the induction medium includes 1 mg/L of naphthylacetic acid, 0.5 mg/L of gibberellin, 0.6 mg/L of kinetin, 1.5 g/L of citric acid, 1 g/L of ascorbic acid, 50 g/L of sucrose, 6 g/L of Phytagel, 1 g/L of a B5 medium and 2.4 g/L of a WPM medium, with a pH of 5.7;
the wild ginseng adventitious roots obtained in the step (1) were inoculated into the same induction medium as that in the step (1) for dark culture under the same conditions for 4-5 weeks; and
the wild ginseng adventitious roots obtained in the step (2) were cut into tissue of about 1 cm in length, and inoculated into a liquid medium for culture on a shaker at 22±1° C. for 3-4 weeks to obtain adventitious roots; wherein the liquid medium contains 4 mg/L of indolebutyric acid, 0.1 g/L of citric acid, 0.05 g/L of ascorbic acid, a B5 medium and 30 g/L of sucrose, with a pH of 5.7.
Similar to the results of Embodiment 1, adventitious roots can be directly induced in one step in the step (1) of this embodiment.
Rhizomes and adventitious roots on rhizomes of centennial wild ginseng were removed, leaving the taproots. The taproots were washed and sterilized, cut into slices having a width of 0.6 cm, a length of 0.7 cm, and a thickness of 0.3 mm, and inoculated into an induction medium for dark culture at 22±1° C. for 4-5 weeks to induce wild ginseng adventitious roots; wherein the induction medium includes 4 mg/L of naphthylacetic acid, 0.6 mg/L of gibberellin, 0.4 mg/L of kinetin, 0.1 g/L of citric acid, 0.05 g/L of ascorbic acid, 30 g/L of sucrose, 3 g/L of Phytagel, 1.55 g/L of a B5 medium and 1.21 g/L of a WPM medium, with a pH of 5.8;
the wild ginseng adventitious roots obtained in the step (1) were inoculated into the same induction medium as that in the step (1) for dark culture under the same conditions for 4-5 weeks; and
the wild ginseng adventitious roots obtained in the step (2) were cut into tissue of about 1 cm in length, and inoculated into a liquid medium for culture on a shaker at 22±1° C. at 130 rpm for 3-4 weeks to obtain adventitious roots; wherein the liquid medium contains 4 mg/L of indolebutyric acid, 0.1 g/L of citric acid, 0.05 g/L of ascorbic acid, a 1/2 MS medium and 30 g/L of sucrose, with a pH of 5.8.
Similar to the results of Embodiment 1, adventitious roots can be directly induced in one step in the step (1) of this embodiment.
(1) The Ginseng Adventitious Roots Prepared in Embodiment 2 were Used;
(2) isolation of stem cells from root tips of adventitious roots: root tips of ginseng adventitious roots in (1) were taken, and subjected to micromanipulation of dissecting and cutting to obtain a root tip stem cell area;
(3) isolation and culture: the stem cell area was inoculated into a stem cell induction medium for dark culture at 22° C. until the inoculated stem cells grow a large number of cell masses; wherein
the stem cell induction medium includes: 3 mg/L of gibberellin, 0.8 mg/L of kinetin, 2.5 mg/L of indoleacetic acid, 50 mg/L of ascorbic acid, 100 mg/L of citric acid, 40 g/L of sucrose, 3 g/L of Phytagel, 1.8 g/L of a 1/2 MS medium, and 1.5 g/L of a B5 medium, with a pH of 5.8;
(4) subculture: part of the stem cell masses obtained in (3) was picked and inoculated into a stem cell subculture medium for dark culture at 22° C. until the inoculated stem cells grow a large number of cell masses; wherein
the stem cell subculture medium includes: 3 mg/L of 2,4-dichlorophenoxyacetic acid, 2 mg/L of gibberellin, 1 mg/L of kinetin, 35 g/L of sucrose, 3 g/L of Phytagel, 1.8 g/L of a 1/2 MS medium and 0.8 g/L of a WPM medium, with a pH of 5.8; and
(5) liquid culture: the stem cells obtained in (4) were inoculated into a stem cell liquid medium for dark culture at 22° C. at 120 rpm for passage once every 3-5 weeks; wherein
the stem cell liquid medium includes 3 mg/L of 2,4-dichlorophenoxyacetic acid, 2 mg/L of gibberellin, 1 mg/L of kinetin, 35 g/L of sucrose, 1.8 g/L of a 1/2 MS medium, and 0.8 g/L of a WPM medium, with a pH of 5.8.
(1) the ginseng adventitious roots prepared in Embodiment 3 were used;
(2) isolation of stem cells from root tips of adventitious roots: root tips of the ginseng adventitious roots in (1) were taken, and subjected to micromanipulation of dissecting and cutting to obtain a root tip stem cell area; (3) isolation and culture: the stem cell area was inoculated into a stem cell induction medium for dark culture at 25° C. until the inoculated stem cells grow a large number of cell masses; wherein
the stem cell induction medium includes: 4 mg/L of gibberellin, 0.6 mg/L of kinetin, 2 mg/L of indoleacetic acid, 75 mg/L of ascorbic acid, 50 mg/L of citric acid, 20 g/L of sucrose, 6 g/L of Phytagel, 2.4 g/L of a 1/2 MS medium, 1 g/L of a B5 medium, with a pH of 5.8;
(4) subculture: part of the stem cell masses obtained in (3) was picked and inoculated into a stem cell subculture medium for dark culture at 25° C. until the inoculated stem cells grow a large number of cell masses; wherein
the stem cell subculture medium includes: 2 mg/L of 2,4-dichlorophenoxyacetic acid, 3 mg/L of gibberellin, 1.2 mg/L of kinetin, 60 g/L of sucrose, 1 g/L of Phytagel, 1 g/L of a 1/2 MS medium and 1.4 g/L of a WPM medium, with a pH 5.8; and
(5) liquid culture: the stem cells obtained in (4) were inoculated into a stem cell liquid medium for dark culture at 25° C. at 150 rpm for passage once every 2 weeks; wherein
the stem cell liquid medium includes 2 mg/L of 2,4-dichlorophenoxyacetic acid, 3 mg/L of gibberellin, 1.2 mg/L of kinetin, 60 g/L of sucrose, 1 g/L of a 1/2 MS medium and 1.4 g/L of a WPM medium, with a pH of 5.8.
(1) the ginseng adventitious roots prepared in Embodiment 4 were used;
(2) isolation of stem cells from root tips of adventitious roots: root tips of the ginseng adventitious roots in (1) were taken, and subjected to micromanipulation of dissecting and cutting to obtain a root tip stem cell area; (3) isolation and culture: the stem cell area was inoculated into a stem cell induction medium for dark culture at 20° C. until the inoculated stem cells grow a large number of cell masses; wherein
the stem cell induction medium includes: 2 mg/L of gibberellin, 1 mg/L of kinetin, 4 mg/L of indoleacetic acid, 15 mg/L of ascorbic acid, 150 mg/L of citric acid, 60 g/L of sucrose, 1 g/L of Phytagel, 1 g/L of a 1/2 MS medium, and 2.5 g/L of a B5 medium, with a pH of 5.8;
(4) subculture: part of the stem cell masses obtained in (3) was picked and inoculated into a stem cell subculture medium for dark culture at 22° C. until the inoculated stem cells grow a large number of cell masses; wherein
the stem cell subculture medium includes 4 mg/L of 2,4-dichlorophenoxyacetic acid, 1 mg/L of gibberellin, 0.8 mg/L of kinetin, 20 g/L of sucrose, 6 g/L of Phytagel, 2.4 g/L of a 1/2 MS medium and 0.6 g/L of a WPM medium, with a pH of 5.8; and
(5) liquid culture: the stem cells obtained in (4) were inoculated into a stem cell liquid medium for dark culture at 22° C. at 120 rpm for passage once every 2 weeks; wherein
the stem cell liquid medium includes 4 mg/L of 2,4-dichlorophenoxyacetic acid, 1 mg/L of gibberellin, 0.8 mg/L of kinetin, 20 g/L of sucrose, 2.4 g/L of a 1/2 MS medium and 0.6 g/L of a WPM medium, with a pH of 5.8.
This comparative example differed from Embodiment 1 in that the induction medium used was different and the other steps were carried out with reference to Embodiment 1. The induction medium in this comparative example includes: 30 g/L of sucrose, 0.5 mg/L of kinetin, 3 mg/L of indolebutyric acid, 1.5 mg/L of 2,4-dichlorophenoxyacetic acid, a 1/2 MS medium, and 3 g/L of Phytagel, with a pH of 5.8.
The adventitious roots produced on the induction medium in the step (1) of this comparative example are shown in
This comparative example differed from Embodiment 1 in that the induction medium used was different and the other steps were carried out with reference to Embodiment 1. The induction medium in this comparative example includes: 30 g/L of sucrose, 0.5 mg/L of kinetin, 3 mg/L of indolebutyric acid, a 1/2 MS medium, and 3 g/L of Phytagel, with a pH of 5.8.
As a result, similar to that shown in the picture in Comparative example 1, adventitious roots cannot be directly induced from mature wild ginseng slices.
This comparative example differed from Embodiment 1 in that the induction medium used was different and the other steps were carried out with reference to Embodiment 1. The induction medium in this comparative example includes: 30 g/L of sucrose, 0.5 mg/L of kinetin, 3 mg/L of indoleacetic acid, WPM, and 3 g/L Phytagel, with a pH of 5.8.
Results: in the first week, the whole body turned yellow, in the third week, the color deepened, and the middle part began to turn brown, and in the fifth week, all became brown and withered.
In this example, the wild ginseng stem cells prepared in Embodiment 7 and the wild ginseng slices used in Embodiment 2 were subjected to saponin detection and polysaccharide detection, respectively.
After pretreatment such as extraction, a sample was separated by a C18 chromatographic column, and detected by a HPLC-UV detector, and the content of ginsenoside components was determined quantitatively by an external standard method.
Methanol (CHO): chromatographically pure, and acetonitrile (C6H1N): chromatographically pure
Standard reagent: ginsenosides Re, Rg1, Ra3, Rb1, Rf, Rb2, Rb3, F3, Rg2, Rd, and F1.
The ginseng stem cell sample obtained in Embodiment 7 was washed for three times with water, ground to a paste in a mortar, sonicated for wall breaking, and lyophilized. The lyophilized sample was ground on a mortar. 50 mg of the ground sample was accurately weighed to be placed in a 10 ml centrifuge tube, a 70% methanol solution was added, and vortex was conducted. Ultrasonic treatment was conducted on an ultrasonic oscillator for 10 min, the above operation was conducted repeatedly twice, and filtration was conducted for later use.
The wild ginseng slices were washed for three times with water, ground to a paste in a mortar, sonicated for wall breaking, and lyophilized. The lyophilized sample was ground on a mortar, 50 mg of the ground sample was accurately weighed to be placed in a 10 ml centrifuge tube, a 70% methanol solution was added, and vortex was conducted. Ultrasonic treatment was conducted on an ultrasonic oscillator for 10 min, the above operation was conducted repeatedly twice, and filtration was conducted for later use.
Preparation of stock solution (0.8 mg/ml): 8.00 mg of ginsenosides Re, Rb1, Rg1, Rd, Rf, F3, Rk2, Rb2, Rb3, Rg2, and F1, which are 11 standards in total, were respectively weighed to be placed in a 10 ml volumetric flask, and the volume was made up to a constant volume with superior pure methanol.
Preparation of use solution (32 μg/ml): 1 ml of the stock solution (0.8 mg/ml) was accurately pipetted into a 25 ml volumetric flask, and made up to a constant volume with superior pure methanol, and filtered through a 0.22 μm organic filter membrane for later use.
The content of ginsenoside components in the sample is calculated according to a formula (1):
The content of ginsenoside in the sample is the sum of those detected in the components.
Detection was performed with reference to a method of the Jilin Province local standard: DB 22/T 1685-2012.
The results of ginsenoside detection and polysaccharide detection are shown in Table 1.
As can be seen from the above results, both the total content of ginsenosides and the content of polysaccharides were increased in the cultured wild ginseng stem cells of the present disclosure compared with mature wild ginseng slices. Therefore, the method for separating and culturing ginseng stem cells of the present disclosure is simple in steps, and time-saving. And the ginseng stem cells grow fast, and have high content of active ingredients.
The above description is only preferred embodiments of the present disclosure, and is not intended to limit the present disclosure in any form. Although the present disclosure has been disclosed above with the preferred embodiments, it is not intended to limit the present disclosure. Any person familiar with this patent can make some changes or modifications to equivalent embodiments by using the technical contents mentioned above without departing from the scope of the technical solution of the present disclosure. However, any simple changes, equivalent variations and modifications made to the above embodiments according to the technical essence of the present disclosure without departing from the content of the technical solution of the present disclosure are still within the scope of the solution of the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110361871.6 | Apr 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/081651 | 3/18/2022 | WO |
