CN113174360A - Isolated culture method of ginseng stem cells - Google Patents

Isolated culture method of ginseng stem cells Download PDF

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CN113174360A
CN113174360A CN202110424326.7A CN202110424326A CN113174360A CN 113174360 A CN113174360 A CN 113174360A CN 202110424326 A CN202110424326 A CN 202110424326A CN 113174360 A CN113174360 A CN 113174360A
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CN113174360B (en
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刘冰
高秀君
闫培生
张明臣
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Shandong Anran Nanometre Ind Development Co ltd
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Abstract

The invention discloses a separation culture method of ginseng stem cells, which comprises the following steps: (1) preparing the adventitious roots of the ginseng by adopting a one-step method; (2) taking a root tip part of the ginseng adventitious root prepared in the step (1), and dissecting and separating to obtain a stem cell area; (3) inoculating the stem cells obtained by separation in the step (2) into a stem cell induction culture medium for dark culture to obtain a stem cell mass, and transferring the stem cell mass into a subculture medium for dark culture; (4) inoculating the stem cells subjected to subculture in the step (3) into a stem cell liquid culture medium, and performing dark culture to obtain the ginseng stem cells. By adopting the liquid culture medium and the culture method, the adventitious roots can be directly generated by the induction of each part of the mature ginseng, the content of ginsenoside in the adventitious roots is improved while the induction steps are simplified and the induction time is shortened, and finally the ginseng stem cells with good indexes are obtained.

Description

Isolated culture method of ginseng stem cells
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a separation culture method of ginseng stem cells.
Background
Ginseng (Panax ginseng c.a. mey.) is a plant of the genus Panax of the family araliaceae, distributed in china, japan and korea, and its rhizome is a rare Chinese medicinal material, called "king of herbaceous plant". Ginseng is sweet, slightly bitter and slightly warm in taste, has the effects of greatly invigorating primordial qi, recovering pulse, relieving depletion, invigorating spleen, benefiting lung, promoting fluid production, nourishing blood, tranquilizing mind, and improving intelligence, and is mainly used for treating loss of body-shirt, spleen deficiency, anorexia, lung deficiency, cough, body fluid deficiency, thirst, palpitation, insomnia, etc. Ginsenoside is the main active component of ginseng, and has the effects of resisting fatigue, delaying aging, regulating central nervous system, improving immunity, improving cardiovascular and cerebrovascular insufficiency, inhibiting tumor cell production, etc. In recent years, ginseng has been widely used in various cosmetics, health products, and drinks, and has a very wide market prospect.
At present, due to excessive mining, environmental damage and the like, wild ginseng resources are almost exhausted, and field cultivation is a main source of ginseng. However, ginseng grows slowly, the planting years are long, the requirements on environmental conditions are strict, the quality of ginseng is easily influenced by climate, cultivation conditions and plant diseases and insect pests, the cultivation technology is complex, and the development prospect of artificial cultivation of ginseng is greatly limited by the problems of pesticide residue exceeding the standard, ginseng land and the like. The ginseng cultivated in the field is difficult to meet the market demand. The tissue culture technology of ginseng has short period, is not limited by seasons, is easy to carry out large-scale industrial production, and has great development prospect.
The existing adventitious roots are generally generated by ginseng callus induction, the callus needs to be induced firstly and then the adventitious roots need to be induced, the required experimental period is long, the operation steps are complex, and the pollution risk is high. In addition, the cultured ginseng also has the problem that the content of ginsenoside is low, so that the clinical application requirement is difficult to meet.
The Chinese patent with the application number of 201410698528.0 discloses a method for inducing the proliferation of adventitious roots of ginseng, which comprises the following steps: cutting tissue culture seedlings of ginseng into tissue small blocks, inoculating the tissue small blocks into a solid induction culture medium to induce and form adventitious roots, cutting the adventitious roots into adventitious root small blocks, and inoculating the tissue small blocks into a liquid multiplication culture medium to carry out multiplication culture of the adventitious roots; wherein the solid induction culture medium and the liquid proliferation culture medium are 1/2MS (-N) culture medium which is used as a basic culture medium and contains indolebutyric acid with the concentration of 1-10 mg/L. In the scheme, the tissue culture seedling with the age of 28-32 days is cut into small blocks to directly induce adventitious roots, the tissue culture seedling is tender and has strong differentiation capability, the tissue culture seedling is actually obtained by seed germination or explant culture, at least 28-32 days are still needed, and callus induction is still needed for the explant culture. Therefore, the cycle is not actually shortened.
Ginseng stem cells are undifferentiated cells having an unlimited division ability, and currently, ginseng-related pharmaceutically active ingredients can be produced by isolating and culturing ginseng stem cells. In the prior art, differentiated tissue organs are used as explants when ginseng stem cells are separated, and the differentiated tissue organs are induced into callus cells with differentiation capacity through three-dimensional culture (dedifferentiation process). However, the cell is essentially derived from differentiated somatic cells, so that the cell has limited division capability and weak stress resistance. In the industrial production, cell line degeneration is easy to occur, and the division capability is weak. In addition, the culture period is long, and the content of effective components in the obtained stem cells needs to be improved.
The ginseng adventitious root has meristem at the root tip part and strong division capability. Therefore, if a method for obtaining ginseng stem cells from the culture of mature ginseng with shorter time and simpler steps can be explored, and the content of effective components in the ginseng stem cells can be increased, the method is beneficial to clinical application and has wide significance.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the prior art and provide a method for separating and culturing ginseng stem cells. The invention obtains the method for obtaining the ginseng stem cells from the culture of the mature ginseng, which has shorter required time and simpler steps, particularly selects a specific liquid culture medium to culture the expanded adventitious roots in the process of preparing the adventitious roots by a one-step method, then dissects and separates the stem cell area of the tip part of the obtained adventitious roots, and then matches the stem cell induction culture medium and the stem cell expansion culture medium with proper component proportion to ensure that the separated stem cells continue to grow, the steps are simpler, and the content of various ginsenosides in the obtained stem cells is high.
In order to solve the technical problems, the invention adopts the technical scheme that:
the invention provides a separation culture method of ginseng stem cells, which comprises the following steps:
(1) cleaning and disinfecting mature ginseng, slicing, and inoculating the ginseng to an adventitious root induction culture medium to induce adventitious roots of the ginseng; inoculating the obtained ginseng adventitious root to an adventitious root induction culture medium again for subculture and propagation; then inoculating the ginseng adventitious roots obtained by propagation into a liquid culture medium for culture to obtain the ginseng adventitious roots;
(2) taking a root tip part of the ginseng adventitious root prepared in the step (1), and dissecting and separating to obtain a stem cell area;
(3) inoculating the stem cells obtained by separation in the step (2) into a stem cell induction culture medium for dark culture to obtain a stem cell mass, and transferring the stem cell mass into a subculture medium for dark culture;
(4) inoculating the stem cells subjected to subculture in the step (3) into a stem cell liquid culture medium, and performing dark culture to obtain ginseng stem cells;
the liquid culture medium used in the step (1) comprises: 10-55g/L of sucrose, 0.6-1.6g/L B5 of culture medium, 0.3-1.2g/L of 1/2MS culture medium, 0-5.4mg/L of 6-benzylamino adenine, 0-5.4mg/L of naphthylacetic acid, 0-8mg/L of gibberellin and 0-5.4mg/L of indoleacetic acid.
In the scheme, the method adopts a one-step method to culture the adventitious roots of the ginseng from the mature ginseng, so that the steps can be saved, and the induction time can be shortened; the root tip part of the ginseng adventitious root has more meristematic tissues and strong activity. The existing adventitious roots are generally generated by ginseng callus induction, the callus needs to be induced firstly and then the adventitious roots need to be induced, the required experimental period is long, the operation steps are complex, and the pollution risk is high. In addition, in the process of inducing the callus to generate adventitious roots, the metabolism degree of the ginseng tissue is higher, so that the content of the generated saponin is reduced, and the ginseng obtained by callus induction culture is difficult to meet the requirements of clinical application and the like.
In the above scheme, in view of the fact that mature ginseng is long in age, high in maturity and not easy to differentiate, there is no report that adventitious roots can be directly induced from mature ginseng at present. However, the inventors of the present invention have unexpectedly found that the mature ginseng slices can be directly induced to produce adventitious roots on a specific induction medium. Therefore, the intermediate step of callus induction is omitted, the adventitious roots can be directly obtained from the mature ginseng by one-step induction, the induction step is simplified, the induction time is shortened, and the problem of saponin content reduction caused by the metabolic process from generation to differentiation of the callus is also avoided.
The further scheme of the invention is as follows: the liquid culture medium in the step (1) also comprises 0-5.4mg/L of indolebutyric acid.
The further scheme of the invention is as follows: the liquid medium of step (1) comprises: 15-45g/L of sucrose, 0.8-1.3g/L B5 of culture medium, 0.5-1.0g/L of 1/2MS culture medium, 0-4.2mg/L of 6-benzylamino adenine, 1.8-5.4mg/L of naphthylacetic acid, 0-8mg/L of gibberellin, 0.6-4.8mg/L of indoleacetic acid and 0-3.6mg/L of indolebutyric acid; preferably, the liquid medium in step (1) comprises: 45g/L of sucrose, 1.28g/L B5 of culture medium, 0.905g/L of 1/2MS culture medium, 4.2mg/L of 6-benzylamino adenine, 3mg/L of naphthylacetic acid, 0.8mg/L of gibberellin, 0.6mg/L of indoleacetic acid and 0.6mg/L of indolebutyric acid.
In the scheme, in the process of inducing adventitious roots of ginseng slices, the components of a liquid culture medium after the propagation step are adjusted, and in order to prevent the MS culture medium and the B5 culture medium from guiding tissues to generate dedifferentiation, the method specifically adopts 1/2MS culture medium to reduce the content of nitrate nitrogen and simultaneously introduces a mixed reagent of cytokinin 6-benzylaminopurine and gibberellin, wherein the 6-benzylaminopurine can effectively inhibit the decomposition of nucleic acid and protein in plant tissues, and simultaneously improves the transport efficiency of amino acid, auxin and inorganic salt, and can induce the generation of the adventitious roots; gibberellin can shorten the G1 phase and the S phase in the cell cycle, so that the cells are biased to the dedifferentiation process, thereby playing a certain role in inhibiting the formation of adventitious roots; although gibberellin has inhibition on adventitious roots and generates a certain antagonistic effect with the 6-benzylamino adenine, under the combined action of 1/2MS and B5 culture media, naphthylacetic acid and indoleacetic acid, the gibberellin has the effects of promoting the transformation of sugar in plant tissues by improving the efficiency of enzyme and increasing the ductility and permeability of cell walls, so that the growth of original cells of roots is promoted. In conclusion, the adventitious root liquid culture medium provided by the invention effectively suppresses the dedifferentiation process of ginseng cells by combining the antagonistic action of cytokinin 6-benzylaminopurine and gibberellin on the growth of adventitious roots in the presence of various culture media and auxins, so that the adventitious roots with higher saponin content can be obtained while the growth multiple can be controlled at a relatively stable level without forming callus in the preparation process.
The further scheme of the invention is as follows: in the step (3), the stem cell induction culture medium comprises 2-4mg/L gibberellin, 0.6-1mg/L kinetin, 2-4mg/L indoleacetic acid, 15-75mg/L ascorbic acid, 50-150mg/L citric acid, 20-60g/L sucrose, 1-6g/L plant gel, 1-2.4g/L1/2MS culture medium and 1-2.5g/L B5 culture medium; preferably, the stem cell induction medium comprises 3mg/L gibberellin, 0.8mg/L kinetin, 2.5mg/L indoleacetic acid, 50mg/L ascorbic acid, 100mg/L citric acid, 40g/L sucrose, 3g/L plant gel, 1.8 g/L1/2MS medium, and 1.5g/L B5 medium.
In the scheme, the stem cell induction culture medium provided by the invention contains indoleacetic acid, gibberellin and kinetin, can induce the growth of stem cells, and ascorbic acid and citric acid can generate a synergistic antioxidant effect. The components in the induction culture medium have synergistic effect, and can promote the rapid growth of stem cells in the initial stage.
The further scheme of the invention is as follows: in the step (3), the stem cell subculture medium comprises 2-4mg/L2, 4-dichlorophenoxyacetic acid, 1-3mg/L gibberellin, 0.8-1.2mg/L kinetin, 20-60g/L sucrose, 1-6g/L plant gel, 1-2.4g/L1/2MS culture medium and 0.6-1.4g/L WPM culture medium; preferably, the stem cell subculture medium comprises 3mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L gibberellin, 1mg/L kinetin, 35g/L sucrose, 3g/L plant gel, 1.8 g/L1/2MS medium and 0.8g/L WPM medium.
The further scheme of the invention is as follows: in the step (4), the stem cell liquid culture medium comprises 2-4mg/L2, 4-dichlorophenoxyacetic acid, 1-3mg/L gibberellin, 0.8-1.2mg/L kinetin, 20-60g/L sucrose, 1-2.4g/L1/2MS culture medium and 0.6-1.4g/L WPM culture medium; preferably, the stem cell liquid culture medium comprises 3mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L gibberellin, 1mg/L kinetin, 35g/L sucrose, 1.8 g/L1/2MS culture medium and 0.8g/L WPM culture medium.
The further scheme of the invention is as follows: the pH value of the stem cell induction culture medium, the stem cell expansion culture medium and the stem cell liquid culture medium is 5.6-6.0. The pH of the induction culture medium, the expansion culture medium and the stem cell liquid culture medium is adjusted by using NaOH or KOH.
The further scheme of the invention is as follows: in step (4), the dark culture is performed at 20-25 ℃ and 100-150rpm for one passage for 3-5 weeks.
The further scheme of the invention is as follows: in the step (2), the root tip part of the adventitious root of the ginseng cultured in the step (1) is taken, the root tip part is observed under a microscope, a stem cell area is determined according to the characteristics of stem cells, and the stem cell area at the root tip is obtained by cutting through a scalpel through micromanipulation.
In the above scheme, the root tip part is observed under a microscope, and the irregular part of the cell shape and arrangement is a growing point, namely a meristem cell aggregation region, wherein the cell contains a plurality of vacuoles. And cutting by a micro-operation scalpel to obtain a root tip stem cell area.
The further scheme of the invention is as follows: in the step (3), the ginseng stem cells after dissection are respectively and sequentially cultured in a stem cell induction culture medium and a stem cell propagation culture medium at the temperature of 20-25 ℃ in a dark place until a large number of cell clusters grow out by inoculating the stem cells.
The further scheme of the invention is as follows: in the step (1), the adventitious root induction culture medium comprises 1-6mg/L of naphthylacetic acid, 0.1-0.6mg/L of kinetin, 0.2-1mg/L of gibberellin, 0.075-1.5g/L of citric acid, 0.03-1g/L of ascorbic acid, 20-60g/L of sucrose, 1-6g/L of plant gel, 1-4g/L B5 of culture medium and 1-2.4g/L of WPM culture medium. Preferably, the adventitious root induction medium includes 4mg/L naphthylacetic acid, 0.6mg/L gibberellin, 0.4mg/L kinetin, 0.1g/L citric acid, 0.05g/L ascorbic acid, 30g/L sucrose, 3g/L plant gel, 1.55g/L B5 medium and 1.21g/L WPM medium.
In the scheme, the naphthylacetic acid is a plant growth regulator, the gibberellin is a plant hormone, and the formation of adventitious roots can be promoted under the combined action. Kinetin is a cytokinin that promotes cell division. Citric acid and ascorbic acid can generate a synergistic antioxidation effect, prevent the in vitro tissue of the mature ginseng from browning, and are beneficial to directly inducing adventitious roots from the in vitro tissue of the mature ginseng. The components in the induction culture medium have synergistic effect, and the aim of directly inducing each part of the mature ginseng to generate adventitious roots is finally realized without an intermediate step of inducing callus. The induction culture medium under the preferable component proportion has the best induction effect on the mature ginseng to generate the adventitious roots, has a large quantity of the generated adventitious roots and good quality, is beneficial to the next step of propagation expansion, and improves the content of active components in the adventitious roots.
In the scheme, when the adventitious roots generated by induction are further subjected to liquid culture, a culture medium for conventional culture, such as 1/2MS culture medium, can be adopted and is consistent with a culture medium for common adventitious roots, so that the technical purpose of shortening the induction time can be realized by adopting the one-step method for inducing the adventitious roots in the invention under the condition of adopting the conventional culture medium for culture, and meanwhile, the pollution risk is reduced compared with the two-step method in the prior art.
In the present invention, WPM medium, N6 medium, B5 medium, 1/2MS medium, and the like are known in the art.
1/2MS culture medium
Ingredient (mg/L): potassium nitrate 950, ammonium nitrate 825, calcium chloride dihydrate 220, magnesium sulfate 185, monopotassium phosphate 85, manganese sulfate 11.15, zinc sulfate 4.3, boric acid 3.1, potassium iodide 0.415, sodium molybdate 0.125, copper sulfate 0.0125, cobalt chloride 0.0125, disodium ethylenediaminetetraacetate 37.3, ferrous sulfate 27.8, inositol 100, glycine 2, hydrochloric acid 0.5, pyridoxine hydrochloride 0.5 and ammonium sulfate hydrochloride 0.1.
B5 Medium
Ingredient (mg/L): potassium nitrate KNO3 2500，MgSO4·7H2O 250，CaCl2·2H2O 150，(NH4)2SO4134，NaH2PO4.H2O 150，KI 0.75，H3BO3 3.0，MnSO4·4H2O 10，ZnSO4·7H2O 2.0，Na2MoO4·2H2O 0.25，CoCl2·6H2O 0.025，CuSO4·5H2O 0.025，Na2-EDTA 37.3，FeSO4·7H2O27.8, inositol 100, nicotinic acid 1.0, pyridoxine hydrochloride 1.0, and ammonium sulfate hydrochloride 10.
Woody Plant Medium (WPM)
Ingredient (mg/L): 400 parts of ammonium nitrate, 556 parts of tetrahydrate calcium nitrate, 990 parts of potassium sulfate, 72 parts of anhydrous calcium chloride, 170 parts of monopotassium phosphate, 0.25 part of sodium molybdate dihydrate, 180 parts of anhydrous magnesium sulfate, 22.4 parts of manganese sulfate monohydrate, 8.6 parts of zinc sulfate heptahydrate, 0.25 part of copper sulfate pentahydrate, 27.8 parts of ferrous sulfate heptahydrate, 37.3 parts of disodium ethylenediamine tetraacetic acid, 100 parts of inositol, 11 parts of vitamin B, 0.5 part of nicotinic acid, 60.5 parts of vitamin B, 2 parts of glycine and 5.2 parts of pH.
N6 culture medium
Ingredient (mg/L): potassium nitrate 2800, ammonium sulfate 463, potassium dihydrogen sulfate 400, magnesium sulfate heptahydrate 185, calcium chloride dihydrate 165, disodium ethylenediaminetetraacetate 37.3, ferrous sulfate heptahydrate 27.8, manganese sulfate hydrate 4.4, zinc sulfate heptahydrate 1.5, boric acid 1.6, potassium iodide 0.8, vitamin B11.0, vitamin B60.5, hydrochloric acid 0.5, glycine 2.0, sucrose 2000, pH 5.8(25 ℃).
The further scheme of the invention is as follows: in the step (1), the inoculating the ginseng adventitious roots obtained by propagation to the liquid medium comprises: cutting the ginseng adventitious roots obtained by propagation into small segments, inoculating the small segments into a liquid culture medium, and culturing the small segments on a shaking table at the temperature of 22 +/-1 ℃ and the rotation speed of 110-.
The further scheme of the invention is as follows: in the step (1), the step of inoculating the ginseng adventitious roots obtained by propagation to a liquid culture medium further comprises: inoculating the adventitious roots cultured on the shaking table for 3-4 weeks into a biological reaction device containing a liquid culture medium, and continuously culturing for 3-4 weeks in a dark place, wherein the ventilation volume of the biological reaction device is 0.01-0.4vvm, and the culture temperature is 22 +/-1 ℃; preferably, the aeration is from 0.15 to 0.25 vvm.
In the scheme, the volume of the sterilized liquid culture medium accounts for 20-80% of the volume of the biological reaction device; the mass of the inoculated adventitious roots accounts for 0.5-2.5% of the volume of the liquid culture medium. The biological reaction device is used for culturing the ginseng adventitious roots and comprises: the tank body is provided with a cover body which can be opened and closed at the top, and an exhaust device is arranged on the cover body or the top of the tank body; at least two air inlet devices are arranged at the bottom of the tank body, and air enters the tank body through the air inlet devices.
In the above scheme, the tank body of the biological reaction device is hollow and is used for containing a liquid culture medium. The bioreactor can be made of any material which is suitable for preparing a fermentation tank and can be used for high-temperature sterilization, such as glass, stainless steel, high-temperature resistant plastic and the like; the stainless steel material is optimized, and the device is durable and long in service life. The cover body on the top of the tank body can be opened or closed and is used for adding liquid culture medium inwards, and the cover body is connected with the tank body in a sealing mode after the liquid culture medium is added. The bottom of the tank body is provided with at least two air inlet devices, and sterile air is introduced into the tank body from different positions, so that cultures in the tank can be fully contacted with the air, and the growth is uniform, and the fast growth of adventitious roots is promoted.
The further scheme of the invention is as follows: in the step (1), the slices are slices with the width of 0.5-0.7cm, the length of 0.5-0.7cm and the thickness of 0.2-0.5 mm; the ginseng age of the mature ginseng is more than 3 years, preferably, the ginseng age of the mature ginseng is more than 6 years.
As a preferred embodiment, the mature ginseng is a centennial ginseng.
The century ginseng is rare in nature, has high edible and medicinal values, can tonify five internal organs, calm spirit, calm soul, stop palpitation, remove pathogenic qi, improve eyesight, and benefit heart and intelligence; the value of the ginseng cultivation method is far higher than that of planted ginseng with short ginseng age. The invention does not need the intermediate step of inducing callus, can directly obtain the adventitious roots by one-step induction from the hundred-year ginseng cut blocks, not only can simplify the induction step and shorten the induction time, but also can obtain the specific functional components in the female parent hundred-year old ginseng, thereby obtaining the adventitious roots with better nutritive value.
Further, the main root, or the reed head, or the part, or the branch root, or the fibrous root of the mature ginseng are cleaned, disinfected, sliced and inoculated into an induction culture medium to induce the adventitious root of the ginseng.
In the scheme, the saponin content in different parts of the ginseng is different, wherein the root part has the highest content, so the part is preferably sliced to improve the saponin content in the induced adventitious root.
In a further scheme, the ginseng is selected from wild ginseng, transplanted ginseng, ginseng under forest and garden ginseng; preferably, the ginseng is wild ginseng.
After adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects:
1. according to the isolated culture method of the ginseng stem cells, the stem cell area is obtained by microscopic observation and dissection of the adventitious roots of the ginseng prepared by the one-step method, and the isolated stem cells are cultured by matching with the stem cell induction culture medium and the stem cell subculture medium with appropriate component proportions, so that the stem cells grow at a very high speed, and the content of active ingredients in the obtained ginseng stem cells is improved;
2. the method for isolated culture of the ginseng adventitious roots realizes the one-step method, and the processed each part of the mature ginseng is inoculated into the induction culture medium to directly induce the ginseng adventitious roots without the intermediate step of inducing callus, so that the induction step can be simplified, the induction time can be shortened, and the pollution risk can be reduced;
3. according to the method for separating and culturing the ginseng stem cells, in the process of culturing the ginseng adventitious roots by the one-step method, the liquid culture medium with specific components is adopted, and the liquid culture medium for the adventitious roots combines the antagonistic action of cytokinin 6-benzylaminopurine and gibberellin on the growth of the adventitious roots under the condition that various culture media and auxins exist, so that the dedifferentiation process of ginseng cells is effectively suppressed, calluses are not formed in the preparation process, the adventitious roots are directly generated, the adventitious roots with high saponin content are obtained, and the growth multiple can be controlled at a relatively stable level.
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention, are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention without limiting the invention to the right. It is obvious that the drawings in the following description are only some embodiments, and that for a person skilled in the art, other drawings can be derived from them without inventive effort. In the drawings:
FIG. 1 is a schematic view of induction of adventitious roots using the induction medium of example 1 of the present invention;
FIG. 2 is a schematic view of induction of adventitious roots using the induction medium of comparative example 1.
It should be noted that the drawings and the description are not intended to limit the scope of the inventive concept in any way, but to illustrate it by a person skilled in the art with reference to specific embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and the following embodiments are used for illustrating the present invention and are not intended to limit the scope of the present invention.
Example 1
In this embodiment, the preparation of the ginseng adventitious roots by the one-step method and the specific liquid medium culture specifically comprises the following steps:
(1) induction of adventitious roots
Removing rhizoma Phragmitis and parts of wild ginseng of 20 ages, cleaning main root, sterilizing, cutting into slices with width of 0.6cm, length of 0.7cm and thickness of 0.3mm, inoculating into induction culture medium, and dark culturing at 22 + -1 deg.C for 4-5 weeks to induce adventitious root of wild ginseng; wherein the induction culture medium comprises 4mg/L naphthylacetic acid, 0.6mg/L gibberellin, 0.4mg/L kinetin, 0.1g/L citric acid, 0.05g/L ascorbic acid, 30g/L sucrose, 3g/L plant gel, 1.55g/L B5 culture medium and 1.21g/L WPM culture medium, and the pH value is 5.8.
(2) Subculture of adventitious roots
Inoculating the mountain ginseng adventitious roots obtained in the step (1) into the same induction culture medium as that in the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) cultivation of adventitious roots
Shearing the mountain ginseng adventitious roots obtained in the step (2) into tissues with the length of about 1cm, inoculating the tissues into a liquid culture medium, and culturing for 3-4 weeks on a shaking table at the temperature of 22 +/-1 ℃ to obtain adventitious roots; wherein the liquid culture medium contains 4mg/L indolebutyric acid, 0.1g/L citric acid, 0.05g/L ascorbic acid, 1/2MS culture medium and 30g/L sucrose, and has pH value of 5.8.
In this example, the adventitious roots produced on the induction medium in the step (1) are shown in FIG. 1, wherein A is a photograph of 1 week of cultivation, B is a photograph of 3 weeks of cultivation, and C is a photograph of 5 weeks of cultivation. As can be seen, after 5 weeks, adventitious roots were induced directly on the slices of the mature wild ginseng.
Example 2
In this embodiment, the preparation of the ginseng adventitious roots by the one-step method and the specific liquid medium culture specifically comprises the following steps:
(1) induction of adventitious roots
Cleaning reed heads of 6-age garden ginseng, sterilizing, cutting into slices with the width of 0.5cm, the length of 0.6cm and the thickness of 0.3mm, inoculating the slices into an induction culture medium, and performing dark culture at the temperature of 22 +/-1 ℃ for 4-5 weeks to induce adventitious roots; wherein the induction culture medium comprises 6mg/L naphthylacetic acid, 0.2mg/L gibberellin, 0.4mg/L kinetin, 1.2g/L citric acid, 0.1g/L ascorbic acid, 20g/L sucrose, 5g/L plant gel, 4g/L B5 culture medium and 1.8g/L LWPM culture medium, and the pH value is 5.6.
(2) Subculture of adventitious roots
Inoculating the adventitious roots obtained in the step (1) into the same induction culture medium as that in the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) cultivation of adventitious roots
Shearing the adventitious roots obtained in the step (2) into tissues with the length of about 1cm, inoculating the tissues into a liquid culture medium, and culturing for 3-4 weeks on a shaking table at the temperature of 22 +/-1 ℃ to obtain the adventitious roots; wherein the liquid culture medium contains 4mg/L indolebutyric acid, 0.1g/L citric acid, 0.05g/L ascorbic acid, 1/2MS culture medium and 30g/L sucrose, and has pH value of 5.6.
Similar to the results of example 1, the adventitious roots can be induced by one step in step (1) of this example.
Example 3
In this embodiment, the preparation of the ginseng adventitious roots by the one-step method and the specific liquid medium culture specifically comprises the following steps:
(1) induction of adventitious roots
Cleaning parts of age 10 of ginseng under forest, sterilizing, cutting into slices with width of 0.7cm, length of 0.7cm and thickness of 0.5mm, inoculating into induction culture medium, and dark culturing at 22 + -1 deg.C for 4-5 weeks to induce adventitious roots; wherein the induction culture medium comprises 5mg/L naphthylacetic acid, 1mg/L gibberellin, 0.1mg/L kinetin, 0.75g/L citric acid, 0.03g/L ascorbic acid, 40g/L sucrose, 4g/L plant gel, 2g/L B5 culture medium and 1g/L WPM culture medium, and the pH value is 6.0.
(2) Subculture of adventitious roots
Inoculating the adventitious roots obtained in the step (1) into the same induction culture medium as that in the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) cultivation of adventitious roots
Shearing the adventitious roots obtained in the step (2) into tissues with the length of about 2cm, inoculating the tissues into a liquid culture medium, and culturing for 3-4 weeks on a shaking table at the temperature of 22 +/-1 ℃ to obtain the adventitious roots; wherein the liquid culture medium contains 4mg/L indolebutyric acid, 0.1g/L citric acid, 0.05g/L ascorbic acid, WPM culture medium and 30g/L sucrose, and has pH of 6.0.
Similar to the results of example 1, the adventitious roots can be induced by one step in step (1) of this example.
Example 4
In this embodiment, the preparation of the ginseng adventitious roots by the one-step method and the specific liquid medium culture specifically comprises the following steps:
(1) induction of adventitious roots
Cleaning main root of 15-age mountain ginseng, sterilizing, cutting into slices with width of 0.5cm, length of 0.6cm and thickness of 0.4mm, inoculating into induction culture medium, and dark culturing at 22 + -1 deg.C for 4-5 weeks to induce mountain ginseng adventitious root; wherein the induction culture medium comprises 1mg/L naphthylacetic acid, 0.5mg/L gibberellin, 0.6mg/L kinetin, 1.5g/L citric acid, 1g/L ascorbic acid, 50g/L sucrose, 6g/L plant gel, 1g/L B5 culture medium and 2.4g/L WPM culture medium, and the pH value is 5.7.
(2) Subculture of adventitious roots
Inoculating the mountain ginseng adventitious roots obtained in the step (1) into the same induction culture medium as that in the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) cultivation of adventitious roots
Shearing the mountain ginseng adventitious roots obtained in the step (2) into tissues with the length of about 1cm, inoculating the tissues into a liquid culture medium, and culturing for 3-4 weeks on a shaking table at the temperature of 22 +/-1 ℃ to obtain adventitious roots; wherein the liquid culture medium contains 4mg/L indolebutyric acid, 0.1g/L citric acid, 0.05g/L ascorbic acid, B5 culture medium and 30g/L sucrose, and has pH value of 5.7.
Similar to the results of example 1, in step (1) of this example, adventitious roots can be induced directly by one step.
Example 5
In this embodiment, the preparation of the ginseng adventitious roots by the one-step method and the specific liquid medium culture specifically comprises the following steps:
(1) induction of adventitious roots
Removing rhizoma Phragmitis and parts of wild ginseng, cleaning main root, sterilizing, cutting into slices with width of 0.6cm, length of 0.7cm and thickness of 0.3mm, inoculating into induction culture medium, and dark culturing at 22 + -1 deg.C for 4-5 weeks to induce adventitious root of wild ginseng; wherein the induction culture medium comprises 4mg/L naphthylacetic acid, 0.6mg/L gibberellin, 0.4mg/L kinetin, 0.1g/L citric acid, 0.05g/L ascorbic acid, 30g/L sucrose, 3g/L plant gel, 1.55g/L B5 culture medium and 1.21g/L WPM culture medium, and the pH value is 5.8.
(2) Subculture of adventitious roots
Inoculating the mountain ginseng adventitious roots obtained in the step (1) into the same induction culture medium as that in the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) cultivation of adventitious roots
Shearing the mountain ginseng adventitious roots obtained in the step (2) into tissues with the length of about 1cm, inoculating the tissues into a liquid culture medium, and culturing for 3-4 weeks on a shaking table at the temperature of 22 +/-1 ℃ to obtain adventitious roots; wherein the liquid culture medium contains 4mg/L indolebutyric acid, 0.1g/L citric acid, 0.05g/L ascorbic acid, 1/2MS culture medium and 30g/L sucrose, and has pH value of 5.8.
Similar to the results of example 1, in step (1) of this example, adventitious roots can be induced directly by one step.
Example 6
In this example, the adventitious roots of ginseng were prepared by further combining the bioreactor with the basis of example 1, and the adventitious roots prepared in step (3) of example 1 were further processed as follows:
adding a liquid culture medium into a tank body (the volume is 5L) of a biological reaction device, wherein the volume of the liquid culture medium in the biological reaction device accounts for 20% of the volume of the biological reaction device; sterilizing at 121 deg.C for 20 min;
cutting the mountain ginseng adventitious roots obtained in example 1 into tissues with a length of about 1 cm; inoculating the adventitious roots into a liquid culture medium of a biological reaction device, wherein the mass of the inoculated adventitious roots accounts for 0.5 percent of the volume of the liquid culture medium; ventilating the biological reaction device, wherein the ventilation rate is 0.01vvm, and culturing in the dark at the temperature of 22 +/-1 ℃ for 3-4 weeks to obtain the adventitious roots.
The liquid medium used in this example was the same as that used in example 1.
Examples 7 to 16
Examples 7 to 16 are obtained by replacing the liquid medium in step (3) with a medium containing 10 to 55g/L of sucrose, 0.6 to 1.6g/L B5 of the medium, 0.3 to 1.2g/L of 1/2MS medium, 0 to 5.4mg/L of 6-benzylaminopurine, 0 to 5.4mg/L of naphthylacetic acid, 0 to 8mg/L of gibberellin, 0 to 5.4mg/L of indoleacetic acid, and 0 to 5.4mg/L of indolebutyric acid, based on example 1, and have a pH of 5.8. The specific content ratio of the medium in each example is shown in Table 1.
Example 17
In this example, the isolated culture of ginseng stem cells using the ginseng adventitious roots obtained in example 10 specifically includes the following steps:
(1) the ginseng adventitious roots prepared in example 10 were used;
(2) taking 0.5mm of the apical part of the adventitious root of the ginseng in the step (1), and dissecting and cutting a stem cell area of the apical part by micromanipulation;
(3) inoculating the stem cells obtained by separation in the step (2) into a stem cell induction culture medium, and carrying out dark culture at the temperature of 22 ℃ until a large number of cell clusters grow out from the inoculated stem cells; the stem cell induction medium comprises: 3mg/L gibberellin, 0.8mg/L kinetin, 2.5mg/L indoleacetic acid, 50mg/L ascorbic acid, 100mg/L citric acid, 40g/L sucrose, 3g/L plant gel, 1.8 g/L1/2MS culture medium, 1.5g/L B5 culture medium, and the pH is 5.8; then collecting the stem cells, transferring the stem cells into a stem cell subculture medium, and continuously culturing the stem cells in a dark place at 22 ℃ until a large number of cell clusters grow from the inoculated stem cells; the stem cell subculture medium comprises 3mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L gibberellin, 1mg/L kinetin, 35g/L cane sugar, 3g/L plant gel, 1.8 g/L1/2MS medium and 0.8g/L WPM medium, and the pH is 5.8;
(4) inoculating the stem cells subjected to subculture in the step (3) into a stem cell liquid culture medium, continuously culturing in a dark place under the conditions of 22 ℃ and 120rpm shaking table, and carrying out passage once for 4 weeks to obtain wild ginseng adventitious root stem cells; the stem cell liquid culture medium comprises 3mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L gibberellin, 1mg/L kinetin, 35g/L cane sugar, 1.8 g/L1/2MS culture medium and 0.8g/L WPM culture medium; the pH was 5.8.
Example 18
In this example, the cultivation of ginseng stem cells using the ginseng adventitious roots obtained in example 11 specifically includes the following steps:
(1) the ginseng adventitious roots prepared in example 11 were used;
(2) taking the apical part of the adventitious root of the ginseng in the step (1) to be 0.8mm, and dissecting and cutting the stem cell area of the apical part by micromanipulation;
(3) inoculating the stem cells obtained by separation in the step (2) into a stem cell induction culture medium, and carrying out dark culture at 25 ℃ until a large number of cell clusters grow out from the inoculated stem cells; the stem cell induction medium comprises: 4mg/L gibberellin, 0.6mg/L kinetin, 2mg/L indoleacetic acid, 75mg/L ascorbic acid, 50mg/L citric acid, 20g/L sucrose, 6g/L plant gel, 2.4g/L1/2MS culture medium, 1g/L B5 culture medium, and the pH is 5.8; then collecting the stem cells, transferring the stem cells into a stem cell subculture medium, and continuously culturing in a dark place at 25 ℃ until a large number of cell clusters grow from the inoculated stem cells; the stem cell subculture medium comprises 2 mg/L2, 4-dichlorophenoxyacetic acid, 3mg/L gibberellin, 1.2mg/L kinetin, 60g/L cane sugar, 1g/L plant gel, 1 g/L1/2MS medium and 1.4g/L WPM medium, and the pH is 5.8;
(4) inoculating the stem cells subjected to subculture in the step (3) into a stem cell liquid culture medium, continuously culturing in a dark place at 22 ℃ under the condition of a 120rpm shaking table, and carrying out passage once for 3 weeks to obtain wild ginseng adventitious root stem cells; the stem cell liquid culture medium comprises 2 mg/L2, 4-dichlorophenoxyacetic acid, 3mg/L gibberellin, 1.2mg/L kinetin, 60g/L cane sugar, 1 g/L1/2MS culture medium and 1.4g/L WPM culture medium, and the pH is 5.8.
Example 19
In this example, the cultivation of ginseng stem cells using the ginseng adventitious roots obtained in example 14 specifically includes the following steps:
(1) the ginseng adventitious roots prepared in example 14 were used;
(2) taking the apical part of the adventitious root of the ginseng in the step (1) to be 0.3mm, and dissecting and cutting the stem cell area of the apical part by micromanipulation;
(3) inoculating the stem cells obtained by separation in the step (2) into a stem cell induction culture medium, and carrying out dark culture at the temperature of 20 ℃ until a large number of cell clusters grow out from the inoculated stem cells; the stem cell induction medium comprises: 2mg/L gibberellin, 1mg/L kinetin, 4mg/L indoleacetic acid, 15mg/L ascorbic acid, 150mg/L citric acid, 60g/L sucrose, 1g/L plant gel, 1 g/L1/2MS culture medium, 2.5g/L B5 culture medium, and the pH is 5.8; then collecting the stem cells, transferring the stem cells into a stem cell subculture medium, and culturing at 22 ℃ in a dark place until a large number of cell clusters grow from the inoculated stem cells; the stem cell subculture medium comprises 4mg/L2, 4-dichlorophenoxyacetic acid, 1mg/L gibberellin, 0.8mg/L kinetin, 20g/L sucrose, 6g/L plant gel, 2.4g/L1/2MS medium and 0.6g/L WPM medium, and the pH is 5.8;
(4) inoculating the stem cells subjected to subculture in the step (3) into a stem cell liquid culture medium, continuously culturing in a dark place at 22 ℃ under the condition of a 120rpm shaking table, and carrying out passage once for 5 weeks to obtain wild ginseng adventitious root stem cells; the stem cell liquid culture medium comprises 4mg/L2, 4-dichlorophenoxyacetic acid, 1mg/L gibberellin, 0.8mg/L kinetin, 20g/L sucrose, 2.4g/L1/2MS culture medium and 0.6g/L WPM culture medium, and the pH is 5.8.
Example 20
In this example, ginseng stem cells were cultured using the ginseng adventitious roots obtained in example 1, and other embodiments of this example are the same as in example 17.
Comparative example 1
This comparative example differs from example 1 in the induction medium used and the other steps are carried out with reference to example 1. The induction culture of this comparative example included: 30g/L of sucrose, 0.5mg/L of kinetin, 3mg/L of indolebutyric acid, 1.5mg/L of 2, 4-dichlorophenoxyacetic acid, 1/2MS culture medium, 3g/L of plant gel and pH value of 5.8.
FIG. 2 shows the results of induction of adventitious roots on the medium in step (1) of this comparative example, wherein A is a photograph taken after 1 week of culture, B is a photograph taken after 3 weeks of culture, and C is a photograph taken after 5 weeks of culture. As can be seen, after 5 weeks, the culture medium of comparative example 1 failed to induce the generation of adventitious roots directly from the mature wild ginseng slices.
Comparative example 2
This comparative example differs from example 1 in the induction medium used and the other steps are carried out with reference to example 1. The induction culture of this comparative example included: 30g/L of sucrose, 0.5mg/L of kinetin, 3mg/L of indolebutyric acid, 1/2MS culture medium, 3g/L of plant gel and pH value of 5.8.
As a result, the adventitious roots cannot be induced directly from the mature wild ginseng slice, similarly to the picture display of comparative example 1.
Comparative example 3
This comparative example differs from example 1 in the induction medium used and the other steps are carried out with reference to example 1. The induction culture of this comparative example included: 30g/L of sucrose, 0.5mg/L of kinetin, 3mg/L of indoleacetic acid, 3g/L of WPM and 3g/L of plant gel, and the pH value is 5.8.
As a result: the whole body turns yellow in the first week, the color deepens in the third week, the middle part begins to turn brown, and the whole body turns brown and is dried up in the fifth week.
Test example 1
In this test example, the fold increase and ginsenoside content of the ginseng adventitious roots obtained in examples 7 to 16 were measured by the following methods:
a method for detecting ginsenoside in adventitious roots of ginseng comprises the following steps:
(1) principle of
The sample is separated by a C18 chromatographic column after pretreatment such as extraction, and is detected by a high performance liquid chromatography-ultraviolet detector, and the content of each component of the ginsenoside is quantitatively measured by an external standard method.
(2) Reagent
Methanol (CH)4O): chromatographically pure acetonitrile (C)6H11N): pure chromatography
(3) Analytical procedure
Taking about 6g (accurate to 0.01g) of the uniformly mixed sample, grinding the sample in a 150mL mortar, transferring the sample into a 50mL centrifuge tube, adding 10mL of water, uniformly mixing, breaking the wall for 3 minutes by 400W on an ultrasonic cell crusher, and freezing the sample in a refrigerator at the temperature of 18 ℃ below zero for 3 hours. Freeze-drying in a freeze-drying machine for 48 hours until no water drops exist outside the cup body.
The sample was ground in a mortar and 50mg was weighed accurately into a 10ml centrifuge tube, 70% methanol solution was added and vortexed. Sonicate on a sonicator for 10 minutes, repeat twice, filter for use.
(4) Reference conditions of the apparatus
A) A chromatographic column: c18 column with column length of 150mm, column inner diameter of 4.6mm, column packing particle size of 5 μm, or equivalent;
B) mobile phase: a: acetonitrile, b: filtering water with 0.45 μm microporous membrane;
C) flow rate: 0.7 mL/min; gradient elution procedure: 0-13 min, 23% -46% acetonitrile, and the volume flow rate is 0.7 mL/min; 13-33 min, 46-68% acetonitrile, and the volume flow rate is 0.7 mL/min; 33-46.5 min, 68-85% acetonitrile, and the volume flow rate is 0.7 mL/min;
D) the column temperature is 30 ℃;
E) the detection wavelength is 203 nm;
F) the injection volume is 10 muL.
(5) Presentation of analytical results
The content of each component of the ginsenoside in the sample is calculated according to the formula (1):
Figure BDA0003028698310000161
in the formula:
x is the content of each component of ginsenoside in the sample, and the unit is milligram per kilogram (mg/kg) or milligram per liter (mg/L);
a1-area of peak of ginsenoside component in sample
A2 peak area of ginsenoside component in standard
Rho-concentration of each component of ginsenoside in standard (ug/ml)
V — final volumetric volume of sample solution in milliliters (mL);
m-sample mass in grams (g);
the content of ginsenoside in the sample is obtained by adding the components.
The content of ginsenoside in the sample is the sum of the components to be detected.
Secondly, the calculation mode of the increase multiple is as follows:
the growth factor is the weight of adventitious roots after the end of growth/the weight of inoculated adventitious root seeds.
The results are shown in table 1:
TABLE 1
Figure BDA0003028698310000171
As can be seen from Table 1, the difference between the growth multiples of the adventitious roots provided in the above examples is stable, which indicates that the adventitious roots prepared by the liquid medium provided by the present invention have stable yield quality. Specifically, according to the descriptions of example 8 and example 11, in the case that the component IBA of indolebutyric acid is removed and the concentration of the carbon source is low in example 11, the increase times of the adventitious roots are the same as that of the medium with the high concentration of the carbon source in example 8, the IAA content of the indoleacetic acid in example 11 is higher, and the ratio of gibberellin in the benzyl gibberellic mixture is reduced compared with that in example 8, so that the adventitious roots in example 11 have higher saponin content, which shows that the synergistic effect of the antagonism of the benzyl gibberellic mixture and the indole acetic acid production is helpful for improving the saponin of the adventitious roots. In example 12, the benzyl gibberellin mixture component is removed, the increase multiple of the benzyl gibberellin is greatly increased due to the lack of inhibition of gibberellin on adventitious roots, but the finally produced saponin content is lower than that of the adventitious roots in example 11 due to the lack of cytokinin 6-benzylaminopurine 6-BA, and the influence of the benzyl gibberellin mixture component on the adventitious root saponin content is further proved. In example 13, the content of gibberellin in the benzyl gibberellin mixture is further reduced on the basis of removing the naphthylacetic acid component, the multiple of growth of the obtained adventitious roots is improved due to the reduction of the ratio of gibberellin, but the content of saponin of the adventitious roots prepared by the naphthylacetic acid-deficient culture medium is greatly reduced due to the decisive factor of the naphthylacetic acid on the selective expression of mRNA. Example 16 was conducted in a manner such that the indole acetic acid in the liquid medium was removed, and as described above, antagonism of the benzyl erythrose mixture failed to produce a synergistic effect with indole acetic acid, resulting in a decrease in the saponin content.
In this experimental example, the liquid culture media prepared in examples 11 and 14 were used, the fold increase in adventitious roots was relatively stable, and the total saponin content was high, whereas in example 10, the fold increase was large, but the saponin content was relatively high, and in the case of culturing stem cells subsequently to adventitious roots, the influence of the fold increase was partially negligible, and examples 10, 11, and 14 having a higher total saponin content were preferably used. Among them, examples 10 and 14 are the most preferable.
Test example 2
In this test example, saponin detection was performed on the wild ginseng adventitious root stem cells obtained in examples 17 to 20 and the wild ginseng slices used in example 1, respectively.
The detection method is as follows:
(1) principle of
The sample is separated by a C18 chromatographic column after pretreatment such as extraction, and is detected by a high performance liquid chromatography-ultraviolet detector, and the content of each component of the ginsenoside is quantitatively measured by an external standard method.
(2) Reagent
Methanol (CH)4O): chromatographically pure acetonitrile (C)6H11N): pure chromatography
Standard reagents: ginsenoside Re, Rg1, Ra3, Rb1, Rf, Rb2, Rb3, F3, Rg2, Rd, F1
(3) Analytical procedure
Preparing a ginseng stem cell sample:
the samples obtained in examples 17 to 20 were washed three times with water, ground into a paste in a mortar, subjected to ultrasonic wall breaking and freeze-drying, then the samples were porphyrized in the mortar, 50mg were accurately weighed into a 10ml centrifuge tube, 70% methanol solution was added, and vortexed. Sonicate on a sonicator for 10 minutes, repeat twice, filter for use. The latter is the same as the adventitious root detection method.
Preparing a wild ginseng sample:
washing the sliced wild ginseng sample with water for three times, grinding the sliced wild ginseng sample into paste in a mortar, ultrasonically breaking the wall, freeze-drying, grinding the sample on the mortar, accurately weighing 50mg in a 10ml centrifuge tube, adding 70% methanol solution, and vortexing. Sonicate on a sonicator for 10 minutes, repeat twice, filter for use.
Preparing a standard substance:
preparation of stock solution (0.8 mg/ml): respectively weighing 11 kinds of standard substances including 8.00mg of ginsenoside Re, Rg1, Ra3, Rb1, Rf, Rb2, Rb3, F3, Rg2, Rd and F1 in a 10ml volumetric flask, and fixing the volume by using high-grade pure methanol.
Preparation of working solution (32 ug/ml): accurately sucking 1ml of stock solution (0.8mg/ml) into a 25ml volumetric flask, fixing the volume with high-grade pure methanol, and filtering through an organic filter membrane of 0.22um for later use.
(4) Reference conditions of the apparatus
A) A chromatographic column: c18 column with column length of 150mm, column inner diameter of 4.6mm, column packing particle size of 5 μm, or equivalent;
B) mobile phase: a: acetonitrile, b: filtering water with 0.45 μm microporous membrane;
C) flow rate: 0.7 mL/min; gradient elution procedure: 0-13 min, 23% -46% acetonitrile, and the volume flow rate is 0.7 mL/min; 13-33 min, 46-68% acetonitrile, and the volume flow rate is 0.7 mL/min; 33-46.5 min, 68-85% acetonitrile, and the volume flow rate is 0.7 mL/min;
D) column temperature: 30 ℃;
E) detection wavelength: 203 nm;
F) sample introduction volume: 10 μ L.
(5) Presentation of analytical results
The content of each component of the ginsenoside in the sample is calculated according to the formula (1):
Figure BDA0003028698310000191
in the formula:
x is the content of each component of ginsenoside in the sample, and the unit is milligram per kilogram (mg/kg) or milligram per liter (mg/L);
a1-area of peak of ginsenoside component in sample
A2 peak area of ginsenoside component in standard
Rho-concentration of each component of ginsenoside in standard (ug/ml)
V — final volumetric volume of sample solution in milliliters (mL);
m-sample mass in grams (g);
the content of ginsenoside in the sample is the sum of the components to be detected.
The results are shown in table 2:
TABLE 2
Figure BDA0003028698310000201
The results show that compared with the mature wild ginseng slice, the content of various ginsenosides in the wild ginseng adventitious root stem cells cultured by the invention is improved, and compared with the scheme of separating the cells by using a penetrant, the wild ginseng adventitious root stem cells dissected and separated from the stem cell area by observing the cell morphology, the cell activity is retained to the maximum extent, and the total content of the ginsenosides in the subsequent preparation of the stem cells is also improved. Therefore, the culture method of the ginseng stem cells is simple and convenient in steps, suitable for large-scale production and high in active ingredient content.
In the liquid phase detection, some ginsenosides peaks were relatively close and could not be separated, and therefore some ginsenosides were mixed and calculated in the table.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (10)

1. A method for isolated culture of ginseng stem cells, which is characterized by comprising the following steps:
(1) cleaning and disinfecting mature ginseng, slicing, and inoculating the ginseng to an adventitious root induction culture medium to induce adventitious roots of the ginseng; inoculating the obtained ginseng adventitious root to an adventitious root induction culture medium again for subculture and propagation; then inoculating the ginseng adventitious roots obtained by propagation into a liquid culture medium for culture to obtain the ginseng adventitious roots;
(2) taking a root tip part of the ginseng adventitious root prepared in the step (1), and dissecting and separating to obtain a stem cell area;
(3) inoculating the stem cells obtained by separation in the step (2) into a stem cell induction culture medium for dark culture to obtain a stem cell mass, and transferring the stem cell mass into a subculture medium for dark culture;
(4) inoculating the stem cells subjected to subculture in the step (3) into a stem cell liquid culture medium, and performing dark culture to obtain ginseng stem cells;
the liquid culture medium used in the step (1) comprises: 10-55g/L of sucrose, 0.6-1.6g/L B5 of culture medium, 0.3-1.2g/L of 1/2MS culture medium, 0-5.4mg/L of 6-benzylamino adenine, 0-5.4mg/L of naphthylacetic acid, 0-8mg/L of gibberellin and 0-5.4mg/L of indoleacetic acid.
2. The isolated culture method of ginseng stem cells according to claim 1, wherein the liquid medium in step (1) further comprises 0-5.4mg/L indolebutyric acid.
3. The isolated culture method of ginseng stem cells according to claim 2, wherein the liquid medium of step (1) comprises: 15-45g/L of sucrose, 0.8-1.3g/L B5 of culture medium, 0.5-1.0g/L of 1/2MS culture medium, 0-4.2mg/L of 6-benzylamino adenine, 1.8-5.4mg/L of naphthylacetic acid, 0-8mg/L of gibberellin, 0.6-4.8mg/L of indoleacetic acid and 0-3.6mg/L of indolebutyric acid;
preferably, the liquid medium in step (1) comprises: 45g/L of sucrose, 1.28g/L B5 of culture medium, 0.905g/L of 1/2MS culture medium, 4.2mg/L of 6-benzylamino adenine, 3mg/L of naphthylacetic acid, 0.8mg/L of gibberellin, 0.6mg/L of indoleacetic acid and 0.6mg/L of indolebutyric acid.
4. The isolated culture method of the ginseng stem cells according to claim 1, wherein in the step (3), the stem cell induction medium comprises 2-4mg/L gibberellin, 0.6-1mg/L kinetin, 2-4mg/L indoleacetic acid, 15-75mg/L ascorbic acid, 50-150mg/L citric acid, 20-60g/L sucrose, 1-6g/L plant gel, 1-2.4g/L1/2MS medium, 1-2.5g/L B5 medium;
preferably, the stem cell induction medium comprises 3mg/L gibberellin, 0.8mg/L kinetin, 2.5mg/L indoleacetic acid, 50mg/L ascorbic acid, 100mg/L citric acid, 40g/L sucrose, 3g/L plant gel, 1.8 g/L1/2MS medium, and 1.5g/L B5 medium.
5. The isolated culture method of the ginseng stem cells according to claim 1, wherein in the step (3), the stem cell subculture medium comprises 2-4mg/L2, 4-dichlorophenoxyacetic acid, 1-3mg/L gibberellin, 0.8-1.2mg/L kinetin, 20-60g/L sucrose, 1-6g/L plant gel, 1-2.4g/L1/2MS medium and 0.6-1.4g/L WPM medium;
preferably, the stem cell subculture medium comprises 3mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L gibberellin, 1mg/L kinetin, 35g/L sucrose, 3g/L plant gel, 1.8 g/L1/2MS medium and 0.8g/L WPM medium.
6. The isolated culture method of the ginseng stem cells according to claim 1, wherein in the step (4), the liquid culture medium of the stem cells comprises 2-4mg/L2, 4-dichlorophenoxyacetic acid, 1-3mg/L gibberellin, 0.8-1.2mg/L kinetin, 20-60g/L sucrose, 1-2.4g/L1/2MS medium and 0.6-1.4g/L WPM medium;
preferably, the stem cell liquid culture medium comprises 3mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L gibberellin, 1mg/L kinetin, 35g/L sucrose, 1.8 g/L1/2MS culture medium and 0.8g/L WPM culture medium.
7. The isolated culture method of ginseng stem cells according to claim 6, wherein the dark culture is performed at 20-25 ℃ and 100-150rpm for one passage at 3-5 weeks in step (4).
8. The isolated culture method of ginseng stem cells according to claim 1, wherein in the step (2), the apical part of the adventitious root of ginseng cultured in the step (1) is taken, the apical part is observed under a microscope, the stem cell region is determined according to the characteristics of the stem cells, and the apical stem cell region is obtained by cutting with a scalpel through micromanipulation.
9. The isolated culture method of the ginseng stem cells according to claim 1, wherein in the step (3), the ginseng stem cells after dissection and separation are respectively cultured in the stem cell induction culture medium and the stem cell subculture medium in sequence at 20-25 ℃ in a dark place until a large number of cell clusters grow from the inoculated stem cells.
10. The isolated culture method of ginseng stem cells according to claim 1, wherein the adventitious root-inducing medium in step (1) comprises 1-6mg/L naphthylacetic acid, 0.1-0.6mg/L kinetin, 0.2-1mg/L gibberellin, 0.75-1.5g/L citric acid, 0.03-1g/L ascorbic acid, 20-60g/L sucrose, 1-6g/L phytogel, 1-4g/L B5 medium and 1-2.4g/L WPM medium.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022206424A1 (en) * 2021-04-02 2022-10-06 山东安然纳米实业发展有限公司 Method for separating and culturing ginseng stem cells
CN115786119A (en) * 2023-02-13 2023-03-14 山东卫康生物医药科技有限公司 Isolated culture control method and system for ginseng stem cells

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101606487A (en) * 2009-07-13 2009-12-23 天津大学 Tissue culture method of ginseng adventitious root
KR20110123934A (en) * 2010-05-10 2011-11-16 주식회사 비트로시스 Method of preparing extracts of wild ginseng stem cell(callus) and adventitious roots and cosmetical compositions for the whitening, uv block and anti-oxidant comprising the same
CN103509749A (en) * 2012-06-29 2014-01-15 鹭港生物药业有限公司 Separation and culture method using ginseng cambium stem cells
CN104472359A (en) * 2014-11-26 2015-04-01 中国医学科学院药用植物研究所 Ginseng adventitious root induced proliferation method
CN108531440A (en) * 2018-04-28 2018-09-14 刘汉石 A kind of ginseng-cell culture medium and its application
CN108739377A (en) * 2018-04-28 2018-11-06 刘汉石 A kind of ginseng adventitious root inducing culture and its application
CN109022343A (en) * 2017-06-12 2018-12-18 厦门鹭港兆康生物科技有限公司 A kind of preparation method of ginseng stem cell
CN109576209A (en) * 2019-01-24 2019-04-05 深圳先声科技发展有限公司 Ginseng forming layer stem cell isolated culture method
CN111876368A (en) * 2020-07-09 2020-11-03 广东岭南职业技术学院 Method for inducing and in vitro culturing plant stem cells derived from apical meristem of vinca root
CN112544430A (en) * 2020-12-10 2021-03-26 中国海洋大学 Transplanting device and construction method for temperate zone rock reef matrix seaweed bed

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101606487A (en) * 2009-07-13 2009-12-23 天津大学 Tissue culture method of ginseng adventitious root
KR20110123934A (en) * 2010-05-10 2011-11-16 주식회사 비트로시스 Method of preparing extracts of wild ginseng stem cell(callus) and adventitious roots and cosmetical compositions for the whitening, uv block and anti-oxidant comprising the same
CN103509749A (en) * 2012-06-29 2014-01-15 鹭港生物药业有限公司 Separation and culture method using ginseng cambium stem cells
CN104472359A (en) * 2014-11-26 2015-04-01 中国医学科学院药用植物研究所 Ginseng adventitious root induced proliferation method
CN109022343A (en) * 2017-06-12 2018-12-18 厦门鹭港兆康生物科技有限公司 A kind of preparation method of ginseng stem cell
CN108531440A (en) * 2018-04-28 2018-09-14 刘汉石 A kind of ginseng-cell culture medium and its application
CN108739377A (en) * 2018-04-28 2018-11-06 刘汉石 A kind of ginseng adventitious root inducing culture and its application
CN109576209A (en) * 2019-01-24 2019-04-05 深圳先声科技发展有限公司 Ginseng forming layer stem cell isolated culture method
CN111876368A (en) * 2020-07-09 2020-11-03 广东岭南职业技术学院 Method for inducing and in vitro culturing plant stem cells derived from apical meristem of vinca root
CN112544430A (en) * 2020-12-10 2021-03-26 中国海洋大学 Transplanting device and construction method for temperate zone rock reef matrix seaweed bed

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
徐春明 等: "药用植物干细胞培养技术及其应用", 《中草药》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022206424A1 (en) * 2021-04-02 2022-10-06 山东安然纳米实业发展有限公司 Method for separating and culturing ginseng stem cells
CN115786119A (en) * 2023-02-13 2023-03-14 山东卫康生物医药科技有限公司 Isolated culture control method and system for ginseng stem cells

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