CN113025554B - Method for culturing ginseng stem cells by using biological reaction device - Google Patents

Abstract

The invention discloses a method for culturing ginseng stem cells by using a biological reaction device, which comprises the following steps: (1) preparing the adventitious roots of the ginseng by adopting a one-step method; (2) taking the root tip part of the adventitious root of the ginseng prepared in the step (1), and placing the root tip part in a penetrating agent solution for treatment; (3) placing the ginseng adventitious root tip treated by the penetrant solution in the step (2) into a stem cell induction culture medium for culture, and then transferring the ginseng adventitious root tip into a propagation culture medium for culture; (4) inoculating the ginseng stem cells cultured in the step (3) to a stem cell liquid culture medium shake flask, and culturing under the condition of 100-140rpm in a dark place; (5) inoculating the ginseng stem cells cultured in the shake flask in the step (4) into a biological reaction device, and culturing in a dark place to obtain the ginseng stem cells; the invention obtains the method for obtaining the adventitious roots from the mature ginseng with shorter time and simpler steps so as to obtain the ginseng stem cells, and adopts a proper biological reaction device for amplification culture, so that the growth multiple of the stem cells is high, and the content of active ingredients in the stem cells is high.

Description

Method for culturing ginseng stem cells by using biological reaction device

Technical Field

The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for culturing ginseng stem cells by using a biological reaction device.

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 small tissue blocks, inoculating the small tissue blocks into a solid induction culture medium to induce and form adventitious roots, cutting the adventitious roots into small adventitious roots, and inoculating the small adventitious roots 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 culturing ginseng stem cells by using a biological reaction device. 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 places the obtained adventitious roots in an osmotic pressure environment, and matches with a stem cell induction culture medium and a stem cell expansion culture medium with proper component proportion, so that the ginseng stem cells can be obtained, 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 method for culturing ginseng stem cells by using a biological reaction device, 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 the root tip part of the adventitious root of the ginseng prepared in the step (1), and placing the root tip part in a penetrating agent solution for treatment;

(3) placing the ginseng adventitious root tip treated by the penetrant solution in the step (2) into a stem cell induction culture medium for culture, and then transferring the ginseng adventitious root tip into a propagation culture medium for culture to obtain ginseng stem cells;

(4) inoculating the ginseng stem cells cultured in the step (3) to a stem cell liquid culture medium shake flask, and culturing under the condition of 100-140rpm in a dark place;

(5) inoculating the ginseng stem cells cultured in the shake flask in the step (4) into a biological reaction device, and culturing in a dark place to obtain the ginseng stem cells;

the liquid culture medium used in the step (1) comprises: 10-55g/L of sucrose, 0.8-1.5g/L of WPM culture medium, 0.6-1.6g/L N6 culture medium, 1-6mg/L of naphthylacetic acid and 0.2-1.2mg/L of kinetin.

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 meristems and strong activity.

The further scheme of the invention is as follows: the liquid culture medium in the step (1) also comprises 0-1.8mg/L of indolebutyric acid.

The further scheme of the invention is as follows: the liquid medium of step (1) comprises: 35g/L of sucrose, 1.35g/L of WPM culture medium, 1g/L N6 culture medium, 1-5mg/L of naphthylacetic acid, 0.2-1mg/L of kinetin and 0.2-1.8mg/L of indolebutyric acid; preferably, the liquid medium in step (1) comprises: 35g/L of sucrose, 1.35g/L of WPM culture medium, 1g/L N6 culture medium, 5mg/L of naphthylacetic acid, 1mg/L of kinetin and 0.2mg/L of indolebutyric acid.

In the scheme, the liquid culture medium components after the propagation step are adjusted in the adventitious root induction process of the ginseng slices, wherein a proper content of WPM culture medium and N6 culture medium is adopted, nitrate nitrogen and ammonium nitrogen in a system reach a relatively balanced proportion, and ammonium in the WPM culture medium and the N6 culture medium plays a role in inhibiting the dedifferentiation process of ginseng cells, so that the tissue after propagation retains the functions of original cells of roots under the action of the liquid culture medium and continues to grow. Furthermore, the liquid culture medium provided by the invention is also added with naphthylacetic acid, kinetin and indolebutyric acid components, wherein the naphthylacetic acid component with higher content regulates and controls the selective expression of original cell mRNA of the root under the condition that the ammonium component inhibits dedifferentiation, and promotes the cells to rapidly divide and grow without forming callus. And less kinetin can remove apical dominance to a certain extent, and further promotes the generation of adventitious roots by cooperating with indolebutyric acid. In conclusion, the adventitious root liquid culture medium provided by the invention provides nitrate nitrogen and ammonium nitrogen with balanced components through the WPM and N6 culture mediums, and simultaneously under the combined action of more naphthylacetic acid for gene expression regulation, less kinetin for removing apical dominance and indolebutyric acid, the preparation process can directly generate adventitious roots without forming callus, so that the adventitious roots with higher saponin content can be obtained, and meanwhile, the multiple of growth of the adventitious roots can be improved and the growth time can be shortened.

The further scheme of the invention is as follows: in the step (5), when the stem cells are inoculated into a stem cell liquid culture medium of a biological reaction device, the mass of the inoculated stem cells accounts for 1.0-10.0% of the volume of the stem cell propagation liquid culture medium; the ventilation volume for culturing the stem cells in the biological reaction device is 0.02-0.2 vvm; preferably, the aeration is 0.05 to 0.15 vvm.

The further scheme of the invention is as follows: the biological reaction device comprises a tank body, and the bottom of the tank body comprises at least two air inlet devices; preferably, the height of the bottom wall of the tank body is gradually reduced from the periphery to the center to form an inverted cone with a large upper part and a small lower part; the discharge gate sets up in the minimum position in center, and air inlet unit surrounds the discharge gate interval evenly distributed on by all around to the diapire that the center gradually reduces.

In the above scheme, in the biological reaction device provided by the invention, the air inlet devices are arranged on the inclined plane of the bottom wall of the inverted cone-shaped tank body with a large upper part and a small lower part, so that the inlet air cannot vertically rise, and further, two or more air inlet devices are uniformly distributed around the center at intervals, which is beneficial to uniform distribution of air in the tank body and uniform growth of stem cells in the tank body.

The further scheme of the invention is as follows: in the step (2), the apical part of the adventitious root of the ginseng is taken and placed in a penetrant solution for low-temperature treatment for 1-10 hours; preferably, the low temperature is 1-6 ℃, preferably 4 ℃; preferably, the length of the apical part of the adventitious root of ginseng is 0.3mm to 0.8 mm.

In the scheme, after the root tip of the adventitious root of the ginseng is treated by the penetrant solution, non-stem cells in the ginseng are not resistant to the death of the hypertonic solution, and only the stem cells can grow; the stem cell growth speed is very fast by matching with a proper stem cell induction culture medium and a stem cell propagation culture medium, and the content of active ingredients in the obtained ginseng stem cells is improved.

The further scheme of the invention is as follows: the osmotic agent solution is at least one of sucrose solution, potassium chloride solution and mannitol solution; preferably, the osmotic agent solution is a sucrose solution with the concentration of 1 mol/L.

The further scheme of the invention is as follows: in the step (3), the stem cell induction culture medium comprises 0.6-1mg/L kinetin, 2-4mg/L indoleacetic acid, 0.5-1.5mg/L naphthylacetic acid, 25-100mg/L ascorbic acid, 50-150mg/L citric acid, 20-60g/L sucrose, 1-6g/L plant gel, 1-2.4 g/L1/2 MS culture medium and 0.6-1.4g/L WPM culture medium; preferably, the stem cell induction medium comprises 1mg/L kinetin, 2.5mg/L indoleacetic acid, 1mg/L naphthylacetic acid, 75mg/L ascorbic acid, 75mg/L citric acid, 30g/L sucrose, 3g/L plant gel, 1.8 g/L1/2 MS medium and 0.8g/L WPM medium.

In the scheme, the stem cell induction culture medium provided by the invention contains indoleacetic acid, naphthylacetic acid and kinetin, can guide the stem cells affected by a penetrant to quickly recover to grow, and meanwhile, the ascorbic acid and the citric acid can also generate a synergistic antioxidation effect to timely remove free radicals which are not beneficial to the growth of the stem cells. 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 propagation medium comprises 2-4mg/L2, 4-dichlorophenoxyacetic acid, 1-3mg/L naphthylacetic acid, 0.8-1.2mg/L kinetin, 20-60g/L cane sugar, 1-6g/L plant gel, 1-2.4 g/L1/2 MS medium and 0.6-1.4g/L WPM medium; preferably, the stem cell propagation medium comprises 3mg/L of 2, 4-dichlorophenoxyacetic acid, 2mg/L of naphthylacetic acid, 1mg/L of kinetin, 35g/L of cane sugar, 3g/L of plant gel, 1.8g/L of 1/2MS medium and 0.8g/L of WPM medium.

The further scheme of the invention is as follows: the pH value of the stem cell induction culture medium and the stem cell propagation culture medium is 5.6-6.0. The pH of the induction culture medium and the propagation culture medium is adjusted by using NaOH or KOH.

The further scheme of the invention is as follows: in the steps (4) and (5), 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, 1-2.4 g/L1/2 MS culture medium and 0.6-1.4g/L WPM culture medium; preferably, the stem cell liquid culture medium comprises 3 mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L gibberellin, 1mg/L kinetin, 35g/L sucrose, 1.8 g/L1/2 MS culture medium and 0.8g/L WPM culture medium.

The further scheme of the invention is as follows: in the step (3), the root tip parts of the adventitious roots of the ginseng are respectively cultured for 25 to 35 days in a light-proof way at the temperature of between 20 and 25 ℃ on a stem cell induction culture medium and a stem cell propagation solid culture medium.

In the scheme, the adventitious roots are generally generated by ginseng callus induction at present, 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 callus to generate adventitious roots, the metabolism degree of the ginseng tissue is high, 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: 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, so that the adventitious roots are induced and generated directly from all parts of the mature ginseng 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 present invention, WPM 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 calcium nitrate tetrahydrate, 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, the top of the tank body is provided with a cover body which can be opened and closed, and the cover body or the top of the tank body is provided with an exhaust device; 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.

In the above embodiment, the volume of the bioreactor provided by the present invention is 1-50L, preferably 2-20L, preferably 3-10L, preferably 5L.

The further scheme of the biological reaction device is as follows: the center of the bottom of the tank body is provided with a discharge hole, and the air inlet devices are uniformly distributed around the discharge hole at intervals; preferably, the height of the bottom wall of the tank body is gradually reduced from the periphery to the center to form an inverted cone with a large upper part and a small lower part; the discharge hole is arranged at the lowest position in the center, and the air inlet devices are uniformly distributed on the bottom wall which is gradually reduced from the periphery to the center at intervals around the discharge hole.

The bioreactor comprises the following further scheme: air inlet unit sets up on the jar body diapire inclined plane of back taper shape big-end-up, so, the gas that gets into can not rise perpendicularly, and is further, and two and more than two air inlet unit distribute around the center interval uniformly, are favorable to the air at jar internal evenly distributed, are favorable to the internal adventitious root of jar to grow evenly.

The bioreactor comprises the following further scheme: the biological reaction device also comprises a supporting structure for supporting the tank body to be vertically placed on a plane. Specifically, the supporting structure comprises supporting legs, the supporting legs are connected with the lower portion of the tank body or integrally arranged, and the tank body is placed on a plane or a platform through the supporting legs. Preferably, the conical bottom of the tank body is positioned in a space surrounded by the supporting feet.

The bioreactor comprises the following further scheme: air inlet unit includes air inlet, intake pipe and air cleaner, the air inlet is located the diapire of the jar body, intake pipe and air inlet sealing connection, air cleaner sets up and filters institute through the air in the intake pipe.

The further scheme of the biological reaction device is as follows: the exhaust device comprises an exhaust port, an exhaust pipe and a filtering device, the exhaust port is arranged on the cover body or the top of the tank body, the exhaust pipe and the exhaust port are communicated in a sealing mode, and the filtering device is arranged on the exhaust pipe; preferably, the filter device is an air filter or a liquid filter.

In a preferred embodiment, the vent is provided in the center of the cover.

In the above scheme, when the filtering device arranged on the exhaust pipe is an air filter, external air can be prevented from entering from the top, and the sterile culture environment in the tank body is ensured. In addition, when the filtering device on the exhaust pipe is replaced with a liquid filter, liquid can be added from the top.

The further scheme of the biological reaction device is as follows: an inoculation port is arranged on the cover body or the top of the tank body and used for inoculation.

The bioreactor comprises the following further scheme: a plurality of transparent observation windows are arranged on the tank body; preferably, the observation window is arranged on the side wall of the middle part and/or the lower part of the tank body.

In the above scheme, when the jar body adopts opaque stainless steel material, the internal portion of jar can be observed from different angles to a plurality of transparent observation windows of different positions that set up, in time masters jar internal stem cell's growth condition.

In a further scheme, a handle is also arranged on the tank body; preferably, the handle comprises at least two.

In the above scheme, the handles arranged on the tank body can be symmetrically arranged, so that a user can conveniently take and move the biological reaction device.

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, and can tonify five internal organs, calm spirit, calm soul, stop palpitation, remove pathogenic qi, improve eyesight, and benefit mind 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 root by one-step induction from the hundred-year ginseng diced piece, not only can simplify the induction step and shorten the induction time, but also can obtain the specific functional components in the maternal hundred-year old ginseng, thereby obtaining the adventitious root 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. the method for culturing the ginseng stem cells by using the biological reaction device provided by the invention adopts the biological reaction device with an improved structure, so that the ventilation is uniform and sufficient, the rapid growth of the stem cells is facilitated, and the production is expanded;

2. the method for culturing the ginseng stem cells by using the biological reaction device, provided by the invention, places the ginseng adventitious roots prepared by the one-step method in a penetrant solution for low-temperature treatment, so that cells which are not resistant to a hypertonic solution die, only grow the stem cells, and then separate and obtain the stem cells, and the stem cells are matched with a stem cell induction culture medium and a stem cell propagation culture medium with proper component proportion, 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;

3. the method for culturing the ginseng stem cells by using the biological reaction device realizes that the ginseng adventitious roots are directly induced by inoculating the processed parts of the mature ginseng into the induction culture medium by adopting a one-step method 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;

4. according to the method for culturing the ginseng stem cells by using the biological reaction device, in the process of culturing the ginseng adventitious root by using the one-step method, the liquid culture medium of the adventitious root provides nitrate nitrogen and ammonium nitrogen with balanced components through the WPM and N6 culture mediums, and meanwhile, under the combined action of gene expression regulation of a large amount of naphthylacetic acid, apical dominance removal of a small amount of kinetin and indolebutyric acid, callus is not formed in the preparation process to directly generate the adventitious root, so that the adventitious root with high saponin content can be obtained, and meanwhile, the multiple of growth of the adventitious root can be improved and the growth time can be shortened.

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 diagram of induction of adventitious roots using the induction medium of comparative example 1;

FIG. 3 is a schematic view showing the structure of the bioreactor of the present invention.

The main components in the figure are: 1-jar body, 2-lid, 3-exhaust apparatus, 4-air inlet unit, 5-discharge gate, 6-intake pipe, 7-air cleaner, 8-blast pipe, 9-filter equipment, 10-observation window, 11-handle, 12-supporting legs.

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.

As shown in FIG. 3, the present invention provides a bioreactor for culturing ginseng adventitious roots, comprising: the tank comprises a tank body 1, wherein the top of the tank body 1 is provided with an openable cover body 2, and an exhaust device 3 is arranged on the cover body 2 or the top of the tank body 1; at least two air inlet devices 4 are arranged at the bottom of the tank body 1, and air enters the tank body 1 through the air inlet devices 4.

The bioreactor is a small bioreactor, has simple structure and convenient operation, can conveniently produce the ginseng stem cells by utilizing the bioreactor, and has fast stem cell growth and high growth multiple.

In the invention, the tank body 1 is hollow and is used for containing 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 2 on the top of the tank body 1 can be opened or closed for adding liquid culture medium, and the cover body 2 is connected with the tank body 1 in a sealing way after the liquid culture medium is added. The bottom of the tank body 1 is provided with at least two air inlet devices 4, and sterile air is introduced into the tank body 1 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 center of the bottom of the tank body 1 is provided with a discharge hole 5, and the air inlet devices are uniformly distributed around the discharge hole 5 at intervals;

preferably, the height of the bottom wall of the tank body 1 is gradually reduced from the periphery to the center to form an inverted cone with a large upper part and a small lower part; the discharge ports 5 are arranged at the lowest position in the center, and the air inlet devices 4 are uniformly distributed on the bottom wall which is gradually reduced from the periphery to the center at intervals around the discharge ports 5.

In the invention, the air inlet devices 4 are arranged on the inclined plane of the bottom wall of the inverted cone-shaped tank body 1 with a large upper part and a small lower part, so that the entering air cannot vertically rise, and furthermore, two or more air inlet devices 4 are uniformly distributed around the center at intervals, which is favorable for the uniform distribution of air in the tank body 1 and the uniform growth of stem cells in the tank body 1.

The bioreactor of the present invention further comprises a support structure for supporting the tank 1 to be vertically placed on a plane. Specifically, the supporting structure comprises supporting legs 12, the supporting legs 12 are connected with the lower part of the tank body 1 or integrally arranged, and the tank body 1 is placed on a plane or a platform through the supporting legs 12. Preferably, the conical bottom of the tank 1 is located in the space enclosed by the support feet 12.

Air inlet unit 4 includes air inlet, intake pipe 6 and air cleaner 7, the air inlet is located the diapire of jar body 1, intake pipe 6 and air inlet sealing connection, air cleaner 7 sets up filters the institute through the air on intake pipe 6.

The exhaust device 3 comprises an exhaust port, an exhaust pipe 8 and a filtering device 9, the exhaust port is arranged on the cover body 2 or the top of the tank body 1, the exhaust pipe 8 is communicated with the exhaust port in a sealing manner, and the filtering device 9 is arranged on the exhaust pipe 8;

preferably, the filter device 9 is an air filter 7 or a liquid filter.

In a preferred embodiment, the exhaust port is provided in the center of the lid body 2.

In the above scheme, when the filtering device 9 arranged on the exhaust pipe 8 is the air filter 7, the external air can be prevented from entering from the top, and the sterile culture environment in the tank body 1 is ensured. In addition, when the filter device 9 on the exhaust pipe 8 is replaced with a liquid filter, it is possible to achieve liquid addition from the top.

An inoculation port is arranged on the cover body 2 or the top of the tank body 1 and used for inoculation.

A plurality of transparent observation windows 10 are arranged on the tank body 1;

the observation window 10 is arranged on the side wall of the middle part and/or the lower part of the tank body 1.

When the tank body 1 is made of opaque stainless steel, the inside of the tank body 1 can be observed from different angles through the plurality of transparent observation windows 10 arranged at different positions, and the growth condition of the ginseng stem cells in the tank body 1 can be mastered in time.

The tank body 1 is also provided with a handle 11; preferably, the handle 11 comprises at least two.

The handles 11 arranged on the tank body 1 can be symmetrically arranged, so that a user can conveniently take and move the biological reaction device.

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 the induction culture medium obtained in the step (1), and carrying out dark culture for 4-5 weeks under the same condition;

(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 the embodiment, the method for preparing the adventitious root of the ginseng by adopting the one-step method and the specific liquid culture medium 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 WPM 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 the step (1), and carrying out dark culture for 4-5 weeks under the same condition;

(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 roots 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 an induction culture medium, and performing dark culture at 22 +/-1 ℃ for 4-5 weeks to induce mountain ginseng adventitious roots; 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 the embodiment, the method for preparing the adventitious root of the ginseng by adopting the one-step method and the specific liquid culture medium 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

Cutting 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 the tissues on a shaker at the temperature of 22 +/-1 ℃ for 3-4 weeks 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 70 percent 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 a tissue having a length of about 1 cm; inoculating into a liquid culture medium of a biological reaction device, wherein the mass of the inoculated adventitious roots accounts for 1.0 percent of the volume of the liquid culture medium; ventilating the biological reaction device, wherein the ventilation rate is 0.8vvm, 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 24

Examples 7 to 24 are obtained by replacing the liquid medium in step (3) with a medium containing 35g/L sucrose, 1.35g/L WPM medium, 1g/L N6 medium, 0 to 1.8mg/L indolebutyric acid (IBA), 1 to 6mg/L naphthylacetic acid (NAA), 0.2 to 1.2mg/L Kinetin (KT) and having a pH of 5.8 based on example 1. The specific content ratio of the medium in each example is shown in Table 1.

Example 25

In this example, the cultivation of ginseng stem cells using the adventitious roots of ginseng obtained in example 17 and the above-mentioned bioreactor, specifically comprises the steps of:

(1) the ginseng adventitious roots prepared in example 17 were used;

(2) taking the apical part of 0.5mm ginseng adventitious root, putting the apical part into 1mol/L sucrose solution, and taking out after 6 hours in a refrigerator at 4 ℃;

(3) placing the treated root tips into a stem cell induction medium comprising: 1mg/L kinetin, 2.5mg/L indoleacetic acid, 1.0mg/L naphthylacetic acid, 75mg/L ascorbic acid, 75mg/L citric acid, 30g/L sucrose, 3g/L plant gel, 1.8 g/L1/2 MS culture medium, 0.8g/L WPM culture medium, and the pH is 5.8; culturing at 22 + -2 deg.C in dark for 25-35 days; then collecting the stem cells, transferring the stem cells into a stem cell propagation solid culture medium, and continuously culturing for 25-35 days in the dark at the temperature of 22 +/-2 ℃ to enable the stem cells to enter a rapid growth period; the stem cell propagation solid culture medium comprises 3 mg/L2, 4-dichlorophenoxyacetic acid, 2mg/L naphthylacetic acid, 1mg/L kinetin, 35g/L cane sugar, 3g/L plant gel, 1.8 g/L1/2 MS culture medium and 0.8g/L WPM culture medium, and the pH value is 5.8;

(4) collecting the ginseng stem cells obtained in the step (3), inoculating the ginseng stem cells into a shake flask containing a stem cell liquid culture medium, and carrying out dark culture at the speed of 120rpm and the temperature of 22 +/-2 ℃;

(5) adding a stem cell liquid culture medium into a tank body (the volume is 5L) of a biological reaction device, wherein the volume of the culture medium in the biological reaction device accounts for 70 percent of the volume of the culture medium; sterilizing at 121 deg.C for 20 min; collecting the ginseng stem cells obtained in the step (4), and inoculating the ginseng stem cells into a biological reaction device containing a stem cell liquid culture medium, wherein the mass of the inoculated stem cells accounts for 8.0% of the volume of the stem cell liquid culture medium; the ventilation volume is 0.15 vvm; culturing at 22 deg.C in dark to obtain Ginseng radix stem cells;

the stem cell liquid culture medium in the steps (4) and (5) comprises: 3mg/L of 2, 4-dichlorophenoxyacetic acid, 2mg/L of gibberellin, 1mg/L of kinetin, 35g/L of cane sugar, 1.8g/L of 1/2MS culture medium and 0.8g/L of LWPM culture medium, and the pH value is 5.8.

Example 26

In this example, the cultivation of ginseng stem cells using the ginseng adventitious roots obtained in example 18 and the above-mentioned bioreactor, specifically comprises the steps of:

(1) the ginseng adventitious roots prepared in example 18 were used;

(2) taking the apical part of 0.8mm ginseng adventitious root, putting the apical part into 1mol/L mannitol solution, and taking out after 6 hours in a refrigerator at 6 ℃;

(3) placing the treated root tips into a stem cell induction medium comprising: 0.6mg/L kinetin, 2mg/L indoleacetic acid, 0.5mg/L naphthylacetic acid, 100mg/L ascorbic acid, 50mg/L citric acid, 20g/L sucrose, 6g/L plant gel, 2.4 g/L1/2 MS culture medium, 1.4g/L WPM culture medium, and the pH is 5.8; culturing at 22 + -2 deg.C in dark for 25-35 days; then collecting the stem cells, transferring the stem cells into a stem cell propagation solid culture medium, and continuously culturing for 25-35 days at 22 +/-2 ℃ in a dark place to enable the stem cells to enter a rapid growth period; the stem cell propagation solid culture medium comprises 2 mg/L2, 4-dichlorophenoxyacetic acid, 3mg/L naphthylacetic acid, 1.2mg/L kinetin, 60g/L cane sugar, 1g/L plant gel, 1 g/L1/2 MS culture medium and 1.4g/L WPM culture medium, and the pH value is 5.8;

(4) collecting the ginseng stem cells obtained in the step (3), inoculating the ginseng stem cells into a shake flask containing a stem cell liquid culture medium, and carrying out dark culture at 130rpm and 22 +/-2 ℃;

(5) adding stem cell liquid culture medium into a tank body (the volume is 5L) of a biological reaction device, wherein the volume of the culture medium in the biological reaction device accounts for 30% of the volume of the biological reaction device; sterilizing at 121 deg.C for 20 min; collecting the ginseng stem cells obtained in the step (4), inoculating the ginseng stem cells into a biological reaction device containing a stem cell liquid culture medium, wherein the mass of the inoculated stem cells accounts for 2% of the volume of the stem cell liquid culture medium, the ventilation volume is 0.05vvm, and the ginseng stem cells are cultured in a dark place at 22 ℃ to obtain the ginseng stem cells;

the stem cell liquid culture medium in the steps (4) and (5) comprises: 2 mg/L2, 4-dichlorophenoxyacetic acid, 3mg/L gibberellin, 1.2mg/L kinetin, 60g/L sucrose, 1 g/L1/2 MS culture medium and 1.4g/L WPM culture medium, and the pH is 5.8.

Example 27

In this example, the cultivation of ginseng stem cells using the ginseng adventitious roots obtained in example 24 and the above-mentioned bioreactor, specifically comprises the steps of:

(1) the ginseng adventitious roots prepared in example 24 were used;

(2) taking the apical part of 0.3mm ginseng adventitious root, putting the apical part into 1mol/L sucrose solution, and taking out after 6 hours in a refrigerator at 1 ℃;

(3) placing the treated root tips into a stem cell induction medium comprising: 1mg/L kinetin, 4mg/L indoleacetic acid, 1.5mg/L naphthylacetic acid, 25mg/L ascorbic acid, 150mg/L citric acid, 60g/L cane sugar, 1g/L plant gel, 1 g/L1/2 MS culture medium, 0.6g/L WPM culture medium, and the pH is 5.8; culturing at 22 + -2 deg.C in dark for 25-35 days; then collecting the stem cells, transferring the stem cells into a stem cell propagation solid culture medium, and continuously culturing for 25-35 days at 22 +/-2 ℃ in a dark place to enable the stem cells to enter a rapid growth period; the stem cell propagation solid culture medium comprises 4mg/L2, 4-dichlorophenoxyacetic acid, 1mg/L naphthylacetic acid, 0.8mg/L kinetin, 20g/L cane sugar, 6g/L plant gel, 2.4 g/L1/2 MS culture medium and 0.6g/L WPM culture medium, and the pH value is 5.8;

(4) collecting the ginseng stem cells obtained in the step (3), inoculating the ginseng stem cells into a shake flask containing a stem cell liquid culture medium, and carrying out dark culture at 130rpm and 22 +/-2 ℃;

(5) adding stem cell liquid culture medium into a tank body (the volume is 5L) of a biological reaction device, wherein the volume of the culture medium in the biological reaction device accounts for 50% of the volume of the biological reaction device; sterilizing at 121 deg.C for 20 min; collecting the ginseng stem cells obtained in the step (4), inoculating the ginseng stem cells into a biological reaction device containing a stem cell liquid culture medium, wherein the mass of the inoculated stem cells accounts for 10% of the volume of the stem cell liquid culture medium, the ventilation volume is 0.2vvm, and the ginseng stem cells are cultured in a dark place at 22 ℃ to obtain the ginseng stem cells;

the stem cell liquid culture medium in the steps (4) and (5) comprises: 4mg/L of 2, 4-dichlorophenoxyacetic acid, 1mg/L of gibberellin, 0.8mg/L of kinetin, 20g/L of cane sugar, 6g/L of plant gel, 2.4g/L of 1/2MS culture medium and 0.6g/L of WPM culture medium, and the pH value is 5.8.

Example 28

In this example, the ginseng stem cells were cultured using the ginseng adventitious roots obtained in example 1 and the bioreactor provided above, and other embodiments of this example are the same as example 25.

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 comparative example 1 picture demonstration.

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, deepens in the third week, the middle part turns brown, and the whole body turns brown and becomes dry 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 24 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 uL.

(5) Presentation of analytical results

The content of each component of the ginsenoside in the sample is calculated according to the formula (1):

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

As can be seen from Table 1, in example 11, no indolebutyric acid IBA is added, while in example 9, 1mg/L of indolebutyric acid is added under the condition that other components are similar to those in example 11, and the comparison of the adventitious roots of ginseng prepared by the two shows that in the case of adding higher concentration of NAA naphthalene acetate and the same content of KT, the increase factor and the saponin content of example 11 lacking the indolebutyric acid IBA are reduced compared with example 9. Further comparing examples 8, 15 and 18, in the case that a small amount of indolebutyric acid is added to all the three, the adding ratio of the naphthylacetic acid and the kinetin is adjusted, and it is found that example 18, in which the naphthylacetic acid and the kinetin are properly reduced, has more excellent fold increase and saponin content; further, it is understood from the descriptions of examples 20 and 23 that when the other components of the liquid culture medium are the same, the fold increase and the saponin content can be increased by decreasing the kinetin content, and in combination with the case of example 18, it is found that when a high content of naphthylacetic acid is present in the liquid culture medium provided by the present invention, the ratio of kinetin needs to be appropriately decreased to increase the synergistic effect of the two. On the basis of the above, according to the descriptions of examples 21 and 24, when the concentration of naphthylacetic acid is further increased to 5mg/L, the contents of indolebutyric acid and kinetin are adjusted at the same time, it is found that the difference between the fold increase of the formation of adventitious roots and the saponin content is smaller by appropriately increasing the content of indolebutyric acid while decreasing kinetin than that of examples 20 and 23, and the synergistic effect of naphthylacetic acid, kinetin and indolebutyric acid on the growth of adventitious roots is further demonstrated. It should be noted that the growth time shown in Table 1 is the time for completing the culture of adventitious roots, i.e., the culture is continued on the basis of the time shown in the table, and the change in the saponin content is not significant any more.

In this experimental example, when the liquid culture media prepared in examples 17, 18, and 24 were used, the growth factor in the adventitious roots was relatively high, and the total amount of saponins was high. The groups with the advantage of a fold increase can be partly ignored due to the need for subsequent stem cell culture of adventitious roots, and examples 17, 18 and 24 with higher total saponins are preferred. Among them, the adventitious roots of examples 17 and 18 have a shorter growth time while having a larger growth multiple and saponin content, which is a more preferable option.

Test example 2

In this test example, saponin detection was performed on the wild ginseng adventitious root stem cells obtained in examples 25 to 28 and the wild ginseng slice 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 25 to 28 were washed three times with water, ground into a paste in a mortar, subjected to ultrasonic wall breaking, freeze-dried, 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):

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

From the above results, it can be seen that the contents of various ginsenosides in the wild ginseng adventitious root stem cells cultured by the present invention are all increased compared with the mature wild ginseng slices, and the total content of ginsenosides is further increased under the addition effect of the bioreactor. 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 (17)

1. A method for culturing ginseng stem cells by using a biological reaction device, comprising:

(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; the adventitious root induction culture medium comprises 1-6mg/L naphthylacetic acid, 0.1-0.6mg/L kinetin, 0.2-1mg/L gibberellin, 0.075-1.5g/L citric acid, 0.03-1g/L ascorbic acid, 20-60g/L cane sugar, 1-6g/L plant gel, 1-4g/L B5 culture medium and 1-2.4g/L WPM culture medium;

(2) taking the root tip part of the adventitious root of the ginseng prepared in the step (1), and placing the root tip part in a penetrating agent solution for treatment;

(3) placing the ginseng adventitious root tip treated by the penetrant solution in the step (2) into a stem cell induction culture medium for culture, and then transferring the ginseng adventitious root tip into a propagation culture medium for culture to obtain ginseng stem cells; the composition of the dry cell induction culture medium is 0.6-1mg/L kinetin, 2-4mg/L indoleacetic acid, 0.5-1.5mg/L naphthylacetic acid, 25-100mg/L ascorbic acid, 50-150mg/L citric acid, 20-60g/L cane sugar, 1-6g/L plant gel, 1-2.4 g/L1/2 MS culture medium and 0.6-1.4g/L WPM culture medium;

the propagation culture medium comprises 2-4mg/L2, 4-dichlorophenoxyacetic acid, 1-3mg/L naphthylacetic acid, 0.8-1.2mg/L kinetin, 20-60g/L sucrose, 1-6g/L plant gel, 1-2.4 g/L1/2 MS culture medium and 0.6-1.4g/L WPM culture medium;

(4) inoculating the ginseng stem cells cultured in the step (3) to a stem cell liquid culture medium shake flask, and culturing under the condition of 100-140rpm in a dark place; the composition of the stem cell liquid culture medium is 2-4mg/L2, 4-dichlorophenoxyacetic acid, 1-3mg/L gibberellin, 0.8-1.2mg/L kinetin, 20-60g/L sucrose, 1-2.4 g/L1/2 MS culture medium and 0.6-1.4g/L WPM culture medium;

(5) inoculating the ginseng stem cells cultured in the shaking flask in the step (4) into a biological reaction device, and culturing in a dark place to obtain the ginseng stem cells;

the liquid culture medium used in the step (1) comprises 10-55g/L of cane sugar, 0.8-1.5g/L of WPM culture medium, 0.6-1.6g/L N6 culture medium, 1-6mg/L of naphthylacetic acid, 0.2-1.2mg/L of kinetin and 0.2-1.8mg/L of indolebutyric acid.

2. The method for culturing the ginseng stem cells using the bioreactor according to claim 1, wherein the composition of the liquid medium in the step (1) is 35g/L sucrose, 1.35g/L WPM medium, 1g/L N6 medium, 1-5mg/L naphthylacetic acid, 0.2-1mg/L kinetin, 0.2-1.8mg/L indolebutyric acid.

3. The method for culturing the ginseng stem cells using the bioreactor according to claim 1, wherein the composition of the liquid medium in the step (1) is 35g/L sucrose, 1.35g/L WPM medium, 1g/L N6 medium, 5mg/L naphthylacetic acid, 1mg/L kinetin, 0.2mg/L indolebutyric acid.

4. The method for culturing ginseng stem cells using a bioreactor according to any one of claims 1 to 3, wherein in the step (5), when the stem cells are inoculated into the stem cell liquid culture medium of the bioreactor, the mass of the inoculated stem cells accounts for 1.0 to 10.0 percent of the volume of the stem cell propagation liquid culture medium; the aeration rate for culturing stem cells in the bioreactor is 0.02-0.2 vvm.

5. The method for culturing ginseng stem cells using a bioreactor according to claim 4, wherein the ventilation amount is 0.05 to 0.15 vvm.

6. The method for culturing ginseng stem cells using a bioreactor according to any one of claims 1 to 3, wherein the bioreactor comprises a tank, and the bottom of the tank comprises at least two gas inlets.

7. The method for culturing the ginseng stem cells using the bioreactor according to claim 6, wherein the height of the bottom wall of the tank is gradually decreased from the periphery to the center to form an inverted cone with a large top and a small bottom; the discharge gate sets up in the minimum position in center, and air inlet unit surrounds the discharge gate interval evenly distributed on by all around to the diapire that the center gradually reduces.

8. The method for culturing ginseng stem cells using a bioreactor apparatus according to any one of claims 1 to 3, wherein, in the step (2), the apical part of the adventitious root of ginseng is taken and put in a penetrant solution to be treated at low temperature for 1 to 10 hours.

9. The method for culturing ginseng stem cells using a bioreactor according to claim 8, wherein the low temperature is 1-6 ℃.

10. The method for culturing ginseng stem cells using a bioreactor according to claim 9, wherein the low temperature is 4 ℃.

11. The method for culturing ginseng stem cells using a bioreactor according to claim 8, wherein the length of the apical part of the adventitious root of ginseng is 0.3mm to 0.8 mm.

12. The method for culturing the ginseng stem cells using the bioreactor according to any one of claims 1 to 3, wherein the osmotic agent solution is at least one selected from the group consisting of a sucrose solution, a potassium chloride solution, and a mannitol solution.

13. The method for culturing ginseng stem cells using a bioreactor according to claim 12, wherein the osmotic agent solution is a sucrose solution of 1 mol/L.

14. The method for culturing ginseng stem cells using a bioreactor according to any one of claims 1 to 3, wherein the composition of the stem cell induction medium in the step (3) is 1mg/L kinetin, 2.5mg/L indoleacetic acid, 1mg/L naphthylacetic acid, 75mg/L ascorbic acid, 75mg/L citric acid, 30g/L sucrose, 3g/L plant gel, 1.8 g/L1/2 MS medium, 0.8g/L WPM medium.

15. The method for culturing the ginseng stem cells using a bioreactor according to any one of claims 1 to 3, wherein the propagation medium in the step (3) is composed of 3mg/L of 2, 4-dichlorophenoxyacetic acid, 2mg/L of naphthylacetic acid, 1mg/L of kinetin, 35g/L of sucrose, 3g/L of plant gel, 1.8g/L of 1/2MS medium and 0.8g/L of WPM medium.

16. The method for culturing ginseng stem cells using a bioreactor according to any one of claims 1 to 3, wherein the composition of the liquid culture medium for stem cells in step (4) is 3mg/L of 2, 4-dichlorophenoxyacetic acid, 2mg/L of gibberellin, 1mg/L of kinetin, 35g/L of sucrose, 1.8g/L of 1/2MS medium and 0.8g/L of WPM medium.

17. The method for culturing ginseng stem cells using a bioreactor according to any one of claims 1 to 3, wherein the ginseng adventitious root tip portion is cultured in the stem cell induction medium and the stem cell propagation solid medium at 20 to 25 ℃ for 25 to 35 days in the absence of light, respectively, in step (3).

Images