CN112806267B

CN112806267B - Method for culturing ginseng adventitious roots by using biological reaction device

Info

Publication number: CN112806267B
Application number: CN202110361903.2A
Authority: CN
Inventors: 张明臣; 刘冰; 闫培生; 高秀君
Original assignee: Shandong Anran Nanometre Ind Development Co ltd
Current assignee: Shandong Anran Nanometre Ind Development Co ltd
Priority date: 2021-04-02
Filing date: 2021-04-02
Publication date: 2022-08-02
Anticipated expiration: 2041-04-02
Also published as: CN112806267A

Abstract

The invention discloses a method for culturing ginseng adventitious roots by using a biological reaction device, which comprises the following steps: (1) cleaning and disinfecting mature ginseng, slicing, and inoculating the ginseng to an induction culture medium to induce adventitious roots of the ginseng; inoculating the ginseng adventitious roots obtained in the step (1) into an induction culture medium again, and performing dark culture; inoculating the adventitious roots of the ginseng obtained in the step (2) into a liquid culture medium, shaking at 100-140rpm, and carrying out dark culture; inoculating the ginseng adventitious roots obtained in the step (3) into a biological reaction device containing a liquid culture medium, and carrying out dark culture to obtain the ginseng adventitious roots; the liquid culture medium 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, 0.2-1.2mg/L of kinetin and 0-1.8mg/L of indolebutyric acid. The method of the invention directly induces and produces the adventitious roots from the mature ginseng, can simplify the induction steps and shorten the induction time, adopts the improved liquid culture medium, and is matched with a biological reaction device and a control process, so that the obtained adventitious roots have high growth multiple.

Description

Method for culturing ginseng adventitious roots 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 adventitious roots 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 requirement on environmental conditions is strict, the quality of ginseng is easily influenced by climate, cultivation conditions and plant diseases and insect pests, the cultivation technology is complex, the pesticide residue exceeds the standard, and the development prospect of artificial cultivation of ginseng is greatly limited by the problems of old 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 application requirement is difficult to meet.

Chinese patent application No. CN201711459037.0 discloses a method for tissue culture of adventitious roots of wild ginseng, comprising the following steps: (1) inducing wild ginseng callus; (2) proliferation of wild ginseng callus; (3) inducing adventitious roots of wild ginseng; (4) the adventitious roots of the wild ginseng in the liquid culture medium proliferate. In the technical scheme, the callus needs to be induced first, then the adventitious root needs to be induced, the required period is long, the process is complex, and the content of the ginsenoside is not high.

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 needs to be obtained through seed germination or explant culture first, at least 28-32 days are still needed, callus induction is still needed in the explant culture, and the period is not shortened actually. Therefore, if a method capable of directly inducing and generating adventitious roots from mature ginseng is sought, the induction period can be shortened, the induction conditions can be simplified, and rapid scale-up production can be carried out by matching with a suitable liquid culture medium and a suitable bioreactor, so that the method has wide economic significance.

The present invention has been made in view of this situation.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for culturing ginseng adventitious roots by using a biological reaction device. The method adopts a one-step method to induce each part of the mature ginseng to generate adventitious roots without the intermediate step of inducing callus, so that the induction step can be simplified, and the induction time can be shortened; the liquid culture medium has relatively simple components, and the obtained adventitious roots have high growth multiple and high ginsenoside content by matching with a biological reaction device and a control process.

In order to solve the technical problems, the invention adopts the technical scheme that:

the first object of the present invention is to provide a method for culturing ginseng adventitious roots using a bioreactor, comprising:

(1) cleaning and disinfecting mature ginseng, slicing, and inoculating the ginseng to an induction culture medium to induce adventitious roots of the ginseng;

(2) inoculating the obtained adventitious root of Ginseng radix into induction culture medium again, and dark culturing;

(3) inoculating the adventitious roots of the ginseng obtained in the step (2) into a liquid culture medium, shaking at 100-140rpm, and carrying out dark culture;

(4) and (3) culturing a biological reaction device: inoculating the ginseng adventitious roots obtained in the step (3) into a biological reaction device containing a liquid culture medium, and carrying out dark culture to obtain the ginseng adventitious roots;

in steps (3) and (4), the liquid medium 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, 0.2-1.2mg/L of kinetin and 0-1.8mg/L of indolebutyric acid.

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 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 induced directly from mature ginseng at present. Through a large number of experiments, the invention unexpectedly discovers that the mature ginseng slices can be directly induced to generate adventitious roots on a specific induction culture medium, so that the adventitious roots can be directly obtained from the mature ginseng by one-step induction without an intermediate step of callus induction, thereby simplifying the induction step and shortening the induction time.

The adventitious roots induced by the one-step method are cultured by adopting the liquid culture medium with the components and the proportion, the growth multiple of the adventitious roots is high, the rapid acquisition of a large number of adventitious roots is facilitated, and the further extraction of the ginsenoside is facilitated.

In a further scheme, in the step (4), the ventilation capacity of the biological reaction device is 0.1-0.45 vvm; preferably, the ventilation is from 0.22 to 0.32 vvm.

By adopting the liquid culture medium, the ventilation capacity of the biological reaction device is 0.1-0.45 vvm; preferably, when the ventilation volume is 0.22-0.32vvm, the growth rate of the adventitious root is high, the growth multiple is high, and the improvement of the content of the ginsenoside is facilitated.

In a further scheme, the biological reaction device comprises a tank body, wherein 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 two at least air inlet unit surround the discharge gate interval evenly distributed by on the diapire that gradually reduces to the center all around.

In the invention, the air inlet devices are arranged on the inclined plane of the bottom wall of the tank body in the shape of the inverted cone 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 are uniformly distributed around the center at intervals, which is favorable for the uniform distribution of air in the tank body and the uniform growth of adventitious roots in the tank body.

In a further aspect, the liquid medium 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 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.

The adventitious roots cultured by the liquid culture medium of the preferred embodiment have high growth multiple, high total saponin content and optimal synthesis.

In the further scheme, in the step (3), the adventitious roots are inoculated into the liquid culture medium, and the mass of the inoculated adventitious roots accounts for 0.5-3.0% of the volume of the liquid culture medium;

preferably, the mass of the inoculated adventitious roots accounts for 0.9-1.8% of the volume of the liquid medium.

In a further proposal, in the step (3), the volume of the sterilized liquid culture medium accounts for 30 to 80 percent of the volume of the biological reaction device,

preferably, the volume of the bioreactor is 45-70%.

In the further scheme, in the steps (1) and (2), dark culture is carried out at the temperature of 22 +/-1 ℃ for 4-5 weeks, and in the step (3), dark culture is carried out at the temperature of 22 +/-1 ℃ for 3-4 weeks.

When the liquid culture medium is matched with the liquid culture medium disclosed by the invention and cultured in a biological reaction device, the addition amount and the inoculation amount of the culture medium are adopted, the growth effect of the adventitious root is better, the growth multiple is high, and meanwhile, the content of ginsenoside is high.

In a further scheme, in the step (1) and the step (2), the induction culture medium comprises 1-6mg/L of naphthylacetic acid, 0.2-1mg/L of gibberellin, 0.1-0.6mg/L of kinetin, 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.

The induction culture medium of the scheme realizes that each part of the mature ginseng is directly induced to generate adventitious roots without an intermediate step of inducing callus, thereby simplifying the induction step and shortening the induction time.

In the scheme, naphthylacetic acid is a plant growth regulator, gibberellin is a plant hormone, and the growth of adventitious roots can be promoted. 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.

In a further embodiment, the induction 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 medium and 1.21g/L WPM medium.

The induction culture medium under the component proportion has the best induction effect on the mature ginseng to generate adventitious roots, has a large number of 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.

The adventitious roots induced and generated by the one-step method can save steps, and when the adventitious roots induced and generated are subjected to further liquid culture, the liquid culture medium can shorten the induction time, reduce the pollution risk and improve the growth multiple of the adventitious roots.

In a further scheme, 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.

In a further scheme, the age of the mature ginseng is more than 3 years;

preferably, the age of the mature ginseng is 6 years or more.

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 promote mentality 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 a further scheme, the ginseng is selected from wild ginseng, transplanted ginseng, ginseng under forest and garden ginseng;

preferably, the ginseng is wild ginseng.

As a specific preferred embodiment, the step of culturing adventitious roots using a bioreactor according to the present invention specifically includes:

(1) induction of adventitious roots

Cleaning main root of mature Ginseng radix, sterilizing, cutting into slices with width of 0.5-0.7cm, length of 0.5-0.7cm and thickness of 0.2-0.5mm, inoculating into induction culture medium containing 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, dark culturing at 22 + -1 deg.C for 4-5 weeks to induce adventitious root of Ginseng radix

(2) Subculture of adventitious roots

Inoculating the 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) propagation culture of adventitious roots

Shearing the adventitious roots of the ginseng obtained in the step (2) into tissues with the length of about 1-2cm, inoculating the tissues into a liquid culture medium, and culturing for 3-4 weeks on a shaking table (100-; wherein the liquid medium 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; (4) culturing adventitious roots by using a biological reaction device:

cutting the ginseng adventitious roots obtained in the step (3) into tissues with the length of about 1-2cm, and inoculating the tissues into a liquid culture medium in a biological reaction device, wherein the liquid culture medium 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.

The volume of the sterilized liquid culture medium accounts for 30-80% of the volume of the biological reaction device; the mass of the inoculated adventitious roots of the adventitious roots accounts for 0.5-3.0% of the volume of the liquid culture medium, and the ventilation capacity of the biological reaction device is 0.1-0.45vvm in the culture process; dark culture is carried out for 4-5 weeks at 22 +/-1 ℃.

In a further embodiment, the pH of the induction medium and the liquid medium of the present invention is 5.6 to 6.0.

The adventitious roots cultured by the liquid culture medium of the preferred embodiment have the best comprehensive growth multiple of the adventitious roots and the total saponin content.

In the present invention, WPM medium, B5 medium, N6 medium, 1/2MS medium and the like are known media in the art.

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.

B5 Medium

Ingredient (mg/L): potassium nitrate KNO ₃ 2500，MgSO ₄ ·7H ₂ O 250，CaCl ₂ ·2H ₂ O 150，(NH ₄ ) ₂ SO ₄ 134，NaH ₂ PO ₄ .H ₂ O 150，KI 0.75，H ₃ BO ₃ 3.0，MnSO ₄ ·4H ₂ O 10，ZnSO ₄ ·7H ₂ O 2.0，Na ₂ MoO ₄ ·2H ₂ O 0.25，CoCl ₂ ·6H ₂ O 0.025，CuSO ₄ ·5H ₂ O 0.025，Na ₂ -EDTA 37.3，FeSO ₄ ·7H ₂ O27.8, inositol 100, nicotinic acid 1.0, pyridoxine hydrochloride 1.0, and ammonium sulfate hydrochloride 10.

N6 Medium (N6 Medium)

Ingredient (mg/L): potassium nitrate 2800, ammonium sulfate 463, monopotassium phosphate 400, magnesium sulfate (MgSO) ₄ ·7H ₂ O)185, calcium chloride (CaCl) ₂ ·2H ₂ O)165, disodium ethylenediaminetetraacetate 37.3, ferrous sulfate (FeSO) ₄ ·7H ₂ O)27.8, manganese sulfate (MnSO) ₄ ·H ₂ O)4.4, zinc sulfate (ZnSO) ₄ ·7H ₂ O)1.5, boric acid 1.6, iodinationPotassium 0.8, vitamin B1 (thiamine hydrochloride) 1.0, vitamin B6 (pyridoxine hydrochloride) 0.5, nicotinic acid 0.5, glycine 2.0, sucrose 20000, pH 5.8, 25 ℃.

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.

It is a second object of the present invention to provide a bioreactor for culturing ginseng adventitious roots, comprising: 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.

The bioreactor is a small bioreactor, has simple structure and convenient operation, can be used for conveniently producing the ginseng adventitious roots, and the adventitious roots can be quickly grown and have high growth times.

In the invention, the tank body 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 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 a further embodiment, the bioreactor has a volume of 1-500L, preferably 1-50L, preferably 2-20L, preferably 3-10L, preferably 5L.

Furthermore, 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.

In a further scheme, a discharge hole is formed in the center of the bottom of the tank body, 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.

In 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 entering air cannot vertically rise, and further, two or more air inlet devices are uniformly distributed around the center at intervals, which is beneficial to the uniform distribution of air in the tank body and the uniform growth of adventitious roots in the tank body.

In a further scheme, the air inlet device comprises an air inlet, an air inlet pipe and an air filter, the air inlet is located on the bottom wall of the tank body, the air inlet pipe is connected with the air inlet in a sealing mode, and the air filter is arranged on the air inlet pipe to filter air.

In a further scheme, 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.

Further, an inoculation port is arranged on the cover body or the top of the tank body and used for inoculation.

In a 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.

When the jar body adopted opaque stainless steel material, a plurality of transparent observation windows of different positions of setting can follow the inside of different angles observation jar bodies, in time master jar internal ginseng adventitious root's the growth condition.

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

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

After adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects:

1. the culture method of the adventitious roots realizes the one-step method, the processed each part of the mature ginseng is inoculated into the induction culture medium to directly induce the adventitious roots of the ginseng, the middle step of inducing callus is not needed, the induction step can be simplified, the induction time is shortened, and the pollution risk is reduced.

2. The invention utilizes the biological reaction device to culture the ginseng adventitious roots, the biological reaction device is a small bioreactor, the structure is simple, the operation and the observation of the internal condition are convenient, the ventilation is sufficient and uniform, the ginseng adventitious roots can be conveniently produced by utilizing the biological reaction device, the adventitious roots are quickly increased, the growth multiple is high, the growth of the adventitious roots is uniform, and meanwhile, the ginsenoside content is high.

3. The improved liquid culture medium has the advantages that the proportion of each component is proper, the synergistic effect can be generated, the growth multiple of the adventitious root is high, and meanwhile, the content of ginsenoside is high.

4. When the invention is cultured in a biological reaction device, culture conditions of proper inoculation amount of 0.5-3.0%, ventilation amount of 0.1-0.45vvm vvm and the like are adopted, which is beneficial to the growth of the adventitious roots, the increase multiple of the adventitious roots and the further beneficial to the improvement of the content of effective components. 5. The method for culturing the adventitious roots adopts an induction culture medium which 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 cane sugar, 1-6g/L of plant gel, 1-4g/L B5 of culture medium and 1-2.4g/L of WPM culture medium, all components have synergistic action, the problem that the adventitious roots are directly induced from mature ginseng in one step is solved, and the number of the generated adventitious roots is large.

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 also be derived from them without inventive effort. In the drawings:

FIG. 1 is a schematic view of the structure of a bioreactor of the present invention;

FIG. 2 is a schematic view of induction of adventitious roots using the induction medium of example 1 of the present invention;

FIG. 3 is a schematic diagram of induction of adventitious roots using the induction medium of comparative example 1;

the device comprises a tank body 1, a cover body 2, an exhaust device 3, an air inlet device 4, a discharge hole 5, an air inlet pipe 6, an air filter 7, an exhaust pipe 8, a filter device 9, an observation window 10, a handle 11 and supporting legs 12.

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.

In the present invention, the preparation method or the detection method is not particularly limited, and any method that can achieve the object in the prior art can be used.

As shown in FIG. 1, 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; the bottom of the tank body 1 is provided with at least two air inlet devices 4, and air enters the tank body 1 through the air inlet devices 4.

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 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.

A discharge hole 5 is formed in the center of the bottom of the tank body 1, and the air inlet devices 4 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 further, two or more air inlet devices 4 are uniformly distributed at intervals around the center, which is beneficial to the uniform distribution of air in the tank body 1 and the uniform growth of adventitious roots in the tank body 1.

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, liquid addition from the top can be achieved.

An inoculation port is arranged on the cover body 2 or the top of the tank body 1 and is 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 adventitious roots of ginseng in the tank body 1 can be grasped 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.

The biological reaction device adopted in the embodiment (for example, the embodiments 24 to 26) of the invention is the biological reaction device of the scheme.

Example 1

(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. 2, in which A is a photograph of 1 week of culture, B is a photograph of 3 weeks of culture, and C is a photograph of 5 weeks of culture. As can be seen, after 5 weeks, adventitious roots were induced directly on the slices of the mature wild ginseng.

Example 2

(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

(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, 0.075g/L citric acid, 0.03g/L ascorbic acid, 40g/L sucrose, 1g/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

(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.

Example 4

(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

(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

(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

(3) cultivation of adventitious roots

In the above examples 1 to 5, the liquid medium was not optimized, had simple composition, and was used to simply verify whether the adventitious roots of ginseng induced in one step could grow normally, and the results showed that it could grow normally.

Examples 6 to 23

Culture methods of examples 6 to 23 the method of example 1 was referenced, except that the liquid medium in example 1 was replaced with the optimized liquid medium of examples 6 to 23, the liquid medium including: 35g/L of sucrose, 1.35g/L of WPM medium, 1g/L N6 medium, 0-1.8mg/L of indolebutyric acid (IBA), 1-6mg/L of naphthylacetic acid (NAA), 0.2-1.2mg/L of Kinetin (KT) and the pH value is 5.8.

In addition, the content of ginsenoside in the adventitious root of the ginseng is detected by adopting the following detection method:

(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) ₄ O): chromatographically pure acetonitrile (C) ₆ H ₁₁ N): pure chromatography

Standard reagents: ginsenoside Re, Rg1, Ra3, Rb1, Rf, Rb2, Rb3, F3, Rg2, Rd, F1

(3) Analytical procedure

Ginseng adventitious root test solution:

taking about 6g (accurate to 0.01g) of the uniformly mixed adventitious root sample, grinding in a 150mL mortar, transferring to a 50mL centrifuge tube, adding 10mL of water, mixing, breaking the wall for 3 minutes by 400W on an ultrasonic cell crusher, and freezing for 3 hours in a refrigerator at-18 ℃. Freeze-drying in a freeze-drying machine for 48 hours until no water drops exist outside the cup body.

The freeze-dried sample is taken and porphyrized in a mortar, 50mg is accurately weighed in a 10ml centrifuge tube, 70% methanol solution is added, and vortex is carried out. 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 standard substances including 8.00mg of ginsenoside Re, Rb1, Rg1, Rd, Rf, F3, Rk2, Rb2, Rb3, Rg2 and F1 in a 10ml volumetric flask, and adding high-grade pure methanol to the volume.

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) 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):

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.

The fold increase was calculated 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 of the experiment are shown in table 1.

TABLE 1

As can be seen from table 1 above, on the basis of determining the usage amount of sucrose, WPM, and N6, the influence of plant hormones IBA, NAA, and KT on the yield of adventitious roots and the saponin content at different addition amounts is examined, so as to select a proper plant hormone and a proper proportion. As can be seen from the table, the compositions of examples 9, 12-13, 15-17 and 20-23 have better adventitious root growth factor and total saponin content, wherein the comprehensive optimal value of the adventitious root growth factor and the total saponin content is obtained under the conditions of example 17.

EXAMPLE 24 culturing of adventitious root of Panax ginseng by means of bioreactor

(1) Induction of adventitious roots

(2) Subculture of adventitious roots

(3) propagation culture of adventitious roots

Shearing the 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 ℃ at 100rpm to obtain adventitious roots; the liquid medium was the same as in example 17.

(4) Culturing adventitious roots in a biological reaction device

The composition of the liquid medium was the same as in example 17; 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;

shearing the adventitious roots obtained in the step (3) into tissues with the length of about 1 cm; inoculating into liquid culture medium of biological reaction device, wherein the mass of inoculated adventitious root is 1.8% of the volume of liquid culture medium; ventilating the biological reaction device, wherein the ventilation rate is 0.32vvm, and culturing in the dark at the temperature of 22 +/-1 ℃ for 3-4 weeks to obtain the adventitious roots.

EXAMPLE 25 culturing of adventitious root of Panax ginseng by means of bioreactor

(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 performing dark culture 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

(3) propagation culture of adventitious roots

Shearing the 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 shaking table at the temperature of 22 +/-1 ℃ and the rpm of 130 for 3-4 weeks to obtain adventitious roots; the liquid medium was the same as in example 16.

(4) Culturing adventitious roots in a biological reaction device

The composition of the liquid medium was the same as in example 16; 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 45 percent of the volume of the liquid culture medium; sterilizing at 121 deg.C for 20 min;

shearing the mountain ginseng adventitious roots obtained in the step (3) into tissues with the length of about 2 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 0.1vvm, and culturing in the dark at the temperature of 22 +/-1 ℃ for 3-4 weeks to obtain the adventitious roots.

Example 26 culturing of adventitious root of Panax ginseng Using a bioreactor

(1) Induction of adventitious roots

(2) Subculture of adventitious roots

(3) propagation culture of adventitious roots

Shearing the ginseng 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 the tissues on a shaking table at the temperature of 22 +/-1 ℃ and the rpm of 140 for 3-4 weeks to obtain adventitious roots; the liquid medium was the same as in example 17.

(4) Culturing adventitious roots in a biological reaction device

The composition of the liquid medium was the same as in example 17; 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 35 percent of the volume of the biological reaction device; sterilizing at 121 deg.C for 20 min;

shearing the mountain ginseng adventitious roots obtained in the step (3) into tissues with the 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 3.0 percent of the volume of the liquid culture medium; ventilating the biological reaction device, wherein the ventilation rate is 0.45vvm, and culturing in the dark at the temperature of 22 +/-1 ℃ for 3-4 weeks to obtain the adventitious roots.

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. 3 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, a WPM culture medium and 3g/L of plant gel, wherein 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.

Comparative example 4

This comparative example differs from example 24 in the bioreactor used.

In the comparative example, the biological reaction device is only provided with an air inlet device (common fermentation tank) at the right center of the conical bottom of the tank body, and the bottom wall of the tank body is in a downward-sunken arc shape; the culture method and the liquid medium were the same as in example 24.

Thus, examining the cases of culturing ginseng adventitious roots using examples 24 to 26 (using the bioreactor of the present invention, having a volume of 5L) and the ordinary fermentation tank of the present comparative example (having a volume of 5L), the ginseng adventitious roots growth results were as follows:

TABLE 2

Therefore, compared with a common fermentation tank, the biological reaction device provided by the embodiment of the invention has the advantages that the weight gain of the adventitious roots is higher, the growth speed is high, the growth is better, the efficiency is improved, and higher content of ginsenoside is obtained, wherein the growth multiple of the adventitious roots is the highest under the condition of the embodiment 24, and meanwhile, the content of ginsenoside is also the highest, and the synthesis is optimal.

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

1. A method for culturing ginseng adventitious roots by using a biological reaction device, which is characterized by comprising the following steps:

in the step (1) and the step (2), the composition of the induction culture medium is 1-6mg/L of naphthylacetic acid, 0.2-1mg/L of gibberellin, 0.1-0.6mg/L of kinetin, 0.075-1.5g/L of citric acid, 0.03-1g/L of ascorbic acid, 20-60g/L of cane sugar, 1-6g/L of plant gel, 1-4g/L B5 of culture medium and 1-2.4g/L of WPM culture medium;

in the steps (3) and (4), the liquid culture medium comprises 10-55g/L of cane sugar, 0.8-1.5g/L of WPM culture medium, 0.6-1.6g/L of LN6 culture medium, 1-6mg/L of naphthylacetic acid, 0.2-1.2mg/L of kinetin and 0-1.8mg/L of indolebutyric acid.

2. The method according to claim 1, wherein the aeration amount of the bioreactor in the step (4) is 0.1 to 0.45 vvm.

3. The method of claim 2, wherein the aeration rate of the bioreactor is 0.22 to 0.32 vvm.

4. The method according to claim 1, wherein in the step (4), the biological reaction device comprises a tank body, 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.

5. The method according to any one of claims 1 to 4, wherein the composition of the liquid medium is 35g/L sucrose, 1.35g/L WPM medium, 1g/L N6 medium, 1 to 5mg/L naphthylacetic acid, 0.2 to 1mg/L kinetin, 0.2 to 1.8mg/L indolebutyric acid.

6. The method according to claim 5, wherein the composition of the liquid medium 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.

7. The method according to any one of claims 1 to 4, wherein in the step (3), the adventitious roots are inoculated into the liquid medium, and the mass of the inoculated adventitious roots accounts for 0.5 to 3.0% of the volume of the liquid medium.

8. The method of claim 7, wherein the mass of the inoculated adventitious roots is 0.9% to 1.8% of the volume of the liquid medium.

9. The method according to any one of claims 1 to 4, wherein in the step (3), the volume of the sterilized liquid medium is 30 to 80% of the volume of the bioreactor apparatus.

10. The method of claim 9, wherein the sterilized liquid culture medium has a volume of 45% to 70% of the volume of the bioreactor apparatus.

11. The method according to any one of claims 1 to 4, wherein the dark culture is performed at 22. + -. 1 ℃ for 4 to 5 weeks in the steps (1) and (2), and the dark culture is performed at 22. + -. 1 ℃ for 3 to 4 weeks in the step (3).

12. The method of claim 1, wherein the induction medium has a composition of 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.

13. The method according to any one of claims 1 to 4, wherein the ginseng is selected from wild ginseng, mountain ginseng, ginseng under forest, and ginseng garden.

14. The method of claim 13, wherein the ginseng is wild ginseng.