CN112806266B

CN112806266B - Method for culturing ginseng adventitious root by using biological reaction device

Info

Publication number: CN112806266B
Application number: CN202110360926.1A
Authority: CN
Inventors: 高秀君; 闫培生; 刘冰; 张明臣
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: 2022-05-10
Anticipated expiration: 2041-04-02
Also published as: CN112806266A

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 a solid induction culture medium to induce adventitious roots of the ginseng; inoculating the ginseng adventitious roots obtained in the step (1) into a solid induction culture medium again, and carrying out dark culture; inoculating the adventitious roots of the ginseng obtained in the step (2) into a liquid culture medium, and performing dark culture at 100-120 rpm; inoculating the ginseng adventitious roots obtained in the step (3) into a biological reaction device containing a liquid culture medium, and performing dark culture to obtain the ginseng adventitious roots; the liquid culture medium comprises: 15-50g/L of sucrose, 0.6-2.4g/L of WPM culture medium, 1-2g/L N6 culture medium, 0-150mg/L of ascorbic acid, 0-225mg/L of citric acid and 0-7mg/L of indolebutyric acid. The method directly induces and generates the adventitious roots from the mature ginseng, has simple steps and short induction time, is matched with an optimized liquid culture medium, a biological reaction device and culture conditions, and has fast growth of the adventitious roots and 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, enhancing immunity, improving cardiovascular and cerebrovascular blood supply 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.

However, on one hand, 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. On the other hand, the cultured ginseng also has the problems of low content of ginsenoside, unstable content and difficulty in meeting application requirements.

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) proliferating adventitious roots of wild ginseng in the liquid culture medium. In the technical scheme, the callus needs to be induced first, and then the adventitious root needs to be induced, so that the required period is long, the process is complex, and the content of ginsenoside is not high.

The Chinese patent with the application number of 201410698528.0 discloses a ginseng adventitious root induced proliferation method, 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 and generate adventitious roots from each part of the mature ginseng, does not need the intermediate step of inducing callus, can simplify the induction step and shorten the induction time, further adopts an optimized liquid culture medium and a corresponding proper culture condition, and has fast growth of the adventitious roots and high growth multiple.

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 a solid induction culture medium to induce adventitious roots of the ginseng;

(2) inoculating the ginseng adventitious roots obtained in the step (1) into a solid induction culture medium again, and carrying out dark culture;

(3) inoculating the adventitious roots of the ginseng obtained in the step (2) into a liquid culture medium, shaking at 100-120rpm, 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 performing dark culture to obtain the ginseng adventitious roots;

in steps (3) and (4), the liquid medium comprises: 15-50g/L of sucrose, 0.6-2.4g/L of WPM culture medium, 1-2g/L N6 culture medium, 0-150mg/L of ascorbic acid, 0-225mg/L of citric acid and 0-7mg/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 addition, the cultured ginseng also has the problems of low ginsenoside content, slow growth and difficulty in meeting the requirements of clinical application.

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.

A large number of experiments show that the adventitious roots induced by the one-step method are cultured by adopting the liquid culture medium with the components and the proportion, and the adventitious roots grow fast and have high growth multiple by matching with a biological reaction device and corresponding culture conditions.

In a further scheme, in the step (4), the ventilation capacity of the biological reaction device is 0.01-0.4 vvm; preferably, the aeration is from 0.15 to 0.25 vvm.

By adopting the liquid culture medium, the ventilation capacity of the biological reaction device is 0.01-0.4 vvm; preferably, when the ventilation amount is 0.15-0.25vvm, the growth rate of the adventitious roots is high and the growth multiple is high.

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 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 aspect, the liquid medium comprises: 35-45g/L of sucrose, 0.6-1.2g/L of WPM culture medium, 1g/L N6 culture medium, 0-50mg/L of ascorbic acid, 75-225mg/L of citric acid and 1-3mg/L of indolebutyric acid;

preferably, the liquid medium comprises: 35g/L of sucrose, 1.2g/L of WPM medium, 1g/L N6 medium, 50mg/L of ascorbic acid, 225mg/L of citric acid and 3mg/L of indolebutyric acid.

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 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 0.5-2.5% of the volume of the liquid culture medium;

preferably, the mass of the inoculated adventitious roots accounts for 0.7-1.5% 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 20 to 80 percent of the volume of the biological reaction device,

preferably, it is 30% to 70%, more preferably 70% of the volume of the bioreactor.

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

When the liquid culture medium is used for culture in a biological reaction device, the culture medium addition amount and the adventitious root inoculation amount are adopted, the adventitious root growth effect is good, and the growth multiple is higher.

In a further scheme, in the step (1) and the step (2), the solid 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, the naphthylacetic acid is a plant growth regulator, and the gibberellin is a plant hormone, so that the formation 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 one-step method has short time for inducing and generating the adventitious roots, and when the induced and generated adventitious roots are further subjected to liquid culture, the liquid culture medium can shorten the induction time, reduce the pollution risk and improve the content of total saponins in the adventitious roots of ginseng.

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 more than 6 years;

as a preferred embodiment, the mature ginseng is a centennial ginseng.

The century ginseng is rare in nature, has high edible and medicinal values, can tonify five internal organs, calm spirit, calm soul, stop palpitation, remove pathogenic qi, improve eyesight, and benefit heart and intelligence; the value of the ginseng cultivation method is far higher than that of planted ginseng with short ginseng age. The invention does not need the intermediate step of inducing callus, can directly obtain the adventitious roots by one-step induction from the hundred-year ginseng cut blocks, not only can simplify the induction step and shorten the induction time, but also can obtain the specific functional components in the female parent hundred-year old ginseng, thereby obtaining the adventitious roots with better nutritive value.

Further, the main root, or the reed head, or the part, or the branch root, or the fibrous root of the mature ginseng are cleaned, disinfected, sliced and inoculated into an induction culture medium to induce the adventitious root of the ginseng.

In 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 ginseng adventitious roots using a bioreactor according to the present invention specifically comprises:

(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 solid 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 solid induction culture medium as the solid induction culture medium obtained in the step (1), and performing 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 (100-120rpm) at the temperature of 22 +/-1 ℃ to obtain the adventitious roots; wherein the liquid medium comprises: 35g/L of sucrose, 1.2g/L of WPM medium, 1g/L N6 medium, 50mg/L of ascorbic acid, 225mg/L of citric acid and 3mg/L of indolebutyric acid.

(4) And (3) culturing a biological reaction device:

cutting the ginseng adventitious roots obtained in the step (3) into tissues with the length of about 1-3cm, and inoculating the tissues into a liquid culture medium in a biological reaction device, wherein the liquid culture medium comprises: 35g/L of sucrose, 1.2g/L of WPM medium, 1g/L N6 medium, 50mg/L of ascorbic acid, 225mg/L of citric acid and 3mg/L of indolebutyric acid;

wherein, 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, and the ventilation capacity of the biological reaction device is 0.01-0.4vvm 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.

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

1/2MS culture medium

Ingredient (mg/L): potassium nitrate 950, ammonium nitrate 825, calcium chloride dihydrate 220, magnesium sulfate 185, monopotassium phosphate 85, manganese sulfate 11.15, zinc sulfate 4.3, boric acid 3.1, potassium iodide 0.415, sodium molybdate 0.125, copper sulfate 0.0125, cobalt chloride 0.0125, disodium ethylenediaminetetraacetate 37.3, ferrous sulfate 27.8, inositol 100, glycine 2, hydrochloric acid 0.5, pyridoxine hydrochloride 0.5 and ammonium sulfate hydrochloride 0.1.

B5 Medium

Ingredient (mg/L): potassium nitrate KNO₃ 2500，MgSO₄·7H₂O 250，CaCl₂·2H₂O 150，(NH₄)₂SO₄134，NaH₂PO₄.H₂O 150，KI 0.75，H₃BO₃ 3.0，MnSO₄·4H₂O 10，ZnSO₄·7H₂O 2.0，Na₂MoO₄·2H₂O 0.25，CoCl₂·6H₂O 0.025，CuSO₄·5H₂O 0.025，Na₂-EDTA 37.3，FeSO₄·7H₂O27.8, inositol 100, nicotinic acid 1.0, pyridoxine hydrochloride 1.0, and ammonium sulfate hydrochloride 10.

Woody Plant Medium (WPM)

Ingredient (mg/L): 400 parts of ammonium nitrate, 556 parts of tetrahydrate calcium nitrate, 990 parts of potassium sulfate, 72 parts of anhydrous calcium chloride, 170 parts of monopotassium phosphate, 0.25 part of sodium molybdate dihydrate, 180 parts of anhydrous magnesium sulfate, 22.4 parts of manganese sulfate monohydrate, 8.6 parts of zinc sulfate heptahydrate, 0.25 part of copper sulfate pentahydrate, 27.8 parts of ferrous sulfate heptahydrate, 37.3 parts of disodium ethylenediamine tetraacetic acid, 100 parts of inositol, 11 parts of vitamin B, 0.5 part of nicotinic acid, 60.5 parts of vitamin B, 2 parts of glycine and 5.2 parts of pH.

N6 Medium (N6 Medium)

Ingredient (mg/L): potassium nitrate 2800, ammonium sulfate 463, monopotassium phosphate 400, magnesium sulfate (MgSO)₄·7H₂O)185, calcium chloride (CaCl)₂·2H₂O)165, disodium ethylenediaminetetraacetate 37.3, ferrous sulfate (FeSO)₄·7H₂O)27.8, manganese sulfate (MnSO)₄·H₂O)4.4, zinc sulfate (ZnSO)₄·7H₂O)1.5, boric acid 1.6, potassium iodide 0.8, vitamin B1 (thiamine hydrochloride) 1.0, vitamin B6 (pyridoxine hydrochloride) 0.5, niacin 0.5, glycine 2.0, sucrose 20000, pH 5.8, 25 ℃.

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

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.

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 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.

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.

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

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

1. the invention utilizes the biological reaction device to cultivate the ginseng adventitious roots, the biological reaction device is a small-sized biological reactor, the structure is simple, the operation and the observation of the internal situation are convenient, the bottom of the tank body of the biological reaction device is conical, the plurality of air inlet devices are arranged on the conical inclined plane, air is introduced in a plurality of directions, the ventilation is sufficient and uniform, the biological reaction device can be utilized to conveniently produce the ginseng adventitious roots, the growth of the adventitious roots is fast, the growth multiple is high, and the growth of the adventitious roots is uniform.

2. The liquid culture medium has proper mixture ratio of the components, can generate synergistic effect, is matched with proper control parameters such as ventilation capacity and inoculation quantity, has high growth multiple of the adventitious root, and is also favorable for improving the content of the ginsenoside.

3. 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.

4. The method for culturing the adventitious roots adopts an induction culture medium which 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 cane sugar, 1-6g/L of plant gel, 1-4g/L B5 of culture medium and 1-2.4g/L of WPM culture medium, and all components have synergistic effect, so that the problem that the adventitious roots are directly induced from mature ginseng by a one-step method is solved, and the quantity 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 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 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 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 are 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 supporting 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 around the center at intervals, which is favorable for 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, 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 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 biological reaction device used in the embodiments (such as the embodiments 14 to 16) of the present invention is the biological reaction device of the above 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 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 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, 4g/L plant gel, 2g/L B5 culture medium and 1g/L WPM culture medium, and the pH value is 6.0.

(2) Subculture of adventitious roots

Inoculating the adventitious roots obtained in the step (1) into the same induction culture medium as that in the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;

(3) cultivation of adventitious roots

Shearing the adventitious roots obtained in the step (2) into tissues with the length of about 2cm, inoculating the tissues into a liquid culture medium, and culturing for 3-4 weeks on a shaking table at the temperature of 22 +/-1 ℃ to obtain the adventitious roots; wherein the liquid culture medium contains 4mg/L indolebutyric acid, 0.1g/L citric acid, 0.05g/L ascorbic acid, WPM culture medium and 30g/L sucrose, and has pH of 6.0.

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 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 13

Culture methods of examples 6 to 13 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 13, which included: 15-50g/L of sucrose, 0.6-2.4g/L of WPM culture medium, 1-2g/L N6 culture medium, 0-150mg/L of ascorbic acid (Vc), 0-225mg/L of citric acid, 0-7mg/L of indolebutyric acid (IBA), 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)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

Preparation of 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 are shown in the following table:

TABLE 1

As can be seen from Table 1 above, the liquid culture medium prepared according to the formulation of examples 10-12 has a high fold increase of adventitious roots and a high total amount of saponins detected, wherein the fold increase and the saponin content of the adventitious roots obtained according to example 10 are optimal.

EXAMPLE 14 cultivation of ginseng adventitious roots Using a 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 dark culturing at 22 + -1 deg.C for 4-5 weeks to induce adventitious root; 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 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 ℃ at 120rpm to obtain adventitious roots; the liquid medium was the same as in example 10.

(4) Bioreactor culture

The composition of the liquid medium was the same as in example 10; 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 1.0% of the volume of liquid culture medium; ventilating the biological reaction device, wherein the ventilation rate is 0.15vvm, and culturing in the dark at the temperature of 22 +/-1 ℃ for 3-4 weeks to obtain the adventitious roots.

EXAMPLE 15 culturing of adventitious root of Panax ginseng with 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 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 110rpm to obtain adventitious roots; the liquid medium was the same as in example 11.

(4) Culturing adventitious roots in a biological reaction device

The composition of the liquid medium was the same as in example 11; adding a liquid culture medium into a tank body (the volume is 5L) of a biological reaction device, wherein the volume of the liquid culture medium in the biological reaction device accounts for 20% of the volume of the biological reaction device; sterilizing at 121 deg.C for 20 min;

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 0.5 percent of the volume of the liquid culture medium; ventilating the biological reaction device, wherein the ventilation rate is 0.01vvm, and culturing in the dark at the temperature of 22 +/-1 ℃ for 3-4 weeks to obtain the adventitious roots.

EXAMPLE 16 culturing of adventitious root of Panax ginseng with 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 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 10. (4) Culturing adventitious roots in a biological reaction device

The composition of the liquid medium was the same as in example 10; 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 80 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 into liquid culture medium of biological reaction device, wherein the mass of inoculated adventitious root is 2.5% of the volume of liquid culture medium; ventilating the biological reaction device, wherein the ventilation amount is 0.4vvm, and culturing for 3-4 weeks in a dark environment at the temperature of 22 +/-1 ℃ 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 14 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 other structures were the same, and the culture method and the liquid medium were the same as in example 14. The method for measuring the total content of the saponin is the same as the above.

Thus, examining the cases of culturing ginseng adventitious roots using examples 14 to 16 (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 the common fermentation tank, the biological reaction device of the invention has better growth and faster speed of the adventitious roots in the examples 14 to 16, and is beneficial to improving the efficiency, and particularly, the liquid culture medium of the invention has higher growth times of the adventitious roots in the examples 14 to 16, which shows that the liquid culture medium can greatly improve the growth times of the adventitious roots by matching with the biological reaction device. Among them, the adventitious roots under the conditions of example 14 had a high growth multiple and the total saponin content was the highest.

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:

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

(3) inoculating the adventitious roots of the ginseng obtained in the step (2) into a liquid culture medium, shaking at 100-120rpm, and culturing in dark;

in the step (1) and the step (2), the solid 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 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 composition of the liquid culture medium is 15-50g/L of sucrose, 0.6-2.4g/L of WPM culture medium, 1-2g/L N6 culture medium, 0-150mg/L of ascorbic acid, 0-225mg/L of citric acid and 1-7mg/L of indolebutyric acid.

2. The method according to claim 1, wherein the aeration amount of the bioreactor in the step (4) is 0.01 to 0.4 vvm.

3. The method of claim 1, wherein the aeration of the bioreactor is 0.15 to 0.25 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 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.

5. The method according to any one of claims 1 to 4, wherein the composition of the liquid medium is 35 to 45g/L sucrose, 0.6 to 1.2g/L WPM medium, 1g/L N6 medium, 0 to 50mg/L ascorbic acid, 75 to 225mg/L citric acid, 1 to 3mg/L indolebutyric acid.

6. The method according to claim 5, wherein the composition of the liquid medium is 35g/L sucrose, 1.2g/L WPM medium, 1g/L N6 medium, 50mg/L ascorbic acid, 225mg/L citric acid, 3mg/L indolebutyric acid.

7. The method according to any one of claims 1 to 4, wherein in the step (4), the adventitious roots are inoculated into the liquid medium, and the mass of the inoculated adventitious roots accounts for 0.5 to 2.5% of the volume of the liquid medium.

8. The method of claim 7, wherein the mass of the inoculated adventitious roots is 0.7% to 1.5% of the volume of the liquid medium.

9. The method according to any one of claims 1 to 4, wherein in the step (4), the volume of the sterilized liquid medium is 20% 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 30% to 70% of the volume of the bioreactor apparatus.

11. The method of claim 10, wherein the sterilized liquid culture medium occupies 70% of the volume of the bioreactor apparatus.

12. 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 steps (1) and (2), and the dark culture is performed at 22 ± 1 ℃ for 3 to 4 weeks in step (3).

13. The method of claim 1, wherein the solid 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.

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

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