CN112544443B - Method for rapidly propagating mountain ginseng adventitious roots - Google Patents
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Abstract
The invention provides a rapid propagation method of mountain ginseng adventitious roots, belonging to the technical field of plant tissue culture, the method takes wild ginseng roots as explants, callus is obtained by the induction culture of the explants, the adventitious roots are obtained by the rooting culture of the callus, the adventitious roots are screened by a specific screening culture medium to obtain high-yield adventitious roots, and the high-yield adventitious roots are propagated and cultured in a propagation culture medium in an airlift bioreactor for 40-50 days to obtain the mountain ginseng adventitious roots for harvesting. The dry weight of the mountain ginseng adventitious roots harvested by the method is as high as 17.31g/L, and the biomass is 20-30 times of that of the mountain ginseng adventitious roots in inoculation. The method can obviously improve the quantity and biomass of the adventitious roots, has short breeding period, and the obtained mountain ginseng adventitious roots have uniform growth, stable heredity and no pollution and have the same nutrient components and nutrient value as the cultivated ginseng.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a method for rapidly propagating mountain ginseng adventitious roots.
Background
The wild ginseng is wild ginseng in the mountain, is a famous and precious traditional Chinese medicine and has a high value. Among the ginseng, wild ginseng is considered to have the best quality and the best efficacy because of its long natural growth period. The application of ginseng which is one of traditional and rare Chinese medicinal materials in China is expanded to a plurality of fields such as medicines, health care products, beauty and skin care products and the like. The wild ginseng has the phenomenon of 'no price and no market' due to extremely scarce resources.
The problem of ginseng resource can not be economically, reasonably and sustainably solved because the ginseng currently occupying the market is lack of ginseng forest land resources, ginseng continuous cropping is obstructed, and the quality standard of artificial cultivation can not be met. The tissue culture technology of ginseng has shown great development potential, and can effectively solve the resource problem of ginseng. At present, a stable ginsenoside source can be obtained by culturing a large amount of ginseng adventitious roots in a bioreactor. The process flow of the adventitious root in vitro culture comprises the technical links of adventitious root induction, solid proliferation and propagation, suspension cascade amplification culture and the like, wherein the high-efficiency induction is the premise of realizing the scale production of the adventitious root.
Therefore, the wild ginseng root is used as an explant, the quantity and biomass of the adventitious roots obtained by induction are obviously increased by optimizing macroelements, trace elements and organic compounds in a culture medium and culturing in an airlift bioreactor, the obtained wild ginseng adventitious roots have uniform growth, stable heredity and no pollution, have the same nutrient components and nutrient values as the cultivated ginseng, and simultaneously provide theoretical basis for further regulating secondary metabolites and expanding production.
Disclosure of Invention
The invention aims to provide a method for rapidly propagating mountain ginseng adventitious roots, which has high propagation efficiency, and the mountain ginseng adventitious roots obtained by the method have the same nutritional value as cultivated ginseng.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for rapidly propagating mountain ginseng adventitious roots, which comprises the following steps:
1) taking wild ginseng roots as explants, inoculating the explants into an induction culture medium, and carrying out induction culture to obtain callus;
2) inoculating the callus into a rooting culture medium, and carrying out rooting culture for 6-8 weeks to obtain adventitious roots;
3) inoculating the adventitious roots into a screening culture medium, carrying out subculture, and taking the adventitious roots with the adventitious root induction number of 36-46 as adventitious root systems;
4) inoculating the adventitious root system into a proliferation culture medium, and performing proliferation culture for 40-50 days to obtain mountain ginseng adventitious roots for harvesting; the enrichment culture is carried out in an airlift bioreactor;
the screening culture medium in the step 3) takes an S1 culture medium as a basic culture medium, and also comprises 2-2.5 g/L gelrite;
the S1 culture medium comprises the following components in mass concentration: NH (NH)4Cl 401.175mg/L、KNO3 3791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 660mg/L、FeNa-EDTA 37.3mg/L、MnSO4·4H2O 22.3mg/L、KI 0.83mg/L、CuSO4·5H2O 0.05mg/L、CoCl2·6H2O 0.025mg/L、Na2MoO4·2H2O 0.25mg/L、ZnSO4·7H2O 8.6mg/L、H3BO36.2mg/L, 100mg/L inositol, 0.375mg/L nicotinic acid, 60.375mg/L vitamin B, 10.075mg/L vitamin B, 1.5mg/L, IBA 5mg/L glycine and 50000mg/L white sugar.
Preferably, the wild ginseng root in the step 1) is disinfected; the disinfection is carried out by adopting the following method: soaking the wild ginseng root in an ethanol water solution for 25-35 s, and then soaking in a sodium hypochlorite water solution for 35-45 min for surface disinfection;
the volume percentage content of the ethanol in the ethanol water solution is 70-75 percent;
the mass concentration of sodium hypochlorite in the sodium hypochlorite aqueous solution is 4%.
Preferably, the induction medium in step 1) takes an MS medium as a basal medium, and further comprises the following components by mass: 28-32 g/L of white sugar, 2, 4D 1-2 mg/L and gelrite 2-2.5 g/L; the pH value of the induction culture medium is 5.6-6.0.
Preferably, the time for the induction culture in the step 1) is 6-8 weeks; the temperature of the induction culture is 21-23 ℃.
Preferably, the rooting medium in step 2) takes an MS culture medium as a basic culture medium, and further comprises the following components in mass concentration: 28-32 g/L, IBA 3-4 mg/L of white sugar and 2-2.5 g/L of gelrite; the pH value of the rooting culture medium is 5.6-6.0.
Preferably, the proliferation medium in step 4) comprises S1 medium.
Preferably, the density of the adventitious root system in the step 4) is 5-7 g/L.
Preferably, the air injection amount in the airlift bioreactor in the step 4) is 0.05 vvm.
The invention has the beneficial effects that: the invention provides a rapid propagation method of mountain ginseng adventitious roots, which comprises the steps of taking wild ginseng roots as explants, obtaining callus through induction culture of the explants, obtaining the adventitious roots through rooting culture of the callus, screening the adventitious roots through a specific screening culture medium to obtain high-yield adventitious roots (the number of the adventitious roots reaches 36-46 per explant, and the induced adventitious roots are uniform, symmetrical and milky), and obtaining the mountain ginseng adventitious roots for harvesting after the high-yield adventitious roots are cultured in a proliferation culture medium for proliferation for 40-50 days in an airlift bioreactor. The dry weight of the mountain ginseng adventitious roots harvested by the method is as high as 17.31g/L, and the biomass is 20-30 times of that of the mountain ginseng adventitious roots in inoculation. The method can obviously improve the quantity and biomass of the adventitious roots obtained by induction, and the obtained mountain ginseng adventitious roots have uniform growth, stable heredity and no pollution and have the same nutrient components and nutrient values as those of cultivated ginseng. In addition, the method has the advantages of simple process, low cost, good repeatability and short period, is not influenced by external environment, and provides a theoretical basis for further regulating secondary metabolites and enlarging production.
Detailed Description
The invention provides a method for rapidly propagating mountain ginseng adventitious roots, which comprises the following steps:
1) taking wild ginseng roots as explants, inoculating the explants into an induction culture medium, and carrying out induction culture to obtain callus;
2) inoculating the callus into a rooting culture medium, and carrying out rooting culture for 6-8 weeks to obtain adventitious roots;
3) inoculating the adventitious roots into a screening culture medium, carrying out subculture, and taking the adventitious roots with the adventitious root induction number of 36-46 as adventitious root systems;
4) inoculating the adventitious root system into a proliferation culture medium, and performing proliferation culture for 40-50 days to obtain mountain ginseng adventitious roots for harvesting; the enrichment culture is carried out in an airlift bioreactor;
the screening culture medium in the step 3) takes an S1 culture medium as a basic culture medium, and also comprises 2-2.5 g/L gelrite;
the S1 culture medium comprises the following components in mass concentration: NH (NH)4Cl 401.175mg/L、KNO3 3791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 660mg/L、FeNa-EDTA 37.3mg/L、MnSO4·4H2O 22.3mg/L、KI 0.83mg/L、CuSO4·5H2O 0.05mg/L、CoCl2·6H2O 0.025mg/L、Na2MoO4·2H2O 0.25mg/L、ZnSO4·7H2O 8.6mg/L、H3BO36.2mg/L, 100mg/L inositol, 0.375mg/L nicotinic acid, 60.375mg/L vitamin B, 10.075mg/L vitamin B, 1.5mg/L, IBA 5mg/L glycine and 50000mg/L white sugar.
The method comprises the steps of taking 100-year Jilin Shaoshan root which is comforted with pine as an explant, inoculating the explant into an induction culture medium, and carrying out induction culture to obtain the callus.
In the invention, the wild ginseng root is preferably a fresh wild ginseng main root; the wild ginseng root is preferably subjected to disinfection treatment; the disinfection is preferably carried out by the following method: soaking the wild ginseng root in an ethanol water solution for 25-35 s, and then soaking in a sodium hypochlorite water solution for 35-45 min for surface disinfection; the soaking time is preferably 30 s; the time for disinfecting the surface is preferably 40 min; soaking in the ethanol water solution, and then cleaning with sterile water for 1-2 times; cleaning the surface with sterile water for 1 time preferably every 10min in the surface disinfection process; after the surface disinfection treatment, preferably, the method further comprises the step of cleaning the wild ginseng root after the surface disinfection by using sterile water; the number of times of cleaning is preferably 4-5 times.
In order to improve the disinfection effect, the disinfectant which is easy to wash away by sterile water or can be decomposed automatically, has little damage to materials and is harmless to human bodies and other organisms is selected, and a disinfection method combining alcohol and sodium hypochlorite is adopted in the specific implementation process of the invention. Typically, plant material is first subjected to removal of soil or dead tissue prior to sterilization and then rinsed with water to remove external contaminants. In the first step, alcohol is used for soaking for 30s to remove air on the material, and simultaneously, the strong wetting effect is realized, so that the disinfectant can be favorably infiltrated. And in the second step, sodium hypochlorite aqueous solution is utilized to disinfect the surface, so that the disinfectant has strong disinfection capability, is not easy to remain and is harmless to the environment. Sterile water shaking cleaning and disinfectant oscillation disinfection are carried out in the disinfection process.
In the invention, the volume percentage content of ethanol in the ethanol aqueous solution is preferably 70-75%, and more preferably 72%; the mass concentration of sodium hypochlorite in the sodium hypochlorite aqueous solution is preferably 4%.
In the invention, the wild ginseng root is preferably a wild ginseng root slice; the size (section area) of the wild ginseng root slice is preferably 0.25-0.5 cm2。
In the invention, the induction culture medium preferably takes an MS culture medium as a basic culture medium, and further comprises the following components in mass concentration: 28-32 g/L of white sugar, 2, 4D 1-2 mg/L and gelrite 2-2.5 g/L, and more preferably, the induction culture medium preferably takes an MS culture medium as a basal culture medium, and further comprises the following components in mass concentration: white sugar 30g/L, 2, 4D 1.5mg/L and gelrite2.3g/L; the pH value of the induction culture medium is preferably 5.6-6.0, and more preferably 5.8.
In the present invention, the MS medium comprises the following components in mass concentration:
NH4NO3 1650mg/L、KNO3 1900mg/L、MgSO4·7H2O 370mg/L、KH2PO4 170mg/L、CaCl2·2H2O 440mg/L、FeNa-EDTA 37.3mg/L、MnSO4·4H2O 22.3mg/L、KI 0.83mg/L、CuSO4·5H2O 0.025mg/L、CoCl2·6H2O 0.025mg/L、Na2MoO4·2H2O 0.25mg/L、ZnSO4·7H2O 8.6mg/L、H3BO36.2mg/L, inositol 100mg/L, nicotinic acid 0.5mg/L, vitamin B60.5 mg/L, vitamin B10.1mg/L and glycine 2.0 mg/L.
The coagulator adopted in the culture medium is gelrite, the gelrite has high purity and small using amount, and the gelled culture medium has high transparency and is beneficial to culture body observation.
In the invention, the temperature of the induction culture is preferably 21-23 ℃; the time for induction culture is preferably 6-8 weeks.
After the callus is obtained, the callus is inoculated into a rooting culture medium for rooting culture to obtain adventitious roots.
In the invention, the rooting culture medium preferably takes an MS culture medium as a basic culture medium, and also comprises the following components in mass concentration: 28-32 g/L, IBA 3-4 mg/L of white sugar and 2-2.5 g/L of gelrite, and more preferably, the rooting medium preferably takes an MS culture medium as a basic culture medium and further comprises the following components in mass concentration: white sugar 30g/L, IBA 3.5.5 mg/L and gelrite2.3g/L; the pH value of the rooting medium is preferably 5.6-6.0, and more preferably 5.8.
In the invention, the rooting culture temperature is preferably 21-23 ℃; the rooting culture time is preferably 6-8 weeks; adventitious roots differentiated from the induced callus.
The adventitious roots obtained by callus differentiation are uniform and stable in growth and high in biological yield, and variation phenomenon rarely occurs in long-term subculture.
After the adventitious roots are obtained, the adventitious roots are inoculated into a screening culture medium for subculture, and the adventitious roots with the adventitious root induction number of 36-46 are taken as adventitious root systems. The adventitious roots hardly form callus during expansion and differentiation, which is beneficial to bioreactor culture, otherwise the adventitious roots are easy to sink to the bottom in the culture process, and the growth is influenced.
In the invention, the screening culture medium takes an S1 culture medium as a basic culture medium and also comprises 2-2.5 g/L gelrite; the pH value of the screening culture medium is preferably 5.6-6.0, and more preferably 5.8.
In the present invention, the IBA acts to enhance the induction of rooting rate.
In the invention, the S1 culture medium comprises the following components in mass concentration: NH (NH)4Cl 401.175mg/L、KNO33791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 660mg/L、FeNa-EDTA 37.3mg/L、MnSO4·4H2O 22.3mg/L、KI 0.83mg/L、CuSO4·5H2O 0.05mg/L、CoCl2·6H2O 0.025mg/L、Na2MoO4·2H2O 0.25mg/L、ZnSO4·7H2O 8.6mg/L、H3BO36.2mg/L, 100mg/L inositol, 0.375mg/L nicotinic acid, 60.375mg/L vitamin B, 10.075mg/L vitamin B, 1.5mg/L, IBA 5mg/L glycine and 50000mg/L white sugar.
The screening culture medium adopts a chelated iron reagent, namely the sodium ferric ethylenediamine tetraacetate, so that the effective absorption of the mountain ginseng adventitious roots to iron elements can be ensured, and the phenomenon of iron deficiency can be prevented.
The screening medium of the present invention includes inositol which functions to provide organic nutrition, contribute to the effect of vitamin B1, and promote adventitious root formation and growth.
In the invention, the time of subculture is preferably 60-70 days; the temperature of the subculture is preferably 21-23 ℃.
After the adventitious root system is obtained, the adventitious root system is inoculated into a proliferation culture medium for proliferation culture for 40-50 days to obtain the mountain ginseng adventitious root for harvesting; the propagation culture is carried out in an airlift bioreactor, preferably an airlift glass bioreactor.
In the present invention, the proliferation medium preferably includes S1 medium.
The mass concentration of macroelements in the multiplication medium is 3/4 (NH) of MS medium4 +：NO3 -Is 7.5: 37.5; CaCl2.2H2The O concentration is 4.5mmol/L, and the mass concentration of the trace elements is 1 time of that of the MS culture medium (CuSO)4.5H2O is 0.2 mu mol/L), the mass concentration of the organic substances is 3/4 (inositol 100mg/L) of MS culture medium, IBA is 5mg/L and white sugar is 50g/L, which is beneficial to the proliferation of the adventitious root system of the mountain ginseng. In the invention, the white sugar is adopted, which is beneficial to saving the cost.
In the invention, the density of the access of the adventitious root system is preferably 5-7 g/L, and more preferably 6 g/L; the preferred amount of air injected in the airlift bioreactor is 0.05 vvm.
In the invention, the airlift bioreactor realizes gas and substance exchange by passing gas through a liquid pool from the bottom through a nozzle or a hole disc; the airlift reactor has the advantages of simple structure, small shearing force, good mass transfer effect, low operation cost and cost, and no stirring device. The amount of air injected during the reactor culture is a very important parameter. And a proper amount of air is introduced into the reactor, so that the culture and the culture solution can be fully mixed, the nutrient can be fully absorbed by the culture, and necessary dissolved oxygen is provided for the growth of plants and the accumulation of metabolites.
In the present invention, the time for the propagation culture is preferably 7 weeks; the temperature of the proliferation culture is preferably 21-23 ℃.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
1. In the method for proliferating the adventitious roots of the wild ginseng, the culture medium concentrations of a solid culture system in a culture dish and a triangular flask suspension culture system are compared when the wild ginseng is proliferated under laboratory conditions:
in order to screen high-yield adventitious roots, 8 roots with a length of 1.5-2 cm and an adventitious root with a fresh weight of 0.42g are transferred to a culture dish with a diameter of 90mm containing 25-30 mL of a solid medium (S2 medium, S3 medium, S4 medium, S5 medium) and a 250mL triangular flask containing 70mL of a liquid medium (S2 medium, S3 medium, S4 medium, S5 medium) for dark culture, wherein 2.3g/Lgelrite is additionally added to the solid medium. After solid culture for 60 days, investigating the number, length, fresh weight and dry weight of differentiated lateral roots; carrying out suspension culture on a shaking table with the rotating speed of 110rpm for 40 days, and then investigating the fresh weight and the dry weight; the results are shown in Table 1;
the culture media are respectively:
the S2 culture medium is prepared by adding the following raw materials into distilled water: NH (NH)4NO3 825mg/L、KNO3950 mg/L、MgSO4·7H2O 185mg/L、KH2PO4 85mg/L、CaCl2·2H2O 220mg/L、FeNa-EDTA 18.65mg/L、MnSO4·4H2O 11.15mg/L、KI 0.415mg/L、CuSO4·5H2O 0.0125mg/L、CoCl2·6H2O 0.0125mg/L、Na2MoO4·2H2O 0.125mg/L、ZnSO4·7H2O 4.3mg/L、H3BO33.1mg/L, 50mg/L inositol, 0.25mg/L nicotinic acid, 60.25mg/L vitamin B, 10.05mg/L vitamin B, 1.0mg/L, IBA 5mg/L glycine and 50000mg/L white sugar;
the S3 culture medium is prepared by adding the following raw materials into distilled water: NH (NH)4NO3 1237.5mg/L、KNO3 1425mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 330mg/L、FeNa-EDTA 27.975mg/L、MnSO4·4H2O 16.725mg/L、KI 0.6225mg/L、CuSO4·5H2O 0.01875mg/L、CoCl2·6H2O 0.01875mg/L、Na2MoO4·2H2O 0.1875mg/L、ZnSO4·7H2O 6.45mg/L、H3BO34.65mg/L, inositol 75mg/L, nicotinic acid 0.375mg/L, vitamin B60.375mg/L, vitamin B10.075mg/L, glycine 1.5mg/L, IBA 5mg/L and white sugar 50000 mg/L;
s4 Medium consisted ofThe raw materials are added into distilled water to prepare: NH (NH)4NO3 1650mg/L、KNO3 1900mg/L、MgSO4·7H2O 370mg/L、KH2PO4 170mg/L、CaCl2·2H2O 440mg/L、FeNa-EDTA 37.3mg/L、MnSO4·4H2O 22.3mg/L、KI 0.83mg/L、CuSO4·5H2O 0.025mg/L、CoCl2·6H2O 0.025mg/L、Na2MoO4·2H2O 0.25mg/L、ZnSO4·7H2O 8.6mg/L、H3BO36.2mg/L, 100mg/L inositol, 0.5mg/L nicotinic acid, 60.5 mg/L vitamin B, 10.1mg/L vitamin B, 2.0mg/L, IBA 5mg/L glycine and 50000mg/L white sugar.
The S5 culture medium is prepared by adding the following raw materials into distilled water: NH (NH)4NO3 2475mg/L、KNO3 2850mg/L、MgSO4·7H2O 555mg/L、KH2PO4 255mg/L、CaCl2·2H2O 660mg/L、FeNa-EDTA 55.95mg/L、MnSO4·4H2O 33.45mg/L、KI 1.245mg/L、CuSO4·5H2O 0.0375mg/L、CoCl2·6H2O 0.0375mg/L、Na2MoO4·2H2O 0.375mg/L、ZnSO4·7H2O 12.9mg/L、H3BO39.3mg/L, inositol 150mg/L, nicotinic acid 0.75mg/L, vitamin B60.75mg/L, vitamin B10.15mg/L, glycine 3.0mg/L, IBA 5mg/L and white sugar 50000 mg/L.
TABLE 1 Effect of Medium concentration on adventitious root growth under solid and suspension culture conditions
Note: the letters in the tables indicate significance of difference at a p <0.05 level, as in the tables below.
As is apparent from Table 1, the ginseng cultured in the S3 medium had the highest indefinite number, length and biomass in the solid culture. The ginseng cultured by the S2 culture medium with low culture medium concentration or the S5 culture medium with high culture medium concentration has the indefinite number and the length which are obviously lower than those of the S3 culture medium. The same results were also obtained when the cells were cultured in a 250mL Erlenmeyer flask in suspension. The biomass was high when the culture was carried out using the S3 medium and the S4 medium. The S3 culture medium is beneficial to obtain high yield of adventitious roots and proliferation of adventitious roots because the plants cannot normally absorb nutrients and water due to high concentration of inorganic salts.
2. In the method for proliferating the adventitious roots of the wild ginseng, NH in a culture medium of S3 in the scheme is adopted in solid culture in a culture dish during propagation under laboratory conditions and in suspension culture in an Erlenmeyer flask4 +With NO3 -Comparison of molar ratio of (a):
the total nitrogen molar concentration in the culture medium is set to 45mmol/L, and NH is used4Cl and KNO3Adjusting NH in culture Medium4 +With NO3 -The molar concentration ratio of (A) to (B) is 7.5: 37.5, 15: 30. 22.5: 22.5, concentrations of other materials were the same as in S3 medium (except NH) described in the above protocol4 +With NO3 -) In the same concentration, i.e. MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 330mg/L、FeNa-EDTA 27.975mg/L、MnSO4·4H2O 16.725mg/L、KI 0.6225mg/L、CuSO4·5H2O 0.01875mg/L、CoCl2·6H2O 0.01875mg/L、Na2MoO4·2H2O 0.1875mg/L、ZnSO4·7H2O 6.45mg/L、H3BO34.65mg/L, inositol 75mg/L, nicotinic acid 0.375mg/L, vitamin B60.375mg/L, vitamin B10.075mg/L, glycine 1.5mg/L, IBA 5mg/L and white sugar 50000 mg/L; 5 different NH's are obtained4 +With NO3 -The molar concentration ratio of the culture medium is as follows:
the S6 culture medium is prepared by adding the following raw materials into distilled water: NH (NH)4Cl 401.175mg/L、KNO3 3791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 330mg/L、FeNa-EDTA 27.975mg/L、MnSO4·4H2O 16.725mg/L、KI 0.6225mg/L、CuSO4·5H2O 0.01875mg/L、CoCl2·6H2O 0.01875mg/L、Na2MoO4·2H2O 0.1875mg/L、ZnSO4·7H2O 6.45mg/L、H3BO34.65mg/L, inositol 75mg/L, nicotinic acid 0.375mg/L, vitamin B60.375mg/L, vitamin B10.075mg/L, glycine 1.5mg/L, IBA 5mg/L and white sugar 50000 mg/L; NH in the S6 medium4 +With NO3 -In a molar ratio of 7.5: 37.5;
the S7 culture medium is prepared by adding the following raw materials into distilled water: NH (NH)4Cl 802.35mg/L、KNO3 3033mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 330mg/L、FeNa-EDTA 27.975mg/L、MnSO4·4H2O 16.725mg/L、KI 0.6225mg/L、CuSO4·5H2O 0.01875mg/L、CoCl2·6H2O 0.01875mg/L、Na2MoO4·2H2O 0.1875mg/L、ZnSO4·7H2O 6.45mg/L、H3BO34.65mg/L, inositol 75mg/L, nicotinic acid 0.375mg/L, vitamin B60.375mg/L, vitamin B10.075mg/L, glycine 1.5mg/L, IBA 5mg/L and white sugar 50000 mg/L; NH in the S7 medium4 +With NO3 -The molar concentration ratio of (a) to (b) is 15: 30, of a nitrogen-containing gas;
the S8 culture medium is prepared by adding the following raw materials into distilled water: NH (NH)4Cl 1203.525mg/L、KNO32274.75mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 330mg/L、FeNa-EDTA 27.975mg/L、MnSO4·4H2O 16.725mg/L、KI 0.6225mg/L、CuSO4·5H2O 0.01875mg/L、CoCl2·6H2O 0.01875mg/L、Na2MoO4·2H2O 0.1875mg/L、ZnSO4·7H2O 6.45mg/L、H3BO34.65mg/L, inositol 75mg/L, nicotinic acid 0.375mg/L, vitamin B60.375mg/L, vitamin B10.075mg/L, glycine 1.5mg/L, IBA 5mg/L and white sugar 50000 mg/L; NH in the S8 medium4 +With NO3 -In a molar ratio of 22.5: 22.5;
in order to screen high-yield adventitious roots, 8 roots 1.5-2 cm long and an adventitious root 0.42g in fresh weight were transferred to a 90 mm-diameter petri dish containing 25-30 mL of a solid medium (S6 medium, S7 medium, and S8 medium) and a 250mL triangular flask containing 70mL of a liquid medium (S6 medium, S7 medium, and S8 medium), respectively, and cultured in the dark. The solid medium needs to be supplemented with 2.3 g/Lgelrite. After solid culture for 60 days, investigating the number, length, fresh weight and dry weight of differentiated lateral roots; carrying out suspension culture on a shaking table with the rotating speed of 110rpm for 40 days, and then investigating the fresh weight and the dry weight; the results are shown in table 2:
TABLE 2 NH in solid and suspension culture conditions4 +With NO3 -Influence of molar concentration ratio on growth of adventitious roots
As can be seen from Table 2, NH4 +With NO3 -The influence of different molar concentrations on the growth of adventitious roots is different, when an S6 solid culture medium is adopted, the growth of the adventitious roots is the best, the number and the biomass of the adventitious roots of the ginseng are both obviously higher than those of the ginseng subjected to other treatments, and the number of the adventitious roots is obviously reduced along with the increase of the proportion of ammonium nitrogen; the lateral roots formed by adopting the S8 solid culture medium hardly grow, which shows that the high-concentration nitrate nitrogen is suitable for the growth of the adventitious roots, and the ammonium nitrogen content is too high to inhibit the growth of the adventitious roots. The same results were also obtained when the cells were cultured in a 250mL Erlenmeyer flask in suspension. Biomass of adventitious roots in NH4 +：NO3 -Is 7.5: 37.5(S6 medium), the biomass decreased as the proportion of ammonium nitrogen increased. Therefore, when the total nitrogen molar concentration is 45mmol/L, it is usedMiddle NH4 +With NO3 -In a molar ratio of 7.5: the S6 culture medium of 37.5 is favorable for obtaining high-yield adventitious root system and adventitious root proliferation.
3. In the method for proliferating the adventitious roots of wild ginseng, the calcium ion concentrations in the culture medium of S6 in the above scheme are compared in the solid culture in a culture dish and the suspension culture in an Erlenmeyer flask under the laboratory conditions:
in order to screen high-yield adventitious roots, 8 roots 1.5-2 cm long and 0.42g fresh adventitious roots are transferred to a 90 mm-diameter petri dish containing 25-30 mL of a solid medium (S6 medium, S9 medium, S10 medium and S11 medium) and a 250mL Erlenmeyer flask containing 70mL of a liquid medium (S6 medium, S9 medium, S10 medium and S11 medium) to be dark-cultured, and the solid medium needs to be additionally added with 2.3 g/Lgelrite. After solid culture for 60 days, investigating the number, length, fresh weight and dry weight of differentiated lateral roots; carrying out suspension culture on a shaking table with the rotating speed of 110rpm for 40 days, and then investigating the fresh weight and the dry weight; the results are shown in Table 3;
the culture media are respectively:
the S9 culture medium is prepared by adding the following raw materials into distilled water: NH (NH)4Cl 401.175mg/L、KNO3 3791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 220mg/L、FeNa-EDTA 27.975mg/L、MnSO4·4H2O 16.725mg/L、KI 0.6225mg/L、CuSO4·5H2O 0.01875mg/L、CoCl2·6H2O 0.01875mg/L、Na2MoO4·2H2O 0.1875mg/L、ZnSO4·7H2O 6.45mg/L、H3BO34.65mg/L, inositol 75mg/L, nicotinic acid 0.375mg/L, vitamin B60.375mg/L, vitamin B10.075mg/L, glycine 1.5mg/L, IBA 5mg/L and white sugar 50000 mg/L; the concentration of calcium ions in the S9 culture medium is 1.5 mmol/L;
the S6 culture medium is prepared by adding the following raw materials into distilled water, as with the S6 culture medium described in the above scheme: NH (NH)4Cl 401.175mg/L、KNO3 3791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 330mg/L、FeNa-EDTA 27.975mg/L、MnSO4·4H2O 16.725mg/L、KI 0.6225mg/L、CuSO4·5H2O 0.01875mg/L、CoCl2·6H2O 0.01875mg/L、Na2MoO4·2H2O 0.1875mg/L、ZnSO4·7H2O 6.45mg/L、H3BO34.65mg/L, inositol 75mg/L, nicotinic acid 0.375mg/L, vitamin B60.375mg/L, vitamin B10.075mg/L, glycine 1.5mg/L, IBA 5mg/L and white sugar 50000 mg/L; the concentration of calcium ions in the S6 culture medium is 2.25 mmol/L;
the S10 culture medium is prepared by adding the following raw materials into distilled water: NH (NH)4Cl 401.175mg/L、KNO33791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 440mg/L、FeNa-EDTA 27.975mg/L、MnSO4·4H2O 16.725mg/L、KI 0.6225mg/L、CuSO4·5H2O 0.01875mg/L、CoCl2·6H2O 0.01875mg/L、Na2MoO4·2H2O 0.1875mg/L、ZnSO4·7H2O 6.45mg/L、H3BO34.65mg/L, inositol 75mg/L, nicotinic acid 0.375mg/L, vitamin B60.375mg/L, vitamin B10.075mg/L, glycine 1.5mg/L, IBA 5mg/L and white sugar 50000 mg/L; the concentration of calcium ions in the S10 culture medium is 3.0 mmol/L;
the S11 culture medium is prepared by adding the following raw materials into distilled water: NH (NH)4Cl 401.175mg/L、KNO33791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 660mg/L、FeNa-EDTA 27.975mg/L、MnSO4·4H2O 16.725mg/L、KI 0.6225mg/L、CuSO4·5H2O 0.01875mg/L、CoCl2·6H2O 0.01875mg/L、Na2MoO4·2H2O 0.1875mg/L、ZnSO4·7H2O 6.45mg/L、H3BO34.65mg/L, inositol 75mg/L, nicotinic acid 0.375mg/L, vitamin B60.375mg/L, vitamin B10.075mg/L, glycine 1.5mg/L, IBA 5mg/L and white sugar 50000 mg/L; the concentration of calcium ions in the S11 culture medium is 4.5 mmol/L;
TABLE 3 Effect of calcium ion concentration on adventitious root growth under solid and suspension culture conditions
As shown in Table 3, the number of ginseng adventitious roots depends on Ca in the medium2+The concentration is increased, and the biomass of the adventitious roots is obviously higher when the S11 solid culture medium is adopted than other treatments. The same results were also obtained when the cells were cultured in a 250mL Erlenmeyer flask in suspension. Calcium ion is an essential nutrient element for promoting growth and biomass accumulation, and can improve the absorption of mineral elements. In conclusion, Ca2+The S11 culture medium with the concentration of 4.5mmol/L is favorable for obtaining high-yield adventitious root system and adventitious root proliferation.
4. In the method for proliferating the adventitious roots of the wild ginseng, the concentrations of trace elements in a culture medium of S11 in the scheme are compared during solid culture in a culture dish and triangular flask suspension culture during propagation under laboratory conditions;
adding trace elements (Mn) in S11 culture medium2+、Zn2+、Co2+、Cu2+、Fe2+、I-、BO3 3-、MoO4 2-) The concentrations were set to 0.25, 0.5, 0.75 and 1 times the concentration of MS medium, respectively, and the concentrations of other raw materials were the same as those of S11 medium (except for Mn) described in the above protocol2+、Zn2+、Co2+、Cu2+、Fe2+、I-、BO3 3-、MoO4 2-) Is the same concentration, i.e. NH4Cl 401.175mg/L、KNO33791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O660 mg/L, inositol 75mg/L, nicotinic acid 0.375mg/L, vitamin B60.375mg/L, vitamin B10.075mg/L, glycine 1.5mg/L, IBA 5mg/L and white sugar 50000 mg/L; obtaining 5 different microelementsThe culture medium with the element concentration is respectively as follows:
the S12 culture medium is prepared by adding the following raw materials into distilled water: NH (NH)4Cl 401.175mg/L、KNO33791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO41 27.5mg/L、CaCl2·2H2O 660mg/L、FeNa-EDTA 9.325mg/L、MnSO4·4H2O 5.575mg/L、KI 0.2075mg/L、CuSO4·5H2O 0.00625mg/L、CoCl2·6H2O 0.00625mg/L、Na2MoO4·2H2O 0.0625mg/L、ZnSO4·7H2O 2.15mg/L、H3BO31.55mg/L, inositol 75mg/L, nicotinic acid 0.375mg/L, vitamin B60.375mg/L, vitamin B10.075mg/L, glycine 1.5mg/L, IBA 5mg/L and white sugar 50000 mg/L; the concentration of the trace elements in the S12 culture medium is 0.25 time of that of the MS culture medium;
the S13 culture medium is prepared by adding the following raw materials into distilled water: NH (NH)4Cl 401.175mg/L、KNO33791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 660mg/L、FeNa-EDTA 18.65mg/L、MnSO4·4H2O 11.15mg/L、KI 0.415mg/L、CuSO4·5H2O 0.0125mg/L、CoCl2·6H2O 0.0125mg/L、Na2MoO4·2H2O 0.125mg/L、ZnSO4·7H2O 4.3mg/L、H3BO33.1mg/L, inositol 75mg/L, nicotinic acid 0.375mg/L, vitamin B60.375mg/L, vitamin B10.075mg/L, glycine 1.5mg/L, IBA 5mg/L and white sugar 50000 mg/L; the concentration of the trace elements in the S13 culture medium is 0.5 time of that of the MS culture medium;
the S11 culture medium is prepared by adding the following raw materials into distilled water, as with the S11 culture medium described in the above scheme: NH (NH)4Cl 401.175mg/L、KNO3 3791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 660mg/L、FeNa-EDTA 27.975mg/L、MnSO4·4H2O 16.725mg/L、KI 0.6225mg/L、CuSO4·5H2O 0.01875mg/L、CoCl2·6H2O 0.01875mg/L、Na2MoO4·2H2O 0.1875mg/L、ZnSO4·7H2O 6.45mg/L、H3BO34.65mg/L, inositol 75mg/L, nicotinic acid 0.375mg/L, vitamin B60.375mg/L, vitamin B10.075mg/L, glycine 1.5mg/L, IBA 5mg/L and white sugar 50000 mg/L; the concentration of the trace elements in the S11 culture medium is 0.75 time of that of the MS culture medium;
the S14 culture medium is prepared by adding the following raw materials into distilled water: NH (NH)4Cl 401.175mg/L、KNO33791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 660mg/L、FeNa-EDTA 37.3mg/L、MnSO4·4H2O 22.3mg/L、KI 0.83mg/L、CuSO4·5H2O 0.025mg/L、CoCl2·6H2O 0.025mg/L、Na2MoO4·2H2O 0.25mg/L、ZnSO4·7H2O 8.6mg/L、H3BO36.2mg/L, inositol 75mg/L, nicotinic acid 0.375mg/L, vitamin B60.375mg/L, vitamin B10.075mg/L, glycine 1.5mg/L, IBA 5mg/L and white sugar 50000 mg/L; the concentration of the trace elements in the S14 culture medium is 1 time of that of the MS culture medium;
in order to screen high-yield adventitious roots, 8 roots 1.5-2 cm long and an adventitious root 0.42g in fresh weight were transferred to a 90 mm-diameter petri dish containing 25-30 mL of a solid medium (S11 medium, S12 medium, S13 medium, and S14 medium) and a 250mL Erlenmeyer flask containing 70mL of a liquid medium (S11 medium, S12 medium, S13 medium, and S14 medium), respectively, and cultured in dark. The solid medium needs to be supplemented with 2.3 g/Lgelrite. After solid culture for 60 days, investigating the number, length, fresh weight and dry weight of differentiated lateral roots; carrying out suspension culture on a shaking table with the rotating speed of 110rpm for 40 days, and then investigating the fresh weight and the dry weight; the results are shown in Table 4:
TABLE 4 Effect of trace element concentration on adventitious root growth under solid and suspension culture conditions
As is clear from Table 4, the ginseng obtained by the solid culture using the S14 medium had the highest indefinite number, length and biomass. The number of adventitious roots cultured by the S12 culture medium is obviously lower than that of other treatments, and the length of the adventitious roots obtained by the S14 culture medium is obviously higher than that of other treatments, which indicates that the low concentration of trace elements is not beneficial to the growth of the adventitious roots. When the culture was carried out in a suspension culture in a 250mL Erlenmeyer flask, the biomass was the highest when the S11 medium was used, but the biomass was not significantly different from that in the S14 medium. Therefore, the S14 medium with the concentration of trace elements 1 time that of the MS medium is beneficial to obtain high-yield adventitious root system and adventitious root proliferation.
5. In the method for proliferating the adventitious roots of the wild ginseng, the concentration of copper ions in a culture medium of S14 in the scheme is compared when solid culture in a culture dish and suspension culture in a triangular flask are carried out under laboratory conditions;
in order to screen high-yield adventitious roots, 8 roots 1.5-2 cm long and 0.42g fresh adventitious roots are transferred to a 90 mm-diameter petri dish containing 25-30 mL of a solid medium (S14 medium, S15 medium, S16 medium and S17 medium) and a 250mL Erlenmeyer flask containing 70mL of a liquid medium (S14 medium, S15 medium, S16 medium and S17 medium) to be dark-cultured, and the solid medium needs to be additionally added with 2.3 g/Lgelrite. After solid culture for 60 days, investigating the number, length, fresh weight and dry weight of differentiated lateral roots; carrying out suspension culture on a shaking table with the rotating speed of 110rpm for 40 days, and then investigating the fresh weight and the dry weight; the results are shown in Table 5;
the culture media are respectively:
the S15 culture medium is prepared by adding the following raw materials into distilled water: NH (NH)4Cl 401.175mg/L、KNO33791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 660mg/L、FeNa-EDTA 37.3mg/L、MnSO4·4H2O 22.3mg/L、KI 0.83mg/L、CuSO4·5H2O 0.01875mg/L、CoCl2·6H2O 0.025mg/L、Na2MoO4·2H2O 0.25mg/L、ZnSO4·7H2O 8.6mg/L、H3BO36.2mg/L, inositol 75mg/L, nicotinic acid 0.375mg/L, vitamin B60.375mg/L, vitamin B10.075mg/L, glycine 1.5mg/L, IBA 5mg/L and white sugar 50000 mg/L; cu in the S15 culture medium2+The concentration is 0.075 mu mol/L;
the S14 culture medium is prepared by adding the following raw materials into distilled water, as with the S14 culture medium described in the above scheme: NH (NH)4Cl 401.175mg/L、KNO3 3791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 660mg/L、FeNa-EDTA 37.3mg/L、MnSO4·4H2O 22.3mg/L、KI 0.83mg/L、CuSO4·5H2O 0.025mg/L、CoCl2·6H2O 0.025mg/L、Na2MoO4·2H2O 0.25mg/L、ZnSO4·7H2O 8.6mg/L、H3BO36.2mg/L, inositol 75mg/L, nicotinic acid 0.375mg/L, vitamin B60.375mg/L, vitamin B10.075mg/L, glycine 1.5mg/L, IBA 5mg/L and white sugar 50000 mg/L; cu in the S14 culture medium2+The concentration is 0.1 mu mol/L;
the S16 culture medium is prepared by adding the following raw materials into distilled water: NH (NH)4Cl 401.175mg/L、KNO33791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 660mg/L、FeNa-EDTA 37.3mg/L、MnSO4·4H2O 22.3mg/L、KI 0.83mg/L、CuSO4·5H2O 0.05mg/L、CoCl2·6H2O 0.025mg/L、Na2MoO4·2H2O 0.25mg/L、ZnSO4·7H2O8.6mg/L、H3BO36.2mg/L, inositol 75mg/L, nicotinic acid 0.375mg/L, vitamin B60.375mg/L, vitamin B10.075mg/L, glycine 1.5mg/L, IBA 5mg/L and white sugar 50000 mg/L; cu in the S16 culture medium2+The concentration is 0.2 mu mol/L;
the S17 culture medium is prepared by adding the following raw materials into distilled water: NH (NH)4Cl 401.175mg/L、KNO33791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 660mg/L、FeNa-EDTA 37.3mg/L、MnSO4·4H2O 22.3mg/L、KI 0.83mg/L、CuSO4·5H2O 0.075mg/L、CoCl2·6H2O 0.025mg/L、Na2MoO4·2H2O 0.25mg/L、ZnSO4·7H2O8.6mg/L、H3BO36.2mg/L, inositol 75mg/L, nicotinic acid 0.375mg/L, vitamin B60.375mg/L, vitamin B10.075mg/L, glycine 1.5mg/L, IBA 5mg/L and white sugar 50000 mg/L; cu in the S17 culture medium2+The concentration is 0.3 mu mol/L;
TABLE 5 Cu in solid and suspension culture conditions2+Effect of concentration on adventitious root growth
As can be seen from Table 5, the indefinite number of roots, length and biomass did not differ significantly between treatments when solid-cultured with the S14 medium, the S16 medium and the S17 medium, but were significantly higher than Cu2+S15 medium at a concentration of 0.075. mu. mol/L. When the culture medium is suspended in a 250mL triangular flask, the culture medium is accompanied by Cu2+The concentration increases and then decreases, when Cu is added2+The highest biomass was obtained at a concentration of 0.2. mu. mol/L. Cu2+Mainly affects the respiration of cells and participates in the electron transfer in the oxidation process. Without addition of Cu2+Can affect synthesis of peroxidase and ascorbate oxidase to inhibit cell division, and excessive Cu is added2+Promote the synthesis of peroxidase and ascorbate oxidase, which promotes the synthesis of lignin and causes cell aging that affects cell proliferation. As described above, Cu2+The S16 culture medium with the concentration of 0.2 mu mol/L is favorable for obtaining high-yield adventitious root system and adventitious root proliferation.
6. In the method for proliferating the adventitious roots of the wild ginseng, the inositol concentration in the culture medium of S16 in the scheme is compared when solid culture in a culture dish and triangular flask suspension culture are carried out under laboratory conditions;
in order to screen high-yield adventitious roots, 8 roots of 1.5-2 cm in length and 0.42g of fresh adventitious roots are transferred to a 90mm petri dish containing 25-30 mL of solid medium (S16 medium, S1 medium and S18 medium) and a 250mL triangular flask containing 70mL of liquid medium (S16 medium, S1 medium and S18 medium) respectively for dark culture, and the solid medium needs to be additionally added with 2.3 g/Lgelrite. After solid culture for 60 days, investigating the number, length, fresh weight and dry weight of differentiated lateral roots; carrying out suspension culture on a shaking table with the rotating speed of 110rpm for 40 days, and then investigating the fresh weight and the dry weight; the results are shown in Table 6;
the culture media are respectively:
the S16 culture medium is prepared by adding the following raw materials into distilled water, as with the S16 culture medium described in the above scheme: NH (NH)4Cl 401.175mg/L、KNO3 3791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 660mg/L、FeNa-EDTA 37.3mg/L、MnSO4·4H2O 22.3mg/L、KI 0.83mg/L、CuSO4·5H2O 0.05mg/L、CoCl2·6H2O 0.025mg/L、Na2MoO4·2H2O 0.25mg/L、ZnSO4·7H2O 8.6mg/L、H3BO36.2mg/L, inositol 75mg/L, nicotinic acid 0.375mg/L, vitamin B60.375mg/L, vitamin B10.075mg/L, glycine 1.5mg/L, IBA 5mg/L and white sugar 50000 mg/L;
the S1 culture medium is prepared by adding the following raw materials into distilled water, as with the S1 culture medium described in the above scheme: NH (NH)4Cl 401.175mg/L、KNO3 3791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 660mg/L、FeNa-EDTA 37.3mg/L、MnSO4·4H2O 22.3mg/L、KI 0.83mg/L、CuSO4·5H2O 0.05mg/L、CoCl2·6H2O 0.025mg/L、Na2MoO4·2H2O 0.25mg/L、ZnSO4·7H2O 8.6mg/L、H3BO36.2mg/L inositol100mg/L, 0.375mg/L nicotinic acid, 60.375mg/L vitamin B, 10.075mg/L vitamin B, 1.5mg/L, IBA 5mg/L glycine and 50000mg/L white sugar;
the S18 culture medium is prepared by adding the following raw materials into distilled water: NH (NH)4Cl 401.175mg/L、KNO33791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 660mg/L、FeNa-EDTA 37.3mg/L、MnSO4·4H2O 22.3mg/L、KI 0.83mg/L、CuSO4·5H2O 0.05mg/L、CoCl2·6H2O 0.025mg/L、Na2MoO4·2H2O 0.25mg/L、ZnSO4·7H2O8.6mg/L、H3BO36.2mg/L, 150mg/L inositol, 0.375mg/L nicotinic acid, 60.375mg/L vitamin B, 10.075mg/L vitamin B, 1.5mg/L, IBA 5mg/L glycine and 50000mg/L white sugar;
TABLE 6 Effect of inositol concentration on adventitious root growth under solid and suspension culture conditions
As can be seen from Table 6, the culture medium S1, which contained inositol in the amount corresponding to the amount of the MS starting formula, was used for the solid culture, and the number of adventitious roots, length and biomass were the highest. When the content of inositol is 0.75 times of the original formula amount of MS, the number and length of the variable elements are not obviously different from the original formula amount; when the inositol content is 1.5 times of the original formula amount of MS, the variable number and length are significantly lower than the original formula amount. When cultured in suspension in 250mL flasks, biomass increased with increasing inositol concentration in the medium. Inositol is a kind of biotin and is an indispensable component in the organism. Compared with a solid culture mode, under the condition of suspension culture, the delivery and supply conditions of the nutrient elements by the liquid culture medium and the intake of the nutrient elements by the explants are obviously superior to those of the solid culture medium. Therefore, the S1 culture medium containing 1 time of inositol compared with the MS original formula is beneficial to obtain high yield of adventitious roots and proliferation of adventitious roots.
7. Effect of different inoculum densities of 3L airlift bioreactor reaction system when proliferating under laboratory conditions in method for adventitious root proliferation of wild ginseng:
respectively completely immersing adventitious roots with different inoculation densities (fresh weights of 4g/L, 6g/L, 8g/L and 10g/L respectively) in an airlift bioreactor with the capacity of 3L containing 2L of S1 liquid culture medium, injecting air with the supply amount of 0.1vvm, and investigating fresh weight, dry weight and growth rate after culturing for 40 days; the results are shown in Table 7; the S1 liquid medium is the same as the S1 medium described in the above protocol;
TABLE 73L Effect of different inoculum densities on adventitious root growth in airlift bioreactor propagation
| Index (I) | Fresh weight (g/L) | Dry weight (g/L) | Rate of increase |
| Inoculation density: 4g/L | 120.75b | 13.71c | 30.88 |
| Inoculation density: 6g/L | 124.45a | 14.94a | 22.34 |
| Inoculation density: 8g/L | 126.97a | 15.35a | 17.06 |
| Inoculation density: 10g/L | 121.89b | 14.28b | 12.47 |
As can be seen from Table 7, the biomass was significantly better treated than the other 2 inoculum densities when the inoculum densities were 6g/L and 8 g/L. At an inoculum size of 4g/L, the growth rate was highest, but the fresh and dry weights were significantly lower than the other treatments, delaying the growth to the limit. In the tissue culture process, proper inoculation density is one of the essential important factors for regulating organ formation and growth in bioreactor culture, and over high inoculation density can accelerate the culture to reach the growth limit, limit the utilization rate of nutrient components and oxygen, inhibit the normal growth of the culture and accelerate aging; and the inoculation density is too low, which wastes culture space and cost, prolongs culture time and is not favorable for the normal growth of the culture. Therefore, the volume of the liquid culture medium containing 2L S1 was 6.0g/L, which is an economical inoculation density suitable for the proliferation and growth of adventitious roots of ginseng in a 3L airlift bioreactor.
8. Influence of different air injection amounts of 3L airlift bioreactor reaction system in proliferation under laboratory conditions in the method for proliferation of adventitious roots of wild ginseng:
setting ventilation volumes as 0.05vvm, 0.1vvm, 0.15vvm, 0.2vvm and 0.05-0.1 vvm respectively (the ventilation volume is 0.05vvm in the first two weeks, and the ventilation volume is always 0.1vvm thereafter), completely immersing 6g/L (fresh weight) of adventitious roots in an airlift bioreactor containing 2L of S1 liquid culture medium and having a capacity of 3L, and examining the fresh weight, dry weight and growth rate after culturing for 40 days; the results are shown in Table 8; the S1 liquid medium is the same as the S1 medium described in the above protocol;
TABLE 83L Effect of different air injection amounts on adventitious root growth in air-lift bioreactor propagation
| Index (I) | Fresh weight (g/L) | Dry weight (g/L) | Rate of increase |
| Air injection amount: 0.05vvm | 124.56a | 14.95a | 22.73 |
| Air injection amount: 0.1vvm | 117.31b | 14.11b | 21.40 |
| Air injection amount: 0.15vvm | 106.67c | 13.56c | 20.53 |
| Air injection amount: 0.2vvm | 107.86c | 13.84c | 20.97 |
| Air injection amount: 0.05 to 0.1vvm | 125.66a | 15.41a | 23.46 |
As is clear from Table 8, when the air injection amounts were 0.05vvm and 0.05 to 0.1vvm, the effect of adventitious root growth was significantly better than that of the other 3 treatments. As ventilation continues to increase, plant growth is inhibited by hydrodynamic stress due to excessive dissolved oxygen rates, and biomass tends to decrease. The reactor is filled with a proper amount of air, so that the culture and the culture solution can be fully mixed, the nutrient can be absorbed by the culture, and dissolved oxygen can be provided for the growth of plants and the accumulation of metabolites. Therefore, when the air injection amount in the airlift bioreactor containing 2L S1 liquid medium and having a capacity of 3L is 0.05vvm and 0.05-0.1 vvm, the ginseng adventitious roots can be proliferated and grown.
9. Influence of the culture method on the proliferation and growth of adventitious roots when the wild ginseng is proliferated under laboratory conditions in the proliferation method of the adventitious roots:
the adventitious roots are respectively cultured in a suspension culture mode and in an airlift bioreactor. The liquid suspension culture is that 6g/L (fresh weight) of adventitious roots are inoculated into a 250mL (containing 70mLS1 liquid culture medium) triangular flask and cultured on a shaking table with the rotating speed of 110 rpm; the bioreactor culture is that 6g/L (fresh weight) of adventitious roots are inoculated into an airlift bioreactor (3L bioreactor contains 2LS1 liquid culture medium, 5L bioreactor contains 2LS1 liquid culture medium, and 5L bioreactor contains 4LS1 liquid culture medium), and the aeration of the reactor is 0.05 vvm. Investigating fresh weight, dry weight and growth rate after culturing for 40 days; the results are shown in Table 9; the S1 liquid medium is the same as the S1 medium described in the above protocol;
TABLE 93 Effect of culture methods in air-lift bioreactor multiplication on adventitious root growth
| Index (I) | Fresh weight (g/L) | Dry weight (g/L) | Rate of increase |
| 70mL culture medium/250 mL triangular flask | 103.43c | 11.57c | 17.08 |
| 2000mL culture medium/3000 mL bioreactor | 126.71a | 15.62a | 23.40 |
| 2000mL culture Medium/5000 mL bioreactor | 120.90b | 14.38b | 21.47 |
| 4000mL culture medium/5000 mL bioreactor | 125.79a | 15.54a | 23.28 |
The ginseng adventitious roots are cultured by utilizing 2 culture modes of triangular flask suspension and airlift bioreactor, and the result shows that the proliferation effect of the ginseng adventitious roots cultured in the airlift bioreactor is obviously better than that of the triangular flask suspension culture. 3L bioreactor with 2L Medium and 5L bioreactor with 4L MediumThe growth vigor of the adventitious roots is better, the proliferation effect has no significant difference, the proliferation coefficients respectively reach 23.40 and 23.28, and are significantly higher than that of a 5L bioreactor containing 2L culture medium. It can be seen that, in the case of the triangular flask suspension culture, no fresh air is supplied and only 0.15h is required-1The air of the ratio circulates, and is easy to agglomerate in the culture process, so that the nutrient and oxygen supply in the center of the agglomerate are insufficient, and the biomass is reduced; the culture in the reactor is realized by introducing a proper amount of air to fully mix the culture and the culture solution, so that the nutrient is absorbed by the culture to be beneficial to adventitious root proliferation. Meanwhile, the condition of culturing the ginseng adventitious roots by using the 3L airlift bioreactor is basically suitable for the enlarged culture of the 5L reactor, and the ratio of the explant inoculation amount to the culture medium amount and the air amount in the reactor system influences the growth and development of the ginseng adventitious roots, so that the proper culture medium volume has direct influence on reaching the highest biomass and improving the space utilization rate. Therefore, the utilization of 4L of culture medium in a 5L bioreactor achieves higher biomass and improves the space utilization rate, and provides theoretical basis and technical support for scale production of mountain ginseng adventitious roots.
10. Influence of growth cycle of 3L airlift bioreactor reaction system in proliferation under laboratory conditions in the method for proliferation of adventitious roots of wild ginseng:
to investigate the dynamic of biomass change during the cultivation in the adventitious roots reactor, 6g/L (fresh weight) of adventitious roots were completely submerged in a 3L capacity airlift bioreactor containing 2L of S1 liquid medium, with a reactor aeration of 0.05 vvm. Culturing for 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, and 10 weeks, then stopping culturing, and investigating fresh weight, dry weight, and growth rate; the results are shown in Table 10; the S1 liquid medium is the same as the S1 medium described in the above protocol;
effect of different growth cycles on adventitious root growth in propagation of airlift bioreactor of Table 103L
| Index (I) | Fresh weight (g/L) | Dry weight (g/L) | Growth Rate (day)-1) |
| 5 weeks | 91.32 | 11.05 | 0.47 |
| 6 weeks | 120.90 | 14.38 | 0.52 |
| 7 weeks | 127.08 | 17.31 | 0.54 |
| 8 weeks | 133.50 | 17.92 | 0.50 |
| 9 weeks | 149.02 | 19.11 | 0.47 |
| For 10 weeks | 156.09 | 21.31 | 0.47 |
As can be seen from Table 10, by examining the growth of the adventitious roots in the reactor, it was found that the fresh weight and the dry weight tended to increase with the culture period. But the adventitious root growth rate showed a decreasing trend after increasing to 7 weeks. This indicates that the adventitious roots begin to enter the senescence phase 7 weeks after growth. Therefore, subculture to a new medium is required 7 weeks after the culture. After 7 weeks of culture, the weight of the obtained dry mountain ginseng adventitious roots is 17.31g/L, and the biomass is 20-30 times.
11. Detection of saponin content, trace elements and heavy metal content of effective ginseng component
Completely immersing 6g/L fresh ginseng adventitious root in an airlift bioreactor containing 4L S1 liquid culture medium and 5L, culturing for 7 weeks, and detecting the saponin content, trace elements and heavy metals content of effective components of ginseng; the S1 liquid medium is the same as the S1 medium described in the above protocol;
after 7 weeks of culture, the contents of different substances in the mountain ginseng adventitious roots are shown in Table 11 by the analysis of special economic animals and plants and a product quality supervision, inspection and test center in the rural area.
TABLE 11 content of different substances in mountain ginseng adventitious roots
| Name of active substance | Content (wt.) | Detection method |
| Total saponins | 1.13％ | GB/T 18765-2015 |
| Monomeric saponin Rb1 | 0.101％ | NY/T 1842-2010 |
| Rb2 | 0.0132％ | NY/T 1842-2010 |
| Rb3 | 0.014％ | NY/T 1842-2010 |
| Rc | 0.0024％ | NY/T 1842-2010 |
| Rd | 0.0245％ | NY/T 1842-2010 |
| Re | 0.00608％ | NY/T 1842-2010 |
| Rg1 | 0.0102％ | NY/T 1842-2010 |
| Rg2 | 0.00226％ | NY/T 1842-2010 |
| Rf | 0.00623％ | NY/T 1842-2010 |
| Selenium | 0.0327mg/kg | GB 5009.268-2016 |
| Inorganic element calcium | 1.17x103mg/kg | GB 5009.92-2016 |
| Zinc | 1.02x102mg/kg | GB 5009.14-2017 |
| Magnesium alloy | 1.46x103mg/kg | GB 5009.241-2017 |
| Manganese oxide | 2.78x102mg/kg | GB 5009.242-2017 |
| Iron | 1.54x102mg/kg | GB 5009.90-2016 |
| Potassium salt | 3.83x103mg/100g | GB 5009.91-2017 |
| Sodium salt | 1.92x103mg/100g | GB 5009.91-2017 |
| Phosphorus (P) | 0.202％ | GB 5009.87-2016 |
| Arsenic (As) | 0.052mg/kg | GB 5009.11-2014 |
| Heavy metal copper | 1.04mg/kg | GB 5009.13-2017 |
| Lead (II) | 0.075mg/kg | GB 5009.12-2017 |
| Mercury | 0.005mg/kg | GB 5009.17-2014 |
The result shows that the total saponin content of the adventitious roots of the wild ginseng obtained by tissue culture reaches 1.13 percent (containing 9 ginsenoside monomers) after the culture is carried out for 40-60 days. The contents of heavy metals and harmful elements such as lead, mercury, arsenic and copper are very low and are within the limit range established in the 'Chinese pharmacopoeia' 2015 edition. The medicinal effect of ginseng is not only related to the organic active components contained in ginseng, but also the inorganic components play an important role. Wherein the ginseng adventitious root also contains 5 microelements of iron, zinc, copper, manganese and selenium, and two major elements of calcium and magnesium, which are necessary for human body related to health chiffon. Therefore, the tissue culture adventitious root of ginseng has the characteristics of high-quality ginseng food such as stable nutrient components, no pesticide residue, no heavy metal, no potential toxicity and the like. In future, higher-content ginsenoside needs to be produced, and two-step culture is carried out to facilitate the accumulation of metabolites.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (6)
1. A method for rapidly propagating mountain ginseng adventitious roots comprises the following steps:
1) taking wild ginseng roots as explants, inoculating the explants into an induction culture medium, and carrying out induction culture to obtain callus;
2) inoculating the callus into a rooting culture medium, and carrying out rooting culture for 6-8 weeks to obtain adventitious roots;
3) inoculating the adventitious roots into a screening culture medium, carrying out subculture, and taking the adventitious roots with the adventitious root induction number of 36-46 as adventitious root systems;
4) inoculating the adventitious root system into a proliferation culture medium, and performing proliferation culture for 40-50 days to obtain mountain ginseng adventitious roots for harvesting; the enrichment culture is carried out in an airlift bioreactor;
the screening culture medium in the step 3) takes an S1 culture medium as a basic culture medium, and also comprises 2-2.5 g/L gelrite;
the S1 culture medium consists of the following components in mass concentration: NH (NH)4Cl401.175mg/L、KNO3 3791.25mg/L、MgSO4·7H2O 277.5mg/L、KH2PO4 127.5mg/L、CaCl2·2H2O 660mg/L、FeNa-EDTA 37.3mg/L、MnSO4·4H2O 22.3mg/L、KI 0.83mg/L、CuSO4·5H2O 0.05mg/L、CoCl2·6H2O 0.025mg/L、Na2MoO4·2H2O 0.25mg/L、ZnSO4·7H2O 8.6mg/L、H3BO36.2mg/L of inositol, 100mg/L of nicotinic acid, 0.375mg/L of vitamin B, 60.375mg/L of vitamin B, 10.075mg/L of vitamin B, 1.5mg/L, IBA 5mg/L of glycine and 50000mg/L of white sugar;
the proliferation medium in step 4) comprises S1 medium;
the density of the access of the adventitious root system in the step 4) is 5-7 g/L;
the air injection amount in the airlift bioreactor in the step 4) is 0.05-0.1 vvm.
2. The method according to claim 1, wherein the wild ginseng root is sterilized in step 1); the disinfection is carried out by adopting the following method: soaking the wild ginseng root in an ethanol water solution for 25-35 s, and then soaking in a sodium hypochlorite water solution for 35-45 min for surface disinfection;
the volume percentage content of the ethanol in the ethanol water solution is 70-75%;
the mass concentration of sodium hypochlorite in the sodium hypochlorite aqueous solution is 4%.
3. The method according to claim 1, wherein the induction medium in step 1) is MS medium as a basic medium, and further comprises the following components in mass concentration: 28-32 g/L of white sugar, 1-2 mg/L of 2, 4-D and 2-2.5 g/L of gelrite; the pH value of the induction culture medium is 5.6-6.0.
4. The method according to claim 1, wherein the time for the induction culture in step 1) is 6-8 weeks; the temperature of the induction culture is 21-23 ℃.
5. The method according to claim 1, wherein the rooting medium in step 2) is MS medium as a basic medium, and further comprises the following components in mass concentration: 28-32 g/L, IBA 3-4 mg/L of white sugar and 2-2.5 g/L of gelrite; the pH value of the rooting culture medium is 5.6-6.0.
6. The method according to claim 1, wherein the air injection amount in the airlift bioreactor in step 4) is 0.05vvm, 0.1vvm, or 0.05 to 0.1 vvm; the 0.05-0.1 vvm means that the ventilation volume is 0.05vvm in the first two weeks and is always 0.1vvm later.
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