CN112956415B - Liquid culture medium for culturing ginseng adventitious roots and culture method - Google Patents
Liquid culture medium for culturing ginseng adventitious roots and culture method Download PDFInfo
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Abstract
The invention discloses a liquid culture medium for culturing ginseng adventitious roots and a culture method, wherein the liquid culture medium comprises: 10-55g/L of sucrose, 0.6-1.6g/L B5 of culture medium, 0.3-1.2g/L of 1/2MS culture medium, 0-5.4mg/L of 6-benzylamino adenine, 0-5.4mg/L of naphthylacetic acid, 0-8mg/L of gibberellin and 0-5.4mg/L of indoleacetic acid. The culture method comprises the following steps: cleaning and disinfecting mature ginseng, slicing, and inoculating the ginseng to an induction culture medium to induce adventitious roots of the ginseng; inoculating the obtained ginseng adventitious root to an induction culture medium again for subculture and propagation; then cutting the obtained ginseng adventitious roots into segments, inoculating the segments into the liquid culture medium, and culturing to obtain the adventitious roots. By adopting the liquid culture medium and the culture method, each part of mature ginseng can be directly adopted to induce and generate adventitious roots, the intermediate step of inducing callus is not needed, the induction step can be simplified, the induction time can be shortened, and meanwhile, the content of ginsenoside in the adventitious roots can be greatly improved.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a liquid culture medium for culturing adventitious roots of ginseng and a culture method.
Background
Ginseng (Panax ginseng c.a. mey.) is a plant of the genus Panax of the family araliaceae, distributed in china, japan and korea, and its rhizome is a rare Chinese medicinal material, called "king of herbaceous plant". Ginseng is sweet, slightly bitter and slightly warm in taste, has the effects of greatly invigorating primordial qi, recovering pulse, relieving depletion, invigorating spleen, benefiting lung, promoting fluid production, nourishing blood, tranquilizing mind, and improving intelligence, and is mainly used for treating loss of body-shirt, spleen deficiency, anorexia, lung deficiency, cough, body fluid deficiency, thirst, palpitation, insomnia, etc. Ginsenoside is the main active component of ginseng, and has the effects of resisting fatigue, delaying aging, regulating central nervous system, improving immunity, improving cardiovascular and cerebrovascular insufficiency, inhibiting tumor cell production, etc. In recent years, ginseng has been widely used in various cosmetics, health products, and drinks, and has a very wide market prospect.
At present, due to excessive mining, environmental damage and the like, wild ginseng resources are almost exhausted, and field cultivation is a main source of ginseng. However, ginseng grows slowly, the planting years are long, the requirements on environmental conditions are strict, the quality of ginseng is easily influenced by climate, cultivation conditions and plant diseases and insect pests, the cultivation technology is complex, and the development prospect of artificial cultivation of ginseng is greatly limited by the problems of pesticide residue exceeding the standard, ginseng land and the like. The ginseng cultivated in the field is difficult to meet the market demand. The tissue culture technology of ginseng has short period, is not limited by seasons, is easy to carry out large-scale industrial production, and has great development prospect.
The existing adventitious roots are generally generated by ginseng callus induction, the callus needs to be induced firstly and then the adventitious roots need to be induced, the required experimental period is long, the operation steps are complex, and the pollution risk is high. In addition, the cultured ginseng also has the problem that the content of ginsenoside is low, so that the clinical application requirement is difficult to meet.
Chinese patent application No. CN201711459037.0 discloses a method for tissue culture of adventitious roots of wild ginseng, comprising the following steps: (1) inducing wild ginseng callus; (2) proliferation of wild ginseng callus; (3) inducing adventitious roots of wild ginseng; (4) the adventitious roots of the wild ginseng in the liquid culture medium proliferate. In the technical scheme, the callus needs to be induced first, 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 method for inducing the proliferation of adventitious roots of ginseng, which comprises the following steps: cutting tissue culture seedlings of ginseng into tissue small blocks, inoculating the tissue small blocks into a solid induction culture medium to induce and form adventitious roots, cutting the adventitious roots into adventitious root small blocks, and inoculating the tissue small blocks into a liquid multiplication culture medium to carry out multiplication culture of the adventitious roots; wherein the solid induction culture medium and the liquid proliferation culture medium are 1/2MS (-N) culture medium which is used as a basic culture medium and contains indolebutyric acid with the concentration of 1-10 mg/L. In the scheme, the tissue culture seedling with the age of 28-32 days is cut into small blocks to directly induce adventitious roots, the tissue culture seedling is tender and has strong differentiation capability, the tissue culture seedling needs to be obtained through seed germination or explant culture first, at least 28-32 days are still needed, callus induction is still needed in the explant culture, and the period is not shortened actually.
Therefore, if a method capable of directly inducing the mature ginseng to generate adventitious roots can be found, the method not only can shorten the induction period and simplify the induction conditions, but also can improve the content of effective components in the adventitious roots of the ginseng has wide 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 provides a ginseng adventitious root and a preparation method thereof. The culture method of the adventitious roots of the invention induces each part of the mature ginseng to generate the adventitious roots without the intermediate step of inducing callus, thereby simplifying the induction step and shortening the induction time. Further, by adopting the liquid culture medium, the growth multiple of the adventitious roots is high, and the content of ginsenoside in the adventitious roots can be greatly improved.
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 liquid medium for ginseng adventitious root culture, comprising: 10-55g/L of sucrose, 0.6-1.6g/L B5 of culture medium, 0.3-1.2g/L of 1/2MS culture medium, 0-5.4mg/L of 6-benzylamino adenine, 0-5.4mg/L of naphthylacetic acid, 0-8mg/L of gibberellin and 0-5.4mg/L of indoleacetic acid.
In a further scheme, the pharmaceutical composition also comprises 0-5.4mg/L of indolebutyric acid.
As a preferred embodiment, the liquid medium includes: 15-45g/L of sucrose, 0.8-1.3g/L B5 of culture medium, 0.5-1.0g/L1/2MS of culture medium, 0-4.2mg/L of 6-benzylamino adenine, 1.8-5.4mg/L of naphthylacetic acid, 0-8mg/L of gibberellin, 0.6-4.8mg/L of indoleacetic acid and 0-3.6mg/L of indolebutyric acid.
The liquid culture medium is suitable for culturing the adventitious roots induced by the mature ginseng by adopting a one-step method; it can also be used to culture adventitious roots produced by the two-step method in the prior art. The adventitious roots induced by the one-step method are cultured by adopting the liquid culture medium with the components and the mixture ratio, the growth multiple of the adventitious roots is high, and meanwhile, the content of ginsenoside in the adventitious roots of ginseng can be greatly improved.
As a more preferred embodiment, the liquid medium includes: the method comprises the following steps: 45g/L of sucrose, 1.28g/L B5 of culture medium, 0.905g/L of 1/2 of MS culture medium, 4.2mg/L of 6-benzylamino adenine, 3mg/L of naphthylacetic acid, 0.8mg/L of gibberellin, 0.6mg/L of indoleacetic acid and 0.6mg/L of indolebutyric acid.
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.
The second purpose of the invention is to provide a method for culturing ginseng adventitious roots, which comprises the steps of cleaning, disinfecting, slicing and inoculating mature ginseng into an induction culture medium to induce the ginseng adventitious roots; inoculating the obtained ginseng adventitious root to an induction culture medium again for subculture and propagation; then the obtained ginseng adventitious root is cut into segments, and inoculated into a liquid culture medium for culture to obtain the adventitious root.
The existing adventitious roots are generally generated by ginseng callus induction, the callus needs to be induced firstly and then the adventitious roots need to be induced, the required experimental period is long, the operation steps are complex, and the pollution risk is high. In addition, the cultured ginseng also has the problem that the content of ginsenoside is low, so that the clinical application requirement is difficult to meet.
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.
In a further embodiment, the induction medium comprises 1-6mg/L naphthylacetic acid, 0.2-1mg/L gibberellin, 0.1-0.6mg/L kinetin, 0.75-1.5g/L citric acid, 0.03-1g/L ascorbic acid, 1-4g/L B5 medium and 1-2.4g/L WPM 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 auxin hormone, so that the growth of adventitious roots can be promoted. Kinetin is a cytokinin that promotes cell division. Citric acid and ascorbic acid can generate a synergistic antioxidation effect, prevent the in vitro tissue of the mature ginseng from browning, and are beneficial to directly inducing adventitious roots from the in vitro tissue of the mature ginseng. The components in the induction culture medium have synergistic effect, and the aim of directly inducing each part of the mature ginseng to generate adventitious roots is finally realized without an intermediate step of inducing callus.
In a further scheme, the induction culture medium also comprises 20-60g/L of sucrose and 1-6g/L of plant gel.
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, 1.55g/L B5 medium and 1.21g/L WPM medium.
In a further scheme, the induction culture medium further comprises 30g/L of sucrose and 3g/L of plant gel.
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.
In a further scheme, the liquid culture medium is B5 culture medium, WPM culture medium or 1/2MS culture medium as basal culture medium, and contains 4mg/L indolebutyric acid, 0.1g/L citric acid, 0.05g/L ascorbic acid and 30g/L sucrose.
The adventitious roots induced by the one-step method have no difference with other two-step methods, and when the adventitious roots induced by the one-step method are subjected to further 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 method for culturing the adventitious roots of the ginseng comprises the following steps:
(1) cleaning and disinfecting mature ginseng, slicing, inoculating the ginseng into an induction culture medium, and performing dark culture at the temperature of 22 +/-1 ℃ for 4-5 weeks to induce adventitious roots of the ginseng;
(2) inoculating the ginseng adventitious roots obtained in the step (1) into the same induction culture medium as that in the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) and (3) shearing the adventitious roots of the ginseng obtained in the step (2) into small segments, inoculating the small segments 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.
In a further scheme, the age of the mature ginseng is more than 3 years; preferably, the age of the mature ginseng is 6 years or more.
As a preferred embodiment, the mature ginseng is a centennial ginseng. The century ginseng is rare in nature, has high edible and medicinal values, and can tonify five internal organs, calm spirit, calm soul, stop palpitation, remove pathogenic qi, improve eyesight, and promote mentality and intelligence. The value of the ginseng cultivation method is far higher than that of planted ginseng with short ginseng age. The invention does not need the intermediate step of inducing callus, can directly obtain the adventitious roots by one-step induction from the hundred-year ginseng cut blocks, not only can simplify the induction step and shorten the induction time, but also can obtain the specific functional components in the female parent hundred-year old ginseng, thereby obtaining the adventitious roots with better nutritive value.
Further, the main root, or the reed head, or the part, or the branch root, or the fibrous root of the mature ginseng are cleaned, disinfected, sliced and inoculated into an induction culture medium to induce the adventitious root of the ginseng.
In a further scheme, the ginseng is selected from wild ginseng, transplanted ginseng, ginseng under forest and garden ginseng;
preferably, the ginseng is wild ginseng.
As a specific preferred embodiment, the step of culturing adventitious roots of the present invention specifically includes:
(1) induction of adventitious roots
Cleaning main roots of mature ginseng, sterilizing, cutting into slices with the width of 0.5-0.7cm, the length of 0.5-0.7cm and the thickness of 0.2-0.5mm, inoculating the slices into an 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 cane sugar, 3g/L plant gel, 1.55g/L B5 culture medium and 1.21g/L WPM culture medium, and carrying out dark culture at the temperature of 22 +/-1 ℃ for 4-5 weeks to induce the adventitious roots of the ginseng;
(2) subculture of adventitious roots
Inoculating the ginseng adventitious roots obtained in the step (1) into the same induction culture medium as that in the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) cultivation of adventitious roots
And (3) 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 at the temperature of 22 +/-1 ℃ to obtain the adventitious roots. Wherein, the liquid culture medium comprises: 45g/L of sucrose, 1.28g/L B5 of culture medium, 0.905g/L of 1/2MS culture medium, 4.2mg/L of 6-benzylamino adenine, 3mg/L of naphthylacetic acid, 0.8mg/L of gibberellin, 0.6mg/L of indoleacetic acid and 0.6mg/L of indolebutyric acid.
In a further embodiment, the pH of the induction medium and the liquid medium of the present invention is 5.6 to 6.0.
The adventitious roots cultured by the liquid culture medium of the preferred embodiment have the best comprehensive growth multiple of the adventitious roots and the total saponin content.
In the present invention, WPM medium, B5 medium, N6 medium, 1/2MS medium, and the like are known 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 ℃.
After adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects:
1. the liquid culture medium has the advantages that the proportion of each component is proper, the synergistic effect can be generated, the growth multiple of the adventitious root is high, and the content of ginsenoside in the adventitious root can be greatly improved.
2. 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.
3. 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, 0.75-1.5g/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 induction of adventitious roots using the induction medium of example 1 of the present invention;
FIG. 2 is a schematic view of induction of adventitious roots using the induction medium of comparative example 1.
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, and the following embodiments are used for illustrating the present invention and are not used for limiting the scope of the present invention.
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. 1, wherein A is a photograph of 1 week of cultivation, B is a photograph of 3 weeks of cultivation, and C is a photograph of 5 weeks of cultivation. As can be seen, after 5 weeks, adventitious roots were induced directly on the slices of the mature wild ginseng.
Example 2
(1) Induction of adventitious roots
Cleaning reed heads of 6-age garden ginseng, sterilizing, cutting into slices with the width of 0.5cm, the length of 0.6cm and the thickness of 0.3mm, inoculating the slices into an induction culture medium, and performing dark culture at the temperature of 22 +/-1 ℃ for 4-5 weeks to induce adventitious roots; wherein the induction culture medium comprises 6mg/L naphthylacetic acid, 0.2mg/L gibberellin, 0.4mg/L kinetin, 1.2g/L citric acid, 0.1g/L ascorbic acid, 20g/L sucrose, 5g/L plant gel, 4g/L B5 culture medium and 1.8g/L WPM culture medium, and the pH value is 5.6.
(2) Subculture of adventitious roots
Inoculating the adventitious roots obtained in the step (1) into the same induction culture medium as that in the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) cultivation of adventitious roots
Shearing the adventitious roots obtained in the step (2) into tissues with the length of about 1cm, inoculating the tissues into a liquid culture medium, and culturing for 3-4 weeks on a shaking table at the temperature of 22 +/-1 ℃ to obtain the adventitious roots; wherein the liquid culture medium contains 4mg/L indolebutyric acid, 0.1g/L citric acid, 0.05g/L ascorbic acid, 1/2MS culture medium and 30g/L sucrose, and has pH value of 5.6.
Similar to the results of example 1, the adventitious roots can be induced by one step in step (1) of this example. Example 3
(1) Induction of adventitious roots
Cleaning parts of age 10 of ginseng under forest, sterilizing, cutting into slices with width of 0.7cm, length of 0.7cm and thickness of 0.5mm, inoculating into induction culture medium, and dark culturing at 22 + -1 deg.C for 4-5 weeks to induce adventitious roots; wherein the induction culture medium comprises 5mg/L naphthylacetic acid, 1mg/L gibberellin, 0.1mg/L kinetin, 0.75g/L citric acid, 0.03g/L ascorbic acid, 40g/L sucrose, 1g/L plant gel, 2g/L B5 culture medium and 1g/L WPM culture medium, and the pH value is 6.0.
(2) Subculture of adventitious roots
Inoculating the adventitious roots obtained in the step (1) into the same induction culture medium as that in the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) cultivation of adventitious roots
Shearing the adventitious roots obtained in the step (2) into tissues with the length of about 2cm, inoculating the tissues into a liquid culture medium, and culturing for 3-4 weeks on a shaking table at the temperature of 22 +/-1 ℃ to obtain the adventitious roots; wherein the liquid culture medium contains 4mg/L indolebutyric acid, 0.1g/L citric acid, 0.05g/L ascorbic acid, WPM culture medium and 30g/L sucrose, and has pH of 6.0.
Similar to the results of example 1, the adventitious roots can be induced by one step in step (1) of this example.
Example 4
(1) Induction of adventitious roots
Cleaning main root of 15-age mountain ginseng, sterilizing, cutting into slices with width of 0.5cm, length of 0.6cm and thickness of 0.4mm, inoculating into induction culture medium, and dark culturing at 22 + -1 deg.C for 4-5 weeks to induce mountain ginseng adventitious root; wherein the induction culture medium comprises 1mg/L naphthylacetic acid, 0.5mg/L gibberellin, 0.6mg/L kinetin, 1.5g/L citric acid, 1g/L ascorbic acid, 50g/L sucrose, 6g/L plant gel, 1g/L B5 culture medium and 2.4g/L WPM culture medium, and the pH value is 5.7.
(2) Subculture of adventitious roots
Inoculating the mountain ginseng adventitious roots obtained in the step (1) into the same induction culture medium as that in the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) cultivation of adventitious roots
Shearing the mountain ginseng adventitious roots obtained in the step (2) into tissues with the length of about 1cm, inoculating the tissues into a liquid culture medium, and culturing for 3-4 weeks on a shaking table at the temperature of 22 +/-1 ℃ to obtain adventitious roots; wherein the liquid culture medium contains 4mg/L indolebutyric acid, 0.1g/L citric acid, 0.05g/L ascorbic acid, B5 culture medium and 30g/L sucrose, and has pH value of 5.7.
Similar to the results of example 1, in step (1) of this example, adventitious roots can be induced directly by one step.
Example 5
(1) Induction of adventitious roots
Removing rhizoma Phragmitis and parts of wild ginseng, cleaning main root, sterilizing, cutting into slices with width of 0.6cm, length of 0.7cm and thickness of 0.3mm, inoculating into induction culture medium, and dark culturing at 22 + -1 deg.C for 4-5 weeks to induce adventitious root of wild ginseng; wherein the induction culture medium comprises 4mg/L naphthylacetic acid, 0.6mg/L gibberellin, 0.4mg/L kinetin, 0.1g/L citric acid, 0.05g/L ascorbic acid, 30g/L sucrose, 3g/L plant gel, 1.55g/L B5 culture medium and 1.21g/L WPM culture medium, and the pH value is 5.8.
(2) Subculture of adventitious roots
Inoculating the mountain ginseng adventitious roots obtained in the step (1) into the same induction culture medium as that in the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) cultivation of adventitious roots
Shearing the mountain ginseng adventitious roots obtained in the step (2) into tissues with the length of about 1cm, inoculating the tissues into a liquid culture medium, and culturing for 3-4 weeks on a shaking table at the temperature of 22 +/-1 ℃ to obtain adventitious roots; wherein the liquid culture medium contains 4mg/L indolebutyric acid, 0.1g/L citric acid, 0.05g/L ascorbic acid, 1/2MS culture medium and 30g/L sucrose, and has pH value of 5.8.
Similar to the results of example 1, in step (1) of this example, adventitious roots can be induced directly by one step.
Comparative example 1
This comparative example differs from example 1 in the induction medium used and the other steps are carried out with reference to example 1. The induction culture of this comparative example included: 30g/L of sucrose, 0.5mg/L of kinetin, 3mg/L of indolebutyric acid, 1.5mg/L of 2, 4-dichlorophenoxyacetic acid, 1/2MS culture medium, 3g/L of plant gel and pH value of 5.8.
FIG. 2 shows the results of induction of adventitious roots on the medium in step (1) of this comparative example, wherein A is a photograph taken after 1 week of culture, B is a photograph taken after 3 weeks of culture, and C is a photograph taken after 5 weeks of culture. As can be seen, after 5 weeks, the culture medium of comparative example 1 failed to induce the generation of adventitious roots directly from the mature wild ginseng slices.
Comparative example 2
This comparative example differs from example 1 in the induction medium used and the other steps are carried out with reference to example 1. The induction culture of this comparative example included: 30g/L of sucrose, 0.5mg/L of kinetin, 3mg/L of indolebutyric acid, 1/2MS culture medium, 3g/L of plant gel and pH value of 5.8.
As a result, the adventitious roots cannot be induced directly from the mature wild ginseng slice, similarly to the picture display of comparative example 1.
Comparative example 3
This comparative example differs from example 1 in the induction medium used and the other steps are carried out with reference to example 1. The induction culture of this comparative example included: 30g/L of sucrose, 0.5mg/L of kinetin, 3mg/L of indoleacetic acid, 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.
Screening of liquid Medium
In order to improve the content of ginsenoside in the adventitious roots of the ginseng, the liquid culture medium is further screened and optimized.
The method for detecting the ginsenoside in the adventitious root of the ginseng comprises the following steps:
(1) principle of
The sample is separated by a C18 chromatographic column after pretreatment such as extraction, and is detected by a high performance liquid chromatography-ultraviolet detector, and the content of each component of the ginsenoside is quantitatively measured by an external standard method.
(2) Reagent
Methanol (CH)4O): chromatographically pure acetonitrile (C)6H11N): pure chromatography
(3) Analytical procedure
Grinding about 6g (accurate to 0.01g) of the uniformly mixed sample in a 150mL mortar, transferring the sample into a 50mL centrifuge tube, adding 10mL of water, uniformly mixing, breaking the wall for 3 minutes by 400W on an ultrasonic cell crusher, and freezing 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 sample was ground in a mortar and 50mg was weighed accurately into a 10ml centrifuge tube, 70% methanol solution was added and vortexed. Sonicate on a sonicator for 10 minutes, repeat twice, filter for use.
(4) Reference conditions of the apparatus
A) A chromatographic column: c18 column with column length of 150mm, column inner diameter of 4.6mm, column packing particle size of 5 μm, or equivalent;
B) mobile phase: a: acetonitrile, b: filtering water with 0.45 μm microporous membrane;
C) flow rate: 0.7 mL/min; gradient elution procedure: 0-13 min, 23% -46% acetonitrile, and the volume flow rate is 0.7 mL/min; 13-33 min, 46-68% acetonitrile, and the volume flow rate is 0.7 mL/min; 33-46.5 min, 68-85% acetonitrile, and the volume flow rate is 0.7 mL/min;
D) the column temperature is 30 ℃;
E) the detection wavelength is 203 nm;
F) the injection volume is 10 muL.
(5) Presentation of analytical results
The content of each component of the ginsenoside in the sample is calculated according to the formula (1):
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 ginsenoside content 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.
EXAMPLE 6 liquid Medium I
The cultivation method of this example was conducted in accordance with the method of example 1 except that the liquid culture medium of example 1 was used in place of the various liquid culture media of this example, and the ginsenoside content in the ginseng adventitious roots was measured, which was conducted in accordance with the above-mentioned method.
The first 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 (Vc), 0-225mg/L of citric acid, 0-7mg/L of indolebutyric acid (IBA), and the pH value is 5.8.
TABLE 1
As can be seen from the above Table 1, the liquid culture medium I with the ratio of the experimental group 5-7 has higher growth multiple of the adventitious roots and higher total amount of the saponin to be detected, wherein the growth multiple of the adventitious roots and the saponin content obtained by the experimental group 5 are optimal comprehensively.
Example 7 liquid Medium II
The cultivation method of this example was conducted in accordance with the method of example 1 except that the liquid culture medium of example 1 was used in place of the various liquid culture media of this example, and the ginsenoside content in the ginseng adventitious roots was measured, which was conducted in accordance with the above-mentioned method.
The liquid culture medium II comprises: 10-55g/L sucrose, 0.3-1.2 g/L1/2MS culture medium, 0.6-1.6g/L B5 culture medium, 0-5.4mg/L indoleacetic acid (IAA), 0-5.4mg/L indolebutyric acid (IBA), 0-5.4mg/L naphthylacetic acid (NAA), 0-5.4 mg/L6-benzylamino adenine (6BA), 0-8mg/L Gibberellin (GA), and the pH value is 5.8.
TABLE 2
As can be seen from the above Table 2, the liquid culture medium II prepared by the ratio of the experimental groups 4 and 6 has a high growth multiple of the adventitious roots and a high total amount of the saponin to be detected, wherein the growth multiple of the adventitious roots and the saponin content obtained by the experimental group 4 are optimal comprehensively.
Example 8 liquid Medium III
The cultivation method of this example was conducted in accordance with the method of example 1 except that the liquid culture medium of example 1 was used in place of the various liquid culture media of this example, and the ginsenoside content in the ginseng adventitious roots was measured, which was conducted in accordance with the above-mentioned method.
The liquid culture medium III comprises: 35g/L of sucrose, 1.35g/L of WPM medium, 1g/L N6 medium, 0-1.8mg/L of indolebutyric acid (IBA), 1-6mg/L of naphthylacetic acid (NAA), 0.2-1.2mg/L of Kinetin (KT) and the pH value is 5.8.
TABLE 3
As can be seen from table 3 above, on the basis of determining the usage amounts of sucrose, WPM, and N6, the influence of plant hormones IBA, NAA, and KT on the yield and saponin content of adventitious roots at different addition amounts is examined, so as to select appropriate plant hormones and mixture ratios. As can be seen from the table, the compositions of the experimental groups 4, 7-8, 10-12 and 15-18 have better adventitious root growth multiple and total saponin content, wherein the comprehensive optimal value of the adventitious root growth multiple and the total saponin content is obtained under the condition of the experimental group 12.
Example 9
Various ginsenosides have different contents in Panax plants and have different pharmacological functions. Some ginsenosides have high content, such as Re, Rb1 and Rg1, while others have low content, called rare ginsenosides, such as Rh1, Rh2, Rh3 and Rg 3.
Many pharmacological studies have shown that rare ginsenosides generally have better pharmacological activity. The antitumor activity of the ginsenoside aglycon is strongest, and the antitumor activity of the ginsenoside is weakened in sequence along with the increase of the number of glycosyl groups, namely: aglycone > monoglycoside > diglycoside > triglycoside > tetraglucoside. By studying the metabolism rule of ginsenoside in vivo, it is also found that most of ginsenoside is poorly absorbed in gastrointestinal tract, and deglycosylated secondary glycoside and aglycone have stronger pharmacological activity and higher bioavailability than saponin. Rare ginsenosides and aglycones are contained in a very small amount in a ginseng original plant, a cell culture, an adventitious root and a hairy root, and are mainly obtained by hydrolyzing saponin glycosidic bonds by a physical method and a chemical method or are converted by a biological method by an enzymatic method and a microbiological method at present.
The test example further optimizes the conversion method of the saponin in the adventitious root of the ginseng, and specifically comprises the following steps:
(1) the mountain ginseng adventitious roots prepared by the culture method of the embodiment 1 of the invention are washed by clean water for 3 times, cut into small segments of 1-2cm, the small segments are placed in a mortar, a proper amount of water is added, the grinding is carried out for 1-2 minutes, the grinding is carried out until more than 80 percent of the small segments are ground, the small segments are divided into six groups with the same weight, one group is a control group without any treatment, and the other five groups are experimental groups 1-5.
(2) The experimental groups 1 to 5 were treated according to the conditions in table 4, specifically: mixing Na2CO3The solution was added to prepared adventitious roots to make Na in each experimental group2CO3And (3) heating to a set temperature (DEG C) at different concentrations, preserving heat for a set time (min), then cooling to room temperature, repeating the processes of heating, preserving heat and cooling, and repeating the set times.
For example, in Experimental group 1, Na2CO3Adding the solution into the ground adventitious root, and adding Na into the sample2CO3The concentration of (A) is as follows: 1M Na2CO38ml/L (equivalent to 8 mmol/L); heating to 85 deg.C, maintaining the temperature for 60min, cooling to room temperature, and repeating the heating, maintaining and cooling processes for 2 times. Other experimental groups were similar.
(3) The sample was treated and the content of ginsenoside was measured by the method in test example 1.
The method of processing a sample comprises: transferring the treated sample into a 50ml centrifuge tube, adding 10ml water, mixing, breaking cell wall with 400W in an ultrasonic cell crusher for 3 minutes, and freezing in a refrigerator at-18 ℃ for 3 hours. Freeze-drying in a freeze-drying machine for 48 hours until no water drops exist outside the cup body.
The sample was ground in a mortar and 50mg was weighed accurately into a 10ml centrifuge tube, 70% methanol solution was added and vortexed. Sonicate on a sonicator for 10 minutes, repeat twice, filter for use.
TABLE 4
As can be seen from the above table, in the samples of Experimental group 4, Na is contained2CO3The concentration is as follows: 1M of Na2CO32ml/L, heating to 115 deg.C, holding for 30min, cooling, and repeating for 4 times to obtain the most transformed saponinThe effect is best. In this way, Na is added2CO3Can avoid alkali pollution, is beneficial to environmental protection, and simultaneously has high content of the obtained rare saponin Rh 2.
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 (4)
1. A liquid culture medium for culturing adventitious roots of ginseng, which is characterized by comprising: 45g/L of sucrose, 1.28g/L B5 of culture medium, 0.905g/L of 1/2MS culture medium, 4.2mg/L of 6-benzylamino adenine, 3mg/L of naphthylacetic acid, 0.8mg/L of gibberellin, 0.6mg/L of indoleacetic acid and 0.6mg/L of indolebutyric acid.
2. A method for culturing ginseng adventitious roots is characterized by comprising the following steps:
cleaning and disinfecting mature ginseng, slicing, and inoculating the ginseng to an induction culture medium to induce adventitious roots of the ginseng; inoculating the obtained ginseng adventitious root to an induction culture medium again for subculture and propagation; then cutting the obtained ginseng adventitious roots into segments, inoculating the segments into the liquid culture medium of claim 1, and culturing to obtain adventitious roots;
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, 1.55g/L B5 culture medium, 1.21g/L WPM culture medium, 30g/L sucrose and 3g/L plant gel.
3. The method for culturing ginseng adventitious roots according to claim 2, comprising the steps of:
(1) cleaning and disinfecting mature ginseng, slicing, inoculating the ginseng into an induction culture medium, and performing dark culture at the temperature of 22 +/-1 ℃ for 4-5 weeks to induce adventitious roots of the ginseng;
(2) inoculating the ginseng adventitious roots obtained in the step (1) into the same induction culture medium as that in the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) and (3) shearing the adventitious roots of the ginseng obtained in the step (2) into small segments, inoculating the small segments 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.
4. The method for culturing ginseng adventitious roots according to claim 2 or 3, wherein the main root, or the reed head, or the part, or the branch root, or the fibrous root of the mature ginseng is washed, sterilized, sliced, and inoculated into an induction medium to induce ginseng adventitious roots.
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