CN113647326B - Method for culturing adventitious roots of ginseng - Google Patents
Method for culturing adventitious roots of ginseng Download PDFInfo
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Abstract
The invention discloses a method for culturing ginseng adventitious roots, which comprises the steps of cleaning, disinfecting, slicing mature ginseng, inoculating the ginseng into an induction culture medium, and inducing 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. Carrying out induction culture on a main root, or a reed head, or a portion of \33404m, or a branch root, or a fibrous root slice of the mature ginseng. The culture method of the adventitious roots can directly adopt each part of the mature ginseng to induce and generate the adventitious roots, does not need the intermediate step of inducing callus, can simplify the induction step and shorten the induction time.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a ginseng adventitious root and a preparation method thereof.
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 supply of ginseng cultivated in a 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; and (4) proliferation of adventitious roots of wild ginseng in the liquid culture medium. In the technical scheme, the callus needs to be induced first, then the adventitious root needs to be induced, the required period is long, the process is complex, and the content of the ginsenoside is not high.
Chinese patent application No. 201410698528.0 discloses a ginseng adventitious root induced proliferation method, which comprises the following steps: cutting tissue culture seedlings of ginseng into small tissue blocks, inoculating the small tissue blocks into a solid induction culture medium to induce and form adventitious roots, cutting the adventitious roots into small adventitious roots, and inoculating the small adventitious roots into a liquid multiplication culture medium to carry out multiplication culture of the adventitious roots; wherein the solid induction culture medium and the liquid proliferation culture medium are 1/2MS (-N) culture medium which is used as a basic culture medium and contains indolebutyric acid with the concentration of 1-10 mg/L. In the scheme, the tissue culture seedling with the age of 28-32 days is cut into small blocks to directly induce adventitious roots, the tissue culture seedling is tender and has strong differentiation capability, the tissue culture seedling is actually obtained by seed germination or explant culture, at least 28-32 days are still needed, and callus induction is still needed for the explant culture. Therefore, the cycle is not actually shortened.
Therefore, if a method capable of directly inducing the mature ginseng to produce adventitious roots can be found, the method capable of shortening the induction period and simplifying the induction conditions has a wide meaning.
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 can induce and generate the adventitious roots from each part of the mature ginseng, does not need the intermediate step of callus induction, and can simplify the induction step and shorten the induction time.
In order to solve the technical problems, the invention adopts the technical scheme that:
the first purpose of the invention is to provide a method for culturing ginseng adventitious roots, which comprises the steps of cleaning, disinfecting, slicing mature ginseng, inoculating the slices into an induction culture medium, and inducing 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 the segments are 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 a plant hormone, so that the growth of the 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, so that the adventitious roots are induced and generated directly from all parts of the mature ginseng 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 a B5 culture medium, a WPM culture medium or a 1/2MS culture medium as a basal culture medium, and contains 4mg/L of indolebutyric acid, 0.1g/L of citric acid, 0.05g/L of ascorbic acid and 30g/L of sucrose.
In the invention, when the adventitious roots generated by induction are further cultured in liquid, a culture medium for conventional culture, such as a 1/2MS culture medium, can be adopted and is consistent with a culture medium for the common adventitious roots, which shows that the adventitious roots generated by the one-step method of the invention have no difference with other two-step methods, can be cultured by adopting the conventional culture medium, and simultaneously, the induction time is shortened, and the pollution risk is reduced.
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 the step (1), and culturing in the dark for 4-5 weeks under the same conditions;
(3) Cutting the ginseng adventitious roots 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 more than 6 years;
as a preferred embodiment, the mature ginseng is a centennial ginseng. The century ginseng is rare in nature, has high edible and medicinal values, and can tonify five internal organs, calm spirit, calm soul, stop palpitation, remove pathogenic qi, improve eyesight, and benefit mind and intelligence; the value of the ginseng cultivation method is far higher than that of planted ginseng with short ginseng age. The invention does not need the intermediate step of inducing callus, can directly obtain the adventitious root by one-step induction from the hundred-year ginseng diced piece, not only can simplify the induction step and shorten the induction time, but also can obtain the specific functional components in the maternal hundred-year old ginseng, thereby obtaining the adventitious root with better nutritive value.
Further, the main root, or the reed head, or the portion of the mature ginseng, or the branch root, or the fibrous root 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, mountain 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 root of mature ginseng, sterilizing, cutting into slices with width of 0.5-0.7cm, length of 0.5-0.7cm and thickness of 0.2-0.5mm, inoculating to induction culture medium containing 4mg/L naphthylacetic acid, 0.6mg/L gibberellin, 0.4mg/L kinetin, 0.1g/L citric acid, 0.05g/L ascorbic acid, 30g/L sucrose, 3g/L plant gel, 1.55g/L B5 culture medium and 1.21g/L WPM culture medium, and dark culturing at 22 + -1 deg.C for 4-5 weeks to induce adventitious root of ginseng.
(2) Subculture of adventitious roots
Inoculating the ginseng adventitious roots obtained in the step (1) into the same induction culture medium as the step (1), and culturing in the dark for 4-5 weeks under the same conditions;
(3) Cultivation of adventitious roots
And (3) cutting the ginseng adventitious roots obtained in the step (2) into tissues with the length of about 1-2cm, inoculating the tissues into a liquid culture medium containing 4mg/L of indolebutyric acid, 0.1g/L of citric acid, 0.05g/L of ascorbic acid, 1/2MS culture medium and 30g/L of sucrose, and culturing for 3-4 weeks on a shaker at the temperature of 22 +/-1 ℃ to obtain the adventitious roots.
Furthermore, in order to improve the content of ginsenoside in the adventitious roots of the ginseng, the liquid culture medium is further screened and optimized.
As a preferred embodiment, the liquid medium comprises: 35g/L of sucrose, 1.2g/L of WPM medium, 1g/L of N6 medium, 50mg/L of ascorbic acid, 225mg/L of citric acid and 3mg/L of indolebutyric acid.
As still another preferred embodiment, the liquid medium comprises: 45g/L of sucrose, 1.28g/L of B5 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.
As another preferred embodiment, the liquid medium includes: 35g/L of sucrose, 1.35g/L of WPM medium, 1g/L of N6 medium, 5mg/L of naphthylacetic acid, 1mg/L of kinetin and 0.5mg/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, a WPM medium, a B5 medium, an N6 medium, a 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 KNO 3 2500，MgSO 4 ·7H 2 O 250，CaCl 2 ·2H 2 O 150，(NH 4 ) 2 SO 4 134，NaH 2 PO 4 .H 2 O 150，KI 0.75，H 3 BO 3 3.0，MnSO 4 ·4H 2 O 10，ZnSO 4 ·7H 2 O 2.0，Na 2 MoO 4 ·2H 2 O0.25，CoCl 2 ·6H 2 O 0.025，CuSO 4 ·5H 2 O 0.025，Na 2 -EDTA 37.3，FeSO 4 ·7H 2 O27.8, inositol 100, nicotinic acid 1.0, pyridoxine hydrochloride 1.0, and ammonium sulfate hydrochloride 10.
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, 1 part of vitamin B, 0.5 part of nicotinic acid, 0.5 part 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 ·7H 2 O) 185, calcium chloride (CaCl) 2 ·2H 2 O) 165, disodium ethylenediaminetetraacetate 37.3, ferrous sulfate (FeSO) 4 ·7H 2 O) 27.8, manganese sulfate (MnSO) 4 ·H 2 O) 4.4, zinc sulfate (ZnSO) 4 ·7H 2 O) 1.5, boric acid 1.6, potassium iodide 0.8, vitamin B1 (thiamine hydrochloride) 1.0, vitamin B6 (pyridoxine hydrochloride) 0.5, nicotinic acid 0.5, glycine 2.0, sucrose 20000, pH 5.8, 25 ℃.
After adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects:
1. the culture method of the adventitious roots realizes the one-step method, the processed each part of the mature ginseng is inoculated into the induction culture medium to directly induce the adventitious roots of the ginseng, the middle step of inducing callus is not needed, the induction step can be simplified, the induction time is shortened, and the pollution risk is reduced.
2. The 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 of B5 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 diagram of induction of adventitious roots using the induction medium of example 1 of the present invention;
FIG. 2 is a schematic diagram of induction of adventitious roots using the induction medium of comparative example 1.
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 the reed head and the portion \33404mfrom the wild ginseng of 20 ages, cleaning the main root, sterilizing, cutting into slices with the width of 0.6cm, the length of 0.7cm 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 the adventitious root of the wild ginseng; wherein the induction culture medium comprises 4mg/L naphthylacetic acid, 0.6mg/L gibberellin, 0.4mg/L kinetin, 0.1g/L citric acid, 0.05g/L ascorbic acid, 30g/L sucrose, 3g/L plant gel, 1.55g/L B5 culture medium and 1.21g/L WPM culture medium, and the pH value is 5.8.
(2) Subculture of adventitious roots
Inoculating the mountain ginseng adventitious roots obtained in the step (1) into the same induction culture medium as the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) Cultivation of adventitious roots
Cutting the mountain ginseng adventitious roots obtained in the step (2) into tissues with the length of about 1cm, inoculating the tissues into a liquid culture medium, and culturing the tissues on a shaker at the temperature of 22 +/-1 ℃ for 3-4 weeks to obtain adventitious roots; wherein the liquid culture medium contains 4mg/L indolebutyric acid, 0.1g/L citric acid, 0.05g/L ascorbic acid, 1/2MS culture medium and 30g/L sucrose, and has pH value of 5.8.
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 garden ginseng of 6 ages, 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 medium as 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 step (1) of this example can induce the generation of adventitious roots in one step, the number of adventitious roots example 3
(1) Induction of adventitious roots
Cleaning, disinfecting and sterilizing a portion of 10-age forest ginseng, cutting the cleaned portion into slices with the width of 0.7cm, the length of 0.7cm and the thickness of 0.5mm, inoculating the slices into an induction culture medium, and carrying out dark culture at the temperature of 22 +/-1 ℃ for 4-5 weeks to induce adventitious roots; wherein the induction culture medium comprises 5mg/L naphthylacetic acid, 1mg/L gibberellin, 0.1mg/L kinetin, 0.75g/L citric acid, 0.03g/L ascorbic acid, 40g/L sucrose, 4g/L plant gel, 2g/L B5 culture medium and 1g/L WPM culture medium, and the pH value is 6.0.
(2) Subculture of adventitious roots
Inoculating the adventitious roots obtained in the step (1) into the same induction medium as 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 radix Ginseng Indici, 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 adventitious root of radix Ginseng Indici; 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 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 of indolebutyric acid, 0.1g/L of citric acid, 0.05g/L of ascorbic acid, B5 culture medium and 30g/L of sucrose, and the pH value is 5.7.
Similar to the results of example 1, the step (1) of this example can directly induce the generation of adventitious roots in one step.
Example 5
(1) Induction of adventitious roots
Removing the reed head and the portion \33404mfrom the centenarian ginseng, cleaning the main root, sterilizing, cutting into slices with the width of 0.6cm, the length of 0.7cm 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 of the centenarian ginseng; wherein the induction culture medium comprises 4mg/L naphthylacetic acid, 0.6mg/L gibberellin, 0.4mg/L kinetin, 0.1g/L citric acid, 0.05g/L ascorbic acid, 30g/L sucrose, 3g/L plant gel, 1.55g/L B5 culture medium and 1.21g/L WPM culture medium, and the pH value is 5.8.
(2) Subculture of adventitious roots
Inoculating the mountain ginseng adventitious roots obtained in the step (1) into the same induction culture medium as the step (1), and carrying out dark culture for 4-5 weeks under the same conditions;
(3) Cultivation of adventitious roots
Cutting the mountain ginseng adventitious roots obtained in the step (2) into tissues with the length of about 1cm, inoculating the tissues into a liquid culture medium, and culturing the tissues on a shaker at the temperature of 22 +/-1 ℃ for 3-4 weeks to obtain adventitious roots; wherein the liquid culture medium contains 4mg/L indolebutyric acid, 0.1g/L citric acid, 0.05g/L ascorbic acid, 1/2MS culture medium and 30g/L sucrose, and has pH value of 5.8.
Similar to the results of example 1, in step (1) of this example, adventitious roots can be induced directly by one step.
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, 3g/L of WPM and 3g/L of plant gel, and the pH value is 5.8.
As a result: the whole body turns yellow in the first week, 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.
Test example 1 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 cultivation method in this test example was conducted in accordance with the method of example 1, except that the liquid medium in example 1 was replaced with various liquid media of the test example described below, and the ginsenoside content in the ginseng adventitious roots was measured.
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) 4 O): chromatographically pure acetonitrile (C) 6 H 11 N): pure chromatography
(3) Analytical procedure
Taking about 6g (accurate to 0.01 g) of the uniformly mixed sample, grinding the sample in a 150mL mortar, transferring the sample into a 50mL centrifuge tube, adding 10mL of water, uniformly mixing, breaking the wall for 3 minutes by 400W on an ultrasonic cell crusher, and freezing the sample in a refrigerator at the temperature of 18 ℃ below zero for 3 hours. Freeze-drying in a freeze-drying machine for 48 hours until no water drops exist outside the cup body.
The sample was ground in a mortar and 50mg was weighed accurately into a 10ml centrifuge tube, 70% methanol solution was added and vortexed. Sonicate for 10 minutes on a sonicator, repeat twice, filter for use.
(4) Reference conditions of the apparatus
A) A chromatographic column: c18 column, column length 150mm, column inner diameter 4.6mm, column packing particle size 5 μm, or equivalent;
b) Mobile phase: a: acetonitrile, b: filtering the water through a 0.45 mu m microporous filter membrane;
c) Flow rate: 0.7mL/min; gradient elution procedure: 0 to 13min,23 to 46 percent of acetonitrile, and the volume flow is 0.7mL/min; 13-33min, 46-68% acetonitrile, and volume flow of 0.7mL/min;33 to 46.5min,68 to 85 percent of acetonitrile, and the volume flow is 0.7mL/min;
d) Column temperature: 30 ℃;
e) Detection wavelength: 203nm;
f) Sample injection volume: 10 μ L.
(5) Presentation of analytical results
The content of each component of the ginsenoside in the sample is calculated according to the formula (1):
in the formula:
x is the content of each component of ginsenoside in the sample, and the unit is milligram per kilogram (mg/kg) or milligram per liter (mg/L);
a1-area of peak of each component of ginsenoside in sample
A2-peak area of each component of ginsenoside in standard
Rho-concentration of each component of ginsenoside in standard (ug/ml)
V — final volumetric volume of sample solution in milliliters (mL);
m-sample mass in grams (g);
the content of ginsenoside in the sample is the sum of the components to be detected.
The fold increase was calculated as follows:
multiple of growth = weight of adventitious roots after growth/weight of inoculated adventitious root seeds.
1. Liquid culture medium one
The first liquid culture medium comprises: 15-50g/L of sucrose, 0.6-2.4g/L of WPM medium, 1-2g/L of N6 medium, 0-150mg/L of ascorbic acid (Vc), 0-225mg/L of citric acid and 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.
2. Liquid culture medium II
The liquid culture medium II comprises: 10-55g/L of sucrose, 0.3-1.2g/L of 1/2MS culture medium, 0.6-1.6g/L of B5 culture medium, 0-5.4mg/L of indoleacetic acid (IAA), 0-5.4mg/L of indolebutyric acid (IBA), 0-5.4mg/L of naphthylacetic acid (NAA), 0-5.4mg/L of 6-benzylamino adenine (6 BA), 0-8mg/L of 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.
3. Liquid culture medium III
The liquid culture medium III comprises: 35g/L of sucrose, 1.35g/L of WPM medium, 1g/L of 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 of 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 of adventitious roots and the content of saponins at different addition amounts is examined, so as to select appropriate plant hormones and mixture ratios. As can be seen from the table, the growth factor of the adventitious roots and the content of the total saponins are better under the formulas of the experimental groups 4, 7-8, 10-12 and 15-18, wherein the comprehensive optimal value of the growth factor of the adventitious roots and the content of the total saponins is obtained under the condition of the experimental group 12.
Test example 2
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 > triglucoside > 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 Na 2 CO 3 The solution was added to prepared adventitious roots to make Na in each experimental group 2 CO 3 And (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, na 2 CO 3 Adding the solution into the grinded adventitious root, and adding Na into the sample 2 CO 3 The concentration of (A) is as follows: 1M Na 2 CO 3 8ml/L (corresponding 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.
A method of processing a sample comprising: 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 sample of Experimental group 4, na is contained 2 CO 3 The concentration is as follows: 1M of Na 2 CO 3 2ml/L, heating to 115 ℃, keeping the temperature for 30min, and repeating the treatment for 4 times after cooling, wherein the converted saponin obtained by the method comprehensively has the most and the best effect. In this way, na is added 2 CO 3 Can 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 the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the present invention.
Claims (11)
1. A method for culturing ginseng adventitious root is characterized in that mature ginseng is cleaned, disinfected, sliced and inoculated into an induction culture medium to induce the ginseng adventitious root; 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, and inoculating the segments into a liquid culture medium for culture to obtain the adventitious roots; the composition of the induction culture medium is 1-6mg/L naphthylacetic acid, 0.1-0.6mg/L kinetin, 0.2-1mg/L gibberellin, 0.75-1.5g/L citric acid, 0.03-1g/L ascorbic acid, 1-4g/L B5 culture medium, 1-2.4g/L WPM culture medium, 20-60g/L cane sugar and 1-6g/L plant gel.
2. A method for culturing adventitious root of Ginseng radix comprises cleaning and sterilizing mature Ginseng radix, slicing, inoculating into induction culture medium, and inducing to obtain adventitious root of Ginseng radix; 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 a liquid culture medium, and culturing to obtain adventitious roots; the composition of the induction culture medium is 1-6mg/L naphthylacetic acid, 0.1-0.6mg/L kinetin, 0.2-1mg/L gibberellin, 0.1g/L citric acid, 0.03-1g/L ascorbic acid, 1-4g/L B5 culture medium, 1-2.4g/L WPM culture medium, 20-60g/L cane sugar and 1-6g/L plant gel.
3. The method for culturing ginseng adventitious roots according to claim 2, wherein the composition of the induction medium is 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, 1.21g/L WPM medium, 30g/L sucrose and 3g/L plant gel.
4. The method for culturing adventitious roots of ginseng according to any one of claims 1 to 3, wherein the liquid medium is a basal medium consisting of B5 medium, WPM medium or 1/2MS medium, and contains 4mg/L of indolebutyric acid, 0.1g/L of citric acid, 0.05g/L of ascorbic acid and 30g/L of sucrose.
5. The method for culturing ginseng adventitious roots according to any one of claims 1 to 3, comprising the steps of:
(1) Cleaning and disinfecting mature ginseng, slicing, inoculating the ginseng into an induction culture medium, and carrying out dark culture at the temperature of 22 +/-1 ℃ for 4 to 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 in the step (1), and carrying out dark culture for 4 to 5 weeks under the same condition;
(3) Cutting the ginseng adventitious roots 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 under the condition of 22 +/-1 ℃ to obtain the adventitious roots.
6. The method for culturing adventitious roots of ginseng according to any one of claims 1 to 3, wherein the age of mature ginseng is 3 years or more.
7. The method for culturing adventitious roots of ginseng according to claim 6, wherein the age of mature ginseng is 6 years or more.
8. The method of claim 6, wherein the mature ginseng is centennial ginseng.
9. The method for culturing ginseng adventitious roots according to any one of claims 1 to 3, wherein the main root, or the reed head, or the portion of \33404m, 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.
10. The method for culturing adventitious roots of ginseng according to any one of claims 1 to 3, wherein the ginseng is selected from the group consisting of wild ginseng, mountain ginseng, under-forest ginseng and garden ginseng.
11. The method for culturing adventitious roots of ginseng according to claim 10, wherein the ginseng is wild ginseng.
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