CN103141391A - Cultural method of American ginseng adventitious root tissue - Google Patents
Cultural method of American ginseng adventitious root tissue Download PDFInfo
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- CN103141391A CN103141391A CN2013100863105A CN201310086310A CN103141391A CN 103141391 A CN103141391 A CN 103141391A CN 2013100863105 A CN2013100863105 A CN 2013100863105A CN 201310086310 A CN201310086310 A CN 201310086310A CN 103141391 A CN103141391 A CN 103141391A
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Abstract
The invention discloses a cultural method of American ginseng adventitious root tissue. The cultural method disclosed by the invention comprises the following steps: (1) inducing American ginseng callus; (2) secondarily culturing the American ginseng callus; (3) inducing the American ginseng adventitious root; (4) secondarily culturing the American ginseng adventitious root; and (5) culturing a reactor of the American ginseng adventitious root. According to the method disclosed by the invention, the growth velocity is much faster than the primary root. The root can be proliferated by 21.76 fold after culturing for a culture cycle; the productivity of the active secondary metabolite is high and stable; and meanwhile, the cultures are uniform and not affected by the natural environment such as natural disaster and the like. The cultural method of American ginseng adventitious root tissue disclosed by the invention has the characteristics of simple technical process, low cost, high inductivity, good repeatability and fast proliferation of the adventitious root.
Description
Technical field
The present invention relates to a kind of American Ginseng cultural method, belong to the Traditional Chinese medicine American ginseng field of tissue culture.
Background technology
American Ginseng (Panax quinquefolium L.) claims again U.S.'s ginseng (American ginseng), originates in eastern united states and Canada, is Araliaceae (Araliaceae) panax species, is used as medicine with root the most common.Modern pharmacology studies show that, American Ginseng has the glycometabolism of adjusting, strengthens immunity, delays senility, antitumor, hypotensive, prevention and Cardiovarscular, liver protecting, enhancing learning ability and the effects such as memory capability, intelligence development.Due to excessively excavating for many years, the forest ecological environment that American Ginseng is depended on for existence is seriously damaged, and wild American Ginseng resource exhaustion is used for the medicinal cultivation American Ginseng that mostly is at present.Yet the cultivation production cycle of American Ginseng is long, and floor space is large, and the impact of the easy climate of its quality, cultivation condition and damage by disease and insect, and supply is difficult to the demand of satisfying the market; The problems such as agriculture is residual to exceed standard, old ginseng have also greatly limited the development of artificial cultivation American Ginseng.The American Ginseng cultural method has the advantages such as be not subjected to season of growth restriction, do not carry virus, cultivation cycle is short, and is easy to carry out large-scale industrialization production, therefore has very large development prospect.China is a lot of about the report of American Ginseng cultivation at present, but mainly concentrates on the cultivation of callus and suspension cell.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of American Ginseng adventive root method for tissue culture is provided.
Technical scheme of the present invention is summarized as follows:
A kind of American Ginseng adventive root method for tissue culture comprises the steps:
(1) inducing of American Ginseng callus:
Get the tissue that the root skin is removed in the fresh elimination of American Ginseng after sterilization, be cut into 0.5～1cm
2Fritter be inoculated on the first solid culture medium, cultivated for 3～4 weeks under aseptic, 23～25 ℃ and lucifuge condition, form callus;
(2) subculture of American Ginseng callus is cultivated:
Callus is inoculated on the second solid culture medium, cultivated for 3～4 weeks under aseptic, 23～25 ℃ and lucifuge condition;
(3) inducing of American Ginseng adventive root:
The callus that the subculture that step (2) is obtained is turned out is inoculated on the third solid culture medium, cultivates for 3～5 weeks under aseptic, 23～25 ℃ and lucifuge condition, turns out the American Ginseng adventive root;
(4) subculture of American Ginseng adventive root is cultivated:
Pick out the American Ginseng adventive root that well-grown step (3) obtains and be inoculated in liquid nutrient medium, on shaking table, cultivated for 4～5 weeks under aseptic, 23-25 ℃ and lucifuge condition, obtain the American Ginseng adventive root;
(5) bioreactor culture of American Ginseng adventive root:
The American Ginseng adventive root that step (4) is obtained is inoculated in the spherical bubbling style reactor of the 3L that contains the 2L liquid nutrient medium, cultivates for 4～5 weeks, results, drying under aseptic, 20-25 ℃ and lucifuge condition;
Described the first solid culture medium is: contain 1.0-2.0mg/L (2,4-dichlorophenoxy) acetic acid (2,4-D), the MS solid culture medium of the pH=5.8-6.0 of 0.1-0.5mg/L kinetin (KT), 30-40g/L sucrose;
Described the second solid culture medium is: contain 1.0-2.0mg/L (2,4-dichlorophenoxy) acetic acid (2,4-D), the MS solid culture medium of the pH=5.8-6.0 of 0.1-0.5mg/L6-benzyladenine (6-BA), 30-40g/L sucrose;
Described the third solid culture medium is: the MS solid culture medium that contains the pH=5.8-6.0 of 1.0-5.0mg/L indolebutyric acid (IBA), 30-40g/L sucrose;
Described liquid nutrient medium is the MS liquid nutrient medium that contains the pH=5.8-6.0 of 3.0-5.0mg/L indolebutyric acid (IBA), 0.5-1.0mg/L methyl α-naphthyl acetate (NAA), 30-40g/L sucrose.
Advantage of the present invention:
The present invention utilizes method for tissue culture to cultivate the American Ginseng adventive root, have more practical significance than cultivating the American Ginseng cell, its growth rate is fast more a lot of than primitive root, (can breed 21.76 times after a cultivation cycle during bioreactor culture) and the productive rate of its active secondary metabolite are high and more stable, the culture homogeneous is not subjected to again the impact of the natural environments such as natural disaster simultaneously.It is simple that the present invention has technical process, with low cost, and inductivity is high, good reproducibility, and adventive root is bred the characteristics such as fast.Provide the indefinite Root tissue culture of a kind of American Ginseng and the method for product development.
Description of drawings
Fig. 1 is that hormon and proportioning are on the impact of root induction.
Fig. 2 is the foundation of American Ginseng adventive root liquid culture system; In figure: the C. adventive root of inducing of A. American Ginseng callus B. adventive root is inoculated in liquid culture medium D. American Ginseng adventive root cultivating system.
Fig. 3 is that hormon is to the adventive root affects on the growth.
Fig. 4 is the adventive root of American Ginseng adventive root bioreactor culture and results; In figure: A American Ginseng adventive root bioreactor culture; The adventive root of B American Ginseng adventive root bioreactor culture results.
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment.
Embodiment 1
A kind of American Ginseng adventive root method for tissue culture comprises the steps:
(1) inducing of American Ginseng callus:
Rinse well with running water for fresh with the cultivation American Ginseng, as for oven dry surface in 45 ℃ of baking ovens, then the ethanol with 75% sprays the surface in super-clean bench, extremely do with sterilization with the calcination of alcolhol burner flame, scalpel with the bacterium of going out cuts the otch of a 1cm on the root surface afterwards, and break into two with one's hands from both sides, then the tissue of inside is cut into 0.5～1cm
2Fritter receive on preprepared the first solid culture medium, cultivated for 4 weeks under aseptic, 23 ℃ and lucifuge condition, form callus;
(2) subculture of American Ginseng callus is cultivated:
Select well-grown callus, be inoculated on ready the second solid culture medium with scalpel and the tweezers of in advance sterilization, cultivated for 4 weeks under aseptic, 23 ℃ and lucifuge condition;
(3) inducing of American Ginseng adventive root:
The callus that the subculture of selecting well-grown step (2) to obtain is turned out is inoculated on the third solid culture medium with scalpel and the tweezers of sterilization in advance, cultivates for 4 weeks under aseptic, 23 ℃ and lucifuge condition, turns out the American Ginseng adventive root;
(4) subculture of American Ginseng adventive root is cultivated:
Pick out American Ginseng adventive root that well-grown step (3) obtains and be inoculated in liquid nutrient medium with scalpel and the tweezers of sterilization in advance, on the shaking table of 100rpm speed, cultivated for 4 weeks under aseptic, 23 ℃ and lucifuge condition, obtain the American Ginseng adventive root; Can set up American Ginseng adventive root liquid culture system (see figure 2), in incubation, hormon is seen Fig. 3 to the adventive root affects on the growth.
(5) bioreactor culture of American Ginseng adventive root:
The American Ginseng adventive root of choosing well-grown step (4) acquisition is inoculated in the spherical bubbling style reactor of the 3L that contains the 2L liquid nutrient medium with scalpel and the tweezers of sterilizing in advance, cultivated for 5 weeks under aseptic, 23 ℃ and lucifuge condition, results American Ginseng adventive root is seen Fig. 4, drying;
Being formulated as of the first solid culture medium: 100mL is contained 2.0mg/L2, the MS solid culture medium of 4-D, 0.5mg/L KT, 30g/L sucrose is transferred pH to 5.8 with the HCl aqueous solution of 1mol/L and the NaOH aqueous solution of 1mol/L, and sterilization is 15 minutes under 121 ℃, the pressure of 0.1MPa;
The second solid culture medium is formulated as: 100mL is contained 2.0mg/L2, the MS solid culture medium of 4-D, 0.5mg/L6-BA, 30g/L sucrose is transferred pH to 5.8 with the HCl aqueous solution of 1mol/L and the NaOH aqueous solution of 1mol/L, and sterilization 15 minutes is standby under 121 ℃, the pressure of 0.1MPa;
The third solid culture medium is formulated as: the MS solid culture medium that 100mL contains 3.0mg/L IBA, 40g/L sucrose is transferred pH to 5.8 with the HCl aqueous solution of 1mol/L and the NaOH aqueous solution of 1mol/L, and sterilization 15 minutes is standby under 121 ℃, the pressure of 0.1MPa;
Liquid nutrient medium is formulated as: the MS liquid nutrient medium that 100mL contains 3.0mg/L IBA, 1.0mg/L NAA, 40g/L sucrose is transferred pH to 5.8 with the HCl aqueous solution of 1mol/L and the NaOH aqueous solution of 1mol/L, and sterilization 15 minutes is standby under 121 ℃, the pressure of 0.1MPa.
Embodiment 2
A kind of American Ginseng adventive root method for tissue culture comprises the steps:
(1) inducing of American Ginseng callus:
Get the tissue that the root skin is removed in the fresh elimination of American Ginseng after sterilization, be cut into 0.5～1cm
2Fritter be inoculated on the first solid culture medium, cultivated for 3.5 weeks under aseptic, 24 ℃ and lucifuge condition, form callus;
(2) subculture of American Ginseng callus is cultivated:
Callus is inoculated on the second solid culture medium, cultivated for 3.5 weeks under aseptic, 24 ℃ and lucifuge condition;
(3) inducing of American Ginseng adventive root:
The callus that the subculture that step (2) is obtained is turned out is inoculated on the third solid culture medium, cultivates for 5 weeks under aseptic, 24 ℃ and lucifuge condition, turns out the American Ginseng adventive root;
(4) subculture of American Ginseng adventive root is cultivated:
Pick out the American Ginseng adventive root that well-grown step (3) obtains and be inoculated in liquid nutrient medium, on shaking table, cultivated for 4.5 weeks under aseptic, 24 ℃ and lucifuge condition, obtain the American Ginseng adventive root;
(5) bioreactor culture of American Ginseng adventive root:
The American Ginseng adventive root that step (4) is obtained is inoculated in the spherical bubbling style reactor of the 3L that contains the 2L liquid nutrient medium, cultivates for 4.5 weeks, results, drying under aseptic, 20 ℃ and lucifuge condition;
The first solid culture medium is: 100mL is contained 1.0mg/L2, the MS solid culture medium of 4-D, 0.1mg/L KT, 35g/L sucrose is transferred pH to 5.9 with the HCl aqueous solution of 1mol/L and the NaOH aqueous solution of 1mol/L, and sterilization is 15 minutes under 121 ℃, the pressure of 0.1MPa;
The second solid culture medium is formulated as: 100mL is contained 1.0mg/L2, the MS solid culture medium of 4-D, 0.1mg/L6-BA, 35g/L sucrose is transferred pH to 5.9 with the HCl aqueous solution of 1mol/L and the NaOH aqueous solution of 1mol/L, and sterilization 15 minutes is standby under 121 ℃, the pressure of 0.1MPa;
The third solid culture medium is formulated as: the MS solid culture medium that 100mL contains 1.0mg/L IBA, 30g/L sucrose is transferred pH to 5.9 with the HCl aqueous solution of 1mol/L and the NaOH aqueous solution of 1mol/L, and sterilization 15 minutes is standby under 121 ℃, the pressure of 0.1MPa;
Liquid nutrient medium is formulated as: the MS liquid nutrient medium that 100mL contains 5.0mg/L IBA, 1.0mg/L NAA, 30g/L sucrose is transferred pH to 5.9 with the HCl aqueous solution of 1mol/L and the NaOH aqueous solution of 1mol/L, and sterilization 15 minutes is standby under 121 ℃, the pressure of 0.1MPa.
Embodiment 3
A kind of American Ginseng adventive root method for tissue culture comprises the steps:
(1) inducing of American Ginseng callus:
Get the tissue that the root skin is removed in the fresh elimination of American Ginseng after sterilization, be cut into 0.5～1cm
2Fritter be inoculated on the first solid culture medium, cultivated for 3 weeks under aseptic, 25 ℃ and lucifuge condition, form callus;
(2) subculture of American Ginseng callus is cultivated:
Callus is inoculated on the second solid culture medium, cultivated for 3 weeks under aseptic, 25 ℃ and lucifuge condition;
(3) inducing of American Ginseng adventive root:
The callus that the subculture that step (2) is obtained is turned out is inoculated on the third solid culture medium, cultivates for 3 weeks under aseptic, 25 ℃ and lucifuge condition, turns out the American Ginseng adventive root;
(4) subculture of American Ginseng adventive root is cultivated:
Pick out the American Ginseng adventive root that well-grown step (3) obtains and be inoculated in liquid nutrient medium, on shaking table, cultivated for 4 weeks under aseptic, 25 ℃ and lucifuge condition, obtain the American Ginseng adventive root;
(5) bioreactor culture of American Ginseng adventive root:
The American Ginseng adventive root that step (4) is obtained is inoculated in the spherical bubbling style reactor of the 3L that contains the 2L liquid nutrient medium, cultivates for 4 weeks, results, drying under aseptic, 25 ℃ and lucifuge condition;
The first solid culture medium is: 100mL is contained 1.5mg/L2, the MS solid culture medium of 4-D, 0.3mg/L KT, 40g/L sucrose is transferred pH to 6.0 with the HCl aqueous solution of 1mol/L and the NaOH aqueous solution of 1mol/L, and sterilization is 15 minutes under 121 ℃, the pressure of 0.1MPa;
The second solid culture medium is formulated as: 100mL is contained 1.5mg/L2, the MS solid culture medium of 4-D, 0.3mg/L6-BA, 40g/L sucrose is transferred pH to 6.0 with the HCl aqueous solution of 1mol/L and the NaOH aqueous solution of 1mol/L, and sterilization 15 minutes is standby under 121 ℃, the pressure of 0.1MPa;
The third solid culture medium is formulated as: the MS solid culture medium that 100mL contains 5.0mg/L IBA, 35g/L sucrose is transferred pH to 6.0 with the HCl aqueous solution of 1mol/L and the NaOH aqueous solution of 1mol/L, and sterilization 15 minutes is standby under 121 ℃, the pressure of 0.1MPa;
Liquid nutrient medium is formulated as: the MS liquid nutrient medium that 100mL contains 3.0mg/L IBA, 1.0mg/L NAA, 35g/L sucrose is transferred pH to 6.0 with the HCl aqueous solution of 1mol/L and the NaOH aqueous solution of 1mol/L, and sterilization 15 minutes is standby under 121 ℃, the pressure of 0.1MPa.
Experiment shows, the American Ginseng adventive root that each embodiment of method of the present invention cultivates, its growth rate is fast more a lot of than primitive root, and (can breed 21.76 times after a cultivation cycle during bioreactor culture) and the productive rate of its active secondary metabolite are high and more stable, the culture homogeneous.
Get dried American Ginseng adventive root, add the distilled water of 15-25 times of weight, standing 5-12h, water-bath 1-5 time, each 1-3h.Repeat water-bath 2-5 time, merging filtrate is concentrated to certain volume, and take absolute ethyl alcohol modulation final concentration as 50-90%, 0-6 ℃ of hold over night removed supernatant, and with sevage method deproteinized, drying is pulverized, and obtains the Radix Panacis Quinquefolii polysaccharide powder.
Get the above-mentioned Radix Panacis Quinquefolii polysaccharide of 100g, 200g HPMC, 150g lactose, after mixing, cross 100 mesh sieves, add the ethanolic solution of the ethyl cellulose of 20g1-5%, then 60 ℃ of dry 5h cross the 20 whole grains of mesh sieve, namely obtain the Radix Panacis Quinquefolii polysaccharide granular preparation.
Claims (1)
1. an American Ginseng adventive root method for tissue culture, is characterized in that comprising the steps:
(1) inducing of American Ginseng callus:
Get the tissue that the root skin is removed in the fresh elimination of American Ginseng after sterilization, be cut into 0.5～1cm
2Fritter be inoculated on the first solid culture medium, cultivated for 3～4 weeks under aseptic, 23～25 ℃ and lucifuge condition, form callus;
(2) subculture of American Ginseng callus is cultivated:
Callus is inoculated on the second solid culture medium, cultivated for 3～4 weeks under aseptic, 23～25 ℃ and lucifuge condition;
(3) inducing of American Ginseng adventive root:
The callus that the subculture that step (2) is obtained is turned out is inoculated on the third solid culture medium, cultivates for 3～5 weeks under aseptic, 23～25 ℃ and lucifuge condition, turns out the American Ginseng adventive root;
(4) subculture of American Ginseng adventive root is cultivated:
Pick out the American Ginseng adventive root that well-grown step (3) obtains and be inoculated in liquid nutrient medium, on shaking table, cultivated for 4～5 weeks under aseptic, 23-25 ℃ and lucifuge condition, obtain the American Ginseng adventive root;
(5) bioreactor culture of American Ginseng adventive root:
The American Ginseng adventive root that step (4) is obtained is inoculated in the spherical bubbling style reactor of the 3L that contains the 2L liquid nutrient medium, cultivates for 4～5 weeks, results, drying under aseptic, 20-25 ℃ and lucifuge condition;
Described the first solid culture medium is: the MS solid culture medium that contains the pH=5.8-6.0 of 1.0-2.0mg/L (2,4-dichlorophenoxy) acetic acid, 0.1-0.5mg/L kinetin, 30-40g/L sucrose;
Described the second solid culture medium is: the MS solid culture medium that contains the pH=5.8-6.0 of 1.0-2.0mg/L (2,4-dichlorophenoxy) acetic acid, 0.1-0.5mg/L6-benzyladenine, 30-40g/L sucrose;
Described the third solid culture medium is: the MS solid culture medium that contains the pH=5.8-6.0 of 1.0-5.0mg/L indolebutyric acid, 30-40g/L sucrose;
Described liquid nutrient medium is the MS liquid nutrient medium that contains the pH=5.8-6.0 of 3.0-5.0mg/L indolebutyric acid, 0.5-1.0mg/L methyl α-naphthyl acetate, 30-40g/L sucrose.
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| CN104885949A (en) * | 2015-06-08 | 2015-09-09 | 安徽农业大学 | Method for inducing panax quinquefolium callus |
| CN108531440A (en) * | 2018-04-28 | 2018-09-14 | 刘汉石 | A kind of ginseng-cell culture medium and its application |
| CN109220801A (en) * | 2018-10-23 | 2019-01-18 | 大连工业大学 | A kind of method for plant tissue culture |
| CN110235784A (en) * | 2019-07-19 | 2019-09-17 | 东北农业大学 | A method of induction Liao Dong Aralia wood adventitious root occurs and proliferation |
| CN111134010A (en) * | 2020-01-06 | 2020-05-12 | 烟台大学 | Method for preparing American ginseng adventitious roots with high antioxidant activity |
| CN112956415A (en) * | 2021-01-18 | 2021-06-15 | 山东安然纳米实业发展有限公司 | Liquid culture medium for culturing ginseng adventitious roots and culture method |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104885949A (en) * | 2015-06-08 | 2015-09-09 | 安徽农业大学 | Method for inducing panax quinquefolium callus |
| CN108531440A (en) * | 2018-04-28 | 2018-09-14 | 刘汉石 | A kind of ginseng-cell culture medium and its application |
| CN109220801A (en) * | 2018-10-23 | 2019-01-18 | 大连工业大学 | A kind of method for plant tissue culture |
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| CN110235784A (en) * | 2019-07-19 | 2019-09-17 | 东北农业大学 | A method of induction Liao Dong Aralia wood adventitious root occurs and proliferation |
| CN111134010A (en) * | 2020-01-06 | 2020-05-12 | 烟台大学 | Method for preparing American ginseng adventitious roots with high antioxidant activity |
| CN112956415A (en) * | 2021-01-18 | 2021-06-15 | 山东安然纳米实业发展有限公司 | Liquid culture medium for culturing ginseng adventitious roots and culture method |
| CN112956415B (en) * | 2021-01-18 | 2021-09-24 | 山东安然纳米实业发展有限公司 | Liquid culture medium for culturing ginseng adventitious roots and culture method |
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Application publication date: 20130612 |