CN109729974A - A kind of method for tissue culture of Low- temperature culture ginseng adventitious root - Google Patents
A kind of method for tissue culture of Low- temperature culture ginseng adventitious root Download PDFInfo
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- CN109729974A CN109729974A CN201910111145.1A CN201910111145A CN109729974A CN 109729974 A CN109729974 A CN 109729974A CN 201910111145 A CN201910111145 A CN 201910111145A CN 109729974 A CN109729974 A CN 109729974A
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Abstract
A kind of method for tissue culture of Low- temperature culture ginseng adventitious root of disclosure of the invention, include the following steps: that (1) sucrose is added in 3/4MS culture medium and makes final concentration of 40g/L, indolebutyric acid, which is added, makes final concentration of 5mg/L, 6-Furfurylaminopurine, which is added, makes final concentration of 0.1mg/L, adjust pH=5.8-5.92, fluid nutrient medium is obtained, is sterilized；(2) segment that ginseng adventitious root is cut into 0.5-1cm is inoculated in the fluid nutrient medium after sterilizing, 4-13 DEG C shaking table culture 1 week, shaking table temperature is adjusted to 23-25 DEG C, 4 weeks harvests of culture.Method of the invention has easy to operate, the advantages such as cultivation cycle is short, and growth cost is low.The present invention is grown by Low temperature regulation ginseng adventitious root, under the premise of not influencing biomass, so that the content of ginsenoside is significantly improved.
Description
Technical field
The present invention relates to field of plant tissue culture technique, and in particular to a kind of tissue training of Low- temperature culture ginseng adventitious root
The method of supporting.
Background technique
Ginseng is araliaceae ginseng plant, is traditional Chinese medicine, is known as King of Herbs, first of the Triratna of northeast.Ginseng
Saponin(e is its main active constituent, has antifatigue, anti-aging, adjusts central nervous system, improve immunity of organisms, improve
Cardiovascular and cerebrovascular blood supply insufficiency, inhibition growth of tumour cell and other effects.Currently, the market demand to ginseng increases year by year, however plant
The problems such as growth cycle needed for training ginseng is longer, affected by environment serious and " old ginseng " is difficult to solve, so that ginseng raw
Material medicine supply is difficult to meet the needs of market.Therefore, plant tissue culture technique is used to part and solves the problems, such as ginseng resource, raw
Produce ginseng medicinal active ingredient.
Currently, it is current urgent problem to be solved that saponin content is lower in tissue culture ginseng adventitious root.
Summary of the invention
The present invention is provided ginsenoside in a kind of raising ginseng adventitious root and is contained by the cultivation temperature of control ginseng adventitious root
The method for tissue culture of the Low- temperature culture ginseng adventitious root of amount.
Technical solution of the present invention is summarized as follows:
A kind of method for tissue culture of Low- temperature culture ginseng adventitious root, includes the following steps:
(1) sucrose is added in 3/4MS culture medium makes final concentration of 40g/L, and indolebutyric acid, which is added, makes final concentration of 5mg/
L, 6-Furfurylaminopurine, which is added, makes final concentration of 0.1mg/L, adjusts pH=5.8-5.92, obtains fluid nutrient medium, sterilizes；
(2) segment that ginseng adventitious root is cut into 0.5-1cm is inoculated in the fluid nutrient medium after sterilizing, is shaken at 4-13 DEG C
Shaking table temperature is adjusted to 23-25 DEG C by bed culture 1 week, is cultivated 4 weeks and is harvested.
Method of the invention has easy to operate, the advantages such as cultivation cycle is short, and growth cost is low.The present invention passes through low temperature tune
Ginseng adventitious root growth is controlled, under the precursor for not influencing biomass, so that the content of ginsenoside is significantly improved.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below.
The method of fluid nutrient medium sterilizing of the invention is preferred: fluid nutrient medium is fitted into the shaking flask of 250mL 121
DEG C, it sterilizes 25 minutes under 0.1MP pressure.
Embodiment 1
A kind of method for tissue culture of Low- temperature culture ginseng adventitious root, includes the following steps:
(1) sucrose is added in 3/4MS culture medium makes final concentration of 40g/L, and indolebutyric acid, which is added, makes final concentration of 5mg/
L, 6-Furfurylaminopurine, which is added, makes final concentration of 0.1mg/L, adjusts pH=5.85, obtains fluid nutrient medium, sterilizes；
(2) segment that ginseng adventitious root is cut into 0.5-1cm is inoculated in the fluid nutrient medium after sterilizing, in 4 DEG C of shaking tables
Shaking table temperature is adjusted to 23 DEG C by culture 1 week, is cultivated 4 weeks and is harvested.
Embodiment 2
A kind of method for tissue culture of Low- temperature culture ginseng adventitious root, includes the following steps:
(1) sucrose is added in 3/4MS culture medium makes final concentration of 40g/L, and indolebutyric acid, which is added, makes final concentration of 5mg/
L, 6-Furfurylaminopurine, which is added, makes final concentration of 0.1mg/L, adjusts pH=5.92, obtains fluid nutrient medium, sterilizes；
(2) segment that ginseng adventitious root is cut into 0.5-1cm is inoculated in the fluid nutrient medium after sterilizing, in 7 DEG C of shaking tables
Shaking table temperature is adjusted to 25 DEG C by culture 1 week, is cultivated 4 weeks and is harvested.
Embodiment 3
A kind of method for tissue culture of Low- temperature culture ginseng adventitious root, includes the following steps:
(1) sucrose is added in 3/4MS culture medium makes final concentration of 40g/L, and indolebutyric acid, which is added, makes final concentration of 5mg/
L, 6-Furfurylaminopurine, which is added, makes final concentration of 0.1mg/L, adjusts pH=5.83, obtains fluid nutrient medium, sterilizes；
(2) segment that ginseng adventitious root is cut into 0.5-1cm is inoculated in the fluid nutrient medium after sterilizing, in 10 DEG C of shaking tables
Shaking table temperature is adjusted to 24 DEG C by culture 1 week, is cultivated 4 weeks and is harvested.
Embodiment 4
A kind of method for tissue culture of Low- temperature culture ginseng adventitious root, includes the following steps:
(1) sucrose is added in 3/4MS culture medium makes final concentration of 40g/L, and indolebutyric acid, which is added, makes final concentration of 5mg/
L, 6-Furfurylaminopurine, which is added, makes final concentration of 0.1mg/L, adjusts pH=5.8, obtains fluid nutrient medium, sterilizes；
(2) segment that ginseng adventitious root is cut into 0.5-1cm is inoculated in the fluid nutrient medium after sterilizing, in 13 DEG C of shaking tables
Shaking table temperature is adjusted to 24 DEG C by culture 1 week, is cultivated 4 weeks and is harvested.
Reference examples
The method for tissue culture of ginseng adventitious root, includes the following steps:
(1) sucrose is added in 3/4MS culture medium makes final concentration of 40g/L, and indolebutyric acid, which is added, makes final concentration of 5mg/
L, 6-Furfurylaminopurine, which is added, makes final concentration of 0.1mg/L, adjusts pH=5.82, obtains fluid nutrient medium, sterilizes；
(2) segment that ginseng adventitious root is cut into 0.5-1cm is inoculated in the fluid nutrient medium after sterilizing, by shaking table temperature
24 DEG C of cultures are adjusted to harvest within 5 weeks.
The measuring method of ginsenoside kind compound content
1) chromatographic condition
Liquid phase chromatogram condition: chromatographic column is Kromasil C18 (4.6mm × 250mm, 5 μm)；Mobile phase (A) is acetonitrile,
Mobile phase (B) is ultrapure water；Gradient elution program: 0 → 35min, A:20%；35 → 40min, A:20% → 30%；40→
50min, A:30% → 31%；50 → 60min, A:31% → 32%；60 → 70min, A:32% → 45%；70→100min,
A:45% → 60%；100 → 110min, A:60% → 60%；Flow velocity 1.0mL/min；Sampling volume is 20 μ L；35 DEG C of column temperature；
Detection wavelength 203nm.
2) preparation of standard solution
Precision weighs the ginsenoside Rg through being dried under reduced pressure 24 hours or more1、Re、Ro、Rf、Rb1、Rg2、Rh1、Rc、Rb2、
Rb3、Rd、Rg3、Rh2Each 1mg of standard items, is placed in 5mL volumetric flask, and a small amount of methanol ultrasonic dissolution, again with methanol constant volume is made into
4 DEG C of refrigerator preservations are set in the ginsenoside hybrid standard product stock solution of 0.2mg/mL, sealing.
3) preparation of sample solution
Precision weighs the dry adventitious root sample of 0.2g and is placed in 100mL triangular flask, and 20mL methanol, 60 DEG C of water are added
1h is bathed, is filtered while hot, repeats to extract primary, merging filtrate.Filtrate volatilizes, and the dissolution of 10mL distilled water is added, with the positive fourth of 20mL
Alcohol is extracted twice, and n-butanol layer is collected, after rotary evaporation is handled, with methanol constant volume to 1mL.With 0.22 μm of miillpore filter mistake
Filter, obtains sample solution.Testing result is shown in Table 1.
1 ginseng adventitious root total saponin content of table
| Group | Embodiment 1 | Example applies 2 | Example applies 3 | Example applies 4 | Reference examples |
| Total saponin content (mg/g) | 25.57±0.31 | 30.23±0.65 | 32.30±0.71 | 22.40±1.11 | 15.59±0.90 |
With reference examples ratio, its total saponin content of product that the method for embodiment 1- embodiment 4 obtains is much higher than reference examples
Total Ginsenosides Content may be up to 32.30mg/g in ginseng adventitious root in content, especially embodiment 3, mention compared with reference examples
It is 2.07 times high.
Although above having used general explanation and specific embodiment party, the present invention is described in detail, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention are fallen within the scope of the claimed invention.
Claims (1)
1. a kind of method for tissue culture of Low- temperature culture ginseng adventitious root, it is characterized in that including the following steps:
(1) sucrose is added in 3/4MS culture medium makes final concentration of 40g/L, and indolebutyric acid, which is added, makes final concentration of 5mg/L, adds
Entering 6-Furfurylaminopurine makes final concentration of 0.1mg/L, adjusts pH=5.8-5.92, obtains fluid nutrient medium, sterilizes；
(2) segment that ginseng adventitious root is cut into 0.5-1cm is inoculated in the fluid nutrient medium after sterilizing, is trained in 4-13 DEG C of shaking table
It supports 1 week, shaking table temperature is adjusted to 23-25 DEG C, cultivate 4 weeks and harvest.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112544443A (en) * | 2020-12-01 | 2021-03-26 | 延边朝鲜族自治州农业科学院（延边特产研究所） | Method for rapidly propagating mountain ginseng adventitious roots |
| CN113647325A (en) * | 2021-01-18 | 2021-11-16 | 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 |
| CN113655130A (en) * | 2021-01-18 | 2021-11-16 | 山东安然纳米实业发展有限公司 | HPLC (high performance liquid chromatography) detection method and application of ginsenoside Ro |
| CN114403005A (en) * | 2022-01-27 | 2022-04-29 | 上海瑞帝安生物科技有限公司 | Ginseng adventitious root induction method and application of extract in cosmetics |
| CN115053810A (en) * | 2022-06-29 | 2022-09-16 | 珲春华瑞参业生物工程股份有限公司 | Culture method for simultaneously increasing contents of multiple ginsenosides in ginseng adventitious roots |
| CN115152628A (en) * | 2022-06-29 | 2022-10-11 | 珲春华瑞参业生物工程股份有限公司 | Adventitious root metaphase cultivation method and culture medium for improving PPD saponin content in ginseng adventitious roots |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112544443A (en) * | 2020-12-01 | 2021-03-26 | 延边朝鲜族自治州农业科学院（延边特产研究所） | Method for rapidly propagating mountain ginseng adventitious roots |
| CN112544443B (en) * | 2020-12-01 | 2022-03-29 | 吉林省农业科学院 | Method for rapidly propagating mountain ginseng adventitious roots |
| CN113647325A (en) * | 2021-01-18 | 2021-11-16 | 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 |
| CN113655130A (en) * | 2021-01-18 | 2021-11-16 | 山东安然纳米实业发展有限公司 | HPLC (high performance liquid chromatography) detection method and application of ginsenoside Ro |
| CN113647325B (en) * | 2021-01-18 | 2022-11-29 | 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 |
| CN113655130B (en) * | 2021-01-18 | 2023-09-22 | 山东安然纳米实业发展有限公司 | HPLC detection method and application of ginsenoside Ro |
| CN114403005A (en) * | 2022-01-27 | 2022-04-29 | 上海瑞帝安生物科技有限公司 | Ginseng adventitious root induction method and application of extract in cosmetics |
| CN115053810A (en) * | 2022-06-29 | 2022-09-16 | 珲春华瑞参业生物工程股份有限公司 | Culture method for simultaneously increasing contents of multiple ginsenosides in ginseng adventitious roots |
| CN115152628A (en) * | 2022-06-29 | 2022-10-11 | 珲春华瑞参业生物工程股份有限公司 | Adventitious root metaphase cultivation method and culture medium for improving PPD saponin content in ginseng adventitious roots |
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