CN107646685A - A kind of method for industrializing Herba Saussureae Involueratae culture of the large-scale production rich in flavones - Google Patents
A kind of method for industrializing Herba Saussureae Involueratae culture of the large-scale production rich in flavones Download PDFInfo
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- CN107646685A CN107646685A CN201711037131.7A CN201711037131A CN107646685A CN 107646685 A CN107646685 A CN 107646685A CN 201711037131 A CN201711037131 A CN 201711037131A CN 107646685 A CN107646685 A CN 107646685A
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- herba saussureae
- saussureae involueratae
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The present invention relates to a kind of method for industrializing Herba Saussureae Involueratae culture of the large-scale production rich in flavones, belong to saussurea involucrata Cultivating techniques field.It is by inducing Herba Saussureae Involueratae callus, screening Herba Saussureae Involueratae culture and large-scale production Herba Saussureae Involueratae culture.The present invention uses sterilized Herba Saussureae Involueratae seed culture aseptic seedling, Herba Saussureae Involueratae callus is induced with aseptic seedling, change culture medium, cultivation temperature, stable propagation, character stabilization, the Herba Saussureae Involueratae culture rich in flavones are filtered out, and uses solid culture/liquid suspension culture and gas-lifting type culture large-scale production Herba Saussureae Involueratae culture.The present invention can be on the basis of flavones content be ensured, stable, high-yielding Herba Saussureae Involueratae culture, and has the advantages of with short production cycle, production quality is unified, adaptation normal temperature growth condition.
Description
Technical field
The present invention relates to a kind of method for industrializing Herba Saussureae Involueratae culture of the large-scale production rich in flavones, belong to saussurea involucrata
Cultivating techniques field.
Background technology
Herba Saussureae Involueratae contains the plurality of active ingredients such as flavones, alkaloid, polysaccharide, has invigorate blood circulation, dispelling cold and removing dampness, hemostasis
Detumescence, isostere endotoxin and other effects.In recent years, Herba Saussureae Involueratae as ethnic drug and Folk medicine in anti-inflammatory and antalgic, Robust speaker feature, anti-ageing
Effect in terms of old and suppression cancer cell hyperplasia receives much concern.
Herba Saussureae Involueratae is born in the rock seam near the high mountain snow line of 2400 ~ 4000 meters of height above sea level, cliff and ice sulphur ruckle, from
Because growing environment is special under the conditions of so, it can just yield positive results within 3 to 5 years, seed flies away with the wind in intermountain during the florescence, only runs into
The seed of control environment is possible to sprout and grown, therefore natural propagation rate is relatively low.Herba Saussureae Involueratae is classified as by China within 1996
Second class protection plant, Herba Saussureae Involueratae are uniquely to be included in《Chinese Plants Red Data Book》Saussurea involucrata plant, be that China national three-level is in imminent danger
Species.No. 13 files of State Council in 2000 have prohibited the wild saussurea involucrata of excavation.
Due to the harsh growth conditions of Herba Saussureae Involueratae and relatively low breeding potential, plus the long-term excessive excavation of people, make day
Mountain saussurea involucrata resource wretched insufficiency.Herba Saussureae Involueratae culture rich in flavones is cultivated by plant tissue culture engineering, on the one hand solved
The problem of Herba Saussureae Involueratae inadequate resource, on the other hand improve the content of active ingredient.
The content of the invention
The purpose of the present invention is to overcome the weak point that flavones content in Herba Saussureae Involueratae culture is low, quality differs, there is provided
It is a kind of industrialize large-scale production rich in flavones Herba Saussureae Involueratae culture method, can on the basis of flavones content is ensured,
Stable, high-yielding Herba Saussureae Involueratae culture.
Technical scheme, a kind of method for industrializing Herba Saussureae Involueratae culture of the large-scale production rich in flavones,
Step is as follows：
（1）Aseptic seedling is induced after sterilization treatment using Herba Saussureae Involueratae seed；
（2）The leaf, root and stem of aseptic seedling are forwarded on the culture medium of MS addition plant hormones, evoked callus；
（3）Pick out that color is most deep, well-grown callus, the squamous subculture on screening and culturing medium, obtain purple, propagation
It hurry up, the Saussurea involucrata culture A that character is stable；
（4）Saussurea involucrata culture A is placed in 10 ~ 12 DEG C of 15 ~ 20d of cultivation, multiple subculture, constantly selects color on screening and culturing medium
Saussurea involucrata culture most purple, character is best, 3 degree of cultures are improved every 2 months temperature, until 23 ~ 27 DEG C, select and obtain saussurea involucrata training
Support thing B；
（5）Saussurea involucrata culture B is subjected to large-scale gas-lifting type culture, it is the Saussurea involucrata culture rich in flavones to collect drying.
Step（1）Concretely comprise the following steps：It is 75% ethanol immersion Herba Saussureae Involueratae seed, 30 ~ 40s with mass concentration, it is then sterile
Water washing 2 ~ 3 times, then 15 ~ 20min is soaked with the mercuric chloride solution that mass concentration is 0.1%, sterile water wash 3 ~ 5 times, it is inoculated in
On MS solid mediums, carry out illumination cultivation and obtain aseptic seedling, illumination condition is 2000 ~ 3000Lux, 10 ~ 12h/d.
Step（2）Comprise the following steps that：By step（1）Leaf, root and the stem of gained aseptic seedling be inoculated in and with the addition of 0.5 ~
On 2mg/L 6-BA and 0.5 ~ 2mg/L NAA MS solid mediums, lucifuge culture.
Step（3）And step（4）Described screening and culturing medium is that with the addition of 0.5 ~ 2mg/L 6-BA and 0.5 ~ 2mg/L
NAA MS solid/liquid culture mediums, illumination condition are 2000 ~ 3000Lux, 10 ~ 12h/d.
Step（3）And step（4）The squamous subculture includes solid culture and liquid suspension culture intersects culture.
Step（5）Concretely comprise the following steps：Saussurea involucrata culture B is subjected to large-scale gas-lifting type culture, gas-lifting type culture medium is
MS fluid nutrient mediums, illumination condition are 2000 ~ 3000Lux, 12h/d, aeration quantity 0.2L/min；Then culture is collected to exist
20-24h is dried at 50-70 DEG C, that is, obtains Herba Saussureae Involueratae culture.
Sucrose and/or agar are added according to proportioning in above-mentioned MS liquid/solids culture medium.
Beneficial effects of the present invention：The present invention changes culture medium and cultivation temperature, and it is steady to filter out stable propagation, character
Fixed, the Herba Saussureae Involueratae culture rich in flavones, day is mass produced using solid culture, liquid suspension culture and gas-lifting type culture
Mountain Saussurea involucrata culture.The present invention can be on the basis of flavones content be ensured, stable, high-yielding Herba Saussureae Involueratae culture, and has production week
Phase is short, production quality is unified, adapts to the advantages of normal temperature growth condition.
Embodiment
Embodiment 1
（1）Aseptic seedling is induced after sterilization treatment using Herba Saussureae Involueratae seed；Sterilization steps include, and are soaked with 75% ethanol
30s, sterile water washing 2 times, 0.1% mercuric chloride solution immersion 15min, sterile water wash 3 times, it is inoculated on MS solid mediums,
Illumination 2000Lux, 12h/d.
（2）By aseptic seedling（Leaf, root, stem）It is forwarded on the culture medium of MS addition plant hormones, evoked callus；Induction
Culture medium is 0.5mg/L 6-BA+0.5mg/LNAA+MS+200g/ sucrose+5.6g/L agar, and condition of culture is illumination
2000Lux, 12h/d.
（3）Pick out color it is most deep, close to aubergine, well-grown callus, subculture is trained on screening and culturing medium
Support, obtain purple, the Saussurea involucrata culture A that propagation is fast, character is stable；Screening and culturing medium is 0.5mg/L 6-BA+0.8mg/LNAA+
MS+200g/ sucrose+5.6g/ agar, condition of culture are illumination 2000Lux, 12h/d.
（4）Saussurea involucrata culture A is placed in into 10 degree to cultivate 20 days, multiple subculture, constantly selects color on screening and culturing medium
Saussurea involucrata culture most purple, character is best, 2 degree of cultures are improved every 2 months temperature, until 27 degree, select and obtain saussurea involucrata culture
Thing B.
（5）Saussurea involucrata culture B is subjected to large-scale gas-lifting type culture, it is that the saussurea involucrata rich in flavones is cultivated to collect drying
Thing.Gas-lifting type culture medium is MS+250g/L sucrose, 2000Lux, 12h/d, aeration quantity 0.2L/min.
Embodiment 2
（1）Aseptic seedling is induced after sterilization treatment using Herba Saussureae Involueratae seed；Sterilization steps include, and are soaked with 75% ethanol
30s, sterile water washing 3 times, 0.1% mercuric chloride solution immersion 15min, sterile water wash 5 times, it is inoculated on MS solid mediums,
Illumination 3000Lux, 10h/d.
（2）By aseptic seedling（Leaf, root, stem）It is forwarded on the culture medium of MS addition plant hormones, evoked callus；Induction
Culture medium is 2.5mg/L6-BA+2.5mg/LNAA+MS+250g/L sucrose+6g/L agar, and condition of culture is illumination 3000Lux,
10h/d。
（3）Pick out color it is most deep, close to aubergine, well-grown callus, subculture is trained on screening and culturing medium
Support, obtain purple, the Saussurea involucrata culture A that propagation is fast, character is stable；Screening and culturing medium is 2mg/L 6-BA+2.5mg/LNAA+MS
+ 250g/L sucrose+6g/L agar, condition of culture are illumination 3000Lux, 10h/d.
（4）Saussurea involucrata culture A is placed in into 15 degree to cultivate 15 days, multiple subculture, constantly selects color on screening and culturing medium
Saussurea involucrata culture most purple, character is best, 3 degree of cultures are improved every 2 months temperature, until 27 degree, select and obtain saussurea involucrata culture
Thing B.
（5）Saussurea involucrata culture B is subjected to large-scale gas-lifting type culture, it is that the saussurea involucrata rich in flavones is cultivated to collect drying
Thing.Gas-lifting type culture medium is MS+250g/L sucrose, 3000Lux, 10h/d, aeration quantity 0.2L/min.
Embodiment 3
（1）Aseptic seedling is induced after sterilization treatment using Herba Saussureae Involueratae seed；Sterilization steps include, and are soaked with 75% ethanol
30s, sterile water washing 2 times, 0.1% mercuric chloride solution immersion 15min, sterile water wash 4 times, it is inoculated on MS solid mediums,
Illumination 3000Lux, 11h/d.
（2）By aseptic seedling（Leaf, root, stem）It is forwarded on the culture medium of MS addition plant hormones, evoked callus；Induction
Culture medium is 1mg/L 6-BA+2mg/LNAA+MS+250g/L sucrose+5.8g/L agar, and condition of culture is illumination 2800Lux,
11h/d。
（3）Pick out color it is most deep, close to aubergine, well-grown callus, subculture is trained on screening and culturing medium
Support, obtain purple, the Saussurea involucrata culture A that propagation is fast, character is stable；Screening and culturing medium is 2mg/L 6-BA+2mg/L NAA+MS+
230g/L sucrose+5.8g/L agar, condition of culture are illumination 2500Lux, 11h/d.
（4）Saussurea involucrata culture A is placed in into 13 degree to cultivate 18 days, multiple subculture, constantly selects color on screening and culturing medium
Saussurea involucrata culture most purple, character is best, 2 degree of cultures are improved every 2 months temperature, until 26 degree, select and obtain saussurea involucrata culture
Thing B.
（5）Saussurea involucrata culture B is subjected to large-scale gas-lifting type culture, it is that the saussurea involucrata rich in flavones is cultivated to collect drying
Thing.Gas-lifting type culture medium is MS+250g/L sucrose, 2800Lux, 12h/d, aeration quantity 0.2L/min.
Claims (6)
- A kind of 1. method for industrializing Herba Saussureae Involueratae culture of the large-scale production rich in flavones, it is characterized in that step is as follows：（1）Aseptic seedling is induced after sterilization treatment using Herba Saussureae Involueratae seed；（2）The leaf, root and stem of aseptic seedling are forwarded on the culture medium of MS addition plant hormones, evoked callus；（3）Pick out that color is most deep, well-grown callus, the squamous subculture on screening and culturing medium, obtain purple, propagation It hurry up, the Saussurea involucrata culture A that character is stable；（4）Saussurea involucrata culture A is placed in 10 ~ 12 DEG C of 15 ~ 20d of cultivation, multiple subculture, constantly selects color on screening and culturing medium Saussurea involucrata culture most purple, character is best, 3 degree of cultures are improved every 2 months temperature, until 23 ~ 27 DEG C, select and obtain saussurea involucrata training Support thing B；（5）Saussurea involucrata culture B is subjected to large-scale gas-lifting type culture, it is the Saussurea involucrata culture rich in flavones to collect drying.
- 2. the method for Herba Saussureae Involueratae culture of the industrialization large-scale production rich in flavones as claimed in claim 1, it is characterized in that Step（1）Concretely comprise the following steps：Herba Saussureae Involueratae 30 ~ 40s of seed is soaked for 75% ethanol with mass concentration, then sterile water washing 2 ~ 3 Time, then 15 ~ 20min is soaked with the mercuric chloride solution that mass concentration is 0.1%, sterile water wash 3 ~ 5 times, it is inoculated in the training of MS solids Support on base, carry out illumination cultivation and obtain aseptic seedling, illumination condition is 2000 ~ 3000Lux, 10 ~ 12h/d.
- 3. the method for Herba Saussureae Involueratae culture of the industrialization large-scale production rich in flavones as claimed in claim 1, it is characterized in that Step（2）Comprise the following steps that：By step（1）Leaf, root and the stem of gained aseptic seedling, which are inoculated in, with the addition of 0.5 ~ 2mg/L 6-BA On 0.5 ~ 2mg/L NAA MS solid mediums, lucifuge culture.
- 4. the method for Herba Saussureae Involueratae culture of the industrialization large-scale production rich in flavones as claimed in claim 1, it is characterized in that Step（3）And step（4）Described screening and culturing medium is that the MS that with the addition of 0.5 ~ 2mg/L 6-BA and 0.5 ~ 2mg/L NAA consolidates Body/fluid nutrient medium, illumination condition are 2000 ~ 3000Lux, 10h/d.
- 5. the method for Herba Saussureae Involueratae culture of the industrialization large-scale production rich in flavones as claimed in claim 1, it is characterized in that Step（3）And step（4）The squamous subculture includes solid culture and liquid suspension culture intersects culture.
- 6. the method for Herba Saussureae Involueratae culture of the industrialization large-scale production rich in flavones as claimed in claim 1, it is characterized in that Step（5）Concretely comprise the following steps：Saussurea involucrata culture B is subjected to large-scale gas-lifting type culture, gas-lifting type culture medium is trained for MS liquid Base is supported, illumination condition is 2000 ~ 3000Lux, 12h/d, aeration quantity 0.2L/min；Then culture is collected at 50-70 DEG C 20-24h is dried, that is, obtains Herba Saussureae Involueratae culture.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108575758A (en) * | 2018-05-28 | 2018-09-28 | 杭州雪域生物技术有限公司 | A kind of culture media composition of Herba Saussureae Involueratae tissue cultures and its application |
| CN110012836A (en) * | 2019-04-29 | 2019-07-16 | 江苏灵福嘉叶生物科技有限公司 | A method of large-scale production Hairy Root of Astragalus membranaceus |
| CN114176004A (en) * | 2020-08-24 | 2022-03-15 | 中国科学院青岛生物能源与过程研究所 | Regeneration culture method of saussurea involucrate |
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| CN1337467A (en) * | 2001-08-13 | 2002-02-27 | 贾景明 | Cell culture process of producing Xinjiang Saussurea involucrate general flavone |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108575758A (en) * | 2018-05-28 | 2018-09-28 | 杭州雪域生物技术有限公司 | A kind of culture media composition of Herba Saussureae Involueratae tissue cultures and its application |
| CN110012836A (en) * | 2019-04-29 | 2019-07-16 | 江苏灵福嘉叶生物科技有限公司 | A method of large-scale production Hairy Root of Astragalus membranaceus |
| CN114176004A (en) * | 2020-08-24 | 2022-03-15 | 中国科学院青岛生物能源与过程研究所 | Regeneration culture method of saussurea involucrate |
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