CN1167332C - Tripterygium wilfordii regenerated plant and its preparation process - Google Patents
Tripterygium wilfordii regenerated plant and its preparation process Download PDFInfo
- Publication number
- CN1167332C CN1167332C CNB021109338A CN02110933A CN1167332C CN 1167332 C CN1167332 C CN 1167332C CN B021109338 A CNB021109338 A CN B021109338A CN 02110933 A CN02110933 A CN 02110933A CN 1167332 C CN1167332 C CN 1167332C
- Authority
- CN
- China
- Prior art keywords
- medium
- plant
- culture
- tripterygium wilfordii
- content
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention discloses a preparation process of tripterygium wilfordii regenerated plants, which mainly utilizes the technique of clonal variation, mutagenesis, chromosome doubling, regeneration, no hormone propagation, etc. Tripterygium wilfordii regenerated plants prepared by the process can obtain mass propagation under the controllable standard conditions, and the propagation coefficient of each generation can achieve five to six times. Besides, the triptolide content is 0.009 to 0.015%, and the total diterpene lactone content is higher than that of tripterygium wilfordii extractum sold on markets. Furthermore, the triptolide content and the total diterpene lactone content can keep stable.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to a kind of preparation technology of tripterygium wilfordii regenerated plant.
Background technology
Thunder godvine (Tripterygium wilfordii Hook f.) is the Celastraceae tripterygium plant.Pharmacological evaluation shows, that thunder godvine has is significantly antitumor, anti-inflammatory, immunosupress and antifertility action.Present result of study shows that tripterygium wilfordii diterpene lactone compounds is its main active ingredient.The thunder godvine complex chemical composition, pharmacological action is extensive, and important clinical application value is arranged.
But because thunder godvine is perennial woody climber, poor growth is widely applied clinically, causes wild resource seriously poor.Therefore, the artificial culture of thunder godvine is to extenuating the biological polymorphism of the nervous situation of resource, protection, satisfying people's needs of medical treatment and foreign exchange earning etc. and play a part duty-bound.
At present, mainly concentrate on the young stem, the leaf that utilize wild thunder godvine for the research of thunder godvine artificial breeding technique and carry out callus induction and cell suspension cultures as explant.Yet also there is weak point in these technology:
1. callus or suspension cell belong to the undifferentiated type culture, and wild thunder godvine all makes medicinal material with plant, and it belongs to the differentiated product, and there is difference in essence in the two.
2. the cultivation yield of callus or suspension cell is lower, is difficult to large-scale culture and is used for industrialization.
3. genetic variation easily takes place in callus or suspension cell in long-term subculture is cultivated, and causes the active ingredient instability of culture.
So, how to develop a kind of can large-scale production, the metastable artificial culture technology of composition of remaining valid again, be the target that the researcher pursued always.
Summary of the invention
Purpose of the present invention is to provide a kind of tripterygium wilfordii regenerated plant, its reproduction coefficient height, and its content of effective is than the height of wild thunder godvine, and the component content of can remaining valid is relatively stable.
Another object of the present invention, be to provide the preparation technology of the high tripterygium wilfordii regenerated plant of a kind of reproduction coefficient and active constituent content, mainly be to utilize cytometaplasia and the totipotent the principles of science of cell, change the low characteristic of its poor growth and active constituent content, and in regeneration plant stably express.
The method for preparing tripterygium wilfordii regenerated plant provided by the invention, be according to the totipotent principle of cell, each cells in-vitro of plant can grow under the suitable culture condition and the duplicate plant of parent, and cytometaplasia principle, be that cell or callus in the cultured in vitro process somaclonal variation can take place, give mutagenesis or chromosome doubling processing to cultured in vitro cell or callus, can increase range of variation and improve variation frequency.Therefore, we at first carry out mutagenesis or a large amount of variations of chromosome doubling processing initiation to thunder godvine cell or callus.
Get wild thunder godvine or seed seedling, as explant,, thinly slice, place in the aseptic inducing culture through surface sterilizing 15～20 minutes with its tender tissue, the cultivation through about 35～45 days, 22～25 ℃ of cultivation temperature obtain callus.Carry out successive transfer culture then 2～3 times, whenever be commissioned to train and supported 25～30 days, 22～25 ℃ of cultivation temperature obtain a large amount of callus.A large amount of callus are inserted liquid nutrient medium, on shaking table, cultivated 25～30 days, and 22～25 ℃ of cultivation temperature, rotating speed is 110～125 rev/mins, forms suspended culture cell or cell mass.
Under the aseptic condition safety and low toxicity mutagen or chromosome doubling agent and suspended culture cell or cell mass were cultivated 12～24 hours altogether.Then with sterile water flush away mutagen or double agent, suspension cell or cell mass inserted repair the medium middle-shallow layer and cultivated 22～25 ℃ of cultivation temperature about 9～12 days.This step can be replaced by the successive transfer culture in 3～4 generations.
Then above-mentioned suspended culture cell or cell mass are placed on the differential medium and cultivate illumination every day 10～12 hours, 22～25 ℃ of cultivation temperature.From various regrowths, select the normal type regrowth after 25～30 days.Regrowth places and forms regeneration plant on the proliferated culture medium.
Select the high and sturdy regeneration plant of reproduction coefficient, get the mensuration that its a part of plant carries out the total diterpene lactone content, and then to the total diterpene lactone content high carry out the triptolide Determination on content.The total diterpene lactone content is measured and is adopted the MDNB colorimetric method, and the triptolide Determination on content adopts high performance liquid chromatography.Behind assay, therefrom select the active constituent content regeneration plant higher than wild thunder godvine.Above-mentioned useful variation can be gone down by reservation of vegetative propagation approach and stable heredity.
Because mutant or variant also can take place to link to each other or disjunct other variations when main variation takes place, and regeneration plant easily produces the characteristic of adaptation reaction to the domestication condition, therefore the regeneration plant stem apex is placed successively hormone (as the 6-benzylaminopurine) concentration from high to low again to zero domestication medium, 22～25 ℃ of illumination 10～12 hours every days were cultivated 25～30 days at every turn.Select fast growth, plant that active constituent content is high from no hormone simple culture media at last.Perhaps also regeneration plant directly can be placed and tame cultivation on the no hormone culture-medium.
Domestication type regeneration plant is placed on the simple culture media, 22～25 ℃ of cultivations, illumination 10～12 hours every days (as using fluorescent lamp), in per generation, can be bred 5～6 times.According to the method described above, to per generation plant carry out the stability that total diterpene lactone content and triptolide Determination on content detect its active ingredient and content thereof, wherein the content of triptolide remains between the 0.009-0.015% substantially.
Regeneration plant reproduction coefficient height, appearance through the large-scale production acquisition is sturdy, active constituent content is close with original regeneration plant, shows that the primary characteristic of regeneration plant is constant substantially after many generations (reaching for 16 generations at present) cultivate.
Description of drawings
Fig. 1 is the preparation flow figure of tripterygium wilfordii regenerated plant
Embodiment
Below in conjunction with specific embodiment, to further specify technical characterictic of the present invention.Should be understood that following embodiment only to be used to the present invention is described and be not used in the scope of the present invention that limits.
Among the following embodiment of the present invention in the used medium,
MS is a kind of medium, and its prescription is (mg/L of unit):
NH
4NO
3 1650 KNO
3 1900
CaCl
2·2H
2O 440 MgSO
4·7H
2O 370
KH
2PO
4 170 H
3BO
3 6.2
MnSO
4·4H
2O 22.3 CoCl
2·6H
2O 0.025
CuSO
4·5H
2O 0.025 ZnSO
4·7H
2O 8.6
NaMoO
4·2H
2O 0.25 KI 0.88
FeSO
4·7H
2O 27.8 Na
2EDTA·2H
2O 37.3
Vitamin B1 0.1 vitamin B6 0.5
Yan acid 0.5 inositol 100
Glycine 2.0
N
6Be a kind of medium, its prescription is (mg/L of unit):
KNO
3 2830 (NH
4)
2SO
4 463
KH
2PO
4 400 MgSO
4·7H
2O 185
CaCl
2·2H
2O 166 FeSO
4·7H
2O 27.8
Na
2EDTA·2H
2O 37.3 MnSO
4·4H
2O 4.4
ZnSO
4·7H
2O 1.5 H
3BO
3 1.6
KI 0.8 glycine 2.0
Vitamin B1 1.0 vitamin B6s 0.5
Yan acid 0.5
The somaclonal variation and the mutagenesis of embodiment 1 thunder godvine cell or callus
Get the tender tissue of wild thunder godvine or seed seedling and make explant, through 75% ethanol sterilization 30 seconds, sterilization ling or bleaching powder sterilization 15 minutes, sterile water is cleaned, thinly slice and carry out inducing culture, inducing culture is 2/3MS, 2,4 dichlorophenoxyacetic acid (2,4-D) 1.0mg/L, methyl (NAA) 0.1mg/L, kinetin (KT) 0.1mg/L, 5% sucrose, 0.6% agar are cultivated 35 days to induce the formation callus.Carry out the successive transfer culture in 2～3 generations then, subculture medium is: 2/3N6, caseinhydrolysate (CH) 500mg/L, 2,4-dichlorphenoxyacetic acid (2,4-D) 0.5mg/L, indolebutyric acid (IBA) 0.5mg/L, kinetin (KT) 0.1mg/L, 4% sucrose, 0.6% agar, whenever be commissioned to train and supported 30 days, obtain a large amount of callus.A large amount of callus are inserted liquid nutrient medium (2/3N
6, 500mg/L caseinhydrolysate (CH), 2,4-dichlorphenoxyacetic acid (2,4-D) 1.0mg/L, heteroauxin (IAA) 0.5mg/L, kinetin (KT) 0.1mg/L, 4% sorbose) in, on shaking table, cultivate, rotating speed is 120rpm, every switching in 15 days once, cultivated 30 days, collect yellow embryonal suspension cultured cell or cell mass.
Can on subculture medium, continue this moment to cultivate for 3～4 generations to bring out somaclonal variation, also can with cultivated altogether 15 hours through the 80mg/L bleomycin A5 of sterilization or the colchicin of 100mg/L, sterile water is cleaned the back and is taken out to insert and repair medium (2/3MS, 15% Coconut Juice, 3% sorbose) middle-shallow layer and cultivated 10 days.
More than the temperature of all kinds of cultivations be 22～25 ℃.
The plant regeneration of embodiment 2 thunder god vine suspending cultured cells or cell mass
To or repair foresythia suspended culture cell or the cell mass cultivated and carry out differentiation culture through successive transfer culture, differential medium is 1/2MS, heteroauxin (IAA) 0.1mg/L, kinetin (KT) 0.5mg/L, 6-benzylaminopurine (6-BA) 1.5mg/L, 3% sucrose, 0.6% agar, cultivation temperature is 22～25 ℃, sturdy field run plant is selected in illumination every day 12 hours from regrowth after 30 days.
The screening of the thunder godvine strain system that embodiment 3 reproduction coefficient and active constituent content are high
Regeneration plant is carried out enrichment culture, proliferated culture medium is 2/3MS, caseinhydrolysate (CH) 500mg/L, kinetin (KT) 0.5mg/L, 6-benzylaminopurine (6-BA) 0.5mg/L, methyl (NAA) 0.1mg/L, 3% sucrose, 0.6% agar, select sturdy, the strain system of growing fast, get the mensuration (Lin Qishou etc. that its part plant carries out the total diterpene lactone content, " medicinal herb components chemistry " 1977,390 pages, Beijing, Science Press), thunder godvine medicinal extract sheet (lot number is 20000505) with Lei Shi medicine company Shanghai Chinese Medicines Factory No.2 is standard items, therefrom screens the higher regeneration plant of content.
Select the high regeneration plant of total diterpene lactone content to carry out triptolide Determination on content (Ye Xiaochuan again, " Pharmaceutical Analysis magazine ", 1997,17 the 5th phases of volume: 319 pages), the standard sample of triptolide is provided by professor Li Yuanchao of Shanghai Pharmaceutical Inst., Chinese Academy of Sciences, therefrom screens the high regeneration plant of content.
Embodiment 4 tripterygium wilfordii regenerated plants do not have the domestication that hormone is cultivated
The stem apex of getting the high-quality regeneration plant places the higher medium of hormone 6-BA content (2/3MS, 6-benzylaminopurine (6-BA) 0.5mg/L, 3% sucrose, 0.6% agar) to go up and cultivated 28 days, and the fast plant of growth is therefrom selected in illumination every day 11 hours; Place the lower medium of hormone-content (2/3MS, 6-benzylaminopurine (6-BA) 0.1mg/L, 3% sucrose, 0.6% agar) to go up again and cultivated 28 days, the fast plant of growth is therefrom chosen in illumination every day 10 hours; Place no hormone culture-medium (2/3MS, 3% sucrose, 0.6% agar) to go up at last and cultivated 30 days, the fast and healthy and strong plant of growth is therefrom chosen in illumination every day 12 hours.Or directly place on the no hormone culture-medium and cultivate, select the fast regeneration plant of growth.Above cultivation temperature is 22～25 ℃.
The stability test of embodiment 5 tripterygium wilfordii regenerated plants
Do the regeneration induction step of three the foregoing descriptions 1～4, all filter out the fast and sturdy tripterygium wilfordii regenerated plant of growth at every turn.To carry out the mensuration of active constituent content through the regeneration plant that 3 generations, 6 generations, 9 generations, 12 generations, the breeding of 15 generations obtain, the result is as shown in table 1:
| For test agent | Total diterpene lactone content (comparing) with thunder godvine medicinal extract | Triptolide content (%) |
| 3 generation regeneration plant | ＞contrast | 0.013 |
| 6 generation regeneration plant | ＞contrast | 0.012 |
| 9 generation regeneration plant | ＞contrast | 0.013 |
| 12 generation regeneration plant | ＞contrast | 0.012 |
| 15 generation regeneration plant | ＞contrast | 0.012 |
After testing, the lactone alcohol content of wild thunder godvine is less than 0.003%.As seen, the regeneration techniques of tripterygium wilfordii regenerated plant of the present invention repeatability is better, and active constituent content height and genetic stability are better.
Claims (3)
1. the preparation technology of a tripterygium wilfordii regenerated plant is characterized in that, comprises the steps:
(1) somaclonal variation of thunder godvine cell or callus and mutagenesis
The tissue that cuts the young tender organ of wild thunder godvine or seed seedling is made explant, sterilizes after 15～20 minutes, thinly slices, and places on the inducing culture, and cultivation temperature is 22～25 ℃, cultivates through 35～40 days, induces the formation callus; Callus is placed subculture medium, 2～3 generations of successive transfer culture, whenever to be commissioned to train and to support 25～30 days, cultivation temperature is 22～25 ℃, forms a large amount of callus; A large amount of callus place liquid nutrient medium, and cultivation temperature is 22～25 ℃, cultivates on shaking table 25～30 days, and rotating speed is 110～130 rev/mins, form suspended culture cell or cell mass; Afterwards cell mass was inserted in the mutagen of sterilization treatment or chromosome doubling agent solution co-incubation 12～24 hours, culture is cleaned mutagen or chromosome doubling agent with sterile water, insert and repair medium middle-shallow layer cultivation 9～12 days, cultivation temperature is 22～25 ℃;
(2) plant regeneration of thunder god vine suspending cultured cell or cell mass
Then suspended culture cell or cell mass are inserted in the differential medium and cultivate, illumination every day 10～12 hours, cultivation temperature are 22～25 ℃, choose the normal type regeneration plant after 25～30 days from regeneration plant, place proliferated culture medium to cultivate, choose the sturdy plant of growth then;
(3) screening of tripterygium wilfordii regenerated plant
Regeneration plant forms strain system by cutting propagation means, therefrom select the strain system sturdy, that reproduction coefficient is high, each strain system gets a certain amount of plant and carries out total diterpene lactone content mensuration, therefrom select the high strain system of content, the strain system that selected total diterpene lactone content is high carries out triptolide again and measures, and therefrom selects the high strain system of lactone alcohol content;
(4) the no hormone of tripterygium wilfordii regenerated plant is cultivated domestication
The fast tripterygium wilfordii regenerated plant of selected active constituent content height and growth rate places 6-benzylaminopurine concentration to be respectively on the domestication medium of 0.5mg/L, 0.1mg/L, 0mg/L successively, at 22～25 ℃, cultivated 25～30 days, illumination every day 10～12 hours, at last, choose the strain system that growth is fast and active constituent content is high from no hormone culture-medium;
In the above-mentioned steps, the inducing culture based formulas is: 2/3MS, 2,4 dichlorophenoxyacetic acid 1.0mg/L, methyl 0.1 mg/L, kinetin 0.1mg/L, 5% sucrose, 0.6% agar;
Liquid nutrient medium is: 2/3N
6, 500mg/L caseinhydrolysate, 2,4 dichlorophenoxyacetic acid 1.0mg/L, heteroauxin 0.5mg/L, kinetin 0.1mg/L, 4% sorbose;
Subculture medium is: 2/3N6,500mg/L caseinhydrolysate, 2,4 dichlorophenoxyacetic acid 0.5mg/L, indolebutyric acid 0.5mg/L, kinetin 0.1mg/L, 4% sucrose, 0.6% agar;
The reparation medium is: 2/3MS, 15% Coconut Juice, 3% sorbose;
Differential medium is 1/2MS, heteroauxin 0.1mg/L, kinetin 0.5mg/L, 6-benzylaminopurine 1.5mg/L, 3% sucrose, 0.6% agar;
Proliferated culture medium is: 2/3MS, 500mg/L caseinhydrolysate, kinetin 0.5mg/L, 6-benzylaminopurine 0.5mg/L, methyl 0.1mg/L, 3% sucrose, 0.6% agar;
The domestication medium is: 2/3MS, 3% sucrose, 0.6% agar.
2. the preparation technology of tripterygium wilfordii regenerated plant as claimed in claim 1, it is characterized in that, also can adopt cell mass to handle without mutagenesis or chromosome doubling with mutagen or chromosome doubling agent mutagenesis in the described step (1), the successive transfer culture that carried out for 3～4 generations at subculture medium reaches somaclonal variation.
3. the preparation technology of tripterygium wilfordii regenerated plant as claimed in claim 1 is characterized in that, no hormone is cultivated and tamed and can directly carry out on no hormone culture-medium in the described step (4).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021109338A CN1167332C (en) | 2002-03-01 | 2002-03-01 | Tripterygium wilfordii regenerated plant and its preparation process |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021109338A CN1167332C (en) | 2002-03-01 | 2002-03-01 | Tripterygium wilfordii regenerated plant and its preparation process |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1366810A CN1366810A (en) | 2002-09-04 |
| CN1167332C true CN1167332C (en) | 2004-09-22 |
Family
ID=4741325
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB021109338A Expired - Fee Related CN1167332C (en) | 2002-03-01 | 2002-03-01 | Tripterygium wilfordii regenerated plant and its preparation process |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1167332C (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100333638C (en) * | 2005-07-22 | 2007-08-29 | 上海延农生物工程有限公司 | Method for inducing, breeding and transplanting triperygium wilfordii cluster buds |
| CN101638669B (en) * | 2009-09-09 | 2011-11-02 | 成都大学 | Method for producing bulbus fritillariae cirrhosae total alkaloid by adopting cell mass suspension culture |
| CN101803518B (en) * | 2010-03-25 | 2011-12-28 | 广州陈李济药厂 | Standardized plating method of Kunming begonia traditional Chinese medicinal materials |
-
2002
- 2002-03-01 CN CNB021109338A patent/CN1167332C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1366810A (en) | 2002-09-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fulzele et al. | Tissue cultures of Artemisia annua: organogenesis and artemisinin production | |
| Paek et al. | High frequency of bulblet regeneration from bulb scale sections of Fritillaria thunbergii | |
| Fulzele et al. | Production of terpenoid from Artemisia annua L. plantlet cultures in bioreactor | |
| CN101766122B (en) | Method for cultivating sweet sorghum tissue and special culture medium thereof | |
| Wadegaonkar et al. | Direct rhizogenesis and establishment of fast growing normal root organ culture of Withania somnifera Dunal | |
| Singh et al. | Technique for rapid in vitro multiplication of Vitis vinifera L. cultivars | |
| CN100346689C (en) | Breeding method for test tube seedling of elaeagnus molllis | |
| Liu et al. | Induction and in vitro alkaloid yield of calluses and protocorm-like bodies (PLBs) from Pinellia ternata: alkaloid yield of in vitro tissues from Pinellia ternata | |
| RU2757318C1 (en) | Method for obtaining microplants of medicinal plant stephania glabra (roxb.) miers | |
| SE1050412A1 (en) | Procedure for stratification and storage of somatic embryos | |
| CN110317772A (en) | A kind of method of drawing material of barley microspore | |
| Nieves et al. | Callus induction in cotyledons of Moringa oleifera Lam. | |
| Tonon et al. | Synchronized somatic embryo development in embryogenic suspensions of Fraxinus angustifolia | |
| CN1167332C (en) | Tripterygium wilfordii regenerated plant and its preparation process | |
| Park et al. | Production of ginkgolides and bilobalide from optimized the Ginkgo biloba cell culture | |
| Chee | Plant regeneration from somatic embryos of Taxus brevifolia | |
| Smiskova et al. | Somatic embryogenesis from zygotic embryos of Schisandra chinensis | |
| CN1275513C (en) | Ironbark dendrobe sprout and preparation process and use thereof | |
| WO1993002204A1 (en) | Production of ginkgolides in cell culture | |
| Ilahi et al. | Enhanced clonal propagation and alkaloid biosynthesis in cultures of Rauwolfia | |
| Nobakht et al. | In vitro mass propagation of Typhonium flagelliforme as affected by plant growth regulators | |
| JP4563493B2 (en) | Efficient production of naphthoquinone anticancer active ingredients | |
| KR100533793B1 (en) | Mass propagation Method for Stock and Adventitious Root of Acanthopanax Koreanum Nakai Using Bioreactor | |
| CN1281050A (en) | Process for preparing ginkgolic polyose by culturing ginkgolic cells | |
| CN111406651B (en) | Tissue culture and rapid propagation method of drynaria fortunei seedlings by somatic embryo approach |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20040922 Termination date: 20130301 |