CN106520662A - Synthetic medium used for herba cistanche cells - Google Patents

Synthetic medium used for herba cistanche cells Download PDF

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Publication number
CN106520662A
CN106520662A CN201611014520.3A CN201611014520A CN106520662A CN 106520662 A CN106520662 A CN 106520662A CN 201611014520 A CN201611014520 A CN 201611014520A CN 106520662 A CN106520662 A CN 106520662A
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herba
culture medium
phytohormone
culture
cells
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夏雪城
裘先临
马丹
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TIANJIN BOAI BIO-PHARMACEUTICAL Co Ltd
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TIANJIN BOAI BIO-PHARMACEUTICAL Co Ltd
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0025—Culture media for plant cell or plant tissue culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/35—Polyols, e.g. glycerin, inositol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/74—Undefined extracts from fungi, e.g. yeasts
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/76—Undefined extracts from plants

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Abstract

According to the invention, nutrient substance used for the growth of herba cistanche cells is widely selected through the experiment means, and the formula of the invention is determined according to the self metabolic properties and practical use effects of the herba cistanche cells. According to the formula, ammonium nitrate and potassium nitrate are taken as nitrogen sources, monopotassium phosphate is taken as phosphorus source, under the basic nutritional ingredients of the MS culture medium, the plant hormones including 2.4-D, 6-BA and NAA are added for the purpose of improving metabolites, meanwhile, the inducers including yeast extract and chitosan are added, through the above improvement, the growth rate of the herba cistanche cells is greatly improved, and the secretion amount of phenylethanoid glycosides is greatly increased. On the basis, according to the accidental discoveries, by adding the traditional Chinese medicinal materials with specific varieties and ingredients, the cell culture density can be improved. Based on the above beneficial discoveries, through the experiment means, the traditional Chinese medicinal materials used for treating the human diseases, such as herba tadehagis triquetri, herba saxifragae stoloniferae, herba ranunculi chinensis and herba abri are selected, and the obtained culture medium obtains the outstanding culture effects.

Description

A kind of synthetic medium for Herba Cistanches cell
Technical field
The present invention relates to plant cell culture technology, designs further to the nutritional condition of plant cell, and in particular to A kind of synthetic medium for Herba Cistanches cell.
Background technology
Plant cell culture technology is with vitro plant cell or protoplast as object, with cell concentration amplification and target generation Thank to the culture process for the purpose of product accumulation.For the cell culture for the purpose of obtaining specific metabolite, which is main Process procedure is not only in that the amplification of cell concentration, meanwhile, the induction synthesis of target metabolic product is even more important to cultivating benefit. In the incubation of plant cell, condition of culture and nutritional condition are the most important factors for affecting yield, and wherein condition of culture is Refer to the control of the environmental conditions such as temperature, dissolved oxygen, illumination, pH, and nutritional condition refers mainly to the culture medium of plant cell.Due in envelope Close the sole nutrition source that culture medium in culture environment is plant cell, therefore growth water of the composition of culture medium to plant cell Flat, metabolite classification has extremely important impact, particularly with the cell culture for the purpose of obtaining specific metabolite For, culture medium often affects the key factor of culture efficiency.
Herba Cistanches are a kind of rare Chinese medicines for being grown on salt-soda soil and Gobi desert, with nourishing kidney tonifying YANG, remove free radical And the effects such as enhancing immunity, its main medicinal ingredient is phenethyl alcohol glycoside, including echinacoside, Syring vulgaris glycosides, 2 '-acetyl Syring vulgaris glycosides and cistanoside A etc..Herba Cistanches are colonized on the root of the plants such as sacsaoul, due to unauthorized and excessive mining, cause shortage of resources, Vegetation deterioration is serious, accelerates desertification of land.
In prior art, the extraction of phenethyl alcohol glycoside is more with natural Herba Cistanches herb as raw material, the growth of one side plant herb Slowly, yield is relatively low, on the other hand as the plant herb of routine growth state, its metabolite more rule, it is difficult to mesh Mark metabolite realizes a large amount of secretion.Herba Cistanches tissue culture and cell suspension cultures technology are arisen at the historic moment in this case, In associated culture techniques, medium component is as its sole nutrition source for vitro growth rates and metabolite classification have Have a major impact, promotion of the conventional culture medium to Herba Cistanches cell own growth and phenethyl alcohol glycoside secretory volume in prior art Effect is not ideal enough.
The content of the invention
It is contemplated that for the technological deficiency of prior art, there is provided a kind of synthetic medium for Herba Cistanches cell, To solve the technical problem of Herba Cistanches cell culture effect on driving birds is not good in prior art.
The invention solves the problems that another technical problem be the Herba Cistanches cell cultivation process of prior art, target metabolic is produced The yield of thing phenethyl alcohol glycoside is relatively low.
To realize above technical purpose, the present invention is employed the following technical solutions：
A kind of synthetic medium for Herba Cistanches cell, the culture medium contains the composition of MS culture medium, while also containing Phytohormone 2.4-D 0.25mg/L, phytohormone 6-BA 2mg/L, phytohormone NAA 1mg/L.
Preferably, the culture medium also yeast extract containing 1% (g/mL).
Preferably, the culture medium also shitosan containing 5mg/L.
Preferably, Herba Desmodii Triquetri (Herba Tadehagi Triquetri) squeezing juice of the culture medium also containing 1g/L.
Preferably, Herba Saxifragae squeezing juice of the culture medium also containing 1g/L.
Preferably, Herba Ranunculi Chinensiss squeezing juice of the culture medium also containing 1g/L.
Preferably, Herba Abri squeezing juice of the culture medium also containing 1g/L.
Preferably, the culture medium contains the composition of MS culture medium, while also contain phytohormone 2.4-D0.25mg/L, Phytohormone 6-BA 2mg/L, phytohormone NAA 1mg/L, yeast extract 1% (g/mL), shitosan 5mg/L, Herba Desmodii Triquetri (Herba Tadehagi Triquetri) pressure Squeeze the juice 1g/L, Herba Saxifragae squeezing juice 1g/L, Herba Ranunculi Chinensiss squeezing juice 1g/L, Herba Abri squeezing juice 1g/L.
In above technical scheme, the MS culture medium can be prepared according to the general technology general knowledge of this area, when The MS culture medium of formula in detail below can also so be adopted：NH4NO31650mg/L, KNO31900mg/L, CaCl2·2H2O 440mg/L, MgSO4·7H2O 370mg/L, KH2PO4170mg/L, KI 0.83mg/L, H3BO36.2mg/L, MnSO4·4H2O 22.3mg/L, ZnSO4·7H2O 8.6mg/L, Na2MoO4·2H2O 0.25mg/L, CuSO4·5H2O 0.025mg/L, CoCl2·6H2O 0.025mg/L, FeSO4·7H2O 27.8mg/L, Na2-EDTA·2H2O 37.3mg/L, inositol 100mg/L, Nicotinic acid 0.5mg/L, vitamin B6 0.5mg/L, vitamin B1 0.1mg/L, glycine 2mg/L, water surplus.
In above technical scheme, Herba Desmodii Triquetri (Herba Tadehagi Triquetri), Herba Saxifragae, Herba Ranunculi Chinensiss, Herba Abri refer to its fresh herb respectively.In the present invention Described squeezing juice, refers to the Fresh sap of each raw material obtained using physical method, including but not limited to squeezing, crushing centrifugation Etc. conventional method.
The present invention has extensively screened the nutrient substance as Herba Cistanches cell growth by laboratory facilities, thin according to Herba Cistanches Born of the same parents' own metabolism characteristic and practical effect determine inventive formulation.The formula using ammonium nitrate, potassium nitrate as nitrogen source, with Potassium dihydrogen phosphate is phosphorus source, under the basal nutrient composition of MS culture medium, with the addition of for the purpose of improving metabolite 2.4-D, The phytohormone such as 6-BA, NAA, while with the addition of the inducers such as yeast extract and shitosan, are greatly carried by above-mentioned improvement The growth rate and the secretory volume of phenethyl alcohol glycoside of high Herba Cistanches cells.The present invention is found surprisingly that by adding on this basis Plus the Chinese crude drug of particular types and composition contributes to lifting cell culture density.The beneficial discovery based on more than, the present invention pass through Laboratory facilities preferred Herba Desmodii Triquetri (Herba Tadehagi Triquetri), Herba Saxifragae, Herba Ranunculi Chinensiss, Herba Abri etc. are originally used for the Chinese medicine composition of human disease treatment, Gained culture medium surprisingly obtains prominent culture effect so that Herba Cistanches cell culture density is obviously improved, while its growth Speed is faster.The present invention obtains superior technique effect with the technological means of novelty, while cost is relatively low, be easily achieved, because This has a good application prospect.
Specific embodiment
Hereinafter the specific embodiment to the present invention is described in detail.In order to avoid excessive unnecessary details, Will not be described in detail to belonging to known structure or function in following examples.In addition to being defined, institute in following examples Technology and scientific terminology are with the identical meanings being commonly understood by with those skilled in the art of the invention.
Embodiment 1
A kind of synthetic medium for Herba Cistanches cell, the culture medium contains the composition of MS culture medium, while also containing Phytohormone 2.4-D 0.25mg/L, phytohormone 6-BA 2mg/L, phytohormone NAA 1mg/L.
Embodiment 2
A kind of synthetic medium for Herba Cistanches cell, the culture medium contains the composition of MS culture medium, while also containing Phytohormone 2.4-D 0.25mg/L, phytohormone 6-BA 2mg/L, phytohormone NAA 1mg/L, 1% (g/ of yeast extract mL)。
Embodiment 3
A kind of synthetic medium for Herba Cistanches cell, the culture medium contains the composition of MS culture medium, while also containing Phytohormone 2.4-D 0.25mg/L, phytohormone 6-BA 2mg/L, phytohormone NAA 1mg/L, shitosan 5mg/L.
Embodiment 4
A kind of synthetic medium for Herba Cistanches cell, the culture medium contains the composition of MS culture medium, while also containing Phytohormone 2.4-D 0.25mg/L, phytohormone 6-BA 2mg/L, phytohormone NAA 1mg/L, Herba Desmodii Triquetri (Herba Tadehagi Triquetri) squeezing juice 1g/L.
Embodiment 5
A kind of synthetic medium for Herba Cistanches cell, the culture medium contains the composition of MS culture medium, while also containing Phytohormone 2.4-D 0.25mg/L, phytohormone 6-BA 2mg/L, phytohormone NAA 1mg/L, Herba Saxifragae squeezing juice 1g/L.
Embodiment 6
A kind of synthetic medium for Herba Cistanches cell, the culture medium contains the composition of MS culture medium, while also containing Phytohormone 2.4-D 0.25mg/L, phytohormone 6-BA 2mg/L, phytohormone NAA 1mg/L, Herba Ranunculi Chinensiss squeezing juice 1g/L.
Embodiment 7
A kind of synthetic medium for Herba Cistanches cell, the culture medium contains the composition of MS culture medium, while also containing Phytohormone 2.4-D 0.25mg/L, phytohormone 6-BA 2mg/L, phytohormone NAA 1mg/L, Herba Abri squeezing juice 1g/L.
Embodiment 8
A kind of synthetic medium for Herba Cistanches cell, the culture medium contains the composition of MS culture medium, while also containing Phytohormone 2.4-D 0.25mg/L, phytohormone 6-BA 2mg/L, phytohormone NAA 1mg/L, 1% (g/ of yeast extract ML), shitosan 5mg/L, Herba Desmodii Triquetri (Herba Tadehagi Triquetri) squeezing juice 1g/L, Herba Saxifragae squeezing juice 1g/L, Herba Ranunculi Chinensiss squeezing juice 1g/L, Herba Abri squeezing Juice 1g/L.
Embodiment 9
The present embodiment be used to evaluating culture medium that above example 1～8 provided as during synthetic medium to Herba Cistanches The impact of the phenethyl alcohol glycoside yield of cell, experimental result are as shown in table 1.
1 different culture media of table is used as impact during synthetic medium to the phenethyl alcohol glycoside yield of Herba Cistanches cell
When as can be seen here, by the use of culture medium provided by the present invention as synthetic medium culture Herba Cistanches cell, to which Phenethyl alcohol glycoside yield has definite facilitation.
Above embodiments of the invention are described in detail, but the content have been only presently preferred embodiments of the present invention, Not to limit the present invention.All any modification, equivalent and improvement made in the application range of the present invention etc., all should It is included within protection scope of the present invention.

Claims (8)

1. a kind of synthetic medium for Herba Cistanches cell, it is characterised in that the culture medium contains the composition of MS culture medium, together When also contain phytohormone 2.4-D 0.25mg/L, phytohormone 6-BA 2mg/L, phytohormone NAA 1mg/L.
2. a kind of synthetic medium for Herba Cistanches cell according to claim 1, it is characterised in that the culture medium is also Yeast extract containing 1% (g/mL).
3. a kind of synthetic medium for Herba Cistanches cell according to claim 1, it is characterised in that the culture medium is also Shitosan containing 5mg/L.
4. a kind of synthetic medium for Herba Cistanches cell according to claim 1, it is characterised in that the culture medium is also Herba Desmodii Triquetri (Herba Tadehagi Triquetri) squeezing juice containing 1g/L.
5. a kind of synthetic medium for Herba Cistanches cell according to claim 1, it is characterised in that the culture medium is also Herba Saxifragae squeezing juice containing 1g/L.
6. a kind of synthetic medium for Herba Cistanches cell according to claim 1, it is characterised in that the culture medium is also Herba Ranunculi Chinensiss squeezing juice containing 1g/L.
7. a kind of synthetic medium for Herba Cistanches cell according to claim 1, it is characterised in that the culture medium is also Herba Abri squeezing juice containing 1g/L.
8. a kind of synthetic medium for Herba Cistanches cell according to claim 1, it is characterised in that the culture medium contains There is the composition of MS culture medium, while also containing phytohormone 2.4-D 0.25mg/L, phytohormone 6-BA 2mg/L, phytohormone NAA 1mg/L, yeast extract 1% (g/mL), shitosan 5mg/L, Herba Desmodii Triquetri (Herba Tadehagi Triquetri) squeezing juice 1g/L, Herba Saxifragae squeezing juice 1g/L, Herba Ranunculi Chinensiss squeezing juice 1g/L, Herba Abri squeezing juice 1g/L.
CN201611014520.3A 2016-11-15 2016-11-15 Synthetic medium used for herba cistanche cells Pending CN106520662A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115281083A (en) * 2022-07-06 2022-11-04 贵州大学 Method for inducing saxifrage callus secondary metabolite
CN115281084A (en) * 2022-07-06 2022-11-04 贵州大学 Method for inducing saxifrage tissue culture seedling secondary metabolite

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1457632A (en) * 2002-05-14 2003-11-26 中国科学院过程工程研究所 Method for producing callus and differentiating sprout by inducing cistanche salsa seed
CN1730651A (en) * 2004-08-06 2006-02-08 天津天士力制药股份有限公司 Broomrape callus induction method and induction medium

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1457632A (en) * 2002-05-14 2003-11-26 中国科学院过程工程研究所 Method for producing callus and differentiating sprout by inducing cistanche salsa seed
CN1730651A (en) * 2004-08-06 2006-02-08 天津天士力制药股份有限公司 Broomrape callus induction method and induction medium

Non-Patent Citations (1)

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Title
成喜雨: "肉苁蓉细胞培养及其过程代谢调控", 《中国优秀硕士学位论文全文数据库(电子期刊)农业科技辑》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115281083A (en) * 2022-07-06 2022-11-04 贵州大学 Method for inducing saxifrage callus secondary metabolite
CN115281084A (en) * 2022-07-06 2022-11-04 贵州大学 Method for inducing saxifrage tissue culture seedling secondary metabolite
CN115281083B (en) * 2022-07-06 2023-07-11 贵州大学 Method for inducing secondary metabolite of saxifraga stolonifera callus
CN115281084B (en) * 2022-07-06 2023-07-14 贵州大学 Method for inducing saxifraga tissue culture Miao Cisheng metabolite

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