CN102618484A - Method for improving saffron pigment synthesizing capability of saffron cells - Google Patents
Method for improving saffron pigment synthesizing capability of saffron cells Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 26
- 235000015655 Crocus sativus Nutrition 0.000 title claims abstract description 18
- 235000013974 saffron Nutrition 0.000 title claims abstract description 16
- 239000004248 saffron Substances 0.000 title claims abstract description 16
- 239000000049 pigment Substances 0.000 title abstract 4
- 230000002194 synthesizing effect Effects 0.000 title abstract 3
- 241000196324 Embryophyta Species 0.000 claims abstract description 23
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 20
- 239000006143 cell culture medium Substances 0.000 claims abstract description 14
- 229920001542 oligosaccharide Polymers 0.000 claims abstract description 10
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for improving saffron pigment synthesizing capability of saffron cells, which includes the following steps: (1) inoculating saffron calluses onto a plant cell culture medium containing 0.5 mg/L of cytokinin and 2 mg/L of archusia at temperature of 20+/-1 DEG C for 25-30 days; and (2) culturing the calluses cultured in the step (1) in a plant cell culture medium containing 60 mg/L or 20 mg/L of oligosaccharide, and adding 0.5 mg/L of cytokinin and 2 mg/L of archusia at temperature of 20+/-1 DEG C for 26 days. The method can remarkably improve synthesizing capability of saffron cells and content of saffron pigment, and the obtained saffron pigment is stable and not apt to brown.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of method that improves the synthetic safran of Stigma Croci cell.
Background technology
Stigma Croci (Crocus sativus L.) has another name called style and stigma of Saffron Crocus, Stigma Croci, is Iridaceae crocus napiform root class per nnial herb, originates in south of europe and Iran, imports Tibet into through India, so be called Stigma Croci.Stigma Croci is as a kind of precious seasonings, spices and the dyestuff long history of the existing centuries in Europe.Stigma Croci also has higher pharmaceutical use, is a kind of famous and precious medicinal material.Abroad be recorded in the earliest about 1550 Christian eras among " Ai Baisi sudd book " as medicinal, have promoting blood circulation and removing blood stasis, the strongly fragrant peculiar effect such as knot of opening of loosing.Continuing to use oneself for a long time as traditional Chinese medicine in China, is its rectification of name with Stigma Croci in the Compendium of Material Medica.China tradition traditional Chinese medical science thinks that Stigma Croci has effects such as promoting blood circulation and removing blood stasis, resolving stagnation for tranquilization, removing pathogenic heat from blood and toxic substance from the body, be used for through close, stasis of blood resistance in postpartum, wound, typhoid fever, hemoptysis haematemesis, maculae caused by violent heat pathogen, disease such as melancholy ruffian is vexed, palpitation with fear is gone mad.Modern pharmacology research shows, Stigma Croci column cap extract and purification component crocin (crocin) thereof, Picrocrocin (picrocrocin), crocetin (crocetin) etc. antitumor, regulate immunity, treatment and prevent cardiovascular and cerebrovascular diseases, depression and aspect such as anti-oxidant to have clear and definite pharmacological effect.Especially remarkable in the anti-tumor aspect effect, to white blood disease, ovarian cancer, colorectal carcinoma, papillosarcoma, pinacocyte knurl and soft tissue sarcoma are all had stronger restraining effect, its toxic side effect is starkly lower than antitumor drug commonly used at present especially.
Stigma Croci originates in ground such as various countries, southern Europe such as Spain, Greece and Iran.Because it is had relatively high expectations to growing environment, only there is a small amount of cultivation in Tibet, Xinjiang, Zhejiang, Jiangxi, Jiangsu, Beijing, Shanghai in China.In addition, the Stigma Croci quality of traditional way cultivation is uneven, output and quality are lower.Therefore, the Stigma Croci resource is extremely limited.The import hyoscine leans in China, seriously restricts its application in medicine industry.
Plant tissue and cell culture technology are widely used in medicinal plant production, for the production of famous and precious medicine and valuable medicine resource provides new approach.Reports such as cells produce alkannin, ginsenoside and Artemisinin such as existing extensive culture successful Asian puccoon, genseng and sweet wormwood.Utilizing plant cell culture technology scale operation crocin isoreactivity material is to solve one of Stigma Croci resources effective method.Domestic investigator has successively carried out the research work that the Stigma Croci cell cultures is produced crocin, but the subject matter that exists is that product yields poorly.The content that how to improve safran in the Stigma Croci cell culture is the key of Stigma Croci cell culture technology commercial application.Discover that elicitor is handled the content that can improve useful secondary metabolite in the plant tissue cultures.Elicitor be can the stimulating plant cell material of synthetic defensive secondary metabolite; It can be through changing the enzyme activity of katalaze(enzyme) in the secondary metabolism approach; Cause the change of metabolic flux and speed of reaction in the secondary metabolism approach; Thereby improve the output of secondary metabolite,, caused people's extensive concern for the synthetic regulation and control of vegetable cell secondary metabolism provide new tool.Utilizing elicitor to improve secondary metabolite, is the method for in the medicinal plant cell cultures, using always at present, and the most frequently used elicitor has fungal cell wall hydrolyzate, chitosan, methyl jasmonate, metals ion etc.
Oligochitosan (also claiming Chitosan poly oligosaccharide, chitin oligo saccharide, formal name used at school β-1,4-oligosaccharides-glycamine) is to pass through β-1 by 2～10 GS molecules, and the alkaline oligosaccharides that the 4-glycosidic link is formed by connecting is obtained by acidolysis or enzymolysis behind the Regitex FA deacetylation.Research shows that oligochitosan is as a kind of external source exciton, but the defensive raction of activated plant, through suppressing the germ invasion, inducing approach such as producing plant protecting chemical, the relevant defensin gene expression of activation to improve the whole resistance of plant.Oligochitosan promotes the effect of Secondary Metabolism of Plant product accumulation also to receive much concern as elicitor.50 with the 200ppm oligochitosan can significantly improve Greece wild marjoram (Origanum vulgare ssp.hirtum) polyphenol content (Heng Y etc., J.Agric.Food Chem., 2012,60,136-143).The 50-75mg/L oligochitosan have the effect of inducing Caulis Sacchari sinensis leaf accumulation polyphenol (Zhou Gui etc., southern agricultural sciences, 2011,42 (8), 874-877).Oligochitosan also can obviously improve under 4 ℃ of storage conditions anthocyanidin content in the cherry (Garry K etc., European Food Research and Technology, 233 (2), 351-358).Adopt seed dressing to combine the foliar spray method with oligomeric oligochitosan processing salvia seeds and blade, oligomeric oligochitosan can promote more significantly Tanshinone II A content raising (Zhang Yuan etc., the Anhui agricultural sciences, 2010,38 (34), 19316-19318).
Summary of the invention
Technical problem to be solved by this invention provides a kind of method that improves the synthetic safran of Stigma Croci cell; The problem that this method mainly yields poorly based on the Stigma Croci cytopigment of cultivating; Proposition utilizes oligochitosan as novel elicitor, is applied in the Stigma Croci cell culture system to improve the synthetic safran ability of Stigma Croci cell.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is following:
A kind of method that improves the synthetic safran of Stigma Croci cell, it comprises the steps:
(1) the Stigma Croci callus is inoculated on the plant cell culture medium that contains 0.5mg/L phytokinin, 2mg/L archusia and 30g/L sucrose, cultivated 25～30 days in 20 ± 1 ℃;
(2) callus culture after step (1) is cultivated is in the plant cell culture medium that contains 60mg/L or 20mg/L oligosaccharides; Add 0.5mg/L phytokinin, 2mg/L archusia and 20g/L sucrose in the described plant cell culture medium, cultivated 26 days in 20 ± 1 ℃.
In the step (1), described Stigma Croci callus is that form corm of saffron or bud are handled the callus of inducing formation with ordinary method.
In step (1) and the step (2), described phytokinin is 6-benzyl aminopurine (hereinafter to be referred as 6-BA).
In the step (1), described archusia is naphthylacetic acid (hereinafter to be referred as NAA).
In the step (1), said plant cell culture medium is the MS substratum.The MS substratum is high salt culture medium, contains high concentration nitrate, potassium ion and ammonium ion, and micro-kind is more, and its prescription can be referring to Murashige T, Skoog, FA.1962, Physiol.Plant 15:473-479.
In the step (2), described archusia is indolylacetic acid (hereinafter to be referred as IAA).
In the step (2), described oligosaccharides is an oligochitosan, molecular weight 3000Da.
In the step (2), described plant cell culture medium is 1/2B5 substratum (wherein SODIUM PHOSPHATE, MONOBASIC, saltpetre, ammonium sulfate, sal epsom consumption reduce by half).The B5 medium prescription can be referring to Gamborg OL, Miller RA, Ojima K.1968, Exp.Cell Res.50:151-158.
In the step (2), oligosaccharides adds choose opportunities in the plant cell culture medium before the cell cultures of step (2) or after the cell cultures of step (2) in 15 days.
Beneficial effect: method of the present invention can obviously promote the ability of the synthetic safran of Stigma Croci cell, reaches the purpose that improves safran output, and the safran of producing is stable, is difficult for brownization.
Embodiment
According to following embodiment, can understand the present invention better.Yet, those skilled in the art will readily understand that the described content of embodiment only is used to explain the present invention, and the present invention that should also can not limit in claims to be described in detail.
Embodiment 1:
Select healthy form corm of saffron, peel off the bulb epithelium, bulb is cleaned back flushing under flowing water spend the night.With 75% (v/v) alcohol immersion 1min, with 0.1% mercuric chloride solution sterilization 15min, use rinsed with sterile water afterwards 4 times again.Be cut into 0.5～1cm after blotting the bulb surface-moisture with sterilization filter paper
3Fritter.Be inoculated in and contain 2.0mg/L 2, on the MS substratum of 4-D and 0.5mg/L 6-BA, cultivation can obtain the form corm of saffron callus in about 35 days under 20 ± 1 ℃.
The form corm of saffron callus that obtains is inoculated on the MS substratum, and wherein additional 0.5mg/L 6-BA, 2mg/LNAA, 30g/L sucrose and 6g/L agar are cultivated down in 20 ± 1 ℃ of dark conditions, and per 25～30 days subcultures once.
26～28 days form corm of saffron callus of succeeding transfer culture is inoculated in the 1/2B5 substratum that contains the 60mg/L oligochitosan; Wherein additional 0.5mg/L 6-BA, 2mg/L IAA and 20g/L sucrose; Shaking culture is 26 days under 20 ± 1 ℃ of dark conditions, rotating speed 130r/min.Utilize the content of metric measurement safran, its content is 22.04mg/gdcw.
Under the same conditions, do not add oligochitosan in the 1/2B5 substratum and be contrast, the content of safran is 12.19mg/gdcw.Can find out, the Stigma Croci cell that uses the inventive method to cultivate, the content comparison of its safran is according to having improved 80.8%.
Embodiment 2:
26～28 days form corm of saffron callus of succeeding transfer culture in the embodiment 1 is inoculated in the 1/2B5 substratum wherein additional 0.5mg/L 6-BA, 2mg/L IAA and 20g/L sucrose, shaking culture under 20 ± 1 ℃ of dark conditions, rotating speed 130r/min.When cultivating the 4th day, add oligochitosan, final concentration is 60mg/L, continues to be cultured to 26 days, and the content comparison of safran is according to having improved 121.3%.
Embodiment 3:
26～28 days form corm of saffron callus of succeeding transfer culture in the embodiment 1 is inoculated in the 1/2B5 substratum wherein additional 0.5mg/L 6-BA, 2mg/L IAA and 20g/L sucrose, shaking culture under 20 ± 1 ℃ of dark conditions, rotating speed 130r/min.When cultivating the 14th day, add oligochitosan, final concentration is 60mg/L, continues to be cultured to 26 days, and the content comparison of safran is according to having improved 85.7%.
Embodiment 4:
Bulb is pressed embodiment 1 method sterilization back and is downcut the bud point; Place on the MS substratum that does not contain hormone, sprouting grows aseptic bud, directly induces explant material with aseptic bud as evoked callus; Be cut into the segment of about 0.5～1cm; Be inoculated in and contain 2.0mg/L 2, on the MS substratum of 4-D and 0.25mg/L 6-BA, cultivate acquisition safranine bud callus in 20 ± 1 ℃.
The safranine bud callus that obtains is inoculated into respectively on the MS substratum, and wherein additional 0.5mg/L 6-BA, 2mg/L NAA and 30g/L sucrose are cultivated down in 20 ± 1 ℃ of dark conditions, and per 25～30 days subcultures once.
26～28 days safranine bud callus of succeeding transfer culture is inoculated in the 1/2B5 substratum that contains the 20mg/L oligochitosan; Wherein additional 0.5mg/L 6-BA, 2mg/L IAA and 20g/L sucrose; Shaking culture is 26 days under 20 ± 1 ℃ of dark conditions, rotating speed 130r/min.The content of safran is 17.78mg/g dcw.
Under the same conditions, do not add oligochitosan in the 1/2B5 substratum and be contrast, the content of its safran is 14.61mg/g dcw.
The Stigma Croci cell that uses the inventive method to cultivate, the content comparison of its safran is according to having improved 21.7%.
Embodiment 5:
26～28 days safranine bud callus of succeeding transfer culture in the embodiment 4 is inoculated in the 1/2B5 substratum wherein additional 0.5mg/L BA, 2mg/L IAA and 20g/L sucrose, shaking culture under 20 ± 1 ℃ of dark conditions, rotating speed 130r/min.When cultivating the 4th day, add oligochitosan, final concentration is 20mg/L, continues to be cultured to 26 days, and the content comparison of safran is according to having improved 51.3%.
Claims (9)
1. a method that improves the synthetic safran of Stigma Croci cell is characterized in that it comprises the steps:
(1) the Stigma Croci callus is inoculated on the plant cell culture medium that contains 0.5mg/L phytokinin, 2mg/L archusia and 30g/L sucrose, cultivated 25～30 days in 20 ± 1 ℃;
(2) callus culture after step (1) is cultivated is in the plant cell culture medium that contains 60mg/L or 20mg/L oligosaccharides; Add 0.5mg/L phytokinin, 2mg/L archusia and 20g/L sucrose in the described plant cell culture medium, cultivated 26 days in 20 ± 1 ℃.
2. the method for the synthetic safran of raising Stigma Croci cell according to claim 1 is characterized in that, in the step (1), described Stigma Croci callus is that form corm of saffron or bud are handled the callus of inducing formation with ordinary method.
3. the method for the synthetic safran of raising Stigma Croci cell according to claim 1 is characterized in that in step (1) and the step (2), described phytokinin is a 6-benzyl aminopurine.
4. the method for the synthetic safran of raising Stigma Croci cell according to claim 1 is characterized in that in the step (1), described archusia is a naphthylacetic acid.
5. the method for the synthetic safran of raising Stigma Croci cell according to claim 1 is characterized in that in the step (1), said plant cell culture medium is the MS substratum.
6. the method for the synthetic safran of raising Stigma Croci cell according to claim 1 is characterized in that in the step (2), described archusia is an indolylacetic acid.
7. the method for the synthetic safran of raising Stigma Croci cell according to claim 1 is characterized in that in the step (2), described oligosaccharides is an oligochitosan.
8. the method for the synthetic safran of raising Stigma Croci cell according to claim 1 is characterized in that in the step (2), described plant cell culture medium is the 1/2B5 substratum.
9. the method for the synthetic safran of raising Stigma Croci cell according to claim 1; It is characterized in that; In the step (2), oligosaccharides adds choose opportunities in the plant cell culture medium before the cell cultures of step (2) or after the cell cultures of step (2) in 15 days.
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| CN105483074A (en) * | 2016-01-09 | 2016-04-13 | 佛山市金蓝领教育科技有限公司 | Saffron suspension cell line establishing method |
| CN106497861A (en) * | 2016-11-15 | 2017-03-15 | 天津市博爱生物药业有限公司 | A kind of culture medium for Stigma Croci cell |
| CN108419679A (en) * | 2018-05-31 | 2018-08-21 | 浙江大学 | The tissue culture method of west safflower |
| CN110999791A (en) * | 2019-12-27 | 2020-04-14 | 青岛大学附属医院 | Saffron crocus culture medium for increasing crocin content |
| CN114058563A (en) * | 2020-07-31 | 2022-02-18 | 伽蓝（集团）股份有限公司 | Composition for suspension culture of nardostachys chinensis bunge cells and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105483074A (en) * | 2016-01-09 | 2016-04-13 | 佛山市金蓝领教育科技有限公司 | Saffron suspension cell line establishing method |
| CN106497861A (en) * | 2016-11-15 | 2017-03-15 | 天津市博爱生物药业有限公司 | A kind of culture medium for Stigma Croci cell |
| CN108419679A (en) * | 2018-05-31 | 2018-08-21 | 浙江大学 | The tissue culture method of west safflower |
| CN110999791A (en) * | 2019-12-27 | 2020-04-14 | 青岛大学附属医院 | Saffron crocus culture medium for increasing crocin content |
| CN114058563A (en) * | 2020-07-31 | 2022-02-18 | 伽蓝（集团）股份有限公司 | Composition for suspension culture of nardostachys chinensis bunge cells and application thereof |
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