CN105104194B - Promote Anji white tea callus proliferation and the method for improving wherein polyphenol content - Google Patents
Promote Anji white tea callus proliferation and the method for improving wherein polyphenol content Download PDFInfo
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Abstract
The invention discloses a kind of promotion Anji white tea callus proliferation and/or the method for improving wherein polyphenol content.The method comprises the following steps：Anji white tea callus is placed in culture medium A and is cultivated, so as to promote Anji white tea callus proliferation and/or improve polyphenol content in the white tea callus of Anji；The culture medium A is that the culture medium obtained after TDZ, IAA and phenylalanine is added in MS solid mediums；Final concentration of 0.1 1.0mg/L of the final concentration of 1.0mg/L of final concentration of 0.5mg/L, IAA of TDZ, phenylalanine in the culture medium A.It is demonstrated experimentally that using culture medium provided by the present invention the fresh weight of Anji white tea callus and dry weight ratio can be made to significantly improve with compareing, and the content of wherein Tea Polyphenols is set to be up to 26.72%.The present invention is significant for the exploitation of Anji white tea resource and the industrialized production of Tea Polyphenols.
Description
Technical field
The invention belongs to field of plant tissue culture, it is related to a kind of promotion Anji white tea callus proliferation and/or raising
The wherein method of polyphenol content.
Background technology
Anji white tea is a kind of Han nationality's well-known tea, originates from Anji County Xi Long townshiies of Zhejiang Province, is tea (Camellia sinensis)
A kind of albino mutation kind of middle energy stabilization heredity.Spring spire is white, and especially with two leaves and a bud as most white, passages through which vital energy circulates is emerald green.With
Temperature rising, illumination enhancing, it is white that leaf color is gradated, and Summer-autumn tea is green.Anji white tea originates in Zhejiang Province Anji
County, Anji white tea is introduced a fine variety on the ground such as Jiangsu, Jiangxi, Henan, Sichuan, Chongqing, Guizhou at present.
Anji white tea has good health-care efficacy.Contain the compound and tealeaves lipid of several amino acids such as Anji white tea
In diphenylamines, can liver-protecting and stomach-protecting, can promote liver synthesize haemoglutinin, there is anti-aging；For another example Anji white tea is rich
Containing aminobutyric acid, can lowering blood pressure and blood fat, hypoglycemic；And for example Anji white tea contains trace element manganese, zinc, selenium and tea polyphenols thing
Matter, can strengthen memory, protect nerve cell, and brain damage is very helpful；Tea Polyphenols also such as Anji white tea can give protection against cancer
Anticancer etc..
Not yet it is related to how to promote Anji white tea callus proliferation at present and/or improves the phase of wherein polyphenol content
Close report.
The content of the invention
Promote Anji white tea callus proliferation and/raising Anji white tea callus it is an object of the present invention to provide one kind
The method of polyphenol content in tissue.
Tea is more in promotion Anji white tea callus proliferation provided by the present invention and/or raising Anji white tea callus
The method of phenol content, specifically includes following steps：Anji white tea callus is placed in culture medium A and is cultivated, so as to promote
Enter Anji white tea callus proliferation and/or improve polyphenol content in the white tea callus of Anji；The culture medium A is in MS
The culture medium obtained after TDZ, IAA and phenylalanine is added in solid medium；The final concentration of TDZ can be in the culture medium A
The final concentration of 0.5mg/L, IAA can be able to be 0.1-1.0mg/L for 1.0mg/L, the final concentration of phenylalanine；pH5.8.
Further, the final concentration of TDZ is specially the final concentration specially 1.0mg/ of 0.5mg/L, IAA in the culture medium A
L, the final concentration of phenylalanine are specially 0.5mg/L；pH5.8.
It is a further object to provide a kind of method of promotion Anji white tea callus proliferation.
The method of promotion Anji white tea callus proliferation provided by the present invention, comprises the following steps：By Anji white tea
Callus is placed in culture medium B and is cultivated, so as to promote Anji white tea callus proliferation；The culture medium B is in MS
The culture medium obtained after TDZ and IAA is added in solid medium；The final concentration of TDZ can be 0.5-4.0mg/ in the culture medium B
The final concentration of L, IAA can be 0.5-4.0mg/L；pH5.8.
Further, the final concentration of TDZ is specially the final concentration specially 1.0mg/ of 0.5mg/L, IAA in the culture medium B
L；pH5.8.
In the above described two methods, the condition of the culture can be：25 ± 2 DEG C of temperature, light application time 12h/ days, illumination
30~40mmolm of intensity-2·s-1.The cycle of the culture can be 45 days.
Further, the Anji white tea callus can be prepared according to the method for comprising the following steps：Anji is white
The blade inoculation of tea obtains the Anji white tea callus group in induction of callus is carried out on callus inducing medium
Knit；The culture medium that the callus inducing medium is obtained after adding TDZ in MS solid mediums；The callus is lured
The final concentration for leading TDZ in culture medium can be 1.0-2.0mg/L (such as 1.0mg/L)；pH5.8；
Wherein, the condition of the induction of callus can be：25 ± 2 DEG C of temperature, light application time 12h/ days, illumination are strong
30~40mmolm of degree-2·s-1；The cycle of the induction of callus can be 45 days.
The step of the above method in (1), the blade of the Anji white tea is the blade after sterilization, size is about 0.5 ×
0.5cm；The sterilization is concretely：The blade of Anji white tea to be sterilized is rinsed into 1h with flowing water, flushed blade is used
Volume fraction is 70% alcohol disinfecting 30s, then with the water-soluble liquid disinfectant 8min of mercuric chloride that mass fraction is 0.1%, finally with nothing
Bacterium water is rinsed (as rinsed 5 times).
A further object of the present invention is to provide a kind of for promoting Anji white tea callus proliferation and/or improving Anji
The culture medium of polyphenol content in white tea callus.
It is provided by the present invention for promote Anji white tea callus proliferation and/or improve Anji white tea callus in
The culture medium of polyphenol content, it is concretely following (A) or (B)：
(A) culture medium A；
(B) complete set of culture medium being made up of the culture medium A and the callus inducing medium.
It is of the invention that a kind of culture medium for promoting Anji white tea callus proliferation of offer is provided.
Culture medium for promoting Anji white tea callus proliferation provided by the present invention, concretely following (C) or
(D)：
(C) the culture medium B；
(D) complete set of culture medium being made up of the culture medium B and the callus inducing medium.
The application of (E) or (F) as follows falls within protection scope of the present invention：
(E) culture medium of (A) or (B) is promoting Anji white tea callus proliferation and/or raising Anji white
Application in tea callus in polyphenol content；
(F) application of the culture medium of (C) or (D) in Anji white tea callus proliferation is promoted.
In the present invention, the carbon source in the MS solid mediums is sucrose；Gel in the MS solid mediums
It is agar.
Final concentration of 30g/L of the sucrose in the MS solid mediums；The agar is in the MS solid cultures
Final concentration of 7g/L in base.
More specific, the pH5.8 of the MS solid mediums, solvent is water, and solute and its concentration are as follows：
A. it is a large amount of：NH4NO31.65g/L, KNO31.9g/L, MgSO4 .7H2O 0.37g/L, KH2PO40.17g/L, CaCl2·
2H2O 0.44g/L；
B. it is micro：KI 0.83mg/L, H3BO36.2mg/L, MnSO4 .4H2O 22.3mg/L, ZnSO4 .7H2O8.6mg/L,
Na2MoO4 .2H2O 0.25mg/L, CuSO4 .5H2O 0.025mg/L, CoCl2 .6H2O 0.025mg/L；
C. vitamin：Nicotinic acid 0.5mg/L, pyridoxine hydrochloride 0.5mg/L, thiamine hydrochloride 0.1mg/L, glycine 2mg/L；
D. molysite：FeSO4 .7H2O 27.8mg/L, Na2EDTA 37.3mg/L；
E. inositol 0.1g/L；
F. sucrose 30g/L；
G. agar 7g/L.
Each concentration is final concentration of the respective components in the MS solid mediums above.
The present invention have studied its callus induction, propagation and Tea Polyphenols synthesis with the tender leaf of Anji white tea as explant
Optimum condition.Result of study shows：MS+TDZ 1.0-2.0mg/mL are the optimal medium of callus induction, inductivity
It is 86.54%-90.21%；Callus takes on a red color, and quality is loose, growth is vigorous.2,4-D, NAA, 6-BA are unfavorable for callus group
The propagation knitted, IAA, TDZ are conducive to it to breed.MS+TDZ 0.5-4.0mg/L+IAA0.5-4.0mg/L are callus proliferations
Optimal medium, fresh weight proliferation times reach as high as 16.89 times, and dry weight proliferation times reach as high as 20.75 times.In culture medium
Addition TDZ, IAA and phenylalanine are conducive to the synthesis and accumulation of Tea Polyphenols in callus.As TDZ for 0.5mg/L, IAA are
When 1.0mg/L, phenylalanine are 0.5mg/L, the content highest of Tea Polyphenols, up to 26.72%.MS+TDZ0.5mg/L+
IAA1.0mg/L+ phenylalanines 0.5mg/L is the appropriate media of synthesis with the accumulation of Tea Polyphenols in the white tea callus of Anji.
The present invention is significant for the exploitation of Anji white tea resource and the industrialized production of Tea Polyphenols.
Specific embodiment
Experimental technique used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
The pH of MS solid mediums involved in following embodiments is 5.8, and Ju Ti Pei Fang is as shown in table 1：
The MS solid mediums of table 1 constitute (pH5.8)
| Compound name | Concentration (mg/L) in culture medium |
| 440 | |
| 170 | |
| 370 | |
| 1650 | |
| 1900 | |
| KI | 0.83 |
| 0.025 | |
| 6.2 | |
| 0.25 | |
| 22.3 | |
| 0.025 | |
| 8.6 | |
| 37.3 | |
| 27.8 | |
| Thiamine hydrochloride | 0.1 |
| Pyridoxine hydrochloride | 0.5 |
| Nicotinic acid | 0.5 |
| Inositol | 100 |
| Glycine | 2.0 |
| Sucrose | 30000 |
| Agar | 7000 |
The induction of embodiment 1, the callus of Anji white tea
First, the selection of explant with disinfect
Anji white tea picks up from Guizhou Province Dejiang County Plain township in May, 2013, and according to original producton location cultivation Anji white tea
Form state characteristic standard is confirmed that material object is consistent with title.Anji white tea tender leaf is taken, running water rinses 1h, in ultra-clean work
Make on platform first with 70% (volume fraction) ethanol disinfection 30s, then with 0.1% (mass fraction) HgCl2Solution sterilization 8min, it is aseptic
Water is rinsed 5 times, stand-by.
2nd, the induction of the callus of Anji white tea
In superclean bench, the blade of the Anji white tea by step one after sterilized is cut into about 0.5 × 0.5cm sizes
Stripping and slicing, is inoculated into containing various concentrations 2 respectively, and callus group is carried out in the MS solid mediums of 4-D, NAA, 6-BA, KT, TDZ
The induction knitted.Condition of culture is 25 ± 2 DEG C of temperature, light application time 12h/ days, 30~40mmolm of intensity of illumination-2·s-1；Training
The cycle of supporting is 45 days.
Research finds that the inducing effect of TDZ is preferably (table 1).MS+TDZ 1.0-2.0mg/L are Anji white tea callus
Optimal inducing culture, inductivity is 86.54%-90.21%.Callus inducing medium (MS+TDZ 1.0-2.0mg/L)
The culture medium obtained after specially adding TDZ in MS solid mediums；The end of TDZ is dense in the callus inducing medium
It is 1.0-2.0mg/L to spend；pH5.8.
Inductive effect of the hormon of table 1 to callus induction
Note：Different lowercase letter indication differences significantly (P<0.05).
The propagation of embodiment 2, the callus of Anji white tea
The good callus of the growth conditions obtained in embodiment 1 is chosen, is cut into scalpel on superclean bench
Fritter, is seeded in different callus proliferation mediums, and every bottle is inoculated with 5 pieces of (fresh weight is 0.8g, dry weight is 0.04g) explants
Body, each is processed as 50 explants, is repeated 3 times, and is harvested after 45 days.Wherein condition of culture is 25 ± 2 DEG C of temperature, light application time
12h/ days, 30~40mmolm of intensity of illumination-2·s-1。
Research finds that cultivation effect is best when the TDZ and IAA of additional 0.5-4.0mg/L in the medium, callus
Fresh weight and the maximum respectively 13.51g and 0.83g of dry weight, are 16.89 times and 20.75 times (table 2) of initial inoculum.MS+TDZ
0.5-4.0mg/L+IAA 0.5-4.0mg/L are the optimal mediums of callus proliferation.Callus proliferation medium (MS+
TDZ 0.5-4.0mg/L+IAA 0.5-4.0mg/L) it is specially the training for being added in MS solid mediums and being obtained after TDZ and IAA
Support base；The final concentration of 0.5-4.0mg/ of final concentration of 0.5-4.0mg/L, IAA of TDZ in the callus inducing medium
L；pH5.8.
The TDZ of table 2 combines the influence to callus proliferation with IAA
Note：Different lowercase letter indication differences significantly (P<0.05).
Influence of the embodiment 3, precursor substance to the propagation and polyphenol content of callus
According to the result of embodiment 2, callus proliferation medium (MS+TDZ 0.5mg/mL+IAA 1.0mg/ are chosen
ML) as minimal medium, precursor substance, growth and polyphenol content of the research precursor substance to callus are added thereto to
Influence.The present embodiment is precursor substance from sodium acetate and phenylalanine.
The good callus of the growth conditions obtained in embodiment 1 is chosen, is cut into scalpel on superclean bench
Fritter, is seeded in callus proliferation medium (the MS+TDZ 0.5mg/mL+ containing various concentrations sodium acetate or phenylalanine
IAA 1.0mg/mL) interior (concentration of sodium acetate or phenylalanine is referring specifically to table 3), (fresh weight is 0.8g, does for every bottle of 5 pieces of inoculation
Weight is 0.04g) explant, each is processed as 50 explants, is repeated 3 times, and is harvested after 45 days, counts the callus group of different disposal
The fresh weight and dry weight knitted and the content for determining wherein Tea Polyphenols.Wherein condition of culture is 25 ± 2 DEG C of temperature, light application time 12h/
My god, 30~40mmolm of intensity of illumination-2·s-1。
Result is as shown in table 3.As seen from table：
Precursor substance sodium acetate is added in the medium, and when the concentration of sodium acetate is 0-1.0mg/L, it is to callus
Fresh weight and dry weight influence it is little, but after its concentration is more than 1.0mg/L, the fresh weight and dry weight of callus begin to decline, when
When sodium acetate is 1.0mg/L, the content of Tea Polyphenols reaches maximum, is 23.48%, but difference is not notable compared with control group, when
After the concentration of sodium acetate is more than 1.0mg/L, the concentration of Tea Polyphenols begins to decline, and is 20.42%.
Precursor substance phenylalanine is added in the medium, and when concentration of phenylalanine is 0-1.0mg/L, it is to callus group
Fresh weight and the dry weight influence knitted are little, but after its concentration is more than 1.0mg/L, the fresh weight and dry weight of callus begin to decline,
And significant difference (p<0.05).But the content of Tea Polyphenols is as the increase of concentration of phenylalanine is in first increasing the trend that drops afterwards, when
When concentration of phenylalanine is 0-0.5mg/L, the content of Tea Polyphenols gradually increases, and when concentration of phenylalanine is 0.5mg/L, reaches
There is significant difference (p between maximum 26.72%, with other treatment<0.05).After concentration of phenylalanine is more than 0.5mg/L, tea
The content of polyphenol begins to decline, and when concentration of phenylalanine is 2.0mg/L, the content of Tea Polyphenols is minimized, and is 22.06%.
Therefore, MS+TDZ 0.5mg/L+IAA 1.0mg/L+ phenylalanines 0.5mg/L is tea in the white tea callus of Anji
The appropriate media of synthesis with the accumulation of polyphenol.Culture medium (MS+TDZ 0.5mg/L+IAA 1.0mg/L+ phenylalanines 0.5mg/
L the culture medium obtained after) adding TDZ, IAA and phenylalanine in MS solid mediums；The end of TDZ is dense in the culture medium A
Spend final concentration of 1.0mg/L, the final concentration of 0.5mg/L of phenylalanine for 0.5mg/L, IAA；pH5.8.
The precursor substance of table 3 is to the growth of callus and the influence of Tea Polyphenols
Claims (7)
1. a kind of promotion Anji white tea callus proliferation and the method for improving polyphenol content in the white tea callus of Anji, wrap
Include following steps：Anji white tea callus is placed in culture medium A and is cultivated, so as to promote Anji white tea callus to increase
Grow and/or improve polyphenol content in the white tea callus of Anji；
The culture medium A is that the culture medium obtained after TDZ, IAA and phenylalanine is added in MS solid mediums；The culture
The final concentration of 0.1-1.0mg/L of the final concentration of 1.0mg/L of final concentration of 0.5mg/L, IAA of TDZ, phenylalanine in base A.
2. method according to claim 1, it is characterised in that：The final concentration of 0.5mg/L of TDZ in the culture medium A,
The final concentration of 1.0mg/L of IAA, the final concentration of 0.5mg/L of phenylalanine.
3. method according to claim 1, it is characterised in that：The condition of the culture is：When 25 ± 2 DEG C of temperature, illumination
Between 12h/ days, 30~40mmolm of intensity of illumination-2·s-1。
4. method according to claim 1, it is characterised in that：The cycle of the culture is 45 days.
5. method according to claim 1, it is characterised in that：The Anji white tea callus is according to including following step
What rapid method was prepared：By the blade inoculation of Anji white tea in carrying out callus induction on callus inducing medium
Culture, obtains the Anji white tea callus；After the callus inducing medium adds TDZ in MS solid mediums
The culture medium for obtaining；The final concentration of 1.0-2.0mg/L of TDZ in the callus inducing medium；
The condition of the induction of callus is：25 ± 2 DEG C of temperature, light application time 12h/ days, intensity of illumination 30~
40mmol·m-2·s-1；The cycle of the induction of callus is 45 days.
6. it is used to promote Anji white tea callus proliferation and/or improves the culture of polyphenol content in the white tea callus of Anji
Base, is following (A) or (B)：
(A) culture medium A described in claim 1 or 2；
(B) the callus inducing medium group described in the culture medium A and claim 5 described in claim 1 or 2
Into complete set of culture medium.
7. culture medium described in claim 6 is promoting Anji white tea callus proliferation and/or is improving Anji white tea callus
Application in middle polyphenol content.
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| CN108112474A (en) * | 2016-11-28 | 2018-06-05 | 山东农业大学 | A kind of method that cultured in vitro improves STEVIA REBAUDIANA RA contents |
| CN107864858B (en) * | 2017-10-30 | 2020-10-16 | 湖南农业大学 | Method for preparing anthocyanin by suspension culture and extraction of tea leaf callus |
| CN108142288A (en) * | 2017-11-21 | 2018-06-12 | 山东农业大学 | A kind of method that cultured in vitro improves chrysanthemum for tea use flower sugariness |
| CN110432147A (en) * | 2019-09-03 | 2019-11-12 | 万源生态股份有限公司 | A kind of var. longistyla callus acquisition methods rich in polyphenol |
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