CN106117175A - A kind of red meat Tissue-cultured apple seedling produces the method for anthocyanin - Google Patents
A kind of red meat Tissue-cultured apple seedling produces the method for anthocyanin Download PDFInfo
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- CN106117175A CN106117175A CN201610495211.6A CN201610495211A CN106117175A CN 106117175 A CN106117175 A CN 106117175A CN 201610495211 A CN201610495211 A CN 201610495211A CN 106117175 A CN106117175 A CN 106117175A
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- anthocyanin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
- C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
- C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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Abstract
The invention discloses a kind of method that red meat Tissue-cultured apple seedling produces anthocyanin, operating procedure is as follows: (1) obtains sterilizable material, (2) initial culture obtains red meat Tissue-cultured apple seedling, (3) successive transfer culture obtains a large amount of red meat Tissue-cultured apple seedling, (4) strong seedling culture, the extraction of (5) anthocyanin and the preparation of crude product.The experiment proves that, utilizing the red meat Tissue-cultured apple seedling that said method is cultivated, its Anthocyanin Content is up to 359.86U g‑1FW, it it is 7.8 times of the Anthocyanin Content in convention sucrose culture medium under room temperature, 12 weeks peels, young leaves after 5 years raw red meat Fructus Mali pumilae bearing-age trees are spent respectively, spend after 12 weeks sarcocarp, colored 1.8,2.8,2.9,3.6 times, the present invention is not lacked by time and raw material and is limited, realize the industrialized production utilizing tissue culture method to carry out anthocyanin, provide a kind of new approach for producing anthocyanin.
Description
Technical field
The present invention relates to biological technical field, particularly relate to plant biotechnology field, particularly relate to tissue culture
Method produces the method for Fructus Mali pumilae anthocyanin.
Technical background
Anthocyanin is the natural water-soluble colorants being widely present in the flower of plant, fruit, and it belongs to flavonoid class chemical combination
Thing, is a kind of antioxidant.The beneficial effect of anthocyanin has: vessel softening, blood pressure lowering, enhances metabolism and prevents heart and brain blood
Pipe disease, antioxidant activity, prevents canceration, improves visual performance etc., is a kind of to generally acknowledge healthy and helpful healthy material, and red
Containing abundant anthocyanin in meat Fructus Mali pumilae.But, the red meat apple variety of current China popularization on producing and the area of cultivation
The most fewer, and lacked by time and raw material and limited, it is impossible to meet the requirement of factorial praluction, and Anthocyanin Content is relatively
Low, therefore, a kind of method exploring large-scale production anthocyanin, to the selection-breeding of functional fruit and opening of the Natural antioxidant
Send out application significant.
The raw material that the present invention uses is that the sarcocarp obtained after the Xinjiang red merit of red meat Fructus Mali pumilae 1 hybridizes with Fuji apple is scarlet
The new the most slightly stem apex of major clique (2010-15), wherein the correlation properties of the maternal Xinjiang red merit of red meat Fructus Mali pumilae 1 describe, and inventor is
Through having delivered article: Morphological characteristic and pollination compatibility ofa
New red flesh apple, HongxunNo.1.Research on Crops, 2013,14 (1): 199-204, this red meat is excellent
System (2010-15) is planted in the breeding base of Qingdao Agricultural University.
Summary of the invention
It is an object of the invention to utilize a kind of new method to extract anthocyanin, solve current red meat apple variety and cultivation
Few by area and by season and raw material lack limited, Anthocyanin Content is too low, can not meet the requirement of factorial praluction
Problem.
For solving above-mentioned technical problem, the present invention provides following solution:
A kind of red meat Tissue-cultured apple seedling produces the method for anthocyanin, it is characterised in that: comprise the following steps:
(1) sterilizable material is obtained:
Major clique (2010-15) the red meat Fructus Mali pumilae that after selecting Xinjiang red meat Fructus Mali pumilae to hybridize with Fuji apple, the sarcocarp of acquisition is scarlet
New slightly stem apex be outer implant, with 1% tween (or detergent) water soaking 15min, then after rinsing 20min with tap water, Yu Chao
On clean workbench, with the alcohol-pickled sterilization 30s of 75%, aseptic water washing 1 time, then with 0.1% mercuric chloride solution soaking disinfection
6-8min, period constantly rocks, and makes each of outer implant all touch disinfectant solution, finally by sterile water wash 5 times；
(2) initial culture acquisition red meat Tissue-cultured apple seedling:
Outer implant being inoculated on inducing culture, described inducing culture, with MS as minimal medium, adds 1.0mg
The 6-BA (benzyladenine) of L-1, the IBA (indolebutyric acid) of 0.1mg L-1,30g L-1 sucrose, 8g L-1 agar, 3g
L-1 activated carbon, 4g L-1PVP, Medium's PH Value is 5.8, after 121 DEG C of high pressure steam sterilization 25min, subpackage, treat outer implant
When growing into the budlet of 0.6-1.3cm, carry out subculture increment and cultivate；
(3) a large amount of red meat Tissue-cultured apple seedling of successive transfer culture acquisition:
Being inoculated on subculture medium by budlet and carry out increment cultivation, described successive transfer culture based component is MS, 1.0mg L-
16-BA, 0.1mg L-1IBA, after about 10-20 days, differentiates 2-3 Multiple Buds, after 25-35 days, Multiple Buds length to about 2cm,
Once, in Subculture, temperature controls at 20-30 DEG C average 4-5 week subculture；
(4) strong seedling culture:
Take the red meat Tissue-cultured apple seedling that successive transfer culture grows fine, be seeded to MS as minimal medium, add 1.0mg
L-16-BA (benzyladenine), 0.1mg L-1IBA (indolebutyric acid), 30g L-1Fructose or sorbitol, 8g L-1Fine jade
In the culture medium of fat, it is placed in 17 DEG C of constant temperature illumination boxs and carries out strong seedling culture；
(5) extraction of anthocyanin and the preparation of crude product:
Choose 17 DEG C of constant temperature culture that fructose or sorbitol process tissue cultured seedling after 14 days, become by liquid nitrogen grinding after shredding
Powder, is placed in the powder obtained in conical flask, and adds acetone soln by solid-to-liquid ratio 1:10, is sealed by conical flask sealed membrane, puts
In ultrasonic cleaner, control temperature is at 30 DEG C, and ultrasonic 30min is placed in dark extraction 10 hours, obtains lixiviating solution, will leaching
The extract buchner funnel sucking filtration of connection vacuum air pump, pours gained filtrate rotation into and steams bottle, on a rotary evaporator with 20-30
DEG C temperature rotation boil off except acetone solvent, then lucifuge stands, then the granule on sucking filtration, filter paper is anthocyanin crude product.
The present invention is reached to provide the benefit that:
(1) when carrying out initial culture, at MS medium supplemented activated carbon and PVP, it is therefore prevented that the generation of brownization, additional
100mg·L-1Carbenicillin, Bacterial Contamination can be avoided, making outer implant body pollution rate and melting brown rate is 0, and the stem apex rate of sprouting is
100%；
(2), after being inoculated into subculture medium, after about 10-20 days, 2-3 Multiple Buds, after 25-35 days, Multiple Buds are differentiated
Long to about 2cm, average 4-5 week can subculture once, make cultivated area increase, solve the restricted problem of raw material；
(3) after the tissue cultured seedling processed through fructose or sorbitol cultivates 14d under 17 DEG C of constant temperature illumination boxs, its cyanine
Glycosides content 359.86U g respectively-1FW and 349.41U g-1FW, is the cyanine under room temperature in (25 DEG C) convention sucrose culture medium
Glycosides content (45.86U g-1FW) 7.8 and 7.6 times, cultivate tissue cultured seedling cyanine after 14d under the conditions of 17 DEG C that wherein fructose processes
Glycosides content is 12 weeks peel (201.3U g after 5 years raw bearing-age trees are spent respectively-1FW), young leaves (124.9U g-1FW) rear 12, are spent
Week sarcocarp (124.0U g-1FW) and flower (98.2U g-1FW) 1.8,2.8,2.9,3.6 times, Anthocyanin Content is greatly improved.
Accompanying drawing explanation
Fig. 1 is the Anthocyanin Content of 5 years raw tree different times Different Organs of red meat Fructus Mali pumilae major clique (2010-15).
Detailed description of the invention
Embodiment:
A kind of red meat Tissue-cultured apple seedling produces the method for anthocyanin, it is characterised in that: comprise the following steps:
(1) sterilizable material is obtained:
Major clique (2010-15) the red meat Fructus Mali pumilae that after selecting Xinjiang red meat Fructus Mali pumilae to hybridize with Fuji apple, the sarcocarp of acquisition is scarlet
New slightly stem apex be outer implant, with 1% tween (or detergent) water soaking 15min, then after rinsing 20min with tap water, Yu Chao
On clean workbench, with the alcohol-pickled sterilization 30s of 75%, aseptic water washing 1 time, then with 0.1% mercuric chloride solution soaking disinfection
6-8min, period constantly rocks, and makes each of outer implant all touch disinfectant solution, finally by sterile water wash 5 times；
(2) initial culture acquisition red meat Tissue-cultured apple seedling:
Outer implant being inoculated on inducing culture, described inducing culture, with MS as minimal medium, adds 1.0mg
The 6-BA (benzyladenine) of L-1, the IBA (indolebutyric acid) of 0.1mg L-1,30g L-1 sucrose, 8g L-1 agar, 3g
L-1 activated carbon, 4g L-1PVP, Medium's PH Value is 5.8, after 121 DEG C of high pressure steam sterilization 25min, subpackage, treat outer implant
When growing into the budlet of 0.6-1.3cm, carry out subculture increment and cultivate；
(3) a large amount of red meat Tissue-cultured apple seedling of successive transfer culture acquisition:
Being inoculated on subculture medium by budlet and carry out increment cultivation, described successive transfer culture based component is MS, 1.0mg L-
16-BA, 0.1mg L-1IBA, after about 10-20 days, differentiates 2-3 Multiple Buds, after 25-35 days, Multiple Buds length to about 2cm,
Once, in Subculture, temperature controls at 20-30 DEG C average 4-5 week subculture；
(4) strong seedling culture:
Take the red meat Tissue-cultured apple seedling that successive transfer culture grows fine, be seeded to MS as minimal medium, add 1.0mg
L-16-BA (benzyladenine), 0.1mg L-1IBA (indolebutyric acid), 30g L-1Fructose or sorbitol, 8g L-1Fine jade
In the culture medium of fat, it is placed in 17 DEG C of constant temperature illumination boxs and carries out strong seedling culture；
(5) extraction of anthocyanin and the preparation of crude product:
Choose 17 DEG C of constant temperature culture that fructose or sorbitol process tissue cultured seedling after 14 days, become by liquid nitrogen grinding after shredding
Powder, is placed in the powder obtained in conical flask, and adds acetone soln by solid-to-liquid ratio 1:10, is sealed by conical flask sealed membrane, puts
In ultrasonic cleaner, control temperature is at 30 DEG C, and ultrasonic 30min is placed in dark extraction 10 hours, obtains lixiviating solution, will leaching
The extract buchner funnel sucking filtration of connection vacuum air pump, pours gained filtrate rotation into and steams bottle, on a rotary evaporator with 20-30
DEG C temperature rotation boil off except acetone solvent, then lucifuge stands, then the granule on sucking filtration, filter paper is anthocyanin crude product.
The acetone solvent selected in the extraction of above-mentioned anthocyanin and the preparation of crude product is volatile, can be gone by rotary evaporation
Removing, the tissue cultured seedling liquid nitrogen grinding after shredding becomes fine powder and extracts by the method for ultrasonic 30 minutes, can be farthest by group
Anthocyanin in seedlings cultivating is leached out, vacuum lyophilization lyophilizing anthocyanin crude product, it is to avoid red meat Fructus Mali pumilae anthocyanin is due to temperature
Degree contacts with the oxygen in air and degrades.
It should be noted that for large-scale industrialized production anthocyanin, improving tissue cultured seedling Anthocyanin Content, inventor exists
With the addition of different types of sugar respectively in the MS culture medium of strong seedling culture to test, sugar type sets 8 kinds altogether, it may be assumed that sorbitol, really
Sugar, glucose, sucrose, galactose, lactose, maltose, mannose, under different treatment temperatures, constant temperature culture is after 14 days
Tissue cultured seedling, its Anthocyanin Content is respectively (it is lethal that mannose is that tissue cultured seedling is withered, therefore can not survey its Anthocyanin Content):
It should be noted that inventor is big with life in 5 years in order to compare the tissue cultured seedling Anthocyanin Content through sugar and Temperature Treatment
The difference of the Anthocyanin Content of the fruit of tree, blade and flower, when determining 5 years raw tree differences of red meat Fructus Mali pumilae major clique (2010-15)
The Anthocyanin Content of phase Different Organs, refers to accompanying drawing 1: wherein, cultivates tissue cultured seedling flower after 14d under the conditions of 17 DEG C that fructose processes
Blue or green glycosides content is 12 weeks peels (201.3U g-1FW), young leaves (124.9U g-1FW), Hua Hou after 5 years raw bearing-age trees are spent respectively
12 weeks sarcocarp (124.0U g-1FW) and flower 1.8,2.8,2.9,3.6 times of (98.2U g-1FW).
It should be understood that the mensuration of Anthocyanin Content is carried out according to Liu et al (2009), by gather blade, flower,
Put into 10ml1%Hcl/ methanol solution after the chopping of peel, sarcocarp in 4 DEG C of refrigerators, extract 24h, extract with spectrophotometric determination
Liquid is light absorption value at 553nm and 600nm, makees with the optical density changing value 53nm-A600nm=0.01 of the extracting solution of every gram of sample
It is an anthocyanin unit, represents with U g-1FW.
Last it is noted that the foregoing is only presently preferred embodiments of the present invention, be not intended to limit the present invention, all
Simple any amendment of being made according to the technical spirit of the present invention, equivalent, modification etc., all should be included in the guarantor of the present invention
Within the scope of protecting.
Claims (1)
1. the method that a red meat Tissue-cultured apple seedling produces anthocyanin, it is characterised in that: comprise the following steps:
(1) sterilizable material is obtained:
Major clique (2010-15) the red meat Fructus Mali pumilae that after selecting Xinjiang red meat Fructus Mali pumilae to hybridize with Fuji apple, the sarcocarp of acquisition is scarlet new
Slightly stem apex is outer implant, with 1% tween (or detergent) water soaking 15min, then after rinsing 20min with tap water, in ultra-clean work
In station, with the alcohol-pickled sterilization 30s of 75%, aseptic water washing 1 time, then with 0.1% mercuric chloride solution soaking disinfection 6-
8min, period constantly rocks, and makes each of outer implant all touch disinfectant solution, finally by sterile water wash 5 times；
(2) initial culture acquisition red meat Tissue-cultured apple seedling:
Outer implant being inoculated on inducing culture, described inducing culture, with MS as minimal medium, adds 1.0mg L-1's
6-BA (benzyladenine), 0.1mg L-1IBA (indolebutyric acid), 30g L-1Sucrose, 8g L-1Agar, 3g L-1Activity
Charcoal, 4g L-1PVP, Medium's PH Value is 5.8, after 121 DEG C of high pressure steam sterilization 25min, subpackage, treat that outer planting bulk-growth becomes
During the budlet of 0.6-1.3cm, carry out subculture increment and cultivate；
(3) a large amount of red meat Tissue-cultured apple seedling of successive transfer culture acquisition:
Being inoculated on subculture medium by budlet and carry out increment cultivation, described successive transfer culture based component is MS, 1.0mg L-16-
BA, 0.1mg L-1IBA, after about 10-20 days, differentiates 2-3 Multiple Buds, and after 25-35 days, Multiple Buds length is to about 2cm, averagely
Once, in Subculture, temperature controls at 20-30 DEG C 4-5 week subculture；
(4) strong seedling culture:
Take the red meat Tissue-cultured apple seedling that successive transfer culture grows fine, be seeded to MS as minimal medium, add 1.0mg L-1's
6-BA (benzyladenine), 0.1mg L-1IBA (indolebutyric acid), 30g L-1Fructose or sorbitol, 8g L-1The training of agar
Support on base, be placed in 17 DEG C of constant temperature illumination boxs and carry out strong seedling culture；
(5) extraction of anthocyanin and the preparation of crude product:
Choose 17 DEG C of constant temperature culture that fructose or sorbitol process tissue cultured seedling after 14 days, after shredding, become powder by liquid nitrogen grinding, will
The powder obtained is placed in conical flask, adds acetone soln by solid-to-liquid ratio 1:10, is sealed by conical flask sealed membrane, is placed in ultrasonic
In ripple washer, control temperature is at 30 DEG C, and ultrasonic 30min is placed in dark extraction 10 hours, obtains lixiviating solution, is used by lixiviating solution
Connect the buchner funnel sucking filtration of vacuum air pump, gained filtrate is poured into rotation and steams bottle, on a rotary evaporator with the temperature of 20-30 DEG C
Degree rotation boils off except acetone solvent, and then lucifuge stands, then the granule on sucking filtration, filter paper is anthocyanin crude product.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108401908A (en) * | 2018-05-07 | 2018-08-17 | 沈阳农业大学 | Culture medium for preventing tissue culture seedlings of gynura bicolor from browning and growing point necrosis and application thereof |
| CN109042319A (en) * | 2018-07-20 | 2018-12-21 | 河南农业大学 | A method of preventing the browning of apple tissue culture and adventitious bud inducing |
| CN109392722A (en) * | 2018-12-25 | 2019-03-01 | 江苏艺轩园林景观工程有限公司 | Make a variation winter red fruit tissue culture method for breeding |
| CN110915471A (en) * | 2019-12-12 | 2020-03-27 | 福建省农业科学院果树研究所 | Method for improving color stability of red pulp peach pulp |
| WO2022199087A1 (en) * | 2021-03-24 | 2022-09-29 | 青岛农业大学 | Use of anthocyanin in suppression of furan production |
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| CN104115747A (en) * | 2014-07-03 | 2014-10-29 | 山东农业大学 | Production of apple flavonoids by using tissue culture method |
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| YUGANG ZHANG等: "he anthocyanins component and the influene factors of contents in red flesh apple ‘Hong-Xun No.1’", 《EUR. J. HORTIC. SCI.》 * |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108401908A (en) * | 2018-05-07 | 2018-08-17 | 沈阳农业大学 | Culture medium for preventing tissue culture seedlings of gynura bicolor from browning and growing point necrosis and application thereof |
| CN108401908B (en) * | 2018-05-07 | 2022-05-13 | 沈阳农业大学 | Culture medium for preventing tissue culture seedlings of gynura bicolor from browning and growing point necrosis and application thereof |
| CN109042319A (en) * | 2018-07-20 | 2018-12-21 | 河南农业大学 | A method of preventing the browning of apple tissue culture and adventitious bud inducing |
| CN109042319B (en) * | 2018-07-20 | 2021-10-19 | 河南农业大学 | Method for preventing tissue culture browning and adventitious bud induction of apples |
| CN109392722A (en) * | 2018-12-25 | 2019-03-01 | 江苏艺轩园林景观工程有限公司 | Make a variation winter red fruit tissue culture method for breeding |
| CN110915471A (en) * | 2019-12-12 | 2020-03-27 | 福建省农业科学院果树研究所 | Method for improving color stability of red pulp peach pulp |
| CN110915471B (en) * | 2019-12-12 | 2021-10-08 | 福建省农业科学院果树研究所 | Method for improving color stability of red pulp peach pulp |
| WO2022199087A1 (en) * | 2021-03-24 | 2022-09-29 | 青岛农业大学 | Use of anthocyanin in suppression of furan production |
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