CN108056021A - A kind of purslane callus induction and the method for producing flavonoids - Google Patents
A kind of purslane callus induction and the method for producing flavonoids Download PDFInfo
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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
- A01H4/001—Culture apparatus for tissue culture
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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
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The present invention provides a kind of purslane callus induction and the method for producing flavonoids, belongs to callus induction field, and seedling plumular axis is seeded in 6 BA+0.5mg/L of MB5+1.0mg/L, 2,4 D+AgNO as explant after sprouting 7d with purslane seed3In 4.0mg/L culture mediums, after cultivating 30d under blue light, growth coefficient and Flavonoid Content highest.The method of the present invention is simple, for purslane factorial praluction flavonoids provide faster with effective approach.
Description
Technical field
The invention belongs to callus induction fields, and in particular to a kind of purslane callus induction and production flavonoids
Method.
Background technology
Purslane（Portulaca oleraceaL）, and longevity greens/mustard green, long life grass etc. can be called, it is interior energy completion then
The meat wild herb of whole growth cycles, Yin Qigen（White）, stem（It is red）, leaf（Green）, flower（Yellow）And seed
（Black）Color it is different,《Bencao Tujing》It is inside otherwise known as " five-element's grass ".Purslane blazons China on both sides of the Changjiang River, adaptability
It is extremely strong, and with the characteristic of the ego-defense as other general wild plants, heat resistanceheat resistant and drought-resistant characteristic are apparent.Purslane
Why obtain liking and paying close attention to for consumers in general and scholar, be because it not only the minerals containing certain medical value,
A variety of chemical compositions such as amino acid, polysaccharide, aliphatic acid, vitamin, alkaloid, acids and Flavonoid substances, also stop with antibacterial
Blood, dissipate blood detumescence, pesticide-germicide, anti-inflammatory diuresis, clearing heat and detoxicating, Li Shui dries, enhancing is immunized and anti-oxidant, anti-aging, resists and swells
The functions such as knurl are one of health foods through health ministry certification.In recent years, purslane is in chemical composition, clinical practice, guarantor
Research in terms of the processing of health food and cultivation technique is more, and because of purslane, it can be served and pharmaceutically acceptable, but wild resource is deficient,
Scholar also strengthens application of the purslane in terms of tissue cultures, to meet the needs of to seed resource and to cultivate high yield excellent
The purslane kind of matter.It reports and comes across 2003 for the first time on the research of Purslane plants tissue cultures in the country.Qiu Ninghong, Lee
Research of the cover to the tissue culture and rapid proliferation of purslane, establishes plant regeneration system, in MS medium supplementeds 0.5-
2.0 mg/L 6-BA and 0.5 mg/L 2,4-D are conducive to carry out the induction of callus using cotyledon, plumular axis as explant.Afterwards
The researcher come also obtains identical result.
A just most important envirment factor in growth and development of plants.It is except being that a kind of energy controls photosynthetic energy
Source or a kind of inducement signal that can influence tissue growth.Different light receptors perceives optical signal in tissue-cultured seedling body, so as to shadow
Ring growth and development, photosynthesis characteristics and the trait expression to tissue-cultured seedling.It is applied for light quality and induces and grow in plant callus
The Portugal of the researchs such as the Saussurea medusas of researchs such as aspect, reports out the research of various plants in this regard, and Ru Zhaode is repaiied, true
No. 47 RHIIZOMA DIOSCOREAE from Henan of China of the researchs such as garlic, the Guo Junli of researchs such as grape, horse beautiful jade etc., these results of study show plant callus
Induction or growth are influenced very big, suitable light quality be subject to light quality and are conducive to callus induction and growth pair.Therefore light quality exists
Purslane callus induction and the influence of growth have certain research significance, by present invention contemplates that being purslane callus
Factorial praluction flavonoids provide faster with effective approach.
The content of the invention
It is an object of the invention to provide a kind of purslane callus induction and the methods for producing flavonoids.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of purslane callus induction and the method for producing flavonoids, the method are seedling after purslane seed sprouting 7d
Plumular axis is seeded in+0.5 mg/L 2 of 1.0 mg/L 6-BA of MB5+, 4-D+AgNO as explant34.0mg/L culture medium
In, 30d is cultivated under blue light.Condition of culture be illumination every day 12h, 25 DEG C of temperature.
The cultural method of seedling is：First the seed of purslane is wrapped up with filter paper, then it is being put into 50-55 DEG C of warm water
Middle immersion 10-15min, carries out disinfection of hoting water treatment of seeds, and then rinses 20 min with flowing water；Then by filter paper in 75% alcohol
The seed disinfection 60s of package；Then in 0.1% HgCl2Middle disinfection 8min, is during which constantly stirred；Then rushed with sterile water
It washes 4-5 times；Finally be inoculated by the processed MS minimal mediums of high pressure sterilization, place it in intensity of illumination 2000 ~
3000lx, light application time 12h/d are cultivated under the conditions of 25 ± 2 DEG C of temperature, obtain aseptic seedlings.
The advantage of the invention is that：
1. establishing purslane callus induction system rapidly and efficiently, incubation time is saved
2. establishing the condition of culture for improving Flavonoid Content in purslane callus, the purslane flavonoids anniversary can be realized
Resource is saved in production.
Specific embodiment
Embodiment 1
1 material and method
1.1 test material
This experiment is using the Wild Ma Chi Xian seed that is produced from Sichuan as examination material.
1.2 test method
1.2.1 the acquisition of aseptic seedling
First the seed of purslane is wrapped up with filter paper, then it is impregnated to 10-15min in the warm water for being put into 50-55 DEG C, carries out temperature
Soup seed soaking disinfection, then rinses 20 min, finally carry out disinfection processing and inoculation in superclean bench with flowing water.Exist first
The seed disinfection 60s for wrapping up filter paper in 75% alcohol；Then in 0.1% HgCl28min is sterilized, is during which constantly stirred；
Then aseptic water washing is used 4-5 times；It is finally inoculated by the processed MS minimal mediums of high pressure sterilization, every bottle connects 10
Seed connects 40 bottles, places it in 2000 ~ 3000lx of intensity of illumination, light application time 12h/d, is trained under the conditions of 25 ± 2 DEG C of temperature
It supports, obtains aseptic seedling.
1.2.2 different seedling ages and influence of the tissue site to purslane callus induction
The aseptic seedling of 4d, 7 d, 10 d, 13 d, 16 d connect cotyledon and plumular axis as material respectively after being sprouted using purslane seed
Kind to being induced in callus inducing medium MB5+6-BA 1.0mg/L+2,4-D0.5mg/L, more different seedling ages and
Influence of the tissue site to purslane callus.
1.2.3 influence of the hormon to purslane callus induction
（1）Influences of the various concentration 6-BA to purslane callus induction
The cotyledon of seedling age 7d is inoculated in the 0.5 mg/L 2,4-D+ AgNO of culture medium MB5+ of evoked callus3 4.0mg/
L, and additional various concentration 6-BA（0.5 mg/L、1.0 mg/L、1.5 mg/L、2.0 mg/L、3.0 mg/L）In induced,
Compare influences of the various concentration 6-BA to purslane callus induction.The observation statistics callus induction rate when cultivating 30d.
Each processing 30 explant of inoculation, 3 repetitions.
（2）Influences of the various concentration IAA to purslane callus induction
The cotyledon of seedling age 7d is inoculated in the 1.0 mg/L 6-BA+AgNO of culture medium MB5+ of evoked callus3 4.0mg/
L, and additional various concentration IAA（0 mg/L、0.2 mg/L、0.5 mg/L、0.8 mg/L、1.0 mg/L）In induced.
（3）Various concentration 2,4-D
The cotyledon of seedling age 7d is inoculated in the 1.0 mg/L 6-BA+AgNO of culture medium MB5+ of evoked callus3 4.0mg/
L, and additional various concentration 2,4-D（0 mg/L、0.2 mg/L、0.5 mg/L、0.8 mg/L、1.0 mg/L）In induced.
（4）Various concentration NAA
The cotyledon of seedling age 7d is inoculated in the 1.0 mg/L 6-BA+AgNO of culture medium MB5+ of evoked callus3 4.0mg/
L, and additional various concentration NAA（0 mg/L、0.2 mg/L、0.5 mg/L、0.8 mg/L、1.0 mg/L）In induced.
1.2.4 influence of the different light medium to purslane callus induction
Select the tissue culture strong sprout for growing fine consistent that blade is cut into 0.5cm to try material2The square of left and right, young stem are cut into about 1-
The stem section of 2cm is inoculated on the optimum medium MS+6-BA1.0mg/L+2,4-D0.5mg/L of induction purslane callus,
In inoculation by the rear-face contact of blade to medium matrix, 18 bottles of often processing inoculation, 5 every bottle, and 3 repetitions of progress.It connects
It is cultivated on kind postposition feux rouges, blue light, white light, yellow light, green light and dark 6 kinds of light processing culturing racks, light application time 12h/d, training
It is 25 DEG C to support temperature.Afterwards, the upgrowth situation of 1 callus is observed every 1d or 2d, records out each light processing callus
Time of occurrence and form, count the callus number induced after 40d.
1.2.5 influence of the different light medium to purslane callus proliferation
Aseptically the purslane callus cultivated in advance is weighed first, is then cut into small stem section inoculation
Feux rouges, blue light, white light, yellow light, green is respectively placed in onto proliferated culture medium MS+6-BA1.0mg/L+IAA1.0mg/L, after inoculation
Squamous subculture is carried out on light and dark 6 kinds of light processing culturing racks, 30 bottles of often processing inoculation, carries out 3 repetitions by 3 every bottle.Culture
Condition be illumination every day 12h, 25 DEG C of temperature.The callus weighed after 30 days after observing.
1.2.6 influence of the different light medium to Flavonoid Content in purslane callus
After purslane callus is inoculated into most suitable proliferated culture medium, feux rouges, blue light, white light, yellow light, green light are respectively placed in
It is cultivated on dark 6 kinds of light processing culturing racks, 30 bottles of often processing inoculation, carries out 3 repetitions by 3 every bottle.Condition of culture is
Illumination every day 12h, 25 DEG C of temperature.Flavonoid Content measure is carried out after 30 days.0.1g is weighed after callus is dried to be placed in
70 DEG C of 85% ethanol water bath constant temperature of 30mL draws supernatant 4mL in test tube after 2h, is then drawn respectively in cuvette
In be placed under 510nm wavelength be divided detection, measure spectrophotometric value, pass through calibration curve formula regression equation α=1.2C+
4.8x10-3, the range of linearity is 0 ~ 0.5 mg, and related coefficient is r=0.9955, calculates Flavonoid Content.
1.3 data process＆analysis
Callus induction rate（%）The total explant number × 100% of=callus explant number/inoculation derived；
Callus growth rate（%）=callus increases weight/callus inoculation weight × 100%.
It is handled and is analyzed for test data using Excel and 19.0 grade statistical softwares of SPSS.
2 results and analysis
2.1 aseptic seedlings obtain and strong sprout
It is in aubergine with plumular axis in 4~5cm, cotyledon using purslane seed as material, the aseptic seedling obtained after 7d, plant height is tried.
2.2 different seedling ages and influence of the tissue site to purslane callus induction
The aseptic seedling of 4d, 7 d, 10 d, 13 d, 16 d connect blade and plumular axis as material respectively after being sprouted using purslane seed
Kind arrives+0.5 mg/L 2,4-D+ AgNO of callus inducing medium MB5+6-BA1.0mg/L3It is lured in 4.0mg/L
It leads, the influence of more different seedling ages and tissue site to purslane callus.
As can be seen from the table, 7d seedling ages are conducive to purslane callus induction, and plumular axis is as explant inductivity
Slightly above cotyledon, the two materials can carry out callus induction as explant.
Influence of 2.3 hormons to purslane callus induction
（1）Influences of the various concentration 6-BA to purslane callus induction
The plumular axis of seedling age 7d is inoculated in the 0.5 mg/L 2,4-D+ AgNO of culture medium MB5+ of evoked callus3 4.0mg/
L, and additional various concentration 6-BA（0、0.5 mg/L、1.0 mg/L、1.5 mg/L、2.0 mg/L）In induced.
Illustrate to be conducive to callus induction when 6-BA concentration is 0.5 mg/L.
（2）Influences of the various concentration IAA to purslane callus induction
The plumular axis of seedling age 7d is inoculated in the 1.0 mg/L 6-BA+AgNO of culture medium MB5+ of evoked callus3 4.0mg/
L, and additional various concentration IAA（0 mg/L、0.2 mg/L、0.5 mg/L、0.8 mg/L、1.0 mg/L）In induced.From
As can be seen that IAA is not suitable for purslane callus induction in table.Highest inductivity just only has 62.3%.
（3）Various concentration 2,4-D
The cotyledon of seedling age 7d is inoculated in the 1.0 mg/L 6-BA+AgNO of culture medium MB5+ of evoked callus3 4.0mg/
L, and additional various concentration 2,4-D（0 mg/L、0.2 mg/L、0.5 mg/L、0.8 mg/L、1.0 mg/L）In induced.
As can be seen from the table, when 2,4-D concentration is 0.5 or 1.0 mg/L, purslane callus induction is all conducive to.
（4）Various concentration NAA
The cotyledon of seedling age 7d is inoculated in the 1.0 mg/L 6-BA+AgNO of culture medium MB5+ of evoked callus3 4.0mg/
L, and additional various concentration NAA（0 mg/L、0.2 mg/L、0.5 mg/L、0.8 mg/L、1.0 mg/L）In induced.From
As can be seen that NAA is also detrimental to purslane callus induction in table.
Influence of 2.4 different light mediums to purslane evoked callus
2.4.1 influence of the different light medium to purslane plumular axis evoked callus
Purslane plumular axis is inoculated on the culture medium of evoked callus, then be placed in feux rouges, blue light, white light, yellow light, green light and
Cultivated in dark 6 kinds of light processings, research different light medium purslane stem section is induced callus earliest time of occurrence and
The influence of inductivity.As a result it is as shown in the table, and light quality has a certain impact to the induction of purslane plumular axis callus, various light qualities
Hypocotyl can induce out callus, wherein needing the time most short with the callus that induces of feux rouges, 6d has callus group
Generation is knitted, inductivity highest reaches 100%, although the inductivity of blue light is also 100%, compared with feux rouges, callus induction
Going out the time has delayed 2d.The order of the inductivity of callus from stem segment from high to low is feux rouges=blue light under each light quality>White light>Secretly
Light>Yellow light>Green light.
Influence of the light quality to the earliest time of occurrence of purslane plumular axis callus and inductivity
* capitalization represents 0.01 level difference conspicuousness in table.
2.4.2 influence of the different light medium to purslane callus quality
Different light medium also has a certain impact to the quality of purslane callus, and from plate II, Portulaca Oleracea Leaves are red
The callus quality generated under light is very fine and close, and color is finer and close for callus quality that is faint yellow, being generated under blue light, color
Closer for the callus quality that is generated under pistac, white light, color is light green, the callus matter that is generated under half-light
Ground is loose, and color is loose for the faint yellow callus quality whitened, generated under yellow light, and color is yellow green, is generated under green light
It is close with callus quality, color is whitened for green.The callus and blade that stem section generates under different light medium processing
It is more loose compared to quality, the slightly micro- brown of color.
From the point of view of above-mentioned test data, light quality is influenced to compare on purslane stem section with Callus of Leaf induction
It arrives：Under each light processing the height order of stem section and Callus of Leaf inductivity is consistent, and stem section goes out under each light processing
More the time, inductivity was also substantially above blade substantially earlier than blade, thus illustrate stem section induce callus it is earliest when
Between it is shorter than blade, callus growth speed is also fast compared with blade.The callus quality that blade induces under each light quality is compared with stem
Duan Hao, browning probability of happening is low compared with stem section, and materials are easier to largely to be operated.Therefore can be used according to experiment specific requirement
Different explants are in terms of evoked callus.
Influence of 2.5 different light mediums to purslane callus proliferation
Purslane callus is inoculated on proliferated culture medium, then is placed in feux rouges, blue light, white light, yellow light, green light and dark 6
Squamous subculture, influence of the research different light medium to Portulaca Oleracea Leaves callus growth are carried out in kind light processing.As a result such as table institute
Show, each light quality can promote the growth of purslane callus, and the wherein fresh increment of blue light is maximum, is 9.71g, growth rate is most
Height reaches 359%.Blue light and other light processing significant differences, blue light are significantly higher than for the growth effect of purslane callus
Other light qualities.Feux rouges and other light processing significant differences, white light, yellow light and the half-light difference in 5% level of signifiance is not notable.It says
Mingguang City's matter has certain positive effect in purslane callus induction.
The influence of mass color confrontation purslane callus growth
* 0.05 level difference conspicuousness of lowercase letter in table.
Influence of 2.6 light qualities to Flavonoid Content in purslane callus
As seen from the table, blue light is conducive to flavonoids accumulation, content highest in purslane callus.
As stated above, the optimal medium of purslane callus induction multiplication and Flavonoid Content accumulation is originally determined
It is with condition of culture：Seedling plumular axis is as explant after purslane seed sprouts 7d, be seeded in 1.0 mg/L 6-BA of MB5++
In 0.5 mg/L 2,4-D+AgNO3 4.0mg/L culture mediums, after cultivating 30d under blue light, growth coefficient and Flavonoid Content
Highest.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification should all belong to the covering scope of the present invention.
Claims (2)
1. a kind of purslane callus induction and the method for producing flavonoids, it is characterised in that：The method is purslane kind
Seedling plumular axis is seeded in+0.5 mg/L 2 of 1.0 mg/L 6-BA of MB5+, 4-D+AgNO as explant after son sprouts 7d3
In 4.0mg/L culture mediums, 30d is cultivated under blue light.
2. a kind of purslane callus induction according to claim 1 and the method for producing flavonoids, it is characterised in that：
The cultural method of seedling is：The seed of purslane is wrapped up with filter paper, then it is impregnated into 10- in the warm water for being put into 50-55 DEG C
15min carries out disinfection of hoting water treatment of seeds, and then rinses 20 min with flowing water；Then the seed wrapped up filter paper in 75% alcohol
Sterilize 60s；Then in 0.1% HgCl2Middle disinfection 8min, is during which constantly stirred；Then aseptic water washing is used 4-5 times；Most
After be inoculated by the processed MS minimal mediums of high pressure sterilization, placing it in 2000 ~ 3000lx of intensity of illumination, during illumination
Between 12h/d, cultivated under the conditions of 25 ± 2 DEG C of temperature, obtain aseptic seedlings.
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| CN112655554A (en) * | 2020-12-16 | 2021-04-16 | 云南省农业科学院园艺作物研究所 | Method for accelerating induction of apple callus and growth based on LED light quality |
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