CN107996402A - A kind of method for preventing tobacco Brown - Google Patents
A kind of method for preventing tobacco Brown Download PDFInfo
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- CN107996402A CN107996402A CN201711332623.9A CN201711332623A CN107996402A CN 107996402 A CN107996402 A CN 107996402A CN 201711332623 A CN201711332623 A CN 201711332623A CN 107996402 A CN107996402 A CN 107996402A
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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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- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The present invention is a kind of method for preventing tobacco Brown, is included the following steps：The disinfection of vegetable seeds, vegetable seeds culture, explant preculture, explantation tissue carry out explant callus anti-browning culture after cultivating, and it is 28 DEG C to control day temperature, and nocturnal temperature is 25 DEG C, and intensity of illumination is 30 50 μm of ol/ (m2S), when light application time is 16 small/day, incubation time is 40 days.The anti-browning culture medium is：Added in MS minimal mediums and prevent brown 4 8mg/L citric acids of additive, 0.2 0.5g/L polyvinylpyrrolidone (PVP), 30g/L sucrose, 4g/L agar, the pH value of culture medium is 5.7,121 DEG C of sterilizing 15min.It after browning culture processing, can effectively alleviate browning, improve the differentiation state of explant entirety.Add citric acid and polyvinylpyrrolidone (PVP) in the medium, have the advantages that easy to use, cost is low, effect is good.
Description
Technical field
The invention belongs to field of plant tissue culture, and in particular to one kind, which is used, contains citric acid and polyvinylpyrrolidone
The culture medium method that prevents tobacco Brown.
Background technology
Browning is a kind of common unfavorable phenomenon produced in plant tissue culture course, is in recent years in terms of plant tissue culture
One of research hotspot.
Physiological status of the internal cause including explant material of influence browning, genotype, growing environment, nutrition condition, growth
Position and age etc.；The component of external cause including culture medium, the species content of additional hormone and ratio, adsorbent, anti-browning agent, have
No Heat thermostability, condition of culture temperature, pH, light application time, intensity of illumination, aeration status etc. can also influence the generation of browning.
In the factors for causing browning, the destruction that Physical Zoneization is distributed in the destruction of membrane structure or cell is the enzymatic of aldehydes matter
The key of browning occurs for oxidation and tissue[1].After explantation tissue is cut, when the cell of incision comes to harm, phenol,
The separation effect of enzyme is broken, intracellular phenolic compound secreting outside, phenol oxidase release or synthesis.In suitable pH, temperature
Under the conditions of degree etc., under the action of phenol oxidase, oxidation reaction occurs for aldehydes matter (substrate) and oxygen, forms brown quinones
Material, this makes section quickly become sepia or crineous.This brown thing is gradually spread in the medium, suppresses other enzymes
Activity, poisons whole explantation tissue, and explant is final dead therewith further browning.
Tobacco is the annual herb plant of Solanaceae Nicotiana, this category there are about 60 kinds.America and Oceania are originated in, China
4 kinds of cultivation：Tobacco, makhorka, light tobacco and Henbane, wherein tobacco planting area are maximum.Tobacco has medicinal, industrial
With, protect the value such as tendon beauty.In addition, one of " model plant " of tobacco as Plant Tissue Breeding, in plant cell engineering
Occupy critical role in research.There are many researchs and report in relation to Tissues of Tobacco and cell culture.Browning this many woody
Common phenomenon in plant tissue culture, and a big obstacle of Tissues of Tobacco culture.
Research shows, can be by controlling relevant biological factor, kinds of culture medium and component, and the absorption that addition is certain
Agent or anti-browning agent reach the effect for mitigating browning.
The present invention is alleviated outside tobacco using addition anti-browning agent citric acid in the medium and polyvinylpyrrolidone (PVP)
Implant browning, correlation technique have no report.
The content of the invention
It is an object of the invention to provide a kind of method for preventing tobacco Brown, this method can be to plant tissue culture
During the browning problem that occurs effectively alleviated, and then play an important roll to Plant Tissue Breeding.
The scheme that the present invention solves technical problem is as follows：
A kind of method for preventing tobacco Brown, includes the following steps：
Step 1：The disinfection of vegetable seeds：Full cigarette seed is taken to move on superclean bench, with 75% alcohol by vegetable seeds
30s, aseptic water washing 2 times are soaked, then uses 1%AgNO310min is sterilized, sterile water soaking and washing 5 times, is blotted with aseptic filter paper
It is inoculated with after vegetable seeds surface moisture, wherein sterile water is through autoclaved ultra-pure water；
Step 2：Vegetable seeds culture：The vegetable seeds of step 1 is inoculated into MS culture mediums, is 25 ± 1 in cultivation temperature
DEG C, 30-50 μm of ol/ (m of intensity of illumination2S), light application time for 16 it is small when/day under conditions of cultivate 60 days, germination grows up to
Explant seedling；The MS culture mediums add the agar that 30g/L sucrose adds 4g/L for MS minimal mediums, and the pH value of culture medium is
5.7 ；
Step 3：Explant preculture：By explant seedling inoculation that step 2 obtains in added with 12g cordate houttuynias and including MS
Minimal medium, 4.5g/L agar, 30g/L sucrose MS preculture solid mediums in, controlled at 25 DEG C, intensity of illumination
For 30-50 μm of ol/ (m2s), light application time for 11 it is small when/day, incubation time is 4 days, then by the transfer of explant seedling simultaneously
It is inoculated in the MS solid mediums immersed with 10g honey, controlled at 24 DEG C, intensity of illumination is 30-50 μm of ol/ (m2
S), when light application time is 10 small/day, incubation time is 3 days, finally by the explant after obtained preculture, is inoculated in repeatedly
MS culture mediums 4-5 times, make the aldehydes matter part in tissue penetrate into culture medium.The MS culture mediums add for MS minimal mediums
30g/L sucrose adds the agar of 4g/L, and the pH value of culture medium is 5.7；
Step 4：Explantation tissue cultivates：The explant seedling that step 3 obtains is placed in MS differential mediums, control temperature on daytime
Spend for 28 DEG C, nocturnal temperature is 25 DEG C, and intensity of illumination is 30-50 μm of ol/ (m2S), when light application time is 16 small/and day, culture
Time is 7 days, obtains explant callus, the MS differential mediums are：The methyl α-naphthyl acetate of MS minimal mediums+0.5mg/L
The agar of the 6-benzyladenine 6-BA+30g/L sucrose+4g/L of NAA+2mg/L, the pH value of culture medium is 5.7；
Step 5：Explant callus anti-browning culture：The explant callus obtained in step 4 is placed in MS anti-brownings
In culture medium, it is 28 DEG C to control day temperature, and nocturnal temperature is 25 DEG C, and intensity of illumination is 30-50 μm of ol/ (m2S), during illumination
Between for 16 it is small when/day, incubation time is 40 days so that explant callus browning rate is 9.32-35.7%, inductivity
91.6-54.6%；The MS anti-browning culture mediums are：Added in MS minimal mediums and prevent brown additive 4-8mg/L citric acids,
0.2-0.5g/L polyvinylpyrrolidone (PVP), 30g/L sucrose, 4g/L agar, the pH value of culture medium are gone out for 5.7,121 DEG C
Bacterium 15min.
The MS anti-brownings culture medium of 1L about can 30, the culture dish of falling 100mm, 10,150mm culture dishes, 300mL blake bottles 15
It is a.
Compared with prior art, the present invention its advantage is：
1st, after browning culture processing, it can effectively alleviate browning, improve the differentiation state of explant entirety.Add citric acid
With polyvinylpyrrolidone (PVP) in the medium, have the advantages that easy to use, cost is low, effect is good.
2nd, tobacco explant is inoculated in the MS trainings that with the addition of different material respectively by the present invention successively before differentiation is cultivated
Support base and carry out preculture, cordate houttuynia has inhibitory action to multiple-microorganism, can be to the surface of the seed disinfection.
3rd, honey is the middle honey that apis cerana is produced, and middle honey has the antioxygens such as ascorbic acid, flavonoids and some enzymes
Chemical conversion point, and the bad smell of cordate houttuynia can be blocked, and the generation of melatonin can be promoted, prevent cell oxidative damage can
With Restrain browning, reduce melting brown rate, while middle honey also has substantial amounts of natural nutrient substance, can supplementing culture medium deficiency,
Significantly improve the inductivity of explant.
Embodiment
With reference to embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and it should not be regarded as limiting this hair
Bright scope.In the examples where no specific technique or condition is specified, according to the described technology of document in the art or condition
Or carried out according to product description.Reagents or instruments used without specified manufacturer, is that can be obtained by buying
Conventional products.
Embodiment 1
Step 1：The disinfection of vegetable seeds：Full tobacco seed is taken to move on superclean bench, with 75% alcohol by plant species
Son immersion 30s, aseptic water washing 2 times, then use 1%AgNO310min is sterilized, sterile water soaking and washing 5 times, is inhaled with aseptic filter paper
It is inoculated with after dry vegetable seeds surface moisture, wherein sterile water is through autoclaved ultra-pure water；
Step 2：Vegetable seeds culture：The vegetable seeds of step 1 is inoculated into MS culture mediums, is 25 ± 1 in cultivation temperature
DEG C, 30-50 μm of ol/ (m of intensity of illumination2S), light application time for 16 it is small when/day under conditions of cultivate 60 days, germination grows up to
Explant seedling；The MS culture mediums add the agar that 30g/L sucrose adds 4g/L for MS minimal mediums, and the pH value of culture medium is
5.7 ；
Step 3：Explant preculture：By explant seedling inoculation that step 2 obtains in added with 12g cordate houttuynias and including MS
Minimal medium, 4.5g/L agar, 30g/L sucrose MS preculture solid mediums in, controlled at 25 DEG C, intensity of illumination
For 30-50 μm of ol/ (m2s), light application time for 11 it is small when/day, incubation time is 4 days, and cordate houttuynia has multiple-microorganism
Inhibitory action, can be to the surface of the seed disinfection；Then seed is shifted and is inoculated in the MS solids training immersed with 10g honey
Support in base, controlled at 24 DEG C, intensity of illumination be 30-50 μm of ol/ (m2s), when light application time is 10 small/day, during culture
Between be 3 days, honey can block the bad smell of cordate houttuynia, and can promote the generation of melatonin, the pre- training that will finally obtain
Explant after supporting, is inoculated in MS culture mediums 4-5 times repeatedly, the aldehydes matter part in tissue is penetrated into culture medium.To reduce
Oxidation reaction occurs for aldehydes matter (substrate) and oxygen, forms brown quinones substance.The MS culture mediums are cultivated substantially for MS
Base adds the agar that 30g/L sucrose adds 4g/L, and the pH value of culture medium is 5.7；
Step 4：Explantation tissue cultivates：The explant that step 3 is obtained is placed in MS differential mediums, controls the day temperature to be
28 DEG C, nocturnal temperature is 25 DEG C, and intensity of illumination is 30-50 μm of ol/ (m2S), when light application time is 16 small/and day, incubation time
For 7 days, explant callus is obtained, the MS differential mediums are：The methyl α-naphthyl acetate NAA of MS minimal mediums+0.5mg/L
The agar of the 6-benzyladenine 6-BA+30g/L sucrose+4g/L of+2mg/L, the pH value of culture medium is 5.7；
Step 5：Explant anti-browning culture：The explant callus obtained in step 4 is placed in MS anti-browning culture mediums
In, it is 28 DEG C to control day temperature, and nocturnal temperature is 25 DEG C, and intensity of illumination is 30-50 μm of ol/ (m2S), light application time 16
Hour/day, incubation time are 40 days, and explant callus browning rate is 9.32%, inductivity 91.6%；The MS prevents brown
Changing culture medium is：Added in MS minimal mediums and prevent brown additive 5mg/L citric acids, 0.3g/L polyvinylpyrrolidone
(PVP), 30g/L sucrose, 4g/L agar, the pH value of culture medium is 5.7.
Embodiment 2
Step（1）To step（4）It is same as Example 1；
Step（5）, explant anti-browning culture：The explant callus obtained in step (4) is placed in MS anti-browning cultures
In base, it is 28 DEG C to control day temperature, and nocturnal temperature is 25 DEG C, and intensity of illumination is 30-50 μm of ol/ (m2S), light application time is
16 it is small when/day, incubation time be 40 days, explant callus browning rate be 15.2%, inductivity 76.4%.MS anti-brownings are trained
It is to add to prevent brown additive 8mg/L citric acids in MS minimal mediums to support base, 0.5g/L polyvinylpyrrolidone (PVP),
30g/L sucrose, the agar of 4g/L, the pH value of culture medium is 5.7；
Embodiment 3
Step（1）To step（4）It is same as Example 1；
Step（5）, explant anti-browning culture：The explant callus obtained in step (4) is placed in MS anti-browning cultures
In base, it is 28 DEG C to control day temperature, and nocturnal temperature is 25 DEG C, and intensity of illumination is 30-50 μm of ol/ (m2S), light application time is
16 it is small when/day, incubation time be 40 days, explant callus browning rate be 28.1%, inductivity 67.6%.MS anti-brownings are trained
Foster base is：Added in MS minimal mediums and prevent brown additive 6mg/L citric acids, 0.4g/L polyvinylpyrrolidone (PVP),
30g/L sucrose, the agar of 4g/L, the pH value of culture medium is 5.7.
Embodiment 4
Step（1）To step（4）It is same as Example 1；
Step（5）, explant anti-browning culture：The explant callus obtained in step (4) is placed in MS anti-browning cultures
In base, it is 28 DEG C to control day temperature, and nocturnal temperature is 25 DEG C, and intensity of illumination is 30-50 μm of ol/ (m2S), light application time is
16 it is small when/day, incubation time be 40 days, explant callus browning rate be 35.7%, inductivity 54.6%.MS anti-brownings are trained
Foster base is：Added in MS minimal mediums and prevent brown additive 4mg/L citric acids, 0.2g/L polyvinylpyrrolidone (PVP),
30g/L sucrose, the agar of 4g/L, the pH value of culture medium is 5.7.
, can be to explant induced tissue culture browning using tobacco explant anti-browning inducing culture of the present invention
Certain inhibitory action is played, melting brown rate is reduced to 9.32%, citric acid and polyvinyl pyrrole can be seen that by the contrast of following table
Pyrrolidone has obvious effect to suppressing browning, and cordate houttuynia and honey also play reduction melting brown rate, improve inductivity
Effect.
The basic principles, main features and the advantages of the invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (2)
- A kind of 1. method for preventing tobacco Brown, it is characterised in that：Include the following steps：Step 1：The disinfection of vegetable seeds;Step 2：Vegetable seeds culture：The vegetable seeds of step 1 is inoculated into MS culture mediums, is 25 ± 1 in cultivation temperature DEG C, 30-50 μm of ol/m of intensity of illumination2S, light application time for 16 it is small when/day under conditions of cultivate 60 days, germination grow up to outside Implant seedling；The MS culture mediums add the agar that 30g/L sucrose adds 4g/L for MS minimal mediums, and the pH value of culture medium is 5.7 ；Step 3：Explant preculture：By explant seedling inoculation that step 2 obtains in added with 12g cordate houttuynias and including MS Minimal medium, 4.5g/L agar, 30g/L sucrose MS preculture solid mediums in, controlled at 25 DEG C, intensity of illumination For 30-50 μm of ol/ (m2s), light application time for 11 it is small when/day, incubation time is 4 days, and then seed is shifted and is inoculated in Immersed with the MS solid mediums of 10g honey, controlled at 24 DEG C, intensity of illumination is 30-50 μm of ol/ (m2s), illumination When time is 10 small/day, incubation time is 3 days, finally by the explant after obtained preculture, is inoculated in MS culture mediums repeatedly 4-5 times, the aldehydes matter part in tissue is set to penetrate into culture medium, the MS culture mediums add 30g/L sucrose for MS minimal mediums Add the agar of 4g/L, the pH value of culture medium is 5.7；Step 4：Explantation tissue cultivates：The explant that step 3 is obtained is placed in MS differential mediums, controls the day temperature to be 28 DEG C, nocturnal temperature is 25 DEG C, and intensity of illumination is 30-50 μm of ol/ (m2S), when light application time is 16 small/and day, incubation time For 7 days, explant callus is obtained, the MS differential mediums are：The methyl α-naphthyl acetate NAA+ of MS minimal mediums+0.5mg/L The agar of the 6-benzyladenine 6-BA+30g/L sucrose+4g/L of 2mg/L, the pH value of culture medium is 5.7；Step 5：Explant anti-browning culture：The explant callus obtained in step 4 is placed in MS anti-browning culture mediums In, it is 28 DEG C to control day temperature, and nocturnal temperature is 25 DEG C, and intensity of illumination is 30-50 μm of ol/ (m2S), light application time 16 Hour/day, incubation time are 40 days so that explant callus browning rate is reduced to 9.32-35.7%, inductivity 91.6- 54.6%；The MS anti-browning culture mediums are：Added in MS minimal mediums and prevent brown additive 4-8mg/L citric acids, 0.2- 0.5g/L polyvinylpyrrolidone (PVP), 30g/L sucrose, 4g/L agar, the pH value of culture medium is 5.7,121 DEG C of sterilizings 15min。
- 2. the method according to claim 1 for preventing tobacco Brown, it is characterised in that：Plant described in step 1 The disinfection of species is：Take full vegetable seeds to move on superclean bench, vegetable seeds soaked into 30s with 75% alcohol, Aseptic water washing 2 times, then use 1%AgNO310min is sterilized, sterile water soaking and washing 5 times, vegetable seeds is blotted with aseptic filter paper It is inoculated with after surface moisture, the sterile water is through autoclaved ultra-pure water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711332623.9A CN107996402A (en) | 2017-12-13 | 2017-12-13 | A kind of method for preventing tobacco Brown |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711332623.9A CN107996402A (en) | 2017-12-13 | 2017-12-13 | A kind of method for preventing tobacco Brown |
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| CN107996402A true CN107996402A (en) | 2018-05-08 |
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| CN201711332623.9A Pending CN107996402A (en) | 2017-12-13 | 2017-12-13 | A kind of method for preventing tobacco Brown |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114902960A (en) * | 2022-05-07 | 2022-08-16 | 武汉生物工程学院 | Method and culture medium for efficiently inducing tobacco seed callus |
| CN114982632A (en) * | 2022-04-21 | 2022-09-02 | 安徽农业大学 | Method for reducing browning rate of regeneration plant of black-bone vegetable microspore |
-
2017
- 2017-12-13 CN CN201711332623.9A patent/CN107996402A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114982632A (en) * | 2022-04-21 | 2022-09-02 | 安徽农业大学 | Method for reducing browning rate of regeneration plant of black-bone vegetable microspore |
| CN114902960A (en) * | 2022-05-07 | 2022-08-16 | 武汉生物工程学院 | Method and culture medium for efficiently inducing tobacco seed callus |
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