CN108142290A - A kind of method that tobacco Brown is prevented with sodium thiosulfate - Google Patents
A kind of method that tobacco Brown is prevented with sodium thiosulfate Download PDFInfo
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- CN108142290A CN108142290A CN201711332624.3A CN201711332624A CN108142290A CN 108142290 A CN108142290 A CN 108142290A CN 201711332624 A CN201711332624 A CN 201711332624A CN 108142290 A CN108142290 A CN 108142290A
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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 is a kind of method that tobacco Brown is prevented with sodium thiosulfate.Include the following steps：The disinfection of vegetable seeds, vegetable seeds culture, plant explant culture, plant explant anti-browning culture, the subculture anti-browning culture of plant explant, obtained plant explant browning rate are 18.7 23.3%.Brown phenomenon can effectively be alleviated, the differentiation state of explant entirety can be improved.Add sodium thiosulfate (Na2S2O3) in the medium and the natural antioxidant content of addition, have many advantages, such as that easy to use, at low cost, effect is good.
Description
Technical field
The invention belongs to field of plant tissue culture, and in particular to a kind of to prevent tobacco Brown with sodium thiosulfate
Method.
Background technology
Tobacco belong in botany Dicotyledoneae (Dicotycoledoneac), Tubiflorae (Tubiflorae),
Solanaceae (Solanaceae), Nicotiana, Nicotiana is the fifth-largest family of plant of Solanaceae.Nicotiana tabacum is removed in Nicotiana species at present
(N. tabacum L.) and makhorka be widely used in outside tobacco industry, remaining is all divided into cigarette category tobacco wild species.There is report
Road claims tobacco to be usually used in the area researches such as basic biochemistry metabolism, genome evolution, transgenosis, and Nicotiana tabacum is as research plant
One of model organism, be the common important tool of plant molecular research field.
Brown is an important factor for influencing tissue cultures in plant tissue culture course, and browning is due in tissue
Polyphenol oxidase is activated, and aldehydes matter generates quinones substance after being aoxidized, and this kind of brown color substance can be diffused into culture, suppression
The activity of other enzymes is made, poisons entire explantation tissue.It can be alleviated in tissue cultures by adding anti-browning agent in the medium
The browning of explant.Although the factor for causing browning is more, suitable explant is selected, screens suitable culture
Base and condition of culture can be effectively prevented browning.
But browning problem of the tobacco in tissue culture procedures be it is multiple common, almost unavoidably, browning meeting
Lead to the death of plant, that is, the plant in culture medium cannot continue to cultivate, so it is in Tissues of Tobacco to prevent browning
It strongly to be solved the problems, such as in culture.
The present invention utilizes addition anti-browning agent sodium thiosulfate (Na in the medium2S2O3) alleviate tobacco Brown, phase
Pass method has not been reported.
Invention content
The purpose of the present invention is to provide a kind of methods that tobacco Brown is prevented with sodium thiosulfate；This method can
Effectively to be alleviated, and then play an important roll to Plant Tissue Breeding to the browning problem occurred during plant tissue culture.
The present invention is as follows using technical problem scheme is solved：
A kind of method that tobacco Brown is prevented with sodium thiosulfate, includes the following steps：
Step（1）, the disinfection of vegetable seeds：Full vegetable seeds is taken, is then moved on superclean bench, it will with 75% alcohol
Seed impregnates 30s, aseptic water washing 2 times, then uses 1%AgNO310min is sterilized, sterile water soaking and washing 5 times uses aseptic filter paper
It is inoculated with after blotting explant surface moisture, wherein sterile water is through autoclaved ultra-pure water；
Step（2）, vegetable seeds culture：Vegetable seeds after the disinfection that step (1) is obtained is inoculated into MS culture mediums, is being trained
It is 25 ± 1 DEG C to support temperature, 30-50 μm of ol/ (m of intensity of illumination2S), light application time is cultivates 60d, seed under conditions of 16h/d
Germination is grown to seedling, and the MS culture mediums are：The agar of MS minimal medium+30g/L sucrose+4g/L, the pH of culture medium
Be worth is 5.7；
Step（3）, plant explant culture：The seed seedling that will be obtained in step (2) is punched using card punch and obtains Tobacco Leaf
Leaf dish is placed in MS differential mediums and cultivates by disk, in 28 DEG C of temperature, illumination cultivation 16h/d, 30-50 μm of ol/ of intensity of illumination
(m2S), 25 DEG C of dark culturing 8h/d cultivate 5 days, obtain tobacco healing tissue, the MS differential mediums are：MS is basic
Culture medium+0.5mg/L methyl α-naphthyl acetate NAA+2mg/L6- benzyladenine 6-BA+30g/L sucrose+4g/L agar, the pH of culture medium
Be worth is 5.7；
Step（4）, plant explant anti-browning culture：By the tobacco healing tissue obtained in step (3) be placed in MS tentatively prevent it is brown
Change and cultivated in culture medium, in 28 DEG C of temperature, illumination cultivation 16h/d, 30-50 μm of ol/ (m of intensity of illumination2S), 25 DEG C of dark
8h/d is cultivated, 20d is cultivated under the conditions of this, obtains tobacco healing tissue, the preliminary anti-browning culture mediums of MS are：In MS minimal mediums
It adds in and prevents brown additive 1.0-5.0g/L sodium thiosulfate (Na2S2O3), 30g/L sucrose, 4g/L agar, the pH values of culture medium are
5.7；
Step（5）, the subculture anti-browning culture of plant explant：
1）The preparation of chamomile extracting solution：Chamomile of the selection shortly past viewing period takes flower and cleans, squeeze juice, by juice mistake
Solid residue is filtered out, in superclean bench using 0.22 μm of filter filtration sterilization, sterile extracting solution is made；
2）The preparation of pot marigold extracting solution：Pot marigold of the selection shortly past viewing period takes flower and cleans, squeeze juice, by juice mistake
Solid residue is filtered out, in superclean bench using 0.22 μm of filter filtration sterilization, sterile extracting solution is made；
3）The preparation of tomato extract solution：Juice is filtered to remove residue, in superclean bench by tomato belt leather squeeze juice
Using 0.22 μm of filter filtration sterilization, sterile extracting solution is made；
4）The preliminary anti-browning plant explant obtained in step (4) is placed in MS subculture anti-browning culture mediums, in culture temperature
When spending 28 DEG C, illumination cultivation 16h/d, 25 DEG C of dark culturing 8h/d, 30-50 μm of ol/ (m of intensity of illumination2S) it is cultivated under conditions of
20d obtains the plant explant that melting brown rate is 18.7-23.3%；The MS subculture anti-browning culture mediums are：MS is cultivated substantially
It is added in base and prevents brown additive 100-200mg/L chamomiles extracting solution, 100-200mg/L pot marigolds extracting solution, 100-200mg/L
Tomato extract solution, 30g/L sucrose, 4g/L agar, the pH values of culture medium are 5.7.
The MS primary of 1L and subculture anti-browning culture medium about can 30, the culture dish of falling 100mm, 10,150mm culture dishes,
15,300mL culture bottles；
Preliminary anti-browning is to utilize sodium thiosulfate (Na2S2O3) as antioxidant, it is influenced and the relevant peroxide of browning
Enzyme, polyphenol oxidase activity, so as to alleviate Brown, subculture anti-browning is by the use of natural antioxidant content as training
Based additive is supported, so as to achieve the effect that anti-browning, Brown phenomenon is alleviated in the combined use of two kinds of antioxidants, and
With remarkable result.
Compared with prior art, the present invention advantage is：Tobacco explant is after anti-browning culture processing, Ke Yiyou
Effect alleviates Brown phenomenon, can improve the differentiation state of explant entirety.Add sodium thiosulfate (Na2S2O3) training
The antioxidant content that in base and addition is natural is supported, has many advantages, such as that easy to use, at low cost, effect is good.
Specific 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 range.In the examples where no specific technique or condition is specified, according to the described technology of document in the art or condition
Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, being can be by buying what is obtained
Conventional products.
Embodiment 1
Step（1）, the disinfection of tobacco seed：Full tobacco seed is taken, is then moved on superclean bench, it will with 75% alcohol
Seed impregnates 30s, aseptic water washing 2 times, then uses 1%AgNO310min is sterilized, sterile water soaking and washing 5 times uses aseptic filter paper
It is inoculated with after blotting explant surface moisture, wherein sterile water is through autoclaved ultra-pure water；
Step（2）, tobacco seed culture：Tobacco seed after the disinfection that step (1) is obtained is inoculated into MS culture mediums, is being trained
It is 25 ± 1 DEG C to support temperature, 30-50 μm of ol/ (m of intensity of illumination2S), light application time is cultivates 60d, seed under conditions of 16h/d
Germination is grown to seedling, and the MS culture mediums are：The agar of MS minimal medium+30g/L sucrose+4g/L, the pH of culture medium
Be worth is 5.7；
Step（3）, tobacco explant culture：The seed seedling that will be obtained in step (2) is punched using card punch and obtains Tobacco Leaf
Leaf dish is placed in MS differential mediums and cultivates by disk, and leaf dish is placed in MS differential mediums, in 28 DEG C of cultivation temperature, illumination
Cultivate 16h/d, 25 DEG C of dark culturing 8h/d, 30-50 μm of ol/ (m of intensity of illumination2S), it cultivates 5 days, obtains tobacco callus group
It knits, the MS differential mediums are：MS minimal medium+0.5mg/L methyl α-naphthyl acetate NAA+2mg/L6- benzyladenines 6-BA+
30g/L sucrose+4g/L agar, the pH values of culture medium are 5.7；
Step（4）, tobacco explant anti-browning culture：The tobacco explant obtained in step (3) is placed in the preliminary anti-brownings of MS
In culture medium, in 28 DEG C of cultivation temperature, illumination cultivation 16h/d, 25 DEG C of dark culturing 8h/d, 30-50 μm of ol/ of intensity of illumination
(m2S) 20d is cultivated under conditions of, obtains tobacco healing tissue, the preliminary anti-browning culture mediums of MS are：Add in MS minimal mediums
Enter anti-brown additive 2.0g/L sodium thiosulfate (Na2S2O3), 30g/L sucrose, 4g/L agar, the pH values of culture medium are 5.7；
Step（5）, the subculture anti-browning culture of tobacco explant：
1）The preparation of chamomile extracting solution：Chamomile of the selection shortly past viewing period takes flower and cleans, squeeze juice, by juice mistake
Solid residue is filtered out, in superclean bench using 0.22 μm of filter filtration sterilization, sterile extracting solution is made；
2）The preparation of pot marigold extracting solution：Pot marigold of the selection shortly past viewing period takes flower and cleans, squeeze juice, by juice mistake
Solid residue is filtered out, in superclean bench using 0.22 μm of filter filtration sterilization, sterile extracting solution is made；
3）The preparation of tomato extract solution：Juice is filtered to remove residue, in superclean bench by tomato belt leather squeeze juice
Using 0.22 μm of filter filtration sterilization, sterile extracting solution is made；
4）The preliminary anti-browning tobacco explant obtained in step (4) is placed in MS subculture anti-browning culture mediums, in culture temperature
When spending 28 DEG C, illumination cultivation 16h/d, 25 DEG C of dark culturing 8h/d, 30-50 μm of ol/ (m of intensity of illumination2S) it is cultivated under conditions of
20d obtains the tobacco explant that melting brown rate is 18.7%；The MS subculture anti-browning culture mediums are：In MS minimal mediums
Add in prevent brown additive 100mg/L chamomiles extracting solution, 100mg/L pot marigolds extracting solution, 100mg/L tomato extract solutions,
30g/L sucrose, 4g/L agar, the pH values of culture medium are 5.7.
Embodiment 2
Step（1）To step（3）It is same as Example 1.
Step（4）, tobacco explant anti-browning culture：It is tentatively anti-that the tobacco explant obtained in step (3) is placed in MS
In browning culture medium, in 28/25 DEG C, time 16/8h of cultivation temperature, 30-50 μm of ol/ (m of intensity of illumination2S) it is trained under conditions of
Nutrient does not cultivate 20d, and the preliminary anti-browning culture mediums of MS are to add in prevent brown additive 4.0g/L thiosulfuric acids in MS minimal mediums
Sodium (Na2S2O3), 30g/L sucrose, the agar of 4g/L, the pH value of culture medium is 5.7；
Step（5）, the subculture anti-browning culture of tobacco explant：
1）To 3）It is same as Example 1；
4）The preliminary anti-browning tobacco explant obtained in step (4) is placed in MS subculture anti-browning culture mediums, in culture temperature
When spending 28 DEG C, illumination cultivation 16h/d, 25 DEG C of dark culturing 8h/d, 30-50 μm of ol/ (m of intensity of illumination2S) it is cultivated under conditions of
20d obtains the tobacco explant that melting brown rate is 20.5%；The MS subculture anti-browning culture mediums are：In MS minimal mediums
Add in prevent brown additive 150mg/L chamomiles extracting solution, pot marigold extracting solution 150mg/L, tomato extract solution 150mg/L,
30g/L sucrose, the agar of 4g/L, culture medium pH values be 5.7.
Embodiment 3
Step（1）To step（3）It is same as Example 1.
Step（4）, tobacco explant anti-browning culture：It is tentatively anti-that the tobacco explant obtained in step (3) is placed in MS
In browning culture medium, in 28/25 DEG C, time 16/8h of cultivation temperature, 30-50 μm of ol/ (m of intensity of illumination2S) it is trained under conditions of
Nutrient does not cultivate 20d, and the preliminary anti-browning culture mediums of MS are to add in prevent brown additive 5.0g/L thiosulfuric acids in MS minimal mediums
Sodium (Na2S2O3), 30g/L sucrose, the agar of 4g/L, the pH values of culture medium are 5.7；
Step（5）, the subculture anti-browning culture of tobacco explant：
1）To 3）It is same as Example 1；
4）The preliminary anti-browning tobacco explant obtained in step (4) is placed in MS subculture anti-browning culture mediums, in culture temperature
When spending 28 DEG C, illumination cultivation 16h/d, 25 DEG C of dark culturing 8h/d, 30-50 μm of ol/ (m of intensity of illumination2S) it is cultivated under conditions of
20d obtains the tobacco explant that melting brown rate is 23.3%；The MS subculture anti-browning culture mediums are：In MS minimal mediums
It adds in and prevents brown additive 200mg/L chamomile extracting solutions, pot marigold extracting solution 200mg/L, tomato extract solution 200mg/L,
30g/L sucrose, the agar of 4g/L, the pH values of culture medium are 5.7.
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 improvements may be made to the invention, 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 that tobacco Brown is prevented with sodium thiosulfate, it is characterised in that；Include the following steps：Step（1）, the disinfection of vegetable seeds；Step（2）, vegetable seeds culture：Vegetable seeds after the disinfection that step (1) is obtained is inoculated into MS culture mediums, is being trained It is 25 ± 1 DEG C to support temperature, 30-50 μm of ol/ (m of intensity of illumination2S), light application time is cultivates 60d, seed under conditions of 16h/d Germination is grown to seedling, and the MS culture mediums are：The agar of MS minimal medium+30g/L sucrose+4g/L, the pH of culture medium Be worth is 5.7；Step（3）, plant explant culture：The seed seedling leaf dish obtained in step (2) is placed in MS differential mediums, During 28 DEG C of cultivation temperature, illumination cultivation 16h/d, 25 DEG C of dark culturing 8h/d, 30-50 μm of ol/ (m of intensity of illumination2S), 5 are cultivated My god, tobacco healing tissue is obtained, the MS differential mediums are：MS minimal medium+0.5mg/L methyl α-naphthyl acetates NAA+2mg/L6- Benzyladenine 6-BA+30g/L sucrose+4g/L agar, the pH values of culture medium are 5.7；Step（4）, plant explant anti-browning culture：The plant explant obtained in step (3) is placed in the preliminary anti-brownings of MS In culture medium, in 28 DEG C of cultivation temperature, illumination cultivation 16h/d, 25 DEG C of dark culturing 8h/d, 30-50 μm of ol/ of intensity of illumination (m2S) 20d is cultivated under conditions of, obtains tobacco healing tissue, the preliminary anti-browning culture mediums of MS are：Add in MS minimal mediums Enter anti-brown additive 1.0-5.0g/L sodium thiosulfate (Na2S2O3), 30g/L sucrose, 4g/L agar, the pH values of culture medium are 5.7；Step（5）, the subculture anti-browning culture of plant explant：1）The preparation of chamomile extracting solution：Chamomile of the selection shortly past viewing period takes flower and cleans, squeeze juice, by juice mistake Solid residue is filtered out, in superclean bench using 0.22 μm of filter filtration sterilization, sterile extracting solution is made；2）The preparation of pot marigold extracting solution：Pot marigold of the selection shortly past viewing period takes flower and cleans, squeeze juice, by juice mistake Solid residue is filtered out, in superclean bench using 0.22 μm of filter filtration sterilization, sterile extracting solution is made；3）The preparation of tomato extract solution：Juice is filtered to remove residue, in superclean bench by tomato belt leather squeeze juice Using 0.22 μm of filter filtration sterilization, sterile extracting solution is made；4）The preliminary anti-browning plant explant obtained in step (4) is placed in MS subculture anti-browning culture mediums, in culture temperature Degree is in 28 DEG C of cultivation temperature, illumination cultivation 16h/d, 25 DEG C of dark culturing 8h/d, 30-50 μm of ol/ (m of intensity of illumination2S) Under the conditions of cultivate 20d, obtain melting brown rate be 18.7-23.3% plant explant；The MS subculture anti-browning culture mediums are： In MS minimal mediums add in prevent brown additive 100-200mg/L chamomiles extracting solution, 100-200mg/L pot marigolds extracting solution, 100-200mg/L tomato extract solutions, 30g/L sucrose, 4g/L agar, the pH values of culture medium are 5.7.
- 2. the method according to claim 1 that tobacco Brown is prevented with sodium thiosulfate, it is characterised in that；It is described The disinfection of vegetable seeds be：Full vegetable seeds is taken, is then moved on superclean bench, is impregnated seed with 75% alcohol 30s, aseptic water washing 2 times, then use 1%AgNO310min is sterilized, sterile water soaking and washing 5 times blots explant with aseptic filter paper It is inoculated with after body surface moisture, wherein sterile water is through autoclaved ultra-pure water.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109105257A (en) * | 2018-08-09 | 2019-01-01 | 华南农业大学 | A kind of method of flue-cured tobacco tissue-culturing rapid propagation nursery |
| CN113207694A (en) * | 2021-06-10 | 2021-08-06 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Culture medium for tobacco callus culture, preparation method thereof and tobacco callus culture method |
-
2017
- 2017-12-13 CN CN201711332624.3A patent/CN108142290A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109105257A (en) * | 2018-08-09 | 2019-01-01 | 华南农业大学 | A kind of method of flue-cured tobacco tissue-culturing rapid propagation nursery |
| CN113207694A (en) * | 2021-06-10 | 2021-08-06 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Culture medium for tobacco callus culture, preparation method thereof and tobacco callus culture method |
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