CN106857252A - The method that disinfection by chlorine dioxide culture medium is used for the quick breeding of potato seedling or Stem covered by vermiculite - Google Patents
The method that disinfection by chlorine dioxide culture medium is used for the quick breeding of potato seedling or Stem covered by vermiculite 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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Abstract
The invention discloses a kind of method that disinfection by chlorine dioxide culture medium is used for the quick breeding of potato seedling or Stem covered by vermiculite, it is characterized in that at room temperature, using liquid chlorine dioxide as disinfectant, aseptic toxicity-removing white potato seedling fast propagating culture medium or toxicity-removing white potato Stem covered by vermiculite culture medium is quickly obtained, is quickly bred or Stem covered by vermiculite for toxicity-removing white potato seedling.The present invention do not need autoclave sterilization pot can quickly realize culture medium it is sterilized, to culture vessel material without specific demand, simplified culture base disinfectant program, lengthening disinfection time are simultaneously greatly reduced energy consumption cost, and then virus-free potato primary stock producing cost is greatly reduced, toxicity-removing white potato introduces a collection large-scale application aborning is helped lend some impetus to, China's potato level of production is improved.
Description
Technical field
The present invention relates to a kind of field of plant tissue culture technique, more particularly to a kind of disinfection by chlorine dioxide culture
Base is used for the new method of the quick breeding of potato seedling or Stem covered by vermiculite.
Background technology
Potato be dicotyledon Solanaceae Solanum it is a kind of with grain, vegetables, feed multi-purpose type crop, it is suitable to habitat
Answering property is relatively broad, is suitable to the environmental condition that other chief crops are difficult to grow, and has plantation in global 155 countries.2016
The beginning of the year, the issue of the Ministry of Agriculture of China《Instruction on propulsion Potato Industry exploitation》, propose to the year two thousand twenty, potato planting
Area is expanded to more than 100,000,000 mu, and the kind planting proportion of suitable staple food processing reaches 30%, and staple food consumption accounts for potato overall consumption
The 30% of amount.Potato staple food grain is China staple food grain crop yield space is small, eco-environmental pressure increase, consumer's nutrition not
The Major Strategic of the guarantee national food security implemented under the backgrounds such as equilibrium.However, China's potato level of production is relatively low, averagely
Per mu yield is not as good as 1000 kg, and the per mu yield of developed country is then more than 2500 kg.Key constraints in potato production are them
Germ plasm resource is degenerated caused by virus is infected in long-term nutrition growth course, has been recorded more than 30 kinds of Potyviruses.In length
During the nutrient growth of phase, potato has met with the invasion and attack of a large amount of viruses, causes yield and quality to be greatly reduced.Viral diagnosis
It was found that, all test kinds are all attacked by least one virus.In this regard, scientist has carried out a large amount of fruitful detoxifications
Research, to recover its yield and quality.Detoxification test tube plantlet is obtained using Potato Shoot-tips tissue cultures, and then induces Potato microtuber,
It is to obtain virus-free potato primary stock basic link.By virus-free culture, the quality of potato is significantly improved, and yield reaches
3000kg, is 3 times before detoxification.However, because tissue culture process causes virus-free potato original silkworm egg cost high, in addition China peasant from
The custom reserved seed for planting, about 80% is band seed culture of viruses source in China's potato actual production so that average product is greatly reduced.Therefore, reduce
Detoxification virus-free potato original silkworm egg production cost will be helpful to greatly improve China's potato yield, promote its staple food grain process.
Using cellular omnipotency, the shoot apical meristem of still uninfecting virus is chosen for explant carries out cultured in vitro acquisition
Regeneration, can effectively remove virus, improve the potato level of production.During potato aseptic seedling virus-free culture, culture medium is high
Warm autoclaving can expend that to account for whole incubation cost ratio high.Therefore, the medium sterilization method for developing low cost is replaced
It is most important to penetration and promotion potato virus-free plantlet for traditional autoclave sterilization method.
First artificial synthetic medium of principal component is set up from the american plant physiologist White thirties in 20th century
Since, plant tissue culture technique is developed rapidly and is widely used in micropropagation of plants, gene function is verified and molecular breeding
Deng field.Tissue culture technique is, using the totipotency of cell, cell or organ to be cultivated into the process of whole plant.In vitro
Under the conditions of, the culture of cell or organ must be carried out in an aseptic environment.Therefore, culture medium has to pass through strict sterilization to protect
Hold gnotobasis.At present, medium sterilization mainly passes through high temperature and high pressure method（The kPa of pressure 102.9,121~126 DEG C of temperature, dimension
Hold 20~30 minutes）Complete.Autoclave sterilization consumption electric energy accounts for whole culture medium and prepares cost（Including reagent, power consumption and people
Member is taken）50% or so, average every bottle culture medium consistent with potato aseptic seedling virus-free culture cost accounting result（33 ml）Disappear
Consuming electric power expense is about 0.083 yuan.Meanwhile, the sterilizing methods are relatively strong to medium container material selectivity, and by several times
Container light transmittance can be significantly reduced after sterilizing, influence culture effect, and the material such as preferable PVC- polyvinyl chloride of translucency is usual
Autoclave sterilization can not be carried out, ethylene oxide sterilizing need to be used, discarded after disposable use, caused expensive and cause huge
Waste.Additionally, in the infrastructure for not possessing the instrument and equipments such as high-temperature high-pressure sterilizing chamber and condition, especially extensive tissue training
Support detoxification field, it is difficult to realize the preparation and operation of aseptic culture medium.Therefore, other simple sterilizing methods replacement high temperature are explored high
Pressure sterilization turns into one of study hotspot of field of plant tissue culture.Scientist explores various disinfectants in plant culture
Application in terms of sterilizing.Domestic and international scientist has investigated the reagents such as plants essential oil and sodium dichloro cyanurate to Plant Tissue Breeding
The sterilization effect of base.But all there is expensive, the problem of disinfectant substance residual after sterilizing, and lack system research.
Chlorine dioxide has compared with strong oxidizing property, is a kind of disinfectant of new environmental sound, in use not
Toxicant can be produced.Chlorine dioxide has been widely used in the closely related numerous areas of production and living.Our seminars exist
Application of the liquid chlorine dioxide in the sterilization of plant explant is have rated in early-stage Study, it is found that chlorine dioxide can effectively kill plant
The microorganism on beyond the region of objective existence implant surface, but explant is active relevant with plant endogenous aldehydes matter content after sterilization treatment.And then, I
Test disinfection by chlorine dioxide plant tissue culture media and be successfully applied to Activities of Some Plants tissue cultures practice
（CN106171970A）.This points out us that chlorine dioxide culture medium is used for into virus-free potato primary stock production very big potential,
Help development energy-saving and environmental protection, easy potato culture compound method, accelerate toxicity-removing white potato in the popularizing planting of China,
Promote the staple food grainization development of potato.
The content of the invention
It is used for Ma Ling it is an object of the invention to overcome the deficiencies in the prior art to provide a kind of disinfection by chlorine dioxide culture medium
The quick breeding of potato seedling or the method for Stem covered by vermiculite, simplified culture base disinfectant program, lengthening disinfection time are simultaneously greatly reduced energy
Expend and use.
The technical solution adopted in the present invention is：At room temperature, it is quick to obtain using liquid chlorine dioxide as disinfectant
Aseptic toxicity-removing white potato seedling fast propagating culture medium or toxicity-removing white potato Stem covered by vermiculite culture medium, for toxicity-removing white potato
Seedling quickly breeds or Stem covered by vermiculite.
Described liquid chlorine dioxide is quickly prepared using sodium chlorite and food grade salt acid reaction, weighs 1.5 g Asias chlorine
Sour sodium adds 50 mL distilled water in brown bottle, after it is completely dissolved, adds 5 mL food grade hydrochloric acids, reacts 10 minutes
Afterwards, 450 mL distilled water are added, is mixed and is obtained 1000 mg/L aqueous solution of chlorine dioxide.
Described liquid chlorine dioxide adds concentration needed for distilled water diluting to 1~150 mg/L before use.
Further, also including carrying out fumigation to culture medium separation container with chlorine dioxide：Chlorine dioxide
Body is generated using sodium chlorite with food grade salt acid reaction, weighs 1.5 g sodium chlorites in brown bottle, adds 50 mL distillations
Water, after it is completely dissolved, adds 5 mL food grade hydrochloric acids, obtains 1000 mg chlorine dioxides, is placed in the closed appearance of respective volume
Device obtains final product required Chlorine Dioxide Gas concentration；Culture medium is dispensed in confined space using 1~150 mg/L chlorine dioxides
Container suffocating sterilization 30 minutes.
Described toxicity-removing white potato seedling fast propagating culture medium is prepared using following steps：Weigh appropriate MS culture mediums into
Point and 40 g/L sucrose be placed in 1~150 mg/L aqueous solution of chlorine dioxide of culture medium cumulative volume 60%, with 100 turns per minute
Rotating speed on magnetic stirring apparatus stir sterilization 20 minutes, then heat 10 minutes to culture medium temperature be 50~60 DEG C；With being matched somebody with somebody
The distilled water of culture medium cumulative volume 40% processed is clear to being completely dissolved in micro-wave oven or heating by electric cooker by coagulator agar or plant gel
It is clear；The medium component sterilized is mixed with the coagulator of dissolving, pH to 5.8~6.0 is adjusted；With culture medium separation container point
Dress, it is standby after cooling.
Described toxicity-removing white potato Stem covered by vermiculite culture medium is prepared using following steps：Weigh appropriate MS medium components
And 50~80 g/L sucrose be placed in 1~150 mg/L aqueous solution of chlorine dioxide of culture medium cumulative volume 60%, with 100 turns per minute
Rotating speed on magnetic stirring apparatus stir sterilization 20 minutes, then heat 10 minutes to culture medium temperature be 50~60 DEG C；With institute
Prepare the distilled water of culture medium cumulative volume 40% by coagulator agar or plant gel in micro-wave oven or heating by electric cooker to being completely dissolved
It is limpid；The medium component sterilized is mixed with the coagulator of dissolving, pH to 5.8~6.0 is adjusted；With culture medium separation container point
Dress, it is standby after cooling.
Described toxicity-removing white potato seedling quickly breeding be by toxicity-removing white potato seedling be cut into about 1 cm it is long with stipes
Stem section, the toxicity-removing white potato seedling fast propagating culture medium that the insertion of morphology lower end is prepared, cultivates, light under the conditions of 25 ± 1 DEG C
According to 14 hours/day, the Lx of intensity of illumination 6000.
Described test tube potato induction is that toxicity-removing white potato seedling is cut into the about 1 cm stem sections with stipes long, shape
Insert toxicity-removing white potato Stem covered by vermiculite culture medium in state lower end；Condition of culture is hour/day of illumination 10, temperature 25 during illumination ±
1 DEG C, the Lx of intensity of illumination 6000,19 ± 1 DEG C of temperature when dark.
Described MS culture mediums include 1900 mg/L potassium nitrate, 1650 mg/L ammonium nitrate, 170 mg/L biphosphates
Potassium, 370 mg/L epsom salts, 440 mg/L calcium chloride dihydrates, 27.85 mg/L green-vitriols, 37.25 mg/L second two
Amine tetraacethyl disodium, the hydrated manganese sulfates of 22.3 mg/L tetra-, 8.6 mg/L white vitriols, 6.2 mg/L boric acid, 0.83 mg/
L KIs, 0.025 mg/L cupric sulfate pentahydrates, the molybdic acid hydrate sodium of 0.25 mg/L bis-, 0.025 mg/L CoCL2 6H2Os, 2.0
Mg/L glycine, 0.1 mg/L thiamine hydrochlorides, 0.5 mg/L pyridoxine hydrochlorides, 0.5 mg/L nicotinic acid, 100 mg/L fleshes
Alcohol.Potato propagating culture medium is that MS culture mediums add 40 g/L sucrose, and Stem covered by vermiculite culture medium is MS culture mediums addition 50
~80 g/L sucrose.Used medium curing agent is 8 g/L agar or 3 g/L plant gels.
Compared with prior art, the beneficial effects of the invention are as follows:Not needing autoclave sterilization pot can quickly realize training
Support base it is sterilized, to culture vessel material without specific demand, simplified culture base disinfectant program, lengthening disinfection time and significantly
Reducing energy consumption expense, and then virus-free potato primary stock producing cost is greatly reduced, help lend some impetus to toxicity-removing white potato introduces a collection and exist
Large-scale application in production, improves China's potato level of production.
Brief description of the drawings
Fig. 1 is disinfection by chlorine dioxide culture medium and the breeding of autoclave sterilization control medium potato " China 1 " seedling photo.
In Fig. 1, the left side is potato " China 1 " seedling of autoclave sterilization culture medium breeding, and the right is disinfection by chlorine dioxide culture medium
Potato " China 1 " seedling of breeding.
Fig. 2 is the potato photo of disinfection by chlorine dioxide culture medium induction in blake bottle.White arrow is illustrated as being induced
Potato.
Fig. 3 is disinfection by chlorine dioxide culture medium induction potato " China 1 " Potato microtuber photo to be harvested.White arrow is shown
Mean potato.
Fig. 4 is disinfection by chlorine dioxide culture medium induction potato " in 5 " Potato microtuber photo to be harvested.White arrow is shown
Mean potato.
Fig. 5 is " China 1 " potato photo that the disinfection by chlorine dioxide culture medium for harvesting is induced.
Fig. 6 is " in 5 " potato photo that the disinfection by chlorine dioxide culture medium for harvesting is induced.
Specific embodiment
The preparation of the disinfection by chlorine dioxide culture medium of embodiment one
According to chemical equation 5NaClO+4HCl=4ClO2+5NaCl+2H2O, using sodium chlorite and food grade salt acid reaction
Realize the quick preparation of chlorine dioxide.
1.5 g sodium chlorites are weighed in brown bottle, 50 mL distilled water are added, after it is completely dissolved, 5 mL foods is added
Grade hydrochloric acid, after reacting 10 minutes, adds 450 mL distilled water, mixes and obtains 1000 mg/L aqueous solution of chlorine dioxide.
Before use with concentration needed for distilled water diluting to 1~150 mg/L.
Toxicity-removing white potato seedling fast propagating culture medium is prepared：Weigh appropriate plant MS medium components and be placed in and prepare training
1~150 mg/L aqueous solution of chlorine dioxide of matrix product 60% is supported, 40 g/L sucrose is added, with 100 turns per minute of rotating speed in magnetic
On power agitator stir sterilization 20 minutes, while heating 10 minutes to culture medium temperature be 50~60 DEG C.By coagulator agar or
Plant gel is dissolved in the respective concentration aqueous solution of chlorine dioxide of prepared culture volume 40%, and micro-wave oven or heating by electric cooker are to complete
CL is limpid.Medium component is mixed with the coagulator of dissolving, regulation pH value uses culture medium separation container to 5.8~6.0
Be can be used after packing cooling.
Toxicity-removing white potato Stem covered by vermiculite culture medium is prepared：Weigh appropriate plant MS medium components and be placed in and prepare culture
Matrix product 60% 1~150 mg/L aqueous solution of chlorine dioxide, add 50~80 g/L sucrose, with 100 turns per minute of rotating speed in
On magnetic stirring apparatus stir sterilization 20 minutes, while heating 10 minutes to culture medium temperature be 50~60 DEG C.By coagulator agar
Or plant gel is dissolved in the respective concentration aqueous solution of chlorine dioxide of prepared culture volume 40%, micro-wave oven or heating by electric cooker are extremely
It is completely dissolved limpid.Medium component is mixed with the coagulator of dissolving, regulation pH value to 5.8~6.0 is dispensed with culture medium and held
Be can be used after device packing cooling.
Culture medium separation container is using 1~150 mg/L chlorine dioxides in confined space suffocating sterilization 30 minutes.Take
1.5 g sodium chlorites add 50 mL distilled water in brown bottle, after it is completely dissolved, add 5 mL food grade hydrochloric acids, i.e.,
1000 mg chlorine dioxides can be obtained.It is placed in respective volume closed container and obtains final product required Chlorine Dioxide Gas concentration.In confined air
Between middle sterilizing of the realization to culture medium separation container.
The disinfection by chlorine dioxide medium culture toxicity-removing white potato seedling of embodiment two
For examination Potato Cultivars include China 1, in 5 and A-R-I.Toxicity-removing white potato seedling is cut into about 1 cm in superclean bench
Stem section with stipes long, the toxicity-removing white potato seedling fast propagating culture medium that morphology lower end insertion embodiment one is prepared.With
It is control that autoclave sterilization method prepares identical component culture medium.Cultivated under the conditions of 25 ± 1 DEG C, hour/day of illumination 14, illumination
The Lx of intensity 6000.After 20 days, potato seedling can be grown to 10 centimeter lengths, and growing way is as good as with autoclave sterilization control medium,
And seedling is more more than control branch（Fig. 1）.Too low chlorine dioxide concentration cannot be realized to the thorough of culture medium and Potatoes
Sterilization, causes partial medium and potato seedling to pollute.And too high chlorine dioxide concentration is then caused seriously to potato cell
Injury, causes that blade is withered and yellow, plant is lopsided, or even dead.To three Potato Cultivars, 1~150 mg/L liquid chlorine dioxide
It is capable of achieving to culture medium and the high-effective disinfecting of potato seedling, while not causing unfavorable injury to potato virus-free plantlet.Show 1
~100 mg/L liquid chlorine dioxide sterilising mediums are applied to the tissue rapid propagation culture of potato virus-free plantlet, are conducive to Ma Ling
The large-scale breeding of potato seedling.
The disinfection by chlorine dioxide culture medium of embodiment three induces test tube potato
With China 1, in 5 and A-R-I be for examination Potato Cultivars.Toxicity-removing white potato seedling is cut into about 1 cm in superclean bench
Stem section with stipes long, the toxicity-removing white potato Stem covered by vermiculite culture medium that morphology lower end insertion embodiment one is prepared.Culture
Condition is hour/day of illumination 10,25 ± 1 DEG C of temperature during illumination, the Lx of intensity of illumination 6000,19 ± 1 DEG C of temperature when dark.30 days
Afterwards, test tube potato is initially formed（Fig. 2）.Try 3 kinds and all successfully induce Potato microtuber, and Stem covered by vermiculite rate reaches
To more than 80%（Fig. 3, Fig. 4）.Potato microtuber is harvested after 50 days and is preserved（Fig. 5, Fig. 6）.
In the present embodiment, 1~150 mg/L liquid chlorine dioxide is realized to the thorough disinfection for examination potato culture
And successfully induce Potato microtuber, and Disinfection Effect to may persist to 60 days or so, growth of potato sets breeding and Stem covered by vermiculite
Can be completed in same blake bottle, simplify the operating procedure of Stem covered by vermiculite, the big rule of toxicity-removing white potato Potato microtuber can be promoted
Mould is produced.
Claims (8)
1. a kind of method that disinfection by chlorine dioxide culture medium is used for the quick breeding of potato seedling or Stem covered by vermiculite, its feature exists
In：At room temperature, using liquid chlorine dioxide as disinfectant, quickly obtain aseptic toxicity-removing white potato seedling and quickly breed training
Base or toxicity-removing white potato Stem covered by vermiculite culture medium are supported, is quickly bred or Stem covered by vermiculite for toxicity-removing white potato seedling.
2. the method for claim 1, it is characterised in that：Described liquid chlorine dioxide uses sodium chlorite and food-grade
Hydrochloric acid reaction is quickly prepared, and weighs 1.5 g sodium chlorites in brown bottle, adds 50 mL distilled water, after it is completely dissolved,
5 mL food grade hydrochloric acids are added, after reacting 10 minutes, 450 mL distilled water is added, is mixed and is obtained 1000 mg/L titanium dioxides
Chlorine water solution.
3. method as claimed in claim 2, it is characterised in that：Described liquid chlorine dioxide adds distilled water diluting extremely before use
Concentration needed for 1~150 mg/L.
4. the method for claim 1, it is characterised in that：Also include entering culture medium separation container with chlorine dioxide
Row fumigation：Chlorine dioxide is generated using sodium chlorite with food grade salt acid reaction, weighs 1.5 g sodium chlorites in palm fibre
In color bottle, 50 mL distilled water are added, after it is completely dissolved, add 5 mL food grade hydrochloric acids, obtain 1000 mg chlorine dioxides
Body, is placed in respective volume closed container and obtains final product required Chlorine Dioxide Gas concentration；Existed using 1~150 mg/L chlorine dioxides
Confined space was to culture medium separation container suffocating sterilization 30 minutes.
5. the method for claim 1, it is characterised in that：Described toxicity-removing white potato seedling fast propagating culture medium is used
It is prepared by following steps：Weigh appropriate MS medium components and 40 g/L sucrose are placed in 1~150 mg/L of culture medium cumulative volume 60%
Aqueous solution of chlorine dioxide, with 100 turns per minute of rotating speed in sterilization 20 minutes is stirred on magnetic stirring apparatus, then heats 10 minutes
It it is 50~60 DEG C to culture medium temperature；With the distilled water of prepared culture medium cumulative volume 40% by coagulator agar or plant gel
It is limpid to being completely dissolved in micro-wave oven or heating by electric cooker；The medium component sterilized is mixed with the coagulator of dissolving, pH is adjusted
To 5.8~6.0；Dispensed with culture medium separation container, it is standby after cooling.
6. the method for claim 1, it is characterised in that：Described toxicity-removing white potato Stem covered by vermiculite culture medium use with
It is prepared by lower step：Weigh appropriate MS medium components and 50~80 g/L sucrose are placed in the 1~150 of culture medium cumulative volume 60%
Mg/L aqueous solution of chlorine dioxide, with 100 turns per minute of rotating speed in sterilization 20 minutes is stirred on magnetic stirring apparatus, then heats 10
Minute to culture medium temperature is 50~60 DEG C；With the distilled water of prepared culture medium cumulative volume 40% by coagulator agar or plant
Gel is limpid to being completely dissolved in micro-wave oven or heating by electric cooker；The medium component sterilized is mixed with the coagulator of dissolving, is adjusted
Section pH to 5.8~6.0；Dispensed with culture medium separation container, it is standby after cooling.
7. the method for claim 1, it is characterised in that：Quickly breeding is by detoxification horse to described toxicity-removing white potato seedling
Bell potato seedling is cut into the about 1 cm stem sections with stipes long, and the toxicity-removing white potato seedling that the insertion of morphology lower end is prepared quickly breeds
Culture medium, cultivates, hour/day of illumination 14, the Lx of intensity of illumination 6000 under the conditions of 25 ± 1 DEG C.
8. the method for claim 1, it is characterised in that：Described test tube potato induction is by toxicity-removing white potato children
Seedling is cut into the about 1 cm stem sections with stipes long, morphology lower end insertion toxicity-removing white potato Stem covered by vermiculite culture medium；Culture bar
Part is hour/day of illumination 10,25 ± 1 DEG C of temperature during illumination, the Lx of intensity of illumination 6000,19 ± 1 DEG C of temperature when dark.
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| CN108552060A (en) * | 2018-07-19 | 2018-09-21 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | The sterilizing methods of sweet potato tissue cultures explant |
| CN110810403A (en) * | 2019-12-10 | 2020-02-21 | 淮北师范大学 | Wheat seed dressing agent and preparation method and application thereof |
| CN113455393A (en) * | 2021-07-21 | 2021-10-01 | 华中农业大学 | Method for inducing tubepotato based on carbon dioxide enrichment |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108552060A (en) * | 2018-07-19 | 2018-09-21 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | The sterilizing methods of sweet potato tissue cultures explant |
| CN110810403A (en) * | 2019-12-10 | 2020-02-21 | 淮北师范大学 | Wheat seed dressing agent and preparation method and application thereof |
| CN113455393A (en) * | 2021-07-21 | 2021-10-01 | 华中农业大学 | Method for inducing tubepotato based on carbon dioxide enrichment |
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| CN106857252B (en) | 2019-03-12 |
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