CN106857252B - Disinfection by chlorine dioxide culture medium is quickly bred for potato seedling or the method for Stem covered by vermiculite - Google Patents
Disinfection by chlorine dioxide culture medium is quickly bred for potato seedling or the method for 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
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Abstract
The invention discloses a kind of disinfection by chlorine dioxide culture mediums quickly to breed or the method for Stem covered by vermiculite for potato seedling, it is characterized in that at room temperature, use liquid chlorine dioxide as disinfectant, it is quickly obtained sterile toxicity-removing white potato seedling fast propagating culture medium or toxicity-removing white potato Stem covered by vermiculite culture medium, is quickly bred or Stem covered by vermiculite for toxicity-removing white potato seedling.The present invention do not need autoclave sterilization pot can fast implement culture medium it is sterilized, to culture vessel material without specific demand, simultaneously energy consumption cost is greatly reduced in simplified culture base disinfectant program, lengthening disinfection time, and then virus-free potato primary stock producing cost is greatly reduced, toxicity-removing white potato provenance large-scale application in production is peomoted, China potato production level 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 quickly bred for potato seedling or the new method of Stem covered by vermiculite.
Background technique
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, and the environmental condition grown is difficult to suitable for other chief crops, and in the whole world, there are plantation in 155 countries.2016
The beginning of the year, the Ministry of Agriculture, China publication " about the instruction for promoting Potato Industry exploitation " propose to arrive the year two thousand twenty, potato planting
Area is expanded to 100,000,000 mu or more, 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 be China staple food grain crop yield space is small, eco-environmental pressure increases, consumer's nutrition not
The Major Strategic for the guarantee national food security implemented under the backgrounds such as equilibrium.However, China's potato production level is lower, it is average
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 caused by virus infection is degenerated in long-term nutrition growth course, has recorded more than 30 kinds Potyvirus.In length
During the nutrient growth of phase, potato has met with the invasion of a large amount of viruses, and yield and quality is caused to be greatly reduced.Viral diagnosis
It was found that invasion of all test kinds all by least one virus.In this regard, scientist has carried out a large amount of fruitful detoxifications
Research, to restore 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, since tissue culture process causes virus-free potato original silkworm egg cost high, in addition China peasant from
The habit 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, it reduces
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 for choosing not yet virus infection is that explant carries out in vitro culture acquisition
Regeneration can effectively remove virus, improve potato production level.During potato aseptic seedling virus-free culture, culture medium is high
Warm high pressure sterilization can expend that account for entire incubation cost ratio high.Therefore, the medium sterilization method for developing low cost is replaced
It is most important to potato virus-free plantlet is popularized for traditional autoclave sterilization method.
The artificial synthetic medium of first principal component is established from american plant physiologist White the 1930s
Since, plant tissue culture technique rapidly develops and is widely used in micropropagation of plants, gene function verifying and molecular breeding
Equal fields.Tissue culture technique is the totipotency using cell, and cell or organ are cultivated to the process at intact plant.In vitro
Under the conditions of, the culture of cell or organ must carry out in an aseptic environment.Therefore, culture medium has to pass through stringent disinfection to protect
Hold gnotobasis.Currently, medium sterilization mainly passes through high temperature and high pressure method, (102.9 kPa of pressure, is tieed up by 121~126 DEG C of temperature
Hold 20~30 minutes) it completes.Autoclave sterilization consumption electric energy accounts for entire culture medium and prepares cost (including reagent, energy consumption and people
Member take) 50% or so, it is consistent with potato aseptic seedling virus-free culture cost accounting result, be averaged every bottle of culture medium (33 ml) disappear
Consuming electric power expense is about 0.083 yuan.Meanwhile the sterilizing methods are stronger to medium container material selectivity, and by several times
Container light transmittance can significantly reduce after sterilizing, influence culture effect, and the materials such as preferable PVC- polyvinyl chloride of translucency are usual
It not can be carried out autoclave sterilization, ethylene oxide sterilizing need to be used, disposably discarded after, caused expensive and cause huge
Waste.In addition, in the infrastructure for not having 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, it is high that other simple sterilizing methods substitution high temperature are explored
Sterilization is pressed to become one of the research hotspot of field of plant tissue culture.Scientist explores a variety of disinfectants in plant culture
The application for the aspect that sterilizes.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 the problem of expensive, disinfectant substance remains after sterilizing, and lack system research.
Chlorine dioxide have be compared with strong oxidizing property a kind of novel environmental sound disinfectant, in use not
Toxicant can be generated.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 disinfection of plant explant is had rated in early-stage study, discovery chlorine dioxide can effectively kill plant
The microorganism on beyond the region of objective existence implant surface, but explant activity is related with plant endogenous aldehydes matter content after sterilization treatment.In turn, I
Test disinfection by chlorine dioxide plant tissue culture media and be successfully applied to Activities of Some Plants tissue cultures practice
(CN106171970A).This, which prompts us that chlorine dioxide culture medium is used for virus-free potato primary stock production, very big potential,
Help develop energy-saving and environmental protection, easy potato culture preparation method, accelerate toxicity-removing white potato China popularizing planting,
Promote the staple food grainization development of potato.
Summary of the invention
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 for Ma Ling
Potato seedling quickly breeds or the method for Stem covered by vermiculite, and simultaneously energy is greatly reduced in simplified culture base disinfectant program, lengthening disinfection time
It expends and uses.
The technical scheme adopted by the invention is that: at room temperature, uses liquid chlorine dioxide as disinfectant, be quickly obtained
Sterile toxicity-removing white potato seedling fast propagating culture medium or toxicity-removing white potato Stem covered by vermiculite culture medium is used for toxicity-removing white potato
Seedling quickly breeds or Stem covered by vermiculite.
The liquid chlorine dioxide is prepared using sodium chlorite and food grade hydrochloric acid rapid reaction, weighs 1.5 Asia g chlorine
50 mL distilled water are added in brown bottle in sour sodium, and after it is completely dissolved, 5 mL food grade hydrochloric acids are added, react 10 minutes
Afterwards, 450 mL distilled water are added, mixes and obtains 1000 mg/L aqueous solution of chlorine dioxide.
Concentration needed for the liquid chlorine dioxide adds distilled water to be diluted to 1~150 mg/L before use.
It further, further include that fumigation: chlorine dioxide is carried out to culture medium separation container with chlorine dioxide
Body reacts generation with food grade hydrochloric acid using sodium chlorite, weighs 1.5 g sodium chlorites in brown bottle, and 50 mL distillation is added
Water is added 5 mL food grade hydrochloric acids, obtains 1000 mg chlorine dioxides, be placed in the closed appearance of respective volume after it is completely dissolved
Device is up to required Chlorine Dioxide Gas concentration；Culture medium is dispensed in confined space using 1~150 mg/L chlorine dioxide
Container suffocating sterilization 30 minutes.
The toxicity-removing white potato seedling fast propagating culture medium using following steps prepare: weigh appropriate MS culture medium at
Point and 40 g/L sucrose be placed in 1~150 mg/L aqueous solution of chlorine dioxide of culture medium total volume 60%, with 100 turns per minute
Revolving speed on magnetic stirring apparatus stirring disinfection 20 minutes, then heat 10 minutes to culture medium temperature be 50~60 DEG C；With being matched
Coagulator agar or plant gel are heated to being completely dissolved clear by the distilled water of culture medium total volume 40% processed in micro-wave oven or electric furnace
It is clear；The medium component of disinfection and the coagulator of dissolution are mixed, pH to 5.8~6.0 is adjusted；With culture medium separation container point
Dress, it is spare after cooling.
The toxicity-removing white potato Stem covered by vermiculite culture medium is prepared using following steps: weighing appropriate MS medium component
And 50~80 g/L sucrose be placed in 1~150 mg/L aqueous solution of chlorine dioxide of culture medium total volume 60%, with 100 turns per minute
Revolving speed on magnetic stirring apparatus stirring disinfection 20 minutes, then heat 10 minutes to culture medium temperature be 50~60 DEG C；With institute
Coagulator agar or plant gel are heated to being completely dissolved by the distilled water for preparing culture medium total volume 40% in micro-wave oven or electric furnace
It is limpid；The medium component of disinfection and the coagulator of dissolution are mixed, pH to 5.8~6.0 is adjusted；With culture medium separation container point
Dress, it is spare after cooling.
The toxicity-removing white potato seedling quickly breed be toxicity-removing white potato seedling is cut into about 1 cm long with stipes
Stem section, the toxicity-removing white potato seedling fast propagating culture medium that the insertion of morphology lower end is prepared, is cultivated, light under the conditions of 25 ± 1 DEG C
According to 14 hours/day, 6000 Lx of intensity of illumination.
The test tube potato induction is the stem section with stipes that toxicity-removing white potato seedling is cut into about 1 cm long, shape
Toxicity-removing white potato Stem covered by vermiculite culture medium is inserted into state lower end；Condition of culture is 10 hour/day of illumination, temperature 25 when illumination ±
1 DEG C, 6000 Lx of intensity of illumination, 19 ± 1 DEG C of temperature when dark.
The MS culture medium includes 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, 22.3 mg/L, tetra- hydrated manganese sulfate, 8.6 mg/L white vitriols, 6.2 mg/L boric acid, 0.83 mg/
L potassium iodide, 0.025 mg/L cupric sulfate pentahydrate, 0.25 mg/L, bis- molybdic acid hydrate sodium, 0.025 mg/L CoCL2 6H2O, 2.0
Mg/L glycine, 0.1 mg/L thiamine hydrochloride, 0.5 mg/L pyridoxine hydrochloride, 0.5 mg/L niacin, 100 mg/L fleshes
Alcohol.Potato propagating culture medium is that MS culture medium adds 40 g/L sucrose, and Stem covered by vermiculite culture medium is MS culture medium 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 present invention are: training can be fast implemented by not needing autoclave sterilization pot
Support base it is sterilized, to culture vessel material without specific demand, simplified culture base disinfectant program, lengthening disinfection time and substantially
Energy consumption cost is reduced, and then virus-free potato primary stock producing cost is greatly reduced, toxicity-removing white potato provenance is peomoted and exists
Large-scale application in production improves China potato production level.
Detailed description of the invention
Fig. 1 is that disinfection by chlorine dioxide culture medium and autoclave sterilization control medium breeding potato " China 1 " seedling are shone
Piece.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 training
Support potato " China 1 " seedling of base breeding.
Fig. 2 is the potato photo that disinfection by chlorine dioxide culture medium induces in culture 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 of harvest is induced.
Fig. 6 is " in 5 " potato photo that the disinfection by chlorine dioxide culture medium of harvest is induced.
Specific embodiment
The preparation of one disinfection by chlorine dioxide culture medium of embodiment
According to chemical equation 5NaClO+4HCl=4ClO2+5NaCl+2H2O is anti-using sodium chlorite and food grade hydrochloric acid
It should 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 food is added
Grade hydrochloric acid adds 450 mL distilled water after reaction 10 minutes, mixes and obtains 1000 mg/L aqueous solution of chlorine dioxide.
Concentration needed for being diluted to 1~150 mg/L with distilled water before use.
Toxicity-removing white potato seedling fast propagating culture medium is prepared: being weighed appropriate plant MS medium component and is placed in and prepares training
1~150 mg/L aqueous solution of chlorine dioxide for supporting matrix product 60%, is added 40 g/L sucrose, with 100 turns of revolving speed per minute in magnetic
On power blender stirring disinfection 20 minutes, while heat 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 electric furnace have been heated to
Fully dissolved is limpid.Medium component and the coagulator of dissolution are mixed, adjust pH value to 5.8~6.0, with culture medium separation container
It can be used after packing is cooling.
Toxicity-removing white potato Stem covered by vermiculite culture medium is prepared: being weighed appropriate plant MS medium component and is placed in and prepares culture
Matrix product 60% 1~150 mg/L aqueous solution of chlorine dioxide, be added 50~80 g/L sucrose, with 100 turns of revolving speed per minute in
On magnetic stirring apparatus stirring disinfection 20 minutes, while heat 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 electric furnace are heated to
It is completely dissolved limpid.Medium component and the coagulator of dissolution are mixed, pH value is adjusted to 5.8~6.0, is dispensed and held with culture medium
It can be used after device packing is cooling.
Culture medium separation container is using 1~150 mg/L chlorine dioxide at confined space suffocating sterilization 30 minutes.It takes
50 mL distilled water are added in brown bottle in 1.5 g sodium chlorites, after it is completely dissolved, 5 mL food grade hydrochloric acids are added, i.e.,
1000 mg chlorine dioxides can be obtained.Respective volume closed container is placed in up to required Chlorine Dioxide Gas concentration.In confined air
Between middle sterilizing of the realization to culture medium separation container.
Two disinfection by chlorine dioxide culture medium culture toxicity-removing white potato seedling of embodiment
For examination Potato Cultivars include China 1, in 5 and A-R-I.Toxicity-removing white potato seedling is cut into about in superclean bench
The stem section with stipes of 1 cm long, the toxicity-removing white potato seedling that morphology lower end insertion embodiment one is prepared quickly breed culture
Base.Preparing identical component culture medium with autoclave sterilization method is control.It is cultivated under the conditions of 25 ± 1 DEG C, illumination 14 hours/
It, 6000 Lx of intensity of illumination.After 20 days, potato seedling can be grown to 10 centimeter lengths, and growing way compares culture with autoclave sterilization
Base is no different, and seedling is more (Fig. 1) than control branch.Too low chlorine dioxide concentration cannot achieve to culture medium and Potatoes
Thorough disinfection, cause partial medium and potato seedling to pollute.And excessively high chlorine dioxide concentration then makes potato cell
At grievous injury, lead to that blade is withered and yellow, plant is lopsided, or even dead.To three Potato Cultivars, 1~150 mg/L liquid two
The high-effective disinfecting to culture medium and potato seedling can be achieved in chlorine monoxid, while not causing unfavorable wound to potato virus-free plantlet
Evil.Show that 1~100 mg/L liquid chlorine dioxide sterilising medium is suitable for the tissue rapid propagation culture of potato virus-free plantlet, has
Conducive to the large-scale breeding of potato seedling.
Three disinfection by chlorine dioxide culture medium of embodiment induces test tube potato
With China 1, in 5 and A-R-I be for try Potato Cultivars.Toxicity-removing white potato seedling is cut into about in superclean bench
The toxicity-removing white potato Stem covered by vermiculite culture medium that embodiment one is prepared is inserted into the stem section with stipes of 1 cm long, morphology lower end.
Condition of culture is 10 hour/day of illumination, 25 ± 1 DEG C of temperature, 6000 Lx of intensity of illumination when illumination, 19 ± 1 DEG C of temperature when dark.
After 30 days, test tube potato initially forms (Fig. 2).It tries 3 kinds and all successfully induces Potato microtuber, and Stem covered by vermiculite rate
Reach 80% or more (Fig. 3, Fig. 4).Potato microtuber is harvested after 50 days and saves (Fig. 5, Fig. 6).
In the present embodiment, 1~150 mg/L liquid chlorine dioxide is realized to the thorough disinfection for trying potato culture
And Potato microtuber is successfully induced, and Disinfection Effect may persist to 60 days or so, growth of potato sets breeding and Stem covered by vermiculite
It can be completed in same culture bottle, simplify the operating procedure of Stem covered by vermiculite, can promote the big rule of toxicity-removing white potato Potato microtuber
Mould production.
Claims (4)
1. a kind of disinfection by chlorine dioxide culture medium is quickly bred for potato seedling or the method for Stem covered by vermiculite, feature exist
In: at room temperature, uses liquid chlorine dioxide as disinfectant, be quickly obtained sterile 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 for toxicity-removing white potato seedling or Stem covered by vermiculite；
The liquid chlorine dioxide is prepared using sodium chlorite and food grade hydrochloric acid rapid reaction, weighs 1.5 g sodium chlorites
In brown bottle, 50 mL distilled water are added, after it is completely dissolved, 5 mL food grade hydrochloric acids are added, react after ten minutes, then
450 mL distilled water are added, mixes and obtains 1000 mg/L aqueous solution of chlorine dioxide；
Concentration needed for the liquid chlorine dioxide adds distilled water to be diluted to 1~150 mg/L before use；
The toxicity-removing white potato seedling fast propagating culture medium using following steps prepare: weigh appropriate MS medium component and
40 g/L sucrose are placed in 1~150 mg/L aqueous solution of chlorine dioxide of culture medium total volume 60%, with 100 turns of revolving speed per minute
In on magnetic stirring apparatus stirring disinfection 20 minutes, then heat 10 minutes to culture medium temperature be 50~60 DEG C；It is trained with preparing
Coagulator agar or plant gel are heated to being completely dissolved limpid by the distilled water of feeding base total volume 40% in micro-wave oven or electric furnace；
The medium component of disinfection and the coagulator of dissolution are mixed, pH to 5.8~6.0 is adjusted；It is dispensed with culture medium separation container, it is cold
But spare after；
The toxicity-removing white potato Stem covered by vermiculite culture medium is prepared using following steps: weighing appropriate MS medium component and 50
~80 g/L sucrose are placed in 1~150 mg/L aqueous solution of chlorine dioxide of culture medium total volume 60%, with 100 turns per minute turn
Speed on magnetic stirring apparatus stirring disinfection 20 minutes, then heat 10 minutes to culture medium temperature be 50~60 DEG C；With being prepared
Coagulator agar or plant gel are heated to being completely dissolved clear by the distilled water of culture medium total volume 40% in micro-wave oven or electric furnace
It is clear；The medium component of disinfection and the coagulator of dissolution are mixed, pH to 5.8~6.0 is adjusted；With culture medium separation container point
Dress, it is spare after cooling.
2. disinfection by chlorine dioxide culture medium as described in claim 1 is quickly bred for potato seedling or Stem covered by vermiculite
Method, it is characterised in that: further include that fumigation: chlorine dioxide is carried out to culture medium separation container with chlorine dioxide
Generation is reacted with food grade hydrochloric acid using sodium chlorite, weighs 1.5 g sodium chlorites in brown bottle, 50 mL distilled water are added,
After it is completely dissolved, 5 mL food grade hydrochloric acids are added, obtains 1000 mg chlorine dioxides, is placed in respective volume closed container
Up to required Chlorine Dioxide Gas concentration；Culture medium is dispensed in confined space using 1~150 mg/L chlorine dioxide and is held
Device suffocating sterilization 30 minutes.
3. disinfection by chlorine dioxide culture medium as described in claim 1 is quickly bred for potato seedling or Stem covered by vermiculite
Method, it is characterised in that: it is the band that toxicity-removing white potato seedling is cut into 1 cm long that the toxicity-removing white potato seedling, which quickly breeds,
The stem section of stipes, the toxicity-removing white potato seedling fast propagating culture medium that the insertion of morphology lower end is prepared, under the conditions of 25 ± 1 DEG C
Culture, 14 hour/day of illumination, 6000 Lx of intensity of illumination.
4. disinfection by chlorine dioxide culture medium as described in claim 1 is quickly bred for potato seedling or Stem covered by vermiculite
Method, it is characterised in that: the described test tube potato induction be toxicity-removing white potato seedling is cut into 1 cm long with stipes
Toxicity-removing white potato Stem covered by vermiculite culture medium is inserted into stem section, morphology lower end；Condition of culture is 10 hour/day of illumination, when illumination
25 ± 1 DEG C of temperature, 6000 Lx of intensity of illumination, 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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| CN101822214A (en) * | 2009-10-28 | 2010-09-08 | 乐陵希森马铃薯产业集团有限公司 | Method for breeding minitype potato seeds by directly using potato stem segments |
| CN104285785A (en) * | 2013-07-19 | 2015-01-21 | 四川农业大学 | Microtuber induction method applicable to potato variety ''Chuanyu 10'' |
| CN106171970A (en) * | 2016-06-20 | 2016-12-07 | 淮北师范大学 | Rapid preparation method of plant culture medium with low phenol content |
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| CN101822214A (en) * | 2009-10-28 | 2010-09-08 | 乐陵希森马铃薯产业集团有限公司 | Method for breeding minitype potato seeds by directly using potato stem segments |
| CN104285785A (en) * | 2013-07-19 | 2015-01-21 | 四川农业大学 | Microtuber induction method applicable to potato variety ''Chuanyu 10'' |
| CN106171970A (en) * | 2016-06-20 | 2016-12-07 | 淮北师范大学 | Rapid preparation method of plant culture medium with low phenol content |
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