CN101933459A - Method for disinfecting explant in drepanostachyum luodianense tissue culture process - Google Patents
Method for disinfecting explant in drepanostachyum luodianense tissue culture process Download PDFInfo
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Abstract
The invention discloses a method for disinfecting an explant in a drepanostachyum luodianense tissue culture process, which comprises the following steps of: (1) totally soaking a vigorous semi-lignified branch, serving as the explant, of drepanostachyum luodianense into 1 percent detergent solution for 30 minutes; (2) soaking the explant into tap water for 30 minutes; (3) flushing the explant by using running water till no foam is generated, and sucking the moisture on the surface of the explant by using filter paper; (4) soaking the flushed explant into 70 to 75 percent alcohol to perform disinfection for 1 minute on a clean bench; (5) soaking the explant into sterile water and flushing the explant for 3 to 4 times with continuous shaking; (6) finally soaking the explant into 0.1 percent mercury bichloride or 2 percent sodium hypochlorite solution to perform disinfection for 6 to 12 minutes with continuous shaking; and (7) flushing the explant by using the sterile water, and sucking the moisture on the surface of the explant by using the sterile filter paper so that the explant can be used of inoculation. The method fills the blank of quick reproduction of drepanostachyum luodianense tissue culture, lays a quick and sufficient foundation for garden planting, large-area greening and karst rocky desertification recovery of the drepanostachyum luodianense, and provides a feasible scheme for factory large-scale production of the drepanostachyum luodianense.
Description
Technical field
The invention belongs to bamboo class tissue culture technique, specifically relate to the sterilization method of explant in a kind of drepanostachyum luodianense tissue culture procedures.
Background technology
Drepanostachyum luodianense (Drepanostachyum Luodianense (Yi et R.S.Wang) Keng f.) is a grass family Bambusoideae sickle preface Sinobambusa bamboo kind, has another name called " rattan bamboo ", is Chinese endemic species, is listed in critical species in " the red register of Chinese species ".Cultivation is only arranged in Luodian, Pingtang and the Changshun in Guizhou at present or wild distribution is arranged, often grow in flakes on the exposed tor of limestone of height above sea level 600～1000m.Pliable and tough cool chair and the farm implements compiled of its bamboo wood also can be used as paper making raw material; The form grace, it is good afforestation bamboo kind, and at the abominable karst well-grown of environment, can be drought-resistant and barren, solid soil, water conservation, fertilizer-preserving ability effect to enhancing soil are very remarkable, be the suitable natural disposition species of karst, in the karst ecosystem, ecological functions such as water source self-restraint, water and soil conservation, nutrient balance brought into play important effect, have very high ecological benefits.
The cyclostage of bamboo class is long, seldom blossoms and bears fruit, and is difficult to obtain the seed of large-scale breeding.And traditional move bamboo, bury whip, there are the problems such as kind of bamboo is many, the seedling transportation is inconvenient, labour intensity is big, reproduction coefficient is low that consume in propagation method such as bamboo branch cuttage.Adopt the method efficient height of tissue culture, condition is controlled, and growth is fast, and the cycle is short, and repeatability is strong, can carry out advantages such as anniversary test or production, can breed seedling fast, is fit to batch production hundred seedlings of bamboo.
Up to now, bamboo tissue culture work has both at home and abroad obtained very big progress, but exists many problems, how to pass through the pollution rate that effective sterilization method reduces explant, needs the problem that at first solves exactly.Compare with other plant, the bamboo explant sterilize this link comparatively the difficulty, be difficult to the sterilization effect that reaches desirable.This specific form structure with bamboo plant is relevant.The epidermal cell of bamboo plant has fine and close mastoid process and thicker cuticula, and the spore of embedding adhered dust and various mushrooms etc. between the material of mastoid process surface wax shape and mastoid process has all hindered the infiltration of thimerosal, thereby influenced the abundant sterilization of explant.If select for use bar bud, twig to do explant, the bamboo stalk that hollow is hard also causes explant cutting difficulty, and bacterium and fungal spore enter bamboo stalk interior tissue easily from the hollow part, cause follow-up pollution.When explant was sterilized, the control of disinfecting time also was a major issue, and the time, too weak point can not killing bacteria virus, and the time is oversize also can to damage plant in kill pathogens, causes brown stain, influenced inducing and breeding of explant.Therefore, seek effective sterilizing methods, reduce abandoning of pollution bottle, for reducing the seedling production cost and realizing that batch production production tool has very important significance.
At present domestic to drepanostachyum luodianense research maximum be drepanostachyum luodianense structure of community, diversity, water and soil conservation function, soil seed bank, soil seed pool, leaf area index with and field such as culture technique research applications, and aspect tissue culture especially the research of explant sterilization method still be blank.
Summary of the invention
Technical problem to be solved by this invention provides an important step in a kind of drepanostachyum luodianense tissue culture procedures, and promptly the sterilization method of explant is bred the low problem of survival rate in difficulty and the tissue culture procedures to overcome drepanostachyum luodianense.
Plant Tissue Breeding is usually according to the following steps:
One, culture medium preparation and sterilization: it is general MS medium that the present invention tests used medium, must sterilize in 24h in medium preparation back, usually the sterilizing methods that adopts is an autoclaving, its principle is by heating, in airtight pressure cooker along with the continuous rising of water vapour pressure, the boiling point of water is improved constantly, thereby kettle temperature also increase.
Two, the processing of explant and sterilization
The target of sterilization is: should eliminate the germ on the material, not damage vegetable material again.
Three, the inoculation of explant
The sterile working equipment that uses in the sterile working laboratory is superclean bench.Should carry out the housekeeping on ground before the inoculation, spray transfer room with the spraying of 75% ethanol, wiping superclean bench table top, culture dish, alcolhol burner and other instruments also will be placed on the superclean bench in advance, and one reinstates ultra violet lamp 20min.To wash one's hands with wipes of alcohol before the operation, will wash one's hands through wipes of alcohol commonly used in the operation.Inaccurate speech is also inaccurately breathed facing to the operating space, so as not in the oral cavity with microbial contamination material, medium and apparatus.Can adopt 250 or the wide-mouth bottle of 500mL in the operation, put into 95% alcohol, so that operation tool is carried out disinfection.When sterile working, in the alcohol of immersions 95% such as tweezers, scissors, scalpel, take out calcination sterilization on alcolhol burner flame before using.The calcination inoculating tool is opened the medium bottleneck after cooling near alcolhol burner, immediately the explant of being got is inserted in the solid culture medium rapidly.When inoculation, can not move and want quick with the scalpel that gets rusty, with cutting, reduce the time that wound exposes in air with connecing.Bottleneck should be burnt on flame before the lid bottle cap, again lid also be burnt on flame, cover then.Again operation all will be sterilized instrument on flame at every turn.
Four, Plant Tissue Breeding:
Condition of culture is: intensity of illumination 1200Lx, illumination every day 12h, 25～27 ℃ of temperature.Observe its growth and pollution condition every day from having inoculated, write down the result after 30 days, add up it and pollute number, survive number, analyze and draw preferred plan.
Preparation, sterile working and the artificial culture environment of explant depended in the success or not of Plant Tissue Breeding.Can the preparation of explant be build up first pass that in-vitro propagate system wanted.So-called explant is meant the activated sterile tissue or the cell of cell of extraneous growing plants and a series of aseptic process formation of tissue process, gathers and processes suitable explant according to the different materials in the work.Sterilization is an important step of Plant Tissue Breeding, as the explant material of plant, all has a lot of bacteriums and mould regardless of inner still outside under field conditions (factors).Therefore for explant can be grown under aseptic condition, sterilized into necessary procedure of tissue culture.The target of sterilization is: should eliminate the germ on the material, not damage vegetable material again.Therefore to consider the killing effect of medicament on the one hand, therefrom select the most effective bactericide all kinds of mushrooms; On the other hand, also should consider the endurance of vegetable material to bactericide.Use what medicament, processing time, test according to vegetable material kind and the position of taking.
The present invention on the basis of experiment, proposes the sterilization method of drepanostachyum luodianense explant according to the characteristics of drepanostachyum luodianense, has selected the most effective bactericide, and according to the drepanostachyum luodianense material patience of bactericide has been tested out drug concentration and processing time.
The sterilization method of explant of the present invention, its disinfection process follow these steps to carry out: (1) makes explant with the semi-lignified branch of drepanostachyum luodianense robust growth, soaks 30min fully with 1% liquid detergent solution, and (2) soak 30min with running water; (3) with flowing water wash produce to non-foam till, blot surface moisture with filter paper; (4) on superclean bench, the explant of having rinsed well is immersed the 1min that sterilizes in the 70-75% alcohol; (5) constantly shake flushing 3～4 times with the sterile water immersion; (6) immerse 0.1% mercuric chloride or the 2% liquor natrii hypochloritis 6-12min that sterilizes at last, constantly shake therebetween; (7) then with behind the aseptic water washing, blot surface moisture, promptly can be used for inoculation with aseptic filter paper.
Explant generally uses concentration of alcohol 70%～75%.The ethanol of this concentration range has stronger penetration power and sterilizing power.When utilizing the Ethanol Treatment material, generally be no more than 30s.Use the Ethanol Treatment explant, except the effect that the sterilization of explant body surface is arranged, also have the effect of invade tissues material.The explant of many plants all needs the infiltration of elder generation by ethanol, and then uses other medicines to do further sterilization.Because the bamboo plant epidermal cell has very fine and close mastoid process, the spore embedding of dust and various mushrooms in intermastoid slit.There is the material of one deck wax shape on the mastoid process surface, and epidermal cell also has thicker cuticula, and these have all hindered the infiltration of thimerosal.Therefore before carrying out disinfection, also want to soak with liquid detergent earlier, the inside wax shape material is dissolved with alcohol.And arrive 1min invade tissues well with the ethanol disinfection time lengthening.
It is 0.1% that the present invention uses mercuric chloride concentration, and proposes the processing time on experiment basis.Material through mercuric chloride was handled because the residual meeting of mercuric chloride in the outer planting surface causes the necrosis of explant in incubation, even makes the cultivation failure.So must be after mercuric chloride be handled, clean with aseptic water washing, generally to wash more than 5 times, the residual mercuric chloride that just can guarantee the explant surface is seldom.
The present invention proposes the processing time equally with 2% clorox on experiment basis, the explant after the processing will be used aseptic water washing 3～4 times equally.
Experimental results show that when step (5) alcohol concentration is 75% step (6) sterilization treatment is handled explant 10min with 0.1% mercuric chloride, in the MS medium, inoculation survival rate is the highest, is below or above 10min, and inoculation survival rate all can reduce.
When step (5) alcohol concentration was 75%, step (6) sterilization treatment was handled explant 10min with 2% clorox, and in the MS medium, inoculation survival rate is the highest, is below or above 10min, and inoculation survival rate all can reduce.
The invention has the beneficial effects as follows and solved drepanostachyum luodianense breeding difficulty, especially tissue culture procedures explant sterilization, the problem of inducing, the explant pollution rate is low, can drop to 13.3%, the untainted explant survival rate in inoculation back reaches 73.3%, having overcome traditional female bamboo transplants, propagation method consumes resources such as bamboo whip transplanting, big to environmental destruction, the cultivation time is long, efficient is low, shortcomings such as cost of transportation height, and be not subjected to the restriction in season, be large-area popularization of drepanostachyum luodianense and utilization, giving full play to its ecological benefits and economic benefit provides fast and the effective way of sufficient supplies.
Below further specify beneficial effect of the present invention by embodiment.
Embodiment
Below in conjunction with preferred embodiment, the sterilization method of the drepanostachyum luodianense tissue culture sterile system that foundation the present invention is proposed, embodiment, feature and effect thereof, describe in detail as after.
(1) test material: for the material of examination pick up from from the Luodian transplant in the Guizhou University nursery, survived and drepanostachyum luodianense plant robust growth, explant is selected the full semi-lignified branch of annual shoot bud for use.
(2) test method
1, the sterilization of medium
Select normally used MS medium for use, must sterilize in 24h in medium preparation back, usually the sterilizing methods that adopts is an autoclaving, its principle is by heating, in airtight pressure cooker along with the continuous rising of water vapour pressure, the boiling point of water is improved constantly, thereby kettle temperature also increase.The article that at first will need to sterilize are put into sterilizing installation, close vent valve, after the energising, when treating that pressure rises to 0.05MPa, open vent valve, deflate, treat that gauge hand makes zero after, close vent valve again.Along with pressure constantly rises, the boiling point of water improves constantly, thereby kettle temperature also increases thereupon.Manometer rises when reaching 0.1～0.15Mpa, and kettle temperature can reach 121 ℃.Under this vapor (steam) temperature, the branch spore of various bacteriums and highly heat-resistant thereof is killed.Stay-in-grade article such as sterile water, cultivation medium, inoculating appliance all can prolong sterilization time as one sees fit or improve pressure behind the autoclaving, to reach good bactericidal effect.But medium must strictly observe the dwell time, and is should pressurize thorough, prevents that again composition in the medium is rotten or renders a service and reduce, arbitrarily time expand.The general high voltage sterilization is kept 20～30min and is got final product.
2, the processing of explant and sterilization
The drepanostachyum luodianense explant adopts the semi-lignified branch of robust growth, soaks 30min fully with 1% liquid detergent solution, soaks 30min with running water then, and flowing water washes to the non-foam generation, blots surface moisture with filter paper.On superclean bench, the explant of having rinsed well is immersed the 1min that sterilizes in 75% alcohol, adopt one of two kinds of thimerosals 0.1% mercuric chloride or 2% liquor natrii hypochloritis to handle explant with the inferior back of aseptic water washing 3～4 (during constantly shake).Every kind of thimerosal adopts different disinfecting time to handle:
1. the sterilization of 0.1% mercuric chloride is 6 minutes; 2. the sterilization of 0.1% mercuric chloride is 8 minutes; 3. the sterilization of 0.1% mercuric chloride is 10 minutes; 4. the sterilization of 0.1% mercuric chloride is 12 minutes; 5. the sterilization of 2% clorox is 6 minutes; 6. the sterilization of 2% clorox is 8 minutes; 7. the sterilization of 2% clorox is 10 minutes; 8. 2% clorox sterilization 12 minutes (during constantly shake).Use aseptic water washing then 5 times (each 4 minutes, during constantly shake), blot surface moisture with aseptic filter paper.The explant clip that disinfects is about the joint branch that 1.5cm is with bud, explant is inoculated on the medium, disinfect 10 of inoculations for every kind, repeat 3 times, survival rate is averaged.
3, the inoculation of explant
The sterile working equipment that uses in the sterile working laboratory is superclean bench.Should carry out the housekeeping on ground before the inoculation, spray transfer room with the spraying of 75% ethanol, wiping superclean bench table top, culture dish, alcolhol burner and other instruments also will be placed on the superclean bench in advance, and one reinstates ultra violet lamp 20min.To wash one's hands with wipes of alcohol before the operation, will wash one's hands through wipes of alcohol commonly used in the operation.Inaccurate speech is also inaccurately breathed facing to the operating space, so as not in the oral cavity with microbial contamination material, medium and apparatus.Can adopt 250 or the wide-mouth bottle of 500mL in the operation, put into 95% alcohol, so that operation tool is carried out disinfection.When sterile working, in the alcohol of immersions 95% such as tweezers, scissors, scalpel, take out calcination sterilization on alcolhol burner flame before using.The calcination inoculating tool is opened the medium bottleneck after cooling near alcolhol burner, immediately the explant of being got is inserted in the solid culture medium rapidly.When inoculation, can not move and want quick with the scalpel that gets rusty, with cutting, reduce the time that wound exposes in air with connecing.Bottleneck should be burnt on flame before the lid bottle cap, again lid also be burnt on flame, cover then.Again operation all will be sterilized instrument on flame at every turn.
4, condition of culture:
The factors such as the main and illumination of artificial culture environment, temperature, osmotic pressure, acid-base value, aeration status have confidential relation.The artificial culture environment is the self-sow environment of simulating plant, i.e. illumination, temp. and humidity and soil.Tissue culture is carried out under scattered beam usually.The explant material of handling well is inoculated on the ready medium cultivates.Condition of culture is: intensity of illumination 1200Lx, illumination every day 12h, 25～27 ℃ of temperature.Observe its growth and pollution condition every day from having inoculated, write down the result after 30 days, add up it and pollute number, survive number, analyze and draw preferred plan.
(3) viewing test phenomenon and interpretation of result, experimental result such as table 1 (explant disinfection treatment method and cultivation results), as can be seen from Table 1: different disinfectants and the pollution rate and survival rate generation different influence of disinfecting time to explant.Mercuric chloride is handled the prolongation along with the time, and pollution rate presents reduction trend, and when handling 12min, pollution rate is minimum, and survival rate then in time prolongation presents trend of rising, and when handling 10min, survival rate is the highest, continue time expand then survival rate descend; Also along with the prolongation of time, pollution rate descends in the clorox processing, and pollution rate is minimum during 12min, and survival rate prolongation in time raises, and when handling 10min, survival rate is the highest.Relatively, same treatment is in the time between two kinds of disinfectants, and mercuric chloride is handled pollution rate and handled lowly than clorox, and mercuric chloride is processed into motility rate and is processed into the motility rate height than clorox, and the Disinfection Effect of mercuric chloride totally is better than clorox.Take all factors into consideration as above result of the test as can be known, the suitable sterilization method of drepanostachyum luodianense explant is that 0.1% mercuric chloride in above-mentioned 8 kinds of processing is handled 10min and 0.1% mercuric chloride is handled 12min, not only pollution rate is lower and 0.1% mercuric chloride is handled 10min, and survival rate is the highest, therefore, the optimum sterilization method of drepanostachyum luodianense explant is that 0.1% mercuric chloride is handled 10min.
Table 1 explant disinfection treatment method and cultivation results
" MS medium " of the present invention is meant the medium of Murashige and Skoog development, and its component prescription sees Table 2.
Table 2MS medium component
The above, it only is preferred embodiment of the present invention, be not that the present invention is done any pro forma restriction, any technical solution of the present invention content that do not break away from,, all still belong in the scope of technical solution of the present invention any simple modification, equivalent variations and modification that above embodiment did according to technical spirit of the present invention.
Claims (3)
1. the sterilization method of explant in the drepanostachyum luodianense tissue culture procedures, it is characterized in that: disinfection process follows these steps to carry out, (1) semi-lignified branch of drepanostachyum luodianense robust growth is made explant, soak 30min fully with 1% liquid detergent solution, (2) soak 30min with running water; (3) with flowing water wash produce to non-foam till, blot surface moisture with filter paper; (4) on superclean bench, the explant of having rinsed well is immersed the 1min that sterilizes in the 70-75% alcohol; (5) constantly shake flushing 3～4 times with the sterile water immersion; (6) invade 0.1% mercuric chloride or the 2% liquor natrii hypochloritis 6-12min that sterilizes at last, constantly shake therebetween; (7) then with behind the aseptic water washing, blot surface moisture, promptly can be used for inoculation with aseptic filter paper.
2. sterilization method as claimed in claim 1 is characterized in that: step (5) alcohol concentration is 75% o'clock, and step (6) sterilization treatment is handled explant 10min with 0.1% mercuric chloride.
3. sterilization method as claimed in claim 1 is characterized in that: step (5) alcohol concentration is 75% o'clock, and step (6) sterilization treatment is handled explant 10min with 2% clorox.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102972176A (en) * | 2012-11-29 | 2013-03-20 | 广西壮族自治区林业科学研究院 | Method for obtaining explants for tissue culture of bamboo |
| CN103314848A (en) * | 2013-05-29 | 2013-09-25 | 南京林业大学 | Rapidly breeding method for ampelocalamus luodianensis |
| CN108651279A (en) * | 2017-04-01 | 2018-10-16 | 国际竹藤中心 | A kind of embryo sterilization method for Bamboo Tissue culture |
| CN114097609A (en) * | 2021-10-21 | 2022-03-01 | 云南省热带作物科学研究所 | Explant sterilization method in plant tissue culture |
-
2010
- 2010-09-01 CN CN 201010268792 patent/CN101933459A/en active Pending
Non-Patent Citations (1)
| Title |
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| 《云南农业科技》 20081231 李云海等 孝顺竹外植体适宜消毒方法及丛芽诱导培养基的筛选 18-19 1-3 , 第2期 2 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102972176A (en) * | 2012-11-29 | 2013-03-20 | 广西壮族自治区林业科学研究院 | Method for obtaining explants for tissue culture of bamboo |
| CN103314848A (en) * | 2013-05-29 | 2013-09-25 | 南京林业大学 | Rapidly breeding method for ampelocalamus luodianensis |
| CN103314848B (en) * | 2013-05-29 | 2014-12-10 | 南京林业大学 | Rapidly breeding method for ampelocalamus luodianensis |
| CN108651279A (en) * | 2017-04-01 | 2018-10-16 | 国际竹藤中心 | A kind of embryo sterilization method for Bamboo Tissue culture |
| CN108651279B (en) * | 2017-04-01 | 2021-10-22 | 国际竹藤中心 | Embryo disinfection method for bamboo tissue culture |
| CN114097609A (en) * | 2021-10-21 | 2022-03-01 | 云南省热带作物科学研究所 | Explant sterilization method in plant tissue culture |
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Open date: 20110105 |