CN104938334B - A kind of method of Nano Silver sterilizing Mongolian oak fern spore - Google Patents
A kind of method of Nano Silver sterilizing Mongolian oak fern spore Download PDFInfo
- Publication number
- CN104938334B CN104938334B CN201510303444.7A CN201510303444A CN104938334B CN 104938334 B CN104938334 B CN 104938334B CN 201510303444 A CN201510303444 A CN 201510303444A CN 104938334 B CN104938334 B CN 104938334B
- Authority
- CN
- China
- Prior art keywords
- spore
- mongolian oak
- nano silver
- oak fern
- sterilized
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The present invention provides a kind of method that Nano Silver sterilizes Mongolian oak fern spore, comprises the following steps：By Mongolian oak fern spore aseptic water washing more than 1 time, sterilized 15 25 minutes with the Nano silver solution of 100 2000ppm, then sterilized 3 10 minutes with 5% liquor natrii hypochloritis, eventually pass aseptic water washing more than 2 times.The method of the present invention can cause that the spore of Mongolian oak fern had both reached optimal sterilizing state, and spore germination rate higher and relatively low abnormal rate are maintained again.
Description
Technical field
The invention belongs to plant tissue culture field, and in particular to a kind of suitable Mongolian oak fern (Drynaria fortunei (Knuze)
J.SM.) the sterilizing methods of sporogenous tissue's culture.
Background technology
Mongolian oak fern is that extremely distinctive one kind is grown nonparasitically upon another plant draft fern in Drynariaceae Mongolian oak Cyclosorus, and the root-like stock of its Mongolian oak fern is rich in shaddock ped
Glucoside (naringin), the chemical composition such as 21- hopenens (hop-21-ene) and oil recovery sterol (campesterol), with controlling tooth
Bitterly, the effect such as finnitus due to the kidney deficiency, deafness, alopecia areata is controlled.
In the production of Mongolian oak fern, its propagation method is an important problem, and traditional modes of reproduction of Mongolian oak fern is divided into plant division
Two kinds of breeding and sporogenesis, division propagation are very easy to the constraint to conditions such as season, planting environments, and plant division breeding potential
It is very low；Sporogenesis is readily available spore, and breeding coefficient is high, is the effective way of fast breeding seedling, but is also easily to receive
The influence of envirment factor；In the last few years, the research of various fern seed sterile culture, sterile culture are constantly launched both at home and abroad
Because having the features such as not influenceed and be efficient by extraneous factors such as environment, times, and widely applied to the production of plant with
In, in the sterile culture of Mongolian oak fern spore, sterilizing methods are a very crucial steps, sterilize it is improper easily cause culture in
Pollution, the influence for different sterilization methods to Mongolian oak fern spore sterile culture is studied, and is that the sterile culture of Mongolian oak fern is established
Theoretical foundation, and for it commercially produces offer technical support.
During the sterile culture of spore, fungi can only be had certain effect with the flushing of sterilized water, but will not kill
Dead bacterium, 70% alcohol is commonly used in order to effectively prevent the germ contamination during sterile culture, in the sterile culture of spore, is risen
Mercury, sodium hypochlorite (NaOCl), calcium hypochlorite Ca (OCl2) ﹒ 4H2The inorganic biocide such as O), the species of inorganic biocide, concentration and
Sterilization time is the study hotspot of spore sterile culture, and there are some researches show sodium hypochlorite concentration brings up to 5%, fungal contamination can subtract
It is few, but can produce the side effect to cause the germination rate to reduce, even if calcium hypochlorite degree reaches 5% does not interfere with spore germination, but very
Bacterium pollution is basic not to be reduced.Different types of spore also can be different to the sensitivity of different inorganic biocides, branch sand-grass
Through sodium hypochlorite, red streptomysin, sterilizing, the spore for only being sterilized with streptomysin can be sprouted fern, and the spore of ciliate desert-grass can but be used
Mercuric chloride sterilizes.Using sodium hypochlorite the sterile culture of Mongolian oak fern more, there are some researches show with 5% liquor natrii hypochloritis sterilize 5 minutes
Can cause that Mongolian oak fern reaches preferable sterilization effect in sterile culture, although also there are some researches show only with sodium hypochlorite sterilizing energy
It is enough effectively to reduce germ contamination of the spore in tissue cultures, but may there is no obvious effect to fungal contamination.
The sterilizing methods for being permitted various plants tissue cultures in the last few years have introduced the antiseptic of organic Nano grade, also
It is a kind of antibacterial agent to be developed with nanometer technology, the active oxygen ion that nano antibacterial agent is produced under light has
Very strong oxidability, can in a short time destroy the multiplication capacity of bacterium and make cell death, and general nano antibacterial agent is matched somebody with somebody
Side is by simple nontoxic metal or metal oxide, such as aluminium of nanocrystal types, zinc, magnesia, calcium oxide, oxidation
Aluminium, zinc oxide etc. are made, and carry active halogen such as MgO.Cl2And MgO.Br2(Olga, 2002), the powder of Nano grade
With formula to bacillus, the spore and the lethal effect of vegetative cell of viral and toxin, and it kills the size of ability
Related to its concentration and time of contact, research has confirmed that in phanerogamous tissue cultures nano-particle solution is more non-
Nano-solution can play stronger disinfective action, such as nano-TiO really2With non-nano TiO2But, it is the same with inorganic biocide,
Its Disinfection Effect also can be different because of explant species, such as the rye grass of water slurry culture, barley, the seed of flax, zeroth order
Nano-iron particle solution (nZVI) produces bacteriostasis in 250mg/L, and Nano silver solution (Ag) can just be produced in 10mg/L
Bacteriostasis；The stem of plant is higher than seed to the susceptibility of nano silver antimicrobials.Improper (the mistake of concentration of nano-particle solution
It is high) or side effect can be also played during process time improper (long), such as suppressing seed and sprout and root growth, active oxygen is formed,
Peroxidating is coerced, serious lipid peroxidation etc..
The sterilizing research of spore focuses mostly in inorganic biocide, but does not only often reach ideal with inorganic biocide sterilizing
Fungi sterilization effect, species is different also different to the sensitivity of bactericidal agent, and the sterilizing research of Mongolian oak fern spore is little.
Nano antibacterial agent is relatively common in the sterile culture of seed as a kind of new organic bactericidal agent, and
Applied in the sterile culture of several explants, but report is had no in the sterile culture of spore.Accordingly, it would be desirable to explore one kind
More efficiently Mongolian oak fern spore sterilizing methods.
The content of the invention
To solve the above problems, the present invention provides a kind of method that Nano Silver sterilizes Mongolian oak fern spore.
The method of the Nano Silver sterilizing Mongolian oak fern spore that the present invention is provided, comprises the following steps：By Mongolian oak fern spore sterilized water
Rinse more than 1 time, sterilized 15-25 minute with the Nano silver solution of 100-2000ppm, then with 5% liquor natrii hypochloritis sterilizing 3-
10 minutes, eventually pass aseptic water washing more than 2 times.
Preferably, Nano silver solution concentration is 300-800ppm.
It is furthermore preferred that Nano silver solution concentration is 500ppm.
In one embodiment of the invention, preferably sterilized 5-6 minutes with 5% liquor natrii hypochloritis.
In another embodiment of the invention, sterilization process is completed in sterile centrifugation tube.
In the present invention, the commercially available acquisition of Nano silver solution.
The present invention a kind of Mongolian oak fern spore aseptic seeding method is also provided, its be according to described in sterilizing methods sterilized,
It is seeded in aseptic 1/2MS culture mediums and cultivates.
The method of the present invention can cause that the spore of Mongolian oak fern had both reached optimal sterilizing state, spore be maintained again higher
Germination rate and relatively low abnormal rate.The inventive method is used in the tissue cultures of Mongolian oak fern spore, former germination rate is 89.5%
Spore, germination rate merely reduces 4.9% after this method, and the abnormal rate in growth course only has 17%, and also assures that
Spore is not contaminated in tissue cultures, and compared with other method, there is the inventive method with low cost, high efficiency to maintain spore
The characteristics of vigor.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
The tissue cultures sterilizing methods of the Mongolian oak fern of embodiment 1 (Drynaria fortunei (Knuze) J.SM.) spore.
Fill 4mg or so Mongolian oak fern spores respectively in 12 5ml centrifuge tubes, and with aseptic water washing 1 time after, respectively with 1%,
5%th, 10% liquor natrii hypochloritis respectively sterilizes 3,5,7,10 minutes.Aseptic water washing 3-4 is eventually passed after, is seeded in aseptic
1/2MS culture mediums in, be put in after sealing culturing room culture.Pollution level as 1 rank is represented with 1 "+", all pollutions are used
5 "+" represent that " 0 " represents pollution-free, pollution condition and the sprouting situation such as table 1 during Spore cultivation, and sterilizing concentration is got over
Low, sterilization time is fewer, pollutes more serious.Sterilizing concentration and sterilization time do not have in the culture of 10% and more than 3 minutes
Pollution, but compared with the germination rate of unpasteurized spore 89.5%, after the concentration that sterilizes reached for 5% time reaches 7 minutes, spore
The sprouting of son begins to be affected, and sterilizing concentration is 10%, and when sterilization time reaches 10 minutes, spore germination is by being influenceed
Most serious, germination rate only has 8.5%, and after the concentration that sterilizes reached for 5% time reaches 10 minutes, spore development is also subject to serious
Influence, abnormal rate is more than 36.6%.
During the hypochlorous acid acid sodium sterilizing different time of the various concentrations of table 1, pollution during Mongolian oak fern Spore cultivation, sprout and
Developmental state
The tissue cultures sterilizing methods of the Mongolian oak fern of embodiment 2 (Drynaria fortunei (Knuze) J.SM.) spore.
Fill 4mg or so Mongolian oak fern spores respectively in 6 5ml centrifuge tubes, and with aseptic water washing 1 time after, respectively with 100,
200th, 300,500,1000, the Nano silver solution of 2000ppm sterilizes 15-25 minute, after eventually passing aseptic water washing 3-4 times,
It is seeded in aseptic 1/2MS culture mediums, culturing room's culture is put in after sealing.Pollution condition and sprouting during Spore cultivation
Situation such as table 2, under various sterilizing concentration, culture has pollution, and sterilizing concentration more low stain is more serious, and sterilizing concentration exists
During 1000ppm, compared with 89.5% germination rate of the spore that do not sterilize is compareed, the sprouting of spore is also smaller by being influenceed, and sprouts
Hair rate is 85%, but the development of spore receives large effect, and abnormal rate reaches 28.8%, and sterilizing concentration reaches
During 2000ppm, the sprouting and development of spore all receive large effect, and germination rate is 77.4%, and abnormal rate is 30.6%.
When the Nano silver solution of the various concentrations of table 2 sterilizes, pollution, sprouting and developmental state during Mongolian oak fern Spore cultivation
| 0 | 100 | 200 | 300 | 500 | 1000 | 2000 | |
| Pollution level | +++++ | +++++ | +++++ | ++++ | +++ | ++ | ++ |
| Germination rate | 89.5% | 89.4% | 90.4% | 89% | 89.2% | 85% | 77.4% |
| Abnormal rate | 13.6% | 17.4% | 17% | 19% | 28.8% | 30.6% |
The tissue cultures sterilizing methods of the Mongolian oak fern of embodiment 3 (Drynaria fortunei (Knuze) J.SM.) spore.
Fill 4mg or so Mongolian oak fern spores respectively in 6 5ml centrifuge tubes, and with aseptic water washing 1 time after, first with 5% chlorine
Acid sodium solution sterilizes 5 minutes and is sterilized 15-25 minutes with the Nano silver solution of 100-2000ppm respectively again, eventually passes sterilized water
3-4 is rinsed after, is seeded in aseptic 1/2MS culture mediums, culturing room's culture is put in after sealing.Dirt during Spore cultivation
Dye situation and sprouting situation such as table 3, when Nano silver solution concentration is less than 500ppm, culture has pollution, and sterilize the lower dirt of concentration
Dye is more serious, and, in 500ppm and the above, culture is without pollution for Nano silver solution concentration.The spore for only being sterilized with sodium hypochlorite is sprouted
Hair rate is 86.8%, and abnormal rate is 21%, and after addition Nano silver solution, the sprouting and development of spore receive significant shadow
Ring, Nano silver solution concentration is higher, influence more serious, when Nano silver solution concentration is 100ppm, spore germination is by being influenceed
Minimum, germination rate is 34.4%, and abnormal rate is 42.2%, when Nano silver solution concentration is 2000ppm, the shadow that spore germination is subject to
Most serious are rung, germination rate is 13.4%, and abnormal rate is 46.8%.
Table 3 is first sterilized 5 minutes with 5% sodium hypochlorite, then when being sterilized 15-25 minutes with various concentrations Nano silver solution, Mongolian oak
Pollution, sprouting and developmental state during fern Spore cultivation
The tissue cultures sterilizing methods of the fern of embodiment 4 (Drynaria fortunei (Knuze) J.SM.) spore.
Fill 4mg or so Mongolian oak fern spores respectively in 6 5ml centrifuge tubes, and with aseptic water washing 1 time after, first use respectively
The Nano silver solution of 100-2000ppm sterilizes 15-25 minutes, then is sterilized 5-6 minutes with 5% liquor natrii hypochloritis, eventually passes
Aseptic water washing 3-4 is seeded in aseptic 1/2MS culture mediums after, and culturing room's culture is put in after sealing.Spore cultivation process
In pollution condition and sprouting situation such as table 4, when Nano silver solution concentration is less than 500ppm, culture has a pollution, and sterilize concentration
More low stain is more serious, and Nano silver solution concentration cultivates pollution-free in 500ppm and the above, when only being sterilized with sodium hypochlorite,
The germination rate of spore be 86.8%, abnormal rate is 21%, Nano silver solution concentration be 500ppm when, the sprouting and development of spore
It is not significantly affected, germination rate is 84.6%, abnormal rate is no appearance pollution in 17%, and incubation, Nano Silver is molten
When liquid reaches 1000ppm and the above, the sprouting and development of spore are influenceed by serious, and Nano silver solution concentration is
During 1000ppm and 2000ppm, germination rate only has 46.6% and 46.8% respectively, and abnormal rate is respectively 28% and 34.2%.
Table 4 is first sterilized 15-25 minutes with various concentrations Nano silver solution, then when being sterilized 5-6 minutes with 5% sodium hypochlorite,
Pollution, sprouting and developmental state during Mongolian oak fern Spore cultivation
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, on the premise of the technology of the present invention principle is not departed from, some improvements and modifications can also be made, these improvements and modifications
Also should be regarded as protection scope of the present invention.
Claims (3)
1. a kind of Nano Silver sterilizes the method for Mongolian oak fern spore, and it comprises the following steps：By Mongolian oak fern spore with aseptic water washing 1 time with
On, sterilized 15-25 minutes with the Nano silver solution of 500ppm, then sterilized 5-6 minutes with 5% liquor natrii hypochloritis, eventually pass nothing
Bacterium water is rinsed more than 2 times.
2. Nano Silver as claimed in claim 1 sterilizes the method for Mongolian oak fern spore, it is characterised in that sterilization process is in sterile centrifugation
Completed in pipe.
3. a kind of Mongolian oak fern spore aseptic seeding method, its method according to claim 1 and 2 is sterilized, and is seeded in nothing
Cultivated in the 1/2MS culture mediums of bacterium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510303444.7A CN104938334B (en) | 2015-06-05 | 2015-06-05 | A kind of method of Nano Silver sterilizing Mongolian oak fern spore |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510303444.7A CN104938334B (en) | 2015-06-05 | 2015-06-05 | A kind of method of Nano Silver sterilizing Mongolian oak fern spore |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104938334A CN104938334A (en) | 2015-09-30 |
| CN104938334B true CN104938334B (en) | 2017-06-16 |
Family
ID=54153880
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510303444.7A Active CN104938334B (en) | 2015-06-05 | 2015-06-05 | A kind of method of Nano Silver sterilizing Mongolian oak fern spore |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104938334B (en) |
-
2015
- 2015-06-05 CN CN201510303444.7A patent/CN104938334B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN104938334A (en) | 2015-09-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102077828A (en) | Compound disinfectant containing quaternary ammonium salt | |
| CN104982516B (en) | A kind of the green ecological preservation method and antistaling agent of citrus | |
| JP5299264B2 (en) | Soil reduction disinfection method, soil reduction disinfectant, soil wetting disinfection method, soil wetting disinfectant and soil disinfectant irrigation system | |
| CN107164262A (en) | A kind of microbial bacterial agent and its bacterial manure available for controlling crop diseases and insect pests | |
| CN101716358A (en) | Environment-friendly high-efficiency soil disinfection method | |
| WO2004100660A1 (en) | Antibacterial antiviral composition | |
| CN106234388B (en) | A kind of composition pesticide of alkene containing benzo fluorine bacterium azoles and jamaicin | |
| ES2717528T3 (en) | Method for the disinfection of agricultural soils | |
| JP2011140463A (en) | Technique for controlling plant disease using waste mushroom bed of edible mushroom | |
| KR20140065566A (en) | Composition containing slightly acidic hypochlorous water for plant diseases and method for controlling plant diseases using the same | |
| CN104938334B (en) | A kind of method of Nano Silver sterilizing Mongolian oak fern spore | |
| CN103858917A (en) | Mildew-resistant liquid used for seeds | |
| Nacheva et al. | Silver nitrate and chlorhexidine gluconate–effective surface sterilization agents in disinfection procedures at initiation of woody shoot tip and embryo culture | |
| IL271831A (en) | Method for disinfecting soils or other agricultural growing media | |
| CN109221223A (en) | A kind of safe and efficient composite disinfectant | |
| CN205249758U (en) | Seed degassing unit | |
| CN107251902A (en) | The composition of dibromo malonamide and its purposes as biocide | |
| CN106070359A (en) | A kind of plant seed germination box sterilization coating agent | |
| Madhuri et al. | Effect of chemical sterilants on surface sterilization of flower stalk during in vitro propagation of Phalaenopsis hybrids cv. Shagan | |
| CN106172414B (en) | A kind of bactericidal composition of the fluorine bacterium azoles of alkene containing benzo | |
| CN105660694A (en) | Bacterial fruit blotch seed disinfectant and application thereof | |
| JP2022036484A (en) | Growth activating liquid for plant seedling rearing | |
| CN114504669A (en) | Safe disinfection equipment and disinfection method for soilless culture substrate | |
| CN105028454B (en) | A kind of antibacterial disinfectant | |
| CN112369325A (en) | Safe, low-toxicity and high-efficiency sterilization method for plant explants |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant |