CN104885941B - Chemical disinfection tissue culture method for miscanthus giganteus - Google Patents
Chemical disinfection tissue culture method for miscanthus giganteus Download PDFInfo
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Abstract
The invention relates to a chemical disinfection tissue culture method for miscanthus giganteus. The method comprises the following steps: culturing container disinfection, medium preparation, callus induction culturing, differentiation culturing, rooting culturing, and bottle seedling transplanting. According to the method, chemical reagents are used for achieving the aim of bacteria killing or bacteria inhibition; in the process of preparing culture media, culture containers and the culture media do not need to be subjected to high-temperature and high-pressure disinfection, so that the work amount and the energy consumption can be reduced, and the step of miscanthus giganteus tissue culture is simplified; the operation is easy as the media are prepared according to different media formulas; the practicability is high, the generalization performance is high, and the tissue culture cost of miscanthus giganteus can be effectively reduced and can be reduced by 10% or more generally.
Description
Technical field
The present invention relates to a kind of method for plant tissue culture, be specifically related to a kind of huge awns chemical disinfection tissue culture method, belong to raw
Thing technical field.
Background technology
Huge awns (Miscanthus giganteus) is also expressed one's surprise, wonder, etc hilllock, is that grass family awns belongs to perennial C4 dogstail, plant height
Up to 4m so being called huge awns.Originate in Japan, introduce Denmark the forties, after by European multiple countries, the U.S. introduce conduct
Energy-source plant research plantation.Recent year many units include that China agricultural university, the Chinese Academy of Sciences etc. successively introduces as energy-source plant
Study.Why huge awns is considered as most potential energy crop in Europe, the U.S., and being endowed that solution is following can the energy
With the star of hope of environmental problem, it is mainly characterized in that: 1, for C4 plant, has specular removal, low breathing, CO2 compensation point contour
Produce the ideal characterisitics of crop；2, perennial herb, plantation in a year, 12-20 can be gathered in the crops continuously；3, yield is high, and dry produces
Amount reaches 12-20 ton/mu.Other crops all it are significantly higher than in European, the U.S. and domestic etc. tests；4, after branch and leaf maturation, internal
Main nutritional labeling will be back to root, can save fertilizer, and energy input and output specific efficiency is that Semen Tritici aestivi, Semen Maydis etc. are mainly made
3-5 times of thing；5, easy firing and ethanol conversion are up to 50%.Miscanthus not only has the highest yield of biomass, and it is received
When obtaining, moisture only has 20% one 30%, and low moisture content is conducive to burning.Additionally, its volatile material is 3 times of coal,
It has more more preferable Ignition Stability than coal；6, strong stress resistance, drought-enduring, tolerance to cold is significantly stronger than chief crop.And disease pest
Evil occurs few., in recent years, European Countries the most extensively plants huge awns, mainly by its combustion power generation.Miscanthus generated energy accounts at present
15 EU Countries always produces the 9% of electricity, and wherein Ireland Miscanthus produces electricity and accounts for the whole nation and produce the 37% of electricity total amount, for EU countries
High.The U.S. is also at Illinois state and Michigan state field planting.Owing to huge awns is less demanding to natural conditions, can be in limit
Soil aerial also obtains high yield, utilizes it to carry out production of energy and not only will not account for arable land and be also fully utilized by limit soil
Ground is huge in application in future China.The ground such as current domestic existing Zhejiang, Fujian, Guangxi, Shandong are planted the most on a small quantity.
Owing to huge awns belongs to triploid, the seed that can not educate.Only with its underground rhizome as seed on Sheng Chaning.But with
Its underground rhizome is as seedling, and its breeding coefficient is extremely low, meanwhile, plants caulome and amasss big, and transport is not only inconvenient, and easy damaged.This
Become the biggest obstacle of implantation in large scale.At present, best solution utilizes tissue culture technique to produce seedling in a large number exactly,
But the key that tissue culture produces seedling is again to reduce cost.The links of tissue culture is both needed to configure corresponding facility and sets
Standby and conventional consumptive material, relatively costly, energy resource consumption is relatively big, and the most whole flow process aseptically to operate, and manual operation becomes
This height.If can be transformed by culture medium, it is thus achieved that both ensured the normal growth of tissue cultured seedling, there is again sterilization, antibacterial or antibacterial merit
The culture medium of energy, so can significantly save the production costs such as tissue cultured seedling, is that the production application cost of solution Seedling in huge Grain in Ear is high
A difficult problem.
Summary of the invention
It is an object of the invention to provide a kind of huge awns chemical disinfection tissue culture method.
It is an object of the invention to be realized by the following method.
The one huge awns chemical disinfection tissue culture method of the present invention, including culture vessel sterilization, culture medium preparation, callus
Inducing culture, differentiation culture, root culture and bottle transplantation of seedlings, it is characterised in that:
1. culture vessel sterilization: by the stainless steel disc of culture bottle, bottle cap and inoculation at 100mg/L sodium hypochlorite+10mg/
The aqueous solution of L nisin+100mg/L calcium propionate soaks no less than 10h, saves backup；
2. culture medium preparation: callus inducing medium is MS+2～5mg/L 2,4-D+0.5～2g/L AC+50～
100mg/L sodium hypochlorite+50～100mg/L Ticarcillin/Clavulanate Acid+0.1～0.5mg/L dodecyl sodium sulfate+30g/L sucrose+3.5g/L
Agar powder, pH5.8；Division culture medium is: MS+3～8mg/L 6-BA+0.1～0.5mg/L NAA+50～100mg/L hypochlorous acid
Sodium+50～100mg/L Ticarcillin/Clavulanate Acid+0.1～0.5mg/L dodecyl sodium sulfate+30g/L sucrose+3.5g/L agar powder, pH5.8；
Root media is 1/2MS+0.1～0.5mg/L NAA+50～100mg/L sodium hypochlorite+50～100mg/L Ticarcillin/Clavulanate Acid+0.1
～0.5mg/L dodecyl sodium sulfate+30g/L sucrose+3.5g/L agar powder, pH5.8；Described MS culture medium is 1962,
MS culture medium disclosed in Murashige and Skoog；Described 6-BA, refers to the 6-fast cry of certain animals of benzyl amino；Described NAA, refers to-naphthalene acetic acid；Institute
State AC, refer to activated carbon；During preparation culture medium, first claim sucrose and agar powder in each culture medium prescription, add culture medium cumulative volume 1/2
Tap water, is heated to agar powder and is completely dissolved, then after assorting other raw materials by each culture medium prescription, constant volume, then use 1mol/L
NaOH or 1mol/L HCl adjust pH value to 5.8, be dispensed in the culture vessel sterilized, sealing, standby after cooled and solidified；
3. the boot leaf phase of induction of callus: Yu Jumang, intercept a length of 10-20cm of children's fringe, remove outer layer bud
Leaf, only stays a piece of internal lobe；First with the ethanol surface sterilization 1min that volume ratio is 70%, distilled water cleans 3 times, then peels off with tweezers
Internal lobe, volume ratio is the hypochlorite disinfectant 10min of 2%, aseptic water washing 3-4 time.Children's fringe is cut into by superclean bench
The segment of 2mm, is placed on callus inducing medium cultivation, transfers weekly once；Culturing room's temperature is 25～28 DEG C, secretly trains
Support；Forwarding illumination cultivation after 4 weeks to, intensity of illumination is 1500～2000lx；
4. differentiation culture: the part yellow green callus that picking inducing culture obtains, is inoculated in division culture medium, training
Supporting and within 4 weeks, become the seedling not having root, every 30d transfers once；Culturing room's temperature is 26 DEG C, illumination 12h, intensity of illumination be 2000～
3000lx；
5. root culture: differentiation culture is not had root seedling, is inoculated on root media, has become after cultivating 25d
Whole plant；Culturing room's temperature is 25 DEG C, illumination 16h, and intensity of illumination is 2000～3000lx；
6. bottle transplantation of seedlings: the whole plant obtained by root culture takes out, and cleans the culture medium of root, with more than 800 times
Bacterium spirit is transplanted to the mold of booth after soaking 30min, and booth upper strata shading net hides, and the ambient temperature of transplanting is 22
～28 DEG C.
The application effect of the present invention:
Due to the chemical disinfection tissue culture method of the present invention, it is to utilize chemical reagent to add in various culture medium, reaches to go out
Bacterium or antibacterial effect.So, the culture medium of preparation is without autoclave sterilization.Therefore, huge awns callus induction is trained
Support, differentiation culture, the impact in each stage of root culture, in addition to considering the evaluation index commonly used, it is also contemplated that pollution rate
Problem.
1. induction of callus: the chemical disinfection tissue culture method of a kind of huge awns of the present invention and the training of conventional huge awns group
Method is compared, survival rate, pollution rate and the mortality rate that the outer implant in the induction of callus stage is cultivated, as shown in table 1,
All there is no too big difference.
The impact on huge Caulis Miscanthi sinensis point inducing culture of the chemical disinfection tissue culture method of table 1 present invention
2. differentiation culture: from table 2 it can be seen that every callus of the huge awns chemical disinfection tissue culture method of the present invention is put down
All differentiation Seedling numbers are apparently higher than conventional huge awns tissue culture method, and pollution rate is lower, and bottle Seedling is the most strong, greener.
The chemical disinfection tissue culture method of table 2 present invention is on huge awns proliferation times and the impact of pollution rate
| Tissue culture mode | Average division Seedling number/ | Average pollution rate (%) | Upgrowth situation |
| Conventional tissue culture method | 5.6 | 0.3 | Seedling is light green, weak |
| Chemical disinfection tissue culture method | 5.8 | 0.1 | Miao Lv, strong |
3. the chemical disinfection tissue culture method of the present invention is on huge awns rooting rate and the impact of pollution rate: in rooting process, this
The chemical disinfection tissue culture method of invention is compared with conventional huge awns tissue culture method, and result is as shown in table 3, and rooting rate and pollution rate are all
There is no too big difference；In terms of the upgrowth situation of bottle Seedling, by the method for the present invention, huge awns tissue cultured seedling stem is thicker, and blade is bigger, leaf color
Dark green.
The huge awns rooting situation comparative result that the chemical disinfection group training of table 3 present invention is trained with conventional group
| Tissue culture mode | Average rooting rate | Average pollution rate | Upgrowth situation |
| (%) | (%) | ||
| Conventional tissue culture method | 100 | 0.3 | Stem is thin, and blade is little, and leaf color is green |
| Chemical disinfection tissue culture method | 100 | 0 | Stem is thick, and blade is big, Ye Senong Green |
4. cost analysis: the chemical disinfection tissue culture method of the present invention is shown in Table 4 with the cost analysis of conventional group training.
The chemical disinfection tissue culture method of the present invention eliminates autoclaving link, simplifies group training program, improves work
Efficiency.Calculate from the expense that can directly calculate, about 50,000 yuan can be saved every year and (prepare in terms of 50L culture medium by every day
Calculate).It addition, also have some to be not easy the project calculated, as pressure cooker keeps in repair, safety, saving space etc.:
The huge awns simplicity tissue culture method of table 4 present invention is cultivated and the cost analysis table of conventional group training
Owing to the culture medium of the present invention is made without autoclave sterilization, the cultivation of huge elongated tissue on the one hand can be reduced right
The requirement of facilities and equipment, especially need not the pressure cooker equipment of the required configuration of autoclave sterilization, has saved cost of investment, electric energy
Consume and also will be greatly lowered；On the other hand, using this culture medium to carry out huge awns virus-elimination seedlings and produce, group training link is the simplest
Changing, manually-operated speed is greatly improved, thus reduces cost of labor.Additionally, due to improved culture medium has self
Sterilization, antibacterial or bacteriostasis, can effectively control the pollution problem in tissue culture procedures, the growth of huge awns will not be produced poison again
Evil or suppression, the most do not affect the normal growth of huge awns, radically simplify huge elongated tissue and cultivates.
There is advantages that
1. in the huge awns chemical disinfection tissue culture method of the present invention, culture vessel and culture medium all without autoclave sterilization,
Decrease workload and energy resource consumption, simplify huge awns group training link, reduce huge awns group training cost.
2. the huge awns chemical disinfection tissue culture method of the present invention, simple to operate, as long as after by different culture medium prescription preparations
, practical, generalization is good.
3. the huge awns chemical disinfection tissue culture method of the present invention and the contrast of conventional huge awns tissue culture method, can reduce cost
More than 10%.
Detailed description of the invention
In order to be further elucidated with the present invention rather than limit the present invention, it is illustrated below in conjunction with embodiment.
Embodiment one: a kind of huge awns chemical disinfection tissue culture method
A kind of huge awns chemical disinfection tissue culture method, comprises the following steps:
1. culture vessel sterilization: by the stainless steel disc of culture bottle, bottle cap and inoculation at 100mg/L sodium hypochlorite+10mg/
The aqueous solution of L nisin+100mg/L calcium propionate soaks no less than 10h, saves backup；
2. culture medium preparation: callus inducing medium is MS+3mg/L 2,4-D+1g/L AC+70mg/L hypochlorous acid
Sodium+0.3mg/L+60mg/L Ticarcillin/Clavulanate Acid dodecyl sodium sulfate+30g/L sucrose+3.5g/L agar powder, pH5.8；Division culture medium
For: MS+5mg/L 6-BA+0.3mg/L NAA+70mg/L sodium hypochlorite+60mg/L Ticarcillin/Clavulanate Acid+0.3mg/L dodecyl sodium sulfonate
Sodium+30g/L sucrose+3.5g/L agar powder, pH5.8；Root media be 1/2MS+0.3mg/L NAA+70mg/L sodium hypochlorite+
60mg/L Ticarcillin/Clavulanate Acid+0.3mg/L dodecyl sodium sulfate+30g/L sucrose+3.5g/L agar powder, pH5.8；During preparation culture medium,
First claim sucrose and agar powder in each culture medium prescription, add the tap water of culture medium cumulative volume 1/2, be heated to agar powder the most molten
Solve, then after assorting other raw materials by each culture medium prescription, constant volume, then adjust pH value with the HCl of NaOH or 1mol/L of 1mol/L
To 5.8, it is dispensed in the culture vessel sterilized, sealing, standby after cooled and solidified；
3. the boot leaf phase of induction of callus: Yu Jumang, intercept a length of 10-20cm of children's fringe, remove outer layer bud
Leaf, only stays a piece of internal lobe.First with 70% ethanol surface sterilization 1min, distilled water cleans 3 times, then peels off internal lobe with tweezers, 2%
Hypochlorite disinfectant 10min, aseptic water washing 3-4 time.Children's fringe is cut into the segment of 2mm by superclean bench, is placed on more
Cultivate on injured tissue inducing culture, transfer weekly once.Culturing room's temperature is 25～28 DEG C, light culture；Illumination is forwarded to after 4 weeks
Cultivating, intensity of illumination is 1500～2000lx.
4. differentiation culture: the part yellow green callus of picking inducing culture, is inoculated in division culture medium, cultivates 4
Zhou Chengwei does not have the seedling of root, and every 30d transfers once；Culturing room's temperature is 26 DEG C, illumination 12h, intensity of illumination be 2000～
3000lx；
5. root culture: do not have root seedling by what differentiation culture obtained, be inoculated on root media, becomes after cultivating 25d
For whole plant；Culturing room's temperature is 25 DEG C, illumination 16h, and intensity of illumination is 2000～3000lx；
6. bottle transplantation of seedlings: the whole plant obtained by root culture takes out, and cleans the culture medium of root, with more than 800 times
Bacterium spirit is soaked after 30min and is transplanted to the mold of booth, and booth upper strata shading net hides, the ambient temperature of transplanting be 22～
28℃。
Claims (4)
1. a huge awns chemical disinfection tissue culture method, including culture vessel sterilization, culture medium preparation, induction of callus,
Differentiation culture, root culture and bottle transplantation of seedlings, it is characterised in that:
(1) culture vessel sterilization: by the stainless steel disc of culture bottle, bottle cap and inoculation at 100mg/L sodium hypochlorite+10mg/L breast
The aqueous solution of acid streptococci element+100mg/L calcium propionate soaks no less than 10h, saves backup；
(2) culture medium preparation: callus inducing medium is MS+2～5mg/L 2,4-D+0.5～2g/L AC+50～
100mg/L sodium hypochlorite+50～100mg/L Ticarcillin/Clavulanate Acid+0.1～0.5mg/L dodecyl sodium sulfate+30g/L sucrose+3.5g/L
Agar powder, pH5.8；Division culture medium is: MS+3～8mg/L 6-BA+0.1～0.5mg/L NAA+50～100mg/L hypochlorous acid
Sodium+50～100mg/L Ticarcillin/Clavulanate Acid+0.1～0.5mg/L dodecyl sodium sulfate+30g/L sucrose+3.5g/L agar powder, pH5.8；
Root media is 1/2MS+0.1～0.5mg/L NAA+50～100mg/L sodium hypochlorite+50～100mg/L Ticarcillin/Clavulanate Acid+0.1
～0.5mg/L dodecyl sodium sulfate+30g/L sucrose+3.5g/L agar powder, pH5.8；Described MS is 1962, Murashige
With Skoog disclosed in MS culture medium；Described 6-BA, refers to 6-benzyl aminopurine；Described NAA, refers to-naphthalene acetic acid；Described AC, refers to live
Property charcoal；During preparation culture medium, first claim sucrose and agar powder in each culture medium prescription, add the tap water of culture medium cumulative volume 1/2, add
Heat is completely dissolved to agar powder, then after assorting other raw materials by each culture medium prescription, constant volume, then with the NaOH of 1mol/L or
The HCl of 1mol/L adjusts pH value to 5.8, is dispensed in the culture vessel sterilized, and sealing is standby after cooled and solidified；
(3) the boot leaf phase of induction of callus: Yu Jumang, intercept a length of 10-20cm of children's fringe, remove outer layer bract,
Only stay a piece of internal lobe；First with the ethanol surface sterilization 1min that volume ratio is 70%, distilled water cleans 3 times, then peels off interior with tweezers
Leaf, volume ratio is the hypochlorite disinfectant 10min of 2%, and children's fringe is cut into 2mm on superclean bench by aseptic water washing 3-4 time
Segment, be placed on callus inducing medium cultivation, transfer weekly once；Culturing room's temperature is 25～28 DEG C, light culture；
Forwarding illumination cultivation after 4 weeks to, intensity of illumination is 1500～2000lx；
(4) differentiation culture: the part yellow green callus that picking inducing culture obtains, is inoculated in division culture medium, cultivates 4
Zhou Chengwei does not have the seedling of root, and every 30d transfers once；Culturing room's temperature is 26 DEG C, illumination 12h, intensity of illumination be 2000～
3000lx；
(5) root culture: differentiation culture is not had root seedling, is inoculated on root media, becomes and completely plant after cultivating 25d
Strain；Culturing room's temperature is 25 DEG C, illumination 16h, and intensity of illumination is 2000～3000lx；
(6) bottle transplantation of seedlings: the whole plant obtained by root culture takes out, and cleans the culture medium of root, with the carbendazim of 800 times
Transplanting to the mold of booth after soaking 30min, booth upper strata shading net hides, and the ambient temperature of transplanting is 22～28
℃。
One the most according to claim 1 huge awns chemical disinfection tissue culture method, it is characterised in that described callus induction
Culture medium is MS+3mg/L 2,4-D+1g/L AC+70mg/L sodium hypochlorite+60mg/L Ticarcillin/Clavulanate Acid+0.3mg/L dodecyl sulphur
Acid sodium+30g/L sucrose+3.5g/L agar powder, pH5.8.
One the most according to claim 1 huge awns chemical disinfection tissue culture method, it is characterised in that described division culture medium is:
MS+5mg/L 6-BA+0.3mg/L NAA+70mg/L sodium hypochlorite+60mg/L Ticarcillin/Clavulanate Acid+0.3mg/L dodecyl sodium sulfate+
30g/L sucrose+3.5g/L agar powder, pH5.8.
One the most according to claim 1 huge awns chemical disinfection tissue culture method, it is characterised in that described root media is
1/2MS+0.3mg/L NAA+70mg/L sodium hypochlorite+60mg/L Ticarcillin/Clavulanate Acid+0.3mg/L dodecyl sodium sulfate+30g/L sucrose
+ 3.5g/L agar powder, pH5.8.
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| CN106818475A (en) * | 2017-01-18 | 2017-06-13 | 福建农林大学 | A kind of utilization micro-organisms base cultivates the tissue culture method of pimento |
| CN106718929A (en) * | 2017-01-18 | 2017-05-31 | 福建农林大学 | A kind of utilization micro-organisms base cultivates the tissue culture method of romaine lettuce |
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| CN1628507A (en) * | 2004-04-21 | 2005-06-22 | 单文修 | Method for cultivating plant tissue under non-aseptic condition |
| CN102090339B (en) * | 2010-12-15 | 2012-11-21 | 湖北光芒能源植物有限公司 | Method for rapidly breeding miscanthus plant miscanthus giganteus embryo by tissue culture |
| CN102308750A (en) * | 2011-07-26 | 2012-01-11 | 天津滨城龙达集团有限公司 | Plant tissue culture bacteriostatic (antiseptic) agent containing botanical fungicide |
| CN102599235A (en) * | 2012-03-14 | 2012-07-25 | 甘肃省农业科学院农产品贮藏加工研究所 | Volatile preservative for potato wedges |
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| JP6041279B2 (en) * | 2013-03-22 | 2016-12-07 | 洋一 水田 | Method for producing plant tissue culture medium, plant tissue culture method, sterilizing agent, bactericidal agent, and plant tissue culturing medium composition |
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