CN102100177A - Efficient degerming method used during rapid production of bulbus fritillariae cirrhosae compost - Google Patents
Efficient degerming method used during rapid production of bulbus fritillariae cirrhosae compost Download PDFInfo
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- CN102100177A CN102100177A CN2010105510996A CN201010551099A CN102100177A CN 102100177 A CN102100177 A CN 102100177A CN 2010105510996 A CN2010105510996 A CN 2010105510996A CN 201010551099 A CN201010551099 A CN 201010551099A CN 102100177 A CN102100177 A CN 102100177A
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- degerming
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- bulbus fritillariae
- fritillariae cirrhosae
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Abstract
The invention discloses an efficient degerming method used during rapid production of a bulbus fritillariae cirrhosae compost. The method is mainly characterized in that a microbiological contamination material during the rapid production of the bulbus fritillariae cirrhosae compost is degermed by adopting special steps according to the inhibition function of antibiotics to the bacterium growth. The special steps include selection of the microbiological contamination material, preprocessing, liquid degerming, first solid degerming, secondary solid degerming and degerming detection. According to the invention, the bacterium contamination during the production of the bulbus fritillariae cirrhosae compost is effectively eliminated under the condition of no damage on the compost, and the maximum degerming rate is up to 92.7 percent.
Description
Technical field
The present invention relates to the effective degerming method in the quick production of a kind of Bulbus Fritillariae Cirrhosae culture.
Background technology
The rare medicinal herbs Bulbus Fritillariae Cirrhosae is the dry bulb of Liliaceae herbaceos perennial Bulbus Fritillariae Cirrhosae (Fritillaria cirrhosa D.Don), be the most frequently used, the most effective relieving cough and reducing sputum medicine, be usually used in lung-heat type cough, the few phlegm of dry cough, deficiency of Yin labor is coughed, cough up sputum streaked with blood, its active ingredient is mainly bulb of fritillary alkaloid and steroid oside.Along with the exploitation of various relieving cough and reducing sputum medicines and a large amount of demands, the consumption of Bulbus Fritillariae Cirrhosae constantly increases, and only depends on the demand that wild Bulbus Fritillariae Cirrhosae can not satisfy medicinal material market far away of excavating.
Plant tissue culture technique not only can effectively solve the dependence of medicinal plant to conditions such as temperature, soil property, weathers, can also produce active components in medicinal plant fast and efficiently.From Routine in 1956 and Nickel cultivate by plant first produce useful secondary metabolite since, the research work of medicinal plant tissue culture has obtained bigger progress, existing hundreds of plant metabolites obtains by cell culture technology, and the content of nearly half secondary metabolite surpasses former plant.Therefore, the active princlple of the tissue culture means acquisition Bulbus Fritillariae Cirrhosae medicinal material of utilization modern biotechnology is to solve the current Bulbus Fritillariae Cirrhosae medicinal material important channel that supply falls short of demand.
Find after deliberation: utilizing tissue culture technique to produce 25 days biomasss of Bulbus Fritillariae Cirrhosae culture can increase about 3.5 times, and alkaloid is about 2 times of wild Bulbus Fritillariae Cirrhosae.But, in the quick aseptic culture production process of Bulbus Fritillariae Cirrhosae culture, owing to some reasons can cause culture materials the situation of microbiological contamination appears.Because in a single day culture materials occurs understanding the accumulation of its growth of severe inhibition and active princlple after the microbiological contamination, and finally causes the death of culture materials, thereby can cause enormous economic loss to quick production.Therefore how removing the microbiological contamination problem that occurs in the quick production of Bulbus Fritillariae Cirrhosae culture effectively has very important significance.Currently used solution generally is after the material of microbiological contamination is taken out, with alcohol and mercuric chloride material to be carried out the secondary sterilization processing.Because Bulbus Fritillariae Cirrhosae culture materials cell children is tender, and lives under the fat city condition of artificial culture for a long time, the ability that cell is resisted external environment reduces, after alcohol and mercuric chloride are disinfected, can cause a large amount of brownization, the death of culture materials once more.Therefore, current in the quick production process of Bulbus Fritillariae Cirrhosae culture, generally all be to take to stop cultivating in case occur after the culture materials microbiological contamination, abandon culture, thereby caused the waste of great amount of manpower and material resources and financial resources.
Summary of the invention
The object of the present invention is to provide the effective degerming method in the quick production of a kind of Bulbus Fritillariae Cirrhosae culture, the effectively degerming of this method can not cause brownization of culture materials, death again.
For achieving the above object, the solution that the present invention adopts comprises the following steps:
(1) choosing of microbiological contamination material: routine observation Bulbus Fritillariae Cirrhosae culture materials upgrowth situation in case be observed visually when having bacterium to produce in material or the medium, be about to culture materials and take out;
(2) preliminary treatment: the culture materials of taking out earlier with aseptic water washing 2～5 times, and is blotted the moisture of material surface with aseptic filter paper repeatedly, and putting into the cefotaxime na concn again is 200～500mgL
-1Antibiotic water in soak 6～12min, failure of oscillation does not swing therebetween, takes out the back is blotted material surface repeatedly with aseptic filter paper moisture;
(3) liquid degerming: pretreated material access is contained antibiotic MS+6-BA2.0mgL
-1+ NAA 0.3mgL
-1+ sucrose 30gL
-1Cultivated for 1～2 week in the liquid nutrient medium, shaking speed 100～120rmin
-1, condition of culture is illumination every day 8～12 hours, intensity of illumination is 1000～1600lx, and 18～22 ℃ of temperature, described antibiotic kind and concentration are Cefotaxime Sodium 300mgL
-1～800mgL
-1Or Cefotaxime Sodium 150mgL
-1～300mgL
-1+ Ceftriaxone Sodium 150mgL
-1～300mgL
-1
(4) solid degerming for the first time: take out material, blot the moisture of material surface repeatedly, insert the antibiotic MS+6-BA 2.0mgL that contains with (3) step identical type and concentration again with aseptic filter paper through the liquid degerming
-1+ NAA 0.3mgL
-1+ sucrose 30gL
-1+ agar 6.5gL
-1Carry out the degerming first time in the solid culture medium and cultivate, incubation time is 25～30 days, and condition of culture is illumination every day 8～12 hours, and intensity of illumination is 1000～1600lx, 18～22 ℃ of temperature;
(5) solid degerming for the second time: take out the material through (4) step degerming after, switching goes into to contain and (4) step identical type but antibiotic MS+6-BA 2.0mgL that concentration reduces by half
-1+ NAA 0.3mgL
-1+ sucrose 30gL
-1+ agar 6.5gL
-1Carry out the degerming second time in the solid culture medium and cultivate, incubation time is 25～30 days, and condition of culture is illumination every day 8～12 hours, and intensity of illumination is 1000～1600lx, 18～22 ℃ of temperature;
(6) degerming detects: take out the material after (5) step degerming, the MS+6-BA 2.0mgL of antibiotic-free is gone in switching
-1+ NAA 0.3mgL
-1+ sucrose 30gL
-1+ agar 6.5gL
-1Solid culture medium in carry out successive transfer culture, after cultivating 25～30 days, observe material degerming situation, and statistics degerming rate, degerming rate=(sterilizable material number after the degerming/degerming material sum) * 100%.
The present invention is according to the inhibitory action of antibiotic to bacterial growth, microbiological contamination material in the quick production Bulbus Fritillariae Cirrhosae culture is carried out degerming by adopting specific step, the Cefotaxime Sodium that is adopted belongs to the third generation cephalosporin of non-oral type, and its mechanism of action is synthesizing of main component peptide glycan to realize bacteria growing inhibiting by what disturb bacteria cell wall.The concrete steps that adopted are that the Bulbus Fritillariae Cirrhosae culture materials of microbiological contamination is soaked certain hour with putting into antibiotic water behind the aseptic water washing earlier, and with insert behind the filter paper wipe dry carry out the liquid degerming in the antibiotic liquid culture contain particular types and concentration and cultivate certain hour after, take out wipe dry and transfer and carry out solid degerming first time in the antibiotic solid culture medium that contains identical type and concentration and cultivate, last switching is again gone into to contain identical type but is finished solid degerming cultivation for the second time in the antibiotic solid culture medium that concentration reduces by half.The present invention can remove the germ contamination that the Bulbus Fritillariae Cirrhosae culture occurs effectively in producing fast under the situation that does not injure culture, its highest degerming rate reaches 92.7%, has reduced the heavy economic losses that the Bulbus Fritillariae Cirrhosae culture causes because of microbiological contamination widely.
Embodiment
Embodiment 1
(1) choosing of microbiological contamination material: routine observation Bulbus Fritillariae Cirrhosae culture materials upgrowth situation in case be observed visually when having bacterium to produce in material or the medium, be about to culture materials and take out;
(2) preliminary treatment: the culture materials of taking out is repaired slightly, use aseptic water washing then 2～5 times, and blot the moisture of material surface repeatedly with aseptic filter paper, putting into the cefotaxime sodium content again is 400mgL
-1Antibiotic water in soak 10min, failure of oscillation does not swing therebetween, takes out the back is blotted material surface repeatedly with aseptic filter paper moisture;
(3) liquid degerming: pretreated material access is contained Cefotaxime Sodium 300mgL
-1With Ceftriaxone Sodium 150mgL
-1MS+6-BA 2.0mgL
-1+ NAA 0.3mgL
-1+ sucrose 30gL
-1Carry out degerming in the liquid nutrient medium and cultivated shaking speed 100～120rmin 10 days
-1, condition of culture is illumination every day 8～12 hours, intensity of illumination is 1000～1600lx, 18～22 ℃ of temperature;
(4) solid degerming for the first time: take out material, blot the moisture of material surface repeatedly, insert again and contain Cefotaxime Sodium 300mgL with aseptic filter paper through the liquid degerming
-1With Ceftriaxone Sodium 150mgL
-1MS+6-BA 2.0mgL
-1+ NAA 0.3mgL
-1+ sucrose 30gL
-1+ agar 6.5gL
-1Carry out the degerming first time in the solid culture medium and cultivate, incubation time is 25～30 days, and condition of culture is illumination every day 8～12 hours, and intensity of illumination is 1000～1600lx, 18～22 ℃ of temperature;
(5) solid degerming for the second time: take out the material after (4) step degerming, switching goes into to contain Cefotaxime Sodium 150mgL
-1With Ceftriaxone Sodium 75mgL
-1MS+6-BA 2.0mgL
-1+ NAA 0.3mgL
-1+ sucrose 30gL
-1+ agar 6.5gL
-1Carry out the degerming second time in the solid culture medium and cultivate, incubation time is 25～30 days, and condition of culture is illumination every day 8～12 hours, and intensity of illumination is 1000～1600lx, 18～22 ℃ of temperature;
(6) degerming detects: take out the material after (5) step degerming, the MS+6-BA 2.0mgL of antibiotic-free is gone in switching
-1+ NAA 0.3mgL
-1+ sucrose 30gL
-1+ agar 6.5gL
-1Solid culture medium in carry out successive transfer culture, cultivating after 25 days statistics degerming rate is 92.7%.
Embodiment 2
Change the antibiotic in the medium of liquid nutrient medium among the embodiment 1 and solid degerming for the first time into Cefotaxime Sodium 600mgL
-1, the antibiotic in the medium of solid degerming changes Cefotaxime Sodium 300mgL into for the second time
-1, other step is with example 1, and the degerming rate is 89.4%.The Cefotaxime Sodium that uses appropriate concentration separately is described, also can realizes effective degerming the Bulbus Fritillariae Cirrhosae culture.
Embodiment 3
Change antibiotic in the medium of liquid nutrient medium in the example 1 and solid degerming for the first time into Cefotaxime Sodium 800mgL
-1, antibiotic changes Cefotaxime Sodium 400mgL in the medium of solid degerming for the second time
-1, other step is with example 1, and the degerming rate is 91.9%; But culture materials begins to occur flavescence, browning, and the growth obviously be suppressed, this explanation high concentration antibiotic in degerming, also culture materials is had certain toxic action.
Claims (1)
1. the effective degerming method during a Bulbus Fritillariae Cirrhosae culture is produced fast is characterized in that comprising the following steps:
(1) choosing of microbiological contamination material: routine observation Bulbus Fritillariae Cirrhosae culture materials upgrowth situation in case be observed visually when having bacterium to produce in material or the medium, be about to culture materials and take out;
(2) preliminary treatment: the culture materials of taking out earlier with aseptic water washing 2～5 times, and is blotted the moisture of material surface with aseptic filter paper repeatedly, and putting into the cefotaxime na concn again is 200～500mgL
-1Antibiotic water in soak 6～12min, failure of oscillation does not swing therebetween, takes out the back is blotted material surface repeatedly with aseptic filter paper moisture;
(3) liquid degerming: pretreated material access is contained antibiotic MS+6-BA 2.0mgL
-1+ NAA 0.3mgL
-1+ sucrose 30gL
-1Cultivated for 1～2 week in the liquid nutrient medium, shaking speed 100～120rmin
-1, condition of culture is illumination every day 8～12 hours, intensity of illumination is 1000～1600lx, and 18～22 ℃ of temperature, described antibiotic kind and concentration are Cefotaxime Sodium 300mgL
-1～800mgL
-1Or Cefotaxime Sodium 150mgL
-1～300mgL
-1+ Ceftriaxone Sodium 150mgL
-1～300mgL
-1
(4) solid degerming for the first time: take out material, blot the moisture of material surface repeatedly, insert the antibiotic MS+6-BA 2.0mgL that contains with (3) step identical type and concentration again with aseptic filter paper through the liquid degerming
-1+ NAA 0.3mgL
-1+ sucrose 30gL
-1+ agar 6.5gL
-1Carry out the degerming first time in the solid culture medium and cultivate, incubation time is 25～30 days, and condition of culture is illumination every day 8～12 hours, and intensity of illumination is 1000～1600lx, 18～22 ℃ of temperature;
(5) solid degerming for the second time: take out the material through (4) step degerming after, switching goes into to contain and (4) step identical type but antibiotic MS+6-BA2.0mgL that concentration reduces by half
-1+ NAA0.3mgL
-1+ sucrose 30gL
-1+ agar 6.5gL
-1Carry out the degerming second time in the solid culture medium and cultivate, incubation time is 25～30 days, and condition of culture is illumination every day 8～12 hours, and intensity of illumination is 1000～1600lx, 18～22 ℃ of temperature;
(6) degerming detects: take out the material after (5) step degerming, the MS+6-BA 2.0mgL of antibiotic-free is gone in switching
-1+ NAA 0.3mgL
-1+ sucrose 30gL
-1+ agar 6.5gL
-1Solid culture medium in carry out successive transfer culture, after cultivating 25～30 days, observe material degerming situation, and statistics degerming rate.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102257966A (en) * | 2011-07-01 | 2011-11-30 | 山东省农业科学院蔬菜研究所 | Method for preventing pollution of garlic ovary culture |
| CN103461137A (en) * | 2013-09-24 | 2013-12-25 | 薛刚 | Method for inducing generation of fritillaria cirrhosa embryoids |
| CN105532482A (en) * | 2016-03-14 | 2016-05-04 | 龙岩市禾康生物科技有限公司 | Antibacterial culture medium for tissue culture of plants and preparation method of culture medium |
| CN105685072A (en) * | 2016-03-14 | 2016-06-22 | 龙岩市禾康生物科技有限公司 | Antibacterial film-coating and growth-promoting agent for explant for plant tissue culture and use method of agent |
| CN106069789A (en) * | 2016-08-19 | 2016-11-09 | 中国科学院华南植物园 | One is carried disease germs in vitro vegetable material tissue culture medium (TCM) and method for tissue culture |
| CN110872564A (en) * | 2019-12-14 | 2020-03-10 | 吉林农业大学 | Tissue separation method for wild agaric |
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2010
- 2010-11-19 CN CN201010551099A patent/CN102100177B/en active Active
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102257966A (en) * | 2011-07-01 | 2011-11-30 | 山东省农业科学院蔬菜研究所 | Method for preventing pollution of garlic ovary culture |
| CN102257966B (en) * | 2011-07-01 | 2013-01-16 | 山东省农业科学院蔬菜研究所 | Method for preventing pollution of garlic ovary culture |
| CN103461137A (en) * | 2013-09-24 | 2013-12-25 | 薛刚 | Method for inducing generation of fritillaria cirrhosa embryoids |
| CN105532482A (en) * | 2016-03-14 | 2016-05-04 | 龙岩市禾康生物科技有限公司 | Antibacterial culture medium for tissue culture of plants and preparation method of culture medium |
| CN105685072A (en) * | 2016-03-14 | 2016-06-22 | 龙岩市禾康生物科技有限公司 | Antibacterial film-coating and growth-promoting agent for explant for plant tissue culture and use method of agent |
| CN105685072B (en) * | 2016-03-14 | 2018-08-21 | 龙岩市禾康生物科技有限公司 | A kind of plant tissue culture explant coating, antibacterial, growth-promoting agent and application method |
| CN106069789A (en) * | 2016-08-19 | 2016-11-09 | 中国科学院华南植物园 | One is carried disease germs in vitro vegetable material tissue culture medium (TCM) and method for tissue culture |
| CN106069789B (en) * | 2016-08-19 | 2018-09-28 | 中国科学院华南植物园 | One kind is carried disease germs in vitro vegetable material tissue culture medium (TCM) and method for tissue culture |
| CN110872564A (en) * | 2019-12-14 | 2020-03-10 | 吉林农业大学 | Tissue separation method for wild agaric |
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