CN103651118A - Method for sterilizing turbinaria conoides rhizoid explant - Google Patents
Method for sterilizing turbinaria conoides rhizoid explant Download PDFInfo
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- CN103651118A CN103651118A CN201310555178.8A CN201310555178A CN103651118A CN 103651118 A CN103651118 A CN 103651118A CN 201310555178 A CN201310555178 A CN 201310555178A CN 103651118 A CN103651118 A CN 103651118A
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Abstract
The invention discloses a method for sterilizing a turbinaria conoides rhizoid explant. The method is characterized by comprising the steps of clearing attachment such as impurity algae on the explant by utilizing a brush and adopting the turbinaria conoides rhizoid as an explant for the tissue culture, placing the explant into 1% to 5% antibiotic to be soaked for 5h to 8h, then taking out the explant, immersing the explant in a potassium iodide solution for 2min to 5min, taking out the explant, sterilizing the explant in povidone iodine for 1min to 3min, and finally washing the explant by utilizing the cooled and filtered high-pressure sterilized seawater. By adopting the method, the application concentration and the application time of the antibiotic can be reduced, and the drug resistance of the explant can be avoided; meanwhile, through the sterilizing effect of the potassium iodide and povidone iodine, not only can the bacteria or fungus on the surface and inside the explant be killed, but also the unrecoverable damage of the sterilizing agent to the explant can be avoided, and 85% bacteria-free feasible rhizoid explant can be obtained through the method.
Description
Technical field
The present invention relates to plant tissue culture technique sterilization field, be specifically related to a kind of sterilization method of intending leaflet loudspeaker algae rhizoid explant.
Background technology
Plan leaflet loudspeaker algae (
turbinaria conoides) be under the jurisdiction of Phaeophyta, Fucales, Sargassaceae, loudspeaker Trentepohlia, be the common a kind of kelp of China.Intend containing abundant marine steroids compound as Marine Sterol in leaflet loudspeaker algae, Marine Sterol is compared with the sterol of terrestrial plant, there is more skeleton and the side chain of horn of plenty, the structure of this uniqueness is given it and is had special efficacy antitumor, antiviral, anti-inflammation and sterilization, and can be used for preparing enzyme inhibitor, therefore, marine steroids compound becomes the element of the first species noticeable in medical treatment and pharmacy industry.
At present, intend leaflet loudspeaker algae and mainly take wild resource as main, but wild resource is not abundant yet; Artificial cultivation technique is also immature, and restriction biological engineering's principal element is that kind of algae source is unstable, germ plasm resource is deficient.Plant tissue culture technique can Fast-propagation, and the anniversary produces, and is not subject to seasonal restrictions; Seedling regularity is high, can carry out rearing new variety, and material requested is less, and reproduction coefficient is large, can in 1 year, by a stem section, turn out millions of seedlings.
The tissue culture technique of sea-plant is relatively backward, studying carefully its main cause is to obtain a large amount of aseptic and feasible explants, marine alga is mainly comprised of cell, although the structure relative complex of tangleweed, but the kind of disinfectant and the dosage highly significant that affects on explant, dosage height easily kills explant, and dosage is low, is not easy to kill bacterium or the fungi that enters into alginic cell inside.
Summary of the invention
The defect existing for prior art, technical problem to be solved by this invention is to provide a kind of sterilization method of intending leaflet loudspeaker algae rhizoid explant, for intending the research of leaflet loudspeaker algae tissue culture technique, improves sufficient aseptic available explant.
In order to solve the problems of the technologies described above, technical scheme of the present invention is as follows:
A kind of sterilization method of intending leaflet loudspeaker algae rhizoid explant: seashore gathers intends after leaflet loudspeaker algae, with seawater, clean up and in ice chest, be transported to operating room, the thallus of intending leaflet loudspeaker algae is removed and left and taken rhizoid as the explant of tissue cultivation use, after the attachments such as the assorted algae adhering on explant being removed totally with hairbrush, it is that 1～5% antibiotic soaks 5～8h that explant is placed in to concentration, and then explant is taken out, in liquor kalii iodide, soak 2～5min, again explant is taken out, 1～3min sterilizes in povidone iodine, finally use cooling filtration autoclaving seawater flushing clean.
Preferably, described antibiotic is any one in penicillin, streptomycin, terramycin.
Preferably, described potassium iodide concentration is 0.5～1%.
Preferably, the concentration of described povidone iodine is 1～5%.
By above-mentioned rhizoid explant, to be cut into length be the segment of 0.2-0.5cm and be inoculated in liquid PES culture fluid, in each blake bottle, inoculate 1 segment, shaking table is cultivated, the speed of shaking table is 100r/min, condition of culture is 18 ℃ of light intensity 5000Lx, light application time 12h, temperature, cultivates the aseptic and the feasibility that detect afterwards explant 2-3 week.
The invention has the beneficial effects as follows: reduce antibiotic working concentration and service time, avoid explant to form pesticide resistance; While is in conjunction with the Disinfection Effect of potassium iodide and povidone iodine, both explant surface and inner bacterium or fungi can have been killed, can avoid again disinfectant to cause expendable injury to explant, through the inventive method, can obtain 85% aseptic, feasible rhizoid explant.
Embodiment
Further set forth by the following examples beneficial effect of the present invention:
Embodiment 1:
Seashore gathers intends after leaflet loudspeaker algae, with seawater, clean up and in ice chest, be transported to operating room, the thallus of intending leaflet loudspeaker algae is removed and left and taken rhizoid as the explant of tissue cultivation use, after the attachments such as the assorted algae adhering on explant being removed totally with hairbrush, it is that 1% penicillin soaks 5h that explant is placed in to concentration, and then explant is taken out, in the liquor kalii iodide that is 0.5% in concentration, soak 2min, again explant is taken out, the 1min that sterilizes in the povidone iodine that is 1% in concentration, finally uses cooling filtration autoclaving seawater flushing clean; By above-mentioned rhizoid explant, to be cut into length be the segment of 0.2-0.5cm and be inoculated in liquid PES culture fluid, in each blake bottle, inoculate 1 segment, shaking table is cultivated, the speed of shaking table is 100r/min, condition of culture is 18 ℃ of light intensity 5000Lx, light application time 12h, temperature, cultivates the aseptic and the feasibility that after 2 weeks, detect explant; Inoculate altogether 40 bottles, wherein, 25 segment surfaces have filamentous to form, and aseptic rate is 62.5%, and has 10 contaminated surfaces of segment to form bacterial clump, has 5 segments unchanged.
Embodiment 2:
Seashore gathers intends after leaflet loudspeaker algae, with seawater, clean up and in ice chest, be transported to operating room, the thallus of intending leaflet loudspeaker algae is removed and left and taken rhizoid as the explant of tissue cultivation use, after the attachments such as the assorted algae adhering on explant being removed totally with hairbrush, it is that 5% terramycin soaks 8h that explant is placed in to concentration, and then explant is taken out, in the liquor kalii iodide that is 1% in concentration, soak 5min, again explant is taken out, the 3min that sterilizes in the povidone iodine that is 5% in concentration, finally uses cooling filtration autoclaving seawater flushing clean; By above-mentioned rhizoid explant, to be cut into length be the segment of 0.2-0.5cm and be inoculated in liquid PES culture fluid, in each blake bottle, inoculate 1 segment, shaking table is cultivated, the speed of shaking table is 100r/min, condition of culture is 18 ℃ of light intensity 5000Lx, light application time 12h, temperature, cultivates the aseptic and the feasibility that after 2 weeks, detect explant; Inoculate altogether 40 bottles, wherein, 25 segment surfaces have filamentous to form, and aseptic rate is 62.5%, and has 2 contaminated surfaces of segment to form bacterial clump, has 13 segments unchanged.
Embodiment 3:
Seashore gathers intends after leaflet loudspeaker algae, with seawater, clean up and in ice chest, be transported to operating room, the thallus of intending leaflet loudspeaker algae is removed and left and taken rhizoid as the explant of tissue cultivation use, after the attachments such as the assorted algae adhering on explant being removed totally with hairbrush, it is that 2% streptomycin soaks 6h that explant is placed in to concentration, and then explant is taken out, in the liquor kalii iodide that is 0.7% in concentration, soak 3min, again explant is taken out, the 2min that sterilizes in the povidone iodine that is 2% in concentration, finally uses cooling filtration autoclaving seawater flushing clean; By above-mentioned rhizoid explant, to be cut into length be the segment of 0.2-0.5cm and be inoculated in liquid PES culture fluid, in each blake bottle, inoculate 1 segment, shaking table is cultivated, the speed of shaking table is 100r/min, condition of culture is 18 ℃ of light intensity 5000Lx, light application time 12h, temperature, cultivates the aseptic and the feasibility that after 2 weeks, detect explant; Inoculate altogether 40 bottles, wherein, 34 segment surfaces have filamentous to form, and aseptic rate is 85%, and has 1 contaminated surface of segment to form bacterial clump, has 5 segments unchanged.
Embodiment 4:
Seashore gathers intends after leaflet loudspeaker algae, with seawater, clean up and in ice chest, be transported to operating room, the thallus of intending leaflet loudspeaker algae is removed and left and taken rhizoid as the explant of tissue cultivation use, after the attachments such as the assorted algae adhering on explant being removed totally with hairbrush, it is that 2% penicillin soaks 6h that explant is placed in to concentration, and then explant is taken out, in the liquor kalii iodide that is 0.7% in concentration, soak 3min, again explant is taken out, the 2min that sterilizes in the povidone iodine that is 2% in concentration, finally uses cooling filtration autoclaving seawater flushing clean; By above-mentioned rhizoid explant, to be cut into length be the segment of 0.2-0.5cm and be inoculated in liquid PES culture fluid, in each blake bottle, inoculate 1 segment, shaking table is cultivated, the speed of shaking table is 100r/min, condition of culture is 18 ℃ of light intensity 5000Lx, light application time 12h, temperature, cultivates the aseptic and the feasibility that after 2 weeks, detect explant; Inoculate altogether 40 bottles, wherein, 32 segment surfaces have filamentous to form, and aseptic rate is 80%, and has 0 contaminated surface of segment to form bacterial clump, has 8 segments unchanged.
Embodiment 5:
Seashore gathers intends after leaflet loudspeaker algae, with seawater, clean up and in ice chest, be transported to operating room, the thallus of intending leaflet loudspeaker algae is removed and left and taken rhizoid as the explant of tissue cultivation use, after the attachments such as the assorted algae adhering on explant being removed totally with hairbrush, it is that 2% terramycin soaks 6h that explant is placed in to concentration, and then explant is taken out, in the liquor kalii iodide that is 0.7% in concentration, soak 3min, again explant is taken out, the 2min that sterilizes in the povidone iodine that is 2% in concentration, finally uses cooling filtration autoclaving seawater flushing clean; By above-mentioned rhizoid explant, to be cut into length be the segment of 0.2-0.5cm and be inoculated in liquid PES culture fluid, in each blake bottle, inoculate 1 segment, shaking table is cultivated, the speed of shaking table is 100r/min, condition of culture is 18 ℃ of light intensity 5000Lx, light application time 12h, temperature, cultivates the aseptic and the feasibility that after 2 weeks, detect explant; Inoculate altogether 40 bottles, wherein, 30 segment surfaces have filamentous to form, and aseptic rate is 75%, and has 3 contaminated surfaces of segment to form bacterial clump, has 7 segments unchanged.
Above-mentioned example just, for explanation technical conceive of the present invention and technical characterstic, can not limit the scope of the invention with this.Equivalent transformation or modification that all essence according to the present invention is done, within all should being encompassed in protection scope of the present invention.
Claims (4)
1. a sterilization method of intending leaflet loudspeaker algae rhizoid explant, it is characterized in that, comprise the steps: that seashore gathers after plan leaflet loudspeaker algae, with seawater, clean up and in ice chest, be transported to operating room, the thallus of intending leaflet loudspeaker algae is removed and left and taken rhizoid as the explant of tissue cultivation use, after the attachments such as the assorted algae adhering on explant being removed totally with hairbrush, it is that 1～5% antibiotic soaks 5～8h that explant is placed in to concentration, and then explant is taken out, in liquor kalii iodide, soak 2～5min, again explant is taken out, 1～3min sterilizes in povidone iodine, finally use cooling filtration autoclaving seawater flushing clean.
2. sterilization method according to claim 1, is characterized in that, described antibiotic is any one in penicillin, streptomycin, terramycin.
3. sterilization method according to claim 1, is characterized in that, described potassium iodide concentration is 0.5～1%.
4. sterilization method according to claim 1, is characterized in that, the concentration of described povidone iodine is 1～5%.
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Citations (5)
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|---|---|---|---|---|
| JPH05103558A (en) * | 1991-10-17 | 1993-04-27 | Kansai Electric Power Co Inc:The | Method for culturing brown algae |
| CN1373633A (en) * | 2000-08-31 | 2002-10-09 | 科学与工业研究委员会 | Improved process for cultivation of algae |
| CN101731147A (en) * | 2009-12-18 | 2010-06-16 | 中国海洋大学 | Gelidium amansii Lamx. culture method by tissue culture breeding |
| CN102771394A (en) * | 2012-07-27 | 2012-11-14 | 山东东方海洋科技股份有限公司 | Method for cloning and culturing seaweed gametophytes |
| CN103202214A (en) * | 2013-04-26 | 2013-07-17 | 上海海洋大学 | Breeding method of sargassum vachellianum artificial seeds |
-
2013
- 2013-11-11 CN CN201310555178.8A patent/CN103651118A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05103558A (en) * | 1991-10-17 | 1993-04-27 | Kansai Electric Power Co Inc:The | Method for culturing brown algae |
| CN1373633A (en) * | 2000-08-31 | 2002-10-09 | 科学与工业研究委员会 | Improved process for cultivation of algae |
| CN101731147A (en) * | 2009-12-18 | 2010-06-16 | 中国海洋大学 | Gelidium amansii Lamx. culture method by tissue culture breeding |
| CN102771394A (en) * | 2012-07-27 | 2012-11-14 | 山东东方海洋科技股份有限公司 | Method for cloning and culturing seaweed gametophytes |
| CN103202214A (en) * | 2013-04-26 | 2013-07-17 | 上海海洋大学 | Breeding method of sargassum vachellianum artificial seeds |
Non-Patent Citations (4)
| Title |
|---|
| C. R. K. REDDY ET AL: "Seaweed micropropagation techniques and their potentials: an overview", 《J APPL PHYCOL》, no. 20, 16 August 2007 (2007-08-16), pages 609 - 617, XP019643754 * |
| G.RAJAKRISHNA KUMAR ET AL.: "Callus induction and thallus regeneration from callus of phycocolloid yielding seaweeds from the Indian coast", 《J APPL PHYCOL》, vol. 19, 10 November 2006 (2006-11-10), XP019480681 * |
| LYDIANE (ANN) KYTE: "Plant Tissue Culture", 《HTTP://OPANSOPANDI.WORDPRESS.COM/2010/02/14/PLANT-TISSUE-CULTURE/》, 14 February 2010 (2010-02-14) * |
| 初少华等: "海藻组织培养研究进展", 《水产科学》, vol. 30, no. 4, 30 April 2011 (2011-04-30) * |
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Application publication date: 20140326 |