CN103609454B - A kind of method reducing tea tree tissue culture procedures China and foreign countries implant body pollution - Google Patents
A kind of method reducing tea tree tissue culture procedures China and foreign countries implant body pollution Download PDFInfo
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- CN103609454B CN103609454B CN201310639348.0A CN201310639348A CN103609454B CN 103609454 B CN103609454 B CN 103609454B CN 201310639348 A CN201310639348 A CN 201310639348A CN 103609454 B CN103609454 B CN 103609454B
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Abstract
The invention discloses a kind of method reducing tea tree tissue culture procedures China and foreign countries implant body pollution, its processing method be the explant chosen is sterile-processed after, the part of explant wound because of variable color of sterilizing is cut, be cut into 1-1.5cm longer, with the stem section of an axillalry bud, stem section be seeded in containing on antibiotic medium, every bottle graft kind 1-2 stem section, inoculation is placed in culturing room cultivates.The present invention utilizes ampicillin and streptomycin sulphate combination, can reduce the pollution rate of explant in tea tree tissue culture procedures, improve survival rate.
Description
Technical field
The invention belongs to technical field of tea tree tissue culture, be specifically related to a kind of method reducing tea tree tissue culture procedures China and foreign countries implant body pollution.
Background technology
Tea tree (
camelliasinensisl.Kuntze.) belonging to Theaceae, Camellia, is a kind of perennial woody, evergreen plant.Tea tree tissue cultures starts from eighties of last century sixties.At present, tea tree tissue cultures obtains complete regeneration plant by cultivating the seed, stem section, axillalry bud, flower pesticide etc. of tea tree.Tea tree is the more Plants of self-contained endophyte, and therefore, carrying out, tissue culture procedures China and foreign countries implant body pollution is serious, seriously governs the development of tea tree tissue cultures.Therefore, study a kind of method reducing tea tree tissue culture procedures China and foreign countries implant body pollution and become this area technical barrier in the urgent need to address at present.
Summary of the invention
The object of the present invention is to provide a kind of method reducing tea tree tissue culture procedures China and foreign countries implant body pollution, the method can reduce the pollution that in tea tree tissue culture procedures, explant occurs, and improves survival rate.
For achieving the above object, the present invention adopts following technical scheme:
Reduce a method for tea tree tissue culture procedures China and foreign countries implant body pollution, it is characterized in that: concrete steps are as follows:
(1) get robust growth in spring, Stem of Tea section with full axillalry bud is explant;
(2) explant being cut into segment, take mass fraction as the alcohol disinfecting 20-25s of 75%, aseptic water washing 2 times; With the mercuric chloride sterilization 4min that mass fraction is 0.1%, aseptic water washing 2 times; Be mercuric chloride sterilization 5min, the aseptic water washing 3-5 time of 0.1% again with mass fraction;
(3) part of explant incision because of variable color of sterilizing is cut, be then cut into 1-1.5cm long, the stem section of a band axillalry bud;
(4) be connected on containing on antibiotic medium by above-mentioned stem section, each bottle inoculation 1-2 stem section, puts into culturing room and cultivates after inoculation.
Described medium is: MS+1.5mg/L6-BA+0.3mg/LNAA+ sucrose 30g+ agar powder 6.5g, pH value is adjusted to 5.6-5.8, and the medium prepared is divided in the wide-mouth triangular flask of 500mL, high-temperature sterilization 20min.
Add antibiotic constituent in medium and concentration as follows:
Ampicillin: 200mg/L;
Streptomycin sulphate: 150mg/L.
Described containing antibiotic medium be by antibiotic after the sterilizing of millipore filter suction filtration, join and be equipped with in the wide-mouth triangular flask of medium, be dispensed into again in blake bottle after shaking up.
Condition of culture is: temperature 23-25 DEG C, intensity of illumination 1500-2000lx, light application time 12h/d, cultivates 30d-35d.
Remarkable advantage of the present invention is:
(1) the present invention effectively can reduce the pollution of explant in tea tree tissue culture procedures, improves the success rate of tissue cultures.
(2) the present invention is by adding ampicillin and streptomycin sulphate in the medium, can reach good fungistatic effect, and make the pollution rate of explant reduce 35%-37%, germination rate reaches 42%-44%.
Embodiment
embodiment 1
Reduce a method for tea tree tissue culture procedures China and foreign countries implant body pollution, its concrete steps are as follows:
(1) get robust growth in spring, Stem of Tea section with full axillalry bud is explant;
(2) explant being cut into segment, take mass fraction as the alcohol disinfecting 20s of 75%, aseptic water washing 2 times, is the mercuric chloride sterilization 4min of 0.1% with mass fraction, aseptic water washing 2 times, then is the mercuric chloride sterilization 5min of 0.1% with mass fraction, aseptic water washing 5 times;
(3) part of explant wound because of variable color of sterilizing is cut, be then cut into 1-1.5cm long, the stem section of a band axillalry bud, stand-by;
(4) prepare medium with MS+1.5mg/L6-BA+0.3mg/LNAA+ sucrose 30g+ agar powder 6.5g, pH value is adjusted to 5.6, and the medium prepared is divided in the wide-mouth triangular flask of 500mL, high-temperature sterilization 20min;
(5) by ampicillin and streptomycin sulphate through the sterilizing of millipore filter suction filtration, the ratio of 200mg ampicillin and 150mg streptomycin sulphate is added in often liter of medium, ampicillin and streptomycin sulphate being joined respectively is equipped with in the wide-mouth triangular flask of medium, is dispensed in blake bottle after shaking up again;
(6) the stem section after process is connected on containing on antibiotic medium, 2 stem sections inoculated by each bottle, inoculate 30 altogether, put into culturing room after inoculation to cultivate, condition of culture is: temperature 23 DEG C, intensity of illumination 1500lx, light application time 12h/d, after cultivating 30d, pollution rate 35.91%, germination rate 42.63%.
embodiment 2
Reduce a method for tea tree tissue culture procedures China and foreign countries implant body pollution, its concrete steps are as follows:
(1) get robust growth in spring, Stem of Tea section with full axillalry bud is explant;
(2) explant being cut into segment, take mass fraction as the alcohol disinfecting 23s of 75%, aseptic water washing 2 times, is the mercuric chloride sterilization 4min of 0.1% with mass fraction, aseptic water washing 2 times, then is the mercuric chloride sterilization 5min of 0.1% with mass fraction, aseptic water washing 4 times;
(3) part of explant wound because of variable color of sterilizing is cut, be then cut into the stem section that 1-1.5cm is long, be with an axillalry bud, stand-by;
(4) prepare medium with MS+1.5mg/L6-BA+0.3mg/LNAA+ sucrose 30g+ agar powder 6.5g, pH value is adjusted to 5.7, and the medium prepared is divided in the wide-mouth triangular flask of 500mL, high-temperature sterilization 20min;
(5) by ampicillin and streptomycin sulphate through the sterilizing of millipore filter suction filtration, the ratio of 200mg ampicillin and 150mg streptomycin sulphate is added in often liter of medium, ampicillin and streptomycin sulphate being joined respectively is equipped with in the wide-mouth triangular flask of medium, is dispensed in blake bottle after shaking up again;
(6) the stem section after process is connected on containing on antibiotic medium, 1 stem section inoculated by each bottle, inoculate 30 altogether, put into culturing room after inoculation to cultivate, condition of culture is: temperature 24 DEG C, intensity of illumination 1800lx, light application time 12h/d, after cultivating 33d, pollution rate 36.64%, germination rate 43.29%.
embodiment 3
Reduce a method for tea tree tissue culture procedures China and foreign countries implant body pollution, its concrete steps are as follows:
(1) get robust growth in spring, Stem of Tea section with full axillalry bud is explant;
(2) explant being cut into segment, take mass fraction as the alcohol disinfecting 25s of 75%, aseptic water washing 2 times, is the mercuric chloride sterilization 4min of 0.1% with mass fraction, aseptic water washing 2 times, then is the mercuric chloride sterilization 5min of 0.1% with mass fraction, aseptic water washing 3 times;
(3) part of explant wound because of variable color of sterilizing is cut, be then cut into the stem section that 1-1.5cm is long, be with an axillalry bud, stand-by;
(4) prepare medium with MS+1.5mg/L6-BA+0.3mg/LNAA+ sucrose 30g+ agar powder 6.5g, pH value is adjusted to 5.8, and the medium prepared is divided in the wide-mouth triangular flask of 500mL, high-temperature sterilization 20min;
(5) by ampicillin and streptomycin sulphate through the sterilizing of millipore filter suction filtration, the ratio of 200mg ampicillin and 150mg streptomycin sulphate is added in often liter of medium, ampicillin and streptomycin sulphate being joined respectively is equipped with in the wide-mouth triangular flask of medium, is dispensed in blake bottle after shaking up again;
(6) the stem section after process is connected on containing on antibiotic medium, 2 stem sections inoculated by each bottle, inoculate 30 altogether, put into culturing room after inoculation to cultivate, condition of culture is: temperature 25 DEG C, intensity of illumination 2000lx, light application time 12h/d, after cultivating 35d, pollution rate 36.87%, germination rate 43.96%.
embodiment 4 variable concentrations ampicillin and streptomycin sulphate are to the inhibition of endophyte
Reduce a method for tea tree tissue culture procedures China and foreign countries implant body pollution, its concrete steps are as follows:
(1) get robust growth in spring, Stem of Tea section with full axillalry bud is explant;
(2) explant being cut into segment, take mass fraction as the alcohol disinfecting 25s of 75%, aseptic water washing 2 times, is the mercuric chloride sterilization 4min of 0.1% with mass fraction, aseptic water washing 2 times, then is the mercuric chloride sterilization 5min of 0.1% with mass fraction, aseptic water washing 5 times;
(3) part of explant wound because of variable color of sterilizing is cut, be then cut into the stem section that 1-1.5cm is long, be with an axillalry bud, stand-by;
(4) prepare medium with MS+1.5mg/L6-BA+0.3mg/LNAA+ sucrose 30g+ agar powder 6.5g, pH value is adjusted to 5.6, and the medium prepared is divided in the wide-mouth triangular flask of 500mL, high-temperature sterilization 20min;
(5) by ampicillin and streptomycin sulphate through the sterilizing of millipore filter suction filtration, by desired concn, ampicillin and streptomycin sulphate are added in medium, are dispensed into again in blake bottle after shaking up;
(6) be connected on containing on antibiotic medium by the stem section after process, 2 stem sections inoculated by each bottle, and put into culturing room after inoculation and cultivate, condition of culture is: temperature 25 DEG C, intensity of illumination 1500lx, light application time 12h/d, cultivate 30d.
As can be seen from Table 1, add ampicillin 200mg/L and streptomycin sulphate 150mg/L to endophyte inhibition better, and germination rate is higher.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (3)
1. reduce a method for tea tree tissue culture procedures China and foreign countries implant body pollution, it is characterized in that: concrete steps are as follows:
(1) get robust growth in spring, Stem of Tea section with full axillalry bud is explant;
(2) explant being cut into segment, take mass fraction as the alcohol disinfecting 20-25s of 75%, aseptic water washing 2 times; With the mercuric chloride sterilization 4min that mass fraction is 0.1%, aseptic water washing 2 times; Be mercuric chloride sterilization 5min, the aseptic water washing 3-5 time of 0.1% again with mass fraction;
(3) part of explant incision because of variable color of sterilizing is cut, be then cut into 1-1.5cm long, the stem section of a band axillalry bud;
(4) be connected on containing on antibiotic medium by above-mentioned stem section, each bottle inoculation 1-2 stem section, puts into culturing room and cultivates after inoculation;
Described medium is: MS+1.5mg/L6-BA+0.3mg/LNAA+ sucrose 30g/L+ agar powder 6.5g/L, and pH value is adjusted to 5.6-5.8, and the medium prepared is divided in the wide-mouth triangular flask of 500mL, high-temperature sterilization 20min;
Add antibiotic constituent in medium and concentration as follows:
Ampicillin: 200mg/L;
Streptomycin sulphate: 150mg/L.
2. the method for reduction tea tree tissue culture procedures China and foreign countries according to claim 1 implant body pollution, it is characterized in that: described containing antibiotic medium be by antibiotic after the sterilizing of millipore filter suction filtration, join and be equipped with in the wide-mouth triangular flask of medium, be dispensed into again in blake bottle after shaking up.
3. the method for reduction tea tree tissue culture procedures China and foreign countries according to claim 1 implant body pollution, is characterized in that: condition of culture is: temperature 23-25 DEG C, intensity of illumination 1500-2000lx, light application time 12h/d, cultivates 30d-35d.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107912302A (en) * | 2017-12-04 | 2018-04-17 | 湖北省农业科学院果树茶叶研究所 | A kind of method for reducing seed contamination in tea seed tissue cultures |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104365478A (en) * | 2014-10-17 | 2015-02-25 | 安徽省农业科学院茶叶研究所 | Primary culture method capable of simultaneously reducing browning and pollution of tea tree axillary bud |
| CN105684905A (en) * | 2016-02-05 | 2016-06-22 | 王少荣 | Method for remedying bacterial contamination of tissue culture seedlings of blueberry |
| CN105613299A (en) * | 2016-02-05 | 2016-06-01 | 王少荣 | Method for eliminating bacterial contamination of chrysanthemum tissue cultured seedlings |
| CN108703068B (en) * | 2018-04-04 | 2021-09-10 | 广西壮族自治区农业科学院生物技术研究所 | Method for removing endophyte in arrowhead culture process, culture method and application |
| CN108812320A (en) * | 2018-07-05 | 2018-11-16 | 济南亚龙生物科技有限公司 | A kind of tea tree detoxification method for tissue culture |
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| CN1428074A (en) * | 2001-12-27 | 2003-07-09 | 中国科学院武汉植物研究所 | Quick propagation method of promising strain line of tea |
| CN101884301A (en) * | 2010-04-08 | 2010-11-17 | 浙江省农业科学院 | Method for preventing pollution in plant tissue culture |
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| CN107912302A (en) * | 2017-12-04 | 2018-04-17 | 湖北省农业科学院果树茶叶研究所 | A kind of method for reducing seed contamination in tea seed tissue cultures |
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