CN1926965A - Process for expelling chrysanthemum flower viroid by cryogenic processing - Google Patents
Process for expelling chrysanthemum flower viroid by cryogenic processing Download PDFInfo
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- CN1926965A CN1926965A CN 200610048344 CN200610048344A CN1926965A CN 1926965 A CN1926965 A CN 1926965A CN 200610048344 CN200610048344 CN 200610048344 CN 200610048344 A CN200610048344 A CN 200610048344A CN 1926965 A CN1926965 A CN 1926965A
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- chrysanthemum
- low temperature
- bud
- viroids
- temperature treatment
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Abstract
The invention relates to a method for using low-temperature treatment to remove chrysanthemum virus. Wherein, it comprises low-temperature treatment, disinfection, cutting off and cultivating stem sharp, viroid detecting, cultivating neuter chrysanthemum. The invention can effectively remove chrysanthemum virus, with simple process and better effect.
Description
Technical field
The invention belongs to the purification and rejuvenation of plant and breed, specifically adopt low temperature treatment to remove the method for chrysanthemum viroids.
Background technology
The chrysanthemum viroids is a mycoplasma viruses, this virus high temperature outbreak, and the chrysanthemum plant that catches an illness dwarfing, the local chlorisis of blade or withered spot, withered, the whole strain death of blade when virus infections is serious, the incidence of disease reaches 20-30%.It is to remove the most frequently used method of plant virus at present that thermal treatment adds Shoot Tip Culture, and effect is better, but there is significant limitation in this method aspect the chrysanthemum detoxification, and promptly one is to remove the chrysanthemum viroids, the 2nd, and thermal treatment easily causes plant withered, dead.
Summary of the invention
The purpose of this invention is to provide a kind of method that adopts low temperature treatment to remove the chrysanthemum viroids.
Overall technology design of the present invention is:
Adopt low temperature treatment to remove the method for chrysanthemum viroids, comprise the steps:
(1) low temperature treatment: chrysanthemum is carried out printing opacity low temperature cultivate, temperature is-2 ℃-3 ℃ or 4 ℃-6 ℃, gets bud point, root turion bud or terminal bud that chrysanthemum has just been sprouted;
(2) disinfect: after the bud point, root turion bud or the terminal bud that obtain in the step (1) are cleaned, sterilization;
(3) cut Shoot Tip Culture;
(4) viroids detects: adopt the RT-PCR method to detect bottle seedling to be checked, obtain to remove the chrysanthemum clone of chrysanthemum viroids.
The tissue culture expanding propagation of (5) detoxification clone chrysanthemum seedling.
The process conditions of each processing step are respectively among the present invention:
Low temperature treatment in the step (1) is on the chrysanthemum root stubble of surviving the winter under field conditions (factors) earthing 3-5 centimetre, covers the transparent heat insulating overcover on it, when its root ground temperature during-2 ℃ of-3 ℃ of scopes, gets bud point or root turion bud that chrysanthemum has just been sprouted.
Because chrysanthemum is a perennial root class herbaceos perennial, in plant part death on the ground in North China winter, the growth of root germinating in next year sprouting according to the characteristic of chrysanthemum perennial root, utilizes the winter natural low temperature to handle.The time of handling is different because of the difference in area, is December-February in the North China.
Low temperature treatment in the step (1) can also select for use the artificial hypothermia to handle, and promptly selects the chrysanthemum test tube bottle seedling of robust growth to place the low temperature incubator of printing opacity, is under 4 ℃ of-6 ℃ of conditions in temperature, carries out 90-120 days low temperature cold treatment, gets terminal bud then.
Disinfecting in the step (2) is bud point, root turion bud or the terminal bud that step (1) is obtained, clean with suds, on superclean bench, used for 70% alcohol surface sterilizing 10-20 second successively, 0.1% mercuric chloride solution sterilization 1～3 minute, use aseptic water washing again 3～5 times, with sterilization filter paper exhaustion sprout surface moisture.
Cutting Shoot Tip Culture in the step (3) is the stem apex that cuts the bud point, root turion bud or the terminal bud that are obtained in the step (2), be inoculated on the medium that 1/3MS+6-BA1.0mg/kg+IBA0.02mg/kg+ sucrose 3%+ agar 5g/L forms and cultivate, obtain chrysanthemum group training clone to be checked, the pH of medium is 5.6-5.8, and the stem apex size is 0.2～0.4cm.Wherein 6-BA is the 6-benzylaminopurine, and IBA is indolebutyric acid (down together).
The tissue culture expanding propagation of detoxification clone chrysanthemum seedling is that the chrysanthemum of virus-free is after testing planted in a steady stream in the step (5), carries out differentiation and proliferation and cultivates.Differential medium consists of MS+6-BA1.0mg/kg+NAA0.2mg/kg+ sucrose 3%+ agar 5% in the tissue culture expanding propagation of wherein detoxification clone chrysanthemum seedling, its pH5.6-5.8, and 25～30 days subcultures are once on differential medium; Carry out culture of rootage when treating chrysanthemum bottle seedling length to 3～4cm, root media is 1/2MS+NAA1.0mg/kg+ sucrose 20g/L, its pH5.6-5.8, condition of culture: temperature 25-28 ℃, illumination 2000-3000Lx; The back hardening of taking root was transplanted in 20-30 days, obtained detoxification chrysanthemum seedling.Wherein NAA is methyl (down together).
The obtained technological progress of the present invention is:
Adopt low temperature that chrysanthemum is handled,, can remove the chrysanthemum viroids effectively in conjunction with Shoot Tip Culture, simple, the easy row of its process, and effect is better.And have certain guidance and reference value for removing the research of other plant viroids.
Embodiment
Below in conjunction with embodiment the present invention is further described:
Embodiment 1
Adopt low temperature treatment to remove the method for chrysanthemum viroids, comprise the steps:
(1) low temperature treatment: on the chrysanthemum root stubble of surviving the winter under field conditions (factors) earthing 3-5 centimetre, cover the little shed of plastics on it, when its root ground temperature during, get bud point or root turion bud that chrysanthemum has just been sprouted-2 ℃ of-3 ℃ of scopes.；
(2) disinfect: bud point, root turion bud or the terminal bud of step (1) acquisition, clean with suds, on superclean bench, used for 70% alcohol surface sterilizing 10-20 second successively, 0.1% mercuric chloride solution sterilization 1～3 minute, use aseptic water washing again 3～5 times, with sterilization filter paper exhaustion sprout surface moisture.；
(3) cut Shoot Tip Culture: the stem apex that cuts the bud point, root turion bud or the terminal bud that are obtained in the step (2), be inoculated on the medium that 1/3MS+6-BA1.0mg/kg+IBA0.02mg/kg+ sucrose 3%+ agar 5g/L forms and cultivate, obtain chrysanthemum group training clone to be checked, the pH of medium is 5.6-5.8, and the stem apex size is 0.2～0.4cm.
(4) viroids detects: adopt the RT-PCR method to detect bottle seedling to be checked, obtain to remove the chrysanthemum clone of chrysanthemum viroids.
The tissue culture expanding propagation of (5) detoxification clone chrysanthemum seedling: with the tissue culture expanding propagation of detoxification clone chrysanthemum seedling in the step (5) is that the chrysanthemum of virus-free is after testing planted in a steady stream, carries out differentiation and proliferation and cultivates.Wherein differential medium consists of MS+6-BA1.0mg/kg+NAA0.2mg/kg+ sucrose 3%+ agar 5%, its pH5.6-5.8, and 25～30 days subcultures are once on differential medium; Carry out culture of rootage when treating chrysanthemum bottle seedling length to 3～4cm, root media is 1/2MS+NAA1.0mg/kg+ sucrose 20g/L, its pH5.6-5.8, condition of culture: temperature 25-28 ℃, illumination 2000-3000Lx; The back hardening of taking root was transplanted in 20-30 days, obtained detoxification chrysanthemum seedling.
Embodiment 2
Step in the present embodiment (1) low temperature treatment is to select the chrysanthemum test tube bottle seedling of robust growth to place the low temperature incubator of printing opacity, is under 4 ℃ of-6 ℃ of conditions in temperature, carries out 90-120 days low temperature cold treatment, gets terminal bud then.All the other steps are with embodiment 1.
Claims (7)
1, adopt low temperature treatment to remove the method for chrysanthemum viroids, it is characterized in that comprising following processing step:
(1) low temperature treatment: chrysanthemum is carried out printing opacity low temperature cultivate, temperature is-2 ℃-3 ℃ or 4 ℃-6 ℃, gets bud point, root turion bud or terminal bud that chrysanthemum has just been sprouted;
(2) disinfect: after the bud point, root turion bud or the terminal bud that obtain in the step (1) are cleaned, sterilization;
(3) cut Shoot Tip Culture;
(4) viroids detects: adopt the RT-PCR method to detect bottle seedling to be checked, obtain to remove the chrysanthemum clone of chrysanthemum viroids.
The tissue culture expanding propagation of (5) detoxification clone chrysanthemum seedling.
2, employing low temperature treatment according to claim 1 removes the method for chrysanthemum viroids, it is characterized in that low temperature treatment in the described step (1) is on the chrysanthemum root stubble of surviving the winter under field conditions (factors) earthing 3-5 centimetre, cover the transparent heat insulating overcover on it, when its root ground temperature during, get bud point or root turion bud that chrysanthemum has just been sprouted-2 ℃ of-3 ℃ of scopes.
3, employing low temperature treatment according to claim 1 removes the method for chrysanthemum viroids, it is characterized in that low temperature treatment in the described step (1) is to select the chrysanthemum test tube bottle seedling of robust growth to place the low temperature incubator of printing opacity, in temperature is under 4 ℃ of-6 ℃ of conditions, carry out 90-120 days low temperature cold treatment, get terminal bud then.
4, remove the method for chrysanthemum viroids according to claim 1,2,3 described employing low temperature treatment, it is characterized in that disinfecting in the described step (2) is bud point, root turion bud or the terminal bud that step (1) is obtained, clean with suds, on superclean bench, used for 70% alcohol surface sterilizing 10-20 second successively, 0.1% mercuric chloride solution sterilization 1～3 minute, use aseptic water washing again 3～5 times, with sterilization filter paper exhaustion sprout surface moisture.
5, employing low temperature treatment according to claim 1 removes the method for chrysanthemum viroids, it is characterized in that cutting in the described step (3) Shoot Tip Culture is the stem apex that cuts the bud point, root turion bud or the terminal bud that are obtained in the step (2), be inoculated on the medium that 1/3MS+6-BA1.0mg/kg+IBA0.02mg/kg+ sucrose 3%+ agar 5g/L forms and cultivate, obtain chrysanthemum group training clone to be checked, the pH of medium is 5.6-5.8, and the stem apex size is 0.2～0.4cm.
6, employing low temperature treatment according to claim 1 removes the method for chrysanthemum viroids, it is characterized in that in the described step (5) that the tissue culture expanding propagation of detoxification clone chrysanthemum seedling is that the chrysanthemum of virus-free is after testing planted in a steady stream, carries out differentiation and proliferation and cultivates.
7, employing low temperature treatment according to claim 1 removes the method for chrysanthemum viroids, it is characterized in that differential medium consists of MS+6-BA1.0mg/kg+NAA0.2mg/kg+ sucrose 3%+ agar 5% in the tissue culture expanding propagation of detoxification clone chrysanthemum seedling in the described step (5), its pH5.6-5.8,25～30 days subcultures are once on differential medium; Carry out culture of rootage when treating chrysanthemum bottle seedling length to 3～4cm, root media is 1/2MS+NAA1.0mg/kg+ sucrose 20g/L, its pH5.6-5.8, condition of culture: temperature 25-28 ℃, illumination 2000-3000Lx; The back hardening of taking root was transplanted in 20-30 days, obtained detoxification chrysanthemum seedling.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2006100483445A CN100444721C (en) | 2006-09-26 | 2006-09-26 | Process for expelling chrysanthemum flower viroid by cryogenic processing |
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| CNB2006100483445A CN100444721C (en) | 2006-09-26 | 2006-09-26 | Process for expelling chrysanthemum flower viroid by cryogenic processing |
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| CN1926965A true CN1926965A (en) | 2007-03-14 |
| CN100444721C CN100444721C (en) | 2008-12-24 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105594590A (en) * | 2016-02-18 | 2016-05-25 | 广西南宁市葛根源原生草药种植农民专业合作社 | Method for cultivating rose polyploidy variety |
| CN111528092A (en) * | 2020-05-26 | 2020-08-14 | 浙江海丰花卉有限公司 | Culture method of chrysanthemum virus-free seedlings |
| CN114680011A (en) * | 2022-03-02 | 2022-07-01 | 浙江海丰生物科技股份有限公司 | Chrysanthemum variety rejuvenation method based on small-batch selection |
-
2006
- 2006-09-26 CN CNB2006100483445A patent/CN100444721C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105594590A (en) * | 2016-02-18 | 2016-05-25 | 广西南宁市葛根源原生草药种植农民专业合作社 | Method for cultivating rose polyploidy variety |
| CN111528092A (en) * | 2020-05-26 | 2020-08-14 | 浙江海丰花卉有限公司 | Culture method of chrysanthemum virus-free seedlings |
| CN111528092B (en) * | 2020-05-26 | 2021-12-14 | 浙江海丰生物科技股份有限公司 | Culture method of chrysanthemum virus-free seedlings |
| CN114680011A (en) * | 2022-03-02 | 2022-07-01 | 浙江海丰生物科技股份有限公司 | Chrysanthemum variety rejuvenation method based on small-batch selection |
| CN114680011B (en) * | 2022-03-02 | 2023-03-31 | 浙江海丰生物科技股份有限公司 | Chrysanthemum variety rejuvenation method based on small-batch selection |
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| CN100444721C (en) | 2008-12-24 |
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