CN102893866A - Strawberry root tip detoxification and tissue culture method - Google Patents
Strawberry root tip detoxification and tissue culture method Download PDFInfo
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- CN102893866A CN102893866A CN2012103893767A CN201210389376A CN102893866A CN 102893866 A CN102893866 A CN 102893866A CN 2012103893767 A CN2012103893767 A CN 2012103893767A CN 201210389376 A CN201210389376 A CN 201210389376A CN 102893866 A CN102893866 A CN 102893866A
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Abstract
The invention discloses a strawberry root tip detoxification and tissue culture method, which comprises the following steps: 1) preparing a medium; 2) preprocessing a material and selecting and sterilizing a root tip explants; 3) culturing a root tip induced regenerated plant; 4) performing bud multiplication culture; 5) performing subculture growth culture; and 6) performing rooting culture. The strawberry root tip detoxification and tissue culture method can solve problems of low efficiency, not thorough disinfection, high contamination rate of tissue culture seedlings and the like in the traditional stem tip detoxification technology for strawberry tissue culture, the operating efficiency is high, the disinfection is thorough, and the contamination rate of the tissue culture seedlings is low.
Description
Technical field
The present invention relates to plant toxic and tissue culture technology field, the detoxification of especially a kind of strawberry tip of a root and tissue culture method.
Background technology
Strawberry is as a kind of economic crops, and the stem apex detoxify tissue-culturing rapid propagation of its seedling is a kind of conventional technology that adopts.But in the process of strawberry stem tip tissue-culturing rapid propagation, have that operation easier is large, inefficiency (at microscopically, peeling off the 0.02mm stem apex with operation tool), be difficult to thorough disinfection, the tissue culture seedling pollution rate is high.To at present, about adopting the technology of carrying out detoxification and group training take the tip of a root of strawberry as explant, there is not yet the reported in literature of mature technology.
Summary of the invention
Technical problem to be solved by this invention provides the detoxification of a kind of strawberry tip of a root and tissue culture method, can solve the problems such as the existing efficient of Strawberry Plantlets tradition Shoot-tip Grafting In Vitro is low, sterilization is difficult thoroughly, tissue culture seedling pollution rate height, operating efficiency is high, sterilization is thorough, the tissue culture seedling pollution rate is low.
For solving the problems of the technologies described above, the technical solution adopted in the present invention is: the detoxification of a kind of strawberry tip of a root and tissue culture method, and the method may further comprise the steps:
1) preparation of medium: take the MS medium as minimal medium, additional 6
–Benayl aminopurine 5～7.5mg/L, 2,4
–Make the pH value behind dichlorphenoxyacetic acid 0.3～0.5mg/L and be 5.8～6.0 tip of a root inducing culture;
Take the MS medium as minimal medium, additional 6
–Benayl aminopurine 5～7.5mg/L, a
–Make the pH value behind methyl α-naphthyl acetate 0.2～0.5mg/L and be 5.8～6.0 shoot proliferation medium;
Take the 1/2MS medium as minimal medium, make the pH value behind additional heteroauxin 0.2～0.5mg/L and be 5.8～6.0 root media;
2) the choosing, sterilize of pretreatment and tip of a root explant: strawberry is put into the sterilization fine sand under 38 ℃ ± 2 ℃, heat-treat 4～6 thoughtful pumpings root system that makes new advances; Get the long main root tip of a root of 1～2cm, blot surface moisture with aseptic filter paper, downcut as explant at the tip of a root of microscopically with the long 1～2mm of main root tip of a root front end;
3) tip of a root regeneration induction plant is cultivated: explant is seeded on the tip of a root inducing culture, under 28 ± 2 ℃, light intensity 800lux, 12 hours/d of illumination, cultivate, the constant light intensity of all the other conditions is strengthened to 1600lux after extremely, light intensity is strengthened to 3200lux after six weeks, light intensity decreasing continues cultivation until induce the regeneration plant young shoot from the tip of a root after eight weeks under 1200lux, and this stage need cultivate 2～3 months altogether;
4) young shoot propagation is cultivated: the plant young shoot is seeded on the shoot proliferation medium, at 28 ± 2 ℃, is cultured under light intensity 2000～3000lux, the 12 hours/d of illumination and grows up to the long seedling of 8～10cm;
5) subculture grown cultures: seedling is cut into the long segment of 2～2.5cm is seeded on the root media, at 28 ± 2 ℃, be cultured under light intensity 2000～3000lux, the 12 hours/d of illumination and grow up to the long seedling of 8～10cm; Every 3～4 weeks that seedling is numerous by carry out a subculture expansion with above-mentioned method later on, count requirement until satisfy seedling;
6) culture of rootage: the subculture seedling that increases is seeded on the root media, at 28 ± 2 ℃, cultivates under light intensity 2000～3000lux, the 12 hours/d of illumination, sending out roots after 1 week to be tied to form is complete test-tube plantlet.
Step 2) in, for putting into 70% alcohol immersion 30 seconds, be 10.5% liquor natrii hypochloritis sterilize 8 minute and aseptic washing 6 time with mass percentage concentration again to the method for long main root tip of a root sterilization.
The composition of MS medium is (mg/L):
ⅰ）NH
4NO
3?33000、KNO
3?38000、CaCl
2·2H
2O?8800、MgSO
4·7H
2O?7400、KH
2PO
4?3400；
ⅱ）KI?166、H
3BO
3?1240、MnSO
4·4H
2O?4460、ZnSO
4·7H
2O?1720、Na
2·MoO
4·2H
2O?50、CuSO
4·5H
2O?5、CaCl
2·6H
2O?5；
ⅲ）FeSO
4·7H
2O?5560、Na
2·EDTA·2H
2O?7460；
To obtain the 1/2MS medium after the MS medium dilute with water twice that prepare.
A kind of strawberry tip of a root provided by the invention detoxification and tissue culture method, beneficial effect is as follows:
1, processing ease, efficient significantly improves: because existing strawberry is adopted the stem apex detoxify group culturation rapid propagating technology of seedling, but in the fast numerous process of Shoot-tip Culture, peeling off the 0.02mm stem apex with operation tool is the larger and ineffective work of difficulty, and outside the tip of a root of strawberry is directly exposed to, the tip of excising its tip of a root at microscopically is just very easy, and efficient improves greatly.
2, sterilization thoroughly, the tissue culture seedling pollution rate is low: stem apex detoxify is because its peripheral stem sheet easy-clear not, and sterilization is difficult to thoroughly cause the tissue culture seedling pollution rate often up to about 60%; And tip of a root detoxification is sterilized easily thoroughly because it is peripheral without any parcel, and the tissue culture seedling pollution rate is low.
3, simplified operation, improved operating efficiency, and inoculum breaks up, the test-tube plantlet reproduction speed is fast, and detoxification is thorough, and strawberry grows fine.
Embodiment
The detoxification of a kind of strawberry tip of a root and tissue culture method, the method may further comprise the steps:
1) preparation of medium: take the MS medium as minimal medium, additional 6
–Benayl aminopurine 5～7.5mg/L, 2,4
–Make the pH value behind dichlorphenoxyacetic acid 0.3～0.5mg/L and be 5.8～6.0 tip of a root inducing culture;
Take the MS medium as minimal medium, additional 6
–Benayl aminopurine 5～7.5mg/L, a
–Make the pH value behind methyl α-naphthyl acetate 0.2～0.5mg/L and be 5.8～6.0 shoot proliferation medium;
Take the 1/2MS medium as minimal medium, make the pH value behind additional heteroauxin 0.2～0.5mg/L and be 5.8～6.0 root media;
2) the choosing, sterilize of pretreatment and tip of a root explant: strawberry is put into the sterilization fine sand under 38 ℃ ± 2 ℃, heat-treat 4～6 thoughtful pumpings root system that makes new advances; Get the long main root tip of a root of 1～2cm, blot surface moisture with aseptic filter paper, downcut as explant at the tip of a root of microscopically with the long 1～2mm of main root tip of a root front end;
3) tip of a root regeneration induction plant is cultivated: explant is seeded on the tip of a root inducing culture, under 28 ± 2 ℃, light intensity 800lux, 12 hours/d of illumination, cultivate, the constant light intensity of all the other conditions is strengthened to 1600lux after extremely, light intensity is strengthened to 3200lux after six weeks, light intensity decreasing continues cultivation until induce the regeneration plant young shoot from the tip of a root after eight weeks under 1200lux, and this stage need cultivate 2～3 months altogether;
4) young shoot propagation is cultivated: the plant young shoot is seeded on the shoot proliferation medium, at 28 ± 2 ℃, is cultured under light intensity 2000～3000lux, the 12 hours/d of illumination and grows up to the long seedling of 8～10cm;
5) subculture grown cultures: seedling is cut into the long segment of 2～2.5cm is seeded on the root media, at 28 ± 2 ℃, be cultured under light intensity 2000～3000lux, the 12 hours/d of illumination and grow up to the long seedling of 8～10cm; Every 3～4 weeks that seedling is numerous by carry out a subculture expansion with above-mentioned method later on, count requirement until satisfy seedling;
6) culture of rootage: the subculture seedling that increases is seeded on the root media, at 28 ± 2 ℃, cultivates under light intensity 2000～3000lux, the 12 hours/d of illumination, sending out roots after 1 week to be tied to form is complete test-tube plantlet.
Step 2) in, for putting into 70% alcohol immersion 30 seconds, be 10.5% liquor natrii hypochloritis sterilize 8 minute and aseptic washing 6 time with mass percentage concentration again to the method for long main root tip of a root sterilization.
The composition of MS medium is (mg/L):
ⅰ）NH
4NO
3?33000、KNO
3?38000、CaCl
2·2H
2O?8800、MgSO
4·7H
2O?7400、KH
2PO
4?3400；
ⅱ）KI?166、H
3BO
3?1240、MnSO
4·4H
2O?4460、ZnSO
4·7H
2O?1720、Na
2·MoO
4·2H
2O?50、CuSO
4·5H
2O?5、CaCl
2·6H
2O?5；
ⅲ）FeSO
4·7H
2O?5560、Na
2·EDTA·2H
2O?7460；
To obtain the 1/2MS medium after the MS medium dilute with water twice that prepare.
Claims (2)
1. strawberry tip of a root detoxification and tissue culture method is characterized in that the method may further comprise the steps:
1) preparation of medium: take the MS medium as minimal medium, additional 6
–Benayl aminopurine 5～7.5mg/L, 2,4
–Make the pH value behind dichlorphenoxyacetic acid 0.3～0.5mg/L and be 5.8～6.0 tip of a root inducing culture;
Take the MS medium as minimal medium, additional 6
–Benayl aminopurine 5～0.75mg/L, a
–Make the pH value behind methyl α-naphthyl acetate 0.2～0.5mg/L and be 5.8～6.0 shoot proliferation medium;
Take the 1/2MS medium as minimal medium, make the pH value behind additional heteroauxin 0.2～0.5mg/L and be 5.8～6.0 root media;
2) the choosing, sterilize of pretreatment and tip of a root explant: strawberry is put into the sterilization fine sand under 38 ℃ ± 2 ℃, heat-treat 4～6 thoughtful pumpings root system that makes new advances; Get the long main root tip of a root of 1～2cm, blot surface moisture after the sterilization, the tip of a root of main root tip of a root front end being grown 1～2mm downcuts as explant;
3) tip of a root regeneration induction plant is cultivated: explant is seeded on the tip of a root inducing culture, under 28 ± 2 ℃, light intensity 800lux, 12 hours/d of illumination, cultivate, the constant light intensity of all the other conditions is strengthened to 1600lux after extremely, light intensity is strengthened to 3200lux after six weeks, light intensity decreasing continues cultivation until induce the regeneration plant young shoot from the tip of a root after eight weeks under 1200lux, and this stage need cultivate 2～3 months altogether;
4) young shoot propagation is cultivated: the plant young shoot is seeded on the shoot proliferation medium, at 28 ± 2 ℃, is cultured under light intensity 2000～3000lux, the 12 hours/d of illumination and grows up to the long seedling of 8～10cm;
5) subculture grown cultures: seedling is cut into the long segment of 2～2.5cm is seeded on the root media, at 28 ± 2 ℃, be cultured under light intensity 2000～3000lux, the 12 hours/d of illumination and grow up to the long seedling of 8～10cm; Every 3～4 weeks that seedling is numerous by carry out a subculture expansion with above-mentioned method later on, count requirement until satisfy seedling;
6) culture of rootage: the subculture seedling that increases is seeded on the root media, at 28 ± 2 ℃, cultivates under light intensity 2000～3000lux, the 12 hours/d of illumination, sending out roots after 1 week to be tied to form is complete test-tube plantlet.
2. a kind of strawberry tip of a root according to claim 1 detoxification and tissue culture method, it is characterized in that: step 2) in, for putting into 70% alcohol immersion 30 seconds, be 10.5% liquor natrii hypochloritis sterilize 8 minute and aseptic washing 6 time with mass percentage concentration again to the method for long main root tip of a root sterilization.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103348918A (en) * | 2013-07-19 | 2013-10-16 | 合肥瑞谷农业科技有限公司 | Efficient detoxification tissue cultivating method of strawberries |
| CN103430845A (en) * | 2013-08-13 | 2013-12-11 | 镇江市农业科学技术实业公司 | Strawberry tissue culturing method |
| CN104304006A (en) * | 2014-09-29 | 2015-01-28 | 苏州家和农业生物科技研究中心 | Method for breeding detoxified strawberry |
| CN106718884A (en) * | 2016-11-29 | 2017-05-31 | 青岛松良基因科技有限公司 | RADIX PRZEWALSKIAE TANGUTICAE seedling and its method for culturing seedlings |
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| JPH01256320A (en) * | 1988-04-07 | 1989-10-12 | Mitsubishi Heavy Ind Ltd | Method for propagating large amount of strawberry young seedling |
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| CN101849509A (en) * | 2010-06-29 | 2010-10-06 | 浙江省农业科学院 | Root tip detoxification and rapid propagation technology for purple potatoes |
| CN101946702A (en) * | 2010-08-13 | 2011-01-19 | 北京金六环农业园 | Special medium for tissue culture of strawberry stem tip and method thereof for producing detoxification seedlings |
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2012
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103348918A (en) * | 2013-07-19 | 2013-10-16 | 合肥瑞谷农业科技有限公司 | Efficient detoxification tissue cultivating method of strawberries |
| CN103430845A (en) * | 2013-08-13 | 2013-12-11 | 镇江市农业科学技术实业公司 | Strawberry tissue culturing method |
| CN104304006A (en) * | 2014-09-29 | 2015-01-28 | 苏州家和农业生物科技研究中心 | Method for breeding detoxified strawberry |
| CN106718884A (en) * | 2016-11-29 | 2017-05-31 | 青岛松良基因科技有限公司 | RADIX PRZEWALSKIAE TANGUTICAE seedling and its method for culturing seedlings |
| CN106718884B (en) * | 2016-11-29 | 2019-01-15 | 青岛松良基因科技有限公司 | RADIX PRZEWALSKIAE TANGUTICAE seedling and its method for culturing seedlings |
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