CN102599057A - Method for removing potato virus by virazole - Google Patents
Method for removing potato virus by virazole Download PDFInfo
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- CN102599057A CN102599057A CN2012100781717A CN201210078171A CN102599057A CN 102599057 A CN102599057 A CN 102599057A CN 2012100781717 A CN2012100781717 A CN 2012100781717A CN 201210078171 A CN201210078171 A CN 201210078171A CN 102599057 A CN102599057 A CN 102599057A
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Abstract
The invention discloses relates to a method for removing plant viruses by virus inhibitor, in particular to a method for removing potato virus by virazole. The method comprising the step of inoculating potato seedlings infected with the virus on the culture medium containing virazole to cultivate for 45-135 days, wherein the concentration of the virazole in the culture medium is 75-150mg/L. The method for removing potato virus by virazole is simple and easy, is convenient to operate, has high removal efficiency for normally potato viruses, can be used during the production, is convenient to treat material in large volumes and is an efficient potato virus removal technology.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method that adopts viral inhibitors to remove plant virus, particularly a kind of method that adopts virazole to remove several frequently seen Potyvirus.
Background technology
Potato culture distributes wider, is the fourth-largest in the world raise crop.Chinese then be the maximum country of potato culture area, potato occupies critical role in the industrial and agricultural production of China, and the potato planting industry of development China then has the important strategic meaning.Yet potato is as asexually propagated crop, the various viruses of its easy infection.Through discovering, kind surplus the virus that the harm potato grows mainly contains 30 is wherein at China's ubiquity and endanger serious having: corium solani (PLRV); Marmor upsilon (PVY); Potato virus X (PVX), marmor solani (PVA), potato virus S (PVS); Marmor angliae (PVM), potato viroids virus or viroidss such as (PSTVd).The accumulation of potato body inner virus can make strain kind sexual involution, and quality and quality reduce, and its growth is caused serious harm.
Have only and adopt modern biotechnology that the virus in the potato seed is removed, recover the physiological function and the industry characteristics of potato kind itself, just can prevent the degeneration of potato, make it to reach the commodity property and the output of kind at the beginning of the cultivation of breeding man.
At present, Potyvirus removal methods commonly used comprises following three kinds:
(1) physical method.Utilize some physical factors such as X ray, ultraviolet ray, processing potato seeds such as ultrashort wave and high temperature or freezing processing make viral passivation, can reach to remove the purpose that virus obtains virus-free seed potato.Wherein the method with thermal treatment passivation virus is the most effective, handles 56 days down at 35 ℃, or handles 39 days at 36 ℃, can remove the PLRV in some potato kind stem tuber.Its advantage is simple to operate, handles large quantities of potato seeds in the short time.Shortcoming is can not eradicate most of viruses, and significant limitation is arranged, and detoxification efficiency is undesirable, can cause material withered or dead simultaneously.
(2) shoot apical meristem detoxification technology.Using the most extensively and in agricultural production and commodity production, obtain the plant toxic technology of immense success at present, is that the plant shoot apical meristem in the biotechnology is cultivated detoxification technology.Degree according to the detoxification difficulty or ease can be arranged as: PLRV＜PVA＜PVY＜PAMV＜PVM＜PVX＜PVS＜PVTVd.But this method operation easier is big, with high content of technology.
(3) chemical method.Research recently shows; Some chemical agents can remove the just virus in the growing plants body effectively; Its principle is that viral inhibitors can stop the viral RNA cap sequence to form under the triphosphoric acid state; Thereby influence duplicating of viral RNA, the normal growth of viral interference reaches the purpose except that virus removal.Common viral inhibitors has at present: ribavirin (virazole), 5-dihydrouracil (DHT), biacetyl-dihydro-5-AzU (DA-DHT) etc.In practical application, people often directly are added to these medicines on the medium of normal plant growth, in order to prophylaxis of viral infections plant itself.Yet the adding of these viral inhibitors has also produced murder by poisoning to plant itself, even under low-down concentration conditions, growing of plant also can be had a strong impact on.Therefore, adopt viral inhibitors to prevent the method for the normal plant infective virus not science that seems very in advance.
Summary of the invention
In view of the deficiency of prior art, the inventor provides a kind of method that adopts virazole to remove Potyvirus through the pluses and minuses of the various poison-removing methods of comprehensive study.This method can the multiple common Potyvirus of effective elimination, and phenomenon can not appear recovering in detoxification plant inner virus content for a long time, and is simultaneously simple, easy to operate, can be widely used in nontoxic plantation and production behind the potato detoxicating.
The objective of the invention is to realize like this:
A kind of method that adopts virazole to remove Potyvirus is characterized in that: the potato set of infective virus is inoculated on the medium that contains virazole cultivated 45-135 days, and the concentration of virazole in said medium is 75-150mg/L.
Preferably, said employing virazole removes the method for Potyvirus, the potato set of infective virus is inoculated on the medium that contains virazole cultivated 90-135 days.
Preferably, said employing virazole removes the method for Potyvirus, and the concentration of virazole in said medium is 75-100mg/L.
Preferably, said employing virazole removes the method for Potyvirus, and the composition of said medium is: the MS+ virazole.
Preferably, said employing virazole removes the method for Potyvirus, and described condition of culture is: intensity of illumination is 50-80mol/m
2/ s, light application time is 16h/D, cultivation temperature is 20 ℃.
Further preferably, described virus is selected from following one or more: corium solani (PLRV), marmor upsilon (PVY); Potato virus X (PVX); Marmor solani (PVA), potato virus S (PVS), marmor angliae (PVM) and potato viroids (PSTVd).
Compared with prior art, the invention property ground adopts virazole to remove Potyvirus, has found out the best working concentration of virazole through a large amount of tests; The several frequently seen Potyvirus of effective elimination in enormous quantities; The virus removal efficiency is high, and phenomenon can not appear recovering in detoxification plant inner virus content for a long time, and is simultaneously simple; Easy to operate, can be widely used in nontoxic plantation and production behind the potato detoxicating.
Description of drawings
Fig. 1 PVX virazole is handled 135 days viral levels and later stage recovery situation
PVX test- tube plantlet 1,2,3 expression was handled 135 days and the potato test-tube plantlet of these 3 test-tube plantlet first segments after being inoculated into successive transfer culture in the common MS medium 3 times through virazole.
Fig. 2 PVM virazole was handled 135 days and the later stage is recovered the viral level situation
PVM test- tube plantlet 1,2,3 expression was handled 135 days and the potato test-tube plantlet of these 3 test-tube plantlet first segments after being inoculated into successive transfer culture in the common MS medium 3 times through virazole.
Fig. 3 PVS virazole was handled 135 days and the later stage is recovered the viral level situation
PVS test- tube plantlet 1,2,3 expression was handled 135 days and the potato test-tube plantlet of these 3 test-tube plantlet first segments after being inoculated into successive transfer culture in the common MS medium 3 times through virazole.
Fig. 4 PLRV virazole was handled 135 days and the later stage is recovered the viral level situation
PLRV test- tube plantlet 1,2,3 expression was handled 135 days and the potato test-tube plantlet of these 3 test-tube plantlet first segments after being inoculated into successive transfer culture in the common MS medium 3 times through virazole.
Fig. 5 PVA virazole was handled 135 days and the later stage is recovered the viral level situation
PVA test- tube plantlet 1,2,3 expression was handled 135 days and the potato test-tube plantlet of these 3 test-tube plantlet first segments after being inoculated into successive transfer culture in the common MS medium 3 times through virazole.
Embodiment
Below be specific embodiment of the present invention, technical scheme of the present invention is done further the description, but protection scope of the present invention is not limited to these embodiment.Every do not deviate from the change of the present invention design or be equal to substitute include within protection scope of the present invention.
The potato test-tube plantlet that infects PVX is inoculated in the MS medium of the virazole that contains finite concentration (0mg/L, 75mg/L, 100mg/L, 150mg/L, 200mg/L), is 50-80mol/m in intensity of illumination
2/ s; Light application time is 16h/D; Cultivation temperature is to cultivate in 20 ℃ the culturing room; A cultivation cycle is 45 days, after one-period the plant top is inoculated into down the cultivation of carrying out following one-period in the concentration medium together, and remaining plant part carries out that ELISA detects and plant plant height, fresh weight are measured (result sees table 1).The malicious source plant that the result shows independent infection PVX was handled 90 days during for 75mg/L-150mg/L in concentration, and viral removal efficiency can reach 100% (result sees table 6).
Table 1 virazole is handled plant height, the fresh weight of the potato test-tube plantlet variable concentrations that infects PVX
Mark: a, b, c are used for representing the significant difference between each data in the form.
The potato test-tube plantlet of the compound MSY of infecting is inoculated in the MS medium of the virazole that contains finite concentration (0mg/L, 75mg/L, 100mg/L, 150mg/L, 200mg/L), is 50-80mol/m in intensity of illumination
2/ s; Light application time is 16h/D; Cultivation temperature is to cultivate in 20 ℃ the culturing room; A cultivation cycle is 45 days, after one-period the plant top is inoculated into down the cultivation of carrying out following one-period in the concentration medium together, and remaining plant part carries out that ELISA detects and plant plant height, fresh weight are measured (result sees table 2).For the compound potato test-tube plantlet that infects MSY, each concentration between 50%-100%, is 0% to the removal efficiency of PVS, PVY to the removal efficiency of PVM when handling 45 days; When handling 90 days, each concentration between 50%-100%, between 40%-67%, is 0% to the removal efficiency of PVY to the removal efficiency of PVS to the removal efficiency of PVM; When handling 135 days, each concentration is 100% to the removal efficiency of PVM, PVS, is 33% (result sees table 7) to the removal efficiency of PVY.
Table 2 virazole is handled compound plant height, the fresh weight that infects the potato test-tube plantlet variable concentrations of YSM
Mark: a, b, c are used for representing the significant difference between each data in the form.
The potato test-tube plantlet that infects PVS is inoculated in the MS medium of the virazole that contains finite concentration (0mg/L, 75mg/L, 100mg/L, 150mg/L, 200mg/L), is 50-80mol/m in intensity of illumination
2/ s; Light application time is 16h/D; Cultivation temperature is to cultivate in 20 ℃ the culturing room; A cultivation cycle is 45 days, after one-period the plant top is inoculated into down the cultivation of carrying out following one-period in the concentration medium together, and remaining plant part carries out that ELISA detects and plant plant height, fresh weight are measured (result sees table 3).The result shows and infects separately when handling 90 days that its removal efficiency can reach more than 75%; When handling 135 days, each concentration all can remove PVS (result sees table 6) fully.
Table 3 virazole is handled plant height, the fresh weight of the potato test-tube plantlet variable concentrations that infects PVS
Mark: a, b, c are used for representing the significant difference between each data in the form.
Embodiment 4 virazoles remove PLRV
The potato test-tube plantlet that infects PLRV is inoculated in the MS medium that contains finite concentration (0mg/L, 75mg/L, 100mg/L, 150mg/L, 200mg/L) virazole, is 50-80mol/m in intensity of illumination
2/ s; Light application time is 16h/D; Cultivation temperature is to cultivate in 20 ℃ the culturing room; A cultivation cycle is 45 days, after one-period the plant top is inoculated into down the cultivation of carrying out following one-period in the concentration medium together, and remaining plant part carries out that ELISA detects and plant plant height, fresh weight are measured (result sees table 4).The result shows the malicious source plant of infecting PLRV separately when handling 90 days, and concentration is 75mg/L, and the virazole of 100mg/L is 33% to the viral removal efficiency of this virus, and concentration is that 150mg/L can remove PLRV fully; When handling 135 days, 75mg/L, the virazole of 100mg/L reaches 100% (result sees table 6) to the removal efficiency of PLRV.
Table 4 virazole is handled plant height, the fresh weight of the potato test-tube plantlet variable concentrations that infects PLRV
Mark: a, b, c are used for representing the significant difference between each data in the form.
Embodiment 5 virazoles remove PVA
The potato test-tube plantlet that infects PVA is inoculated in the MS medium of the virazole that contains finite concentration (0mg/L, 75mg/L, 100mg/L, 150mg/L, 200mg/L), is 50-80mol/m in intensity of illumination
2/ s; Light application time is 16h/D; Cultivation temperature is to cultivate in 20 ℃ the culturing room; A cultivation cycle is 45 days, after one-period the plant top is inoculated into down the cultivation of carrying out following one-period in the concentration medium together, and remaining plant part carries out that ELISA detects and plant plant height, fresh weight are measured (result sees table 5).The result shows the malicious source plant of infecting PVA separately when handling 45 days, and concentration is that the virazole of 75mg/L is 33% to the viral removal efficiency of this virus, and concentration is that the viral removal efficiency of 100mg/L, 150mg/L is 66%; The virazole of 100mg/L, 150mg/L can remove PVA (result sees table 6) fully when handling 90 days.
Table 5 infects plant height, the fresh weight of the potato test-tube plantlet variable concentrations of PVA
Mark: a, b, c are used for representing the significant difference between each data in the form.
Table 6 virazole is handled the viral removal efficiency of the malicious source plant of infecting separately
Table 7 virazole is handled the viral removal efficiency of the malicious source plant of compound dip-dye
Need to prove that the potato virus-free plantlet that above embodiment obtains shows through the genetic stability analysis, gene mutation does not appear in the potato test-tube plantlet of handling through the inventive method; Simultaneously, the recovery situation testing result was after virus removed: phenomenon (result sees Fig. 1-5) does not appear recovering in plant inner virus content in 3 months later stages.
Claims (6)
1. adopt virazole to remove the method for Potyvirus, it is characterized in that: the potato set of infective virus is inoculated on the medium that contains virazole cultivated 45-135 days, and the concentration of virazole in said medium is 75-150mg/L.
2. method according to claim 1 is characterized in that: the potato set of infective virus is inoculated on the medium that contains virazole cultivated 90-135 days.
3. method according to claim 1 is characterized in that: the concentration of virazole in said medium is 75-100mg/L.
4. method according to claim 1 is characterized in that: the composition of said medium is: the MS+ virazole.
5. method according to claim 1 is characterized in that: described condition of culture is: intensity of illumination is 50-80mol/m
2/ s, light application time is 16h/D, cultivation temperature is 20 ℃.
6. according to each described method of claim 1-5, it is characterized in that: described virus is selected from following one or more: PVX, PVY, PVM, PVS, PLRV, PSTVd and PVA.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106718887A (en) * | 2016-11-30 | 2017-05-31 | 云南省农业科学院生物技术与种质资源研究所 | A kind of method of fast eliminating Potyvirus |
| CN110393154A (en) * | 2019-08-21 | 2019-11-01 | 徐州中农薯科农业发展有限公司 | A kind of fresh type Sweetpotato Viruses Elimination tissue-cultured seedling cultural method |
| CN110651709A (en) * | 2019-08-22 | 2020-01-07 | 桂林莱茵生物科技股份有限公司 | Method for improving detoxification efficiency of siraitia grosvenorii seedlings |
| CN111837949A (en) * | 2020-07-10 | 2020-10-30 | 佛山市粤山生物科技有限公司 | Contaminated seedling degerming culture medium for plant tissue culture and culture method |
| CN113317320A (en) * | 2021-04-26 | 2021-08-31 | 山东省农业科学院作物研究所 | Biological agent for improving virus-free rate of sweet potatoes and application thereof |
| CN115216486A (en) * | 2021-04-21 | 2022-10-21 | 浙江大学 | Negative strand RNA viral vector and plant genome editing method without transformation |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106718887A (en) * | 2016-11-30 | 2017-05-31 | 云南省农业科学院生物技术与种质资源研究所 | A kind of method of fast eliminating Potyvirus |
| CN110393154A (en) * | 2019-08-21 | 2019-11-01 | 徐州中农薯科农业发展有限公司 | A kind of fresh type Sweetpotato Viruses Elimination tissue-cultured seedling cultural method |
| CN110651709A (en) * | 2019-08-22 | 2020-01-07 | 桂林莱茵生物科技股份有限公司 | Method for improving detoxification efficiency of siraitia grosvenorii seedlings |
| CN111837949A (en) * | 2020-07-10 | 2020-10-30 | 佛山市粤山生物科技有限公司 | Contaminated seedling degerming culture medium for plant tissue culture and culture method |
| CN115216486A (en) * | 2021-04-21 | 2022-10-21 | 浙江大学 | Negative strand RNA viral vector and plant genome editing method without transformation |
| WO2022223010A1 (en) * | 2021-04-21 | 2022-10-27 | 浙江大学 | Negative-strand rna viral vector and plant genome editing method without transformation |
| CN115216486B (en) * | 2021-04-21 | 2024-04-16 | 浙江大学 | Negative strand RNA virus vector and plant genome editing method without transformation |
| CN113317320A (en) * | 2021-04-26 | 2021-08-31 | 山东省农业科学院作物研究所 | Biological agent for improving virus-free rate of sweet potatoes and application thereof |
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Application publication date: 20120725 |