CN104813935A - Method for removing PVS viruses of potato test-tube plantlets - Google Patents
Method for removing PVS viruses of potato test-tube plantlets Download PDFInfo
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- CN104813935A CN104813935A CN201510187562.6A CN201510187562A CN104813935A CN 104813935 A CN104813935 A CN 104813935A CN 201510187562 A CN201510187562 A CN 201510187562A CN 104813935 A CN104813935 A CN 104813935A
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Abstract
The invention provides a method for removing PVS viruses of potato test-tube plantlets. The method comprises the steps of stripping the stem tips of the pretreated test-tube plantlets and then putting the test-tube plantlets into a stem tip culture medium with the stem tip facing upwards and the section facing downwards for culture, next, culturing the stem tips for 40-50 days under the conditions of a temperature within the range of 22+/-2 DEG C, the illumination time of 12h-15h/d and the illumination intensity of 2000-3000lx, in the period, transferring the stem tips above 0.5cm long to an MS culture medium, and continuously culturing the undifferentiated stem tip callus or small plantlets less than 0.5cm long, and finally, detecting the stem tip seedlings by use of enzyme linked immunosorbent assay, and remaining the satisfied stem tip seedlings, thereby obtaining the potato seedlings without PVS through the whole period of 120-180 days. The method is high in PVS virus removal rate, suitable for batch operations of removing the PVS viruses of the potato variety resources, and capable of obtaining the satisfied seedlings at a time and avoiding repeated labor; besides, the method is high in virus removal rate and stable in virus removal effect, and the detection cost can be reduced.
Description
Technical field
The invention belongs to technical field of agricultural seedling-raising, particularly relate to a kind of method removing potato test-tube plantlet PVS virus.
Background technology
Potatoes cultivated area has more than 570 ten thousand hm
2, occupy first place in the world, but the lower (15.45thm of yield level
-2), not only lower than the average per unit area yield (17.26thm in the world
-2), more not as good as World Developed Countries (50.25thm
-2).Virus disease harm is one of principal element affecting potatoes output, because potato is vegetative reproduction, virus accumulates year by year with potato breeding, cause potato seed sexual involution, research proves that Potyvirus causes the potato underproduction 30% ~ 50%, if different virus mixed infection, then can cause the production loss of more than 80%.It is reported, the virus of potato-infecting reaches more than 30 kinds, Chinese hazard ratio more serious mainly contain potato virus X (PVX) (underproduction 10 ~ 20%), marmor upsilon (PVY) (underproduction 50 ~ 80%), potato virus S (PVS) (underproduction 10 ~ 20%), marmor solani (PVA) (underproduction more than 40%), marmor angliae (PVM) (the weak strain underproduction 10 ~ 20%, the strong strain underproduction 60 ~ 70%) and corium solani (PLRV) (underproduction 40 ~ 70%).In potato test-tube plantlet and breeder's stock are produced, PVS endangers the heaviest one virus, and recall rate is the highest.
Detoxification technology the most frequently used is at present still traditional Shoot-tip Grafting In Vitro, the method is mainly carried out stem apex to the bud on the potato tubers of alternating temperature or high temperature treatment and is peeled off to obtain detoxic seedling, for most of virus, the method is convenient and effective, but is but difficult to obtain satisfied effect for the method the Potatoes carrying PVS.Removing of PVS requires higher to stem apex lift-off technology, the growing point (0.1 ~ 0.3mm) of 1 leaf primordium is only with just likely to take off PVS, and stripping stem apex size is proportionate with stem apex planting percent but becomes negative correlation with detoxification efficiency, the stem apex of the bud of potato tubers is relatively large, PVS from growing point very close to, more not easily remove, so same kind often carries out large-scale stem apex repeatedly for several years peel off the seedling that could obtain not with PVS, add working feature and testing cost, be difficult to meet Production requirement.The effect that same single application thermal treatment and virazole process remove potato test-tube plantlet PVS is also not obvious, also have combination of two in three kinds of technology to remove the processing method of PVS, but because the Temperature Treatment cycle is different, drug concentration is different and the not equal factor of method operating sequence, there is very big-difference in result, virus elimination rate 0 ~ 83% is not etc.
Summary of the invention
The object of the present invention is to provide a kind of method removing potato test-tube plantlet PVS virus, be intended to solve existing Shoot-tip Grafting In Vitro for the poor problem of the Potatoes treatment effect carrying PVS.
The present invention is achieved in that a kind of method removing potato test-tube plantlet PVS virus, comprises the following steps:
(1) stem apex of pretreatment test-tube plantlet is peeled off
The aseptically seedling point of the test-tube plantlet that clip length 0.5 ~ 1cm is pretreated, be placed in the culture dish being covered with wetting sterilizing filter paper, meristematic tissue stripping is carried out under anatomical lens, cutting length is the stem apex of 0.1 ~ 0.3mm with 1 ~ 2 leaf primordium, with stem apex upwards, cut prone direction and be placed in Shoot Tip Culture base and cultivate;
(2) Shoot Tip Culture
Under temperature 22 ± 2 DEG C, light application time 12h ~ 15h/d, intensity of illumination 2000 ~ 3000lx condition, by Shoot Tip Culture 40 ~ 50d, period, be transferred to by the stem sharp of more than length 0.5cm in MS medium, the seedling undifferentiated stem apex callus or length being less than 0.5cm continues to be transferred in Shoot Tip Culture base to be cultivated;
(3) the PVS Viral diagnosis of stem apex seedling
Adopt enzyme linked immunosorbent assay to detect to stem apex seedling, retain qualified stem apex seedling, whole cycle 120 ~ 180d can obtain the potato seed not with PVS.
Preferably, in step (1), described pretreatment specifically comprises: go top segment to be placed in the blake bottle of the MS solid culture medium of interpolation 30 ~ 40mg/L virazole and 10 ~ 20mg/L daminozide the test-tube plantlet after testing containing PVS, 15 every bottle, in temperature 23 ~ 25 DEG C, light application time 15h/d, 15 ~ 20d is cultivated under the condition of light intensity 2000 ~ 3000lx, reject the bottle seedling of bacterium and fungal contamination, when the longest root of newborn seedling reaches 0.5cm, blake bottle is transferred in illumination box and carries out alternating temperature alternate treatment, temperature 40 DEG C/6h and 25 DEG C/6h, light application time 12h/d, light intensity 2000 ~ 3000lx, after cultivating 60 ~ 80d, get seedling point and carry out stem apex stripping.
Preferably, in step (1), described Shoot Tip Culture base comprises following component: basal medium MS, 0.05mg/LNAA, 0.1mg/L6-BA, 0.2mg/LGA3,0.2mg/L calcium pantothenate, 30g/L sucrose and 6g/L agar.
Compared to the shortcoming and defect of prior art, the present invention has following beneficial effect: the method that the alternating temperature that integrated application of the present invention improves, chemical reagent and stem-apex Meristem culture combine removes potato test-tube plantlet PVS virus, and using modified stem apex special culture media cultivates stem apex, guarantee that the stem apex planting percent of intermittent warming seedling is more than 60%, whole detoxification cycle 120 ~ 180d can obtain the potato seed not with PVS.10 kind PVS removal efficiency averages are 66%, and the highest reaches 100%, are significantly higher than traditional stem apex stripping means.The present invention is applicable to batch operation potato germplasm resources being removed to PVS virus, disposablely can obtain qualified seedling, and avoid the duplication of labour, virus elimination rate is high, and detoxification efficiency is stablized, and reduces testing cost.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Remove a method for potato test-tube plantlet PVS virus, it is characterized in that, comprise the following steps:
(1) pretreatment of test-tube plantlet material
Top segment is gone to be placed in the blake bottle of the MS solid culture medium of interpolation 30 ~ 40mg/L virazole and 10 ~ 20mg/L daminozide the test-tube plantlet after testing containing PVS, 15 every bottle, in temperature 23 ~ 25 DEG C, light application time 15h/d, 15 ~ 20d is cultivated under light intensity 2000 ~ 3000lx condition, reject the bottle seedling of bacterium and fungal contamination, when the longest root of newborn seedling reaches 0.5cm, blake bottle is transferred in illumination box and carries out alternating temperature alternate treatment, temperature 40 DEG C/6h and 25 DEG C/6h, light application time 12h/d, light intensity 2000 ~ 3000lx, after cultivating 60 ~ 80d, get seedling point and carry out stem apex stripping,
(2) stem apex of pretreatment test-tube plantlet is peeled off
The seedling point of the test-tube plantlet aseptically using scissors clip length 0.5 ~ 1cm pretreated, be placed in the culture dish being covered with wetting sterilizing filter paper, meristematic tissue stripping is carried out under 30 ~ 40 times of anatomical lens, cutting length is the stem apex of 0.1 ~ 0.3mm with 1 ~ 2 leaf primordium, with stem apex upwards, cut prone direction and be placed in stem apex special culture media and cultivate, Shoot Tip Culture base is the stem apex one-step culture base MS+0.05mg/LNAA+0.1mg/L6-BA+0.2mg/LGA3+0.2mg/L calcium pantothenate+30g/L sucrose+6g/L agar of improvement.Stem apex peels off hourly velocity must be fast, and must by bud point or the sharp filter paper being placed on aseptic humidity of seedling, in case the immature stem apex dehydration of peeling off is dead;
(3) Shoot Tip Culture
Shoot Tip Culture temperature controls in (22 ± 2) DEG C, light application time 12h ~ 15h/d, intensity of illumination 2000 ~ 3000lx.To the stem sharp of more than length 0.5cm be transferred in MS medium during Shoot Tip Culture 40 ~ 50d, the seedling undifferentiated stem apex callus or length being less than 0.5cm continues to be transferred in Shoot Tip Culture base to be cultivated, avoid the newborn seedling of stem apex to cause death because of poor nutritional old and feeble or pollution in advance, must reject in switching process by the abnormal stem sharp of bacterium, fungal contamination and growth.In addition, in the process of stem apex switching, avoid different stem apex cross-infection, the numbering plan of strict implement stem sharp " No. one, a point ".
(4) the PVS Viral diagnosis of stem apex seedling
Adopt enzyme linked immunosorbent assay (ELISA) to detect to stem apex seedling, retain qualified stem apex seedling, whole cycle 120 ~ 180d can obtain the potato seed not with PVS.
The method that the alternating temperature that integrated application of the present invention improves, chemical reagent and stem-apex Meristem culture combine removes potato test-tube plantlet PVS virus, and using modified stem apex special culture media cultivates stem apex, guarantee that the stem apex planting percent of intermittent warming seedling is more than 60%, whole detoxification cycle 120 ~ 180d can obtain the potato seed not with PVS.10 kind PVS removal efficiency averages are 66%, and the highest kind reaches 100%, are significantly higher than traditional stem apex stripping means.The present invention is applicable to batch operation potato germplasm resources being removed to PVS virus, disposablely can obtain qualified seedling, and avoid the duplication of labour, virus elimination rate is high, and detoxification efficiency is stablized, and reduces testing cost.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (3)
1. remove a method for potato test-tube plantlet PVS virus, it is characterized in that, comprise the following steps:
(1) stem apex of pretreatment test-tube plantlet is peeled off
The aseptically seedling point of the test-tube plantlet that clip length 0.5 ~ 1cm is pretreated, be placed in the culture dish being covered with wetting sterilizing filter paper, meristematic tissue stripping is carried out under anatomical lens, cutting length is the stem apex of 0.1 ~ 0.3mm with 1 ~ 2 leaf primordium, with stem apex upwards, cut prone direction and be placed in Shoot Tip Culture base and cultivate;
(2) Shoot Tip Culture
Under temperature 22 ± 2 DEG C, light application time 12h ~ 15h/d, intensity of illumination 2000 ~ 3000lx condition, by Shoot Tip Culture 40 ~ 50d, period, be transferred to by the stem sharp of more than length 0.5cm in MS medium, the seedling undifferentiated stem apex callus or length being less than 0.5cm continues to be transferred in Shoot Tip Culture base to be cultivated;
(3) the PVS Viral diagnosis of stem apex seedling
Adopt enzyme linked immunosorbent assay to detect to stem apex seedling, retain qualified stem apex seedling, whole cycle 120 ~ 180d can obtain the potato seed not with PVS.
2. remove the method for potato test-tube plantlet PVS virus as claimed in claim 1, it is characterized in that, in step (1), described pretreatment specifically comprises: go top segment to be placed in the blake bottle of the MS solid culture medium of interpolation 30 ~ 40mg/L virazole and 10 ~ 20mg/L daminozide the test-tube plantlet after testing containing PVS, 15 every bottle, in temperature 23 ~ 25 DEG C, light application time 15h/d, 15 ~ 20d is cultivated under the condition of light intensity 2000 ~ 3000lx, reject the bottle seedling of bacterium and fungal contamination, when the longest root of newborn seedling reaches 0.5cm, blake bottle is transferred in illumination box and carries out alternating temperature alternate treatment, temperature 40 DEG C/6h and 25 DEG C/6h, light application time 12h/d, light intensity 2000 ~ 3000lx, after cultivating 60 ~ 80d, get seedling point and carry out stem apex stripping.
3. remove the method for potato test-tube plantlet PVS virus as claimed in claim 1, it is characterized in that, in step (1), described Shoot Tip Culture base comprises following component: basal medium MS, 0.05mg/LNAA, 0.1mg/L6-BA, 0.2mg/LGA3,0.2mg/L calcium pantothenate, 30g/L sucrose and 6g/L agar.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105493972A (en) * | 2015-12-04 | 2016-04-20 | 中国农业科学院柑桔研究所 | Method for efficiently removing CTLV (citrus tatter leaf virus) |
| CN105532479A (en) * | 2016-02-04 | 2016-05-04 | 内蒙古农业大学 | Potato stem tip differentiation medium and preparation method thereof |
| CN105532455A (en) * | 2015-12-16 | 2016-05-04 | 王伟 | Nanometer seedling growing method for potato shoot tip |
| 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 |
| CN107568066A (en) * | 2017-10-10 | 2018-01-12 | 四川省农业科学院作物研究所 | A kind of potato stem tip detoxification method |
| CN109329026A (en) * | 2018-11-30 | 2019-02-15 | 青岛农业大学 | A kind of method that high temperature-intermittent warming obtains Sweetpotato Viruses Elimination seedling |
| CN109463067A (en) * | 2018-11-27 | 2019-03-15 | 甘肃省农业科学院马铃薯研究所 | A method of improving Potyvirus removal efficiency |
| CN115281089A (en) * | 2022-08-08 | 2022-11-04 | 镇江金豆豆种业有限公司 | Method for improving potato virus disease removal efficiency |
| CN116267622A (en) * | 2023-04-24 | 2023-06-23 | 石家庄市农林科学研究院 | Method for detoxification and rapid seedling formation of potato stem tip |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105493972A (en) * | 2015-12-04 | 2016-04-20 | 中国农业科学院柑桔研究所 | Method for efficiently removing CTLV (citrus tatter leaf virus) |
| CN105532455A (en) * | 2015-12-16 | 2016-05-04 | 王伟 | Nanometer seedling growing method for potato shoot tip |
| CN105532479A (en) * | 2016-02-04 | 2016-05-04 | 内蒙古农业大学 | Potato stem tip differentiation medium and preparation method thereof |
| 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 |
| CN107568066A (en) * | 2017-10-10 | 2018-01-12 | 四川省农业科学院作物研究所 | A kind of potato stem tip detoxification method |
| CN107568066B (en) * | 2017-10-10 | 2020-03-31 | 四川省农业科学院作物研究所 | Potato stem tip detoxification method |
| CN109463067A (en) * | 2018-11-27 | 2019-03-15 | 甘肃省农业科学院马铃薯研究所 | A method of improving Potyvirus removal efficiency |
| CN109329026A (en) * | 2018-11-30 | 2019-02-15 | 青岛农业大学 | A kind of method that high temperature-intermittent warming obtains Sweetpotato Viruses Elimination seedling |
| CN115281089A (en) * | 2022-08-08 | 2022-11-04 | 镇江金豆豆种业有限公司 | Method for improving potato virus disease removal efficiency |
| CN115281089B (en) * | 2022-08-08 | 2023-04-18 | 镇江金豆豆种业有限公司 | Method for improving potato virus disease removal efficiency |
| CN116267622A (en) * | 2023-04-24 | 2023-06-23 | 石家庄市农林科学研究院 | Method for detoxification and rapid seedling formation of potato stem tip |
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