CN104823851A - Cherry seedling ultralow temperature virus eliminating method - Google Patents
Cherry seedling ultralow temperature virus eliminating method Download PDFInfo
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- CN104823851A CN104823851A CN201510229959.7A CN201510229959A CN104823851A CN 104823851 A CN104823851 A CN 104823851A CN 201510229959 A CN201510229959 A CN 201510229959A CN 104823851 A CN104823851 A CN 104823851A
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Abstract
The invention discloses a cherry seedling ultralow temperature virus eliminating method. The method comprises the following steps: (1) obtaining cherry seedlings through a layering technology; (2) cutting stem tips with a length of about 1 mm from single buds of cherry seedlings; (3) pre-culturing the stem tips on a solid culture medium for 3d, and then culturing tissue-cultured seedlings for 30 to 35 days at a temperature of 26 to 42 DEG C under a light strength of 1000-2000 lx; (4) pre-culturing and loading; (5) carrying out vitrification; (6) freezing; (7) defrosting after freezing, then carrying out a recovery treatment for 1 to 2 times, and preparing for inductive seedling; (8) carrying out inductive seedling; (9) detecting the virus. The provided method can overcome the shortages of conventional methods, the vitrification and ultralow temperature preservation can eliminate the cherry seedling viruses, the virus removing rate is 90.6%, while the virus removing rate of conventional stem tip culture is 63.5%. Compared with the prior art, the provided method has the advantages of higher virus removing rate, bigger stem tip, and convenient and simple operation.
Description
Technical field
The present invention relates to fruit tree virus Prevention Technique field, specifically belong to a kind of cherry seedling ultralow temperature treatment method for detoxication.
Background technology
Cherry is China's current adjustment planting fruit trees structure, realizes the first-selected seeds of increasing peasant income.Virus disease has a strong impact on the seed output and quality of sweet cherry, has become one of major obstacle of restriction China cherry industry development.Prunus necrotic ring spot virus is that the stone fruits virus the most general, harm is more serious occurs, and can cause the decline of tree body, yellowing leaf or necrosis, florescence postpone, the fructescence postpones or the underproduction, have no harvest time serious.According to investigations, in China's sweet cherry cultural area, PNRSV generally occurs, and the ground such as Shandong, Liaoning, Beijing all detect that PNRSV infects.Zong Xiaojuan etc. identify display to sweet cherry production garden, Shandong virus disease RT-PCR, and PNRSV is with malicious rate to be 46.8%.
Existing employing super low temperature technology, techniques for ultra-low temperature preserve a whole set of biotechnology of plant germplasm under referring to the condition of ultralow temperature below-80 DEG C.Because liquid nitrogen temperature can reach-196 DEG C, and economical convenient, be commonly used to the Excised Embryos of plant at present.At present, Excised Embryos enjoys people to pay close attention to as a kind of new detoxification technology, and is confirmed in various plants.1997, Brison etc. were with Excised Embryos in conjunction with shoot tip in vitro culture, and successfully eliminate the plumpox virus on Prunus root-like stock, virus elimination rate reaches 50%, more than the virus elimination rate of simple Shoot Tip Culture more than 2 times.
But traditional Shoot Tip Culture and combined with heat treatment Shoot Tip Culture detoxification efficiency extremely low.Dai Hongyan etc. prove that sweet cherry test-tube plantlet Micro-stem tip is cultivated and effectively can not be removed PNRSV.(2) complicated operation, inefficiency.Tradition Shoot Tip Culture requires that stripping stem apex is less than 0.5mm, and operation easier is large, and seedling efficiency is low.
Summary of the invention
For the problems referred to above, the object of this invention is to provide a kind of cherry seedling ultralow temperature treatment method for detoxication, overcome the deficiencies in the prior art, cryopreservation by vitrification can remove cherry seedling diseases poison, and virus elimination rate is 90.6%.And the Shoot Tip Culture of routine, virus elimination rate is only 63.5%.Not only virus elimination rate is high, and stem apex is drawn materials large, simple to operation.
The technical solution used in the present invention is as follows:
A kind of cherry seedling ultralow temperature treatment method for detoxication, comprises step as follows: 1) get seedling and take the method for layer to obtain cherry seedling;
2) cut and obtain the stem apex that cherry seedling simple bud is about 1mm;
3) taro with red buds stem apex preculture 3d on additional solid medium, plantlet in vitro is cultivated 30 ~ 35 days under temperature 26 ~ 42 DEG C, light intensity 1000-2000lx condition;
4) preculture, loading: by step 3) plantlet in vitro after thermal treatment strips the stem-tip tissue of 1 ~ 2mm at superclean bench, by stem-tip tissue preculture 3 ~ 5 days, then load 20 ~ 30min through room temperature;
5) vitrifying: laden stem-tip tissue goes to ice bath process 60 ~ 90min in cryoprotection solution PVS2,
6) freezing: the stem-tip tissue after vitrifying process divides and is filled in cryopreservation tube, cryovial is dropped into rapidly freezing 20 ~ 24h in liquid nitrogen;
7) thaw after freezing, then carry out Recovery processing 1-2 time, then carry out induction seedling;
8) seedling is induced: the stem-tip tissue after Recovery processing goes to MS and to increase in seedling medium first light culture 3 ~ 7 days, then proceeds to illumination cultivation, squamous subculture after seedling,
9) Viral diagnosis: adopt RT-PCR to detect seedling to be measured, filter out detoxic seedling, to the plant containing this virus, allocation stem apex does Initial culture, and subculture expands after numerous 4 times, the stem apex of allocation about 1mm, vitrification ultra-low temperature liquid nitrogen treatment technology is utilized to remove SMYEV, in viral subtractive process, load and be treated to 20 DEG C, 1h; Vitrifying is treated to 0 DEG C, 2h; After liquid nitrogen process 1h, carry out 45 DEG C of water-bath 2min.
Described step 4) by stem-tip tissue preculture 2 days in the liquid nutrient medium of interpolation 0.2 ~ 0.3mol/L sucrose concentration, stem-tip tissue after preculture is processed 28 ~ 30min in loading liquid under room temperature, and described loading liquid is the glycerine of interpolation 1 ~ 3mol/L and the liquid nutrient medium of 0.3 ~ 0.5mol/L sucrose concentration.
Described described step 6), freezing step drops to-30 DEG C from 0 DEG C ,-35 DEG C or-40 DEG C, and cooling per minute 15 DEG C, immerses liquid nitrogen immediately, or drops to-196 DEG C continuously with this kind of speed.
Described described step 6), freezing step drops to-30 DEG C from 0 DEG C ,-35 DEG C or-40 DEG C, cooling per minute 15 DEG C, and constant temperature pre-freeze 1 hour, then immerses liquid nitrogen afterwards, or drops to-196 DEG C continuously with this kind of speed.
Compared with the prior art, beneficial effect of the present invention is as follows:
The present invention can remove cherry seedling diseases poison by cryopreservation by vitrification, and virus elimination rate is 90.6%, and the Shoot Tip Culture of routine, virus elimination rate is only 63.5%.Therefore, adopt cryopreservation by vitrification detoxicity method of the present invention to carry out detoxification to cherry seedling, not only virus elimination rate is high, and stem apex is drawn materials large, simple to operation; Superfreeze and resuscitation process simplify, time saving and energy saving, efficiency is high, detoxification is thorough, cost is low, less demanding to instrument and equipment.
Embodiment
Embodiment 1, a kind of cherry seedling ultralow temperature treatment method for detoxication, comprises step as follows: 1) get seedling and take the method for layer to obtain cherry seedling;
2) cut and obtain the stem apex that cherry seedling simple bud is about 1mm;
3) taro with red buds stem apex preculture 3d on additional solid medium, plantlet in vitro is cultivated 30 ~ 35 days under temperature 26 ~ 42 DEG C, light intensity 1000-2000lx condition;
4) preculture, loading: by step 3) plantlet in vitro after thermal treatment strips the stem-tip tissue of 1 ~ 2mm at superclean bench, by stem-tip tissue preculture 3 ~ 5 days, then load 20 ~ 30min through room temperature;
5) vitrifying: laden stem-tip tissue goes to ice bath process 60 ~ 90min in cryoprotection solution PVS2,
6) freezing: the stem-tip tissue after vitrifying process divides and is filled in cryopreservation tube, cryovial is dropped into rapidly freezing 20 ~ 24h in liquid nitrogen;
7) thaw after freezing, then carry out Recovery processing 1-2 time, then carry out induction seedling;
8) seedling is induced: the stem-tip tissue after Recovery processing goes to MS and to increase in seedling medium first light culture 3 ~ 7 days, then proceeds to illumination cultivation, squamous subculture after seedling,
9) Viral diagnosis: adopt RT-PCR to detect seedling to be measured, filter out detoxic seedling, to the plant containing this virus, allocation stem apex does Initial culture, and subculture expands after numerous 4 times, the stem apex of allocation about 1mm, vitrification ultra-low temperature liquid nitrogen treatment technology is utilized to remove SMYEV, in viral subtractive process, load and be treated to 20 DEG C, 1h; Vitrifying is treated to 0 DEG C, 2h; After liquid nitrogen process 1h, carry out 45 DEG C of water-bath 2min.
Described step 4) by stem-tip tissue preculture 2 days in the liquid nutrient medium of interpolation 0.2 ~ 0.3mol/L sucrose concentration, stem-tip tissue after preculture is processed 28 ~ 30min in loading liquid under room temperature, and described loading liquid is the glycerine of interpolation 1 ~ 3mol/L and the liquid nutrient medium of 0.3 ~ 0.5mol/L sucrose concentration.
Described described step 6), freezing step drops to-30 DEG C from 0 DEG C ,-35 DEG C or-40 DEG C, and cooling per minute 15 DEG C, immerses liquid nitrogen immediately, or drops to-196 DEG C continuously with this kind of speed.
Embodiment 2, its other step is identical, wherein said step 6), freezing step drops to-30 DEG C from 0 DEG C ,-35 DEG C or-40 DEG C, cooling per minute 15 DEG C, and constant temperature pre-freeze 1 hour, then immerses liquid nitrogen afterwards, or drops to-196 DEG C continuously with this kind of speed.
Can remove cherry seedling diseases poison by cryopreservation by vitrification, virus elimination rate is 90.6%, and the Shoot Tip Culture of routine, virus elimination rate is only 63.5%.Therefore, adopt cryopreservation by vitrification detoxicity method of the present invention to carry out detoxification to cherry seedling, not only virus elimination rate is high, and stem apex is drawn materials large, simple to operation; Superfreeze and resuscitation process simplify, time saving and energy saving, efficiency is high, detoxification is thorough, cost is low, less demanding to instrument and equipment.
More than show and describe general principle of the present invention, principal character and advantage of the present invention.The technical staff of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (4)
1. a cherry seedling ultralow temperature treatment method for detoxication, is characterized in that: comprise step as follows: 1) get seedling and take the method for layer to obtain cherry seedling;
2) cut and obtain the stem apex that cherry seedling simple bud is about 1mm;
3) taro with red buds stem apex preculture 3d on additional solid medium, plantlet in vitro is cultivated 30 ~ 35 days under temperature 26 ~ 42 DEG C, light intensity 1000-2000lx condition;
4) preculture, loading: by step 3) plantlet in vitro after thermal treatment strips the stem-tip tissue of 1 ~ 2mm at superclean bench, by stem-tip tissue preculture 3 ~ 5 days, then load 20 ~ 30min through room temperature;
5) vitrifying: laden stem-tip tissue goes to ice bath process 60 ~ 90min in cryoprotection solution PVS2,
6) freezing: the stem-tip tissue after vitrifying process divides and is filled in cryopreservation tube, cryovial is dropped into rapidly freezing 20 ~ 24h in liquid nitrogen;
7) thaw after freezing, then carry out Recovery processing 1-2 time, then carry out induction seedling;
8) seedling is induced: the stem-tip tissue after Recovery processing goes to MS and to increase in seedling medium first light culture 3 ~ 7 days, then proceeds to illumination cultivation, squamous subculture after seedling,
9) Viral diagnosis: adopt RT-PCR to detect seedling to be measured, filter out detoxic seedling, to the plant containing this virus, allocation stem apex does Initial culture, and subculture expands after numerous 4 times, the stem apex of allocation about 1mm, vitrification ultra-low temperature liquid nitrogen treatment technology is utilized to remove SMYEV, in viral subtractive process, load and be treated to 20 DEG C, 1h; Vitrifying is treated to 0 DEG C, 2h; After liquid nitrogen process 1h, carry out 45 DEG C of water-bath 2min.
2. cherry seedling ultralow temperature treatment method for detoxication according to claim 1, it is characterized in that: described step 4) by stem-tip tissue preculture 2 days in the liquid nutrient medium of interpolation 0.2 ~ 0.3mol/L sucrose concentration, stem-tip tissue after preculture is processed 28 ~ 30min in loading liquid under room temperature, and described loading liquid is the glycerine of interpolation 1 ~ 3mol/L and the liquid nutrient medium of 0.3 ~ 0.5mol/L sucrose concentration.
3. cherry seedling ultralow temperature treatment method for detoxication according to claim 1, is characterized in that: described described step 6), freezing step drops to-30 DEG C from 0 DEG C,-35 DEG C or-40 DEG C, cooling per minute 15 DEG C, immerses liquid nitrogen immediately, or drops to-196 DEG C continuously with this kind of speed.
4. cherry seedling ultralow temperature treatment method for detoxication according to claim 1, it is characterized in that: described described step 6), freezing step drops to-30 DEG C from 0 DEG C,-35 DEG C or-40 DEG C, cooling per minute 15 DEG C, constant temperature pre-freeze 1 hour, then immerses liquid nitrogen afterwards, or drops to-196 DEG C continuously with this kind of speed.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111084046A (en) * | 2019-12-24 | 2020-05-01 | 河北理查德农业科技有限公司 | Cherry breeding method |
Citations (3)
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|---|---|---|---|---|
| CN104012406A (en) * | 2014-04-04 | 2014-09-03 | 大连大学 | Regeneration in-vitro method for sweet cherry variety wanhongzhu |
| CN104429976A (en) * | 2014-12-26 | 2015-03-25 | 山东省果树研究所 | Efficient detoxification method for sweet cherry rootstocks |
| CN104488854A (en) * | 2014-12-02 | 2015-04-08 | 山东省果树研究所 | Vitrification ultralow-temperature preservation method for prunus avium dwarf rootstock gisela |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104012406A (en) * | 2014-04-04 | 2014-09-03 | 大连大学 | Regeneration in-vitro method for sweet cherry variety wanhongzhu |
| CN104488854A (en) * | 2014-12-02 | 2015-04-08 | 山东省果树研究所 | Vitrification ultralow-temperature preservation method for prunus avium dwarf rootstock gisela |
| CN104429976A (en) * | 2014-12-26 | 2015-03-25 | 山东省果树研究所 | Efficient detoxification method for sweet cherry rootstocks |
Non-Patent Citations (1)
| Title |
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| 杨智等，: "超低温处理植物脱毒研究进展", 《北方园艺》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111084046A (en) * | 2019-12-24 | 2020-05-01 | 河北理查德农业科技有限公司 | Cherry breeding method |
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Application publication date: 20150812 |