CN113796312A - Method for ultra-low temperature detoxification of strawberry stem tips by droplet vitrification method - Google Patents
Method for ultra-low temperature detoxification of strawberry stem tips by droplet vitrification method Download PDFInfo
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- CN113796312A CN113796312A CN202010570753.1A CN202010570753A CN113796312A CN 113796312 A CN113796312 A CN 113796312A CN 202010570753 A CN202010570753 A CN 202010570753A CN 113796312 A CN113796312 A CN 113796312A
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- strawberry
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- 235000016623 Fragaria vesca Nutrition 0.000 title claims abstract description 27
- 235000011363 Fragaria x ananassa Nutrition 0.000 title claims abstract description 27
- 238000001784 detoxification Methods 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000004017 vitrification Methods 0.000 title claims abstract description 14
- 240000009088 Fragaria x ananassa Species 0.000 title 1
- 241000220223 Fragaria Species 0.000 claims abstract description 31
- 238000012258 culturing Methods 0.000 claims abstract description 10
- 230000008929 regeneration Effects 0.000 claims abstract description 10
- 238000011069 regeneration method Methods 0.000 claims abstract description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims abstract description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 21
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 17
- 229930006000 Sucrose Natural products 0.000 claims description 17
- 239000005720 sucrose Substances 0.000 claims description 17
- 239000012879 subculture medium Substances 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 10
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 6
- 229910052782 aluminium Inorganic materials 0.000 claims description 6
- 239000011888 foil Substances 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 239000002352 surface water Substances 0.000 claims description 3
- 238000010257 thawing Methods 0.000 claims description 3
- 238000005286 illumination Methods 0.000 claims description 2
- 238000009630 liquid culture Methods 0.000 claims description 2
- 241000700605 Viruses Species 0.000 abstract description 14
- 235000021012 strawberries Nutrition 0.000 abstract description 5
- 241000196324 Embryophyta Species 0.000 abstract description 4
- 230000001172 regenerating effect Effects 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 description 7
- 241000710036 Strawberry mild yellow edge virus Species 0.000 description 5
- 241000413494 Strawberry mottle virus Species 0.000 description 5
- 241000218632 Strawberry vein banding virus Species 0.000 description 5
- 241000947772 Strawberry crinkle virus Species 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for ultra-low temperature detoxification of strawberry stem tips by a droplet vitrification method, belonging to the technical field of strawberry detoxification. The method for ultralow-temperature detoxification of strawberry stem tips by a droplet vitrification method specifically comprises the following steps: taking a strawberry explant infected with virus, cutting a stem tip, pre-culturing, performing small droplet vitrification treatment, putting into liquid nitrogen, unfreezing, recovering and culturing, regenerating a plant and performing subculture. The method for ultralow-temperature detoxification of the stem tips of the strawberries by the droplet vitrification method has the advantages of simplicity in operation, short consumed time, high detoxification rate and high regeneration rate, and has good technical effect and application prospect.
Description
Technical Field
The invention belongs to the technical field of plant virology, and particularly relates to a method for removing strawberry viruses.
Background
Strawberries (Fragaia ananasa) are used as herbaceous plants, which are mainly propagated by means of stolon seedlings formed by mother plants as seedling. Once the strawberries are infected by virus, 100% of mother strains are directly transmitted to offspring, so that the serious variety degradation phenomenon is caused, the yield is generally reduced by 20% -80%, and the remarkable quality reduction is caused. Strawberries, once infected with virus, cannot be resolved by the necessary field management. The use of the virus-free seedlings is one of the most economical and effective means for preventing and treating the strawberry virus disease. However, the research and production of strawberry detoxification and rapid propagation in China are seriously disconnected, the conventional detoxification technology has low detoxification efficiency, and the production cost of the detoxified seedlings is high. Therefore, the method is very necessary for improving the strawberry detoxification efficiency, standardizing the high-efficiency and low-cost rapid propagation technology of the detoxified seedlings and reducing the cost of the detoxified seedlings. The strawberry stem tip ultra-low temperature therapy detoxification technology established by the research team in recent years can effectively solve the problems.
Disclosure of Invention
The research aims at the current situations that strawberry virus diseases are seriously damaged and the detoxification method is laggard in China, and provides a method for ultralow temperature detoxification of strawberry stem tips by a droplet vitrification method.
The invention can be realized by the following technical scheme: a method for ultralow-temperature detoxification of strawberry stem tips by a droplet vitrification method specifically comprises the following steps:
peeling off about 2mm of strawberry stem tips, culturing on a subculture medium for 1 day, transferring to a liquid culture medium containing glycerol and sucrose, culturing for another 1 day, transferring the stem tips to PVS2, treating on ice for 40 minutes, transferring to droplets on sterilized aluminum foil strips, then immersing in liquid nitrogen for freezing for 1 hour, and then quickly taking out the aluminum foil strips and immersing in a liquid sucrose culture medium for unfreezing. Thawing for 20min, taking out stem tip, sucking surface water with filter paper, transferring into subculture medium for regeneration culture, dark treatment for 3 days, and beginning 10 μmol s for 4 days-1m-2Culturing under weak light, and regenerating the stem tip in a new subculture medium under normal light after one week of regeneration.
The subculture medium comprises: MS +30g/L sucrose +7.5g/L agar +0.5 mg/L6-BA +0.1mg/L NAA, pH 5.8.
The formula of the culture medium containing the glycerol and the sucrose is as follows: MS +2.0M glycerol +0.8M sucrose.
The formula of the liquid sucrose culture medium is as follows: MS +1.2M sucrose.
The method for ultralow-temperature detoxification of the stem tips of the strawberries by the droplet vitrification method has the advantages of simplicity in operation, short consumed time, high detoxification rate and high regeneration rate, and has good technical effect and application prospect.
Drawings
FIG. 1 shows the results of RT-PCR detection of 4 viruses: A-D are respectively the detection results of strawberry mottle virus (SMoV), Strawberry Vein Banding Virus (SVBV), strawberry shrunken virus (SCV) and Strawberry Mild Yellow Edge Virus (SMYEV); m: DNA molecular weight standard; 1-10 are respectively 10 strawberry seedlings regenerated after ultralow temperature detoxification of stem tips; wherein the virus was removed for 10 samples of SMoV; for SVBV, samples Nos. 1-2 and 5-10 all removed the virus. The virus was removed for 10 samples of both SCV and SMYEV.
FIG. 2 shows the regenerated tissue culture seedling of strawberry for red color after ultra-low temperature detoxification of stem tip.
Detailed Description
The present invention is further illustrated below by way of examples for the understanding of the present invention, but the following examples do not limit the present invention.
The invention provides a method for ultra-low temperature detoxification of strawberry stem tips by a droplet vitrification method, which comprises the following steps: peeling 50 strawberry stem tips with the diameter of about 2mm, culturing on a subculture medium (MS +30g/L sucrose +7.5g/L agar +0.5 mg/L6-BA +0.1mg/L NAA, pH5.8) for 1 day, then culturing on an MS +2.0M glycerol +0.8M sucrose culture medium for another 1 day, transferring the stem tips into PVS2, treating on ice for 40 minutes, then transferring into droplets on a sterilized aluminum foil strip, then soaking into liquid nitrogen for freezing for 1 hour, and then quickly taking out the aluminum foil strip and soaking into the MS +1.2M sucrose culture medium for unfreezing. Thawing for 20min, taking out stem tip, sucking surface water with filter paper, transferring into subculture medium for regeneration culture, dark treatment for 3 days, and beginning 10 μmol s for 4 days-1m-2Culturing under weak light, and regenerating the stem tip in a new subculture medium under normal light after one week of regeneration. Counting the number of surviving seedlings after one week of regeneration, and calculating the survival rate. The result shows that 50 stem tips finally survive 26, and the survival rate is 52%.
And (3) continuously carrying out illumination culture on the surviving seedlings, carrying out subculture once after 3 weeks, carrying out subculture for 4 weeks after subculture, and carrying out RT-PCR detection on 4 strawberry viruses including SMoV, SVBV, SCV and SMYEV. The detection primer is synthesized by Beijing Olympic Biotechnology GmbH, and the specific primer name and sequence are shown in Table 1.
TABLE 14 detection primers for strawberry virus
The specific detection steps comprise the extraction of total RNA of a sample, RT-PCR and agarose gel electrophoresis detection. After detection, the detoxification rate of the 4 viruses is counted, and the results show that the detoxification rate of SMoV, SCV and SMYEV is 100%, and the detoxification rate of SVBV is 80%.
Claims (4)
1. A method for ultra-low temperature detoxification of strawberry stem tips by a droplet vitrification method is characterized by comprising the following steps: comprises the following steps: peeling off about 2mm of strawberry stem tips, culturing on a subculture medium for 3 days, transferring to a liquid culture medium containing glycerol and sucrose, culturing for 1 day again, transferring the stem tips to PVS2, treating on ice for 40 minutes, transferring to droplets on sterilized aluminum foil strips, then immersing in liquid nitrogen for freezing for 1 hour, and then quickly taking out the aluminum foil strips and immersing in a liquid sucrose culture medium for unfreezing. Thawing for 20min, taking out stem tip, sucking surface water with filter paper, transferring into subculture medium for regeneration culture, dark treatment for 3 days, and beginning 10 μmol s for 4 days-1m-2And (4) performing weak light culture, transferring the stem tip into a new subculture medium after regeneration for one week, and performing regeneration culture under normal illumination.
2. The method for ultra-low temperature detoxification of strawberry stem tips by droplet vitrification, according to claim 1, wherein: the formula of the subculture medium is as follows: MS +30g/L sucrose +7.5g/L agar +0.5 mg/L6-BA +0.1mg/L NAA, pH 5.8.
3. The method for ultra-low temperature detoxification of strawberry stem tips by droplet vitrification, according to claim 1, wherein: the formula of the culture medium containing the glycerol and the sucrose is as follows: MS +2.0M glycerol +0.8M sucrose.
4. The method for ultra-low temperature detoxification of strawberry stem tips by droplet vitrification, according to claim 1, wherein: the formula of the liquid sucrose culture medium is as follows: MS +1.2M sucrose.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010570753.1A CN113796312A (en) | 2020-06-15 | 2020-06-15 | Method for ultra-low temperature detoxification of strawberry stem tips by droplet vitrification method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010570753.1A CN113796312A (en) | 2020-06-15 | 2020-06-15 | Method for ultra-low temperature detoxification of strawberry stem tips by droplet vitrification method |
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| CN113796312A true CN113796312A (en) | 2021-12-17 |
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| CN202010570753.1A Pending CN113796312A (en) | 2020-06-15 | 2020-06-15 | Method for ultra-low temperature detoxification of strawberry stem tips by droplet vitrification method |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101418350A (en) * | 2008-10-28 | 2009-04-29 | 南京农业大学 | Method for removing strawberry light yellow edge virus by ultra low temperature technique |
| CN104604685A (en) * | 2015-01-31 | 2015-05-13 | 四川农业大学 | Ultralow temperature detoxification method for strawberry stem tip |
-
2020
- 2020-06-15 CN CN202010570753.1A patent/CN113796312A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101418350A (en) * | 2008-10-28 | 2009-04-29 | 南京农业大学 | Method for removing strawberry light yellow edge virus by ultra low temperature technique |
| CN104604685A (en) * | 2015-01-31 | 2015-05-13 | 四川农业大学 | Ultralow temperature detoxification method for strawberry stem tip |
Non-Patent Citations (7)
| Title |
|---|
| 吴昀等: "超低温保存植物种质资源的新途径――小滴玻璃化法", 《植物生理学报》 * |
| 吴雨霏等: "草莓超低温脱毒技术体系的优化探究", 《中国第八次草莓大会暨第十三届中国草莓文化节论文集》 * |
| 徐启红等: ""童子一号"草莓玻璃化法超低温保存技术研究", 《北方园艺》 * |
| 罗娅等: "超低温疗法在草莓病毒脱除中的应用", 《分子植物育种》 * |
| 蔡斌华等: "通过玻璃化超低温处理脱除草莓轻型黄边病毒(SMYEV)研究", 《果树学报》 * |
| 邱静: "草莓茎尖超低温疗法脱毒技术体系的建立", 《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》 * |
| 陈曦等: "‘福莓1号’草莓茎尖超低温脱毒技术", 《东南园艺》 * |
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Application publication date: 20211217 |