CN103283595B - Primary culture method of stem tip tissue of strawberry - Google Patents
Primary culture method of stem tip tissue of strawberry Download PDFInfo
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- CN103283595B CN103283595B CN201310182708.9A CN201310182708A CN103283595B CN 103283595 B CN103283595 B CN 103283595B CN 201310182708 A CN201310182708 A CN 201310182708A CN 103283595 B CN103283595 B CN 103283595B
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- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 229910052801 chlorine Inorganic materials 0.000 claims description 3
- 239000000460 chlorine Substances 0.000 claims description 3
- 230000000249 desinfective effect Effects 0.000 claims description 3
- 238000007598 dipping method Methods 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
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- 241000710036 Strawberry mild yellow edge virus Species 0.000 description 2
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to a primary culture method of stem tip tissue of strawberry. The method comprises the following steps of: (1) collecting stolon, wherein the stolon collecting time is 4-7 months, and the stolon of which the tip is between the un-spread leaf and a first spread leaf and which robustly grows is selected; (2) treating the stolon: cleaning and sterilizing the collected stolon, and carrying out low-temperature treatment under 3-8 DEG C, wherein the treatment time is 72h; (3) sterilizing and inoculating in a bechtop. The primary culture method provided by the invention has the advantages that (1) the generation of the brown stain of an explant can be effectively changed, and the survival rate of the explant can be more than 90%; (2) the explant tissue can directly form into buds by an adopted culture medium, so that the happening of heteromorphosis can be avoided, and the purity of the strain characters of the later generation can be guaranteed; (3) the diameter of the explant can be effectively controlled to be less than 0.5mm, the explant can be free from virus, and the bred offspring is completely free from virus.
Description
Technical field
The present invention relates to a kind of primary culture method of stem tip tissue of strawberry.
Background technology
Plant Tissue Breeding refers to from plant corpus isolates the tissue, organ or the cell that suit the requirements, protoplast etc., by sterile working, under manual control condition, carry out cultivating to obtain the technology that the whole plant of regeneration or production have other products of economic worth.This technology has wide practical use in the preservation etc. of the plant of Fast-propagation rare plant or high economic worth, plant corpus virus-free and plant germplasm resource.
Strawberry is asexually propagated plant, and maternal plant easily produces stolon, the joint of stolon can produce new plant, breed offspring.But strawberry is easily by virus infection, and the virus of susceptible parent can be infected by stolon, makes Progeny plants band poison, cause the yield and quality of Progeny plants to decline.Utilize strawberry stem tip tissue to carry out artificial culture and propagation offspring, effectively can prevent the infection of virus, guarantee that offspring is not with virus.Stem-tip tissue cultivates point Initial culture, a Multiplying culture, 4 steps such as culture of rootage and domestication, and wherein Initial culture is the important step obtaining virus-free plant, is also the key technology of strawberry stem tip tissue cultures.
Strawberry stem tip is organized Initial culture to refer to and strawberry stem tip tissue is inoculated into the process specific medium obtaining virus-free plant, and in current strawberry stem tip Initial culture, Problems existing is:
1, stem-tip tissue explant (by living plant body cutting the part stem-tip tissue getting off to carry out to cultivate) survival rate is low, and inoculation is difficult to successfully.
2, the plant aberration rate that goes out of stem-tip tissue explant induction is high, can not ensure the purity of kind.
3, the plant bred can not realize anosis poisoning.
Summary of the invention
The object of this invention is to provide a kind of strawberry stem tip Initial culture method, the explant survival rate that the method can make strawberry stem tip apical meristem inoculate is to more than 90%, and the complete detoxification of Progeny plants, nothing variation occur.
Applicant finds after deliberation:
1, after the inoculation of strawberry stem tip tissue, explant generation brown stain causes the low main cause of survival rate.A large amount of through applicant
Research and experiment, before discovery inoculation, stolon is processed at 5-8 DEG C 72h and the rear light culture 72h of inoculation, can reduce the generation of brown stain, the survival rate of explant can bring up to more than 90%.
2, the generation made a variation during strawberry stem tip Initial culture and the forming feature of bud closely related.The approach that explantation tissue forms sprout has two kinds, and one is: the first dedifferentiation of postvaccinal explantation tissue forms callus, then forms bud by Calli Differentiation; Two are: explantation tissue directly forms bud; The former can produce variation, and the latter produces without variation.Realize producing without variation, key is medium.Applicant is through large quantifier elimination and experiment, have developed the good medium of a kind of effect: MS+NAA(0.1-0.3mg/L)+BA(0.2-0.5mg/L)+sucrose (3%)+agar (8%) pH is 5.8-6.0, this medium can make explantation tissue directly form bud, prevent the generation made a variation, ensure that the purity of sprout kind
3, the height of the sprout virus elimination rate of Initial culture formation is by the impact inoculating explant size, and explant is less than the effect that 0.5mm just can reach complete detoxification.
Based on above-mentioned research, the present invention is by low temperature treatment, and the method for light culture prevents explantation tissue's brown stain; By adopting, explant being seeded on specific medium, making its direct development become the method for bud to prevent the generation of variation; Improve virus elimination rate by the size controlling explant, its concrete technical scheme is as follows:
1, a primary culture method of stem tip tissue of strawberry, is characterized in that comprising the steps:
1) collection of stolon: stolon acquisition time is the 4-7 month, choose top be in do not open up between leaf and first exhibition leaf, the stolon of robust growth;
2) stolon process: after the stolon cleaning gathered, sterilizing, low temperature treatment at 3-8 DEG C, 72 hours processing times;
3) sterilization and inoculation: sterilize and inoculate and all carry out in superclean bench, specific as follows:
3.1 sterilizations:
3.1.1 by the stolon of above-mentioned process, clip 5-8cm from top.
3.1.2 the alcohol disinfecting 15-20 second of 75% is used;
3.1.3 effective chlorine density is the liquor natrii hypochloritis of 1%, sterilization 5-10 minute;
3.1.4 after aqua sterilisa cleaning, for subsequent use;
3.2 inoculations:
3.2.1 the winning of explant: ecto-entad divests the blade of growing point periphery parcel successively under anatomical lens, until growing point exposes.
3.2.2 with dissecting needle needle point picking growing point top tissue, be inoculated in medium:
Cultivate: the explant inoculated, at 20 DEG C, secretly descends cultivation 72 hours, and then cultivates under being positioned over light, condition of culture 25 DEG C, 2000lx, 16hr; Cultivate 10-15d and form sprout, 20-30d opens up leaf, takes root, and when 50-60d plant size is 3-5cm, completes Initial culture; The composition of described medium is: MS+NAA(0.1-0.3mg/L)+BA(0.2-0.5mg/L)+sucrose (3%)+agar (8%), pH is 5.8-6.0;
In step 1), in order to ensure the purity of kind, stolon collection will be carried out in kind garden, and the acquisition length of stolon is 10-15cm, to guarantee the quality of stolon.
Step 2) in sterilization be with 400 times of 84 medicining liquid dipping stolon 10-15 minute, then use distilled water flushing 10-
15 minutes, aforementioned 400 times referred to and dilute 84 thimerosals with water by the volume ratio of 400:1.
In step 3) from the reason of head clip stolon 5-8cm be convenient to sterilization inoculation time easy to operate;
With aqua sterilisa cleaning 3-5 time in step 3).
The multiplication factor of anatomical lens described in step 3) is 10-40x, so that clear observation growing point, and convenient sampling.
In step 3), be ensure explant size ﹤ 0.5mm, described dissecting needle pin rugosity is Φ=0.2-0.4mm, as contrast during operation, needle point get the rugosity that growing point tissue is no more than pin.
In step 3), during described Initial culture, culture vessel is test tube Φ=2.5cm, and often pipe medium addition is 20-25ml.
Tool of the present invention has the following advantages:
1, low temperature treatment stolon before inoculation, carries out light culture after inoculation, effectively can prevent the generation of Explant browning, ensure that explant survives and reach more than 90%.
2, the medium Neng Shi explantation tissue adopted directly forms sprout, prevents the generation made a variation, ensure that the purity of offspring's kind property.
3, adopt the dissecting needle of rugosity Φ=0.2-0.4mm, as contrast during sampling, the diameter that effectively can control explant is less than 0.5mm, ensure that explant is without virus, offspring's totally nontoxic that it breeds.
Embodiment
1, the collection of stolon: in order to ensure that the purity stolon collection of kind will be carried out in kind garden, stolon acquisition time is the 4-7 month, choose top be in do not open up between leaf and first exhibition leaf, the stolon of robust growth, acquisition length 10-15cm.
2, stolon process: the stolon gathered is used after rinsing 15-20 minute from the beginning, with 400 times of 84 medicining liquid dipping 10-15 minute, then use distilled water flushing 10-15 minute, put into freshness protection package, the refrigerator low temperature treatment of 5-8 DEG C is put into, 72 hours processing times after being sealed by freshness protection package.
3, sterilization and inoculation: sterilize and inoculate and all carry out in superclean bench
3.1 sterilizations:
3.1.1 by the stolon of above-mentioned process, clip 5-8cm from top.
3.1.2 the alcohol disinfecting 15-20 second of 75% is used;
3.1.3 effective chlorine density is the liquor natrii hypochloritis of 1%, sterilization 5-15 minute
3.1.4 aqua sterilisa is for subsequent use after cleaning 3-5 time.
3.2 inoculations:
3.2.1 the winning of explant: ecto-entad divests the blade of growing point periphery parcel successively under 10-40x anatomical lens,
Until growing point exposes.
3.2.2 be the dissecting needle needle point picking growing point top tissue of 0.2-0.4mm with pin rugosity Φ, be inoculated in medium, during operation with dissecting needle be contrast, needle point get the rugosity that growing point tissue is no more than pin:
Cultivate: the explant inoculated, at 20 DEG C, secretly descends cultivation 72 hours, and then cultivates under being positioned over light, condition of culture 25 DEG C, 2000lx, 16hr; Cultivate 10-15d and form sprout, 20-30d opens up leaf, takes root, and when 50-60d plant size is 3-5cm, completes Initial culture; The composition of described medium is: MS+NAA(0.1-0.3mg/L)+BA(0.2-0.5mg/L)+sucrose (3%)+agar (8%), pH is 5.8-6.0; During Initial culture, culture vessel is test tube Φ=2.5cm, and often pipe medium addition is 20-25ml.
Application examples 1
Strawberry cultivars: " Feng Xiang "
To infect the stolon of virus disease plant, after adopting the method for the invention inoculated and cultured, the survival rate of inoculation explant is 97.5%.Multiple RT-PCR detection method is adopted to detect virus, result shows Strawberry mottle virus (SMOV), strawberry yellow edge poison (SMYEV), strawberry crinkle virus, (SCV), strawberry veinbanding virus (SVBV) virus waits the viral disease poison testing result of 4 kinds of harm strawberries to be feminine gender, is 100% with before inoculation, plant compares virus elimination rate.Breed seedling in Yangzhou, Zhenjiang, Lianyun Harbour, the ground experimental cultivation such as Suqian result, show kind of property pure, bloom, result uniformity, produce without variation plant, compared with the seedling of local Sterile culture, output increased 50-60%.
Application examples 2
Strawberry cultivars: " red cheek "
Gather the stolon that two kinds infect virus disease plant respectively, the survival rate of explant is 100%.Multiple RT-PCR detection method is adopted to detect virus, result shows Strawberry mottle virus (SMOV), strawberry yellow edge poison (SMYEV), strawberry crinkle virus, (SCV), strawberry veinbanding virus (SVBV) virus waits the viral disease poison testing result of 4 kinds of harm strawberries to be feminine gender, is 100% with before inoculation, plant compares virus elimination rate.Seedling is at the ground experimental cultivation such as 10 Duo Ge cities and counties and Shaanxi, Beijing, Shanghai, Shandong, Anhui, the Inner Mongol in Jiangsu, result shows: various places all occur without variation, bloom, result uniformity, compared with the seedling of local Sterile culture, output increase is respectively Jiangsu 35-65%, Shaanxi 50-7-%, Beijing 50-70%, Shanghai 40-50%, Shandong 50-60%, Anhui 40-70%, Inner Mongol 50-70%.
Claims (6)
1. a primary culture method of stem tip tissue of strawberry, is characterized in that comprising the steps:
1) collection of stolon: stolon acquisition time is the 4-7 month, choose top be in do not open up between leaf and first exhibition leaf, the stolon of robust growth;
2) stolon process: after the stolon cleaning gathered, sterilizing, low temperature treatment at 3-8 DEG C, 72 hours processing times;
3) sterilization and inoculation: sterilize and inoculate and all carry out in superclean bench, specific as follows:
3.1 sterilizations:
3.1.1 by the stolon of above-mentioned process, clip 5-8cm from top;
3.1.2 the alcohol disinfecting 15-20 second of 75% is used;
3.1.3 effective chlorine density is the liquor natrii hypochloritis of 1%, sterilization 5-10 minute;
3.1.4 after aqua sterilisa cleaning, for subsequent use;
3.2 inoculations:
3.2.1 the winning of explant: ecto-entad divests the blade of growing point periphery parcel successively under anatomical lens, until growing point exposes;
3.2.2 with dissecting needle needle point picking growing point top tissue, be inoculated in medium, the diameter being inoculated into the explant in medium is less than 0.5mm;
Cultivate: the explant inoculated is at 20 DEG C, and secretly descend cultivation 72 hours, and then cultivate under being positioned over light, condition of culture 25 DEG C, 2000lx, the photoperiod is 16 h light 8 h dark; Cultivate 10-15d and form sprout, 20-30d opens up leaf, takes root, and when 50-60d plant size is 3-5cm, completes Initial culture; The composition of described medium is: MS+NAA0.1-0.3mg/L+BA0.2-0.5mg/L+ sucrose 3%+ agar 8%, pH is 5.8-6.0;
Described dissecting needle pin rugosity Φ is 0.2-0.4mm, needle point get the rugosity that growing point tissue is no more than pin.
2. primary culture method of stem tip tissue of strawberry as claimed in claim 1, is characterized in that: step 1) in the acquisition length of stolon be 10-15cm.
3. primary culture method of stem tip tissue of strawberry as claimed in claim 1, it is characterized in that: step 2) in sterilization be with 400 times of 84 medicining liquid dipping stolon 10-15 minute, use distilled water flushing 10-15 minute again, aforementioned 400 times refer to and dilute 84 thimerosals with water by the volume ratio of 400:1.
4. primary culture method of stem tip tissue of strawberry as claimed in claim 1, is characterized in that: step 3) in aqua sterilisa cleaning 3-5 time.
5. primary culture method of stem tip tissue of strawberry as claimed in claim 1, is characterized in that: step 3) in, the multiplication factor of described anatomical lens is 10-40x.
6. primary culture method of stem tip tissue of strawberry as claimed in claim 1, is characterized in that: step 3) in, during described Initial culture, culture vessel is the test tube of Φ=2.5cm, and often pipe medium addition is 20-25ml.
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103975854A (en) * | 2014-04-29 | 2014-08-13 | 卞佳林 | Culture medium for culturing strawberry stem tips and inducing plant differentiation and culture method thereof |
| CN105325298B (en) * | 2015-11-18 | 2017-12-22 | 扬州大学 | A kind of lotus rhizome stem-tip tissue Initial culture method |
| CN105969794A (en) * | 2016-06-06 | 2016-09-28 | 江苏农林职业技术学院 | Transgenic method adopting strawberry stem tip growth point |
| CN107135949A (en) * | 2017-06-16 | 2017-09-08 | 黔东南民族职业技术学院 | Strawberry adventitious bud induction culture base and preparation method and application |
| CN112586350A (en) * | 2020-12-02 | 2021-04-02 | 蚌埠海上明珠农业科技发展有限公司 | Strawberry virus-free seedling culture method |
| CN114885837B (en) * | 2022-04-07 | 2023-04-25 | 南充市农业科学院 | Hormone-free culture method for citrus stem tip |
| CN114788495B (en) * | 2022-04-07 | 2023-04-14 | 南充市农业科学院 | Hormone-free strawberry stem tip culture method |
| CN116686707A (en) * | 2023-06-12 | 2023-09-05 | 江苏省农业科学院 | A kind of pretreatment method of plant shoot tip tissue culture |
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|---|---|---|---|---|
| WO1999041975A1 (en) * | 1998-02-19 | 1999-08-26 | Cotton Incorporated | A method for the production of transgenic plants using apical shoot tips |
| 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 |
| CN102257961A (en) * | 2011-05-27 | 2011-11-30 | 东海县黄川春风草莓生产合作社 | Tissue culture method for strawberry seedling |
| CN102422816A (en) * | 2011-10-25 | 2012-04-25 | 上海航育种子基地场 | Method for quickly culturing shoot tips in vitro |
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Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH05244839A (en) * | 1992-03-05 | 1993-09-24 | Iseki & Co Ltd | Method for mass-producing strawberry and method for regenerating plant derived from multiple shoots |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999041975A1 (en) * | 1998-02-19 | 1999-08-26 | Cotton Incorporated | A method for the production of transgenic plants using apical shoot tips |
| 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 |
| CN102257961A (en) * | 2011-05-27 | 2011-11-30 | 东海县黄川春风草莓生产合作社 | Tissue culture method for strawberry seedling |
| CN102422816A (en) * | 2011-10-25 | 2012-04-25 | 上海航育种子基地场 | Method for quickly culturing shoot tips in vitro |
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