CN101790961A - Method for inducing and detecting peppermint autopolyploid - Google Patents
Method for inducing and detecting peppermint autopolyploid Download PDFInfo
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- CN101790961A CN101790961A CN 201010017950 CN201010017950A CN101790961A CN 101790961 A CN101790961 A CN 101790961A CN 201010017950 CN201010017950 CN 201010017950 CN 201010017950 A CN201010017950 A CN 201010017950A CN 101790961 A CN101790961 A CN 101790961A
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- peppermint
- colchicine
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- autopolyploid
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Abstract
The invention provides a technique for inducing autopolyploid by using peppermint shoot tips, which can be used for breeding peppermint autopolyploid. The invention is characterized in that colchicine is used for controlling concentration, time and method, a large amount of peppermint autopolyploid plants can be obtained in a short time, and the ploidy of the plants is detected by using a flow cytometry method. By adopting the method to induce peppermint autopolyploid, the induction rate is high (up to 15%), the survival rate of variant plants is high (more than 90%), the identification method is easy, and the breeding cycle of peppermint can be shortened greatly.
Description
Technical field
The invention belongs to agricultural technology field, relate to multiploid induction and Fast Detection Technique in the peppermint breeding work.
Background technology
Be rich in volatile oil in the stem of peppermint (Mentha haplocalyx Briq.), the leaf, be widely used in industry such as food, medicine, cosmetics, spices, tobacco.The peppermint all herbal medicine is used for the treatment of anemopyretic cold, wind-warm syndrome from the beginning of, headache, hot eyes, larynx numbness, abscess of throat, aphthae, toothache, nettle rash, rubella etc.Peppermint is one of the important spices of China and medicinal plant, in Jiangsu, ground such as Anhui, Jiangxi widely cultivate, and have become local important agriculture extraordinary economic crops.But single, the deterioration of variety phenomenon of kind that occurs in peppermint produces makes the breeding work of peppermint more and more noticeable.Cultivate the good new varieties of peppermint, it is a job of demanding urgently carrying out that the kind in the existing production is upgraded.Polyploid has the unexistent advantage proterties of a lot of dliploids, as the huge property of plant, resistance increase, organic synthesis speed is accelerated and overcome source far away intersterility, the breeding of new variety that polyploid breeding is used for peppermint has good prospect.Polyploid breeding is successfully utilization on many vegetable materials, but the application on peppermint does not appear in the newspapers as yet.The present invention derives from the years of researches result, and inducing and detection technique of a kind of peppermint autopolyploid is provided, and can be used for the polyploid breeding of peppermint.
Summary of the invention
The object of the present invention is to provide inducing and detection method of a kind of peppermint autopolyploid.Adopt this method to carry out the multiploid induction of peppermint, inductivity height (can reach 13%), variant survival rate height (more than 90%) is identified and is expanded and numerously carries out synchronously, can shorten breeding cycle greatly.Main contents comprise as follows:
1. the foundation of sterile system.As explant, on the initial culture base, set up sterile system with the peppermint stem apex.The initial culture base is MS minimal medium+1.0mgL
-16-BA+0.2mgL
-1NAA+5.5gL
-1Agar+30gL
-1Sucrose, pH5.5～5.8, condition of culture is 25 ℃ of temperature, light application time 8～10h/d, intensity of illumination 1500～2000lx.
2. POLYPLOID INDUCEMENT.Get the aseptic stripped indefinite bud of initial culture, behind 0.1%～0.2% colchicine solution immersion, 24～48h, be seeded on the subculture medium that does not contain colchicine; Or the stripped indefinite bud of the aseptic peppermint of getting initial culture, be seeded in and add 20mgL
-1In the subculture medium of colchicine, change over to behind the cultivation 30d and continue in the subculture medium that does not contain colchicine to cultivate.The medium of successive transfer culture, condition of culture are identical with initial culture.
3. the evaluation of polyploid.After handling strain successive transfer culture 20d, get its blade, detect the chromosomal variation situation with flow cytometer.
4. the enrichment culture of polyploid plant.Being seeded in from nearest preceding two stem-segment with node of stem apex of chromosome doubling plant contained 2.0mgL
-16-BA+0.2mgL
-1On the MS proliferated culture medium of NAA, contain 5.5gL in the medium
-1Agar, 30gL
-1Sucrose, pH 5.5～5.8, and condition of culture is the same.Cultivate 20d, switching propagation 2 times.
5. the culture of rootage of polyploid plant.To breed seedling is seeded in and contains 0.5mgL
-16-BA+0.2mgL
-1On the 1/2MS root media of NAA, contain 5.5gL in the medium
-1Agar, 30gL
-1Sucrose, pH 5.5～5.8, and condition of culture is the same.
Hardening with become seedling.Behind the culture of rootage 20d, the seedling blake bottle of will taking root shifts out culturing room, open bottle cap, water spray is preserved moisture, room temperature hardening 4～5d, taking-up doubles strain, the flush away medium is planted in the cave dish that is filled with humus soil, perlite and sand (4: 3: 2), is placed on indoor sheltering from heat or light and preserves moisture, behind 3～5d the cave dish is moved in the shade of field the conventional method management.
Embodiment
Embodiment 1: with peppermint kind ' 68-7 ' is example.
Cut the land for growing field crops seedling stem apex of peppermint kind ' 68-7 ', suds clean 15min, flowing water flushing 30min, 75% alcohol immersion 30s, 0.1%HgCl
2Soak 7min, aseptic water washing 3 times is inoculated in and contains 1.0mgL
-16-BA and 0.2mgL
-1The MS initial culture base of NAA contains 5.5gL in the medium
-1Agar, 30gL
-1Sucrose, pH 5.5～5.8.Condition of culture is 25 ℃ of temperature, light application time 8～10h, intensity of illumination 1500～2000lx.
Getting the aseptic seedling indefinite bud behind the initial culture 30d, place 0.2% colchicine solution, keep 25 ℃ ± 1, is 90rmin at rotating speed
-1Shaking table on soak 24h after, clean with aseptic water washing, be seeded on the subculture medium and cultivate.The medium of successive transfer culture is identical with initial culture with condition of culture.After cultivating 20d, get blade and carry out the ploidy evaluation.
Ploidy is identified the cells were tested by flow cytometry method that adopts.Get spire 50mg, extracting buffer solution [100mmolL
-1Citric acid+0.5% (V/V) Tween-20 (pH 2.30)] middle pulverize, 500 order nylon net filters, centrifugal (1000rm
-1), collect deposition of cells, add dyeing liquor and [extract buffer solution+400mmolL
-1Na
2HPO
412H
2O (pH about 8.9)+3000UmL
-1RNA enzyme A+10ugmL
-1PI], dark place low temperature is dyeing 30min down.500 order nylon net filters, filtrate is detected with Coulter Epics XL type flow cytometer (U.S. Beckman company), and whether observation of cell chromosome doubles.
Testing result shows that the plant of chromosome doubling is autopolyploid.Getting these plant is seeded in from nearest preceding two stem-segment with node of stem apex and contains 2.0mgL
-16-BA and 0.2mgL
-1On the MS proliferated culture medium of NAA, contain 5.5gL in the medium
-1Agar, 30gL
-1Sucrose, pH 5.5～5.8.Condition of culture is the same, cultivates 20d, switching propagation 2 times.
To breed seedling is seeded in and contains 0.5mgL
-16-BA and 0.2mgL
-1On the 1/2MS root media of NAA, contain 5.5gL in the medium
-1Agar, 30gL
-1Sucrose, PH 5.5～5.8.Condition of culture is the same.
The seedling blake bottle of will taking root behind the 20d shifts out culturing room, opens bottle cap, and water spray is preserved moisture, room temperature hardening 4～5d takes out back flush away root medium, plants in the cave dish that is filled with humus soil, perlite and sand (4: 3: 2), behind indoor placement 3～5d, move in the outdoor shade, move into the land for growing field crops after 1 month.
Embodiment 2: with peppermint kind ' 73-8 ' is example.
Outside distinguishing down, other steps are identical with embodiment 1 with method.
Get the aseptic seedling indefinite bud behind the initial culture 30d, be seeded in and add 20mgL
-1In the medium of colchicine (with the initial culture base), behind the cultivation 30d, transfer on the subculture medium that does not contain colchicine and cultivate 20d, be used for ploidy and detect.
Claims (7)
1. peppermint autopolyploid of the present invention induces and detection method, it is characterized in that may further comprise the steps: the stripped stem apex of peppermint is set up sterile system as explant on the initial culture base, behind the stripped aseptic indefinite bud of certain density colchicine processing, carry out successive transfer culture, detecting ploidy with the flow cytometer method changes, the chromosome doubling material that detects affirmation can obtain polyploid plant through enrichment culture, culture of rootage, hardening, transplanting.
2. according to claim 1, initial culture is set up the method for sterile system, it is characterized in that: with the stem apex that exsomatizes as explant; The initial culture base is MS minimal medium+1.0mgL
-16-BA+0.2mgL
-1NAA+5.5gL
-1Agar+30gL
-1Sucrose, pH 5.5～5.8; Condition of culture is 25 ℃ of temperature, light application time 8～10h, intensity of illumination 1500～2000lx.
3. according to claim 1, the colchicine processing method is characterized in that: the material that colchicine is handled is the stripped indefinite bud of tissue culture; Behind 0.1%～0.2% colchicine solution immersion, 24～48h, be seeded on the subculture medium that does not contain colchicine; Or the stripped indefinite bud of the aseptic peppermint of getting initial culture, be seeded in and add 20mgL
-1In the subculture medium of colchicine, change over to behind the cultivation 30d and continue in the subculture medium that does not contain colchicine to cultivate.The medium of successive transfer culture, condition of culture are identical with initial culture.
4. according to claim 1, the authentication method of polyploid is characterized in that: the material of handling through colchicine is got blade behind successive transfer culture 20d, carry out ploidy with flow cytometer and detect.The test-tube plantlet of chromosome doubling is autopolyploid.
5. according to claim 1, the enrichment procedure of ploidy material is characterized in that: proliferated culture medium is MS minimal medium+2.0mgL
-16-BA+0.2mgL
-1NAA+5.5gL
-1Agar+30gL
-1Sucrose, pH 5.5～5.8; Condition of culture is 25 ℃ of temperature, light application time 8～10h, intensity of illumination 1500～2000lx; Cultivate 20d, switching propagation 2 times.
6. according to claim 1, the culture of rootage method of ploidy material is characterized in that: root media is 1/2MS+0.5mgL
-16-BA+0.2mgL
-1NAA+5.5gL
-1Agar+30gL
-1Sucrose, pH 5.5～5.8; Condition of culture is 25 ℃ of temperature, light application time 8～10h, intensity of illumination 1500～2000lx; Cultivate 20d.
7. according to claim 1, hardening and the method that becomes seedling, it is characterized in that: the seedling blake bottle of will taking root shifts out culturing room, opens bottle cap, and water spray is preserved moisture, room temperature hardening 4～5d, take out back flush away root medium, plant in the cave dish that is filled with humus soil, perlite and sand (4: 3: 2), behind indoor placement 3～5d, move in the outdoor shade, move into the land for growing field crops after 1 month.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010017950 CN101790961B (en) | 2010-01-18 | 2010-01-18 | Method for inducing and detecting peppermint autopolyploid |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010017950 CN101790961B (en) | 2010-01-18 | 2010-01-18 | Method for inducing and detecting peppermint autopolyploid |
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| Publication Number | Publication Date |
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| CN101790961B CN101790961B (en) | 2012-05-09 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105393920A (en) * | 2015-12-18 | 2016-03-16 | 江苏省中国科学院植物研究所 | Method for establishing mint leaf high-efficiency regeneration system |
| CN108719046A (en) * | 2017-04-13 | 2018-11-02 | 北京林业大学 | A kind of method of induction culturing hybrid sweetgum tetraploid |
| CN116267623A (en) * | 2023-05-23 | 2023-06-23 | 北京花乡花木集团有限公司 | Tissue culture propagation method for peppermint |
-
2010
- 2010-01-18 CN CN 201010017950 patent/CN101790961B/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105393920A (en) * | 2015-12-18 | 2016-03-16 | 江苏省中国科学院植物研究所 | Method for establishing mint leaf high-efficiency regeneration system |
| CN108719046A (en) * | 2017-04-13 | 2018-11-02 | 北京林业大学 | A kind of method of induction culturing hybrid sweetgum tetraploid |
| CN108719046B (en) * | 2017-04-13 | 2021-12-24 | 北京林业大学 | Method for induced cultivation of hybrid liquidambar formosana tetraploid |
| CN116267623A (en) * | 2023-05-23 | 2023-06-23 | 北京花乡花木集团有限公司 | Tissue culture propagation method for peppermint |
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| Publication number | Publication date |
|---|---|
| CN101790961B (en) | 2012-05-09 |
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