CN104297034A - Method applied to chromosome production of single tobacco plant by tender ovary - Google Patents
Method applied to chromosome production of single tobacco plant by tender ovary Download PDFInfo
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- CN104297034A CN104297034A CN201410557520.2A CN201410557520A CN104297034A CN 104297034 A CN104297034 A CN 104297034A CN 201410557520 A CN201410557520 A CN 201410557520A CN 104297034 A CN104297034 A CN 104297034A
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Abstract
The invention discloses a method applied to chromosome production of a single tobacco plant by tender ovary. The method comprises the following steps: (1) drawing materials; (2) preprocessing and fixing; and (3) subsequently preparing tobacco chromosome by methods of directly smearing, carrying out enzymolysis and then sheeting and smearing. According to the method, the tobacco tender ovary as the material is used for chromosome production; as the tobacco is high in quantity of flowers and long in flower period, a large quantity of materials can be selected; a large quantity of split phases are prepared; part of the split phases has good chromosome morphology and is applied to subsequent test analysis processes such as karyotype analysis and genomic in-situ hybridization. By virtue of the method, the defects of tiny and few materials during chromosome production of tobacco root tip can be overcome; and the method is especially suitable for the chromosome production of the single tobacco plant by the tender ovary.
Description
Technical field
The invention belongs to agricultural technology field, the present invention relates to and a kind ofly utilize young tender ovary to carry out the method for tobacco chromosome sectioning, be specially adapted to carry out chromosome sectioning to single tobacco plant.
Background technology
Plant chromosome film-making is the basis of carrying out the cytogenetical studies such as karyotyping, band analysis and Chromosomal in situ hybridization, along with the application of cytogenetics in Plant Taxonomy and plant breeding in recent years, the application of plant chromosome film-making is more and more extensive.Particularly in breeding, in a lot of situation, chromosome sectioning need be carried out to single (strain) material, and slice-making quality requires, something which increases the requirement to chromosome sectioning, too increases the difficulty of chromosome sectioning undoubtedly very high.Especially the vegetable material that some seeds are tiny, its film-making difficulty is larger.
At present, plant chromosome film-making adopts the tip of a root mostly, and minority adopts the different piece of young leaflet tablet and floral organ.Mostly adopt the tip of a root to carry out chromosome sectioning in tobacco chromosome research, but it may need a large amount of tip of a root as handling object, can obtain more excellent chromosome specimen.But in tobacco breeding process, the part often obtained has the special material that chromosome structure and number morph.In research process, require to carry out chromosome sectioning to each individual plant of this part material.Because tobacco seed is trickle, the root after sprouting is also less, and this causes great difficulty to carrying out chromosome sectioning to single plant, and cutting of the tip of a root often causes adverse influence to plant, as affected growth potential, bacteria infection etc.
Therefore, exploitation one is drawn materials conveniently, and the flaking method that can obtain more split coil method has boundless application prospect to screening chromosomal variation strain and carry out cytogenetical study to it in tobacco breeding.There is the method that scholar reports employing blade, tissue cultures material carries out tobacco chromosome sectioning.But due to the many intercalary meristems of blade, a large amount of split coil method cannot be obtained, subsequent analysis needs can not be met; And the material moisture content of tissue cultures is higher, and ageing tissues and tender tissue mixing, be also difficult to obtain good chromosome sectioning.Because tobacco inflorescence development is very fast, before the mononuclear microspore phase, ovary growth rapidly.Therefore, suitable when selecting the ovary in this period to carry out chromosome sectioning as material.And the tobacco florescence is longer, have that lot of materials is available carries out chromosome sectioning.At present, yet there are no the report about utilizing the tender ovary of tobacco children to carry out chromosome sectioning.
Summary of the invention
The object of this invention is to provide one draws materials conveniently, can obtain more split coil method, flaking method that split coil method chromosome is comparatively unfolded.For achieving the above object, the present invention intends adopting following technical scheme:
The present invention relates to and a kind ofly utilize young tender ovary to carry out the method for tobacco chromosome sectioning, its feature comprises the steps:
(1) draw materials: draw materials in the florescence to single tobacco plant, the time of drawing materials is that between 8:00-9:00 in sunny morning, object of drawing materials is immature bud, is good with meiosis of microspore mother cell phase bud, because different materials bud size there are differences;
(2) pre-service and fixing: taken off by bud with ophthalmic tweezers, put into the cuvette that distilled water is housed after removing anthocaulus, vibration makes bud immerse in solution gently.Be placed on 4 DEG C of refrigerators and place 18-24h (temperature and time can according to chromosome morphology from Row sum-equal matrix), sucking-off distilled water, adds Ka Luoshi immobile liquid (methyl alcohol: glacial acetic acid=3:1), fixedly spends the night;
(3) after follow-up employing direct smear, enzymolysis, compressing tablet or smear methods obtain tobacco chromosome sectioning.
Optionally, directly observing after compressing tablet, obtain good division phases, as needed follow-up test and long-term preservation, conventional method peel can be adopted; After direct smear and enzymolysis, smear also comprises staining procedure: dye with 5% Giemsa solution, chromosome time controling is between 30 seconds to 1 minute.
In a preferred embodiment of the present invention, described direct smear comprises the steps: from vial, take out the bud fixed, and peels off calyx, corolla, stamen and column cap with ophthalmic tweezers; Making it cracked by tweezers gripping ovary is fritter, with tweezer point gripping fritter tissue and to drip a small amount of Ka Luoshi immobile liquid sharp in tweezer, directly smear on clean, dry microslide, after material coating evenly, drip an immobile liquid on the microslide of coated material, immobile liquid is dispelled out gently along microslide one end; On spirit lamp flame micro-bake to immobile liquid be droplet-like.
In a preferred embodiment of the present invention, described enzymolysis compressing tablet, smear comprise the steps:
(4) enzymolysis: take out the bud fixed from vial, calyx is peelled off with ophthalmic tweezers, corolla, stamen and column cap, excision holder, and ovary is decomposed into suitable size (length of side is about the square block of 1-1.5 millimeter) and is placed in centrifuge tube, add distilled water and clean 2 times, after add distilled water immersion about 20 minutes, sucking-off distilled water, add mixed enzyme solution (3% cellulase+0.03% pectase, different manufacturers, batch enzyme liquid suitably can regulate concentration), enzyme and ovary volume ratio are 8-12:1, submergence material, concussion makes tissue block suspend in enzyme liquid, place in 35 DEG C of constant temperature ovens, within 1.0-1.5 hour, (can adjust according to material) takes out afterwards,
(5) Hypotonic treatment: after the material after enzymolysis takes out from constant temperature oven, sharp mouth suction pipe sucking-off enzyme liquid, adds distilled water, places sucking-off distilled water after 10-15 minute, add Ka Luoshi immobile liquid;
(6) compressing tablet: with the careful gripping material of tweezers, as for clean drying microslide central authorities, drip a card bag moral training liquid, with tweezers repeatedly gripping material make it be scattered in dye liquor, dye after about 1 minute, cover glass conventional method compressing tablet is cleaned in gripping.
Selectively:
(7) smear: with tweezers gripping material, and drip Ka Luoshi immobile liquid in tweezer point, rapid application on clean microslide, after material coating evenly, drip the central authorities that a Ka Luoshi immobile liquid is coated with material area on microslide, immobile liquid is dispelled out gently along surrounding, on spirit lamp flame micro-bake to immobile liquid be droplet-like.
It is material that this method chooses the tender ovary of children in florescence, carries out chromosome sectioning, and because tobacco amount is large, and the florescence is long, has lot of materials to supply alternative.And the method such as compressing tablet and smear carries out film-making after providing direct smear, enzymolysis, wherein direct smear chromosome is unfolded, background is shallow, form is good, can carry out karyotyping, and shortcoming is that chromosome is easily lost, impurity is more; Compressing tablet after enzymolysis, it is many that few division phases obtained lost by material, and chromosome morphology is better, is comparatively suitable for carrying out the subsequent analysis such as karyotyping and Chromosomal in situ hybridization process; Smear after enzymolysis, can obtain more division phases, chromosome is comparatively unfolded, and background is more shallow, is suitable for carrying out the subsequent analysis such as karyotyping and Chromosomal in situ hybridization process.
Accompanying drawing illustrates:
Fig. 1 pentaploid tobacco (2n=5x=58) is drawn materials signal;
Fig. 2 pentaploid tobacco (2n=5x=58) direct smear acquisition division phases (1000 ×, scale: 5 microns);
Compressing tablet effect after Fig. 3 pentaploid tobacco (2n=5x=58) enzymolysis (left: 200 ×, scale: 50 microns; Right: 1000 ×, scale: 10 microns), the visible a large amount of division phases (arrow indication) of left figure;
After Fig. 4 pentaploid tobacco (2n=5x=58) enzymolysis, smear obtains division phases (1000 ×, scale: 10 microns);
Fig. 5 smear after enzymolysis ovary obtains hexaploid tobacco (2n=6x=72) and wild diploid tobacco (2n=2x=20) division phases (1000 ×, scale: 10 microns).
Embodiment:
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1 (chromosome sectioning of pentaploid tobacco, hexaploid tobacco and wild diploid tobacco)
1, draw materials: draw materials in initial bloom stage to single tobacco plant, the time of drawing materials is about 8:30 in sunny morning, and object of drawing materials is immature bud (corolla length is about 2/3 of calyx length for good, the about meiosis of microspore mother cell phase).
2, pre-service and fixing: taken off by bud with ophthalmic tweezers, put into the cuvette that distilled water is housed after removing anthocaulus, vibration makes bud immerse in solution gently.Be placed on 4 DEG C of refrigerators and place 24h, sucking-off distilled water, adds Ka Luoshi immobile liquid (methyl alcohol: glacial acetic acid=3:1), fixedly spends the night.
Follow-up film-making is divided into 3 methods:
Method one: direct smear
Smear: take out the bud fixed from vial, carefully peels off calyx, corolla, stamen and column cap with ophthalmic tweezers.Making it cracked by tweezers gripping ovary is fritter, with tweezer point gripping fritter tissue and to drip a small amount of Ka Luoshi immobile liquid sharp in tweezer, directly smear on clean, dry microslide, after material coating evenly, drip a Ka Luoshi immobile liquid on the microslide of coated material, immobile liquid is dispelled out gently along microslide one end.On spirit lamp flame micro-bake to immobile liquid be droplet-like.
5% Giemsa stain dyeing, rear microscopy to be dried, takes pictures.
Method two: enzymolysis compressing tablet
Enzymolysis: take out the bud fixed from vial, calyx, corolla, stamen and column cap is carefully peelled off with ophthalmic tweezers, excision holder, and be positioned over after ovary being decomposed into 1 millimeter of square size in 1.5 milliliters of centrifuge tubes, add about 1 ml distilled water and clean 2 times, after add 1 ml distilled water and soak 20 minutes, sucking-off distilled water, add mixed enzyme solution (3% cellulase+0.03% pectase, enzyme and ovary volume ratio are 10:1) submergence material, slight concussion ovary makes it suspend once in enzyme liquid.Place in 35 DEG C of constant temperature ovens, take out after 1.5 hours.
Hypotonic treatment: after the material after enzymolysis takes out from constant temperature oven, sharp mouth suction pipe careful sucking-off enzyme liquid, carefully adds about 200 microlitre distilled water, places sucking-off distilled water after 15 minutes, carefully adds the Ka Luoshi immobile liquid of about 200 microlitres.
Compressing tablet: with the material after the careful gripping enzymolysis of tweezers, as for clean, dry microslide central authorities, drip a card bag moral training liquid, with tweezers repeatedly gripping material make it be scattered in dye liquor, dye after about 1 minute, cover glass conventional method compressing tablet is cleaned in gripping.
Microscopy, to take pictures.
Method three: enzymolysis smear
, enzymolysis: with method two 3.
Hypotonic treatment: with method two 4.
Smear: by tweezers careful gripping ovary and in a small amount of Ka Luoshi immobile liquid of tweezer point dropping, rapid application on clean microslide, after material coating evenly, drip the central authorities that an immobile liquid is coated with material area on microslide, immobile liquid is dispelled out gently along surrounding.On spirit lamp flame micro-bake to immobile liquid be droplet-like.
Dyeing: use 5% Giemsa staining, dry microscopy afterwards, takes pictures.
The above is the preferred embodiments of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from principle of the present invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (3)
1. utilize young tender ovary to carry out the method for chromosome sectioning to the single plant of tobacco, it is characterized in that: comprise the steps:
(1) draw materials: draw materials within the florescence, the time of drawing materials is that between 8:00-9:00 in sunny morning, object of drawing materials is immature bud, is good with meiosis of microspore mother cell phase bud, because different materials bud size there are differences;
(2) pre-service and fixing: bud is taken off with tweezers, the cuvette that distilled water is housed is put into after removing anthocaulus, vibration makes bud immerse in solution gently, be placed in 4 DEG C of refrigerators fast and place 18-24h (temperature and time can regulate voluntarily according to chromosome morphology), sucking-off distilled water, add Ka Luoshi immobile liquid (methyl alcohol: glacial acetic acid=3:1), fixedly spend the night;
(3) after follow-up employing direct smear, enzymolysis, compressing tablet or smear methods obtain tobacco chromosome sectioning;
Optionally, directly observing after compressing tablet, obtain good division phases, as needed follow-up test and long-term preservation, conventional method peel can be adopted; After direct smear and enzymolysis, smear also comprises staining procedure: dye with 5% Giemsa solution, chromosome time controling is between 30 seconds to 1 minute.
2. the young tender ovary of utilization according to claim 1 carries out the method for chromosome sectioning to the single plant of tobacco, and described direct smear comprises the steps: from vial, take out the bud fixed, and peels off calyx, corolla, stamen and column cap with ophthalmic tweezers; Making it cracked by tweezers gripping ovary is fritter, with tweezer point gripping fritter tissue and to drip a small amount of Ka Luoshi immobile liquid sharp in tweezer, directly smear on clean, dry microslide, after material coating evenly, drip an immobile liquid on the microslide of coated material, immobile liquid is dispelled out gently along microslide one end; On spirit lamp flame micro-bake to immobile liquid be droplet-like.
3. the young tender ovary of utilization according to claim 1 carries out the method for chromosome sectioning to the single plant of tobacco, and described enzymolysis compressing tablet and smear comprise step (4)-(6):
(4) enzymolysis: take out the bud fixed from vial, calyx is peelled off with ophthalmic tweezers, corolla, stamen and column cap, excision holder, and ovary is decomposed into suitable size (length of side is about the square block of 1-1.5 millimeter) and is placed in centrifuge tube, add distilled water and clean 2 times, after add distilled water immersion about 20 minutes, sucking-off distilled water, add mixed enzyme solution (3% cellulase+0.03% pectase, different manufacturers, batch enzyme liquid suitably can regulate concentration), enzyme and ovary volume ratio are 8-12:1, submergence material, concussion makes tissue block suspend in enzyme liquid, place in 35 DEG C of constant temperature ovens, within 1.0-1.5 hour, (can adjust according to material) takes out afterwards,
(5) Hypotonic treatment: after the material after enzymolysis takes out from constant temperature oven, sharp mouth suction pipe sucking-off enzyme liquid, adds distilled water, places sucking-off distilled water after 10-15 minute, add Ka Luoshi immobile liquid;
(6) compressing tablet: with the careful gripping material of tweezers, be placed in clean dry microslide central authorities, drip a carbolfuchsin dyeing liquor, with tweezers repeatedly gripping material make it be scattered in dye liquor, dye after about 1 minute, gripping be clean, dried lid slide conventional method compressing tablet;
Selectively:
Smear: with tweezers gripping material, and drip Ka Luoshi immobile liquid in tweezer point, rapid application on clean microslide, after material coating evenly, drip the central authorities of an immobile liquid coated material ranges on microslide, immobile liquid is dispelled out gently along surrounding, on spirit lamp flame micro-bake to immobile liquid be droplet-like.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105838786A (en) * | 2016-03-24 | 2016-08-10 | 西南大学 | Method of chromosome smearing of adult plants of loquat with young tender flower buds |
| CN107631921A (en) * | 2017-09-14 | 2018-01-26 | 河南科技学院 | A kind of big premium camellia filigree chromosome flaking method |
| CN108132252A (en) * | 2017-12-29 | 2018-06-08 | 青岛袁策生物科技有限公司 | A kind of genome identification method of colored rice |
| CN108267410A (en) * | 2017-12-29 | 2018-07-10 | 青岛袁策生物科技有限公司 | A kind of genome identification method of high protein rice strain |
| CN109652507A (en) * | 2019-02-21 | 2019-04-19 | 华中农业大学 | The in-situ hybridization method of Chinese China pink |
| CN110926896A (en) * | 2019-12-04 | 2020-03-27 | 怀化学院 | Method for metaphase mitosis phase mounting of rhizosphere chromosome of zingiber plant and application thereof |
| CN111323288A (en) * | 2020-04-03 | 2020-06-23 | 贵州省烟草科学研究院 | Winged tobacco monosome identification method |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105838786A (en) * | 2016-03-24 | 2016-08-10 | 西南大学 | Method of chromosome smearing of adult plants of loquat with young tender flower buds |
| CN107631921A (en) * | 2017-09-14 | 2018-01-26 | 河南科技学院 | A kind of big premium camellia filigree chromosome flaking method |
| CN108132252A (en) * | 2017-12-29 | 2018-06-08 | 青岛袁策生物科技有限公司 | A kind of genome identification method of colored rice |
| CN108267410A (en) * | 2017-12-29 | 2018-07-10 | 青岛袁策生物科技有限公司 | A kind of genome identification method of high protein rice strain |
| CN109652507A (en) * | 2019-02-21 | 2019-04-19 | 华中农业大学 | The in-situ hybridization method of Chinese China pink |
| CN110926896A (en) * | 2019-12-04 | 2020-03-27 | 怀化学院 | Method for metaphase mitosis phase mounting of rhizosphere chromosome of zingiber plant and application thereof |
| CN111323288A (en) * | 2020-04-03 | 2020-06-23 | 贵州省烟草科学研究院 | Winged tobacco monosome identification method |
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Application publication date: 20150121 |