CN106092677B - Sweet potato and its relative genus plant cell division phases sample fast preparation method - Google Patents
Sweet potato and its relative genus plant cell division phases sample fast preparation method Download PDFInfo
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- CN106092677B CN106092677B CN201610377477.0A CN201610377477A CN106092677B CN 106092677 B CN106092677 B CN 106092677B CN 201610377477 A CN201610377477 A CN 201610377477A CN 106092677 B CN106092677 B CN 106092677B
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- 244000017020 Ipomoea batatas Species 0.000 title claims abstract description 32
- 235000002678 Ipomoea batatas Nutrition 0.000 title claims abstract description 32
- 241000196324 Embryophyta Species 0.000 title claims abstract description 17
- 230000032823 cell division Effects 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000010494 dissociation reaction Methods 0.000 claims abstract description 11
- 230000005593 dissociations Effects 0.000 claims abstract description 11
- 239000011521 glass Substances 0.000 claims abstract description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000008367 deionised water Substances 0.000 claims abstract description 10
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 10
- 238000004140 cleaning Methods 0.000 claims abstract description 5
- 239000006185 dispersion Substances 0.000 claims abstract description 5
- 238000000879 optical micrograph Methods 0.000 claims abstract description 5
- 238000010186 staining Methods 0.000 claims abstract description 5
- 210000003813 thumb Anatomy 0.000 claims description 8
- 238000004659 sterilization and disinfection Methods 0.000 claims description 7
- 238000004043 dyeing Methods 0.000 claims description 4
- 210000003811 finger Anatomy 0.000 claims description 4
- 208000030208 low-grade fever Diseases 0.000 claims description 4
- 238000005464 sample preparation method Methods 0.000 abstract description 2
- 238000011160 research Methods 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 8
- 238000000034 method Methods 0.000 description 5
- 244000061456 Solanum tuberosum Species 0.000 description 4
- 235000002595 Solanum tuberosum Nutrition 0.000 description 4
- 210000000349 chromosome Anatomy 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 241000207782 Convolvulaceae Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 244000257782 Ipomoea triloba Species 0.000 description 2
- 206010039509 Scab Diseases 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- OCJBOOLMMGQPQU-UHFFFAOYSA-N 1,4-dichlorobenzene Chemical compound ClC1=CC=C(Cl)C=C1 OCJBOOLMMGQPQU-UHFFFAOYSA-N 0.000 description 1
- 239000005725 8-Hydroxyquinoline Substances 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 235000021506 Ipomoea Nutrition 0.000 description 1
- 241000207783 Ipomoea Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 208000020584 Polyploidy Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 230000002559 cytogenic effect Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000012214 genetic breeding Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000031864 metaphase Effects 0.000 description 1
- 239000002366 mineral element Substances 0.000 description 1
- 229960003540 oxyquinoline Drugs 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- MCJGNVYPOGVAJF-UHFFFAOYSA-N quinolin-8-ol Chemical compound C1=CN=C2C(O)=CC=CC2=C1 MCJGNVYPOGVAJF-UHFFFAOYSA-N 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Immunology (AREA)
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The invention discloses a kind of sweet potato and its relative genus plant cell division phases sample fast preparation methods, include the following steps：(1) 2 millimeters of the tip of a root is taken；(2) it dissociates：The tip of a root is put into the hydrochloric acid of the 1.2mol/L newly configured, in 55 DEG C of water-baths, the tip of a root after being dissociated；(3) it rinses and rear hypotonic：The tip of a root after dissociation is taken out from dissociation solution, it is twice using deionized water cleaning, finally hypotonic 24 minutes latter in deionized water；(4) film-making：The tip of a root after will be hypotonic takes out, and is placed on glass slide, obtains tabletting after treatment；(5) tabletting is placed in optical microphotograph under the microscope, the dispersion of selective staining body, clearly cell carries out PHOTOGRAPHIC ANALYSIS, is marked on the cover slip with glass graver, and sample, which is positioned over -80 DEG C of refrigerators, to be preserved.The present invention establishes a set of simple, efficiently, practical, economic sweet potato and its relative genus plant cell division phases sample preparation method, realizes and completes film-making in 1 hour.
Description
Technical field
The present invention relates to plant cytology investigative technique more particularly to a kind of sweet potato and its relative genus plant cell mid-terms point
Split phase sample fast preparation method.
Background technology
Gan Shu [Ipomoea batatas (L.) Lam., 2n=6x=-90]Belong to Convolvulaceae (Convolvulaceae) sweet potato
Belong to (Ipomoea) plant, originate in one band of Peru, Ecuador and Mexico of tropical South America, now plants extensively in the world
The torrid zone, subtropical zone and Temperate Region in China.Statistics shows that sweet potato in the whole world is more than 110 countries and has plantation, wherein China
Sweet potato cultivated area accounts for about global 65.4%, and yield accounts for about global 85.9%.Sweet potato has both the characteristics of food and cash crops,
It is China or even grain important in the world, feed and insutrial crop.It is rich in starch, mineral element, cellulose and β-
Carrotene and multivitamin are known as " vegetables queen " by reputation.
Sweet potato genome is essentially 6X at complexity, the basic number of chromosome 15, cultigen, and relative genus has 2X, 4X, 6X tri-
Kind ploidy, certain kinds of more than one ploidies.Its chromosome is small, and quantity is more, and cytoplasm is dense, colouring power is weak, it is more difficult to obtain clear
The research and development of clear clean chromosome sectioning, cytogenetics level is slower, seriously lags behind the works such as wheat, corn, rice
Object.Research is concentrated mainly in the observation of chromosome number and meiotic behavior at present, to the genome of sweet potato polyploid
At less with structural research.For sweet potato origin and evolve it is still indefinite so far, cultivation sweet potato be autopolyploid, it is heterologous more
Times body or autoallopolyploid always exist dispute.Presently, there are several different hypothesis, each hypothesis all lacks enough
Experimental evidence.As what sweet potato basic research in recent years and breeding research worked deepens continuously, there is an urgent need in cellular level
More in-depth study is carried out to sweet potato, further clear sweet potato origin and evolution, promote sweet potato basic research and genetic breeding
Development.
Although at present about sweet potato and its relevant report of relative genus cytology research, these researchs make extensively
Division phases preparation method still has some shortcomings, includes mainly：(1) use 8-hydroxyquinoline, paracide full
Need the time long with the pretreatment tip of a root, enrichment division phases such as aqueous solution, colchicines；(2) generally pretreatment drug is to people
Body harm is larger；(3) although treated, metaphase cell quantity is less handled and be increased, and accelerating still has
Limit；(4) 12-24 hours must be fixed, preparation process is cumbersome, and cost of labor is higher；(5) cellulase and pectin enzymatic hydrolysis are used
The tip of a root, processing time is long, and is difficult to grasp dissociation degree；(6) preferable sample need to can just be made by a large amount of practices for a long time.
Invention content
The present invention is to provide a kind of sweet potato and its relative genus plant cell division phases mark to solve above-mentioned deficiency
This fast preparation method.
The above-mentioned purpose of the present invention is realized by technical solution below：
For in current sweet potato and its relative genus plant cytology research division phases sample prepare there are the problem of, this
Invention research obtained it is easy be quickly obtained the method containing a large amount of cell division phases samples, with high efficiency, practicability and
With the characteristics of operability etc., the technology of a whole set of complete sweet potato and its preparation of relative genus plant division phases sample is provided
Method realizes and completes film-making in 1 hour, and obtains the division phases of a large amount of sweet potatoes and its relative genus plant.
A kind of sweet potato and its relative genus plant cell division phases sample fast preparation method, it is characterised in that：Including
Following steps：
(1) tip of a root is taken：The materials time directly determines the experiment effect of this experiment, and the step that the present invention is most crucial, must
Noon 12 must be strict controlled in：20 to noon 1:Between 00, eugonic new root is chosen, root is cut using disinfection scalpel
2 millimeters or so of point；
(2) it dissociates：The tip of a root is put into the hydrochloric acid of the 1.2mol/L newly configured, in 55 DEG C of water-baths, stringent timing, water
Bath is taken out after 18 minutes, the tip of a root after being dissociated；
(3) it rinses and rear hypotonic：The tip of a root after dissociation is taken out from dissociation solution, twice using deionized water cleaning, most
It is hypotonic 24 minutes latter in deionized water afterwards；
(4) film-making：The tip of a root after will be hypotonic takes out, and is placed on glass slide, removes the tip of a root most with clean scalpel blade
About 0.5-0.8 millimeters of front end root cap area, dissecting needle smash the tip of a root to pieces, drip about 15 microlitres of Giemsa stains, after dyeing 5-8 minutes, lid
The side of upper coverslip, coverslip is fixed with thumb and index finger, is gently tapped coverslip with pencil, is removed bubble removing, makes tissue
As a thin layer, it then is baked to low-grade fever on the flame of alcolhol burner, a blotting paper is added on the coverslip, uses thumb squeezes
The coverslip keeps cell fully dispersed, obtains tabletting；
(5) tabletting is placed in optical microphotograph under the microscope, the dispersion of selective staining body, clearly cell carries out photograph point
Analysis, is marked on the cover slip with glass graver, and sample, which is positioned over -80 DEG C of refrigerators, to be preserved.
Compared with the prior art, the present invention has the advantages of：The present invention establishes a set of simple, efficiently, practical, economic sweet potato
And its relative genus plant cell division phases sample preparation method, a large amount of cell division phases samples are quickly obtained, are sweet
Potato and its research of relative genus plant cytology and distant hybridization breeding work provide support, realize and complete film-making in 1 hour,
And obtain the division phases of a large amount of sweet potatoes and its relative genus plant.
Specific implementation mode
The present invention is described in further detail with reference to embodiment.
Embodiment 1：Concrete operation method prepared by 20 cell division phases sample of sweet potato variety river potato is as follows：
(1) material prepares：The potato wedge for selecting the full sweet potato variety river potato 20 without small holes caused by worms and scab, uses 75% alcohol
Disinfection 8 minutes, is flushed three times with sterile distilled water.After rinsing, potato wedge is placed in the plastic tub equipped with distilled water at 25 DEG C
It is cultivated in incubator, midway notices that observation, such as water deficient add rapidly water；
(2) tip of a root is taken：Etc. root tubers culture after a week, after growing a large amount of roots, take the tip of a root.The tip of a root time is taken directly to determine this reality
The experiment effect tested, it is necessary to be strict controlled in noon 12：20 to noon 1:Between 00, eugonic new root is chosen, is used
Disinfection scalpel cuts 2 millimeters or so of the tip of a root；
(3) it dissociates：The tip of a root is put into the hydrochloric acid of the 1.2mol/L newly configured, in 55 DEG C of water-baths, stringent timing, water
Bath is taken out after 18 minutes, the tip of a root after being dissociated；
(4) it rinses and rear hypotonic：The tip of a root after dissociation is taken out from dissociation solution, twice using deionized water cleaning, most
It is hypotonic 24 minutes latter in deionized water afterwards；
(5) film-making：The tip of a root after will be hypotonic takes out, and is placed on glass slide, removes the tip of a root most with clean scalpel blade
About 0.5-0.8 millimeters of front end root cap area, dissecting needle smash the tip of a root to pieces, drip about 15 microlitres of Giemsa stains, after dyeing 5-8 minutes, lid
The side of upper coverslip, coverslip is fixed with thumb and index finger, is gently tapped coverslip with pencil, is removed bubble removing, makes tissue
As a thin layer, it then is baked to low-grade fever on the flame of alcolhol burner, a blotting paper is added on the coverslip, uses thumb squeezes
The coverslip keeps cell fully dispersed, obtains tabletting；
(6) tabletting is placed in optical microphotograph under the microscope, the dispersion of selective staining body, clearly cell carries out photograph point
Analysis, is marked on the cover slip with glass graver, and sample, which is positioned over -80 DEG C of refrigerators, to be preserved.
Embodiment 2：Concrete operation method prepared by sweet potato relative genus I.triloba (2X) cell division phases sample is such as
Under：
(1) material prepares：The seed for selecting the full sweet potato relative genus I.triloba (2X) without small holes caused by worms and scab,
75% alcohol disinfecting is used on superclean bench 8 minutes, place into 12% liquor natrii hypochloritis and sterilize 6 minutes, last nothing
Bacterium distilled water flushing is three times.After rinsing, 1-2 millimeters of kind skins are cut away using the scalpel after disinfection.After kind of skin removal is clean,
After kind of skin removal is clean, seed is placed in the disinfection culture for being lined with 2 layers of filter paper, 4-8 ml deionized waters are added, then will
Culture dish is placed in 28 DEG C of incubators and is cultivated, and midway notices that observation, such as water deficient add rapidly water；
(2) tip of a root is taken：Etc. root tubers culture after a week, after growing a large amount of roots, take the tip of a root.The tip of a root time is taken directly to determine this reality
The experiment effect tested, it is necessary to be strict controlled in noon 12：20 to noon 1:Between 00, eugonic new root is chosen, is used
Disinfection scalpel cuts 2 millimeters or so of the tip of a root；
(3) it dissociates：The tip of a root is put into the hydrochloric acid of the 1.2mol/L newly configured, in 55 DEG C of water-baths, stringent timing, water
Bath is taken out after 18 minutes, the tip of a root after being dissociated；
(4) it rinses and rear hypotonic：The tip of a root after dissociation is taken out from dissociation solution, twice using deionized water cleaning, most
It is hypotonic 24 minutes latter in deionized water afterwards；
(5) film-making：The tip of a root after will be hypotonic takes out, and is placed on glass slide, removes the tip of a root most with clean scalpel blade
About 0.5-0.8 millimeters of front end root cap area, dissecting needle smash the tip of a root to pieces, drip about 15 microlitres of Giemsa stains, after dyeing 5-8 minutes, lid
The side of upper coverslip, coverslip is fixed with thumb and index finger, is gently tapped coverslip with pencil, is removed bubble removing, makes tissue
As a thin layer, it then is baked to low-grade fever on the flame of alcolhol burner, a blotting paper is added on the coverslip, uses thumb squeezes
The coverslip keeps cell fully dispersed, obtains tabletting；
(6) tabletting is placed in optical microphotograph under the microscope, the dispersion of selective staining body, clearly cell carries out photograph point
Analysis, is marked on the cover slip with glass graver, and sample, which is positioned over -80 DEG C of refrigerators, to be preserved.
Example the above is only the implementation of the present invention is not intended to limit the scope of the invention, every to utilize this hair
Equivalent structure or equivalent flow shift made by bright specification and embodiment content, it is relevant to be applied directly or indirectly in other
Technical field is included within the scope of the present invention.
Claims (1)
1. a kind of sweet potato and its relative genus plant cell division phases sample fast preparation method, it is characterised in that：Including with
Lower step：
(1) tip of a root is taken：At noon 12：20 to noon 1:Between 00, eugonic new root is chosen, is cut using disinfection scalpel
Take 2 millimeters of the tip of a root；
(2) it dissociates：The tip of a root is put into the hydrochloric acid of the 1.2mol/L newly configured, in 55 DEG C of water-baths, stringent timing, water-bath 18
It is taken out after minute, the tip of a root after being dissociated；
(3) it rinses and rear hypotonic：The tip of a root after dissociation is taken out from dissociation solution, twice using deionized water cleaning, is finally existed
Hypotonic 24 minutes after in deionized water；
(4) film-making：The tip of a root after will be hypotonic takes out, and is placed on glass slide, removes the front end of the tip of a root with clean scalpel blade
0.5-0.8 millimeters of root cap area, dissecting needle smash the tip of a root to pieces, drip 15 microlitres of Giemsa stains, after dyeing 5-8 minutes, covered,
The side of coverslip is fixed with thumb and index finger, is gently tapped coverslip with pencil, is removed bubble removing, so that tissue is become one thin
Layer, is then baked to low-grade fever on the flame of alcolhol burner, and a blotting paper, the lid glass described in thumb squeezes are added on the coverslip
Piece keeps cell fully dispersed, obtains tabletting；
(5) tabletting being placed in optical microphotograph under the microscope, the dispersion of selective staining body, clearly cell carries out PHOTOGRAPHIC ANALYSIS,
It is marked on the cover slip with glass graver, sample, which is positioned over -80 DEG C of refrigerators, to be preserved.
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| CN201610377477.0A CN106092677B (en) | 2016-05-30 | 2016-05-30 | Sweet potato and its relative genus plant cell division phases sample fast preparation method |
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| CN106092677B true CN106092677B (en) | 2018-10-26 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109100189B (en) * | 2018-07-10 | 2020-12-04 | 焦明远 | Cell tissue sample film-making device |
| CN109856330B (en) * | 2019-01-29 | 2021-05-28 | 南京农业大学 | Method for improving mitotic phase of chrysanthemum root tip through artificial regulation |
| CN111175102A (en) * | 2020-01-13 | 2020-05-19 | 沈阳农业大学 | Method for preparing slices of root tip chromosomes of Paeonia plants |
| CN111398275B (en) * | 2020-03-18 | 2023-06-09 | 湖北和诺生物工程股份有限公司 | Root tip tabletting analysis method based on hairy root culture |
| CN112067410A (en) * | 2020-09-16 | 2020-12-11 | 山东农业大学 | Fringe-based chromosome karyotype analysis method for Chinese fringetree |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103993095A (en) * | 2014-06-10 | 2014-08-20 | 中国科学院西北高原生物研究所 | Preparation method of cell chromosome metaphase specimen of psathyrostachys nevski plant |
| CN104374618A (en) * | 2014-10-20 | 2015-02-25 | 山东省果树研究所 | Plant chromosome tablet observation method |
| CN105021433A (en) * | 2015-06-05 | 2015-11-04 | 中国农业科学院草原研究所 | Squash slide preparation method for root tip chromosome of Elymus plant |
| CN105510095A (en) * | 2015-11-30 | 2016-04-20 | 首都师范大学 | Slide preparation method for discriminating chromosome number of Avena magna, Triticum aestivum or filial generation of Avena magna and Triticum aestivum |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103993095A (en) * | 2014-06-10 | 2014-08-20 | 中国科学院西北高原生物研究所 | Preparation method of cell chromosome metaphase specimen of psathyrostachys nevski plant |
| CN104374618A (en) * | 2014-10-20 | 2015-02-25 | 山东省果树研究所 | Plant chromosome tablet observation method |
| CN105021433A (en) * | 2015-06-05 | 2015-11-04 | 中国农业科学院草原研究所 | Squash slide preparation method for root tip chromosome of Elymus plant |
| CN105510095A (en) * | 2015-11-30 | 2016-04-20 | 首都师范大学 | Slide preparation method for discriminating chromosome number of Avena magna, Triticum aestivum or filial generation of Avena magna and Triticum aestivum |
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