CN110132691A - A kind of wild siberian wildrye chromosome flaking method in High-cold regions - Google Patents
A kind of wild siberian wildrye chromosome flaking method in High-cold regions Download PDFInfo
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- 210000000349 chromosome Anatomy 0.000 title claims abstract description 63
- 241000511977 Elymus sibiricus Species 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 37
- 238000004043 dyeing Methods 0.000 claims abstract description 47
- 239000000463 material Substances 0.000 claims abstract description 32
- 238000010494 dissociation reaction Methods 0.000 claims abstract description 30
- 230000005593 dissociations Effects 0.000 claims abstract description 30
- 238000000386 microscopy Methods 0.000 claims abstract description 10
- 239000003643 water by type Substances 0.000 claims abstract description 8
- 230000006835 compression Effects 0.000 claims abstract description 3
- 238000007906 compression Methods 0.000 claims abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 36
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 29
- 239000012153 distilled water Substances 0.000 claims description 22
- 230000000694 effects Effects 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 17
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 claims description 16
- 210000004027 cell Anatomy 0.000 claims description 14
- 229960000583 acetic acid Drugs 0.000 claims description 13
- 239000007864 aqueous solution Substances 0.000 claims description 12
- 239000011521 glass Substances 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000012362 glacial acetic acid Substances 0.000 claims description 10
- 239000012530 fluid Substances 0.000 claims description 8
- 238000011010 flushing procedure Methods 0.000 claims description 8
- 238000012545 processing Methods 0.000 claims description 7
- MCJGNVYPOGVAJF-UHFFFAOYSA-N quinolin-8-ol Chemical compound C1=CN=C2C(O)=CC=CC2=C1 MCJGNVYPOGVAJF-UHFFFAOYSA-N 0.000 claims description 6
- 239000005725 8-Hydroxyquinoline Substances 0.000 claims description 5
- XGGLLRJQCZROSE-UHFFFAOYSA-K ammonium iron(iii) sulfate Chemical compound [NH4+].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O XGGLLRJQCZROSE-UHFFFAOYSA-K 0.000 claims description 5
- 230000032823 cell division Effects 0.000 claims description 5
- 239000006185 dispersion Substances 0.000 claims description 5
- 229960003540 oxyquinoline Drugs 0.000 claims description 5
- 230000000007 visual effect Effects 0.000 claims description 5
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 4
- 230000035784 germination Effects 0.000 claims description 3
- 230000031864 metaphase Effects 0.000 claims description 3
- 239000003595 mist Substances 0.000 claims description 3
- 210000003813 thumb Anatomy 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 241000570445 Elymus nutans Species 0.000 description 6
- 230000000877 morphologic effect Effects 0.000 description 6
- 238000004040 coloring Methods 0.000 description 5
- 210000004940 nucleus Anatomy 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- 239000000975 dye Substances 0.000 description 4
- 244000025254 Cannabis sativa Species 0.000 description 2
- 241000744304 Elymus Species 0.000 description 2
- 208000035199 Tetraploidy Diseases 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000000249 desinfective effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000012286 potassium permanganate Substances 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 206010050789 Hypochromasia Diseases 0.000 description 1
- 241000209504 Poaceae Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003322 aneuploid effect Effects 0.000 description 1
- 208000036878 aneuploidy Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000010429 evolutionary process Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 description 1
- 229910000360 iron(III) sulfate Inorganic materials 0.000 description 1
- 230000031852 maintenance of location in cell Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 210000000745 plant chromosome Anatomy 0.000 description 1
- 230000006920 protein precipitation Effects 0.000 description 1
- 238000007665 sagging Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 210000002262 tip cell Anatomy 0.000 description 1
- 238000012549 training Methods 0.000 description 1
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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
- G01N1/30—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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Abstract
The invention discloses a kind of wild siberian wildrye chromosome flaking methods in High-cold regions, are related to agricultural technology field.The method of the present invention includes: that (1) prepares tip of a root material；(2) material is pre-processed；(3) pretreated material is fixed, saved, set time 18-24h；(4) material after fixation is dissociated, Dissociation time is 50-80min at room temperature, and Dissociation time is 8-18min under 60 DEG C of waters bath with thermostatic control；(5) mordant dyeing and dyeing, dyeing time 2-10h are carried out to the material after dissociation；(6) material after dyeing is softened, the softening time is 30-60min；(7) tabletting is carried out to the material after softening；(8) microscopy is carried out to press sheet compression.The method of the present invention is simple to operate, keeps the Chromosome spread obtained uniform, and background is shallow, is easy to observe, and the chromosome number accuracy obtained is high.
Description
Technical field
The present invention relates to the wild siberian wildrye chromosome flaking methods of agricultural technology field more particularly to a kind of High-cold regions.
Background technique
Siberian wildrye (Elymus sibiricus L.) is grass family Tribe Triticeae Elymuss not familiar this plant of sod grass for many years,
It is the Wild ornamental resources for being distributed widely in Qinghai-Tibet High-cold regions.Ecological environment of the siberian wildrye to this area's extreme cold arid
With good adaptability and extensive ecological plasticity, and high yield and high quality, therefore have become this area's improvement of natural pasture and people
The most commonly used main herbage grass seeds that work grass-land cultivation utilizes.A large amount of Elymus nutans is also distributed with simultaneously in Qinghai-Tibet Platean
Wild ornamental resources.Siberian wildrye and Elymus nutans are all the sagging class Elymuss species of inflorescence, due to morphological feature by
The long-term influence of growing environment is accurately distinguished the two relatively difficult.Siberian wildrye and Elymus nutans are adapted to for a long time
In evolutionary process, two kinds of different ecotypes are intersected in morphological characters with more character, so that taxonomic identification
It is highly difficult, or even will cause many scholars mistake identification not of the same race similar to Morphologic Characters.
It is reported that the chromosome number of Elymus nutans (E.nutans Griseb.) is 2n=6x=42, belong to six times
Body, genome StStHHYY, and the chromosome number of siberian wildrye is 2n=4x=28, belongs to tetraploid, genome is
StStHH.It is found in previous research, siberian wildrye is in addition to extracellular with typical euploid, and there are also some aneuploids are thin
Born of the same parents occur.Variation in these cytology chromosome numbers is the basis of plant morphological characteristic differentiation.Many research reports,
There is character intersections on morphological feature for siberian wildrye and Elymus nutans, it is necessary to will be difficult to the germplasm materials identified progress
The analysis of chromosome number and caryogram is that breeder utilizes one as the supplement and evidence of Morphological Identification, and by karyotyping
A little specificity germplasm materials provide more full and accurate data.
The researchs such as Sun Yikai report that siberian wildrye karyotype formulas is 20+8sm (4SAT), and the siberian wildrye caryogram of the reports such as Wang Qin is public
Formula is 2n=4x=28=24m (2AST)+4sm, and old bodyguard is brave etc., and to research and propose the karyotype formulas of siberian wildrye be 2n=4x=28=
22m+6sm(2SAT).Dewey is studies have shown that siberian wildrye is that there are two the genomic tetraploid of entitled S and H, 2n=for tool
28, genome becomes StStHH.For forefathers to siberian wildrye chromosome research result there are result disunity, controversial is larger,
And chromosome flaking method is also not quite similar.
The wild siberian wildrye of Qinghai-Tibet Platean distribution, long term growth is in the higher psychro-environment of height above sea level, and form and structure are all
There is apparent difference with other siberian wildryes.The present inventor has found fixation in the prior art, solution when studying siberian wildrye chromosome
From, dyeing and softening the methods of time, chromosome morphology and number can not be obviously observed.
Summary of the invention
In view of this, the embodiment of the invention provides a kind of wild siberian wildrye chromosome flaking method in High-cold regions, main mesh
Be to solve the problems, such as that existing chromosome flaking method can not obviously observe chromosome morphology and number.
In order to achieve the above objectives, invention broadly provides following technical solutions:
On the one hand, the embodiment of the invention provides a kind of wild siberian wildrye chromosome flaking method in High-cold regions, the methods
The following steps are included:
(1) prepare tip of a root material；
(2) material is pre-processed；
(3) pretreated material is fixed, saved, set time 18-24h；
(4) material after fixation is dissociated, Dissociation time is 50-80min at room temperature, is dissociated under 60 DEG C of waters bath with thermostatic control
Time is 8-18min；
(5) mordant dyeing and dyeing, dyeing time 2-10h are carried out to the material after dissociation；
(6) material after dyeing is softened, the softening time is 30-60min；
(7) tabletting is carried out to the material after softening；
(8) microscopy is carried out to press sheet compression.
Preferably, the set time is 22-24h.
Preferably, the Dissociation time at room temperature is 70-80min；Dissociation time is 12- under 60 DEG C of waters bath with thermostatic control
15min。
Preferably, the dyeing time is 5-7h.
Preferably, the softening time is 40-45min.
Preferably, the set time is for 24 hours；Dissociation time is 12min under 60 DEG C of waters bath with thermostatic control.
Preferably, the dyeing time is 6h；The softening time is 40min.
Preferably, step (1) concrete operations include: to select the uniform wild siberian wildrye kind of full grains
Son is placed in the training for being lined with wet filter paper with distilled water flushing 5~6 times with 5~10min of disinfecting solution of potassium permanganate of concentration 1%
It supports in ware, is placed in germination in 25 DEG C of insulating box；
Step (2) concrete operations include: to select the sturdy tip of a root, clip 0.5cm or so to 1~1.5cm of root long,
It immerses in the 8-hydroxyquinoline solution that concentration is 2mmol/L, is put into dark place pretreatment time 2h, 4h, handle time 2h, 4h；4h
When processing, it is divided into direct processing 4h and period every two hours replaces a solution.
Preferably, step (3) concrete operations include: by pretreated young root tip of a root distilled water flushing 3~5
It is secondary, it is put into carnoy fluid, i.e. dehydrated alcohol: in glacial acetic acid=3:1, fixed 18h, 19h, 20h, 21h, 22h, 23h, for 24 hours；It will fix
The tip of a root afterwards is thoroughly cleaned up with distilled water, is then placed in 70% alcohol, is saved backup in 4 DEG C of refrigerators；
Step (4) concrete operations include: that young root that is fixation is complete or being stored in alcohol is thoroughly cleaned with distilled water
It is put it into 1mol/L HCl after clean；It dissociates at room temperature, Dissociation time 50min, 60min, 70min, 80min；?
It is dissociated under the conditions of water bath with thermostatic control at 60 DEG C, Dissociation time 8min, 10min, 12min, 14min, 16min, 18min；
Step (5) concrete operations include: that the young root that will dissociate is clean wash with distilled water, then with 4% ferric sulfate
After aqueous ammonium mordant dyeing 4h, by the young root distilled water flushing after mordant dyeing it is clean after, with 0.5% hematoxylin aqueous solution dyeing 2h,
4h、6h、8h、10h。
Preferably, step (6) concrete operations include: completely to be placed on stained young root wash with distilled water
Soften 30min, 35min, 40min, 45min, 50min, 55min, 60min in 45% glacial acetic acid；
Step (7) concrete operations include: to cut tip of a root 1mm or so on clean glass slide, add 45% second of a drop
Acid, covered；Two filter paper doublings are taken, the glass slide for being placed with the tip of a root is placed wherein, coverslip is touched with rubber, makes root
Tip Cells scatter, and with thumb squeezes coverslip, make it that mist be presented on glass slide, are then placed on microscopically observation；
Step (8) concrete operations include: that glass slide is placed on objective table, and dispersion is then first looked under 10 times of mirrors
Preferable cell simultaneously moves into visual field center, then changes and finds the clearly image in metaphase in cell division under 40 times of mirrors and seen
It examines, all visual field of the observation tip of a root, using the readability of chromosome, dispersion and bunching degree etc. as judging the sight of chromosome effect
Examine the standard of figure.
Compared with prior art, the beneficial effects of the present invention are:
The technical issues of present invention can not obviously observe chromosome morphology and number for existing Chromosome Technique,
The flaking method that a kind of wild siberian wildrye chromosome in High-cold regions simple to operate is found by many years research, makes the dye obtained
Colour solid is uniformly dispersed, and background is shallow, is easy to observe, and the chromosome number accuracy obtained is high.
Detailed description of the invention
Fig. 1 is the microscope figure for the pretreatment 4h+ dyeing 6h that the embodiment of the present invention 1 provides；
Fig. 2 is pretreatment 4h (period replaces a solution)+dyeing 6h microscope figure that the embodiment of the present invention 1 provides；
Fig. 3 is that 10min microscope figure is dissociated under 6h+60 DEG C of water bath with thermostatic control of dyeing that the embodiment of the present invention 1 provides；
Fig. 4 is that 12min microscope figure is dissociated under 6h+60 DEG C of water bath with thermostatic control of dyeing that the embodiment of the present invention 1 provides；
Fig. 5 is 8h+60 DEG C of water bath with thermostatic control 12min microscope figure of dyeing that the embodiment of the present invention 1 provides；
Fig. 6 is that the mordant dyeing 4h+ hematoxylin that the embodiment of the present invention 1 provides dyes 6h+60 DEG C of water bath with thermostatic control 12min+ softening
40min microscope figure.
Specific embodiment
For further illustrate the present invention to reach the technical means and efficacy that predetermined goal of the invention is taken, below with compared with
Good embodiment, to specific embodiment, technical solution, feature and its effect applied according to the present invention, detailed description is as follows.Under
Stating the special characteristic, structure or feature in multiple embodiments in bright can be combined by any suitable form.
Embodiment 1
A kind of wild siberian wildrye chromosome flaking method in High-cold regions, comprising the following steps:
Test reagent: 0.002M 8-hydroxyquinoline, Ka Nuoshi fixer (dehydrated alcohol: glacial acetic acid=3:1), 1mol/L
HCl, 4% ammonium ferric sulfate aqueous solution, 0.5% hematoxylin aqueous solution, 45% glacial acetic acid aqueous solution.
Test method: mainly using plant root tip pressed disc method, and colouring method is siderotil-hematoxylin (NH4Fe(S04)2—
C16H14O6) decoration method.
Prepare tip of a root material: selecting the uniform wild siberian wildrye seed of full grains, (this research material therefor comes from
Qinghai-Tibet height above sea level 2850-4650 meters of High-cold regions), with 5~10min of disinfecting solution of potassium permanganate of concentration 1%, rushed with distilled water
It washes 5~6 times, is placed in the culture dish for being lined with wet filter paper, be placed in germination in 25 DEG C of insulating box.
Tip of a root pretreatment: to 1~1.5cm of root long or so (9~10 days), selecting the sturdy tip of a root, clip 0.5cm or so,
It immerses in the 8-hydroxyquinoline solution that concentration is 2mmol/L, is put into dark place pretreatment time 2h, 4h, handle time 2h, 4h.(4h
When processing, it is divided into direct processing 4h and period every two hours replaces a solution).
It is fixed to save: by the pretreated young root tip of a root with distilled water flushing 3~5 times, be put into carnoy fluid (dehydrated alcohol:
Glacial acetic acid=3:1) in fix 18h, 19h, 20h, 21h, 22h, 23h, for 24 hours.The tip of a root after fixation is thoroughly cleaned with distilled water
Completely, it is then placed in 70% alcohol, is saved backup in 4 DEG C of refrigerators.
Dissociation: young root that is fixation is complete or being stored in alcohol is thoroughly cleaned up rear (to prevent cleaning not with distilled water
Completely, it is soaked in distilled water after 20min and cleans again one time after being rinsed at second), it puts it into 1mol/L HCl.
(1) it dissociates at room temperature, Dissociation time 50min, 60min, 70min, 80min.
(2) dissociated under the conditions of water bath with thermostatic control at 60 DEG C, Dissociation time 8min, 10min, 12min, 14min, 16min,
18min。
Mordant dyeing and dyeing: the young root dissociated is clean wash with distilled water, then with 4% ammonium ferric sulfate aqueous solution mordant dyeing 4h
Afterwards, by the young root distilled water flushing after mordant dyeing it is clean after, with 0.5% hematoxylin aqueous solution dye 2h, 4h, 6h, 8h, 10h.
Softening: stained young root is completely placed on wash with distilled water in 45% glacial acetic acid soften 30min, 35min,
40min、45min、50min、55min、60min。
Tabletting: tip of a root 1mm or so is cut on clean glass slide, adds 45% acetic acid of a drop, covered.Take two
Filter paper doubling, the glass slide for being placed with the tip of a root is placed wherein, touches coverslip with rubber, root-tip cells is made to scatter, squeezed with thumb
Gland slide makes it that mist be presented on glass slide, is then placed on microscopically observation.
Microscopy: glass slide is placed on objective table, is then first looked for well dispersed cell under 10 times of mirrors and is moved into view
Yezhong centre is then changed to find under 40 times of mirrors and clearly be observed in the image of metaphase in cell division, and the observation tip of a root is all
The visual field, using the readability of chromosome, dispersion and bunching degree etc. as the standard for judging chromosome effect observation figure.
Interpretation of result:
(1) influence of different pretreatments time
There are two pretreated effects, first is that therefore being formed but not influencing the cell division of early period for inhibition spindle fiber makes
More cellular retentions provide largely available material in metacinesis state, for experiment；It further contracts second is that can induce chromosome
It is short to straighten, it is apparent convenient for Chromosome spread and chromosome morphology.Pretreatment fluid employed in this test is 0.002M 8-
Oxyquinoline, test result is as shown in table 1, and when handling 2h with treatment fluid, chromosome shortening is unobvious, observes under the microscope not
Obviously.And when directly handling 4h with treatment fluid, although chromosome shortens and more obviously largely gets together in rodlike,
It is difficult to observe.But when a solution is replaced in centre, chromosome is in rodlike and largely disperses, and observation is easier to, microscopy effect
Preferably.
Siberian wildrye tip of a root stained preparation effect after the 1. different pretreatments time of table
(2) influence of different set times
Fixed purpose is to be killed material rapidly with the strong fixer of penetration, makes protein precipitation, and make it as far as possible
Keep original state.Test result is as shown in table 2, when handling 18-21h with Ka Nuoshi fixer, because cell does not kill completely,
Chromosome many places are difficult to observe under the microscope in form is changeable or the incomplete state of form.And works as and handle 22- with treatment fluid
When 23, cell has been killed, and chromosome morphology is stablized, and microscopically observation is easier to, but acquired chromosome morphology is imperfect.
Mutually compared with for, when being handled for 24 hours with treatment fluid, chromosome morphology is stable and form is complete, and microscopically observation is relatively clear, mirror
It is preferable to examine effect.
Siberian wildrye tip of a root stained preparation effect after the different set times of table 2.
(3) influence of different Dissociation times
The purpose of dissociation is that cell is made mutually to scatter.Test result handles 50- as shown in table 3, table 4 at room temperature
When 70min, cell degree of scatter is lower, and tabletting is more difficult, and when microscopically observation, cell overlap is more serious, it is difficult to observe.I.e.
Make to handle 80min at room temperature, the most of still difficult dispersion of cell, microscopically observation is not easy.In contrast, by 60 DEG C of constant temperature
The tip of a root dissociated under water bath condition, in addition to faster than under room temperature in time, dissociation effect is also than under room temperature
It is better.When dissociating 8min in thermostat water bath at 60 DEG C, tabletting is easier to, but when observing under the microscope, cell overlap degree
Higher, microscopy effect is poor.When dissociating 12min in thermostat water bath at 60 DEG C, dissociation effect is preferable, and cell holds when tabletting
Easily pressure dissipates, and chromosome is differentiated relatively clear.When dissociating 14min or more in thermostat water bath at 60 DEG C, part cell can be made
Rupture influences microscopy effect so that chromosome deficiency or mixing.
3. room temperature of table dissociates siberian wildrye tip of a root stained preparation effect
Siberian wildrye tip of a root stained preparation effect after table 4. different time, 60 DEG C of waters bath with thermostatic control
(3) influence of different dyeing times
Test result is as shown in table 5, after with 4% ammonium ferric sulfate aqueous solution mordant dyeing 4h, with 0.05% hematoxylin aqueous solution
After dyeing 6h, nucleus coloring is obvious, and cytoplasm is not colored or light coloring, it can be observed that clearly chromosome under microscope.
If dyeing time is too short, nucleus coloring is unobvious, it is difficult to observe clearly chromosome.If dyeing time is too long, nucleus
Coloring degree is deeper, and cytoplasm is also colored, and is equally difficult to observe by clearly chromosome.In addition, it should also be noted that dyeing
Whether the preparation and use of agent hematoxylin aqueous solution are proper, most important to production effect.
Siberian wildrye tip of a root stained preparation effect after the different dyeing times of table 5.
(4) influence of different softening times
Test result is as shown in table 6, when dyeing 6h, due to nucleus color mistake when handling 30-35min with glacial acetic acid
It is deep, be not easy to observe relatively clear chromosome, and when dyeing 40min, observed chromosome clear, microscopy effect compared with
It is good.When soften the time be more than 45min when, due to processing the time it is longer, nucleus fade seriously, in microscopy not it is observed that compared with
For clearly chromosome.
Siberian wildrye tip of a root stained preparation effect after the different softening times after 6. 6h of table dyeing
It was found from the experimental result of above-mentioned table 1- table 6:
(1) draw materials the time: under normal circumstances, the cell division animated period of plant cell chromosome is 8:00-11 in morning:
00, and when temperature is higher, the observable cell in metacinesis period is more.But all due to the tip of a root used in this experiment
Be in constant incubator culture obtained by, therefore draw materials the time it is unaffected.
(2) dissociation process: dissociation process is a link very crucial in plant chromosome tabletting technology.Dissociation solution
Treatment effect is affected to microscopic examination result.This test result shows the wild siberian wildrye young root fixed to be placed in 60
Chromosome when with 1mol/L HCl dissociation 12min finding its microscopy under the conditions of water bath with thermostatic control at DEG C is more dispersed and form is clear
It is clear, convenient for observation.
(3) softening process: since siderotil-hematoxylin staining needs to overstain to test material, so needing
Color separation and softening are carried out to the material section for crossing dye, therefore softening process is also very important a step.The discovery of this test result,
When the material to excess processes carries out sofening treatment with 45% glacial acetic acid, due to different, the later period softening of dyeing time
Degree for the treatment of also will appear larger difference.If dyeing is deeper, the softening time can be extended, if hypochromasia, the softening time must be shortened.
It is however noted that the relationship of softening time and dyeing time has certain control range, if it exceeds this range, then need
It redeterminates.
The embodiment of the present invention the result shows that: wild siberian wildrye young root is used to the 8-hydroxyquinoline solution of 0.002M at normal temperature
4h is pre-processed, a solution is during which every two hours replaced, then after Ka Nuoshi fixer is fixed for 24 hours, the thermostatted water at 60 DEG C
12min is dissociated with 1mol/L HC under the conditions of bath, the young root after dissociation is then placed into mordant dyeing 4h in 4% ammonium ferric sulfate aqueous solution
Afterwards, 6h is dyed with 0.5% hematoxylin aqueous solution, young root is finally placed to film-making observation after softening 40min in 45% acetic acid to be obtained
Obtain form clearly chromosome tabletting.
Above-mentioned flaking method of the invention is simple to operate, it is determined that the best film-making of wild siberian wildrye chromosome sectioning
Method keeps the Chromosome spread obtained uniform, and background is shallow, is easy to observe, and the chromosome number accuracy obtained is high.
Place, those skilled in the art can not select from the prior art to the greatest extent in the embodiment of the present invention.
Disclosed above is only a specific embodiment of the invention, but scope of protection of the present invention is not limited thereto, is appointed
What those familiar with the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, answer
It is included within the scope of the present invention.Therefore, protection scope of the present invention should be with above-mentioned scope of protection of the claims
It is quasi-.
Claims (10)
1. a kind of wild siberian wildrye chromosome flaking method in High-cold regions, which is characterized in that the described method comprises the following steps:
(1) prepare tip of a root material；
(2) material is pre-processed；
(3) pretreated material is fixed, saved, set time 18-24h；
(4) material after fixation is dissociated, Dissociation time is 50-80min, Dissociation time under 60 DEG C of waters bath with thermostatic control at room temperature
For 8-18min；
(5) mordant dyeing and dyeing, dyeing time 2-10h are carried out to the material after dissociation；
(6) material after dyeing is softened, the softening time is 30-60min；
(7) tabletting is carried out to the material after softening；
(8) microscopy is carried out to press sheet compression.
2. a kind of wild siberian wildrye chromosome flaking method in High-cold regions as described in claim 1, which is characterized in that the fixation
Time is 22-24h.
3. a kind of wild siberian wildrye chromosome flaking method in High-cold regions as described in claim 1, which is characterized in that the room temperature
Lower Dissociation time is 70-80min；Dissociation time is 12-15min under 60 DEG C of waters bath with thermostatic control.
4. a kind of wild siberian wildrye chromosome flaking method in High-cold regions as described in claim 1, which is characterized in that the dyeing
Time is 5-7h.
5. a kind of wild siberian wildrye chromosome flaking method in High-cold regions as described in claim 1, which is characterized in that the softening
Time is 40-45min.
6. a kind of wild siberian wildrye chromosome flaking method in High-cold regions as described in claim 1, which is characterized in that the fixation
Time is for 24 hours；Dissociation time is 12min under 60 DEG C of waters bath with thermostatic control.
7. a kind of wild siberian wildrye chromosome flaking method in High-cold regions as described in claim 1, which is characterized in that the dyeing
Time is 6h；The softening time is 40min.
8. a kind of wild siberian wildrye chromosome flaking method in High-cold regions as described in claim 1, which is characterized in that the step
(1) concrete operations include: to select the uniform wild siberian wildrye seed of full grains, with the liquor potassic permanganate of concentration 1%
5~10min of disinfection is placed in the culture dish for being lined with wet filter paper, is placed in 25 DEG C of insulating box with distilled water flushing 5~6 times
Germination；
Step (2) concrete operations include: to select the sturdy tip of a root, clip 0.5cm or so to 1~1.5cm of root long, are immersed
Concentration is to be put into dark place pretreatment time 2h, 4h in the 8-hydroxyquinoline solution of 2mmol/L, handles time 2h, 4h；4h processing
When, it is divided into direct processing 4h and period every two hours replaces a solution.
9. a kind of wild siberian wildrye chromosome flaking method in High-cold regions as described in claim 1, which is characterized in that the step
(3) concrete operations include: to be put into carnoy fluid, i.e. dehydrated alcohol for pretreated young root tip of a root distilled water flushing 3~5 times:
In glacial acetic acid=3:1, fixed 18h, 19h, 20h, 21h, 22h, 23h, for 24 hours；The tip of a root after fixation is thoroughly cleaned with distilled water
Completely, it is then placed in 70% alcohol, is saved backup in 4 DEG C of refrigerators；
Step (4) concrete operations include: that young root that is fixation is complete or being stored in alcohol is thoroughly cleaned up with distilled water
After put it into 1mol/L HCl；It dissociates at room temperature, Dissociation time 50min, 60min, 70min, 80min；At 60 DEG C
It is dissociated under the conditions of lower water bath with thermostatic control, Dissociation time 8min, 10min, 12min, 14min, 16min, 18min；
Step (5) concrete operations include: that the young root that will dissociate is clean wash with distilled water, then with 4% ammonium ferric sulfate water
After solution mordant dyeing 4h, by the young root distilled water flushing after mordant dyeing it is clean after, with 0.5% hematoxylin aqueous solution dyeing 2h, 4h,
6h、8h、10h。
10. a kind of wild siberian wildrye chromosome flaking method in High-cold regions as described in claim 1, which is characterized in that the step
Suddenly (6) concrete operations include: stained young root is completely placed on wash with distilled water in 45% glacial acetic acid soften 30min,
35min,40min,45min,50min,55min,60min；
Step (7) concrete operations include: to cut tip of a root 1mm or so on clean glass slide, add 45% acetic acid of a drop, cover
Upper coverslip；Two filter paper doublings are taken, the glass slide for being placed with the tip of a root is placed wherein, coverslip is touched with rubber, keeps the tip of a root thin
Born of the same parents scatter, and with thumb squeezes coverslip, make it that mist be presented on glass slide, are then placed on microscopically observation；
Step (8) concrete operations include: that glass slide is placed on objective table, are then first looked under 10 times of mirrors well dispersed
Cell and move into visual field center, then change to find under 40 times of mirrors and clearly be observed in the image of metaphase in cell division,
All visual field of the tip of a root is observed, using the readability of chromosome, dispersion and bunching degree etc. as judging chromosome effect observation
The standard of figure.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110849686A (en) * | 2019-11-28 | 2020-02-28 | 江苏省农业科学院 | Tabletting method for rapidly obtaining strawberry root tip chromosome and prepared tablet |
| CN112255069A (en) * | 2020-10-22 | 2021-01-22 | 南京农业大学 | Dendrobium huoshanense root tip specimen tablet and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101161056A (en) * | 2007-09-28 | 2008-04-16 | 中国科学院新疆理化技术研究所 | A method for evoking yili caladium polyploid under culture in vitro |
| CN102183394A (en) * | 2011-03-08 | 2011-09-14 | 沈阳农业大学 | Method for making hyacinth root tip chromosome slice |
| CN109030131A (en) * | 2018-06-12 | 2018-12-18 | 中国水产科学研究院黑龙江水产研究所 | A kind of refined dragonfish method of chromosome preparation |
-
2019
- 2019-06-18 CN CN201910526616.5A patent/CN110132691A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101161056A (en) * | 2007-09-28 | 2008-04-16 | 中国科学院新疆理化技术研究所 | A method for evoking yili caladium polyploid under culture in vitro |
| CN102183394A (en) * | 2011-03-08 | 2011-09-14 | 沈阳农业大学 | Method for making hyacinth root tip chromosome slice |
| CN109030131A (en) * | 2018-06-12 | 2018-12-18 | 中国水产科学研究院黑龙江水产研究所 | A kind of refined dragonfish method of chromosome preparation |
Non-Patent Citations (3)
| Title |
|---|
| 张晓燕: "偃麦草染色体核型与遗传多样性分析", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 薛娟: "四川引种大花蕙兰品种观赏价值评价及倍性鉴定研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 赵增煜: "《常用农业科学试验法》", 31 October 1981, 农业出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110849686A (en) * | 2019-11-28 | 2020-02-28 | 江苏省农业科学院 | Tabletting method for rapidly obtaining strawberry root tip chromosome and prepared tablet |
| CN112255069A (en) * | 2020-10-22 | 2021-01-22 | 南京农业大学 | Dendrobium huoshanense root tip specimen tablet and preparation method and application thereof |
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