CN106932256A - A kind of method for preparing asparagus root tip cell chromosome division phases sample - Google Patents
A kind of method for preparing asparagus root tip cell chromosome division phases sample Download PDFInfo
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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
- G01N1/30—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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- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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- Pathology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of method for preparing asparagus root tip cell chromosome division phases sample, belong to plant cytogenetics technical field.Technical scheme main points are：Using hydroxycarbamide and N2The double blocked methods of O induce the mitosis of synchronizing of Cells of asparagus to obtain a large amount of division phases cells, and it is clear, well dispersed and suitable for the chromosome slide sample of subsequent cell research largely to obtain form by hypotonic, fixed and flaking step.The present invention uses HU as blocking agent, and combines renewal cultivation and N2O treatment, greatly increases asparagus division phases cell, using conventional compression medium cell can be made to account for more than the 50% of TCS, meanwhile, through N2The metaphase chromosome volume morphing of O treatment is good, greatly increases the success rate and stability of asparagus chromosome sectioning, is that the application of the technologies such as asparagus karyotyping, FISH is provided a great convenience.
Description
Technical field
The invention belongs to plant cytogenetics technical field, and in particular to one kind prepares asparagus root tip cell chromosome
The method of division phases sample.
Background technology
Chromosome is most important part in nucleus, and the variation of chromosome number and structure is provided for plant breeding
Abundant genetic resources.Root-tip cells film-making mitosis metaphase is a basic cytogenetical study technology and hand
Section, the technology such as the technology and the karyotyping based on this technology and FISH is in species identification and discriminating, thing
It is widely used in the researchs such as Interspecific relationship determination, species Ploidy Identification and crossbreeding.
Asparagus popular name asparagus, is under the jurisdiction of Liliaceae Asparagus, and its tender stem is rich in amino acid, protein and vitamin etc.
Multiple nutritional components, are one of world-renowned vegetables, and the good reputation of " kings of vegetables " is enjoyed in the world.Asparagus is typical female
Male dioecian plant, its genetic constitution is 2n=2x=20.The sex of asparagus is determined by sex chromosome, with XX sex chromosome
Show as female, and there is the male that shows as of XY sex chromosome, but, its X and Y chromosome are homotype.In recent years, it is right
The research of asparagus is concentrated mainly on the aspects such as nutritional ingredient, molecular biology research, and its cytological research is relatively lagged behind,
This is dense mainly due to the cytoplasm of asparagus, and cell membrane is harder, and chromosome easily trails and chromosome is smaller, it is difficult to obtain
Obtain clear, the well dispersed split coil method of form.Also, although meristem zone's separatist activities of the asparagus tip of a root is more vigorous,
But the cell of metaphase only occupies a little part, still in a phase, this preferably dyes system to most cells to obtain
Piece causes very big obstacle, generally require to carry out substantial amounts of chromosome sectioning can just pick out it is a small amount of preferably can be used for analysis and
The slide of later experiments, wastes substantial amounts of human and material resources and time.Induction plant cell cycle is synchronized and can made in division
The cell proportion of mid-term is greatly increased, and greatly improves the success rate of chromosome sectioning and stability.But for asparagus, very
To Asparagus also without related report.Current most common method is to use hydroxycarbamide（HU）And methyl thymol blue complecon（APM）
Double blocked method induction plant cell mitosis are synchronized, and can obtain high-frequency mitosis metaphase index.But, HU and APM
It is all toxic, and through APM process after, asparagus root tip cell chromosome easily trails, and form is bad, is unfavorable for carrying out the later stage
Deeper into research and analysis.
The content of the invention
Present invention solves the technical problem that there is provided one kind prepares asparagus root tip cell chromosome division phases mark
This method.
The present invention adopts the following technical scheme that one kind prepares asparagus root tip cell chromosome to solve above-mentioned technical problem
The method of division phases sample, it is characterised in that：Using hydroxycarbamide and N2The double blocked method induction Cells of asparagus of O are synchronized
Change mitosis to obtain a large amount of division phases cells, then through hypotonic, fixed and flaking step largely obtain form it is clear, point
Dissipate chromosome slide sample that is good and being studied suitable for subsequent cell.
The method for preparing asparagus root tip cell chromosome division phases sample of the present invention, it is characterised in that tool
Body step is：
（1）Root tip culture：Asparagus seed is soaked into 24h in 30 DEG C of water, then seed is placed on paving layer 2-3 water-soaked
In the culture dish of filter paper, then cultivated 4 days to a length of 0.8-1.5cm of root in 25 DEG C；
（2）Hydroxycarbamide treatment：Under the conditions of 25 DEG C a length of 0.8- of root is processed with the hydroxycarbamide that molar concentration is 2.5mmol/L
The asparagus seed 18h of 1.5cm；
（3）Renewal cultivation：Hydroxycarbamide is removed, seed is cleaned with clear water three times, tip of a root 4- is then cultivated in 25 DEG C of clear water
6h；
（4）N2O is pre-processed：Cut the tip of a root and be put into N21-3h is processed in O air chambers；
（5）It is hypotonic：30-90min is processed with the KCl solution that molar concentration is 0.075mol/L；
（6）It is fixed：Be with volume fraction 90% Acetic Acid Glacil on fix tip of a root 10min；
（7）Drop piece：10min is washed on ice with deionized water after fixation, Root apical meristem area is then cut, and is placed in mass fraction
Pectase and mass fraction for 1% are 2% mixed liquor of cellulase, and 37 DEG C of enzymolysis 2h add TE to delay after the completion of enzymolysis
Fliud flushing is blown and beaten to remove root cap and tip of a root outer tissue repeatedly, then the tip of a root is washed twice with the ethanol that volume fraction is 70%, 40
The tip of a root is smashed to pieces with dissecting needle, vortex suspension cell, centrifugation reservation is precipitated and adds 30 μ L anhydrous acetic acids, inhaled after mixing in μ L ethanol
5-8 μ L drop pieces, room temperature can microscopy after placing 5min.
Further preferably, step（1）Middle asparagus root is long to be preferably 1cm.
Further preferably, step（3）The time of the middle clear water culture tip of a root is preferably 5h.
Further preferably, step（4）Middle N2The treatment pressure of O is 1.01MPa, N2The process time of O is preferably 2h.
Further preferably, step（5）The process time of middle KCl solution is preferably 60min.
The present invention has the advantages that compared with prior art：
1st, cells blocks can be made in the interkinesis using HU, renewal cultivation is carried out after removal HU, inducible synchronizing of cell,
By the renewal cultivation of 5h, a large amount of cells are made to be located at fissional mid-term, after preprocessed, fixed and compressing tablet, mitosis
The ratio of medium cell is greatly increased；
2nd, the present invention uses N2O pre-processes the tip of a root, can help to remove cytoplasm background, makes chromosome morphology good, and method
Simply, in addition, during film-making, the pollution to environment and human body can be substantially reduced and risk is poisoned；
3rd, the present invention is in N2Hypotonic step is increased after O treatment, Chromosome spread can be made good；
4th, the present invention adds TE buffer solutions to blow and beat repeatedly after tip of a root enzymolysis, can remove root cap and tip of a root surface texture, makes dye
Colour solid sample clear background, clean free from admixture；
5th, substantial amounts of asparagus division phases can be obtained using the present invention, the success rate for preparing asparagus chromosome specimen is big
It is big to improve, save substantial amounts of human and material resources and time；
6th, using the method for the present invention can provide facility for the preparation of asparagus chromosome specimen, for asparagus chromosome thing
Study on Evolution of reason map construction, genetic breeding, evolution of sex chromosomes research and asparagus platymiscium etc. is with important reason
By and more practical value.
Brief description of the drawings
Fig. 1 is the cytological map observed by Conventional compression after the asparagus tip of a root is synchronized；
Fig. 2 is the cytological map observed by Conventional compression after the asparagus tip of a root is not synchronized；
Fig. 3 drips the chromosome map that piece method is obtained after being synchronized by the asparagus tip of a root；
Fig. 4 is positioning figures of the 45S rDNA on asparagus chromosome（The chromosome that A is redyed for DAPI, B is Alexa Fluor-
The signal site of 488 fluorescein-labeled 45S rDNA, C is composite diagram）.
Specific embodiment
The above of the invention is described in further details by the following examples, but this should not be interpreted as this
The scope for inventing above-mentioned theme is only limitted to following embodiment, and all technologies realized based on the above of the present invention belong to this hair
Bright scope.
Embodiment 1
A kind of method for inducing asparagus root-tip cells mitotic synchronization, comprises the following steps：
1st, root tip culture：Asparagus seed is soaked into 24h in 30 DEG C of water, then seed is placed on paving layer 2-3 water-soaked
In the culture dish of filter paper, then cultivated 4 days to a length of 1cm of root in 25 DEG C；
2nd, hydroxycarbamide treatment：Stone with a length of 1cm of hydroxycarbamide treatment root that molar concentration is 2.5mmol/L under the conditions of 25 DEG C is tricky
Cypress seed 18h；
3rd, renewal cultivation：Hydroxycarbamide is removed, seed is cleaned with clear water three times, seed 5h is then cultivated in 25 DEG C of clear water；
4、N2O is pre-processed：Cut the tip of a root and be put into N22h is processed in O air chambers, the N2Pressure in O air chambers is 10atm（1.01MPa）；
5th, it is hypotonic：60min is processed with the KCl solution that molar concentration is 0.075mol/L；
6th, it is fixed：Be with volume fraction 90% Acetic Acid Glacil on fix tip of a root 10min；
7th, compressing tablet：Isolation liquid is put into after the tip of a root washing that will be fixed（Volume ratio is 1:1 concentrated hydrochloric acid and absolute ethyl alcohol mixing
Liquid）Middle isolation 1.5min, cuts 2mm tip of a root growing point parts long and is placed on slide after washing removal isolation liquid, fully cuts
The pinkish red dye liquor dyeing 15min of improvement is added dropwise after broken, adding cover glass carries out Conventional compression；
8th, microscopy：Slide is placed in basis of microscopic observation, mitosis metaphase division Phase Proportion is taken pictures and calculate.
After using the method, as shown in figure 1, asparagus metacinesis Phase Proportion can reach more than 50%, and it is not synchronized
Change, the asparagus metacinesis Phase Proportion obtained by conventional treatment is about 1%（Fig. 2）.
Embodiment 2
Drop piece method prepares asparagus metaphase chromosome sample
The asparagus tip of a root after synchronization process is cut into Root apical meristem area, it is 1% to be placed in 20 μ L volume fractions containing quality
In pectase and the mixed liquor that quality volume fraction is 2% cellulase, 2h is digested in 37 DEG C.TE bufferings are added after the completion of enzymolysis
Liquid is blown and beaten to remove root cap and tip of a root outer tissue repeatedly, then washes the tip of a root twice with the ethanol that volume fraction is 70%, in 40 μ L
The tip of a root is smashed to pieces with dissecting needle, vortex suspension cell, centrifugation reservation is precipitated and adds 30 μ L anhydrous acetic acids, and 5- is inhaled after mixing in ethanol
8 μ L drop pieces, room temperature can microscopy after placing 5min.The good slice, thin piece of microscopy is put in UV-crosslinked instrument by 120-125mJ/cm2
It is standby that treatment 2min fixes chromosome.As shown in figure 3, asparagus Chromosome spread is good, form is clear, cytoplasm for microscopy result
Background is relatively low.
Embodiment 3
Asparagus metaphase chromosome FISH
45S probes are marked：By following component in centrifuge tube is added on ice：Corn 45S rDNA 2 μ g, 10 × nick
Translation buffer 2 μ L, Labeled-dNTP（Texas red-dCTP or Alexa Fluor-488-dUTP）0.5µ
2 μ L, DNA polymerase I of L, Non-labeled-dNTPs 5,0.5 μ L of μ L, DNaseI, are mended to 20 μ L with deionized water.
2h is processed in 15 DEG C in PCR instrument after mixing, plus 5 × the μ L of TAE+140ng/ μ L salmon sperm DNAs 175, addition volume fraction 90% ethanol-
The μ L of 10% sodium acetate of volume fraction 500, mix precipitation more than probe 2h, are subsequently placed in centrifuge the centrifugation speed in 13000rpm
Rate is centrifuged 30min, and absolute ethyl alcohol is washed twice, and lucifuge room temperature preservation 30min plus 2 × the μ L dissolution precipitations of 1 × TE of SSC 20.
FISH：Prepare probe solution：The probe that 0.5 μ L are marked adds 2 × SSC, the 1 × TE of 5.5 μ L, will
Probe solution is added dropwise the place for having cell on slide, gently puts plastic coverslip.Slice, thin piece is placed in and is covered with moist tissue
In canister, then be put into slice, thin piece in hybridizing box by boiling water bath 5min, 55 DEG C of hybridization more than 3h.Hybridize slice, thin piece after terminating
Take out, be put into 5min in 2 × SSC, remove cover plate.
Signal detection：Add DAPI fluorescence dye liquors of the 20 μ L containing anti-quencher, lucifuge dyeing on chromosome slide sample
10min, covers cover plate long（22cm×40cm）Microscopy can be carried out under fluorescence microscope afterwards.Microscopy uses Olympus BX63
Fluorescence microscope, CCD carries out chromosome analysis with Metamorph softwares after taking pictures, and Photoshop carries out image procossing.As a result
As shown in figure 4, being observed that bright hybridization signal by fluorescence microscope.
Embodiment above describes general principle of the invention, principal character and advantage, the technical staff of the industry should
Understand, the present invention is not limited to the above embodiments, simply original of the invention is illustrated described in above-described embodiment and specification
Reason, under the scope for not departing from the principle of the invention, various changes and modifications of the present invention are possible, and these changes and improvements each fall within
In the scope of protection of the invention.
Claims (6)
1. a kind of method for preparing asparagus root tip cell chromosome division phases sample, it is characterised in that：Using hydroxycarbamide
And N2The double blocked methods of O induce the mitosis of synchronizing of Cells of asparagus to obtain a large amount of division phases cells, then through low
Ooze, fixed and flaking step largely obtains that form is clear, well dispersed and chromosome slide that studied suitable for subsequent cell
Sample.
2. it is according to claim 1 prepare asparagus root tip cell chromosome division phases sample method, its feature
It is to concretely comprise the following steps：
（1）Root tip culture：Asparagus seed is soaked into 24h in 30 DEG C of water, then seed is placed on paving layer 2-3 water-soaked
In the culture dish of filter paper, then cultivated 4 days to a length of 0.8-1.5cm of root in 25 DEG C；
（2）Hydroxycarbamide treatment：Under the conditions of 25 DEG C a length of 0.8- of root is processed with the hydroxycarbamide that molar concentration is 2.5mmol/L
The asparagus seed 18h of 1.5cm；
（3）Renewal cultivation：Hydroxycarbamide is removed, seed is cleaned with clear water three times, tip of a root 4- is then cultivated in 25 DEG C of clear water
6h；
（4）N2O is pre-processed：Cut the tip of a root and be put into N21-3h is processed in O air chambers；
（5）It is hypotonic：30-90min is processed with the KCl solution that molar concentration is 0.075mol/L；
（6）It is fixed：Be with volume fraction 90% Acetic Acid Glacil on fix tip of a root 10min；
（7）Drop piece：10min is washed on ice with deionized water after fixation, Root apical meristem area is then cut, and is placed in mass fraction
Pectase and mass fraction for 1% are 2% mixed liquor of cellulase, and 37 DEG C of enzymolysis 2h add TE to delay after the completion of enzymolysis
Fliud flushing is blown and beaten to remove root cap and tip of a root outer tissue repeatedly, then the tip of a root is washed twice with the ethanol that volume fraction is 70%, 40
The tip of a root is smashed to pieces with dissecting needle, vortex suspension cell, centrifugation reservation is precipitated and adds 30 μ L anhydrous acetic acids, inhaled after mixing in μ L ethanol
5-8 μ L drop pieces, room temperature can microscopy after placing 5min.
3. it is according to claim 2 prepare asparagus root tip cell chromosome division phases sample method, its feature
It is：Step（1）Middle asparagus root is long to be preferably 1cm.
4. it is according to claim 2 prepare asparagus root tip cell chromosome division phases sample method, its feature
It is：Step（3）The time of the middle clear water culture tip of a root is preferably 5h.
5. it is according to claim 2 prepare asparagus root tip cell chromosome division phases sample method, its feature
It is：Step（4）Middle N2The treatment pressure of O is 1.01MPa, N2The process time of O is preferably 2h.
6. it is according to claim 2 prepare asparagus root tip cell chromosome division phases sample method, its feature
It is：Step（5）The process time of middle KCl solution is preferably 60min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN114062087A (en) * | 2021-11-07 | 2022-02-18 | 福建省热带作物科学研究所 | Method for preparing chromosome of melastoma plant based on FISH hybridization |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN102876801A (en) * | 2012-10-22 | 2013-01-16 | 中国科学院遗传与发育生物学研究所 | Method for identifying exogenous chromosomes and chromosome segments in plant distant hybrids |
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| CN114062087A (en) * | 2021-11-07 | 2022-02-18 | 福建省热带作物科学研究所 | Method for preparing chromosome of melastoma plant based on FISH hybridization |
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