CN104099416A - FISH (fluorescence in situ hybridization) method of sesame chromosome - Google Patents
FISH (fluorescence in situ hybridization) method of sesame chromosome Download PDFInfo
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Abstract
The invention belongs to the field of biotechnologies, and particularly relates to an FISH (fluorescence in situ hybridization) method for sesame chromosome. A sesame medium-term chromosome sample is manufactured by adopting a smear technology, wherein the chromosome is good in dispersing effect and high in smear manufacturing efficiency; based on the foundation, fluorescein is adopted to mark a BAC probe to create the in situ hybridization technology for chromosome of the sesame BAC; after the probe is conducted by hybridization with the chromosome, the fluorescent signal can be directly detected, so as to determine quantity and position of an objective DNA segment on the chromosome. The chromosome sample manufactured by the method can be used for repeated hybridization; utilization ratio and experimental efficiency of the sample are improved; a great amount of time is saved; cost is lowered; blank of BAC-FISH of sesame chromosome in China is filled.
Description
technical field
The invention belongs to biological technical field, be specifically related to a kind of sesame chromosome sectioning and BAC fluorescence in-situ hybridization method.
Background technology
Chromosome sectioning and bacterial artificial chromosome fluorescence in situ hybridization (BAC-FISH) technology are the important technical of carrying out the cytogenetical studies such as biological DNA fragmentation chromosomal localization, physical map structure.In the research of sesame chromosome karyotype analysis, the chromosome flaking method using is traditional tabletting method at present; And there is not yet the application report of any tabletting technology in sesame BAC-FISH research.In the time making chromosome specimen, traditional pressed disc method need to be struck sheet, cover plate processing to chromosome specimen, and karyomit(e) is easily out of shape; In the time carrying out in situ hybridization, chromosome specimen is because of in tenuigenin parcel, and often its background signal is stronger, disturbs larger to the observation of hybridization signal; And peel link in intermediary operation process very likely causes chromosome elimination, thereby cause film-making efficiency low, time-consuming.In addition, traditional pressed disc technique is unfavorable for being repeatedly used of film-making, and this has also affected the application of hybridization in situ technique in sesame physical map structure and the research of specific fragment chromosomal localization largely.Therefore, current urgent need is inquired into a kind of stability and high efficiency sesame chromosome flaking method and BAC-FISH hybridization technique fast, provides technical support for accelerating sesame cytogenetics and genomics research.
Summary of the invention
For overcoming prior art deficiency, the object of the invention is to provide efficiently sesame chromosome fluorescence in-situ hybridization technological method easily of one.
For achieving the above object, the present invention adopts following technical scheme:
A kind of sesame chromosome fluorescence in-situ hybridization method, it comprises the steps:
The preparation of A, chromosome specimen;
B, the preparation of BAC probe:
1) BAC extraction of plasmid DNA
From sesame BAC storehouse, choose object BAC clone, after cultivation, adopt plasmid extraction kit to extract plasmid DNA, plasmid DNA concentration 100-500 ng/ μ L;
2) probe mark
Get 20 μ L BAC DNA, at 0.1Kpa, 120 DEG C, place 5min, then ice bath is placed 5min, and random priming carries out probe mark, 37 DEG C of water-bath 20h, and 65 DEG C of deactivation 5min ,-20 DEG C save backup; Mark system is in table 1;
Table 1 fluorescence probe labeled reactant system
C, fluorescence in situ hybridization
Probe good mark is used for to fluorescence in situ hybridization with 10 times of hybridization solution dilutions as working fluid; The each component of working fluid is in table 2;
Table 2 fluorescence in situ hybridization working fluid composition used
1) chromosome sectioning processing
Chromosome sectioning is soaked in to 1-2min in 45% acetic acid, takes out nature and dry, take off Giemsa stain color; At 37 DEG C, process film-making mark zone 1h with 2 × SSC containing 100 μ g/mL RNase A; Then use 2 × SSC to develop a film three times, each 5min; Use 70%, 85% and dehydrated alcohol dewater step by step to film-making, every grade of 3-5min;
Naturally after drying, process film-making mark zone 30min at 37 DEG C with 1% stomach en-(10mM HCl preparation), 1 × PBS washes 2 times, each 5min; Then process film-making mark zone 10min with 1% paraformaldehyde, 2 × SSC washes 2 times, and each 5min, dries naturally;
Add 70% deionized formamide (2 × SSC) in film-making mark zone, with cover plate, 70 DEG C of sex change 2min; Film-making after sex change is dewatered, and 70%, 85% and the dehydrated alcohol that are placed in successively-20 DEG C of precoolings dewater step by step, and every grade of 3-5min, dries for subsequent use naturally;
2) probe sex change
Get 20 μ L working fluids, 95 DEG C of sex change 10min, more than ice bath 5min, for subsequent use rapidly;
3) hybridization
In film-making mark zone, add the probe that working fluid 8 μ L(can equivalent add 2-3 kind fluorescein), with cover plate, glue edge sealing, 37 DEG C of hybridization are spent the night;
4) signal detection
Under room temperature, brewed of 2 × SSC immersion, washes away cover plate, and 2 × SSC(is containing 0.1 wt % SDS) embathe each 5min 3 times; Then use distilled water flushing one time, lucifuge is dried; Splash into the Vectashield H1000 of 4 μ L containing 4 μ g/mL DAPI in film-making mark zone, be placed under Nikon 80i fluorescent microscope with cover plate, determine target chromosome according to film-making coordinate, observe hybridization signal, under cold light source CCD, carry out IMAQ, adopt Spot Rtke 4.1 softwares to carry out image and synthesize, and utilize Adobe Photoshop 7.0 softwares to adjust image.
The preparation of steps A chromosome specimen is specially:
1) root tip culture
Choose healthy sesame seed, be sowed in the culture dish that is covered with wet filter paper, 21 DEG C are secretly cultured to the preferred 1.5cm of the long 1.2-1.8cm(of root);
2) tip of a root processing
Cut 4-5mm and be placed in glass bottle containing the tip of a root of meristematic zone, immerse 0.002 M oxine in 21 DEG C of dark places reason 1.5h, pouring liquids, 4 DEG C of fixing 1h of stationary liquid (matching while using) that the anhydrous methanol that directly to add by volume ratio be 3:1 and glacial acetic acid form;
Take out the tip of a root cleans 2-3 time in distilled water, cut Meristernatic zone (about 1mm, i.e. " the little tip of a root ") in distilled water, soak 10-20min, then add mixed enzyme solution in 37 DEG C water-baths in the enzymolysis 2-3h of 20 μ l containing 2.5% cellulase and 2.5% polygalacturonase by every 10 little tips of a root; Take out the little tip of a root, distilled water washes away enzyme liquid, then in distilled water, soaks 10-20min; Then the little tip of a root is moved to again to 4 DEG C of fixing 1h in stationary liquid;
3) smear
Draw the little tip of a root with glue head dropper, be placed on the clean slide of 4 DEG C of precoolings, by tip forceps tips, the little tip of a root is broken into pieces and made local cells suspension fast; Then drip stationary liquid and make suspension be scattered in whole slide, naturally dry;
4) dyeing
Slide is inverted (scribbling the tip of a root one faces down), and, on a sheet glass, two ends are propped with kapillary, make to leave space between slide and sheet glass, and Giemsa stain is injected to this space, dyeing 15min, and then tap water rinses, and naturally dries;
5) microscopy
Under Nikon 80i fluorescent microscope, select the chromosome sectioning in mitosis metaphase, mark object karyomit(e) in-scope overleaf, i.e. mark zone, records its coordinate position corresponding on slide.
In the inventive method, film-making sample after hybridization can be carried out to aftertreatment in order to recycling, be specially: throw off after cover plate, with embathing 10min containing 2 × SSC of 0.2% Tween20, then distilled water flushing once, naturally dry, repeat sex change, processed in above-mentioned C step, the in situ hybridization that can be used for another group probe detects.
The inventive method has following innovative point: 1) adopt smear method to carry out sesame chromosome sectioning, select 0.002M oxine pre-treatment 1.5h, select the mixed enzyme solution enzymolysis containing 2.5% cellulase and 2.5% polygalacturonase, can obtain quality phase karyomit(e) in good mid-term, tenuigenin clean background, technological method has novelty.2) method of employing autoclaving (0.1KPa, 120 DEG C) 5min, interrupts BAC plasmid DNA at random into small segment, can meet the requirement of fluorescence probe mark, by probe mark system boil down to 5 μ L, method steps is simplified simultaneously, there is novelty.3) utilize high quality stains system sheet to carry out sesame BAC-FISH hybridization, the treatment steps such as RNase A, stomach en-and the paraformaldehyde to chromosome specimen before hybridization, can increase the recycling number of times (the same film-making of experiment results proved can be stablized hybridization more than 8 times) of chromosome specimen, fill up the blank of sesame BAC-FISH hybridization technique.
The present invention adopts smear technique to make sesame Metaphase Chromosome sample, and Chromosome spread is effective, and film-making efficiency is high; On this basis, adopt fluorescein-labelled BAC probe, set up the Chromosomal in situ hybridization technology of sesame BAC.After probe hybridization karyomit(e), fluorescent signal can be by direct-detection, thereby determines number of copies and the position of target DNA fragment on karyomit(e).The chromosome specimen that adopts the inventive method to make can be used for repeated crossing, has improved utilization ratio and the test efficiency of sample, and has saved plenty of time and cost; In BAC in situ hybridization process, probe mark volume is little, and tolerance range is high, can save a large amount of reagent.The method has been avoided the problems such as endocellular chromosome in conventional tabletting method is easily out of shape, loss, in situ hybridization signal background is dark, crossbreeding effect is poor, fill up the blank of China sesame karyomit(e) BAC hybridization in situ technique, can be used for the researchs such as sesame chromosome karyotype analysis, BAC physical map structure and the location of specific DNA large fragment on karyomit(e), for technical foundation has been established in Sesame group research from now on.
Compared to the prior art, the invention has the advantages that: (1) the present invention adopts smear technique to make chromosome specimen, compared with traditional pressed disc technique, method is simple, and chromosome morphology is better, and film-making efficiency is high, result is stable, and hybrid context signal significantly reduces.(2) the present invention sets up probe mark method and BAC-FISH technology are in use easy to operate, and result is reproducible, and probe and reagent cost reduce greatly.(3) adopt the inventive method repeatedly to stablize hybridization (same film-making can be stablized hybridization more than 8 times) to same chromosome sectioning, utilize certain BAC probe not only 26 karyomit(e)s of sesame can be positioned to differentiation, can also on same group chromosome, locate multiple BAC clones, for the structure of sesame BAC physical map provides technical support.
Brief description of the drawings
Fig. 1 is the root tip chromosomes picture (2n=26) of sesame Henan sesame No. 11;
Fig. 2 is the BAC plasmid DNA electrophorogram after high pressure interrupts; Swimming lane 1:DL, 2000Marker; Swimming lane 2-13: 12 BAC plasmid DNA agarose electrophoresiss after interrupting;
Fig. 3 is the in situ hybridization results of two pairs of BAC probes on sesame karyomit(e); A in figure: the chromosome image after hybridization; B:BAC probe B103DA11(Green Marker) hybridization signal figure (arrow instruction); C:B101CD7(red-label) hybridization signal figure (arrow instruction); D:BAC probe B103DA11(Green Marker) and B101CD7(red-label) hybridization signal composite diagram. ruler units: 5 μ m;
Fig. 4 is the in situ hybridizations of eight pairs of BAC probes on sesame karyomit(e); A:B003CF8(green in figure) and B003AD6(redness) Probe In Situ Hybridization; B:B101CH9(green) and B102CD10(redness) Probe In Situ Hybridization; C:B102DA4(green) and B065AC6(redness) Probe In Situ Hybridization; D:B102DF11(green) and B119G1(redness) Probe In Situ Hybridization; E:B103DA11(green) and B101CD7(redness) Probe In Situ Hybridization; F:B103AB11(green) and B003DF1(redness) Probe In Situ Hybridization; G:B003DD8(green) and B003CD8(redness) Probe In Situ Hybridization; H:B063AG2(green) and B062CD2(redness) Probe In Situ Hybridization; I:8 is to BAC Probe In Situ Hybridization signal composite diagram. ruler units: 5 μ m.
embodiment
Below in conjunction with embodiment, technical scheme of the present invention is described further, but protection scope of the present invention is not limited to this.
embodiment 1:
A kind of sesame chromosome fluorescence in-situ hybridization method, it comprises the steps:
The preparation of A, chromosome specimen
1) root tip culture
Choose No. 11 sesame seeds of healthy Henan sesame, be sowed at paving and have three layers in the culture dish of wet filter paper, 21 DEG C are secretly cultured to the root about 1.5cm that grows up.
2) tip of a root processing
Cut about 4-5mm(containing meristematic zone) the tip of a root be placed in small-sized glass bottle, immerse 0.002M oxine in 21 DEG C of dark places reason 1.5h, pouring liquids, directly adds stationary liquid (V
anhydrous methanol: V
glacial acetic acid=3:1) 4 DEG C of fixing 1h.
Take out the tip of a root repeatedly cleans 3 times in distilled water, cut Meristernatic zone (about 1mm, be the little tip of a root) in distilled water, soak 15min, then add 20 μ l mixed enzyme solutions (containing 2.5wt% cellulase and 2.5wt% polygalacturonase) enzymolysis 2 hours 20 minutes in 37 DEG C of water-baths by every 10 little tips of a root.Take out the little tip of a root, put into distilled water and soak 15min to wash away remaining enzyme liquid, then the little tip of a root is moved to 4 DEG C of fixing 1h in stationary liquid.
3) smear
Draw the little tip of a root with glue head dropper, be placed on the clean slide of precooling (4 DEG C), by tip forceps tips, the little tip of a root is broken into pieces fast, make local cells suspension; Then drip 2 stationary liquids and make suspension be scattered in whole slide, naturally dry.
4) dyeing
Slide is inverted (scribbling the tip of a root one faces down), and, on a sheet glass, two ends are propped with kapillary, make to leave space between slide and sheet glass, and Giemsa stain is injected to this space, dyeing 15min, and then tap water rinses, and naturally dries.
5) microscopy
Under Nikon 80i fluorescent microscope, select in mitosis metaphase, form better and the chromosome sectioning (as Fig. 1) of background relative clean, use overleaf a glass mark object karyomit(e) in-scope, i.e. mark zone, records its coordinate position corresponding on slide.
B, the preparation of sesame BAC probe
1) BAC extraction of plasmid DNA
From sesame BAC storehouse, choose at random 50 BAC clones, after cultivation, adopt plasmid extraction kit method to extract plasmid DNA.Plasmid DNA concentration is adjusted to 500 ng/ μ L.
2) probe mark
Get 20 μ L BAC DNA, 0.1KPa(120 DEG C) descend placement 5min to interrupt at random processing, ice bath 5min subsequently.It is that 100-500bp(is shown in Fig. 2 that BAC plasmid DNA interrupts rear size).Carry out mark (Rhodamine mark red fluorescence, fluorescein Fluorescein is green fluorescence), 37 DEG C of water-bath 20h with DNA fragmentation red, that the plain air exercise of green fluorescence is had no progeny respectively.65 DEG C of deactivation 5min ,-20 DEG C save backup.Labeled reactant system is in table 3.
Table 3 fluorescence probe labeled reactant system
C, fluorescence in situ hybridization
Probe good mark is become to working fluid with 10 times of hybridization solution dilutions, for fluorescence in situ hybridization.The each component of working fluid is in table 4.
Table 4 fluorescent in situ working fluid composition
1) chromosome sectioning processing
Chromosome sectioning is soaked in to 2min in 45v% acetic acid, takes out nature and dry, take off Giemsa stain color.At 37 DEG C, process film-making mark zone 1h with 2 × SSC containing 100 μ g/mL RNase A.Then use 2 × SSC to develop a film three times, each 5min.Use 70v%, 85v% and dehydrated alcohol to dewater step by step to film-making, every grade of 4min.
Naturally after drying, at 37 DEG C with 1wt% stomach en-(10mM HCl preparation) processing film-making mark zone 30min.1 × PBS washes 2 times, each 5min.Then process film-making mark zone 10min with 1% paraformaldehyde, 2 × SSC washes 2 times, and each 5min, dries naturally.
Add 70% deionized formamide (2 × SSC) in film-making mark zone, with cover plate, 70 DEG C of sex change 2min.Film-making after sex change is dewatered, and 70v%, the 85v% and the dehydrated alcohol that are placed in successively-20 DEG C of precoolings dewater step by step, and every grade of 3-5min, dries for subsequent use naturally.
2) probe sex change
Get 20 μ L working fluids, 95 DEG C of sex change 10min, more than ice bath 5min, for subsequent use rapidly.
3) hybridization
In film-making mark zone, add the red probe of working fluid 8 μ L(Rhodamine and the each 4 μ L of the green probe working fluid of Fluorescein), with cover plate, glue edge sealing, 37 DEG C of hybridization are spent the night.
4) signal detection
Under room temperature, brewed of 2 × SSC immersion, washes away cover plate, and 2 × SSC (containing 0.1%SDS) embathes 3 times, each 5min.Then use distilled water flushing one time, lucifuge is dried.Splash into 4 μ L Vectashield H1000(in film-making mark zone containing 4 μ g/mL DAPI), be placed under Nikon 80i fluorescent microscope with cover plate, determine target chromosome according to film-making coordinate.Karyomit(e) is taken pictures and (seen in Fig. 3 a).Then conversion filter successively, takes pictures and (sees respectively b in Fig. 3, c) the hybridization signal of green probe and red probe respectively; Adopt Spot Rtke 4.1 softwares that karyomit(e) and hybridization signal are carried out to image synthetic, with Adobe Photoshop 7.0 softwares, image is adjusted, add scale and (see in Fig. 3 d).
According to above-mentioned steps C, choose at random 50 BAC probe packet are carried out to In situ hybridization, finishing screen is selected 16 and is had single hybridization site BAC probe, and BAC numbering is respectively B003CF8, B003AD6, B101CH9, B102CD10, B102DA4, B065AC6, B102DF11, B119G1, B103DA11, B101CD7, B103AB11, B003DF1, B003DD8, B003CD8, B063AG2 and B062CD2.
D, the aftertreatment of film-making sample and recross
After hybridization, good multiple quality film-making sample is carried out to aftertreatment, be specially: throw off after cover plate, embathe 10min with 2 × SSC, 0.2% Tween20, then distilled water flushing once, dries naturally.For distinguishing the position of above-mentioned 16 BAC probes on karyomit(e), select one group of film-making to carry out repeated fluorescence in situ hybridization 8 times.In a film-making, 8 hybridization all has clearly signal (seeing a to h in Fig. 4) therein.According to result in figure, can find out that 16 BAC are distributed on 10 pairs of karyomit(e)s, wherein B102CD10, B065AC6 and B103DA11 are positioned on same dyad; B003CF8, B101CH9 and B102DA4 are positioned on same dyad; B119G1 and B103AB11 be arranged on same dyad (see Fig. 4 i).
Claims (3)
1. a sesame chromosome fluorescence in-situ hybridization method, is characterized in that, comprises the steps:
The preparation of A, chromosome specimen;
B, the preparation of BAC probe:
1) BAC extraction of plasmid DNA
From sesame BAC storehouse, choose object BAC clone, after cultivation, adopt plasmid extraction kit to extract plasmid DNA, plasmid DNA concentration 100-500 ng/ μ L;
2) probe mark
Get 20 μ L BAC DNA, at 0.1Kpa, 120 DEG C, place 5min, then ice bath is placed 5min, and random priming carries out probe mark, 37 DEG C of water-bath 20h, and 65 DEG C of deactivation 5min ,-20 DEG C save backup; Mark system is in table 1;
Table 1 fluorescence probe labeled reactant system
C, fluorescence in situ hybridization
Probe good mark is used for to fluorescence in situ hybridization with 10 times of hybridization solution dilutions as working fluid; The each component of working fluid is in table 2;
Table 2 fluorescence in situ hybridization working fluid composition used
1) chromosome sectioning processing
Chromosome sectioning is soaked in to 1-2min in 45% acetic acid, takes out nature and dry, take off Giemsa stain color; At 37 DEG C, process film-making mark zone 1h with 2 × SSC containing 100 μ g/mL RNase A; Then use 2 × SSC to develop a film three times, each 5min; Use 70%, 85% and dehydrated alcohol dewater step by step to film-making, every grade of 3-5min;
Naturally after drying, with 1% pepsin film-making mark zone 30min, 1 × PBS washes 2 times at 37 DEG C, each 5min; Then process film-making mark zone 10min with 1% paraformaldehyde, 2 × SSC washes 2 times, and each 5min, dries naturally;
Add 70% deionized formamide in film-making mark zone, with cover plate, 70 DEG C of sex change 2min; Film-making after sex change is dewatered, and 70%, 85% and the dehydrated alcohol that are placed in successively-20 DEG C of precoolings dewater step by step, and every grade of 3-5min, dries for subsequent use naturally;
2) probe sex change
Get 20 μ L working fluids, 95 DEG C of sex change 10min, more than ice bath 5min, for subsequent use;
3) hybridization
In film-making mark zone, add working fluid 8 μ L, with cover plate, glue edge sealing, 37 DEG C of hybridization are spent the night;
4) signal detection
Under room temperature, brewed of 2 × SSC immersion, washes away cover plate, embathes 3 times containing 2 × SSC of 0.1 wt % SDS, each 5min; Then use distilled water flushing one time, lucifuge is dried; Splash into the Vectashield H1000 of 4 μ L containing 4 μ g/mL DAPI in film-making mark zone, be placed under Nikon 80i fluorescent microscope with cover plate, determine target chromosome according to film-making coordinate, observe hybridization signal, under cold light source CCD, carry out IMAQ, adopt Spot Rtke 4.1 softwares to carry out image and synthesize, and utilize Adobe Photoshop 7.0 softwares to adjust image.
2. sesame chromosome fluorescence in-situ hybridization method as claimed in claim 1, is characterized in that, the preparation of steps A chromosome specimen is specially:
1) root tip culture
Choose healthy sesame seed, be sowed in the culture dish that is covered with wet filter paper, 21 DEG C are secretly cultured to the long 1.2-1.8cm of root;
2) tip of a root processing
Cut 4-5mm and be placed in glass bottle containing the tip of a root of meristematic zone, immerse 0.002 M oxine in 21 DEG C of dark places reason 1.5h, pouring liquids, 4 DEG C of fixing 1h of stationary liquid that the anhydrous methanol that directly to add by volume ratio be 3:1 and glacial acetic acid form;
Take out the tip of a root and clean 2-3 time in distilled water, cut Meristernatic zone and in distilled water, soak 10-20min, then add mixed enzyme solution in 37 DEG C water-baths in the enzymolysis 2-3h of 20 μ l containing 2.5% cellulase and 2.5% polygalacturonase by every 10 Meristernatic zone; Take out Meristernatic zone, distilled water washes away enzyme liquid, then in distilled water, soaks 10-20min; Then Meristernatic zone is moved to again to 4 DEG C of fixing 1h in stationary liquid;
3) smear
Draw Meristernatic zone with glue head dropper, be placed on the clean slide of 4 DEG C of precoolings, Meristernatic zone is broken into pieces and made local cells suspension by tip forceps tips; Then drip stationary liquid and make suspension be scattered in whole slide, naturally dry;
4) dyeing
Slide is inverted on a sheet glass, and two ends are propped with kapillary, makes to leave space between slide and sheet glass, and Giemsa stain is injected to this space, dyeing 15min, and then tap water rinses, and naturally dries;
5) microscopy
Under Nikon 80i fluorescent microscope, select the chromosome sectioning in mitosis metaphase, mark object karyomit(e) in-scope overleaf, i.e. mark zone, records its coordinate position corresponding on slide.
3. sesame chromosome fluorescence in-situ hybridization method as claimed in claim 1, it is characterized in that, film-making sample after hybridization is carried out to aftertreatment in order to recycling, be specially: throw off after cover plate, with embathing 10min containing 2 × SSC of 0.2% Tween20, then distilled water flushing once, dries naturally, repeat sex change, processed in above-mentioned C step, the in situ hybridization that can be used for another group probe detects.
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| CN107541553A (en) * | 2017-09-08 | 2018-01-05 | 河南省农业科学院芝麻研究中心 | One group is used to distinguish BAC mark of the sesame 13 to chromosome |
| CN109576348A (en) * | 2019-01-29 | 2019-04-05 | 南京林业大学 | A kind of processing method for Hyacinthus plant root tip chromosome fluorescence in-situ hybridization chromosome sectioning |
| CN109825553A (en) * | 2019-03-06 | 2019-05-31 | 南京林业大学 | A method of poplar whole chromosome is identified using oligonucleotide probe |
| CN109856330A (en) * | 2019-01-29 | 2019-06-07 | 南京农业大学 | A method of chrysanthemum tip of a root Mitotic phase is improved by artificial regulatory |
| CN110220904A (en) * | 2019-06-24 | 2019-09-10 | 广州甘蔗糖业研究所湛江甘蔗研究中心 | A kind of analysis method of the sharp leaf Cinnamomum kanahirai hay karyotype based on the tip of a root |
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| 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 |
| CN109576348B (en) * | 2019-01-29 | 2022-02-18 | 南京林业大学 | Processing method for chromosome slide of root tip chromosome fluorescence in situ hybridization of hyacinth plants |
| CN109825553A (en) * | 2019-03-06 | 2019-05-31 | 南京林业大学 | A method of poplar whole chromosome is identified using oligonucleotide probe |
| CN110220904A (en) * | 2019-06-24 | 2019-09-10 | 广州甘蔗糖业研究所湛江甘蔗研究中心 | A kind of analysis method of the sharp leaf Cinnamomum kanahirai hay karyotype based on the tip of a root |
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