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Method for preparing asparagus root tip cell chromosome metaphase split-phase specimen

A technology of chromosomes and rhizoma rhizoma, applied in the field of plant cytogenetics, can solve problems such as unfavorable research and analysis, easy tailing of chromosomes, bad shape, etc., and achieve the effect of reducing the risk of pollution and poisoning, clear background, and good shape.

Inactive Publication Date: 2017-07-07
HENAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, both HU and APM are toxic, and after being treated with APM, the chromosomes of the root tip cells of A. chinensis are easy to tail and the shape is not good, which is not conducive to further research and analysis in the later stage.

Method used

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  • Method for preparing asparagus root tip cell chromosome metaphase split-phase specimen
  • Method for preparing asparagus root tip cell chromosome metaphase split-phase specimen
  • Method for preparing asparagus root tip cell chromosome metaphase split-phase specimen

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Experimental program
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Effect test

Embodiment 1

[0031] A method for inducing the mitotic synchronization of root tip cells of Lithophora chinensis, comprising the following steps:

[0032] 1. Root tip culture: Soak the seeds of Cypress cypress in 30°C water for 24 hours, then put the seeds in a petri dish with 2-3 layers of filter paper soaked in water, and then cultivate them at 25°C for 4 days until the root length is 1cm;

[0033] 2. Hydroxyurea treatment: Treat cypress seeds with a root length of 1 cm with hydroxyurea at a molar concentration of 2.5 mmol / L at 25°C for 18 hours;

[0034] 3. Recovery culture: remove hydroxyurea, wash the seeds three times with clean water, and then cultivate the seeds in clean water at 25°C for 5 hours;

[0035] 4. N 2 O pretreatment: cut the root tip and put it into N 2 Treated in O gas chamber for 2h, the N 2 The pressure in the O gas chamber is 10atm (1.01MPa);

[0036] 5. Hypotonicity: Treat with KCl solution with a molar concentration of 0.075mol / L for 60 minutes;

[0037] 6. F...

Embodiment 2

[0042] Preparation of Metaphase Chromosome Specimens of Phyllostachys spp.

[0043] The apical meristem area of ​​the synchronously treated Rhizoma cypress root was excised, placed in 20 µL of a mixture containing 1% pectinase and 2% cellulase, and incubated at 37°C. Solution 2h. After enzymatic hydrolysis, add TE buffer and pipette repeatedly to remove the outer layer of the root cap and root tip, then wash the root tip twice with 70% ethanol by volume, mash the root tip with a dissecting needle in 40 μL of ethanol, and vortex Suspend the cells, centrifuge to retain the precipitate, add 30 μL of anhydrous acetic acid, mix well, suck 5-8 μL droplet, and place it at room temperature for 5 minutes before microscopic examination. Films with good microscopic examination are placed in the ultraviolet cross-linking instrument through 120-125mJ / cm 2 Treat for 2 minutes to fix the chromosomes for later use. Microscopic examination results such as image 3 It can be seen that the c...

Embodiment 3

[0045]Fluorescence in situ hybridization of metaphase chromosomes

[0046] 45S probe labeling: Add the following components to a centrifuge tube on ice: corn 45S rDNA 2µg, 10×nicktranslation buffer 2µL, Labeled-dNTP (Texas red-dCTP or Alexa Fluor-488-dUTP) 0.5µL, Non-labeled -dNTPs 2µL, DNA polymerase I 5µL, DNaseI 0.5µL, make up to 20µL with deionized water. After mixing, treat in a PCR instrument at 15°C for 2 hours, add 5×TAE+140ng / µL salmon sperm DNA 175µL, add volume fraction 90% ethanol-volume fraction 10% sodium acetate 500µL, mix well to precipitate the probe for more than 2 hours, and then set Centrifuge in a centrifuge at 13000rpm for 30min, wash with absolute ethanol twice, store at room temperature in the dark for 30min, add 2×SSC 1×TE 20µL to dissolve the precipitate.

[0047] Fluorescence in situ hybridization: Prepare probe solution: add 0.5 µL of labeled probe to 5.5 µL of 2×SSC 1×TE, drop the probe solution onto the cells on the glass slide, and gently place ...

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Abstract

The invention discloses a method for preparing an asparagus root tip cell chromosome metaphase split-phase specimen, and belongs to the technical field of plant cell genetics. The method adopts the technical scheme that hydroxyurea and a N2O dual-block method are used for inducing asparagus cells to be subjected to synchronous mitosis so as to obtain massive metaphase split-phase cells, and through the steps of low-seeping, fixation and making, massive chromosome glass specimens being clear in shape, favorable in dispersibility and suitable for subsequent cytology research are obtained. According to the method disclosed by the invention, HU is used as a blocker, and in combination with renewal cultivation and N2O treatment, the asparagus metaphase split-phase cells are greatly increased; a conventional tabletting method is used, so that the metaphase cells are 50% or above of the total number of cells, and besides, metaphase chromosome after the N2O treatment is favorable in shape; and therefore, the asparagus chromosome tableting success rate and the stability are greatly increased, and the method provides great convenience for the application of techniques of asparagus karyotype analysis, fluorescence in situ hybridization and the like.

Description

technical field [0001] The invention belongs to the technical field of plant cytogenetics, and in particular relates to a method for preparing samples of metaphase mitotic phases of chromosomes of root tip cells of A. chinensis. Background technique [0002] Chromosomes are the most important components of the nucleus, and the variation of chromosome number and structure provides abundant genetic resources for plant breeding. Root tip cell mitotic metaphase production is a basic cytogenetics research technique and method. This technique, as well as techniques such as karyotype analysis and fluorescence in situ hybridization based on this technique, play an important role in species identification and identification, and determination of genetic relationship between species. , species ploidy identification and hybrid breeding and other research has a wide range of applications. [0003] Asparagus, commonly known as asparagus, belongs to the genus Asparagus of the family Lili...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/30
CPCG01N1/30
Inventor 李书粉苏婷程广前李莎邓传良高武军
Owner HENAN NORMAL UNIV
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