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Dripping piece preparation method of plant chromosome

A chromosome and plant technology, which is applied to the field of plant chromosome drop piece preparation, can solve the problems of poor chromosome dispersion effect, increase the number of chromosomes in the metaphase, unfavorable chromosome observation, etc., and achieves easy separation of chromosomes or cells, saving processing time, and processing process. Simple and fast effects

Inactive Publication Date: 2017-01-25
YANGTZE NORMAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

[0003] Aiming at the above-mentioned deficiencies in the prior art, the technical problem to be solved by the present invention is: for the existing chromosome preparation method, the experimental drugs have high toxicity, long processing time, limited dispersion and easy damage to chromosomes, which are not suitable for current molecular and To solve the technical problems of cell biology requirements for chromosome production, and provide a droplet of plant chromosomes with low toxicity, short production time, good chromosome dispersion and integrity, and suitable for in situ hybridization, chromosome separation and microdissection tablet preparation method
Aiming at the technical problem that the reagents used in the pretreatment of plant chromosome preparation in the prior art are highly toxic and have a poor effect on chromosome dispersion, the present invention creatively proposes the use of nitrous oxide for pretreatment of plants through research. Dinitrogen can destroy the spindle silk of plants, and then can increase the number of chromosomes in the middle stage, which is convenient for chromosome observation in the later stage, and after the plant cells treated with such a pretreatment method are enzymatically hydrolyzed, the chromosomes are more likely to disperse, and then the present invention adopts droplet The way of film production does not need to apply too much external force, and the cells can be broken only by the natural gravity when dropping, and the chromosome specimens with circular or oval dispersion, uniform distribution, less chromosome crossover and good dispersion can be obtained. It is easier to carry out biological research work such as single chromosome or cell separation, and overcomes the disadvantages of the smear method in the existing chromosome preparation method that the cells are easily attached to the glass slide and cannot obtain complete chromosomes. The dissecting needle is easy to damage the chromosome, and the high temperature will also damage the DNA structure when baking the slice, which is not conducive to the technical problem of chromosome observation

Method used

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  • Dripping piece preparation method of plant chromosome
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  • Dripping piece preparation method of plant chromosome

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 Chromosome drip preparation for DP17 corn variety:

[0031] (1) Put the DP17 corn seeds in a disk filled with fine sand, put them in an artificial climate box, and cultivate at 28°C. When the root grows to 1.5 cm, cut the root tip and put it into a 5 mL centrifuge tube;

[0032] (2) Cut 1.5cm root and put it in ddH 2 O double-distilled water to wash, moisten the roots and put them into a 0.5 mL EP tube (keep the lid open), let the wet roots stick to the tube wall, but the water cannot flow down in strands;

[0033] (3) Put the EP tube into the nitrous oxide (N 2 O) Treat in the treatment room of the treatment device for 2 hours; the nitrous oxide treatment device decompresses the nitrous oxide in the nitrous oxide tank and passes it into the treatment room, so that the N 2 The pressure of O was 0.5 atmospheres, and the root tips were placed in the treatment chamber at 0.5 atmospheres for 2 h. The purpose was to perform pretreatment, using nitrous gas to des...

Embodiment 2

[0043] Example 2 Chromosome drip preparation for DP46 corn variety:

[0044] (1) Put the DP46 corn seeds in a disk filled with fine sand, put them in an artificial climate box, and cultivate at 28°C. When the root grows to 1.5 cm, cut the root tip and put it into a 5 mL centrifuge tube;

[0045] (2) Cut 1.5cm root and put it in ddH 2 O double-distilled water to wash, moisten the roots and put them into a 0.5 mL EP tube (keep the lid open), let the wet roots stick to the tube wall, but the water cannot flow down in strands;

[0046] (3) Put the EP tube into the nitrous oxide (N 2 O) Treat in the treatment room of the treatment device for 3 hours; the nitrous oxide treatment device decompresses the nitrous oxide in the nitrous oxide tank and then passes it into the treatment room, so that the N 2 The pressure of O is 1 atmosphere;

[0047] (4) Add 90% acetic acid aqueous solution to the EP tube and fix for 2.5 h;

[0048] (5) Remove the roots from the acetic acid and use dd...

Embodiment 3

[0056] Example 3 Preparation of chromosome droplets for DP47 corn variety:

[0057] (1) Put the DP47 corn seeds in a disk filled with fine sand, put them in an artificial climate box, and cultivate at 28°C. When the root grows to 1.5 cm, cut the root tip and put it into a 5 ml centrifuge tube;

[0058] (2) Cut 1.5cm root and put it in ddH 2 O double-distilled water to wash, moisten the roots and put them into a 0.5 mL EP tube (keep the lid open), let the wet roots stick to the tube wall, but the water cannot flow down in strands;

[0059] (3) Put the EP tube into the nitrous oxide (N 2 O) Treat in the treatment room of the treatment device for 3 hours; the nitrous oxide treatment device decompresses the nitrous oxide in the nitrous oxide tank and then passes it into the treatment room, so that the N 2 The pressure of O is 0.75 atmospheres;

[0060] (4) Add 90% acetic acid aqueous solution to the EP tube and fix for 3 h;

[0061] (5) Remove the roots from the acetic acid a...

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Abstract

The invention provides a dripping piece preparation method of plant chromosome. The dripping piece preparation method includes steps of placing a plant organ in nitrous oxide gas for pretreatment, and then fixing the plant organ after pretreatment by stationary liquid, and performing enzymolysis on a cell wall of the plant organ by enzyme, and breaking the plant organ after enzymolysis, and preparing the cell suspension liquid; dripping the cell suspension liquid on a glass slide or a cover glass; forming a plant chromosome specimen. The reagent applied to the method is low in toxicity; the crack obtained through treatment is dispersed in a circular or ellipse form by comparing with regular piece making method, the distribution is uniform, and chromosome intersection is few, and dispersing degree is good; a complete chromosome can be obtained; the preparation method can prepare the chromosome slice with stable quality, clear chromosome and good dispersity; the prepared chromosome slice can be applied to cytological identification, manufacturing of permanent slice, hybridization in-situ, microdissection, and microscope separation, and so on.

Description

technical field [0001] The invention belongs to the technical field of chromosome preparation, and in particular relates to a method for preparing drop slices of plant chromosomes. Background technique [0002] Chromosome preparation is the basis for observation of chromosome morphology, in situ hybridization and chromosome separation. The commonly used methods of chromosome preparation mainly include compression method and smear method. However, the tableting method and the smearing method mainly have the following disadvantages: (1) The experimental drugs are highly toxic. The tableting method and smearing method generally use α-bromine, 8-hydroxyquinoline and colchicine as pretreatment solutions, and the drugs are highly carcinogenic; (2) The treatment time is relatively long, and the general pretreatment needs 3-5 hours, fixed processing 1-2 days, it generally takes 1.5-2.5 days from material collection to production, and the processing time is long. Moreover, it is m...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N1/06
CPCC12N1/06C12Q1/6806C12Q1/6841
Inventor 陈发波李红艳姚启伦何莲王武琼邓丽李玉洁冉佐
Owner YANGTZE NORMAL UNIVERSITY
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