Method for analyzing wheat root tip cell cycle through flow cytometry

A technology of flow cytometry and cell cycle, which is applied in the field of wheat root tip cell cycle (G1 phase), which can solve the problems of difficulty in grasping the concentration of non-ionic detergent, many impurities and sample fragments, and a small number of nuclei, etc., to achieve detection The effect of stable rate, good effect and wide application range

Inactive Publication Date: 2016-07-20
TIANJIN NORMAL UNIVERSITY
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  • Application Information

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Problems solved by technology

Although flow cytometry has been used to study the DNA content of nuclei in corn, sweet potato, kiwi, wheat and other plants, these studies all use different dissociation solutions to extract the nuclei of different tissue cells of these plants, impurities and sample debris More, it is easy to aggregate with the nucleus, and the concentration of non-ionic detergent is difficult to control. It is easy to destroy the nuclear membrane while destroying the cell membrane, which results in a small number of extracted nuclei.

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  • Method for analyzing wheat root tip cell cycle through flow cytometry
  • Method for analyzing wheat root tip cell cycle through flow cytometry
  • Method for analyzing wheat root tip cell cycle through flow cytometry

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Embodiment 1

[0024] A method of cell cycle analysis in the root tip of Nannong 9918 wheat by flow cytometry

[0025] Firstly, the cell cycle analysis of Nannong 9918 wheat root tip cells is carried out with the method of the present invention, including the following steps:

[0026] (1) Select well-developed wheat seeds and use tap water for seed soaking and germination. When the roots of the wheat seedlings grow to 3cm, use a blade to cut the root tip of about 0.2cm length, and continue to use the blade to cut the cut root tip as small as possible The tissue pieces were fixed in freshly prepared Carnot's fixative (methanol: glacial acetic acid in a volume ratio of 3: 1 solution) for 30 minutes, and rinsed with pH 7.4 phosphate buffer for 3 times, each time after standing for 5 minutes , Use a pipette to aspirate the upper buffer solution, then transfer it to 70% ethanol by volume, and store it in a refrigerator at 4°C;

[0027] (2) Place the fixed root tip sample in 0.1-0.2% glycerol for 2.5 ho...

Embodiment 2

[0032] The method of cell cycle analysis in the root tip of Jingdong 8 wheat by flow cytometry

[0033] Firstly, the cell cycle analysis of Jingdong No. 8 wheat root tip cells is carried out using the method of the present invention, including the following steps:

[0034] (1) Select well-developed wheat seeds and use tap water for seed soaking and germination. When the roots of the wheat seedlings grow to 4cm, use a blade to cut the root tips of about 0.2cm length, and continue to use the blade to cut the cut root tips as small as possible The tissue pieces were fixed in freshly prepared Carnot's fixative (methanol: glacial acetic acid in a volume ratio of 3: 1 solution) for 30 minutes, and rinsed with pH 7.4 phosphate buffer for 3 times, each time after standing for 5 minutes , Use a pipette to aspirate the upper buffer solution, then transfer it to 70% ethanol by volume, and store it in a refrigerator at 4°C;

[0035] (2) Place the fixed root tip sample in 0.1-0.2% glycerol for 3...

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Abstract

The invention discloses a method for analyzing the cycle G1, the cycle S and the cycle G2 / M of wheat root tip cells through flow cytometry and belongs to the technical field of biology.The method comprises the steps of immersing wheat seeds and accelerating germination, obtaining a wheat root tip sample through cutting, and fixing the sampling with Carnoy fixing liquid; rinsing the sample thoroughly with a phosphate buffer solution; placing the sample in a mixed enzyme liquid of cellulose and pectinase for conducting enzymolysis for 1-2 hours; after the sample is rinsed thoroughly, conducting grinding, so that single-cell suspension is obtained; after rinsing is conducted, conducting dyeing away from light on the wheat root tip cells through DAPI dye liquor; after rinsing and filtering are conducted, differentiating and counting the cells of cycle time phases including the cycle G1, the cycle S and the cycle G2 / M which are different in DNA content through a flow cytometer, and learning about the proliferation condition of the cells.The method is simple and easy to operate, separated materials are easy to obtain, the enzymolysis method is simple, the obtained single-cell suspension is high in purity, the flow cytometer is good in detection effect, and the result is stable.

Description

[0001] This invention was funded by the National Natural Science Foundation of China Youth Project (31501234). technical field [0002] The invention belongs to the field of biotechnology, and relates to a method for analyzing wheat root tip cell cycle (G1 phase, S phase, G2 / M phase) by flow cytometry. Background technique [0003] Flow Cytometer (FCM) is a new high-tech instrument integrating laser technology, photoelectric measurement technology, computer technology, flow dynamics technology, electronic physics, cell immunofluorescence chemistry technology and monoclonal antibody technology. It can perform qualitative and quantitative analysis and sorting at the single-cell level, and is mainly used in basic research and clinical trials in hematology, immunology, oncology, molecular biology, and cell biology. Compared with animals and microorganisms, due to the complex structure of plant tissues and cells (such as cell walls, special organelles, etc.), it is difficult to p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N15/14G01N1/28G01N1/30
CPCG01N15/1434G01N1/28G01N1/30
Inventor 岳洁瑜王利谈论语
Owner TIANJIN NORMAL UNIVERSITY
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