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Microscopic observation method of plant tissues

A technology for plant tissue and microscope detection, applied in the biological field, can solve problems affecting the analysis of plant cell structure and function, achieve good transparency, improve observation depth, and save costs

Active Publication Date: 2017-05-31
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the microscopic observation of plant cells in the past was limited to a few layers of cells with a depth of about 30 μm, which greatly affected the analysis of plant cell structure and function.

Method used

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  • Microscopic observation method of plant tissues

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1, transparent treatment and microscope observation of Arabidopsis leaves

[0060] 1. Reagents required for transparent treatment of leaves

[0061] The fixative solution is composed of glutaraldehyde, paraformaldehyde and PHEM solution, wherein the volume percentage of glutaraldehyde is 4%, and the mass percentage of paraformaldehyde is 4%.

[0062] The PHEM solution was prepared with a final concentration of 40mM PIPES, 15mM HEPES, 5mM EGTA, 1mM MgCl 2 Composed of water with a pH of 6.9;

[0063] The transparent solution is composed of 4M urea, 20% glycerol (volume percentage), 0.1 volume% TritonX-100 and PHEM solution with final concentration.

[0064] 2. Transparency treatment and microscope observation of Arabidopsis leaves

[0065] 1. Transparency treatment of Arabidopsis leaves

[0066] Seeds of Arabidopsis (Columbian ecotype) were planted on 1 / 2 MS medium, vernalized at 0-4°C for two days, germinated in an incubator at 23±2°C, and moved to culture s...

Embodiment 2

[0074] Embodiment 2, the distribution of chloroplast in the mesophyll cell of Shallot onion

[0075] 1. Reagents required for transparent treatment of leaves

[0076] The fixative solution is composed of glutaraldehyde, paraformaldehyde and PHEM solution, wherein the volume percentage of glutaraldehyde is 4%, and the mass percentage of paraformaldehyde is 8%.

[0077] The PHEM solution was prepared with a final concentration of 60mM PIPES, 25mM HEPES, 10mM EGTA, 2mM MgCl 2 Composed of water with a pH of 6.9;

[0078] The transparent liquid is composed of 6M urea, 30% glycerol (volume percent), 0.1 volume percent TritonX-100 and PHEM solution with a final concentration of 6M.

[0079] 2. Transparency treatment and microscope observation of the leaves of scallion

[0080] Cultivate the scallions to a height of 20-30cm, and get the aerial parts of the seedlings;

[0081] 1) Cut the above-mentioned scallion leaves into 0.8cm long segments, wash with deionized water until the s...

Embodiment 3

[0089] Example 3, the distribution of REMORIN protein in tobacco mesophyll cells

[0090] 1. Reagents required for transparent treatment of leaves

[0091] The fixative solution is composed of glutaraldehyde, paraformaldehyde and PHEM solution, wherein the volume percentage of glutaraldehyde is 5%, and the mass percentage of paraformaldehyde is 5%.

[0092] The PHEM solution was prepared with a final concentration of 60mM PIPES, 20mM HEPES, 10mM EGTA, 2mM MgCl 2 Composed of water with a pH of 6.9;

[0093] The transparent liquid is composed of 6M urea, 20% glycerol (volume percentage), 0.1 volume% TritonX-100 and PHEM solution with final concentration.

[0094] 2. Transparency treatment and microscope observation of tobacco leaves

[0095] Tobacco seeds are germinated at 20-25°C. After the seeds germinate, they are planted in flower soil in a greenhouse at 25±2°C, and cultivated to a plant height of 5-10cm.

[0096] 1) Shake culture the Agrobacterium strain carrying the RE...

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Abstract

The invention discloses a microscopic observation method of plant tissues. According to the microscopic observation method of the plant tissues, disclosed by the invention, after fixing treatment is carried out, potassium hydroxide is used for digesting; transparent treatment is carried out; enzymolysis or other operations are avoided so that the cost is saved; the transparent effect is good and the compatibility with fluorescent staining and marking operation is good: an inner structure of plant deep-layer tissues can be clearly observed through the fluorescent staining under a fluorescence microscope. The microscopic observation method of the plant deep-layer tissues, which combines digestion treatment and a transparent technology, lays a foundation for phenotypic identification of advanced plants and deep researches on a gene expression mode, especially plays an important role in researches of observing the structure of the plant deep-layer tissues and has a high actual application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a microscopic observation method of plant tissue. Background technique [0002] With the continuous deepening of functional genomics and molecular biology research, the expression sites of specific genes in plants and their functions at specific developmental stages have received extensive attention. Fluorescence microscopy and three-dimensional imaging technologies provide important tools for plant phenotype analysis, plant-pathogen interaction, and gene expression pattern analysis. Compared with animal cells, plant cell walls and cytoplasm have different refractive indices, and the abundant pigment and aromatic substances in cells reduce the signal-to-noise ratio of acquired images. Therefore, the microscopic observation of plant cells in the past was limited to a few layers of cells with a depth of about 30 μm, which greatly affected the analysis of plant cell structure and funct...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28
CPCG01N1/28
Inventor 田世平陈彤秦国政徐勇
Owner INST OF BOTANY CHINESE ACAD OF SCI
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