Fast fluorescent staining method of cruciferae crop pollen

A cruciferous crop, fluorescent dyeing technology, applied in the preparation and sampling of scientific instruments, samples for testing, etc., can solve the problems of time-consuming, cumbersome, labor-intensive, etc., and achieve obvious contrast, good repeatability, and reduced cytoplasmic and nuclear staining. Effects of Environmental Pollution

Active Publication Date: 2018-10-19
GUANGDONG KING ZUO AGRI SCI & TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the deficiencies of the prior art, the present invention aims to provide a rapid fluorescent dyeing method for cruciferous crop pollen, which solves the problems of cumbersome

Method used

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  • Fast fluorescent staining method of cruciferae crop pollen
  • Fast fluorescent staining method of cruciferae crop pollen
  • Fast fluorescent staining method of cruciferae crop pollen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Fluorescence dyeing method of mature pollen of rape blossoms

[0026] 1. Staining solution preparation: first dissolve Hoechst33258 fluorescent dye and sucrose in phosphate buffer saline (PBS, pH=7.4), prepare Hoechst33258 concentration 20μg / L, sucrose concentration 10wt% solution 1, and then prepare 60% ethanol ( v / v), ethyl acetic acid 10% (v / v), glycerol 30% (v / v) solution two, mix solution one and solution two in a volume ratio of 1:1 to get the dyeing solution, and put it together Store in a refrigerator at 4°C and protected from light.

[0027] 2. Staining: Use a micropipette to suck 15 μL of the staining solution obtained in step 1 onto the slide, and then pick the open flower that needs to be stained and observed from the inflorescence, use tweezers to pick 1 anther and put it directly on the slide In the staining solution, gently squeeze the anthers to disperse an appropriate amount of pollen, remove the broken anther wall and other debris, apply for a w...

Embodiment 2

[0030] Example 2: Fluorescence dyeing method of Chinese cabbage flower bud pollen

[0031] 1. Staining solution preparation: first dissolve Hoechst33258 fluorescent dye and sucrose in phosphate buffer saline (PBS, pH=7.4), prepare Hoechst33258 concentration 30μg / L, sucrose concentration 8wt% solution 1, and then prepare 70% ethanol ( v / v), ethyl acetic acid 10% (v / v), glycerol 20% (v / v) solution two, mix solution one and solution two in a volume ratio of 1:1 to obtain the staining solution, place Store in a refrigerator at 4°C and dark.

[0032] 2. Staining: Use a micropipette to suck 17μL of the staining solution prepared in step 1 onto the slide, then pick the flower buds to be stained and observed from the inflorescence, and use tweezers to peel 1 anther and put it directly on the slide In the staining solution, gently squeeze the anthers to disperse an appropriate amount of pollen, remove the broken anther wall and other debris, apply for a while (5-10 seconds) to mix the poll...

Embodiment 3

[0035] Example 3: Fluorescence dyeing method of flower bud pollen of Chinese cabbage

[0036] 1. Preparation of staining solution: first dissolve Hoechst33258 fluorescent dye and sucrose in phosphate buffer saline (PBS, pH=7.4) to prepare a solution of Hoechst33258 concentration of 40μg / L and sucrose concentration of 9wt%, and then prepare a solution containing 65% ethanol ( v / v), ethyl acetic acid 10% (v / v), glycerol 25% (v / v) solution two, mix solution one and solution two in a volume ratio of 1:1 to get the dyeing solution, and put it together Store in a refrigerator at 4°C and protected from light.

[0037] 2. Staining: Use a micropipette to suck 20μL of the staining solution obtained in step 1 onto the glass slide, and then pick the flower buds to be stained and observed from the inflorescence, and use tweezers to pick 2 anthers and put them directly on the glass slide In the staining solution, gently squeeze the anthers to disperse an appropriate amount of pollen, remove the...

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PUM

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Abstract

The invention discloses a fast fluorescent staining method of cruciferae crop pollen. The method comprises the following steps of S1, staining liquid preparation: firstly, dissolving Hoechst33258 fluorescent dye and cane sugar into a PBS (phosphate buffer solution); preparing a first solution with the Hoechst33258 concentration being 20 to 40mug/L and the cane sugar concentration being 8 to 10 weight percent; then, preparing a second solution containing 60 to 70 percent (v/v) of ethanol, 10 percent (v/v) of ethyl acetate and 30 to 20 percent (v/v) of glycerol; mixing and uniformly mixing the first solution and the second solution according to a volume ratio of 1:1 to obtain staining liquid; performing low-temperature light avoiding storage; S2, staining: taking staining liquid prepared inthe steps S1 on a glass slide; then, picking blossoming flowers or flower buds to be stained and observed from the inflorescence; picking the anther by tweezers to be directly put into staining liquidon the glass slide; slightly pinching the anther; spreading out pollen; taking out sundries; performing smearing so that the pollen and the staining liquid are uniformly mixed; then, cover glass is covered. The problems of complexity, time waste and labor waste of the existing pollen fluorescent staining method can be solved; meanwhile, the stability and the repeatability of the fluorescent staining method are improved.

Description

Technical field [0001] The invention relates to a pollen fluorescent dyeing method, in particular to a fast fluorescent dyeing method for cruciferous crop pollen. Background technique [0002] Plant pollen wall is a special cell wall composed mainly of sporopollenin and cellulose, which is composed of two layers of outer wall and inner wall. Among them, the outer wall is divided into inner and outer layers. The outer layer of the outer wall is honeycomb or mesh-shaped, and the inner layer of the outer wall is a flat structure. The main component of the outer wall is sporopollenin. The inner wall structure is relatively simple, mainly containing cellulose, pectin and protein. Because plant pollen walls are more complex in structure than normal plant cell walls, and dense and thick, especially mature pollen grains, their permeability and light permeability are extremely poor. Therefore, most plant pollen is difficult to observe under a microscope without transparent technology aft...

Claims

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Application Information

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IPC IPC(8): G01N1/30
CPCG01N1/30G01N2001/302
Inventor 李智军曾晶谭铭许谢景
Owner GUANGDONG KING ZUO AGRI SCI & TECH CO LTD
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