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Active oxygen rapid staining positioning method for plant root system

A plant root system, rapid dyeing technology, applied in the preparation of test samples, sampling devices, etc., can solve the problems of high loss of active oxygen, high wax melting temperature, inability to active oxygen, etc., to maintain the living form, accurate slice results, The effect of improving accuracy

Pending Publication Date: 2022-07-29
GANSU AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional paraffin sections have to go through the steps of fixation, embedding, sectioning and staining, which takes a long time, the temperature of melting wax is high, and the loss of active oxygen is too much, so it cannot be used for the study of active oxygen localization

Method used

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  • Active oxygen rapid staining positioning method for plant root system
  • Active oxygen rapid staining positioning method for plant root system
  • Active oxygen rapid staining positioning method for plant root system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] The steps of a method for rapid dyeing and positioning of plant root reactive oxygen species of the present invention are as follows:

[0061] 1. Root samples and treatment

[0062] Cut the plant root system with a diameter of 5 mm to a length of 1 cm, and quickly use a weight to fix the root system to the bottom of a 10 mL centrifuge tube.

[0063] 2. Staining of root samples

[0064] The vacuum tank of the vacuum device was pre-cooled to 0°C, and a pre-cooled ice bag was placed in it to keep the low temperature, and then the centrifuge tube was placed vertically into the vacuum tank.

[0065] Add detection O pre-cooled to 0°C 2 - The staining solution did not cover the sample.

[0066] After covering the vacuum tank, adjust the vacuum degree to -0.05MPa to reach the negative pressure state, and keep it for 2h for dyeing.

[0067] 3. Root sample fixation

[0068] After staining, take out the centrifuge tube quickly, discard the staining solution, rinse the sample...

Embodiment 2

[0082] The difference between this embodiment and Embodiment 1 is that: in step 2, the dyeing solution is used to detect H 2 O 2 In step 3, the buffer is: 100mmol / L potassium phosphate buffer at pH 6.9. The rest of the steps are the same as in Example 1.

[0083] Taking the root system of wolfberry as an example, the above-mentioned plant root active oxygen H 2 O 2 The rapid staining localization experiment of , the specific experiment is:

[0084] Setting: normal root group: no acupuncture, directly stained wolfberry root; CK group: stained wolfberry root after acupuncture for 72h; FL group: inoculated with Fusarium oxysporum after acupuncture, and then stained wolfberry root after 72h; FL+DPI Group: Diphenylene iodide chloride (DPI, active oxygen inhibitor) and Fusarium oxysporum were inoculated after acupuncture, and the roots of Lycium barbarum were stained after 72 hours. The dyeing of the above groups all used the method of Example 2 of the present invention. The e...

Embodiment 3

[0089] The steps of a method for rapid dyeing and positioning of plant root reactive oxygen species of the present invention are as follows:

[0090] 1. Root samples and treatment

[0091] Cut the plant root system with a diameter of 8 mm to a length of 0.8 cm, and quickly use a clamp to fix the root system to the bottom of a 10 mL centrifuge tube.

[0092] 2. Staining of root samples

[0093] The vacuum bucket was pre-cooled to 4°C, and a pre-cooled ice bag was placed in it to keep the low temperature, and then the centrifuge tube was placed vertically into the vacuum bucket.

[0094] Add detection O pre-cooled to 4°C 2 - The staining solution did not cover the sample.

[0095] After covering the vacuum barrel, adjust the vacuum degree to -0.08MPa to reach the negative pressure state, and keep it for 4h for dyeing.

[0096] 3. Root sample fixation

[0097] After staining, take out the centrifuge tube quickly, discard the staining solution, rinse the sample several times...

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Abstract

The invention provides a plant root system active oxygen rapid staining positioning method which comprises the following steps: (1) fixing a plant root system at the bottom of a container, adding a staining solution corresponding to active oxygen to be detected to submerge a sample, putting the sample into a vacuum container, and carrying out low-temperature vacuum staining; (2) discarding the staining solution, adding a fixing solution, putting into a vacuum container, and carrying out low-temperature vacuum fixing; (3) discarding the stationary liquid, adding a glycerol aqueous solution, putting into a vacuum container, and carrying out low-temperature vacuum filling and shaping; (4) pre-cooling a cold chamber and a slicing cutter, embedding the plant root system discarded with the glycerol aqueous solution by using an OTC embedding medium, adjusting the temperature of a fixed table, and slicing; and (5) after the plant root system returns the temperature, sealing. According to the method, a large amount of manufacturing time is saved, and rapid slicing is achieved; the influence of temperature on active oxygen metabolism is reduced, and the slicing accuracy is greatly improved; the tissue can be prevented from shrinking, the original living form is kept, and the authenticity and reliability of the section are ensured.

Description

technical field [0001] The invention relates to a rapid dyeing and positioning method for active oxygen in plant roots. Background technique [0002] The metabolism of reactive oxygen species in plants is very rapid, O 2 - Metabolic time in milliseconds, the slowest metabolized H 2 O 2 It remains in the body for only a few minutes. The instability of reactive oxygen species should be considered in the staining and localization of plant tissue, and the higher the temperature and the longer the treatment time, the more unstable the results are, and the less accurate the results will be. [0003] The plant root system is cylindrical, and it is impossible to refer to the method of reactive oxygen dyeing and transparency of plant leaves, which must be observed after slicing. The traditional paraffin section has to go through the steps of fixation, embedding, sectioning and staining, which takes a long time, the temperature of melting wax is high, and the loss of reactive oxy...

Claims

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Application Information

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IPC IPC(8): G01N1/30G01N1/36G01N1/28G01N1/06
CPCG01N1/30G01N1/36G01N1/286G01N1/06G01N2001/2873
Inventor 李捷冯丽丹李栋何静
Owner GANSU AGRI UNIV
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