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Wheat tissue active oxygen fluorescence labeling method

A fluorescent labeling, reactive oxygen species technology, applied in the field of plant stress and physiological response, can solve the problems of lack of scientific detection method for the comprehensive production of reactive oxygen species, large differences in production, difficult plant stress resistance or aging process, etc. Achieve precise resistance or aging process, easy operation, improve the effect of fixing and loading

Inactive Publication Date: 2012-07-18
CROP RES INST SHANDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are many kinds of reactive oxygen species in plant tissues, and their production varies greatly. Therefore, it is difficult to explain the stress resistance or aging process of plants with the change of 1-2 kinds of reactive oxygen species content.
More importantly, there is currently no scientific method for the comprehensive production of reactive oxygen species in wheat tissue, especially in larger volumes.

Method used

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  • Wheat tissue active oxygen fluorescence labeling method
  • Wheat tissue active oxygen fluorescence labeling method
  • Wheat tissue active oxygen fluorescence labeling method

Examples

Experimental program
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Effect test

Embodiment 1

[0029] A fluorescent labeling method for reactive oxygen species in plant tissue, applied to wheat (Jimai 22) stem tissue under nutrient stress (nitrogen deficiency), the specific operation is as follows:

[0030] a. Prepare wheat tissue sample marker loading buffer, pipette 5 ml of sample marker loading buffer into a vessel with a volume of 10 ml;

[0031] Prepare 10 mM Tris-HCl buffer solution (pH 7.2); prepare DCFH-DA stock solution with DMSO at a concentration of 5 mM; prepare fluorescent labeling loading solution with buffer solution, containing DCFH-DA at a final concentration of 50 μM, and a final concentration of 50 mM KCl and glutaraldehyde at a final concentration of 2.5% (volume percent).

[0032] b. Cut the wheat stalks into tissue blocks with a length of 0.5 cm, cut them evenly along the longitudinal axis, and divide them into two;

[0033] c. Rinse the tissue block prepared in step b with the sample marker loading buffer twice, then place it in the vessel contai...

Embodiment 2

[0040] A fluorescent labeling method for reactive oxygen species in plant tissue, applied to leaf tissue of wheat (Jimai 22) in the senescence stage of grain filling, the specific operation is as follows:

[0041] a. Prepare wheat tissue sample marker loading buffer, pipette 5 ml of sample marker loading buffer into a vessel with a volume of 10 ml;

[0042] The preparation method of the above label loading buffer is as follows: prepare 10 mM PBS buffer solution (pH 7.4); prepare DCFH-DA mother solution with ethanol at a concentration of 3 mM; prepare fluorescent label loading solution with buffer solution at a final concentration of 30 μM DCFH-DA, KCl at a final concentration of 50 mM, and glutaraldehyde at a final concentration of 2.5% (volume percent);

[0043] b. Cut the wheat leaves into 0.5 cm tissue pieces;

[0044] c. Rinse step (2) with the sample marker loading buffer to prepare the tissue block twice, then put it into the container prepared in step (1), seal it and pu...

Embodiment 3

[0049] A fluorescent labeling method for reactive oxygen species in plant tissue, applied to leaf tissue of wheat (Jimai 22) under drought stress, the specific operation is as follows:

[0050] a. Prepare wheat tissue sample marker loading buffer, pipette 5 ml of sample marker loading buffer into a vessel with a volume of 10 ml;

[0051] Prepare 10 mM PBS buffer (pH 7.2); prepare DCFH-DA stock solution with methanol at a concentration of 3 mM; use buffer to prepare a fluorescently labeled loading solution, containing DCFH-DA at a final concentration of 30 μM, and a final concentration of 50 mM KCl and glutaraldehyde at a final concentration of 2.2-2.8% (volume percent).

[0052] b. Cut the wheat leaves into 0.5 cm tissue pieces;

[0053] c. Rinse step (2) with the sample marker loading buffer to prepare the tissue block twice, then put it into the container prepared in step (1), seal it and pump air, so that all the plant tissue blocks sink to the bottom of the container;

...

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Abstract

The invention belongs to the technical field of plant stress and physiologic response, and particularly relates to the labeling method for active oxygen generation, generation positions and relative content under biotic stress and abiotic stress during the growth and development process of wheat. The labeling method comprises the following steps of: preparing a label load buffer solution, preparing a material tissue block, rinsing the tissue block with the load buffer solution, placing the tissue block in a dark place at 25 DEG C, exciting the tissue block by using a laser excitation device, observing the tissue block and taking a photo. Compared with the traditional labeling method of DAB (Dimethylaminoazobenzene), NBT (Nitroblue Tetrazolium) and other active oxygen species, the labeling method saves time and avoids the distortion of active oxygen content and positions of a sample during long-time labeling load period. When the active oxygen is labeled by DCFH-DA (Dichlorofluorescein Diacetate), the material is fixed, and the accuracy of active oxygen labeling is ensured. The used device has simple requirements and is easy to operate.

Description

technical field [0001] The invention belongs to the technical field of plant adversity stress and physiological response, and relates to a fluorescent labeling method for the location and relative content detection of active oxygen in plant tissues, and in particular to a method for detecting active oxygen under biotic and abiotic stress during the growth and development of wheat Marking method of production, production site and relative content. Background technique [0002] During the growth and development of plants, they are constantly subjected to various adversities and the inevitable aging process, which leads to metabolic disorders of reactive oxygen species in plant cells, that is, reactive oxygen species in plants such as superoxide anions (OH· - ), hydrogen peroxide (H 2 o 2 ), hydroxyl radical (-OH), singlet oxygen ( 1 o 2） and other bursts; and active oxygen scavengers, such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD), carotenoids, vit...

Claims

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Application Information

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IPC IPC(8): G01N1/28
Inventor 孔令安王法宏冯波李升东张宾司纪升
Owner CROP RES INST SHANDONG ACAD OF AGRI SCI
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