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Rapid micro-determination method for soluble sugars in fresh plant leaf

A micro-measurement and soluble technology, which is applied in the field of plant biology, can solve the problems of large amount of reaction reagents, long crushing and extraction reaction time, and limited number of samples for one-time determination, and achieve the effect of saving sample processing time

Inactive Publication Date: 2018-10-19
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although some literature reports have optimized the method for the determination of soluble sugar by anthrone colorimetry (references: Ding Xuemei, Zhang Xiaojun, Zhao Yun, et al. Improvement of the test method for the determination of soluble sugar content by anthrone colorimetry[J]. Heilongjiang Animal Husbandry and Veterinary Medicine ,2014(23):230-233; Wei Jie, Wu Cuiyun, Jiang Yuan, et al. Optimization of conditions for the determination of soluble sugar content in jujube by anthrone method[J]. Food Science, 2014,35(24):136-140;) , but there are still some points to be improved in practical applications, such as samples need to be dried for measurement, the reaction time for pulverization and extraction is long, and the amount of reaction reagents is large. Due to the very limited number, it is urgent to establish a method for the fresh leaves of plants, optimize the sample processing process, and quickly determine the soluble sugar content in plants.

Method used

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  • Rapid micro-determination method for soluble sugars in fresh plant leaf
  • Rapid micro-determination method for soluble sugars in fresh plant leaf
  • Rapid micro-determination method for soluble sugars in fresh plant leaf

Examples

Experimental program
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Effect test

Embodiment 1

[0051] A kind of improved classical anthrone-sulfuric acid colorimetric method of the present invention quickly micro-measures the method for the soluble sugar content in the fresh leaf of plant, comprises the following steps:

[0052] A) Cut the leaves of the plant (generally, the tip of the second leaf or the third leaf is preferred), weigh 10-100 mg, and put it into a 2.0ml centrifuge tube;

[0053] B) Add a little liquid nitrogen into the centrifuge tube, and quickly grind it into powder with a grinding rod;

[0054] C) After the liquid nitrogen evaporates to dryness, add 1.0ml ultrapure water and mix well

[0055] D) put into boiling water bath (100℃) and heat for 10min

[0056]E) Take out the centrifuge tube and centrifuge at 10000rpm for 3min

[0057] F) Carefully pipette 50 μl of the supernatant solution into another clean 2.0ml centrifuge tube

[0058] G) Add 450μl of ultrapure water, 100μl of anthrone (20mg / ml) and concentrated sulfuric acid (1.84g / cm 3 )1000μl, ...

Embodiment 2

[0066] A kind of optimization system of the present invention adopts microcentrifuge tube to carry out color reaction for classical anthrone-sulfuric acid colorimetric method, comprises the following steps

[0067] 1) Optimization of color reaction temperature: Accurately draw 400 μl glucose standard solution (0.5 μg / μl) into a 2.0ml centrifuge tube, add 100 μl ultrapure water, mix well, then add 100 μl anthrone ethyl acetate solution (20 mg / μl) in sequence ml) and 1000 μl concentrated sulfuric acid (specific gravity 1.84), cover tightly and mix thoroughly, then place at different temperatures including 16°C (room temperature), 25°C (water bath), 80°C (water bath), 100°C (water bath) for color development For the reaction, multiple tubes were set at each temperature, and the heating time was 10 minutes. The test tube rack was taken out and placed in a wet ice-water bath to cool rapidly to room temperature. After 10 minutes, it was taken out and placed in a 96-well transparent p...

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Abstract

The invention relates to the field of plant biotechnology, particularly to an improved rapid micro-determination method for soluble sugars in fresh plant leaf. The method comprises: directly carryingout micro sampling, simply extracting, and rapidly determining by using an enzyme labeled plate. According to the present invention, the method is suitable for the high-throughput screening of large quantities of materials; the method does not require the drying process, wherein grinding crushing is performed after rapid freezing with liquid nitrogen so as to substantially save the sample treatment time; a set of the optimized determination system for treating the sample by the micro-centrifuge tube and determining by the enzyme labeled plate is established so as to substantially save the consumption of anthrone, concentrated sulfuric acid and other chemical reagents; the method can be matched to the pipetting workstation so as to achieve the high-quality detection; and a set of the methodfor determining the micro sample based on the fresh plant tissue is established so as to provide the simple and effective evaluation method for the research on the temporal and spatial distribution law of the soluble sugar content in living plant leaves, and provide the useful clues for the stress tolerance research and quality improvement of plants.

Description

technical field [0001] The invention relates to the field of plant biotechnology, in particular to an improved method for rapidly and micro-determining soluble sugars in fresh leaves of plants. Background technique [0002] Sugar is a kind of important organic matter in plants, which plays an important role in plant growth, development and morphogenesis. Sugar not only serves as a respiratory substrate to provide energy for the growth and development of plants, but also serves as a metabolic intermediate to synthesize the carbon frame of plant cell structure substances. At the same time, sugar is also an important signaling molecule in plants, and it also plays a role in regulating gene expression. Soluble sugars refer to sugars that are easily soluble in water, such as glucose, fructose, maltose and sucrose. Soluble sugar is an important osmotic adjustment substance in plants, which is closely related to the stress resistance of plants (such as drought resistance, cold re...

Claims

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Application Information

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IPC IPC(8): G01N21/78
CPCG01N21/78
Inventor 刘成洪宗营杰张述伟郭桂梅何婷陆瑞菊黄剑华
Owner SHANGHAI ACAD OF AGRI SCI
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