This experiment was mainly used to determine plant root vigor.
Operation method
Experimental determination of plant root vigor by alpha-naphthylamine method
Principle
The α-naphthylamine adsorbed on the root surface will be oxidized by the plant roots to produce red 2-hydroxy-1-naphthylamine precipitated on the root surface with oxidizing power, which stains this part of the root red. Root oxidation of α-naphthylamine and its respiratory strength, mainly with the respiratory enzyme peroxidase activity has a close relationship, it is believed that the α-naphthylamine oxidation process is catalyzed by peroxidase, the more the enzyme's vitality, the stronger the oxidation of α-naphthylamine, the more staining will be deeper. Therefore, the root vigor can be determined qualitatively according to the staining depth.
Materials and Instruments
Hydroponic Rice Move I. Material Instrumentation and Reagents Crop root vigor determination (α-naphthylamine oxidation value) Record Sheet Caveat Alpha-naphthylamine is auto-oxidized in air, store with care. Common Problems 1. Reference for α-naphthylamine standard curve: http://img.dxycdn.com/trademd/upload/userfiles/image/2015/01/A1420622582png_small.jpg 2. Formula for calculating the oxidation value of α-naphthylamine http://img.dxycdn.com/trademd/upload/userfiles/image/2015/01/B1420621392png_small.jpg 3. Crop Root Vigor Determination (α-Naphthylamine Oxidation Value) Record Sheet http://img.dxycdn.com/trademd/upload/userfiles/image/2015/01/A1420622588png_small.jpg 4. More can be found at https://wenku.baidu.com/view/6535e846591b6bd97f192279168884868662b87f.html For more product details, please visit Aladdin Scientific website.
alpha-naphthylamine mother liquor
Spectrophotometer Electronic analytical balance Oscillator Graduated test tubes Test tube racks Pipettes Pipette racks Absorbent paper Earwash balls Black paper Triangular vials Measuring cylinders
1. Materials: hydroponic rice and other plant roots
2. Instruments and equipments: spectrophotometer, electronic analytical balance, oscillator, graduated test tubes (or ordinary test tubes), test tube racks, pipettes (10 ml, 2 ml, 1 ml), pipette racks, blotting paper, ear-washing balls, black paper, 100 ml triangular flasks, 100 ml measuring cylinders.
3. Reagents and preparation:
Preparation of 50 μg- ml-1 α-naphthylamine mother liquor: weigh 0.1000 g of analytically pure α-naphthylamine and dissolve it in about 50 ml of distilled water (slightly heated), then cool it down and condense it to 100 ml, i.e., 1000 μg- ml-1 α-naphthylamine solution. Naphthylamine solution is 1000μg-ml-1α-Naphthylamine solution, stored in a brown bottle in a dark place at low temperature. Before use, take up 50 ml of 1000μg- ml-1α-naphthylamine solution and dilute it to 1000 ml with distilled water, i.e. 50μg- ml-1α-naphthylamine mother liquor.
Preparation of 1% p-aminobenzenesulfonic acid solution: Weigh 1g of p-aminobenzenesulfonic acid and dissolve it in 100 ml of 30% acetic acid.
Preparation of 100μg- ml-1 sodium nitrite solution: weigh 0.1g of sodium nitrite and dissolve in 1000 ml of distilled water.
0.1 mol-L-1 phosphate buffer (pH 7.0):
Solution A: Weighing pure Na2HPO4-2H2O11.876g dissolved in distilled water into 1000 ml.
Solution B: Weigh KH2PO49.078g dissolved in distilled water to form 1000 ml.
Take 60 ml of liquid A and 40 ml of liquid B and mix them together.
Experimental steps
1. α-Naphthylamine standard curve production
(1) Take six 20 ml graduated test tubes, numbered, according to the table below to prepare the content of each tube of 0-50μg of α-naphthylamine standard solution. 
Add the reagents in the table, shake well, and put it at room temperature (20-25 ℃) for 5 min to develop the color, then add distilled water so that the total volume of each tube is 20 ml, shake well, and then measure the absorbance (A) value of each tube at 520 nm within 20-60 min, using the tube No. 0 as the blank control.
(2) Standard curve
The α-naphthylamine content of tubes No. 1-5 was taken as the horizontal coordinate, and the absorbance value was taken as the vertical coordinate to draw the standard curve.
2. Root treatment and blank experiment
(1) Wash the rice roots to be measured with water, then absorb the water on the roots with absorbent paper, weigh 1~2g, put it into a 100 ml triangular flask, and add 50 μg- ml-1 of α-naphthylamine solution and phosphate buffer in an equal mixture of 50 ml with gentle shaking.
(2) After 10 min of standing, the rapid adsorption of roots was completed, and 2 ml of solution was taken from the bottle and put into a 20 ml graduated test tube to determine the content of α-naphthylamine (see Method 3 below), which was taken as the starting value (the first time the solution was taken). The rest of the solution is corked and placed on an oscillator, and the reaction is oscillated for 3-6 h at 25°C (if there is no oscillator, it should be shaken regularly during the reaction). After the reaction time is completed then take 2 ml of solution into a graduated test tube and leave it to be measured (the second take). Because the naphthylamine solution will auto-oxidize, do the root treatment along with a blank experiment of the same operation without roots.
3. Determination
Add 10 ml of distilled water to each of the 2 ml of assay solution obtained in the second take and the second blank experiment above, mix well, then add 1 ml of 1% p-aminobenzenesulfonic acid and 1 ml of 100 μg- ml-1 sodium nitrite solution. mix well, let the color develop for 5 min at room temperature (20～25℃), then add distilled water to make the total volume of 20 ml, shake well, and then analyze the color of the solution by the colorimetric analysis at room temperature (20～60℃) within 20～60min, then add the colorimetric analysis at the same time. Within 20-60min, the absorbance (A) value was measured at 520nm with the standard curve No.0 tube as blank control.
Calculation
According to the absorbance value of the first and second sample solution, find out the content of α-naphthylamine from the standard curve, and calculate the oxidation value of α-naphthylamine according to the following formula. 
Record the experimental data and results according to the following table.
