Introduction
Plant roots are active organs for absorption and synthesis. The growth status and metabolic level of roots-known as root activity-directly affect the growth, nutritional status, and final yield of the above-ground parts of plants, making it a key physiological indicator. Root activity (root dehydrogenase activity) can be measured by methods such as TTC reduction, naphthylamine oxidation, and methylene blue adsorption. Roots can oxidize naphthylamine to produce red naphthylamine compounds, and their oxidizing capacity toward naphthylamine is closely related to their respiratory intensity.
The detection principle of this kit is based on the reaction in a weakly acidic environment, using naphthylamine as the substrate. Naphthylamine reacts with sulfanilic acid and nitrite to form a stable red azo compound. By measuring the amount of unoxidized naphthylamine in the solution-indicated by the coloration intensity on the root surface-root activity can be quantitatively determined using a microplate reader at 510 nm. This product is intended for research use only and is not suitable for clinical diagnosis or other purposes.
Kit Contents
P1515879 | Components | Appearance | 100 T | Storage | Quantity Per Test |
P1515879A | Reagent 1 | Liquid | 10 mL | 2-8℃. | As needed |
P1515879B | Reagent 2 | Liquid | 110 mL | 2-8℃. | As needed |
P1515879C | Reagent 3 | Liquid | 10 mL | 2-8℃. Store in the dark. | 75 μL |
P1515879D | Reagent 4 | Liquid | 10 mL | 2-8℃. Store in the dark. | 75 μL |
P1515879E | Standard | Liquid | 2 mL | 2-8℃. Store in the dark. | As needed |
Note: The standard is 0.5 mg/mL α-naphthylamine standard.
User-Prepared Instruments and Reagents:
1. Microplate reader or visible spectrophotometer, water bath/constant-temperature incubator, adjustable pipettes, filter paper, micro glass cuvettes/96-well plates, distilled water.
Experimental Procedure
1. Sample Preparation: Wash the root tissue thoroughly to remove soil, gently blot dry, and avoid squeezing or damaging the root cells. Weigh 0.2 g of the sample into a 5 mL EP tube.
2. Assay Procedure
2.1. Preheat the visible spectrophotometer/microplate reader for at least 30 min, set the wavelength to 510 nm, and zero the instrument with distilled water.
2.2. Dilution of the standard: Dilute the 0.5 mg/mL α-naphthylamine standard with Reagent 1 to prepare standards at 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.0156, 0.0078, and 0 mg/mL. (The 0 mg/mL solution is recorded as the standard blank tube.)
2.3. Standard diluent:
Tube | Standard Volume | Reagent 1 Volume (μL) | Concentration (μmol/mL) |
Std.1 | 200 µL of Standard | 200 | 0.5 |
Std.2 | 100µL of Std.1 | 100 | 0.25 |
Std.3 | 100µL of Std.2 | 100 | 0.125 |
Std.4 | 100µL of Std.3 | 100 | 0.0625 |
Std.5 | 100µL of Std.4 | 100 | 0.03125 |
Std.6 | 100µL of Std.5 | 100 | 0.0156 |
Std.7 | 100µL of Std.6 | 100 | 0.0078 |
Blank | 0 | 100 | 0 |
Note: In the following procedure, 75 µL of standard is required for each standard tube (do not measure the absorbance of the standard directly at this step).
2.4. Pipette the reagents into 5 mL EP tubes as described in the table below.
Reagent (μL) | Test Tube | Blank Tube | Standard Tube |
Sample | 0.2 g | - | - |
Reagent 2 | 2000 | 2000 | - |
Incubate at 37°C in the dark. At 10 min and 3 h 10 min of the reaction, transfer 75 μL of the reaction mixture from each test tube and blank tube to perform the second-step reaction described below. For the second-step reaction, first add Reagent 3 and Reagent 4 to new 1.5 mL EP tubes according to the table below, then transfer 75 μL of the first-step reaction mixture into the EP tubes. Note: Reagent 3 and Reagent 4 must be added first, followed by the reaction mixture. The order must not be reversed! | - |
Reagent 3 | 75 | 75 | 75 |
Reagent 4 | 75 | 75 | 75 |
First-step reaction mixture | 75 | 75 | - |
Standard | - | - | 75 |
Mix thoroughly and allow the color to develop at room temperature for 20 min. Transfer 200 μL to a micro glass cuvette or a 96-well plate, and measure the absorbance at 510 nm. The measurement must be completed within 20 min after color development. Record the absorbance at 10 min as A1 and the absorbance at 3 h 10 min as A2. Calculate ΔAtest = A1test - A2test, ΔAblank = A1blank - A2blank, ΔA = ΔAtest - ΔAblank , and ΔAstandard = Astandard - Astandard blank. (The standard curve and blank tube need only be run once or twice.)
3. Calculation of Root Activity
3.1. Preparation of the standard curve: Plot the standard curve based on the concentration of the standard tubes (x, mg/mL) and the absorbance ΔAstandard (y, ΔAstandard). Using the standard curve, substitute ΔA (y, ΔA) into the formula to calculate the sample concentration (x, mg/mL).
3.2. Calculation based on sample mass:
Root activity is expressed as the amount of oxidized α-naphthylamine. Root activity [μg/(g·h)] = x × V Reagent 2 × 10³ ÷ (W × T) = 666.67 × x ÷ W
10³: unit conversion factor, 1 mg/mL = 10³ μg/mL; V Reagent 2: volume of Reagent 2, 2 mL; W: root mass, g; T: reaction time, 3 h.
Notes
1. The order of adding Reagent 3, Reagent 4, and the sample into the EP tube/96-well plate must not be changed.
2. If the ΔA value is outside the range of the standard curve, either decrease the sample mass (or shorten the first-step reaction time) or increase the sample mass (or lengthen the first-step reaction time).
3. The measurement must be completed within 20 min after color development.
