Plant Root Activity Assay Kit (TTC, Colorimetric Method)

Cat. No.: P1505878

GRADE & PURITY BioReagent

Overview

Plant root systems are active organs for absorption and synthesis. The growth status and metabolic level of roots, known as root system activity, directly affect the growth, nutritional status, and final yield of the aerial parts of plants, making it one of the important physiological indicators of plant growth. TTC (2,3,5-Triphenyltetrazolium Chloride) is a redox substance, a redox pigment with a standard oxidation potential of 80 mV. It is colorless when dissolved in water but turns red upon reduction, generating water-insoluble Triphenylformazan (TTF). TTF is relatively stable and is not auto-oxidized by air. Therefore, TTC is often used as a hydrogen acceptor in enzyme assays.

Assay Principle

When determining plant root system activity, TTC is used as the substrate and incubated for 1-4 hours. Dehydrogenases in the root system can reduce TTC, generating insoluble red TTF. TTF is then extracted from the roots using an organic solvent (such as ethyl acetate or acetone). The absorbance at 485 nm is measured using a spectrophotometer or microplate reader, and the amount of TTC reduced is calculated. This reduction amount represents dehydrogenase activity and serves as an indicator of plant root system vitality. This kit is primarily used for the quantitative determination of plant root system activity or dehydrogenase activity. This product is for research use only and is not suitable for clinical diagnosis or other purposes.

P1505878	Component	50T	Storage
P1505878A	TTC	1 g	RT. Store in the dark.
P1505878B	TTC Assay Buffer	250 mL	RT
P1505878C	TTC Stop Solution	100 mL	RT
P1505878D	Na₂S₂O₄ Powder	0.5 g	RT

Required Materials Not Provided

1. Distilled water, Ethyl acetate or Acetone

2. Precision balance, Centrifuge tubes, Volumetric flasks, Filter paper, Water bath, Mortar and pestle or homogenizer, Spectrophotometer, Cuvettes

Experimental Procedure

1. Preparation of TTC Solution (0.4%)

• Weigh 0.4 g of TTC and dissolve completely in 100 mL of distilled water.

• Store the prepared solution at 4°C protected from light. Discard if it turns red.

2. Preparation of TTC Assay Buffer Working Solution

• Mix the TTC Solution (0.4%) with TTC Assay Buffer in equal proportions.

• Prepare fresh before use. Store at 4°C protected from light. Discard if it turns red.

3. Preparation of TTF Standard Working Solution

• Pipette 0.25 mL of TTC Solution (0.4%) into a 10 mL centrifuge tube or volumetric flask.

• Add a small amount of Na₂S₂O₄ powder (approx. 30 mg) and mix. Red, water-insoluble TTF particles will form.

• Add 10 mL of ethyl acetate, mix thoroughly to dissolve the particles, and let stand for phase separation (the lower 0.25 mL colorless aqueous layer is discarded).

• The TTF concentration in the upper red solution is 100 μg/mL.

• Prepare serial standard tubes according to the table below by pipetting the upper red solution and diluting with ethyl acetate to 10 mL.

Reagent (mL)	Std.0	Std.1	Std.2	Std.3	Std.4	Std.5
TTF Std. Working Sol. (100 μg/mL)	0	0.25	0.5	1	1.5	2
Ethyl Acetate	10	9.75	9.5	9	8.5	8
Equivalent TTF (μg)	0	25	50	100	150	200

4. Sample Preparation

• Take 0.2~0.5 g of plant fibrous roots, wash, blot dry with filter paper.

• Completely immerse the roots in the TTC Assay Buffer Working Solution.

• Incubate at 37°C protected from light for 1~3 hours.

• Add 2 mL of TTC Stop Solution to terminate the reaction.

• Prepare a Blank Control: Immerse roots first in 2 mL TTC Stop Solution to kill the root sample, then add 10 mL TTC Assay Buffer Working Solution. Incubate at 37°C protected from light for 1~3 hours.

5. Extraction

• Remove the roots from both the test and blank control groups, blot dry with filter paper.

• Place the roots in a mortar or homogenizer, add 3~4 mL of ethyl acetate.

• Grind or homogenize thoroughly to extract TTF.

• Transfer the red extract (TTF) to a centrifuge tube.

• Rinse the residue 2-3 times with a small amount of ethyl acetate and combine the washes into the centrifuge tube.

• Adjust the final volume to 10 mL with ethyl acetate and mix well.

6. Measurement

• Using a 1.0 cm light path cuvette:

  • Zero the spectrophotometer using the "Std.0" tube.

  • Measure the absorbance of Std.1 to Std.5 tubes at 485 nm.

  • Zero the instrument using the blank control group extract.

  • Measure the absorbance of the test sample extract at 485 nm using a spectrophotometer or microplate reader.

7. Calculation

• Plot the standard curve with the TTF content of the standard solutions (25, 50, 100, 150, 200 μg) on the X-axis and the corresponding absorbance on the Y-axis.

• Use the regression equation to calculate the TTF content or TTC reduction amount (μg) in the test sample, which represents the root system activity or dehydrogenase activity.

• Root System (Dehydrogenase) Activity [mg/(g·h)] = m / (1000 × W × t)

Parameter Definitions:

• m: TTF content (μg) of the sample extract obtained from the standard curve = TTC reduction amount (μg)

• W: Weight of the plant fibrous roots (g)

• t: Incubation time (h)

8. Standard Curve Reference

• Typical Blank Control Absorbance: 0.03 - 0.07

• A standard curve can be constructed by measuring the absorbance of TTF standards in the range of 5-500 μg.

*Note: Reference ranges may vary due to differences in instruments and operational techniques. For precise quantification, use a multi-point standard curve with replicate measurements. Empirical data suggests deviations in the standard curve at TTF contents below 5 μg and above 500 μg.*

Precautions

1. Prepared TTC Solution (0.4%) and TTC Assay Buffer Working Solution should be stored protected from light. Discard if they turn red.

2. The sample amount may vary depending on the root type.

3. Roots should be blotted dry but not squeezed to damage cells for accurate measurement.

4. The mortar/homogenizer, tubes, and cuvettes used for extraction should be kept dry, free of moisture.

5. Since organic solvents (ethyl acetate or acetone) are used to extract TTF, disposable plastic cuvettes or microplate strips cannot be used. Use quartz or glass cuvettes. Clean them with ethanol after use, followed by water rinsing and drying.

6. If a spectrophotometer is unavailable, a microplate reader can be used, but note the detection volume per well.

7. Use reagents promptly after opening to avoid affecting subsequent experimental results.

8. For your safety and health, wear lab coats and disposable gloves during operation.

Storage and Shipping

Storage

Protected from light,Room temperature

Shipped In

Normal

Stability And Storage

Store at room temperature long term (12 months). Store in the dark.