Experiments for the determination of root vigor by the TTC method
Triphenyltetrazolium chloride (TTC) is a redox substance with a standard redox potential of 80 millivolts. It is a colorless solution when dissolved in water, but after reduction it produces a red, water-insoluble triphenylformazan by the following reaction.
Operation method
Experimental determination of root vigor by TTC method
Principle
Triphenyltetrazolium chloride (TTC) is a redox substance with a standard redox potential of 80 millivolts. It is a colorless solution when dissolved in water, but after reduction, it produces red, water-insoluble triphenylformazan by the following reaction. The reaction formula is as follows: the generated dirty methyl is stable red and will not be automatically oxidized by oxygen in the air. Therefore, TTC is widely used as a hydrogen acceptor for enzyme tests. The reduction of TTC induced by plant roots can be strengthened by the addition of succinic acid, fenugreek acid and malic acid; and it is severely inhibited by malonic acid and iodoacetic acid. Therefore, it is capable of determining dehydrogenase activity. Root vigor was indicated by this enzyme activity.
Materials and Instruments
Corn Wheat root Move I. Instrumentation: For more product details, please visit Aladdin Scientific website.
Ethyl acetate Sodium dithionite TTC solution Phosphate buffer 2N sulfuric acid
Spectrophotometer Model 721 Warm box Mantle Funnel Pipette Volumetric flasks
Model 721 spectrophotometer; warming chamber; mortar; funnel;
Pipettes: 0.5 ml 1, 5 ml 3, 2 ml 1; volumetric flasks (10 ml) 8;
II. Reagents:
1. Ethyl acetate (analytically pure);
2. Sodium dithionite (Na2S2O4), analytically pure, powder;
3. 1% TTC solution: accurately weigh 1.0000 g of TTC, dissolve in a small amount of water, and then dilute to 100 ml. Dilute to the required concentration when used.
4. 0.1 M pH7.5 phosphate buffer.
5. 2N sulfuric acid: take 55 ml of concentrated sulfuric acid with specific gravity of 1.84 in a measuring cylinder and add it into a beaker of 500 ml distilled water while stirring, then dilute it to 1000 ml after cooling.
III. Materials:
Hydroponically or sand-cultivated corn or wheat root plant material (need to be washed).
IV.Method Steps:
TTC standard curve preparation:
Take 0.2 ml of 0.5% TTC solution added to 10 ml stoppered graduated test tube, and then add a little Na2S2O4 powder, shaking well, that is, the production of red dirty. And then use ethyl acetate to the gradient, shake well. Then take 0.25, 0.50, 1.00, 1.50, 2.00 ml of this solution to 10 ml stoppered graduated test tube, with ethyl acetate to the gradient, that is, containing methyl dirty 0.025, 0.05, 0.10, 0.15, 0.20 mg of the standard colorimetric series. Take the blank as the reference, determine the optical density at 485n M and draw the standard curve.
Qualitative determination method:
Take 0.25g of the sample into a triangular vial, add 0.5% TTC solution and 0.1 M phosphate buffer (pH 7.5) 5 ml each, mix thoroughly, and the root tip was completely immersed in the solution, to 37 ℃ incubator for 1 hour, insulation time to immediately add 2N H2SO4 solution 2 ml to terminate the reaction. If the root tip showed red color, it indicated the presence of active dehydrogenase.
Quantitative assay method:
The qualitatively colored root segments were removed, drained of external water with filter paper, transferred to a mortar and pestle, and ground with 3-4 ml of ethyl acetate. Pour the extracted red TTF into a graduated test tube carefully, rinse the residue with ethyl acetate for 2-3 times until the washings are not red, all the washings are combined in a test tube, and finally diluted to 10 ml with ethyl acetate; after shaking well, use ethyl acetate as the control for colorimetry at 485n M, and record the optical density value. Check the standard curve to find out the amount of TTC reduction.