Experimental determination of malondialdehyde content in plants

Summary

Through the experiment, to master the principle and method of the determination of malondialdehyde content in plants

Operation method

Experimental determination of malondialdehyde content in plants

Principle

Malondialdehyde (MDA) is produced as a result of peroxidation of membrane lipids in tissues or organs of plants due to official senescence or injury under adverse conditions. Its content is closely related to plant senescence and adversity injury. To determine the content of malondialdehyde in plants, thiobarbituric acid (TBA) is usually used to produce a chromogenic reaction with malondialdehyde in tissues when heated under acidic conditions, resulting in the production of reddish-brown trimethoprim (3,5,5-trimethyloxazolidine 2,4-dione), which has a maximum absorption wavelength of 532 nm.However, the determination of MDA in plant tissues is interfered with by a variety of substances. However, the determination of MDA in plant tissues is interfered by a variety of substances, the most important of which is soluble sugar, sugar and thiobarbituric acid color reaction product of the maximum absorption wavelength at 450nm, also absorbed at 532nm. Soluble sugar increases when plants are subjected to drought, high temperature, low temperature and other adversity stresses, so the interference of soluble sugar must be excluded when determining the content of malondialdehyde and thiobarbituric acid reaction products in plant tissues. In addition, the effect of non-specific background absorption at 532 nm should also be excluded. Low concentrations of iron ions can significantly increase the absorbance values of thiobarbituric acid and sucrose or malondialdehyde colorimetric reactants at 532 and 450 nm, so a certain amount of iron ions is required in the colorimetric reaction of sucrose, malondialdehyde and thiobarbituric acid, and the content of iron ions in plant tissues usually ranges from 100-300 µg-g- 1Dw, according to the amount of plant samples and the volume of the extract, the final concentration of Fe3+ was added as 0.5 nmol- L-1. The malondialdehyde content was calculated by measuring the absorbance values at 532 nm, 600 nm and 450 nm.

Materials and Instruments

Plant leaves
Trichloroacetic acid Thiobarbituric acid (TBA) solution Quartz sand
Centrifuges Spectrophotometers Electronic Analytical Balances Constant Temperature Baths Mason jars Test tubes Pipettes Test tube racks Pipette racks Earwash balls Scissors

Move

I. Materials, instrumentation and reagents

1. Material: plant leaves.

2. Instrumentation: centrifuge, spectrophotometer, electronic analytical balance, constant temperature water bath, mortar and pestle, test tubes, pipettes (1 ml, 5 ml), test tube racks, pipette racks, ear washer balls, scissors.

3. Reagents: 10% trichloroacetic acid, 0.6% thiobarbituric acid (TBA) solution, quartz sand.

Experimental steps

1. Extraction of malondialdehyde

Weigh 1 g of plant leaves subjected to drought, high temperature, low temperature and other adversity stress, add a small amount of quartz sand and 10% trichloroacetic acid 2 ml, grind until homogenized, then add 8 ml 10% trichloroacetic acid for further grinding, homogenize the homogenate by centrifugation at 4000 r/min for 10 min, and its supernatant is malondialdehyde extract.

2. Color reaction and determination

Take four clean test tubes, numbered, three for the sample tube (three replicates), each add 2 ml of extract, the control tube with distilled water 2 ml, and then each tube then add 2 ml of 0.6% thiobarbituric acid solution. The mixture was shaken well and reacted in boiling water bath for 15 min, cooled rapidly and then centrifuged. The supernatant was taken and the absorbance (A) values were determined at 532, 600 and 450 nm, respectively.

III.Calculation of malondialdehyde content:

Since the maximum absorption wavelength of sucrose-TBA reaction product was 450 nm and the millimolar absorption coefficient was 85.4×10-3, the millimolar absorption coefficients of MDA-TBA reaction product at 532 nm were 7.4×10-3 and 155 × 10-3.The 532 nm nonspecific absorbance value can be represented by the absorbance value at 600 nm.

According to the principle of two-component spectrophotometry, a system of equations was established, and the concentrations of MDA and soluble sugar were found by solving this system of equations.

System of equations:


1. Calculate the MDA concentration in the extract according to the following equation

2. Calculate the MDA content in the sample according to the following equation

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