Colorimetric Assay for the Quantification of Non-Structural Carbohydrates in Plant Tissues

Digestible carbohydrates are the non-structural components of plant tissue, and they function as a reserve energy source for plant growth and metabolism.

To quantify the non-structural carbohydrates, take a measured amount of plant tissue in a tube. Add diluted sulfuric acid to the tube, and heat the sample. Upon heating, sulfuric acid hydrolyzes the polysaccharides of the digestible carbohydrates into their respective monosaccharide subunits.

Cool the sample. Centrifuge the tube to pellet the undigested plant material and obtain a supernatant containing the monosaccharides.

Aspirate the supernatant into a fresh tube, and treat the monosaccharides with phenol solution, followed by the addition of sulfuric acid. Vortex the tube to mix the content and incubate.

During incubation, sulfuric acid dehydrates the monosaccharides to produce furfural derivatives. These furfural derivatives react with the phenol to produce a yellow-gold solution.

Spectroscopically determine the absorbance of the yellow-gold solution at 490 nanometers, which corresponds to the carbohydrate content in the plant sample.

Next, take various concentrations of glucose solutions and repeat the phenol-sulfuric acid treatment to yield different color intensities, and plot a standard curve. Compare the absorbance value of the unknown carbohydrate sample with the glucose standard to obtain the content of digestible carbohydrates in the plant tissue.

First, weigh out replicates from each tissue sample into glass 15-milliliter tubes. Label the tubes, and record the exact mass of each sample.

Next, add 1 milliliter of 0.1 molar sulfuric acid to each tube, and close the caps tightly. Then, place the tubes in a boiling water bath for 1 hour.

Transfer the tubes to a tepid water bath to cool. Then, pour the contents of the tubes into labeled 1.5-milliliter microcentrifuge tubes. After this, centrifuge the tubes at 15,000 g for 10 minutes. Use a micropipette to transfer the supernatant to new labeled 1.5-milliliter tubes.

Using a pipette, transfer 15 microliters of each unknown sample to its own test tube. Then, add 385 microliters of distilled water to each tube.

In a fume hood, add 400 microliters of 5% phenol to each standard and unknown sample test tube. Immediately thereafter, add 2 milliliters of sulfuric acid to each tube.

Make sure to add the sulfuric acid to the surface of the solution.

After incubating the tubes for 10 minutes, vortex the tubes. Then, incubate the tube for an additional 30 minutes.

Transfer 800 microliters from each sample tube to three polystyrene 1.5-milliliter semi-micro cuvettes. Then, set the spectrophotometer to read at 490 nanometers, and calibrate it with a blank cuvette.

Finally, run each cuvette through the spectrophotometer, and record the absorbance.