Lignin is a complex heteropolymer made up of three monolignols. It is the second most abundant polymer on earth next to cellulose. Here, we demonstrate thioglycolic acid method, which is the most reliable method for the estimation of lignin content.
This method is based on the principle that lignin binds to thioglycolic acid through thioether bonds. Further, we divided this protocol into five phases. In the first phase, we prepare the plant material.
In the second phase, we isolate the alcohol insoluble extract. In the third phase, we precipitate lignin using thioglycolic acid and acidic conditions. In the fourth phase, we prepare lignin standard curve using industrial bamboo lignin.
Finally, in the fifth phase, we estimate the lignin content using the standard curve prepared in the fourth phase. Plant material is collected, flipped the pots to separate the roots, washed them thoroughly in water, air dried for two days, transferred them into labeled containers and incubated them in the incubator at 49 degrees centigrade for one week to 10 days. Using scissors, the tissue is further cut into 1 mm to 3 mm size pieces.
Alternatively biomass grinder is used to crush the plant tissues. Use the root tissue Turn on the biomass grinder collect the crushed powder into pre-labeled containers. Similarly, repeat for the stem tissue and leaf tissue.
The crushed powder is loaded into the grinding vase which are then placed in the freezer mill And run the freezer mill at the rate of 10 CP speed for three cycles. Weigh 20 mg of the ground tissue powder and transfer to a pre made 2ml tube. Make a note of the empty tube and the tube with tissue powder and the tissue powder in your lab notebook.
Incubate the tubes with cap open at 60 degrees centigrade for one hour. After incubation, add 1.8 ml of water to each sample vortex centrifuge at 15, 000 RPM for 10 minutes. Discard the supernatant Add 1.8 ml of methanol.
Vortex and incubate in a preheated 60 degrees centigrade block for 20 minutes After incubation centrifuge at 15, 000 RPM for 10 minutes. Repeat this step one more time. After centrifugation discard the supernatant and dry the pellets in the vacuum dryer for two to three hours or until the pallet is completely dry.
Measure and record the weight of each tube in your lab notebook for the estimation of lignin content at the end. Also include positive control industrial bamboo lignin at this point starting from 0.5 mg to 4 mg in triplicates. Add 1 ml of 3 normal hydrochloric acid, along with hundred microliters of glycolic acid to each sample Vortex and incubate at 80 degrees centigrade for 3 hours.
After 3 hours incubation transfer them to a rack and allow it to cool for 10 minutes. then centrifuge at 15, 000 RPM for 10 minutes. After centrifugation, the color of the solution looks like this.
Discard the supernatant. Add 1 ml of water. Vortex Centrifuge at 15, 000 RPM for 10 minutes.
Discard the supernatant. Add 1 ml of 1 normal sodium hydroxide. Vortex and incubate at 37 degrees centigrade shaker overnight.
After overnight incubation centrifuge the tubes at 15, 000 RPM for 10 minutes. Transfer the supernatant fresh pre-labeled 2 ml tubes add 200 microliters of concentrated hydrochloric acid to the supernatant. Mix them by gentle inversion as shown here.
Incubate them in the refrigerator at 4 degrees centigrade overnight. After incubation centrifuge at 15, 000 RPM for 10 minutes. Discard the supernatant.
Add 1 ml of sodium hydroxide. Vortex and incubate in the shaker at room temperature for 10 to 15 minutes. Use spectrophotometer to collect the absorbance readings 280 nanometers, Take 2 microliters of sodium hydroxide as blank since the lignin precipitated is dissolved in sodium hydroxide.
Make the blank to zero. Similarly, repeat for the samples. Use two microliters of each sample to collect the absorbance readings in your lab notebook.
Take three readings for each sample for three technical replicates. In the first column, we have the sample number. In the second column, we have biological replicates where R1, R2, R3 represent three biological replicates of one experimental line.
In the third column, we averaged the absorbance readings obtained at 280 nanometers. We calculated the lignin content in the fourth column using the formula y=mx-c where x is the average OD.m and c values are plotted from the regression line of the standard curve prepared. Fifth column is the weight after the methanol and water washes.
So this is the way that is used for the estimation of lignin content in mg. So we divide the value obtained in the fourth column by fifth column to obtain the value per mg of lignin content in the sixth column. And this value is multiplied by 1000 in order to obtain per gram cell wall weight.
And the percent lignin content is calculated by dividing this value by 1000 and multiplying by 100. Finally, we obtain the average lignin by averaging the lignin of three biological replicates of each experimental line which will be compared with average of another experimental line in the form of a bar graph to understand the differences in the lignin content. We can also apply student t-test to test their level of significance.
In column A, we have commercial bamboo lignin, and then the weight of the bamboo lignin that was taken before TGA and their respective absorbance readings at 280 nanometers. These values in table A were used to plot a scattered graph in column B, which gives us the regression line with m and c values. In C, we compared the values obtained by using the standard curve and the industrial bamboo lignin experimental line one and two were compared in the form of a bar graph and the result is they show difference between each other.
