Here, we demonstrate Updegraff method, the world's most widely used method for the estimation of crystalline cellulose content. Cellulose is basically made up of glucose monomers linked by beta-1, 4 glycosidic bonds. We divided this protocol into five phases.
In the first phase, we prepare the plant biomass material. In the second phase, we extract the crude cell wall. In the third phase, we use Updegraff treatment in which Updegraff is made up of acetic acid and nitric acid that helps in the removal of hemicellulose and lignin from our samples.
In the fourth phase, we subject it to acid hydrolysis with the help of sulfuric acid that produces the glucose monomers. Finally, in the fifth phase, we use anthrone assay to estimate the crystalline cellulose content. Collect the plant material from the greenhouse, wash them thoroughly with water, air dry for two days, separate the tissue and transfer the tissue to the individually labeled containers and incubate in the incubator at 49 degrees Centigrade for 10 days.
Then finally, cut into pieces using scissors or blade. Freeze the tissue and load into mortar and pestle for fine grinding into uniform powder or alternatively use Freezer/Mill. Here, we demonstrate using Freezer/Mill, so we load our tissue samples into these grinding vials and place them in the Freezer/Mill and run the 10 CP speed for three cycles.
The tissue powder is collected in the Falcon tube as shown here. Weigh 20 mg of the tissue powder and transfer into a pre-weighed two mL tube. Add one mL of protein solubilization buffer to remove proteins.Vortex.
Centrifuge at 15, 000 RPM for five minutes. Discard the supernatant. Repeat this step two more times.
Add one mL of sterile water to the retained pellet.Vortex. Centrifuge at 15, 000 RPM for five minutes. Repeat this step two more times.
Add one ML of 70%ethanol to the retained pellet.Vortex. Transfer to a preheated 70 degrees Centigrade block with simultaneous shaking for one hour. After one-hour incubation, centrifuge at 15, 000 RPM for five minutes.
Repeat this step one more time. To the saved pellet, add one mL of 100%methylone.Vortex. Centrifuge at 15, 000 RPM for five minutes.
Add one mL of chloroform methanol.Vortex. Centrifuge at 15, 000 RPM for five minutes. Discard the supernatant and add one mL of acetone.Vortex.
Incubate at room temperature for five minutes. Centrifuge at 15, 000 RPM for five minutes. Re-discard the supernatant and air dry them at 37 degrees Centigrade overnight or continue by vacuum drying.
Load your samples into the vacuum dryer and air dry for two to three hours or until the pellet is completely dry. Weigh five mg of the cell wall extract obtained from the previous step. Transfer into a two mL screw cap tube.
Also include positive control at this point. Use two mg of Whatman filter paper for that purpose. Transfer to a two mL screw cap tube.
Add 1.5 mL of Updegraff reagent to each weighed cell wall extract. Vortex and incubate at 100 degrees Centigrade in a preheated block for 30 minutes. After 30 minutes incubation, transfer to a rack.
Allow it to cool on bench for 10 minutes. Centrifuge at 15, 000 RPM for 10 minutes. Discard the supernatant without disturbing the pellet.
Add one mL of sterile water.Vortex. Centrifuge at 15, 000 RPM for 10 minutes. Discard 500 microliters of the supernatant.
Add one mL of acetone.Vortex. Centrifuge at 15, 000 RPM for five minutes. Discard one mL of the supernatant.
Add one mL of acetone.Vortex. Centrifuge at 15, 000 RPM for five minutes. Discard the supernatant completely.
Add one mL of acetone.Vortex. Centrifuge at 15, 000 RPM for five minutes. Discard the supernatant.
Dry at 37 degrees Centigrade overnight. After a one-night incubation, transfer to a rack. Add one mL of 67%sulfuric acid.Vortex.
Incubate at 25 degrees Centigrade for one hour with simultaneous shaking. After one-hour incubation, make sure the pellet is completely dissolved. Transfer to a rack.
This is the final step of the cellulose extraction. Now the samples are ready for sample dilution. For sample dilution, use 10 microliters of each sample from the previous step and add 490 microliters of sterile water.
Repeat this for all the experimental samples. For the glucose standard curve preparation, prepare a fresh stock of one mg per mL glucose and prepare zero micrograms to 200 micrograms concentrations by adding one mg per mL glucose in 20 microliter increments and make up the final volume to one mL with sterile water. Take 500 microliter of each prepared standard in a screw cap tube and add one mL 0.2%anthrone.
And now the samples are ready for anthrone assay. Add one mL of freshly prepared anthrone. Mix immediately by vortexing and transfer to ice.
Repeat for all the standards and samples in the same way. Then transfer to a preheated 100 degrees Centigrade block for 10 minutes. After 10 minutes incubation, transfer to ice and incubate for five minutes on ice.
After incubation, take 200 microliters of each sample in three replicates. Repeat for both standards and samples in the same way. And a loaded 96-well plate was loaded into a Multimode Detector.
Using Smart Max Pro program, we set the filter setting to 620 nanometers. Plate type to 96-well type. Select all the read area.
Click OK.Read. Read plate. You obtain the absorbance readings of both standards and samples, which will be collected in the Excel sheet for further analysis and estimation of cellulose content.
Glucose standard curve is prepared by using the glucose concentrations in micrograms per mL on x-axis and the normalized absorbance values of the respective concentration at 620 nanometers on the y-axis. We get the regression line with m and c values in the plotted scatter graph we use to estimate the sample crystalline cellulose content. In the first step, we averaged the OD values at 620 nanometers.
And then we normalized the data by subtracting the blank value. We calculate the glucose concentration using the formula x is equal to OD minus c/m where we plug in c and m values from the previous step of the regression line in the standard curve. Since the total volume that was used for anthrone assay is 500 microliters, we divide it by dilution factor two.
Since the sample volume that was used was only 10 microliters, we'll further divide by 10 to get micrograms per microliter concentration of glucose which is equal to mg per mL concentration. And then we used the moisture factor 1.11 which is the moisture gain in the process. And finally, we calculate the percent crystalline cellulose content by dividing it with the cell wall weight, which is close to five mg, and multiply by 100 to get the percent.
Then we averaged the crystalline cellulose content of three biological replicates to get the value for individual samples, which will be compared against another experimental line average of three biological replicates. And t-test can be used to test their level of significance. Here in table A, we show the table values of the concentration and the respective OD at 620 nanometers, which was further used to plot a scatter graph and a regression line with the m and c values that were used in the C for obtaining the cellulose content differences between the two experimental lines.
In conclusion, both samples show cellulose differences. Thanks for watching.
