Extraction of Intercellular Wash Fluid from Wheat Apoplast for Cell Wall-Related Enzymes Analysis

To begin, rinse the wheat leaf pieces twice in distilled water to remove any cytosolic contamination from the cut ends. Insert the leaf pieces into a thick-walled glass tube containing an extraction buffer. Using a water jet pump, apply vacuum infiltration for five minutes to impregnate leaves with the extraction buffer.

Remove the leaf pieces from the glass tube and blot dry them with a paper towel. Insert the dried leaf pieces vertically into a pre-cooled centrifuge tube fitted with a perforated disc. Centrifuge the leaves at 500G for 10 minutes at 4 degrees Celsius.

Using a 100-microliter pipette, transfer the supernatant into a pre-cooled 1.5-milliliter micro-centrifuge tube. Store the collected supernatant at minus 20 degrees Celsius until use.