2.9. Scanning Electron Microscopy Analysis of Leaf and Culm

At 55 and 75 DAT, the middle section of the flag leaves (approximately 0.5 cm in width) and the section of internodes at 18 to 22 cm from the basal (approximately 0.3 cm in width) from each treatment were cut with a sharp blade and fixed in fixative (4% glutaraldehyde) for 2 days at 4°C. The samples were washed in a buffer (0.1 M sodium cacodylate) with three changes of 30 minutes each. The samples were postfixed in osmium tetraoxide for 2 hours at 4°C and were washed again in the buffer (0.1 M sodium cacodylate) with three changes of 30 minutes each. The samples were then dehydrated in each graded acetone series of 35, 50, 75, and 95% (30 minutes) and 100% (1 hour each for three changes) followed by critical point drying (Baltec CPD 030). The leaves and culm specimens were mounted on aluminium stubs, covered with double-sided adhesive tape, and sputter-coated with gold with a sputter coater (Baltec SCD 005). They were viewed under a scanning electron microscope (JEOL JSM 6400, Japan) at an accelerating voltage of 15 kV. Three replications were taken for each of the parameters.