Endosperm ultrastructure observation by scanning electron microscope (SEM)

Mature grains from both treatment and control groups were put in the fixative (5 ml 38 % formalin, 5 ml glacial acetic acid, 90 ml 70 % ethyl alcohol) for a minimum of 12 h. Then the grains were dehydrated sequentially in 70 % ethanol solutions (20 min), 80 % ethanol solutions (20 min), 90 % ethanol solutions (overnight) and 100 % ethanol solutions (20 min). The samples were treated stepwise for 20 min in mixtures of ethanol and isoamyl acetate with ratios 3:1, 1:1 and 1:3 before soaking in isoamylacetate. Finally, critical point drying was done for SEM observation. Grain endosperm ultrastructures were observed by scanning electron microscope S-4800 FESEM (Hitachi, Japan).