Leaf anatomy and scanning electron microscopy (SEM)
For mesophyll structure imaging, leaf sections samples were prefixed in 3% glutaraldehyde solution in 0.1 M cacodylate buffer (during 3 hours at 4°C), rinsed in 0.1 M cacodylate buffer and 0.1 M sucrose, then kept overnight. The next day, samples were fixed in 1% tetroxide (during 2 hours) and rinsed again in 0.1 M cacodylate buffer and 0.1 M sucrose and kept overnight. The fixed material was dehydrated with an acetone series (30%, 50%, 70%, 90% and 100%) for 10 minutes at each concentration. Samples were dried in the critical point dryer (LEICAEM CPD 030) and coated with gold, before being examined under SEM (JEOL-6100 model). Stomatal density and epidermal cell size were determined in the adaxial and abaxial surface of mature fully expanded leaves using SEM micrographs at 330x magnification.