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.