Transmission Electron Microscopy
Small pieces of leaves were cut and fixed in 3% Glutaraldehyde in 0.1M
Cacodylate buffer (pH 7.4) for 10 hours at room temperature in a
desiccator and then transferred to 40° C for a continuation of fixation
overnight. Tissues were washed in cacodylate buffer and post-fixed and
stained with 2% osmium tetroxide, and 1.5% potassium ferricyanide in
0.1M cacodylate buffer for 2 hours. Tissues were then washed in
cacodylate buffer and dehydrated through a graded series of ethanol
treatments for 10 min each step followed by 100% anhydrous ethanol 3
times, 20 min each, and propylene oxide 2 times, 10 minutes each. The
tissues were infiltrated with increasing concentrations of Agar 100
resin in propylene oxide for 16 hours at each step. The tissues were
embedded in fresh resin at 60°C oven for 48 hours. Ultrathin sections,
approximately 80 nm thick, were cut on a Leica Reichert Ultracut S
microtome, collected onto 200 Mesh carbon-formvar coated copper grids
and stained with Uranyl acetate followed by Reynold’s Lead Citrate for
10 min. The sections were examined using Tecnai 12 TEM 100kV (Phillips,
Eindhoven, the Netherlands) equipped with MegaView II CCD camera and
Analysis® version 3.0 software (SoftImaging System GmbH, Münster,
Germany).
Statistical analyses
The experiments were conducted in a completely randomized design with
3-5 replicates of each genotype. Data were statistically examined using
analysis of variance and tested for significant (P ≤ 0.05)
differences using Student’s t test. All statistical analyses were
performed using the algorithm embedded into Microsoft Excel.