2.2 Leaf anatomy
Five mature leaves were used to determine the area occupied by the
vascular elements of the leaf midrib: consecutive hand transverse
sections were cut with a micro scalpel at three different positions in
the leaves: the petiole, the mid part of mid vein, and about 2cm back
from leaf tip. Each section was stained with 0.1% w/v aniline blue in
PO4K3 (pH 10) (Linskens & Esser, 1957),
observed with a Zeiss Axiophot epifluorescent microscope using the DAPI
narrow filter band (excitation 365 nm, bandpass 12 nm; dichroic mirror
FT 395 nm; barrier filter LP397 nm), and photographed with an AxioCam
512 Color linked to the AxioVision software (Zeiss, Oberkoche, Germany).
The cross-sectional area of xylem and phloem was measured (totaling 45
measures for each tissue) and, for the 27 cross sections in which all
vascular elements were visible, the number of vascular elements in each
tissue were counted
Five additional leaves were selected for measurements of individual
sieve tubes and xylem vessels. Longitudinal sections of the petiole,
midrib, second, third, and fourth order veins were obtained with a
scalpel, placed in a solution containing acetic acid: hydrogen peroxide
1:1 v/v, left incubating at 60ÂșC for two days, and then mounted onto
glass slides for microscopy observation and image acquisition
(n>250 total vessel elements measured). Similar areas were
used to obtain fresh longitudinal sections (less than 1mm thick),
mounted onto slides, stained with 0.1% aniline blue, counterstained
with calcofluor white for cellulose (Hughes & McCully, 1975),
photographed and measured (at least 35 sieve tubes per vein order,
n=250).