2.6. Root anatomy
Root samples for anatomy were taken using the same criterion
for all samples: we selected 2 cm of the main root or one of the
larger diameters (in the case of there not being any main root)
at approximately 5 cm from stem-root connection. Samples were
fixed with FAA (acetic formaldehyde:alcohol:acetic acid:distilled
water; 10:50:5:35) until its processing in laboratory. Fixed material
was cut by hand with razor blades raised under a binocular
magnifying glass (Olympus SZ61). The slides were made on a
cross section to longitudinal root axis taking about 100–200 m
in thickness. Cross section samples were stained for 1 min with
toluidine blue solution (0.5% in distilled water), which stains lignin
blue and cellulose purple, and they were then washed in water
to eliminate colorant excess. Stained samples were placed on a
glass slide with water. Images for analysis were photographed
using a camera (Olympus Altra 20) connected to a microscope
(Olympus BX41). Measurements were made over a subsection
along a symmetry axis so that relative values refer to the surface
measured (50%). The variables calculated were: the percentage of
xylem per cross section area (CSA), the number and proportion of
xylem vessels over total xylem CSA and the diameter of the xylem
vessels.