Measurement of stomatal aperture
Main stems of the invasive species were detached between 09:00 and 10:00
h and brought into the lab with the cut end submerged in distilled
water. Epidermal layer of the stems of the third internode and mature
leaves (including adaxial side and abaxial side) of the invasive species
were torn off with fine-tipped tweezers, made wet mount microscope
slides and viewed using a Leica DM6000 microscope (Leica, Wetzlar,
Germany). Digital micrographs of the epidermal surfaces (20× and 40×
magnification) were captured and used to quantify stomatal density and
stomatal aperture. The image dimensions were calibrated using the
objective micrometer. Stomatal densities were analyzed using the images
captured through a the 20x objective and expressed on an area basis
(stomata mm−2). The stomatal density of each sample
was calculated as the average of four images that were captured from
different fields of view. The stomatal lengths and widths were
determined using the images captured through the 40× objective, and for
each treatment, at least 20 stomata were measured.
Observations of chloroplast
ultrastructure
Fresh stems of the third internode and mature leaves were cut into
approximately 1 mm×1 mm pieces, vacuum infiltrated and fixed in 2.5%
glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH
7.0). The samples were then dehydrated in a graded series of ethanol and
embedded in Epon 812 epoxy resin. The embedded samples were sliced into
ultrathin sections approximately 70 nm in thickness using an
ultramicrotome (Leica UC7, Leica), double-stained with uranium lead (a
2% uranyl acetate and lead citrate saturated aqueous solution) and
observed under a transmission electron microscope (HT7700, Hitachi
Japan). The length and width of the chloroplasts were determined from
the images captured at 1200× magnification, and 10 chloroplasts were
measured for each treatment.