Size and density of stomata and guard cells measurement
To estimate of suitability of the stomata size and density as criteria for determination of
ploidy level in basil, stomatal measurement was done after morphological and pollen grain
examination between diploid and autotetraploid regenerant plants. For this purpose, 10
plants of diploid control and 10 plants of putative tetraploids randomly were selected.
Measurement and scoring were performed for five well expanded leaves of each plant.
Three samples of epidermal cells were obtained from lower surface (abaxial side) by nail
varnish technique. A small area of abaxial side of leaves was covered with a thin layer of
clear nail polish and left to dry (Hamill et al., 1992). After drying the polish, it was
removed with a tip forceps then placed on a glass slide and observed through the light
microscope (BX50; Olympus Optical Co. Ltd.) at 400 × magnification (for study stomata
density) and 1000 × magnification (for study of stomata and guard cells measurement and
chloroplast number).