a colchicine solution (100μl) was applied by using a micropipette onto the apical
growing point of each seedling. A range of alternative colchicine concentrations
was applied namely, 0.0, 0.1, and 0.2% (w/v pH 6) four times for about 24 (with
6 h intervals) and 48 h (with 6 h intervals). The treated seedlings were maintained
under the same conditions of growth. The data reported here were obtained from the
original treatment of 60 seedlings, for each colchicine level.
17.2.3 Detection of Tetraploid Plants Following
Colchicine Application to Seedling Apical Tips
Selection of tetraploid plants was done on the basis of morphology (leaf shape)
followed by a selection of size of stomata and the guard cells measurement and
finallyflow cytometry. The putative tetraploids were examined two months later to
validate ploidy stability.
17.2.4 Preliminary Morphological Screening
for Putative Tetraploids
Numerous seedlings were generated from the apical tip application method, so we
conducted an initial screening on the basis of stomatal size and density in order to
isolate a smaller population of putative tetraploids. Leaf samples were taken from
plants when they reached the 5– 6 ‘true-leaf’stage. Samples of epidermal cells were
obtained from lower surface (abaxial side) by nail varnish technique (Hamill et al.
1992 ). A small area (5 mm10 mm) of abaxial side of leaves was covered with a
thin layer of clear nail polish and left to dry. After drying the polish, it was removed
with a tip forcep and then placed on a glass slide and observed through the light
microscope (BX50; Olympus Optical Co. Ltd.) at 100and 400magnification
for study of stomata parameters. To determine stomata density in prepared slides,
each sample was measured fromfivefield of microscopic view.
17.2.5 Ploidy Level Determination by Flow Cytometry
Theflow cytometry performed on tetraploid plants was employed to give an
accurate estimation of nuclear DNA content. In this study, 50 days after treatment,
small pieces of size 0.5 cm^2 were obtained from leaves of tetraploid and diploid
plants. A 400μl of nuclear extraction buffer (solution A kit) was poured on them
and with a sharp blade to prevent crushing the tissue, leaf sections were thorn as
well. The resulting solution was passed through thefilter apparatus, and 1600μlof
370 H. Mansouri and M. Bagheri