THESIS

pollen grains from randomly selected fields were observed under at 100 X
magnification. Three replicates per plant from each species were investigated.
To determine pollen fertility, darkly stained pollen grains were recorded as
fertile and viable, and unstained or very lightly stained ones were considered as sterile
or non-viable. Pollen fertility was calculated by dividing the number of viable pollen
grains by the total number of grains counted in the field of view. Pollen viability was
expressed as pollen fertility percentage in each plant species.
2.2 Ploidy determination
The parents and F1 hybrids were evaluated using flow cytometry. The
samples were prepared for using the protocol of Arumunganathan and Erale (1991).
The young leaves from each cross were collected from the field. Approximately 400
mg of leaf tissue was used in each sample. The leaves were placed in a polystyrene
petri dish containing 1.4 ml of PBS buffer (2.04 g Na2HPO4, 0.5944 g sodium
phosphate, 16.36 g NaCl dissolved in 2 L, pH of the buffer was adjusted to 7.2 with
HCl or NaOH). The leaves were cut into fine strips with a razor blade. The samples
were filtered through a 167 µm nylon mesh into 50 ml falcon tube. A solution of 200
ml PI [6 mg Propidium iodide dissolved in 6 ml of distilled water and take 6 ml of
solution PI mixed with 24 ml of DTT solution (40 mg DTT dissolved in 40 ml PBS
buffer and 2.4 ml triton X100 dissolved in 40 ml of DTT solution)] was added. The
samples were then stirred, incubated at room temperature for 15 min and then kept in
ice until analyses were done on a FACS Calibur flow cytometer.
Approximately 10,000 nuclei were counted for each sample. Ploidy levels
of gynogenic plants were assigned based on the DNA content of their nuclei in
relation to the standards.
26