2.4 Histo-cytological observations
Anthers at six different developmental stages: meiotic (MEI), tetrad
(TED), early uninucleate microspore (EUM), vacuolated uninucleate
microspore (VUM), binucleate (BIN)) and mature pollen (MP) adapted from
Polowick and Sawhney, (1992,
1993a, b)
were sampled from WW and DS plants after 4 d of DS and at different days
after rewatering (DARW). The samples were fixed and post fixed in 2.5%
glutaraldehyde and 1% Osmium tetroxide (OsO4) in
phosphate buffer (PBS 0.1M, pH7.0) respectively, dehydrated in a graded
series of ethanol and absolute acetone. Samples were infiltrated
consecutively with 1:1 mixture of absolute acetone and final spur resin
mixture, 1:3 mixture of absolute acetone and final resin mixture, and
lastly with final resin. Samples were embedded in spur resin and heated
at 70°C for more than nine hours. 2-3µm and 6µm thick sections were cut
using LEICA EM UC7 utratome (LKB) and stained with uranyl acetate and
potassium iodide solution respectively, observed and photographed using
Nikon Eclipse 90i microscope. Thin sections from the same samples were
double-stained with uranyl acetate and alkaline lead citrate and viewed
using Hitachi Model H-7650 transmission electron microscope (TEM).