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).