MICROTUBULAR ORGANIZATION DURING ASYMMETRICAL DIVISION OF THE GENERATIVE CELL IN GAGEA-LUTEA

Video microscopy and conventional or Confocal Laser Scanning Microscop y after DAPI staining and anti-alpha-tubulin labelling were used to st udy the asymmetrical division of the generative cell (GC) in Gagea lut ea. Pollen was cultured for up to 8 hr in a medium containing 10% poly (ethylene glycol), 3.0% to 3.8% sucrose, 0.03% casein acid hydrolysat e, 15 mM 2-(N-morpholinoethane)sulphonic acid-KOH buffer (pH 5.9) and salts, In the pollen grain, the GC had a spherical or ovoid shape and contained a fine network of intermingled microtubules. As the GC enter ed into the pollen tube, it assumed a cylindrical shape with a length often exceeding 250 mu m. A cage of microtubules then developed around the nucleus. The presence of dense and thick microtubular bundles in front of the generative nucleus within the GC coincided with the trans location of the nucleus to the leading end of the GC. No pre-prophase band was ever detected, but a distinct prophase spindle of microtubule s was formed. In some GCs a tubulin-rich dot became visible at each po le of the spindle. After nuclear envelope breakdown, the bundles of mi crotubules spread between the chromosomes and became oriented into par allel arrays. The spindle became shorter at metaphase, and there was n o tubulin labelling at the site of the metaphase plate. At anaphase, t he microtubular apparatus lost its spindle-shape and a bridge of promi nent bundles of microtubules connected the two daughter nuclei. At tel ophase, the site of the cell plate remained unstained by the anti-alph a-tubulin antibody, but a distinct phragmoplast of microtubules was fo rmed more closely to the leading nucleus, resulting in the formation o f unequal sperm cells (SCs). The leading SC was up to 2.5 times smalle r than the following SC and it contained a smaller or equal number of nucleoli.