Optical and ultrastructuralinvestigations have shown that purified digitonin, a non-ionic, steroid-type saponin, dissociates the formation and function of the mitotic spindle from the phragmoplast (Olah 1963(Olah , 1965Underbrink and Olah 1965, 1968).The course of digitonin mitosis was described as similar to colchicine mitosis because it arrests the mitotic division at metaphase.However, during sub sequent phases of digitonin mitosis, a vigorously functioning phragmoplast develops among the paralyzed and scattered chromosomes, resulting in a branched or amor phously shaped cell plate and wall system which remains associated with the re stituted tetraploid nucleus.Our earlier studies using only KMnO4 fixation were extended to include glutaraldehyde-osmium tetroxide fixation, in order to clarify whether the effect of digitonin is related to the formation and function of the microtubules which constitute the mitotic spindle and the phragmoplast.
Materials and methodsCloves of Allium sativum were rooted in distilled water. Rootlets, approxi mately 1cm long, were treated for 15 hours in 0.25% and 0.5% purified digitonin solutions. Appropriate controls, placed in distilled water, accompanied the treat ments. For electron microscopy, root tips were prefixed for 2 hours in 6% glutar aldehyde in 0.15M phosphate buffer (pH7.4) containing 3 drops of 0.1M CaCl2/ 10ml. The root tips were then rinsed for 30 minutes with the buffer and postfixed for 2 hours in 1% 0.15M phosphate buffered OsO4 at pH 7.4. Both fixations were carried out at room temperature. After 3-4 washes in distilled water, root tips were dehydrated in a graded methanol series, passed through propylene oxide, and embedded in Epon. Sections were cut with a diamond knife on a LKB ultra microtome, placed on uncoated grids, stained with uranyl acetate and lead citrate, and examined with a RCA EMU-3C or Hitachi HU-11A electron microscope.
ResultsIn untreated cells, the microtubules showed their characteristic cortical,
