Section 6 — Cytology 



6.4. Effects of Digitonin on Cellular Division Part I. 



L. V. Olah (Carbondale, U.S.A.). 



A unique, complex effect of digitonin on 

 Allium sativum, previously reported by the 

 author, (1 ) was analyzed in detail. Digitonin dis- 

 organizes the spindle, and during nuclear resti- 

 tution a highly refractive, viscous, hyaline sub- 

 stance appears. This is always closely associated 

 with the restituting nucleus. The chromatin 

 surrounds this substance, entirely trapping it 

 at times. Simultaneously, synthesis of cell wall 

 material (PAS and Iodine-H2S04 positive) 

 originates within the hyaline body, increasing its 

 size and refringency. 



Thus, the shape of the restituting nucleus is 

 determined by the location, shape, size, and 

 viscosity of the hyaline body. This mutual 

 association results in indented, crescent-shaped or 

 "hollow" nuclei. 



Once polysaccharide synthesis begins, it 

 spreads into the cytoplasm in a haphazard, 

 ramified manner. 



In some cases, semi-recovered cells are able 

 to form a normal spindle. However, the hyaline 

 body appears in the same cell, at ana-telophase, 

 associated with the restituting group of chromat- 

 ids. PAS testing indicated polysaccharide syn- 

 thesis in this body, which results in a ramified 

 cell wall located near the polar region. 



The hypothesis is accepted that the phragmo- 

 plast consists of two categories of elements; 

 elements of the interzonal area of the spindle and 

 elements which originate from the polar-nuclear 

 area. These latter elements were observed in the 

 area of the restituting nuclei, and according to 

 Porter etal., < 3 ) ought to migrate to the equator. 

 Association of both these elements at the equator, 

 changes the structure of the spindle, resulting in 

 the appearance of the barrel-shaped phragmo- 

 plast. 



It appears that digitonin hampers the migration 

 of the "nuclear" elements from the nuclear area. 

 If a spindle is not present, they appear in the 

 area of the restituting tetraploid nucleus, and 

 remain associated with it. In cells able to form 

 spindles, digitonin blocks the migration of the 

 polar-nuclear elements to the equator, and they 

 remain at the polar region, forming a hyaline 

 body associated with the chromatids of the 

 daughter nuclei. 



Rosette-crystals, formed /'// vivo, similar to 

 digitonide crystals, were detected in the treated 

 cells. The role of sterols and the effect digitonin 

 may have as an anionic detergent is discussed. 



Fitogeneticistas e Fitoparasitologistas, p. 10, 

 1952. Brazil. 



2. Olah, L. V. Proceedings of the IX Inter- 

 national Congress of Genetics. Part II. Caryo- 

 logia VI, Suppl., pp. 836-838, 1954. 



3. Porter, K. R. and Machado, R. D. 

 J. Biophysical Biochem. Cytology.!, 167-180, 1960. 



6.5. Effects of Digitonin on Cellular Division Part II. 

 Electron Microscope Studies. A. G. Under- 

 brink and L. V. Olah (Carbondale, U.S.A.). 



Allium sativum root tips were treated with digi- 

 tonin to study the complex effects described by 

 L. V. Olah. 



These root tips were fixed 2 hr in aqueous 2 

 per cent KMnO-i and treated with 1 per cent 

 uranium acetate for 1 hr. 



The process of the copious and ramifying cell 

 wall formation and the development of the hya- 

 line body induced by digitonin were analyzed. 

 However, using this fixative the spindle substance 

 was not observable. 



Densely packed., circular aggregates of profiles 

 and vesicular-like elements were found which had 

 the size and location of the hyaline bodies seen 

 with the light microscope. In some cells these 

 aggregates were morphologically identical to the 

 elements previously termed "phragmoplast" by 

 Porter et al. (*) Some reconstructing nuclei had 

 completely enclosed these aggregates and in these 

 enclosures polysaccharide synthesis was observed. 



Most reconstructed nuclei were indented on 

 one side having one or more branches of the 

 ramified cell wall system almost always located in 

 the nuclear indention. Thick and profusely 

 ramifying cell walls, anastomizing haphazardly 

 throughout the cytoplasm were observed with 

 high frequency. Portions of cytoplasm assumed to 

 be completely encased by cell wall and lacking 

 chromatin material appeared to be disintegrating. 

 In other cells the cell wall encircled the nucleus 

 with a portion of cytoplasm, giving the appear- 

 ance of formation of a cell within the "mother" 

 cell. 



Many cells were found in which large quanti- 

 ties of polysaccharide materials occupied large 

 portions of the volume of the cell. 



The identity of the aggregated profiles and 

 vesicular-like elements with the digitonin- 

 induced hyaline body described by L. V. Olah 

 and the relationship of these aggregates to the 

 synthesis of the unusually large amounts of 

 polysaccharides is discussed. 



1. Olah, L. V. II. Reuniao Latino-Amevicana de 



1. Porter, K. R. and Machado, R. D. 

 /. Biophysical Biochem. Cytology. 7, 167-180, 1960. 



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