2-hydroxyethyl disulfide effect on spindle and chromosomes
--M. C. Molina and E. A. Bernatené

Cytological evidence of the basic number x=5 in the genus Zea were obtained by Molina and Naranjo (TAG 73:542-550, 1987) and Naranjo and Molina (MNL61:62-63, 1987).

One observation was the formation of a double spindle with 5 II each. From the (C-banding) analysis of the chromosome distribution in each spindle it was concluded that they do not distribute at random. The formation of quadrivalents up to a maximum number of 5 IV was also observed when treating Zea mays with a diluted solution of colchicine. Similarly an increase in the number of quadrivalents up to a maximum of 10 IV was detected when treating Z. perennis and hybrids between Z. perennis x Z. diploperennis (2n=40) with the same solution (Poggio et al., TAG 79:461-464, 1990; Naranjo et al., Res. XXI Cong. Arg. de Genet., C. del Uruguay:62, 1990).

The protein called tubulin is the basic structural unit of the spindle. This protein frequently appears as dimers linked by S-S linkages of 40-80 A size. The mitotic apparatus only disrupts with compounds able to break the S-S linkages, for example 2-hydroxyethyl disulfide or with urea which breaks the hydrogen linkages (Zimmerman, p. 159 in The Cell in Mitosis, Levine, ed., Academic Press, New York, 1963).

With the purpose of studying in further detail the spindle and the chromosome distribution in the genus Zea, the cultivar "Ever Green" was treated with two different 2-hydroxyethyl disulfide diluted solutions (1x10-4M and 2x10-4M) during 1991, whilst immature tassels of "Ever Green" and "Colorado Klein" were treated with three diluted solutions (2x10-4M, 3x10-4M and
4x10-4M) during 1992. All treatments were practiced by putting the tassels into diluted solutions during 18 hours (keeping the submerged portion of the stem in absolute darkness). Thereafter tassels were washed by placing them in pots with tap water for 7 hours. Untreated tassels were cut simultaneously and were placed into tap water for 25 hours. Later on, treated and untreated tassels were fixed in 3:1 (absolute alcohol:acetic acid) solution and kept in a refrigerator until their study. Anthers were squashed in 2% acetic haematoxylin (Nu&ntildeez;, 1988).

When comparing the treated and untreated tassels, it was seen that 2-hydroxyethyl disulfide concentrations at 1x10-4M and 2x10-4M did not affect any cells. Higher concentrations (3x10-4M and 4x10-4M) showed significant differences at diplotene, diakinesis and metaphase I. 65% of the cells in metaphase I showed a clear differentiation in two spindles with 5 chromosomes each. In the remaining 35% of the cells this configuration was not clear. In some cells the apparent formation of two spindles was observed, with 8-9 chromosomes in one of them and only 1-2 chromosomes in the other.

A 5 II association into a group of compact appearance was observed. In others 5 II were distributed in a circle. This characteristic kept or was deeply observed in prometaphase I.

A remarkable difference never observed before in the untreated material is the appearance of a high number of quadrivalents, up to a maximum of five. This effect is similar to that produced when treating "Colorado Klein" with colchicine diluted solutions (0.5x10-4M).

Other effects were the chromosome contraction and sticking among themselves, the differentiation amongst chromosomes inside each group being sometimes difficult. High numbers of univalents and endomitosis were also observed. 

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