Producing good somatic metaphase spreads is hindered by the combined difficulties of obtaining large numbers of dividing cells and of spreading and staining the chromosomes well. If good cells were routinely obtainable it would enhance the recognition of B chromosomes, the identification of translocations and aneuploids, and help with the routine counting of chromosomes. The present contribution describes a chemical prefixation technique using monobromonaphthalene and dimethyl sulfoxide (DMSO).
Over the years many methods have been developed for preparing somatic chromosomes, and they are generally made up of four stages. First, the seed is germinated and the actively growing root tip is collected. Second, a treatment prior to fixation (i.e., chemical or temperature) is used to inhibit spindle formation, preventing the congression of chromosomes to the metaphase plate. Third, fixation in any of a large range of solutions. Fourth, preparation of the root tips by hydrolysis, followed by staining.
Assuming that the material to be examined contains dividing cells it would be desirable to have a prefixation method that resulted in a quick and uniform penetration of the prefixative. Consequently, experiments were conducted utilizing several species in which the prefixative was combined with DMSO (Sallee and Kimber, in press, Cereal Res. Commun.) During these experiments it was determined that the use of DMSO along with monobromonaphthalene reduces the amount of prefixative needed and the time of treatment required, and increases the proportion of good analyzable cells.
Seeds are placed in Petri dishes lined with moist filter paper and germinated from two to three days at 30 C. Three to four cm long root tips are collected and placed in the prefixation solution consisting of five drops of monobromonaphthalene, four drops of DMSO and 100 cc tap water for two to three hours at room temperature and then transferred into glacial acetic acid for fixation. It is important that the DMSO be added to the water after the monobromonaphthalene. The minimum time of fixation is two hours; ideally, they should be left overnight. If the roots are kept longer than two days, the acetic acid should be replaced by 70% ethyl alcohol. When ethyl alcohol is used, the root tips should be rinsed in tap water for ten minutes prior to hydrolysis. Roots are hydrolyzed for fourteen minutes in 1N HCl at 60 C. After hydrolysis the roots are placed into Feulgen stain for ten to fifteen minutes. Slides are made from squashes of small portions of the root tips in propionic orcein. Plastic cover slips proved to be preferable to glass due to the tendency of glass to break under vigorous tapping needed to ensure proper cell separations.
P. J. Sallee
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