The distribution of specific chromosomes in the nucleus has been of interest to us for some time. Reports of our work with maize have appeared in this Newsletter and elsewhere over the last 15 years. Of several major problems attendant to this work, such as preparation of the material, choice and interpretation of statistical analyses, and reconstruction from serial sections, none has been more limiting than the availability of unambiguous cytological markers. One such marker we have utilized is the nucleolus. Since it is difficult to establish the position of one large nucleolus, or occasionally two smaller nucleoli in somatic interphase cells, we have attempted to analyze the position of nucleoli in recently formed microspore quartets. The choice of quartets has included only those that are intact and within which the limits of the microspore nucleus can be reasonably outlined. The nucleolus stains rapidly with carmine and other stains. Little or no squashing (other than the weight of the coverslip) was utilized.

Photomicrographs of quartets in squash preparations were taken and traced onto acetate sheets. The size of the quartets was standardized and the centroids were aligned. A composite tracing of twenty-five quartets was obtained. The maximum area covered by the nucleolus in the composite tracing was only one-eighth of the maximum area covered by the microspore. The position of the nucleoli was analyzed using four concentric circles drawn about the centroid of the quartet: circle 1 of equal radius to a circle circumscribed about the quartet tracing, circle 2 of radius three-quarters that of circle 1, circle 3 of radius one-half that of circle 1 and circle 4 of radius one-quarter that of circle 1. Eighty-nine percent of the nucleoli were found within the band encompassed by circles 2 and 3. The remaining eleven percent were found within the band encompassed by circles 4 and 3. No nucleoli were found external to circle 2. The location of the nucleolus was also examined using a circle of radius equal to that of circle 4 and divided into twelve sections, each making an angle of 30 degrees to the center. Ninety-three percent of the nucleoli were found in the sections 30 degrees to 60 degrees from the X axis.

Based on these analyses it would appear that the nucleolus does not have a random position inside the microspore in quartets in squash preparations. It is found in an area one-eighth the size of the microspore, in the central region of the cell. In the photomicrographs of quartets used, the extent of the nucleus in each microspore could be drawn as the area of the microspore in which chromatin was found. The area of the nucleus was estimated as one-quarter of the area of the microspore. In 82 percent of the microspores the nucleolus was seen at the perimeter of the nucleus.

C. A. Rees-Farrell and D. B. Walden

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