[Background Papers]

Date:: 1966-1976

Reference:: PP/CRI/H/6/2/4

Credit: [Background Papers]. Source: Wellcome Collection.

6/131

$materials and methods The broad bean Vicia faba has 5 acrocentric chromosomes (S chromosomes) and i metacentric chromosome (M chromosome). We have studied both ends of the M chromosome and the ends of the long arms of the S chromosomes. Seeds of Vicia faba (var. Suttons' Prolific Longpod) were cultured and treated with colchicine as described elsewhere 4 . To produce chromatid fragments we irradiated io-day-old seedlings 4 h before fixation with 100 R X-rays applied in 1 min (150 kV; half-value layer, 6 mm aluminium). The tips of the primary roots were fixed in 2% formalin in neutral 0.066 M phosphate buffer for 1.25 h. Metaphase chromosomes were isolated by a pressing method developed for isolating nuclei 2 by which the rigid wall of the plant cell is cracked. The suspension containing the chromosomes was centrifuged at 193 g for 5 min. The supernatant was discarded. To the remaining 1 ml suspension 1 ml trypsin solution—1 mg trypsin per ml phosphate buffer (see above)— was added immediately. After digestion of the chromosomes at 37 o the chromosome suspension was dropped on slides and dried in air. The preparations obtained in this way could be kept for several weeks. The optimum time of digestion* was between 0.5 h and 1.5 h. Because of the variable activity of the trypsin preparations this time had to be redetermined in each experiment. For this purpose the chromosome sus pension was dropped on slides at 10-min intervals. The chromosomes were then stained with orcein; the orcein solution was prepared as described elsewhere 3 . Micro- photographs were made of chromosomes which (as the result of the digestion with trypsin) were more or less stretched and, in addition, well stained. Although in a special series we found that Feulgen staining yielded the same results as staining with orcein, we carried out most of our experiments with the simpler orcein technique. The microphotographs belonging to this paper are all made of orcein-stained chromo somes. results Unirradiated chromosomes Confirming the results of other authors 9 ' 10 Figs. 1-4 demonstrate that there are two length structures per chromatid and that the trypsin treatment has resulted in a considerable increase of the chromosome length. These figures as well as those obtained from irradiated chromosomes (see below) represent only a fraction of the total number of microphotographs made. As Figs. 1-4 further show the chromatid ends seem to be closed, i.e. they have a U-shaped structure. This result was observed after staining both with orcein and by the Feulgen technique (see materials and methods). The somewhat stronger colour of this structure obtained after staining with these dyes may be caused by a relatively weak despiralization of the chromatid end. Whereas all chromatid ends studied by us so far, provided that they could be seen clearly, were U-shaped, the end of the chromatid fragment shown in Fig. 4 seems to be open, i.e. not U-shaped. This fragment originates from a chroma tid break that arose either spontaneously or as the result of the preparation. As will * By time of digestion we mean the interval between the addition of trypsin and the dropping of the chromosome suspension on the slide.$

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