[Background Papers]

Date:
1966-1976
Reference:
PP/CRI/H/6/2/4
Catalogue details

Licence: In copyright

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

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    Mutation Research Elsevier Publishing Company, Amsterdam Printed in The Netherlands 253 Visualization by scanning electron microscopy of achromatic lesions (gaps) induced by X-rays in chromosomes of Vicia faba With the technique of scanning electron microscopy pictures of the three-dimen sional appearance of biological and other specimens can be obtained. In the following study the scanning electron microscope Stereoscan of the Cambridge Instrument Co. Ltd. was used. Although by this technique impressive pictures of human meta- phase chromosomes were recently obtained 1 ' 3 , only occasionally have chromosomes of other objects been studied 4 . To the best of our knowledge the following experiments with the broad bean Vicia faba are the first scanning electron microscopical studies devoted to plant chromosomes. One of the problems of chromosome research which can be investigated by this method is the visualization of achromatic lesions or gaps. These are small non- staining bands traversing (mostly completely) the diameter of the chromatid but without interrupting its longitudinal continuity, i.e. they are not breaks. They represent the most frequent microscopically visible changes induced by X-rays in chromosomes of Vicia faba. (See ref. 7 for a more detailed characterization of X-ray- induced gaps.) The term gap has been used exclusively because these lesions are not stained by the Feulgen test, a result for which the following explanations are possible: (1) DNA loss, (2) loss of the specificity of DNA for this staining reaction, or (3) decrease of the concentration (without involving loss) of DNA, e.g. by a local despiralization of the chromatid. Scanning electron microscopy seemed to us to be a method for answering the question whether gaps represent notches in the chromatid. In this case the second explanation mentioned above could be excluded because such notches would represent either local losses of chromosomal material (apparently the simplest explanation) or local despiralizations of the chromatid. Seeds of Vicia faba (var. Sutton's Prolific Longpod) were cultured, and the seedlings treated with colchicine as described elsewhere 5 » 6 . The chromosomes had to be put on the slide naked, i.e. as free from residual cytoplasm as possible. This isolation of the chromosomes was achieved as described recently 7 , with the following modification of the final step of that procedure. After a drop of the solution containing the isolated chromosomes had been put on a slide and the preparation had been dried in air, a drop of orcein solution was added in order to facilitate the recognition of suitable chromosomes. The orcein solution was prepared as described in ref. 2. After light microscopical inspection, the slides were put for 10 min in absolute ethanol and then dried in vacuum. Areas of about 10 mmx 10 mm containing the selected chro mosomes were cut with a diamond and then overlaid with thin conducting layers of evaporated carbon and gold. In order to induce gaps, io-day-old seedlings were irradiated with 100 Rapplied in i min (150 kV X-rays, 6 mm Al, h.v.l.). Gaps were observed at metaphase but induced (mostly) in late interphase. For the scanning electron microscopical analysis only gaps completely traversing the diameter of the chromatid were selected (by in spection with an ordinary light microscope). In Fig. i an unirradiated chromosome, the so-called M-chromosome, is rep. resented. The nucleolar constriction and the centromere region can be clearly seen- Mutation Res., 11 (1971) 253-255