Fluorescence banding of constitutive heterochromatin in Zea

C. G. Vosa and D. J. Mogford (MNL 54:95, 1980) described one technique to locate knob regions in mitotic chromosomes of maize with the use of fluorochrome 'Hoechst 33258' following B.S.G. procedure. A simpler and direct procedure to obtain fluorescence bands for maize and teosinte chromosomes is described below. The experimental procedure adopted here does not involve any alkali digestion.

Pretreatment: Excised root tips were pretreated in 0.002 M 8-hydroxyquinoline at 14-18 C for 3.5 hr.

Fixation: In 1:3 acetic-alcohol at 10 C for 12-24 hr. After rinsing with 70% alcohol the roots were preserved in it.

Maceration: In 1:1 mixture of 1N HCl and 45% acetic acid at 60 C for 45-60 sec.

Washing: In distilled water for 10-15 min with 3-5 changes.

Squash: Root tips were squashed in a drop of 45% acetic acid.

Air drying: Cover glasses were flipped off after freezing in liquid nitrogen and slides were air dried and stored for 24-48 hr.

Staining: Before staining, the slides were immersed in 95% alcohol for 2-5 min and air dried. The preparations were stained in 5-10 ug/ml aqueous solution of 'Hoechst 33258' for 15-20 min. After a brief rinsing in distilled water, slides were mounted in 1:1 mixture of glycerol and water.

Observations: Fluorescent microscopic observations were made with a Leitz photomicroscope equipped with HBO 200W hg-lamp. Fluorescence was monitored and examined with exciting filters BG 12, BG 3 in combination with barrier filter OG 1. Photographs were taken on Kodak Tri-X pan film.

Remarkably clear fluorescence bands were observed in both maize and teosinte chromosomes. The terminal bands in Guatemalan teosinte may be seen in Figure 1.

The success may lie in the right choice of fresh roots, maceration in 1:1 mixture of 1N HCl and 45% acetic acid, and alcoholic treatment before staining. Maceration in HCl and acetic acid mixture produced the brightest bands. Satisfactory results were also obtained when roots were macerated in cellulase-pectinase enzyme mixture. Other maceration procedures did not yield satisfactory results.

Figure 1.

J. K. S. Sachan, K. R. Sarkar, R. P. Sharma and Andy Pereira


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