Sachan and Tanaka (Japan J. Genet. 51:139-141, 1976) have described a Giemsa staining procedure to discern constitutive heterochromatin in Zea chromosomes. This modified procedure differs from the original one basically at three steps. (1) the use of monobromonaphthalene in place of 8-hydroxyquinoline for pretreatment, (2) the use of glacial acetic acid for fixation in place of Carnoy's Fluid I, and (3) a relatively higher concentration of barium hydroxide for a longer period of time. The procedure laid down here produces much more distinct bands compared to the earlier one.
Pretreatment: Seeds are germinated at 25 C and excised root tips are pretreated in saturated monobromonaphthalene solution (2 drops per 5 ml of water) for 3-4 h at 10 C.
Fixation: In glacial acetic acid for 24 h at 10 C. Fixed root tips are stored in 70% ethanol till further use.
Maceration: In 5% (1:1 cellulase and Pectinase, pH 6.8) at room temperature for 30-90 min.
Washing: In water for 10-15 min at room temperature.
Squash: Roots were squashed in 45% acetic acid and pressed hard.
Air drying: Coverglass is flipped off by dry ice or after freezing in liquid nitrogen, and slides were air dried for 24-48 h.
Alkali treatment: Slides were placed in 7% barium hydroxide aqueous solution for 10-12 min at 50 C.
Washing: Alkali was thoroughly washed with water for 10-15 min with 3-5 changes.
Saline treatment: Slides were incubated in 2xSSC (0.3M NaCl - 0.03M Sodium Citrate) at 60 C for 75 min.
Washing: Slides were washed in water for 10-15 min with 3-5 changes.
Staining: Washed slides were soaked in phosphate buffer pH 6.8 for 1 min and stained in 1.5% Giemsa (E. Merck) for 10-15 min at room temperature.
Air drying: After brief rinsing in water slides were air dried for 12-24 h.
Mounting: Slides were transferred to xylene for 5-10 min and mounted in Canada balsam.
J. K. S. Sachan and K. R. Sarkar
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