Vosa and de Aguiar (MGN 46:165-7, 1972) described a Giemsa staining technique by means of which the positions of chromosome knobs may be located in mitotic chromosomes. The present work describes a fluorescence technique which indicates their positions with even greater clarity. Like the Giemsa method, it is a heterochromatin staining technique based on denaturation-reannealing, but this is followed in the present case not by Giemsa staining but by treatment with the AT-binding fluorochrome "Hoechst 33258."
Actively growing root tips are pretreated by immersion in saturated, aqueous ?-monobromonaphthalene for 4-5 hours, and fixed in 3:1 ethanol:glacial acetic acid for 8-24 hours. They are then hydrolyzed in 0.2 N HCl at 600 C for 2.5 minutes, and squashed in 45% acetic acid under an albumenized coverslip, which is floated off in absolute ethanol and air-dried. The coverslips are then immersed for 6 minutes in a saturated aqueous solution of barium hydroxide at room temperature, rinsed in running deionized water, incubated in 2X saline sodium citrate buffer at 600 C for one hour, rinsed again in deionized water, and air-dried. Following this the preparations are stained for 5 minutes in a 0.02% ethanolic solution of Hoechst 33258, rinsed in ethanol, air-dried, mounted in 50% glycerol, and viewed using a Zeiss fluorescence microscope with exciter filter BG12 and barrier filters 50 plus 53. If desired, the preparations may then be made permanent by Giemsa staining according to the method of Vosa and de Aguiar.
The knob regions are revealed by the fluorescence technique as regions of intense fluorescence, and by the Giemsa technique as regions of heavy staining. Both methods yield good results, though for the most part the fluorescence technique is the more consistent, being less sensitive to minor variations in the temperature of denaturation and the duration of staining. Compared to conventional techniques, both methods possess the important advantage of employing mitotic material, hence eliminating the necessity of procuring meiotic material of the plants to be examined.
C. G. Vosa (Oxford) and D. J. Mogford (Grahamstown)
Return to the MNL 54 On-Line Index
Return to the Maize Newsletter Index
Return to the Maize Genome Database Page