A new orcein technique for mitotic chromosome analysis

In our laboratory several chemicals were tested for analysing the somatic chromosome morphology of maize. The following pretreatment and staining schedule was found to yield satisfactory preparations.

The seeds were allowed to germinate on moist filter paper in petri plates. The primary and secondary roots were collected when they were about 1.0 - 2.0 cm in length. Both types of roots gave equally good preparations by slightly varying the maceration timings. The optimum time of collection was between 12:00 PM. and 1:00 PM. in summer months. The root tips were pretreated with an aqueous solution (0.2 percent) of calcium chloride for 2 hrs. at 10-12 C. This included one minute chilling time in the freezer chest. The concentration of CaCl2 solution and the duration of pretreatment were very critical. After pretreatment, the thoroughly washed root tips were fixed in 1:3 acetic ethanol for overnight and preserved in 70 percent ethanol. They were macerated in a 5 percent aqueous solution of pectinase for 2 hrs. at 37 C and then cleared in 45 percent acetic acid for 10 minutes. They were stained in 2 percent aceto-orcein overnight and squashed gently. The resultant preparation was well scattered with proper condensation of chromosomes, followed by spindle dissolution, centromere exaggeration and metaphase arrest.

Calcium chloride exists in the form of Ca and Cl- ions in solution. The treatment with an aqueous solution of CaCl2 causes a change in the ionic environment and upsets the ionic balance, resulting in viscosity change and spindle dissolution. CaCl2 is a well known dehydrating agent. It is possible that DNA-histone binding is enhanced by differential (unilateral) dehydration, resulting in chromosome condensation and clarification of chromosome morphology (This work was carried out under the supervision of Prof. A. K. Sharma, at the Department of Botany, University of Calcutta, Calcutta, India).

J. S. P. Sarma

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors.

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