A Pollen Grain Squash Technique forSaccharumand Related Genera

Abstract

Anthers collected between 9 and 10 AM were treated for 1 hr at 26-28 C with a 0.5% solution of colchicine, washed for 2-4 min in water, placed in 0.002 M 8-hydroxyquinoline for 1 hr, washed in water for 10 min and fixed in: methanol, 60 ml; chloroform, 30 ml; distilled water, 20 ml; picric acid, 1 gm and mercuric chloride 1 gm, for 24 hr. After washing they were hydrolysed in 1 N HCl for 15 min at 60 C, stained in leuco basic fuchsin for 30 min, then smeared on a slide in a drop of acetocarmine. The slides were sealed, stored overnight, the paraffin was removed, and the slide passed through a 1:1 mixture of n-butyl alcohol and acetic acid, then through pure n-butyl alcohol and mounted in Canada balsam. The significant features of this procedure are: (1) use of chromosomes in the haploid condition for karyotype analysis, (2) better exaggeration of constrictions for easier interpretation of chromosome types and (3) good spreading in plants with a large chromosome number.