A Root Squash Technique for Counting Somatic Chromosomes in Sugar Cane

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Abstract
Sugar cane cuttings after treatment with 0.75% Aeratan (organic mercurial fungicide) were germinated in a soil mixture at 26-28 C and transferred to an incubator at 33-34 C for 1 day before collecting roots. The excised roots were pretreated with a saturated solution of a-bromonaphthalene in 0.05% saponin for 3 hr. at 8-10 C, fixed in 1:3 acetic-alcohol for 48-72 hr. at room temperature, hydrolyzed in 1 N HC1 for 7-8 min. at 60 C, treated with 3% pectinase solution in pH 3.6 acetate buffer for 60-90 min, stained by the Feulgenprocedure, and squashed in 1% acetocarmine. Following this procedure, excellent preparations were obtained for counting chromosomes, in which the details of chromosome morphology were often clearly visible. The important precautions for the successful application of this technique include: avoidance of excessive watering ofthe germinating cuttings, avoidance of sudden temperature changes during processing of unfixed roots, ensuring correct acid temperature during hydrolysis and, finally, use of relatively fresh pectinase solution stored for not more than 2 weeks under refrigeration.