The softened root tips were again washed with 1X enzyme buffer, followed by transfer to 45% acetic acid. The fixative from the root tips was removed by washing in 1X enzyme buffer (citric acid, trisodium citrate buffer) and then macerated in enzyme solution at 37 ☌ (Mixture of 2% w/ v cellulase and 3% v/ v pectinase from Aspergilus niger) until the root tips get softened. Hexandrum from the seeds were excised and pretreated with 0.1% colchicine at 4 ☌ for 4 h for metaphase arrest and subsequently fixed in Carnoy’s fixative for 24 h and stored in 70% ethanol at 4 ☌ afterwards. Preparation of chromosome spread Growing root tips of P. Since chromosome identification constitutes the first step in genetic manipulation of a species, the present investigation was therefore undertaken to provide meaningful landmarks to facilitate unequivocal chromosome identification of individual chromosomes employing DNA: DNA in situ hybridization. ), Pseudotsuga (Amarsinghe and Carlson ), Lupinus (Naganowska and Zielinska ), Trifolium (Ansari et al.
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