The chromosome spreads were prepared by conventional squashing. 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. Subsequently, high-resolution images are directly imported into the Ikaros software for digital karyotyping. ), Pseudotsuga (Amarsinghe and Carlson ), Lupinus (Naganowska and Zielinska ), Trifolium (Ansari et al. With the Metafer software and suitable imaging hardware, metaphase finding and image acquisition is automated.
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