Optimization of Giemsa staining protocol for metaphase chromosome preparation using leaf bud meristem of Garcinia indica(Thouars) Choisyfor karyotype analysis

Garcinia indica is polygamodaceous tree. Being woody plant chromosome count is often limited by their small and huge chromosome number. In any plant obtaining well spread and less distorted chromosome preparations by conventional squash spread method is intricate due to presence of cell wall. Present study described in detailed method of enzyme digestion mediated protoplast dropping for preparing metaphasechromosomes from plant leaf tissues. Pretreatment of samples with 2 mM 8- hydroxyquinoline solution was seen to be best to accumulate maximum (Mitotic index: 71.4%) number of metaphases. The best chromosomal morphology and spreading were obtained after methanol: Glacial acetic acid (3:1) fixative treatment with clear cytoplasmic background. Among the various concentrations of cellulase / pectinase used in the study, maximum number of protoplast were released after 3 hours treatment with 5 % cellulase / pectinase mixture. Chromosomes staining with giemsa stain for 10 min was observed to be sufficient to stained the chromosomes more intensely with cleared morphological details. The methodology described here offers a reliable method for chromosome preparation and is suitable for routine karyotype analysis of large sample sizes.