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Polyploid breeding is an effective tool to induce variability in the existing population, specifically in the crops having very narrow genetic base such as acid lime. Hence, an experiment was conducted to induce polyploidy in acid lime through colchicine treatment. Higher frequency of triploids were obtained in 250 μM colchicine treatment (27.27%), whereas combination of 3× (21.42%), 4× (13.33%) and aneuploid (6.67%) were induced at 500 μM colchicine treatment. Measurement of stomatal density and guard cell size of colchiploids revealed that the former one has negative relation with ploidy level and a positive relation was observed between guard cell size and ploidy level. Further, area and size of nuclei also had a positive relation with ploidy level. Hence, stomatal density, guard cell and nuclei size can be used effectively as morphological marker for preliminary polyploid screening. From the experiment, it can be concluded that 500 μM colchicine treatment of in vitro derived shoot tip explants of acid lime is optimum for creating maximum possible variability through polyploidization in the existing acid lime population. [ABSTRACT FROM AUTHOR]