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A sterile culture system suitable for in vitro chromosome doubling using cluster buds or shoot tips from in vitro L.indica Bicolor seedlings was developed.After treated with various concentrations of liquid colchicines (0.2%,0.5%, 0.8%) and incubated for 6, 12 or 24 h on an orbital shaker at 100 rpm, cluster buds and shoot tips were then cultured on half Murashige and Skoog (MS) medium supplemented with 1.0 mg 1-1 BA and 0.1 mg 1-1 IBA for the induction of cluster buds, and subsequently transferred to half MS solid medium supplemented with 0.5 mg 1-1 NAA for rooting.The highest frequencies (36%) of mutative plants were obtained after immersing the shoot tips in 0.5% colchicines for 12 h.Tetraploid plantlets developed darker and rougher green leaves, larger stomata guard cell and lower density of stomata.The ploidy level of putative tetraploid plants was further verified by flow cytometry and chromosome counts.Seven plantlets have been selected for propagation, cultivating seedling and breeding into new varieties.