In vitro chromosome doubling and tetraploid identification in Lagerstroemia indica

We developed a sterile culture system suitable for in vitro chromosome doubling using cluster buds or shoot tips of Lagerstroemia indica 'Bicolor'. Cluster buds and shoot tips were treated with various concentrations of colchicine solution (0.2%, 0.5%, 0.8%) and incubated for 6, 12 or 24 h on an orbital shaker at 100 rpm. They were then cultured on half Murashige and Skoog (MS) medium supplemented with 1.0 mg l(-1) 6-benzyladenine (BA) and 0.1 mg l(-1) indole-3-butyric acid (IBA) for the induction of cluster buds, and subsequently transferred to half MS medium supplemented with 0.05 mg l(-1) 1-naphthaleneacetic acid (NAA) for rooting. The highest frequency (36%) of mutated plants was obtained by immersing the shoot tips in 0.5% colchicine for 12 h. The ploidy level of putative tetraploid plants was verified by flow cytometry and chromosome counts. Tetraploid plantlets showed darker and rougher green leaves, larger stomata guard cells and lower density of stomata.