In vivo-induced polyploidy in Dendrobium ‘Sonia’ in a bubble bioreactor system using colchicine and oryzalin

Abstract

Development of polyploidy in Dendrobium ‘Sonia’ using colchicine and oryzalin is described. Five-month-old plantlets derived from protocorm-like bodies produced in vitro were used. These plantlets were immersed into a bubble bioreactor containing different concentrations of colchicine (0.05, 0.10 and 0.15% for 3 days) and oryzalin (14.40 and 28.90 µM for 4 days) together with Nonidet (P-40) as a surfactant. Plantlets were aerated to prevent hypoxia. Tetraploidy was successfully induced by 0.10 and 0.15% colchicine. Polyploidy levels were estimated using flow cytometry and then confirmed by chromosome counting, and through anatomical, cytological and morphological studies. The highest percentage (26.60%) of polyploids was induced with 0.15% colchicine. Oryzalin treatment did not yield tetraploid plantlets. The relative fluorescence intensity emitted by propidium iodide (PI), a fluorescent intercalating agent or a DNA-binding dye, was almost twofold higher in tetraploids and nearly fourfold higher in octaploid as compared to diploids. Plantlets with changed chromosome number were obtained at 0.05–0.15% colchicine concentrations (tetraploid induction) and 14.40 µM oryzalin concentration (mixoploid induction). The chromosome number was 2n = 38 in diploids and 2n = 76 in the tetraploids. The tetraploid and diploid plantlets exhibited significant differences in morphological, anatomical and cytological parameters. Stomata of tetraploid plantlets were longer than diploids. Survival rates from treatments were above 70%. In all treatments with colchicine and oryzalin, 0.10 and 0.15% of colchicine proved the most effective for generation of polyploid plantlets. This is beneficial for further breeding purposes of D. ‘Sonia.’ Oryzalin was found lethal. New ornamental varieties with improved traits can be produced by polyploidy using colchicine.