A preliminary investigation on the effect of seed physiological stage, concentration and duration of exposure to calcium hypochlorite on in vitro germinability and seedling development of Phalaenopsis amabilis orchids

For commercial propagation, orchid seeds are germinated aseptically in vitro. Successful establishment is dependent on harvesting seeds that are sufficiently mature to develop into protocorms and sanitization procedures that minimize contamination in culture. However, there are no standardized procedures for sanitizing orchid seeds and there is insufficient information about the optimum state of orchid seed development for best germination. Phalaenopsis amabilis fruits 90, 105, and 120 days after pollination (DAP), surface treated with 15% calcium hypochlorite (CH) for 30 min, were compared with seeds treated with 0, 5, 10, or 15% CH for 5, 10, or 15 min. Ninety six percent of untreated seeds from 90 DAP fruit, surface sanitized with 15% CH, produced protocorms within 40 days after sowing (DAS). Seed sanitization with 5% CH and exposure durations greater than 5 min significantly reduced protocorm percentages. Seeds treated with 10 or 15% CH showed inhibited protocorm development at all exposure times. Protocorms developed root hairs and shoot primodia by 50 DAS and an average of one leaf and root by 85 DAS after treatment with either 0 or 5% CH. Only 27 and 22% of seeds developed into protocorms but not roots or shoots following treatment with 10 or 15% CH, respectively. P. amabilis flowers were hand pollinated and fruits harvested 90, 105, and 120 DAP while still green for seed developmental analysis. Cell number per seed was estimated by counting nuclei stained with 4′-6-diamidino-2-phenylindole using confocal microscopy. Germination percentage and cell number per embryo increased from 14 to 61% and 41 to 66%, respectively, during fruit development from 90 to 120 DAP. Harvesting fruits 120 DAP while still green and using fruit sanitization before plating seeds produced the highest percentage of protocorms. Seed sanitation with greater than 5% CH dramatically reduced protocorm and seedling development.