Induction of repetitive embryogenesis from seed-derived protocorms of Phalaenopsis amabilis var. formosa Shimadzu

An in vitro culture procedure was established for repetitive embryogenesis and plant regeneration from seed-derived protocorms of Phalaenopsis amabilis var. formosa Shimadzu (Orchidaceae). Seed-derived protocorms were cultured on modified half-strength Murashige and Skoog (1962) basal medium (! MS) devoid of plant growth regulators. After 45d, 28.1% of protocorms formed embryos from their posterior regions. 1-Phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ; 0.45, 4.54, and 13.62 muM) promoted direct embryo formation. The best response was at 13.62 muM TDZ, and 100% of the protocorms formed a mean number of 13.5 embryos after 45d of culture. By contrast, naphthaleneacetic acid (NAA) at 0.54 and 5.37 muM inhibited direct embryo formation. On basal medium devoid of plant growth regulators, 18.8% of primary proliferating embryos could form more embryos. TDZ (0.45, 4.54, and 13.62 muM) also promoted this process. Proliferating embryos/protocorms were transferred to basal medium devoid of plant growth regulators for plantlet formation. Plantlets were successfully obtained from the embryos after 4-6 wk. Following subculture every 6 wk for three passages, the plantlets were transferred to sphagnum moss in a container for acclimatization in the greenhouse. The survival rate was 100%.