Plant regeneration from protocorm-derived callus of Cypripedium formosanum

Totipotent callus of Cypripedium formosanum, an endangered slipper orchid species, was induced from seed-derived protocorm segments on a quarter-strength Murashige and Skoog medium containing 4.52 muM 2,4-dichlorophenoxyacetic acid and 4.54 muM 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (thidiazuron). This callus proliferated well and was maintained by subculturing on the same medium. On average, 13 protocorm-like bodies could be obtained from a piece of 4 mm callus after being transferred to the medium with 4.44 muM N-6-benzyladenine after 8 wk of culture. The regenerated protocorm-like bodies formed shoots and roots on medium containing 1 g l(-1) activated charcoal and 20 g l(-1) potato homogenate. After 24 wk of culture on this medium, well-developed plantlets ready for potting were established.