Physical wounding and ethylene stimulated embryogenic stem cell proliferation and plantlet regeneration in protocorm-like bodies of Phalaenopsis orchids.

Phalaenopsis orchids have been regenerated by inducing protocorm-like bodies (PLBs) from etiolated leaf sections. However, the physiological and molecular mechanisms of secondary PLB development and subsequent proliferation have not been explored. Bisectionally cutting primary PLBs resulted in more secondary PLBs at 5 weeks, suggesting an embryogenic stem cell property imposed by wounding of primary PLB tissues. The ethylene precursors ethephon and 1-aminocyclopropanecarboxylic acid and the ethylene perception inhibitor silver nitrate increased PLB formation, while aminoethoxyvinylglycine decreased PLB formation. Ethylene content in wounded PLB explants increased over culture time in media containing ethylene precursors or inhibitors. mRNA levels of PhACS2, PhACS3, and PhACO were increased by ethephon and decreased by ethylene inhibitors. Expression of genes in the ethylene signaling pathway was enhanced following ethylene-precursor treatment and was mitigated by ethylene inhibitors during PLB proliferation. Transcription of PhETR and PhEIN3, as well as PhERS, PhCTR, and PhGTP, was significantly increased 12 h after ethylene treatment. Ethylene and physical wounding stimulated secondary PLB formation in Phalaenopsis, probably through ethylene biosynthesis and signal transduction.