In vitro germination,protocorm formation and plantlet development of mature versus immature seeds from several <Emphasis Type="Italic">Ophrys</Emphasis> species (Orchidaceae)

We investigated the effect of genotype, seed maturity and culture medium on the in vitro germination and development of protocorms and plantlets from seeds of 13 different Ophrys species (O. apifera, O. attica, O. cornuta, O. delfinensis, O. ferrum-equinum, O. lutea, O. mammosa, O. speculum, O. spruneri, O. umbilicata, O. argolica, O. irricolor and O. tenthredinifera) collected in Greece, some of which are endemic to this country. Mature seeds (10 months after collection) and immature seeds (2 months after anthesis) were cultured in a coconut milk-enriched or a pineapple-enriched medium (CEM or PEM, respectively). The highest percentage of callogenesis (96%) was observed in immature seeds of O. delphinensis in the CEM, while the highest percentage of protocorm formation (52%) was observed in mature seeds of O. spuneri in the CEM. Protocorm formation was significantly lower in immature seeds than in mature seeds in both culture media. Eventually almost all of the transferred protocorms developed to plantlets, which later formed minitubers. PEM appeared to be the most suitable for the development of minitubers from plantlets. All of the factors investigated—as well as their interactions—significantly affected callogenesis and protocorm formation. The results are discussed with the perspective of applying an improved protocol for in vitro seed germination and plantlet formation in several under-utilized Ophrys species.Abbreviations CEM Coconut milk-enriched medium - CLT Callus-like tissue - PEM Pineapple-enriched medium - PPFD Photosynthetic photon flux density