Asymbiotic seed germination of hand-pollinated terrestrial orchids

Orchids are known for their adaptation to different environmental and biotic factors, especially pollinators and mycorrhizal fungi. Association with symbiotic fungi has a crucial impact on orchid growth and metabolism, from germination through the seedling stage and in many cases throughout their life. Negative abiotic and biotic impacts on ecosystems are one of the reasons of the decreasing number of orchids in their habitats. In vitro germination is often the best way to propagate beautiful hybrids and endangered species, but asymbiotic seed germination is possible if medium offers nutrients that in the natural habitat would be guaranteed by fungi. In this work, a protocol for ex situ conservation of nine threatened terrestrial orchid entities has been developed. After 20-45 days (depending on species) from hand pollination, seeds were collected, sterilized and then sowed on Petri dishes in Malmgren modified medium and cultivated at 21±2°C in the dark. The percentage of contamination and the percentage of germination were evaluated for every species. The absence of ammonium phosphate monobasic resulted in a good percentage of germination of species typical of dry-grassland and growing on poor soils (i.e., Gymnadenia conopsea and Neotinea maculata). On the other hand, Serapias neglecta, Barlia robertiana and other species typical of cultivated fields and roadsides, rich of ammonium, showed a more stunted and longer time of germination. Our results showed the effectiveness of this protocol on dry-grassland species.