In vitro mycorrhization infection of orchid plantlets obtained by asymbiotic cultures: a method to improve ex vitro acclimatisation

In vitro techniques play an important role in ex situ conservation programs concerning threatened native orchids, especially if applied to propagation by seed. Unfortunately, the in vitro propagation methods applied to terrestrial orchids are bristling with difficulties, first and foremost because terrestrial orchids are very dependent on mycorrhizal relationships. The aim of this work is to test an in vitro mycorrization system using plantlets of terrestrial orchids, obtained by asymbiotic germination, and strains of fungi isolated from the same species of orchids, growing in natural habitat, Orchis morio L. e Serapias vomeracea (N.L. Burm.) Briquet. Co-cultures were carried out using both S. vomeracea seedlings with strains of Ceratobasidium sp. and Tulasnella sp. and O. morio seedlings with Rhizoctonia sp. Different times of co-cultures, three, seven and fourteen days, were tested and then the roots were processed, using Confocal laser microscope and SEM, in order to evaluate the establishment of a useful mycorrhizal symbiosis. Pelotons were often observed into the cells of cortex, both in Serapias and Orchis roots. All the plantlets were transferred on loam in pots and maintained outdoor during spring and summer seasons. In vivo plantlets survival varied in dependence by orchids species and fungal strains scheme.