To develop an effective propagation method, symbiotic cultures were attempted in six threatened species of Japanese terrestrial orchids. Among the 30 fungus strains tested, R. repens was a useful symbiont for Ponerorchis graminifolia var. suzukiana. The binucleate Rhizoctonia fungal group was suitable for the four species, Cephalanthera erecta, C. falcata, C. longibracteata, and Gymnadenia conopsea. There were numerous effective fungal strains for the above five species; Coeloglossum viride var. virescens developed a symbiotic relationship with only one of the fungal isolates tested.
Following the investigation, symbiotic seedlings of P. graminifolia var. suzukiana acclimated to ex vitro condition. With over 12 weeks of cold pre-treatment at 5° C, approximately 90 % of in vitro plantlets formed shoots and roots in the greenhouse. About two years after sowing, symbiotic seedlings P. graminifolia var. suzukiana flowered.