From Seed to Flowering Tuberous Orchid Using Ex Vitro Symbiotic Seed Germination: A Breakthrough Study With Anacamptis Sancta

Abstract

The most effective way to both protect terrestrial orchids and also to meet commercial expectations is via reproduction of orchids and then successful reintroduction of the seedlings. To this end, Anacamptis sancta, which had faced heavy depletion in its natural distribution areas, was reproduced from seed and reintroduced into the natural environment in this study. Molecular identification of the fungus isolated from the roots of A. sancta was performed and it was established that it belongs to Ceratobasidiaceae (ON939729). In order to determine the most compatible fungus for seed germination and seedling development, fungal isolate from roots of A. sancta, as well as Tulasnella calospora (MK250656) from Serapias vomeracea, Tulasnella helicospora (MT612363) from Anacamptis laxiflora were inoculated with A. sancta seeds in sterile soil mixture under ex vitro conditions. Fifteen days after the fungal isolate inoculation, the seeds both in the control group, and the seeds inoculated with T. calospora and T. helicospora were swollen, the seed coat was ruptured (S1), but growth did not continue. Seeds inoculated with Ceratobasidiaceae isolate obtained from A. sancta roots were incubated for 30 days. Of these seeds, 90.46% germinated and 16.98% developed into leafy and rooted seedlings (S5). Twenty one seedlings (stage 4-5) obtained with A. sancta isolate (Ceratobasidiaceae) were transferred to the natural environment and all developed a first true tuber. These first tubers were left in the soil to determine the flowering age and it was determined that flowering occurred 30 months after seed germination. Thus, the life cycle of a tuberous orchid from seed to flowering tuberous mature form under natural conditions has been fully achieved for the first time. This result demonstrates that Ceratobasidiaceae isolate is fully compatible with Anacamptis sancta. Additionally, in the presence of a compatible fungal partner, ex vitro symbiotic seed germination can be recommended as an effective method of protection of endangered temperate orchids, and their reintroduction into the wild.