Abstract
Symbiotic orchid seed germination in an in vitro system allows the growth of mycorrhizal protocorms and plantlets for scientific purposes. Orchids in nature need to establish a mycorrhizal symbiosis with fungal partners to germinate and develop into adult plants. Here we present a protocol for symbiotic germination of the terrestrial Mediterranean green meadow orchid Serapias vomeracea. The fungal symbiont Tulasnella calospora (T. calospora) (Basidiomycetes, Cantharellales) was chosen because of its common occurrence (Girlanda et al., 2011), its ability to grow in culture and compatibility in germination assays. T. calospora is one of the most common rhizoctonia-like fungi associated with terrestrial as well as epiphytic orchids.
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Representative scheme of germination assay [modified from Ercole (2014)] Figure 2. Developed protocorms after 15-30 days of dark incubation Figure 3. Developed protocorms after 45 days (30 days of darkness and 15 days of light exposure) The reproducibility of the symbiotic seed germination of S. vomeracea with T. calospora is usually successful. In some cases we noted low germination percentage due to seed viability mainly. Viability test on seeds allow to evaluating a priori the success rate. Percentage of germination can be evaluated by count of all seeds at time zero (all seeds sown) and the germinated seeds at time 15, 30 and 45 days. Usually we reported high percentage of germination (from 95% at time 15 days, to 60% at time 45 days). The decrease of the percentage of germination from time 15 days to time 45 days is mainly due to “natural selection” and/or competition between seeds (seeds with a high growth rate grow at the detriment of slower).
Notes
Note that this protocol can be applied to many different orchid and fungus species, with some modification in the sterilization time of seeds. Every orchid species has different seeds with different teguments, so the sterilization time depends on this. In some cases success of germination depends on compatibility between orchid and fungus; indeed, not all fungi (also in the Rhizoctonia-form genus) can germinate orchid seeds.
Recipes
Acknowledgments
This protocol was adapted from the following publications: Perotto et al. (2014); Ercole et al. (2013); Ercole (2014). Research on orchid mycorrhiza was partly funded by the Italian MIUR and by local funding from the University of Torino.
References
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