Comparative transcriptomics reveals differences mainly in magnitude, not pattern, of orchid protocorms responses to field and in vitro growth conditions in Dactylorhiza majalis (Orchidaceae)

In vitro techniques have become key to studying orchid symbiosis, particularly mycoheterotrophic germination. Physiological responses at this early stage have traditionally been assessed by comparing symbiotic and asymbiotic protocorms grown in vitro, though this simplified model does not fully capture environmental complexity. Here, we compared, for the first time, the transcriptomic (RNA-seq) profiles of in vitro-grown symbiotic and asymbiotic protocorms of the autotrophic orchid Dactylorhiza majalis with those grown in the field, enabling analysis of responses to both symbiosis and environmental factors. We showed that environmental conditions modulate protocorm physiology to some extent, subtly influencing the transcriptomic landscape but without undermining the general validity of the in vitro model. Field response (FS/IVA) showed stronger upregulation in nutrient and ion transporters (sugars, amino acids, phosphorus) than in vitro symbiotic response (IVS/IVA). Moreover, they both appear to maintain compatibility with mycorrhizal fungi and cope with environmental stressors, but through distinct regulatory strategies shaped by environmental context. FS prioritized transcriptional regulation and immune repression (via TIFY/JAZ/NINJA/ERF), whereas IVS prioritizes oxidative buffering (OPDA-skewed oxylipins + antioxidant metabolism) to cope with culture/light-driven stress. The presented research provides a more comprehensive picture of physiology of mycoheterotrophic germination of orchids seeds. Overall design: The seeds were disinfected in syringes according to the method by ?(Ponert et al., 2012)?. Subsequently they were incubated for five minutes in 70% ethanol, washed with deionised water, incubated for five minutes in a Ca(OCl)2 solution (10 g of chlorinated lime in 50 mL of deionised water), washed with sterile deionised water, and then injected on the surface of a solid medium as a suspension in 9 cm plastic Petri dishes. For symbiotic experiments, we have used simple medium OMA as described in ?(Figura et al., 2021)?. This medium contained 1 % agar (w/v, Sigma-Aldrich) and 0,3% oatmeal powder (Himedia). For asymbiotic germination, the sugar-amended complex ¼-2 medium was used ?(Figura et al., 2018)?. In this case, the pH was adjusted to 5.8 using KOH and HCl. Media of both types were autoclaved at 144 kPa, 121 °C (Tuttnauer 2540 EK-N) for 20 min and poured into 9 cm plastic Petri dishes.