ORIGINAL ARTICLE

The Effect of Different Nitrate Concentrations on the In Vitro Growth and Development of Temperate Orchids

Many orchid species of the temperate climate are threatened and declining without apparent reason. One of the drivers for the decline in orchid populations seems to be increased nitrate levels in soil, which affects the survival of both orchid seeds and mycorrhizal microflora, which normally improves the access of nutrients to orchid plants.

When introducing orchids into in vitro culture, inorganic nitrogen sources used in nutrient media can directly inhibit germination, complicating the selection of an appropriate medium for the conservation of rare species. Some nutrient media are completely devoid of nitrates, replacing them with various organic nitrogen sources. Previous studies have shown that nitrates can directly inhibit the germination of sensitive species in vitro, making the selection of a suitable medium challenging in conservation practice for rare orchids. However, whether the inhibitory effect of nitrates on the further development of orchid seedlings is an adaptive trait that reduces post-germination mortality and influences their resilience and tuber formation remains unclear.

The aim of the study was to determine the sensitivity to nitrates in vitro in seedlings of three species of tuberoid orchids, taking into account the characteristics of their growth under nitrate load and changes in the physiological state of seedlings using histological methods. It was found that elevated nitrate levels did not suppress orchid seedling survival, resulting in the highest rates of necrotic explants in Platanthera bifolia seedlings (44%), as well as in Dactilorhiza incarnata (35%) and D. fuchsii (27%), respectively. On the other hand, the differentiation of root tubers was influenced by an increased level of NO₃⁻ addition (50 mg L^-1), which turned out to be the most effective in stimulating the tuberization with a simultaneous decrease in growth parameters and biomass in all studied orchid species.

Longitudinal sections revealed numerous air chambers in the tuberoid aerenchyma, along with intense accumulation of starch granules in the vascular tissues. Based on the developmental characteristics of seedlings of three orchid species at elevated nitrate levels in vitro, it can be assumed that the high nitrate ion levels observed in soils of natural populations (130-840 mg Kg ^-1) may lead to a decrease in the number of germinating seedlings.

Research focusing on the effects of nitrates could help create suitable nutrient conditions that mimic natural soil environments, ensuring the survival of rare orchid species outside their natural habitats.