Micropropagation of Groenlandia densa (L.) Fourr.: an aquatic plant critically endangered in Poland

The present study provides the first comprehensive protocol for the in vitro propagation of Opposite-leaved Pondweed ( Groenlandia densa (L.) Fourr.), a critically endangered aquatic macrophyte in Poland that requires active conservation measures. Submerged shoots of G. densa were surface-sterilised by immersion in 70% (v/v) ethanol for 5 s, followed by treatment with 0.5%, 1%, or 2% NaClO for 5 min, and then cultured on Murashige and Skoog (MS) medium supplemented with 0.5 mg⋅dm⁻³ 6-BAP. At the multiplication stage, explants were cultured for six weeks on MS media containing BAP or mT at concentrations ranging from 0.5 to 2.5 mg·dm⁻³. In a subsequent experiment, the combined effects of mT (0.5–2.5 mg·dm⁻³) and IBA (1 mg·dm⁻³) were evaluated. For rooting, shoots previously proliferated on MS medium with 1.5 mT and 1 mg∙dm − 3 IBA were transferred to MS or half-strength MS media supplemented with IAA, IBA, NAA at 1 mg·dm⁻³. Morphological traits, including shoot length, fresh biomass, root development, and multiplication coefficient, were evaluated. Among cytokinins, 1 mg·dm⁻³ mT promoted the greatest biomass accumulation and shoot elongation, outperforming BAP. The combination of 1.5 mg·dm⁻³ mT with 1 mg·dm⁻³ IBA significantly enhanced proliferation, root formation, and overall plant quality, yielding the highest multiplication coefficient (10.1). At the rooting stage, auxins combined with reduced mineral salts (½ MS) markedly improved rhizogenesis and ex vitro survival, with 1 mg·dm⁻³ IBA + ½ MS ensuring 100% acclimatization success. The results demonstrate that a sequential application of optimized cytokinin–auxin treatments, followed by rooting under reduced mineral salt conditions, provides a reliable micropropagation protocol for G. densa . This approach supports both high multiplication efficiency and successful acclimatization, offering an effective strategy for conservation and restoration of endangered aquatic plants.