Application of nanoparticles as biostimulator for growth of Digitalis ferruginea subsp. ferruginea L. under in vitro conditions

Abstract

This study examined the physiological, biochemical, ultrastructural, and cytological effects of zinc (Zn), copper (Cu), zinc oxide (ZnO), copper oxide (CuO), and titanium dioxide (TiO2) nanoparticles (NPs) on Digitalis ferruginea subsp. ferruginea L. under in vitro conditions. Germination, stomatal conductance, photosynthetic rate, chlorophyll and carotenoid content, anatomical structures and cytotoxicity were analyzed. Germination percentages were not significantly affected by NP exposure (p=0.800), although minor variations were measured between treatments. ZnO NPs at 20 µg/mL increased photosynthetic rate (6.85±1.01 µmol m⁻ 2 s⁻ 1 ), while CuO NPs at 5 µg/mL induced strong inhibitory effects (0.79±0.45 µmol m⁻ 2 s⁻ 1 ). CuO NPs at 7.5 µg/mL significantly reduced chlorophyll a and b contents (p<0.001), and CuO at 2.5 µg/mL caused a decrease in stomatal conductance (p=0.009). TEM analysis showed that lower NP concentrations preserved cell wall and chloroplast integrity, while higher doses caused thylakoid disorganization, mitochondrial swelling, and vacuolization. Cytological observations presented that CuO, TiO2, and high ZnO concentrations triggered chromosomal abnormalities, such as anaphase bridges, spindle disturbances, and chromosome fragmentation. Root viability assays confirmed the membrane damage in CuO 2.5 µg/mL (p<0.001), while ZnO 20 µg/mL retained integrity. In conclusion, NP effects on Digitalis ferruginea subsp. ferruginea L. were dose- and type-dependent, with ZnO NPs exhibiting biostimulant properties at optimal concentrations and CuO NPs demonstrating cyto- and genotoxic effects. These findings point out the dual role of NPs as growth promoters and stress inducers, emphasizing the importance of NP exposure in plant biotechnological applications.