Effect of biofabricated zinc oxide nanoparticles on callus and in vitro regenerated shoots of <i>R</i> <i>eseda lutea</i> , and assessment of biochemical responses, polyphenolic content, and genetic stability

The conservation of rare and endangered plant species has progressed with the advent of nanotechnology, enabling their large-scale production with desirable traits. The present study was focused on the synthesis of zinc oxide nanoparticles (ZnO-NPs) using the aqueous extract of <i>Convolvulus arvensis</i> and their characterization using various techniques (UV spectra, FTIR, transmission electron microscopy (TEM), and zeta potential), and further, their impact was assessed on callus and <i>in vitro</i> raised shoots of <i>R</i><i>eseda</i> <i>lutea</i>. Low concentrations of ZnO-NPs (15 and 30 mg/L) increased the fresh weight of shoots by 35.38% and 17.43%, respectively. In contrast, a high concentration of ZnO-NPs (60 mg/L) in MS medium resulted in a 29% decrease in shoot biomass. The different concentrations of ZnO-NPs (15, 30, and 60 mg/L) increased the callus biomass by 70.7%, 62.6%, and 24.8%, respectively, compared to the control. The total phenolic content (TPC) and flavonoid content (TFC) in both regenerated stages were varied, and they were increased in callus by 15.5% with 60 mg/L of ZnO-NPs, whereas TPC and TFC were reduced in shoot, and a greater reduction was observed in TFC with the same concentration of ZnO-NP treatment than the control. The biochemical analysis performed on callus and shoot revealed a dose-dependent accumulation of proline and TBARS content. The accumulation of total soluble protein improved in both regeneration stages, and its content varied with different treatment doses of ZnO-NPs. A close relationship was observed in protein accumulation by 26.24%, and chlorophyll contents by 36.4% in shoots with 15 mg/L ZnO-NPs than the control, while both parameters decreased with 60 mg/L ZnO-NPs. The activities of antioxidant enzymes, including GR, SOD, and APX, varied under different treatment doses of ZnO-NPs. The flow cytometry (FCM) results of callus and shoot with ZnO-NPs treatment confirmed the genetic stability by genome size (2C DNA content). The results of this study show that biogenic ZnO-NPs positively influence various attributes of the callus and shoot stages and may support the mass production of <i>R. lutea</i> with abiotic stress tolerance.