Controlling the biosynthesis rate and properties of green-synthesized silver nanoparticles via in vitro calcium chloride treatments of Salvia spinosa L

Green biosynthesis of silver nanoparticles (AgNPs) using plant extracts offers a sustainable approach; however, achieving control over size, morphology, and stability remains challenging. This study introduces a pre-synthesis bio-engineering strategy using in vitro-grown Salvia spinosa L. to tailor AgNPs properties via calcium chloride (CaCl₂) modulation in the growth medium. Three CaCl₂ treatments were assessed: 220 mg/L (Treatment 1), 440 mg/L (Treatment 2, standard MS), and 880 mg/L (Treatment 3). Biosynthesis was monitored via Ultraviolet-Visible (UV-Vis) spectroscopy, X-ray diffraction (XRD), Field Emission Scanning Electron Microscopy (FESEM), Fourier Transform Infrared (FTIR) spectroscopy, and Dynamic Light Scattering (DLS). Antibacterial activity was evaluated against E. coli , B. subtilis , and B. vallismortis . UV-Vis spectroscopy confirmed the Surface Plasmon Resonance (SPR) band at ~ 450 nm for Treatments 1 and 2, with no statistically significant difference between their biosynthesis rates ( p > 0.05), both being significantly higher than Treatment 3. XRD revealed face-centered cubic (FCC) metallic silver crystals (34.9 nm for Treatment 1; 23.5 nm for Treatment 2), while Treatment 3 yielded AgCl predominantly, with no SPR peak. FESEM showed Treatment 1 produced spherical, triangular, and oval morphologies, whereas Treatment 2 yielded uniform spheres. FTIR and DLS analyses suggested the presence of a phytochemical capping layer and reversible aggregation in Treatment 2, which may be favorable for sustained Ag⁺ release. ANOVA detected no statistically significant difference in antibacterial efficacy between Treatments 1 and 2 ( p > 0.05), both being superior to Treatment 3. Modulating CaCl₂ provides a tunable platform for partially engineering AgNPs with pre-defined morphologies and sizes. This green strategy enables the production of AgNPs populations with differing properties that could be further explored for biomedical applications, opening avenues for plant-derived nanomedicines.