Investigation on the effect of selected nano structures on accumulation of biomass and secondary metabolites in In Vitro cultures of Rubia Cordifolia L

Abstract

Rubia cordifolia L., a medicinal plant from the Rubiaceae family, contains several secondary metabolites, such as alizarin, purpurin, rubiadin, and manjishtin, which gives significant medicinal and industrial values. In vitro propagation of this plant can be a convenient method for commercial production, preventing overexploitation of natural populations. When combined with in vitro techniques, nanotechnology offers an alternative for enhanced production of targeted secondary metabolites in crops. The study examined the effects of metal oxide nanostructures on in vitro morphogenic responses and secondary metabolite production of R. cordifolia using MS medium in an in vitro system. The first objective was to develop protocols for synthesising nanostructures and their characterisation. Biocompatible hexagonal-shaped Zinc oxide nanostructures (ZnO NSs) and spherical Copper oxide nanostructures (CuO NSs) were synthesised using stem extract of R. cordifolia. The average particle sizes were 17.9 nm for ZnO NSs and 28.35 nm for CuO NSs. The biosynthesised NSs were characterised using UV- Vis, FTIR, XRD, SEM-EDX, TEM, Raman spectroscopy, and DTG analysis. The study then aimed to develop protocols for direct organogenesis of R. cordifolia using varying concentrations and combinations of plant growth regulators. Nodal explants were selected for direct organogenesis and inoculated to the MS media containing different combinations and concentrations of Kinetin, BA, NAA, IBA and IAA. Kinetin at 0.5 mg/L was found to be the most effective plant growth regulator for the multiplication of shoots. The HPTLC analysis showed the highest percentage of Alizarin and Purpurin were present in all three parts (leaf, stem, and root) of the Methanolic extract of in vitro grown R. cordifolia. The effect of different concentrations of biosynthesised ZnO NSs and CuO NSs on R. cordifolia in vitro cultures was also studied. It was found that the morphogenic responses of the plant varied for each stress signal used. Comprehensive phytochemical analyses, including HRLC-MS and HPTLC fingerprinting, along with assessments of antioxidant properties, indicated that treating plants with various concentrations of metallic oxide NSs led to significant changes in both the quality and quantity of secondary metabolites. Notably, treatment with 0.1 mg/L of CuO NSs resulted in the highest concentrations of secondary metabolites such as Alizarin and Purpurin. The antioxidant activity and chlorophyll content of extracts from plants treated with CuO NSs were significantly enhanced due to the higher concentration of some phytochemical. This method of using NSs presents a promising in vitro approach to boost the production of plant-derived bioactive compounds, which have the potential to revolutionise the pharmaceutical and textile industries.