In vitro regeneration, phytochemical profiling and antioxidant activity in Ruta chalepensis plants established from alginate encapsulated synthetic seeds

Abstract

We developed a highly efficient protocol for the short-term conservation and in vitro regeneration of Ruta chalepensis L. This involved encapsulating nodal segments (NS) in a sodium-alginate solution (3% w/v) and calcium chloride (100 mM) to produce synthetic seeds. The effect of various concentrations of plant growth regulators (PGRs) on the conversion of synthetic seeds into plantlets was examined and the best results, with a conversion rate of 96.8%, were obtained on Murashige and Skoog (MS) medium supplemented with 5.0 μM 6-benzyladenine (BA) and 0.5 μM α-naphthalene acetic acid (NAA) after 12 weeks of culture. When stored at 4 °C for different durations (2-12 weeks), encapsulated and non-encapsulated NS displayed distinct conversion responses. Encapsulated NS stored at 4 °C for 10 weeks exhibited a regeneration rate of 72.7%, while non-encapsulated NS did not survive under the same conditions. The proliferated shootlets derived from cold-stored NS were successfully rooted in agar-solidified MS medium containing 0.5 μM indole-3-butyric acid (IBA) and successfully acclimatized to ex vitro conditions. UV-Visible spectroscopy (UV), gas chromatography-mass spectrometry (GC–MS), and high-performance liquid chromatography (HPLC) were used to identify bioactive chemicals in the aqueous methanolic extracts of the acclimatized plants and compared with the donor plant. The bioactive chemicals were found considerably higher in the acclimatized plants (Phenols 95.0 mg GAE/g DW, flavonoids 99.2 mg QE/g DW, tannins 38.7 mg TAE/g DW, hesperidin 94.5 µg/mg, and quercetin 25.4 µg/mg). The antioxidant potential was also evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and demonstrated high scavenging activity at a concentration of 800 µg/mL in the acclimatized plants (83.0%). This study represents the first successful demonstration of in vitro regeneration, phytochemical profile analysis, and assessment of antioxidant activity in R. chalepensis plants established from synthetic seeds following short-term cold storage.