Calendula officinalis L. is an important medicinal and ornamental plant possessing multiple bioactive compounds. The in vitro plant regeneration method has recently replaced traditional field cultivation practices of calendula due to its fascinating phytochemical profile. In this study, callus formation and indirect organogenesis were described to establish an effective in vitro propagation strategy in C. officinalis. Using a gas chromatography-mass spectrometry (GC-MS) approach, the phytochemical content of tissues developed in vitro and field-grown was studied, and the biochemical contents were quantified and compared in various tissues. The incidence of callus formation from leaf explants was highest (94.44%) on MS medium fortified with 1.0 mg/L BAP and 1.0 mg/L NAA, which later became organogenic. On MS, 1.0 mg/L BAP and 1.0 mg/L NAA showed the highest indirect shoot proliferation (88.88%) efficiency. After being sub-cultured, the regenerated shootlets were cultured onto rooting medium containing different IAA/IBA concentrations; the best rooting percentage (94.44%) was achieved with 1.0 mg/L IBA. The biochemical parameters, like total phenolic content, flavonoid content, and DPPH scavenging activity, were measured. When compared to callus and field-grown developed leaf (donor) samples, all the biochemical characteristics of in vitro-produced leaf were noted to be higher. The methanolic extracts of leaf-callus and field-grown and in vitro-developed leaf tissues were subject to GC-MS-based phytocompound investigation. More than 45 therapeutically significant bioactive chemicals, like n-hexadecanoic acid, vitamin E, stigmasterol, and squalene were found in these samples. These results showed that the callus that is formed from in vitro leaf is a reliable and powerful source of several bioactive compounds with a wide range of medicinal uses. The successful stimulation of callus development, indirect organogenesis, biochemical analysis, and GC-MS confirmation of the presence of significant phytocompounds are all described in this study. This work provides a different avenue for ongoing and sustained synthesis of chemicals without endangering the surrounding ecosystem or native vegetation.
