Secondary metabolites are gaining an increasing importance in various industries, such as pharmaceuticals, dyes, and food, as is the need for reliable and efficient methods of procuring these compounds. To develop sustainable and cost-effective approaches, a comprehensive understanding of the biosynthetic pathways and the factors influencing secondary metabolite production is essential. These compounds are a unique type of natural product which recognizes the oxidative damage caused by stresses, thereby activating the defence mechanism in plants. Various methods have been developed to enhance the production of secondary metabolites in plants. The elicitor-induced in vitro culture technique is considered an efficient tool for studying and improving the production of secondary metabolites in plants. In the present review, we have documented various biosynthetic pathways and the role of secondary metabolites under diverse environmental stresses. Furthermore, a practical strategy for obtaining consistent and abundant secondary metabolite production via various elicitation agents used in culturing techniques is also mentioned. By elucidating the intricate interplay of regulatory factors, this review paves the way for future advancements in sustainable and efficient production methods for high-value secondary metabolites.

Medicinal plants are valuable sources of botanical drugs, extracts and pure compounds. Although several species can be propagated or collected, the access to herbal material is limited in certain cases. The protection of natural habitats and biodiversity demands new sources to provide plant secondary metabolites of medical importance. Adventitious root culture is used to harvest the secondary metabolites from the medicinally important plants, thereby offering an alternative to collection and propagation of medicinal plants. In this review, we comprehensively summarize the previously published data on the use of adventitious root cultures for numerous therapeutic plants. Adventitious roots showed elevated growth rates and production of pharmaceutically important metabolites under sterilized condition with optimized plant-growth regulators in culture media. In the present study, major influencing factors, such as the stages involved in the process of adventitious root formation, medium composition and type of growth regulators, specifically the effect of different auxins on the initiation and formation of roots, are discussed. Elicitation strategies using biotic (yeast extracts, chitosan and pectin) and abiotic factors (MJ, SA, CuSO₄, AgNO₃, NaCl) that affect the in vitro growth of adventitious roots and the role of bioreactors, which are new advancements in the scale-up process, are also highlighted. The development of adventitious root cultures for the production of secondary metabolites of medicinal importance is a perspective that is advantageous from ecological and economical aspects as well.

An efficient in vitro propagation protocol has been established for a valuable medicinal plant, Salix tetrasperma using mature nodal explants. The investigation aimed to observe the influence of various combinations and concentrations of cytokinins (mT, BA, and Kn) and auxins (NAA, IAA, and IBA) on regeneration potential using the Murashige and Skoog (MS) medium. Among individual cytokinin treatments, 5.0 µM mT resulted highest response of 92% with maximum shoot number (11.6 ± 0.08) per explant and shoot length (4.5 ± 0.22 cm) after 12 weeks of culture. However, synergistic treatment of mT (5.0 µM) and NAA (0.5 µM) further improved proliferation with (21.3 ± 0.40) shoots per explant and (6.9 ± 0.13 cm) shoot length in 96% cultures after 12 weeks of incubation. Rooting from in vitro raised microshoots was achieved on ½ MS medium supplemented with various concentrations of low-dose auxins. The highest number of roots (10.4 ± 0.20) per shoot with mean root length (5.7 ± 0.11 cm) with maximum rooting frequency (97%) was observed in 0.5 µM IBA, after 4 weeks of culture. The rooted plantlets achieved a remarkable 86% survivability rate, when transferred to ex vitro conditions during acclimatization. Analysis of photosynthetic parameters and their characteristics during the acclimatization phase revealed a gradual decline in photosynthetic attributes during initial weeks; however, a significant improvement was noted as the growth proceeded. SEM analysis revealed the ultra-morphological structural differences between in vivo and in vitro derived leaves of S. tetrasperma. Moreover, DPPH assay observed differential antioxidant activity of in vitro raised plantlets throughout the acclimatization period. The GC-MS analysis from leaf extracts of donor plants and in vitro derived plantlets has revealed a broad spectrum of phytochemical compounds with significant pharmacological properties. No polymorphism in the banding pattern was found when the genetic fidelity of the regenerated plants was evaluated using SCoT primers, indicating the clonal stability of micropropagated plants. This study is the first to explore the use of mT in regeneration of S. tetrasperma, showing its more effectiveness than BA and Kn. A reliable and more advanced in vitro protocol was established for Salix tetrasperma using cytokinin (mT), a novel aromatic cytokinin. This method led to significant improvement in multiple shoot formation and elongation, root development. Moreover, the assessment of transpiration rate (E), intercellular CO2 concentrations (Ci), net photosynthetic rate (PN), stomatal conductance (gs), chlorophyll (Chl a/b), carotenoid content, phytochemical profile and total antioxidant activity during acclimatization ensures enhanced survivability of in vitro raised plants. Furthermore, the application of mT did not compromise the genetic fidelity of the regenerated plants.

The coral tree ( Erythrina variegata ) is a multipurpose horticultural plant with a plethora of medicinally important alkaloids. Regeneration via tissue culture can provide an efficient alternative to seed-grown plantlets and reduce the cost of the plant significantly. Thidiazuron (TDZ) is an efficient plant growth regulator and is effective in numerous species. However, the response to it varies with the type and position of the tissue on the plantlet treated. This study was carried out to ascertain the best tissue types for micropropagation of the coral tree using TDZ. Three tissue types (shoot tip, nodal, and hypocotyl), originating from different strata of the plantlet were evaluated. Adventitious shoots were observed in all three explants at the tested concentrations. However the quality and the shoot number varied significantly with the type of explant. Explants with a meristematic zone (shoot tip and nodal) were more responsive to the treatment compared with hypocotyl tissue lacking preexisting meristem. Nodal explants produced the maximum number of shoots (about eight) per explant after 4 weeks of culture, whereas shoot tips produced about only five shoots per explant at an equimolar concentration (1.5 µ m ). Approximately three shoots were observed in hypocotyl explants. Moreover, growth and rooting of the regenerated shoots was influenced by the origin of the explants. The molecular characterization of the regenerants using intersimple sequence repeat (ISSR) markers revealed genetic homogeneity among regenerants. An efficient micropropagation method for the coral tree is described.

Synthetic seed technology is an innovative in vitro technique that provides improved storage capabilities for vegetative propagules. Its success mostly depends on the encapsulation matrix’s composition and the encapsulation procedure. The present study focuses on optimizing an encapsulation protocol for short-term storage and germplasm exchange using micro-cuttings of Salix tetrasperma. Among the different synthetic seed types evaluated, double-layered synthetic seeds (DLSs) exhibited the highest re-growth (93.6%) on MS medium supplemented with meta-Topolin (mT) (5.0 µM) and α-naphthalene acetic acid (NAA) (0.5 µM) after 8 weeks of culture. Viability assessment of non-embryogenic synthetic seeds during low-temperature storage (4 °C) demonstrated the enhanced resilience of double-layered synthetic seeds (DLSs) compared to their single-layered (SLS) counterparts. Following acclimatization in Soilrite^®-filled cups, 82% of the plantlets were successfully established in a greenhouse after four weeks. The increased activity and concentration of antioxidants in DLS-derived plantlets suggest the potential role of the extra layer of alginate in mitigating the effects of low-temperature stress during storage. SCoT molecular analysis confirmed the genetic integrity of the synthetic seed-derived plants.