Strategies for the Production of Commercially Important Secondary Metabolites in Cell Cultures of Fagonia Indica

Abstract

Fagonia indica, commonly known as “true herb”, is famous for its variety of medicinal activities especially anti-cancer potential. It is a member of the genus Fagonia and family Zygophyllaceae. The species of the genus Fagonia, especially, F. indica is famous for its diverse class of medicinally important compounds including terpenoids, flavonoids, and other polyphenolic compounds. The medicinal activities of F. indica especially antioxidant and anticancer activities may be attributed to its phenolic compounds. However, isolation only from wild grown F. indica does not guarantee sustainable production of these metabolites. This is because of limitations with wildgrown plants such as over-harvesting, endangerment, seasonal and geographic dependence and variations in metabolic profiles of the plant. In vitro cultures promise to deal with these limitations as they are independent of seasons and geography. Especially, cell cultures promise sustainable, uniform and homogeneous production of secondary metabolites. The present work was aimed to devise various strategies for the enhancement of phenolic compounds through the establishment of feasible cell cultures of F. indica. The following experiments were performed for setting up in vitro cultures of F. indica. The first set of experiments was set to handle the microbial contamination we observed during seed germination in vitro. The microbial growth was affecting normal plantlet growth in vitro conditions. Once the type and endophytic nature of the contaminants were confirmed through different tests, they were characterized as bacteria on a molecular level through polymerase chain reaction and sequencing techniques. Eight different bacterial strains; most of them belonging to the Bacillus genus were isolated and characterized from in vitro grown plantlets of F. indica. Nine different antibiotics were applied to eliminate this bacterial growth during seed germination and plantlet development. Tobramycin showed the lowest minimum inhibitory concentration (<8 mg/L) and the highest efficacy in terms of plantlet development. In the subsequent experiments, callus cultures were established for applications of further strategies to enhance secondary metabolite production. To obtain the ideal cell line, two different explant types (stem and leaf) from a 50 days old F. indica plantlet were used. Callus induction was observed in response to different plant growth regulators (PGRs). Among the explants and PGRs tested, the highest fresh biomassproduced was for stem explants in response to 1.0 mg/L of Thidiazuron (TDZ) after 6 weeks of culture. The highest total phenolic content (TPC) and total flavonoid content (TFC) were recorded in stem derived callus cultures compared to control cultures (lacking TDZ). The highest antioxidant potential (69.45 %) was recorded in stemderived callus treated with 1.0 mg/L of TDZ. Similarly, important polyphenolic compounds like gallic acid, quercetin, apigenin, catechin were detected in elevated levels and were quantified through high-performance liquid chromatography (HPLC) in the optimized callus lines. In further experiments, callus cultures were elicited through manipulation in the carbohydrate source. Carbohydrate source provided in the medium has a significant effect on the growth and metabolism of cell cultures. Different carbohydrate sources (sucrose, glucose, fructose, and maltose) were used in different concentrations (1, 3 and 5 %) in the medium for their effects on biomass accumulation, TPC, antioxidant activity and photosynthetic activity of callus cultures. In accordance with the previous study, the optimum level of sucrose was the highest driver of biomass accumulation followed by maltose which means that disaccharides were feasible for biomass formation. However, glucose proved to be the key elicitor of TPC [1.677 mg gallic acid equivalent (GAE)/g DW] and antioxidant activity (82.11%) of callus cultures. Like glucose, the other monosaccharide (fructose) also significantly elicited the total phenolic compound (1.625 mg GAE/g DW) in comparison to sucrose. Later, these callus cultures were used to produce adventitious roots. Different elicitation strategies such as transient and prolonged elicitation were used with adventitious roots grown on solid as well as a liquid medium as suspension cultures. Interestingly, explants treated with 0.5 mg/L Methyl Jasmonate (Me-J) for 2 hours and incubated on Murashige and Skoog (MS) medium supplemented with 1.0 mg/L α-naphthalene acetic acid (NAA) produced the highest adventitious root induction frequency (88%) and biomass accumulation (6.9 g/L DW). The growth kinetics of roots in suspension cultures showed that maximum biomass was produced on the 27th day in log phase of culture supplemented with 1.0 mg/L NAA. The secondary metabolites were produced in significantly enhanced quantities compared to our previous experiments on TDZ and carbohydrate-induced elicitation. The highest TPC (6.0 mg GAE/g of DW) and TFC [5.0 mg Quercetin Equivalent (QE)/g of DW] were produced by 0.5 mg/L Me-J in roots from the lag phase of growth. This finding was further justified by the quantification of important phenolic compounds through HPLC. In the final set of experiments, the interplay between light, PGRs, and elicitors during cell cultures of F. indica was evaluated. Different PGRs (auxins and cytokinin) and elicitors were applied to cell cultures on solid medium grown in three light regimes (continuous light; photoperiod of 16 hours light/8 hours dark; continuous darkness). The results revealed that 1.0 mg/L benzyl aminopurine (BA), 0.5 mg/L Me-J and 1.0 mg/L Phenylacetic acid (PAA) were the optimum regulators in terms of biomass, production of phenolic compounds and antioxidant activity. Similarly, the continuous light produced the highest biomass (13.2 g/L DW) in cell cultures provided with 1.0 g/L BA. Me-J-treated dark-grown cultures responded with the highest TPC and TFC in both solid medium and cell suspension cultures. Furthermore, the TPC and TFC in cell suspension cultures were higher (6.8 mg GAE/g of DW and 5.2 mg QE/g of DW, respectively) compared to our previous findings. The antioxidant activity was found dependent on TPC and TFC and thus higher activity (88%) was produced in cell suspension cultures grown in Me-J. Conclusively, different elicitation strategies in the form of PGRs, carbohydrate sources, elicitors and different light regimens proved worthy of the enhancement of phenolic compounds in cell cultures of F. indica.