Elicitation of Ocimum basilicum L. var. thyrsiflorum In Vitro Culture for Production of Novel Metabolites

Abstract

Ocimum basilicum L. var. thyrsiflorum is considered as the most important plant in the Lamiaceae family due to its significant medicinal and commercial value. This plant is also known as Thai basil and its parts are well known for their culinary, industrial and medicinal applications. It is common in sub-tropical and tropical zones of America (South and Central), Africa and Asia. The plant is compact, glabrous, erect and 1.5-2 feet high with ovate leaves, green stems and white purple flowers. Thai basil is blessed with different types of bioactive compounds with various functions, including phenols, proteins, flavonoids, rosmarinic acid, chicoric acid, caffeic acid, anthocyanins and essential oil components. Significance of several family members to the essential oil and food sectors has been commercially studied over 75 years. Essential oils extracted from this family have been used to cure various disorders like asthma, intestinal disorder, bronchitis, cancer and heart strokes. This research attempted to increase the accumulation of these biologically active compounds (flavonoid and phenolic) in in vitro cultures of Ocimum basilicum by using different treatments. In the first experiment, Ocimum basilicum callus cultures were established under various treatments and examined for biomass and phenolics production and their antioxidant capacity. For callus cultures establishment, leaf explants were inoculated on Murashige and Skoog (MS) media fortified with different concentrations (0.25-10.0 mg/l) of gibberellic acid (GA3) and 6-benzylaminopurine (BAP) alone or along with α naphthalene acetic acid (NAA). Among all tested plant growth regulators, combination of BAP+NAA (5 mg/L+1 mg/L) yields the maximum biomass accumulation (fresh weight (FW): 190 g/L and dry weight (DW): 13.05 g/L) as well as enhanced phenolic (346.08 mg/L), chicoric acid (35.77 mg/g DW) and rosmarinic acid (7.35 mg/g DW) production. Antioxidant activities revealed maximum in vitro antioxidant activity DPPH (93.2%) and ABTS (1322 µM TEAC) for the extracts from callus cultures grown in optimal conditions as compared to commercial leaves. xii In the second experiment, the synergistic effect of salicylic acid and different light regimes was investigated on antioxidative metabolites production in callus culture of Ocimum basilicum. Optimized callus at BAP + NAA (5 mg/L+1 mg/L) was inoculated with different concentration of salicylic acid (5, 10, 25, 50, 100 µm) and grown under 3 light regimes i.e. photoperiod (16/8 hrs. light/dark), constant white light 24 hrs and dark 24 hrs. Results showed that continuous light with moderate salicylic acid concentration (10 µM) significantly increased total phenolic content (18.79 mg/g DW), flavonoid content (7.22 mg/g DW), eugenol (0.56 mg/g DW), rosmarinic acid (54.35 mg/g DW), chicoric acid (64.45 mg/g DW) cyanidin (0.42 mg/g DW) and peonidin (0.32 mg/g DW) while salicylic acid treatment (25 µM) under photoperiod showed maximum biomass (FW: 226.6 g/L and DW: 16.44 g/L) and caffeic acid production (0.53 mg/g DW). Moreover in vitro antioxidant potential (DPPH and ABTS) and cellular antioxidant capacity was also found higher in continuous light treated callus along with 10 µM SA treatment as compared to photoperiod and dark. In the third experiment, comparative potential of copper oxide (CuO) and manganese oxide (MnO) nanoparticles to elicit the biosynthesis of bioactive metabolites and antioxidative capacity of Ocimum basilicum callus culture was investigated. Optimized callus at BAP + NAA (5 mg/L+1 mg/L) was inoculated with different concentration of CuO and MnO NPs (1, 5, 10, 25, 50, 100 mg/L). Results revealed that callus grown on MS media added with 10 mg/L CuO-NPs resulted in the highest biomass accumulation (FW: 172.8 g/L, DW: 16.7 g/L) TPC: 27.5 mg/g DW and TFC: 9.1 mg/g DW along with antioxidant activities (DPPH: 94%, ABTS: 881 μM TEAC, FRAP: 386μM TEAC) as compared to MnO-NPs and control. Peroxidase (POD: 0.48 nM/min/mg FW) and superoxide dismutase (SOD: 1.28 nM/min/mg FW) activities were also recorded maximum in CuO-NPs elicited cultures. Moreover, the HPLC results showed that rosmarinic acid (11.4 mg/g DW), chicoric acid (16.6 mg/g DW), eugenol (0.21 mg/g DW) was found optimum in cultures at 10 mg/L CuO-NPs.