Elicitation of industrially promising vanillin type aromatic compound 2-hydroxy 4-methoxy benzaldehyde (MBAlD) yield in the in-vitro raised medicinal crop Hemidesmus indicus (L) R. Br. by methyl jasmonate and salicylic acid

Abstract

Growth promoting and secondary metabolite enhancing roles of the elicitors, methyl jasmonate (MeJA) and salicylic acid (SA) were investigated in the popular medicinal crop Hemidesmus indicus (L) R. Br. or Indian Sarsaparilla grown in-vitro. In-planta elicitor treatment (MeJA; 25, 50, and 75 μM and SA; 0.75, 1.5, and 2 mg/mL) induced growth of nodular meristemoids, promoted overall growth and demonstrated enhanced bioactivity and accumulation of root specific alkaloid 2-hydroxy 4-methoxy benzaldehyde (MBAld). A fast and effective micropropagation protocol including both direct and indirect organogenesis and regeneration was standardized for H. indicus (HI-KOL). Murashige and Skoog (MS) media fortified with 6-benzylaminopurine (BAP) (1.5 mg/L) produced the highest (6.7) number of shoots/ explant and average shoot length was 11.7 cm. It was followed by thiodiazuron (TDZ) (1.5 mg/L) (avg. shoot/explant 6.5 and avg. shoot length 11.1 cm). The percentage of emergence of shoot initials from the light green-compact base callus was highest (100%) in MS media supplemented with TDZ (2 mg/L) followed by indole-3-butyric acid (IBA) or indole-3-acetic acid (IAA) (0.50 mg/L). Highest average callus diameter (3.7 cm) was noted in MS media fortified with TDZ (2.0 mg/L) + IBA (0.50 mg/L). For root initiation, IBA (1.5 mg/L) fortified MS medium demonstrated the best result (average root length 9.1 cm). Best responsive in-vitro raised plant materials also demonstrated significant total phenolics, flavonoid, tannin, and photosynthetic pigment contents when compared with other samples and in-vitro grown plantlets. Competitive antioxidant activity 1,1-diphenyl2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays, activity of antioxidant enzymes [superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT)], and elicitor stimulated pancreatic lipase inhibitory activities were also noted. In addition, a rapid, validated, and sensitive densitometric protocol for high performance thin layer chromatography (HPTLC) quantification of MBAld was also standardized and it was noted that both MeJA and SA stimulated accumulation of MBAld in in-vitro samples but the effect was more prominent in MeJA treated plant derived root extracts with 75 μM MeJA treatment produced 3.41 ± 0.8 mg/g MBAld which was higher than the mother plant (MP) (3.30 ± 0.2 mg/g). HPTLC analyses also revealed phytochemical fidelity among the MPs and the treated and non-treated micropropagated plant samples.