Biomass and terpenoids profile of callus extract of Piper betle L. var. Nigra with abiotic elicitor cobalt (II) chloride

Black betel (Piper betle L. var. Nigra) is traditional medicinal plant potentially produce secondary metabolites. Its secondary metabolite content can be promoted using callus culture with abiotic elicitor. This study was aimed to determine the effect of cobalt (II) chloride (CoCl₂) concentration towards biomass and terpenoids profile of leaf callus of Piper betle L. var. Nigra. Leaf explants of Piper betle L. var. Nigra were cultured in solid Murashige and Skoog (MS) medium supplemented with growth regulators 2,4-D (0.5 mg/L) and BAP (2.0 mg/L) to induce callus formation. Elicitation was performed after callus reached five weeks of age by performing subculture on MS medium added with CoCl₂ (0.5 mg/L; 1.0 mg/L; 2.5 mg/L). Calli of Piper betle L. var. Nigra were harvested after one, two, or three weeks before they were extracted using methanol and analyzed of its terpenoids content using Gas Chromatography-Mass Spectrometry (GC-MS) method. Results showed that calli had compact and crumb texture with green, white, brown, and white color. Callus added 1.0 mg/L CoCl₂ at two weeks age produce terpenoids at 5.95%. Callus with 1.0 mg/L CoCl₂ at three weeks of age produced higher fresh and dry weight at 916.4±162.3 mg and 93.3±11.7 mg respectively. Callus supplemented with 1.0 mg/L CoCl₂ at two weeks of age had higher terpenoids percentage compared to control. Two of the highest terpenoid types found in the callus of black betel leaf explant were 1,2-epoxy-1-vinylcyclododecene and hexadecanoic acid.