In Vitro Cultures and Volatile Organic Compound Production in Chiliadenus montanus (Vhal.) Brullo

Callus and microshoot cultures were established for Chiliadenus montanus (Vhal.) Brullo. (Asteraceae), a medicinal plant known for producing volatile organic compounds (VOCs). Callus induction was achieved successfully by culturing leaf explants on full-strength Murashige and Skoog medium (MS) supplemented with 2.2 µM 2, 4-dichlorophenoxy acetic acid (2,4-D) and 6.9 µM kinetin (Kin). Successful direct shoot regeneration was achieved using nodal explants cultured onto half-strength MS media supplemented with 1.4 μM Gibberellic Acid (GA₃) and 4.4 μM 6-Benzylaminopurine (BAP). Indirect microshoots were successfully regenerated using callus cultured on MS media supplemented with 8.8 μM BAP, 2.2 μM Zeatin, and 1.4 μM GA₃ followed by culturing on MS media supplemented with 8.8 μM BAP and 0.5 μM naphthalene acetic acid (NAA). Using wild plant aerial parts, callus and microshoots samples, VOCs were extracted successfully using Headspace Solid-Phase Micro-Extraction (HS-SPME) and analyzed by gas chromatography-mass spectrometry (GC-MS). In wild plant extracts, sesquiterpene hydrocarbons were found to be predominant with the following principal components: Alloaromadendrene (11.92%), trans-Cadina-1(6),4-diene (7.54%), and α-caryophyllene (6.77%). The analysis of in vitro microshoots revealed high levels of oxygenated monoterpenes with cis-Myrtanol (16.62%), and β-Cyclocitral (14.3%) as the main components. Callus extract was dominated by monoterpene hydrocarbons and the main compounds identified were (Z)-β-Ocimene (22.27%), p-Cymene (15.13%), and α-pinene (13.78%). In conclusion, an efficient in vitro production system of VOCs in C. montanus was established that can be used in the future for boosting their production without endangering wild plants.