The study investigated the stimulation of callus tissue from explants (growing apex, true leaves, and hypocotyl segments) of medicinal Pimpinella anisum plant using an L2 medium supplemented with different levels of growth regulators 2,4-D, AgNO₃, and biotic elicitors (Aspergillus flavus, Mucor spp., and yeast extract) to the secondary metabolite induction. Callus formation induced from the single node explants had culture on the L2 medium. The addition of yeast extract 750 mg L^-1 to the medium was superior in the highest average fresh and dry weight of 435.79 and 0.616mg, respectively. The treatment at 300mg L^-1 of Mucor spp. recorded the highest rate of fresh and dry weight of the callus tissue. When cultured on 500 mg L^-1 of Aspergillus flavus, fresh and dry callus weights were 376.09 and 0.628mg, respectively. The different levels of biotic elicitors (Aspergillus flavus, Mucor spp., and yeast extract) stimulate the production of essential oil t-anethole from the callus tissue when added to the medium. The results also showed that the highest increase in the amount of vital oil t-anethole occurred when the medium included 500 mg L^-1 of the yeast extract. It recorded 2.969 mg L^-1 dry weight (DW). The MS medium supplied with 500 mg L^-1 Aspergillus flavus provided a maximum value in the t-anethole essential oil, reaching 3.756 mg L^-1 DW of the callus. Meanwhile, the 300 mg L^-1 of Mucor spp. recorded the utmost amount of crucial oil t-anethole at 3.945 mg L^-1 DW.

Keywords: Pimpinella anisum L., biotic elicitors, growth regulators, yeast extract, secondary metabolite, callus, in vitro

Key findings: The secondary metabolite compounds estimation ensued by quantitative and qualitative analysis using the High-Performance Liquid Chromatography (HPLC) device for extract samples separated from seedlings growing in vitro. For induction and multiplication of callus tissue, applying the seedling explants of the Pimpinella anisum plant occurred. The single-node explants had a high ability to induce callus, one of the best explants used to stimulate callus tissue, followed by hypocotyl segments, then dicotyledonous leaves. The addition of yeast extract led to a response affecting the average fresh and dry weight and the properties of the callus.