Optimization of Callus Induction and Cell Suspension Culture for the Endemic Species Verbascum hasbenlii

Verbascum hasbenlii Aytaç & H. Duman is a narrowly distributed endemic species native to Çanakkale, Türkiye. This study aimed to establish and optimize callus induction and cell suspension culture systems for V. hasbenlii. Leaf explants obtained from 10-week-old seed-derived in vitro plants were cultured on six Murashige and Skoog (MS) media containing different combinations of α-naphthaleneacetic acid (NAA; 0.5 or 1.0 mg/L) and 6-Benzylaminopurine (BAP; 0.5, 1, 2 or 3 mg/L). After four weeks, callus induction was achieved in all treatments (96–100%), although significant differences were observed in explant browning, callus biomass, diameter, and morphology. The medium supplemented with 0.5 mg/L BAP + 0.5 mg/L NAA produced the highest callus biomass (1.245 g) and diameter (5.06 mm), while maintaining low explant browning and a compact-friable texture suitable for suspension culture establishment. Cell suspension cultures exhibited a typical growth pattern with lag, exponential, and stationary phases. On day 9, cultures showed increased growth parameters, including packed cell volume (PCV: 7.50%), fresh weight (FW: 0.0580 g), and dry weight (DW: 0.0052 g), with relatively high cell viability (80.72%). Biomass accumulation reached maximum levels between days 18–21, while cell viability decreased to 66.82%. These findings provide an optimized in vitro culture system for future studies on secondary metabolite production in V. hasbenlii.