Effects of <i>In Vitro</i> Culture Conditions on Growth and Overwintering Bud Formation in a Gentian Leaf Culture

In vitro culture is essential for propagating F1 cultivar parents and vegetatively propagated gentian cultivars. In vitro overwintering bud (OWB) formation is also crucial to maintain and propagate these plants. This study examined the effects of temperature, medium composition, and photoperiod on in vitro growth, including OWB formation, using Gentiana triflora and G. scabra as model species in leaf cultures, a practical propagation method. The growth parameters of the G. triflora line TY1-11-4-9 were assessed under different temperature and sugar composition conditions, revealing positive and negative correlations between OWB and rosette-shaped shoot (RSS) formation and between OWB and flower bud formation, respectively. Consequently, further investigations focused on these growth parameters. Temperature effects on four cultivars/lines revealed interspecific differences: at all temperatures, G. triflora had a higher rate of flower bud formation than G. scabra, whereas G. scabra showed increased OWB and RSS formation. Culturing under low temperature (15°C/15°C: regeneration stage/OWB induction stage, which was also applicable below) conditions, which may be impractical owing to slow growth effects, suppressed flower bud formation in G. triflora, but promoted RSS and OWB formation. Similar effects, albeit milder, were observed at 20°C/15°C, and these conditions were further explored as a practical method combined with other treatments to induce OWB formation. The medium sugar composition had negligible effects on in vitro growth, whereas gibberellin at 1.0 mg·L−1 in the medium composition suppressed OWB and RSS formation in G. scabra. Photoperiod variations affected several growth parameters, with a 12-h photoperiod promoting a high rate of OWB formation in the G. triflora line TY1-11-4-9. In conclusion, low-temperature treatments after a certain amount of growth, along with combined treatments such as photoperiod adjustments and gibberellin inhibitor administration, show promise for promoting OWB formation in gentians. Furthermore, the interspecific differences observed in flower bud formation, RSS production, and OWB formation contribute to a better understanding of gentian physiology and genetics.