Overwintering buds (OWBs) are dormant organs produced by perennial plants. After flowering, Japanese gentians (Gentiana triflora, G. scabra and their hybrids) produce OWBs in autumn and go dormant to survive winter. Although dormant OWBs have been reported to have strong cold and freezing tolerances, no details about mechanisms and regulation are known. We attempted to produce in vitro OWBs (IOWBs) from gentian plantlets to facilitate the study of OWBs. We found that high-sucrose (6%) culturing media induced IOWB formation. IOWBs showed an enhanced freezing tolerance together with high levels of proteins and transcripts know to specifically accumulate in OWBs. Furthermore, IOWBs showed endodormancy release and bud break in the same manner as OWBs. Bud breaking in OWBs and IOWBs was promoted by red light and inhibited by far-red light. These results indicated that IOWBs possess similar physiological characteristics as OWBs, at least regarding stress tolerance and dormancy. Therefore, our artificial IOWB induction technique will be useful for the study of physiological mechanisms in field-grown OWBs.