Vernalization, Photoperiod, and Gibberellin Coordinately Regulate Flower Bud Differentiation in Oenothera biennis L.

Oenothera biennis L. is an economically important oil and ornamental crop. However, the regulatory mechanisms underlying its flower bud differentiation remain poorly understood. To elucidate the interactions between the environmental cues and molecular networks governing this process, this study investigated natural populations from the Lesser Khingan Mountains. The results demonstrate that exposure to 0–10 °C for at least 15 d and long-day (LD) conditions correlated strongly with floral induction. Notably, 200 mg·L−1 of exogenous GA3 effectively substituted for vernalization, significantly promoting bolting and advancing flower development. Expression profiling revealed an antagonistic expression pattern between floral activators such as ObFT and ObAP2 and repressors such as ObFLC. Both vernalization and GA3 upregulated floral activators while suppressing the repressors, and these processes were coincident with the transition to flowering. Flower bud differentiation goes through six continuous stages, spanning from inflorescence primordia to gynoecium primordia formation. Hybrid fruit setting, the pollen–ovule ratio (98) and the outcrossing index (OCI = 3) confirmed that O. biennisis is a facultative selfer, with reproduction predominantly relying on self-pollination. This study provides a systematic characterization of the integrated regulatory network coordinating vernalization, photoperiod, and gibberellin in Oenothera flowering, proposing a framework for the putative FT-FLC antagonism. These findings establish a molecular foundation for the precision management of flowering time and informed breeding strategies.