In vitro asymbiotic germination and micropropagation of Vanilla rivasii (endemic species to biogeographic Chocó): Bases for its mass multiplication and conservation

Abstract The fragmentation of habitats in the Colombian biogeographical Choco threatens endemic species of high value such as the Vanilla rivasii orchid. To safeguard its ecological and biotechnological potential, it is crucial to develop specific in vitro propagation methods. This study established for the first time an asymbiotic germination and micropropagation protocol for this species. Germination was evaluated in 50% Murashige and Skoog medium (MS) supplemented with 0.02 mg L⁻ 1 1-naphthaleneacetic acid, 0.04 and 0.7 mg L⁻ 1 6-benzylaminopurine, 0.05 and 0.5 mg L⁻ 1 gibberellic acid, and 0.05 mg L⁻ 1 indole-3-butyric acid, using seeds at three phenological stages of the pod (immature, intermediate, mature). For micropropagation, nodal segments of different sizes were grown (small: 1.25 to1.56 cm; medium: 1.57 to 1.70 cm; large: 1.71 to1.90 cm). Only one of the bioregulatory combinations induced germination (0.02 mg L⁻ 1 1-naphthaleneacetic acid, 0.04 mg L⁻ 1 6-benzylaminopurine, and 0.05 mg L⁻ 1 gibberellic acid). The phenological stage of the pod was a determining factor. The seeds of pods at the intermediate stage showed an increase in germination (33%), significantly higher than those of green pods (16%) and mature pods (4%). In the multiplication phase, large explants showed the longest shoot length (2.25 cm at 30 d) and rhizogenesis (81% rooting), while small explants had the highest cumulative growth rate (133%). In conclusion, the study identifies an optimal phenological window for the germination of V. rivasii and demonstrates that the size of the explant defines divergent development trajectories. These findings offer novel and evidence-based criteria to optimize conservation and micropropagation protocols for this species.