| Summary: | <i>Peumus boldus</i> Mol (<i>P. boldus</i>), a Chilean endemic tree species occurring in sclerophyllous or evergreen forests, has historically played a significant role in indigenous ancestral medicine. Recently, <i>P. boldus</i> has garnered renewed attention, spurred by the growing interest in its leaves and bark. These parts contain a wealth of bioactive agents, including alkaloids, flavonoids, essential oils, and potent antioxidant properties attributed to their high phenolic compound content. However, the species’ regeneration within its native habitat has been hindered by a confluence of factors such as climate change, agricultural activities, and shifts in land use. Coupled with the seeds’ low germination rate and protracted emergence period, the necessity to develop large-scale propagation methods to bolster <i>P. boldus</i> population numbers has become increasingly evident. Furthermore, the widespread use of <i>P. boldus</i> for medicinal purposes renders it vulnerable to overexploitation, thereby underscoring the need for a comprehensive mass propagation protocol to support conservation efforts. Thus, the main objective of this study was to formulate an in vitro protocol for mass regeneration of <i>P. boldus</i>. The explants excised from nodal sections demonstrated an average survival rate of 74%, while the application of 6-benzylaminopurine (4.44 μM) yielded an average shoot length of 5.9 cm. In vitro shoot rooting achieved a success rate of 80% using perlite supplemented with indole-3-butyric acid (9.84 μM). During the acclimation phase, in vitro rooted plants displayed a remarkable 100% survival rate at the 30-day mark after being transplanted into a substrate consisting of perlite and peat in a (1:1 <i>v</i>/<i>v</i>) ratio.
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