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<p><i>Psychotria viridis</i> is an Amazonian shrub traditionally used in the psychoactive brew ayahuasca due to its content of N,N-dimethyltryptamine (DMT), a potent indole alkaloid with emerging therapeutic interest. Despite its significance, in vitro culture systems for this species remain underexplored, limiting sustainable DMT production alternatives. This study aimed to establish a protocol for callus induction, and in vitro DMT production in <i>P. viridis</i>. Leaf segments from greenhouse seedlings were cultured on Murashige and Skoog (MS) or Gamborg B5 media, supplemented with various concentrations of auxins and cytokinins across a total of 29 treatments under light and dark conditions. Callus formation, biomass accumulation, spontaneous regeneration, and alkaloid content were assessed. DMT quantification was performed using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), and histochemical analysis with Dittmar’s reagent confirmed alkaloid presence. Successful callus induction was achieved, with B5 medium supplemented with 1.0 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) yielding the best combination of biomass accumulation (0.842 <i>± 0.202</i> cm² area; 0.473 <i>± 0.183</i> cm³ volume) and DMT content (34.46 ng/mg). Organized cell aggregates with vacuoles containing alkaloids were observed histologically under this treatment. Additionally, spontaneous shoot regeneration occurred in several treatments, and plantlets grown in a temporary immersion bioreactor exhibited up to 80% survival post-acclimatization. This study represents the first successful establishment of an in vitro culture system for <i>P. viridis</i>, demonstrating the feasibility of callus-based DMT production and plantlet regeneration. The findings provide a novel and scalable biotechnological approach for sustainable alkaloid production, contributing to DMT research, genetic conservation, and potential pharmaceutical applications.</p>

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Establishment of Psychotria viridis Ruiz & Pav. callus cultures, plantlet regeneration and in vitro production of the hallucinogenic indole alkaloid N,N-dimethyltryptamine

  • 期刊论文

  • 发表日期：

    2025年5月20日

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  • Camila Moreno Lopes de Andrade,
  • Beatriz Aparecida Passos Paranhos Bismara,
  • Fabiana Pereira Santos,
  • Maurício Yonamine,
  • José Alexandre de Souza Crippa,
  • Jaime Eduardo Cecílio Hallak,
  • Fernando Henrique Ferrari Alves,
  • Moacir Pasqual,
  • Vanessa Cristina Stein

摘要

Psychotria viridis is an Amazonian shrub traditionally used in the psychoactive brew ayahuasca due to its content of N,N-dimethyltryptamine (DMT), a potent indole alkaloid with emerging therapeutic interest. Despite its significance, in vitro culture systems for this species remain underexplored, limiting sustainable DMT production alternatives. This study aimed to establish a protocol for callus induction, and in vitro DMT production in P. viridis. Leaf segments from greenhouse seedlings were cultured on Murashige and Skoog (MS) or Gamborg B5 media, supplemented with various concentrations of auxins and cytokinins across a total of 29 treatments under light and dark conditions. Callus formation, biomass accumulation, spontaneous regeneration, and alkaloid content were assessed. DMT quantification was performed using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), and histochemical analysis with Dittmar’s reagent confirmed alkaloid presence. Successful callus induction was achieved, with B5 medium supplemented with 1.0 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) yielding the best combination of biomass accumulation (0.842 ± 0.202 cm² area; 0.473 ± 0.183 cm³ volume) and DMT content (34.46 ng/mg). Organized cell aggregates with vacuoles containing alkaloids were observed histologically under this treatment. Additionally, spontaneous shoot regeneration occurred in several treatments, and plantlets grown in a temporary immersion bioreactor exhibited up to 80% survival post-acclimatization. This study represents the first successful establishment of an in vitro culture system for P. viridis, demonstrating the feasibility of callus-based DMT production and plantlet regeneration. The findings provide a novel and scalable biotechnological approach for sustainable alkaloid production, contributing to DMT research, genetic conservation, and potential pharmaceutical applications.