Machine Learning Unmasked Nutritional Imbalances on the Medicinal Plant Bryophyllum sp. Cultured in vitro.

Plant nutrition is a crucial factor that is usually underestimated when designing plant in vitro culture protocols of unexploited plants. As a complex multifactorial process, the study of nutritional imbalances requires the use of time-consuming experimental designs and appropriate statistical and multiple regression analysis for the determination of critical parameters, whose results may be difficult to interpret when the number of variables is large. The use of machine learning (ML) supposes a cutting-edge approach to investigate multifactorial processes, with the aim of detecting non-linear relationships and critical factors affecting a determined response and their concealed interactions. Thus, in this work we applied artificial neural networks coupled to fuzzy logic, known as neurofuzzy logic, to determine the critical factors affecting the mineral nutrition of medicinal plants belonging to Bryophyllum subgenus cultured in vitro. The application of neurofuzzy logic algorithms facilitate the interpretation of the results, as the technology is able to generate useful and understandable "IF-THEN" rules, that provide information about the factor(s) involved in a certain response. In this sense, ammonium, sulfate, molybdenum, copper and sodium were the most important nutrients that explain the variation in the in vitro culture establishment of the medicinal plants in a species-dependent manner. Thus, our results indicate that Bryophyllum spp. display a fine-tuning regulation of mineral nutrition, that was reported for the first time under in vitro conditions. Overall, neurofuzzy model was able to predict and identify masked interactions among such factors, providing a source of knowledge (helpful information) from the experimental data (non-informative per se), in order to make the exploitation and valorization of medicinal plants with high phytochemical potential easier.

Machine Learning Technology Reveals the Concealed Interactions of Phytohormones on Medicinal Plant In Vitro Organogenesis.

Organogenesis constitutes the biological feature driving plant in vitro regeneration, in which the role of plant hormones is crucial. The use of machine learning (ML) technology stands out as a novel approach to characterize the combined role of two phytohormones, the auxin indoleacetic acid (IAA) and the cytokinin 6-benzylaminopurine (BAP), on the in vitro organogenesis of unexploited medicinal plants from the Bryophyllum subgenus. The predictive model generated by neurofuzzy logic, a combination of artificial neural networks (ANNs) and fuzzy logic algorithms, was able to reveal the critical factors affecting such multifactorial process over the experimental dataset collected. The rules obtained along with the model allowed to decipher that BAP had a pleiotropic effect on the Bryophyllum spp., as it caused different organogenetic responses depending on its concentration and the genotype, including direct and indirect shoot organogenesis and callus formation. On the contrary, IAA showed an inhibiting role, restricted to indirect shoot regeneration. In this work, neurofuzzy logic emerged as a cutting-edge method to characterize the mechanism of action of two phytohormones, leading to the optimization of plant tissue culture protocols with high large-scale biotechnological applicability.

Combining Medicinal Plant In Vitro Culture with Machine Learning Technologies for Maximizing the Production of Phenolic Compounds.

We combined machine learning and plant in vitro culture methodologies as a novel approach for unraveling the phytochemical potential of unexploited medicinal plants. In order to induce phenolic compound biosynthesis, the in vitro culture of three different species of Bryophyllum under nutritional stress was established. To optimize phenolic extraction, four solvents with different MeOH proportions were used, and total phenolic content (TPC), flavonoid content (FC) and radical-scavenging activity (RSA) were determined. All results were subjected to data modeling with the application of artificial neural networks to provide insight into the significant factors that influence such multifactorial processes. Our findings suggest that aerial parts accumulate a higher proportion of phenolic compounds and flavonoids in comparison to roots. TPC was increased under ammonium concentrations below 15 mM, and their extraction was maximum when using solvents with intermediate methanol proportions (55-85%). The same behavior was reported for RSA, and, conversely, FC was independent of culture media composition, and their extraction was enhanced using solvents with high methanol proportions (>85%). These findings confer a wide perspective about the relationship between abiotic stress and secondary metabolism and could serve as the starting point for the optimization of bioactive compound production at a biotechnological scale.