Using machine learning to link the influence of transferred Agrobacterium rhizogenes genes to the hormone profile and morphological traits in Centella asiatica hairy roots

Abstract

Hairy roots are made after the integration of a small set of genes from Agrobacterium rhizogenes in the plant genome. Little is known about how this small set is linked to their hormone profile, which determines development, morphology, and levels of secondary metabolite production. We used C. asiatica hairy root line cultures to determine the putative links between the rol and aux gene expressions with morphological traits, a hormone profile, and centelloside production. The results obtained after 14 and 28 days of culture were processed via multivariate analysis and machine-learning processes such as random forest, supported vector machines, linear discriminant analysis, and neural networks. This allowed us to obtain models capable of discriminating highly productive root lines from their levels of genetic expression (rol and aux genes) or from their hormone profile. In total, 12 hormones were evaluated, resulting in 10 being satisfactorily detected. Within this set of hormones, abscisic acid (ABA) and cytokinin isopentenyl adenosine (IPA) were found to be critical in defining the morphological traits and centelloside content. The results showed that IPA brings more benefits to the biotechnological platform. Additionally, we determined the degree of influence of each of the evaluated genes on the individual hormone profile, finding that aux1 has a significant influence on the IPA profile, while the rol genes are closely linked to the ABA profile. Finally, we effectively verified the gene influence on these two specific hormones through feeding experiments that aimed to reverse the effect on root morphology and centelloside content.

Insights into enhancing Centella asiatica organ cell biofactories via hairy root protein profiling

“…These elongation factors play a fundamental role in the process of translation, which constitutes a fundamental step in the intricate process of protein synthesis ( Xu et al., 2022 ). This observation aligns well with the morphological traits characterizing the HIGH line, as reported by ( Alcalde et al., 2022 ), which demonstrated superior elongation, branching, and biomass production.…”

“…Understanding the relationship between transgene copy numbers and gene expression is crucial for developing strategies to improve the stability and performance of transformed lines in various biotechnological applications. Our work significantly extends the findings reported by Alcalde et al. (2022) , where distinctive morphological and metabolic variations were observed among different C. asiatica hairy root lines, likely due to the random insertion of a limited number of genes from the T-DNA, particularly the rol and aux genes.…”

“…The Adv roots were collected from in vitro -grown C. asiatica seedlings. The transformed roots, which were morphologically and phytochemically characterized by ( Alcalde et al., 2022 ), were categorized as HIGH, MID, or LOW based on their respective capacities for the production of centellosides – the key bioactive compounds of C. asiatica plants.…”

“…Moreover, strong precautions were exercised to regulate the growth conditions of these roots, including light shielding, to ensure a robust basis for comparison with the conventionally grown transformed roots. The specific transformed lines were selected according to their previous characterization by ( Alcalde et al., 2022 ), which benchmarked their performance for biotechnological production application.…”

“…The hairy root lines utilized in this study were established and morphologically characterized by Alcalde et al. (2022) .…”