Aprimoramento da obtenção de calos e suspensões celulares de Duroia macrophylla Huber (Rubiaceae) e avaliação dos elicitores NaCl, KCl, AlCl3, SNP(NO), ABA e luz UV na produção de metabólitos secundários

Abstract

Duroia macrophylla Huber (Rubiaceae) is a species that produces important secondary metabolites, highlighting the indole alkaloids with antitumoral and antituberculosis activities. The objective of this work was to establish seedling cultures, calluses and cell suspensions in vitro of the species, as well as to evaluate the effect of various elicitors on the chemical profile of calluses of D. macrophylla. Seeds from D. macrophylla fruits were used as explants for in vitro germination and seedling growth. The seeds were submitted to different treatments and were tested at concentrations of 0, 2, 5 and 10 mg.L-1 of gibberellic acid (GA3) for the induction of germination in Murashige and Skoog (MS) medium. The germinated seedlings were then used as explants in which different concentrations of plant growth regulators (PGRs) were tested, such as α-naphthalene acetic acid (ANA) and 2,4-dichlorophenoxyacetic acid ( 2,4-D) and the cytokinins N-Benzyl-9- (2-tetrahydropyranyl)-adenine (BAP) and kinetin (KIN) for callus induction. All treatments were exposed to two light conditions: 24 h in the dark and photoperiod of 16/8 h (light / dark), both with a constant temperature of 26 ºC. The best response (70%) for the induction of semi-friable callus (SC) was in the presence of growth regulators BAP and ANA at the concentration of 2 mg.L-1 for both. The 24 h condition in the dark was significant for the formation of white and semi-friable callus for all combinations of PGRs and from these callus cell suspensions were established. Growth curves were defined and the phytochemical analyzes of both cultures were performed by TLC and 1H NMR showing differences in growth and chemical profile of both. From the fractionation of the methanolic extract of the callus was isolated an iridoide-glycoside that is still in the process of elucidation. This is the first report of the presence of iridoids in the species. The effect of different concentrations of the NaCl, KCl, AlCl3, SNP (NO), ABA and different UV exposure times on the chemical profile and iridoid production in the callus was evaluated. The results were evaluated by chemometrics analysis: Principal Component Analysis (PCA)/ Fuzzy and Hierarchical Cluster Analysis (HCA) and fuzziThe elicitors SNP and AlCl3 were shown to have a greater effect on the chemical profile and the production of the iridoid in the calluses, whereas NaCl, KCl and ABA inhibited its production. The results obtained in this work demonstrate the significant potential of the use of elicitation in calluses of the species D. macrophylla and encourage the continuation of the study aiming the induction of the production of the active metabolites of the plant.