Culturas de tecidos de Deguelia duckeana: obtenção de calos semi-friáveis e isolamento de triterpenos em calos mantidos in vitro

Abstract

The Amazonian species Deguelia duckeana (Fabaceae) has demonstrated pharmacological potential, especially due to the presence of flavonoids with anticancer activity, previously isolated from dichloromethane (DCM) extracts of the roots. Plant tissue culture represents a promising alternative for the sustainable production of secondary metabolites under controlled conditions, without the need for intensive collection from the natural environment. This research aimed to develop tissue culture protocols for D. duckeana, seeking the production of friable calli and the in vitro regeneration of roots and shoots, as well as to perform phytochemical analyses of methanolic extracts obtained from calli maintained in culture. For the induction of friable calli, five tests were conducted with different culture medium compositions, including variations in growth regulators and their concentrations, substitution of the carbon source (fructose, glucose, and sucrose), reduction of sucrose concentration, pH adjustments, and different light conditions — a photoperiod of 16 hours of light and 8 hours of darkness, as well as cultivation in complete absence of light. The fifth test consisted of combining factors that individually demonstrated influence on friability. Calli were initially induced from leaf explants of a pre-established culture (Rescarolli, 2018). For root induction tests, previously established calli were used, evaluating the response to different combinations and concentrations of the auxins IAA (indole-3-acetic acid), IBA (indole-3-butyric acid), 2,4-D, and the cytokinin BAP. For regeneration tests via indirect organogenesis, calli were also used as explants. In these tests, explants were cultured on MS medium containing 100% mineral salts, supplemented with different combinations and concentrations of the growth regulators NAA, BAP, and KIN. Previously established maintenance calli kept in culture were subjected to successive subcultures, collected, weighed, identified, lyophilized, extracted with methanol, and fractionated. Phytochemical analyses by thin-layer chromatography and ¹H NMR allowed the isolation of the triterpenes lupeol, betulinaldehyde, and betulinic acid — bioactive compounds that reinforce the potential of the species as a constant source of substances with pharmacological relevance.Despite the absence of friable calli in the tests and the low efficiency in the regeneration of roots and shoots, the results obtained provide important support for adjustments in future protocols and demonstrate the feasibility of tissue culture as a complementary tool in the prospecting of secondary metabolites in D. duckeana. The cultures were maintained through successive subcultures totaling 12 years of culture follow-up, and consistency in metabolite production was observed. These results demonstrate the robustness of the plant species Deguelia duckeana in continuing the in vitro production of metabolites even under different stimuli.