Bio nanoparticles as elicitors increase accumulation of betulin and betulinic acid in callus cultures

Abstract

This study aimed to develop a protocol for callus induction and also to evaluate the effects of chitosan nano-fiber (CTS-N), chitin nano-fiber (CT-N), and cellulose nano-fiber (Cellul-N) as elicitors on betulin and betulinic acid production alterations in the produced stem cells of a hybrid of Betula pendula. Inner birch bark samples were cultured in three different WPM, NT, and B5 media applying three hormonal treatments. Callogenesis was repeated in the selected medium providing the maximum percentage of callus induction (i.e., NT medium enriched with 1.5 mg.L−1 of 2,4-D + 0.5 mg.L−1 of BAP) and was observed for 5 months. Calluses were re-cultured in the medium with CTS-N, CT-N, and Cellul-N as elicitors at different concentrations of 0.5, 0.75, and 1 mg.L−1 and were extracted after specific time intervals (i.e., one, two, and three days) followed by determining their fresh and dry weights. Also, betulin and betulinic acid levels were determined by High Performance Liquid Chromatography (HPLC). As the results revealed, the heaviest fresh and dry weights (1.5-fold heavier than the control) were obtained for calli treated with 1% CTS-N for three days. Furthermore, one day elicitation using 0.5% Cellul-N induced the highest amount of betulin (0.7 mg.g-1), and the highest amount of betulinic acid (0.98 mg.g-1) was obtained in the callies treated by 0.5% CT-N for 3 days, while the corresponding weights for the control treatments were measured 0.08 mg.g-1 and 0.002 mg.g-1, respectively. And the maximum antioxidant activity of 93.9% was achieved from the elicitation utilizing 0.5% CTS-N for two days. This is the first report on the noticeable impact of nanomaterials, i.e., CTS-N, CT-N, and Cellul-N, on increasing botulin and betulinic acid accumulation along with the antioxidant activity, which, of course, requires further scrutiny.