|
Abstract:
|
Abstract: Cedrela fissilis Velloso (Meliaceae) is a fast growing native tree from the Atlantic Forest economically important for its medicinal properties and for producing valuable hardwood. The objectives of this work were to study the effects of carbon sources (sucrose, glucose, fructose), glutamine, explant types (leaf node, leaf, epicotyl, cotyledonary node, cotyledon, hypocotyl, root), growth regulators (BAP and NAA), light and dark on the levels of total proteins, total soluble sugars, starch, total phenolics, flavonoids, carotenoids (content and types), chlorophylls a and b, metabolic profiles, antioxidant activity of in vitro cultured calluses, and to evaluate the morphology and hystochemistry of callus originated from the different explants type cultured on glucose. Calluses were induced on Murashige and Skoog culture medium supplemented with 0,2% Phytagel from all types of explant tested (except from leaf explant cultured on 118 mM sucrose, 2,73 mM glutamine in the light), cultured on different carbon sources (sucrose, fructose, glucose), in all combinations of BAP and NAA concentration (2,5 µM e 5 µM), in different glutamine concentration (0; 2,73 e 5,46 µM), either in light or in the dark. The manipulation of sucrose (59 mM e 118 mM) and glutamine concentration (0; 2,73 e 5,46 mM) in Murashige & Skoog, supplemented with 0,2% de Phytagel, 2,5 µM BAP and 5 µM NAA allowed the establishment of optimal callus culture condition for cotyledonary node which promoted the production of the highest levels of total proteins (59 mM sucrose and 5,46 mM glutamine), total soluble sugars (118 mM sucrose and 5,46 mM glutamine), starch (118 mM sucrose and 5,46 mM glutamine), total phenolics (118 mM sucrose and 2,73 mM de glutamine), flavonoids (118 mM sucrose and 2,73 mM glutamine), carotenoids (118 mM sucrose and 5,46 mM glutamine), chlorophylls a (59 mM sucrose and 5,46 mM de glutamine) e b (59 mM de sucrose and 5,46 mM glutamine, 118 mM sucrose and 2,73 mM glutamine) and highest percentage of DPPH free radical inhibition (118 mM sucrose and 2,73 mM de glutamine).The manipulation of carbon sources (118 mM sucrose, fructose, glucose) in combination with BAP and NAA (2,5 µM e 5 µM), in MS culture medium supplemented with 0,2% de Phytagel and 2,73 mM glutamine optimized the biosynthesis of total proteins (sucrose, 5 µM BAP and 5 µM NAA; fructose 2,5 µM de BAP and 5 µM NAA), total soluble sugars (sucrose, 2,5 µM BAP and 2,5 µM NAA), starch (sucrose, 2,5 µM BAP and 2,5 µM NAA), total phenolics (sucrose, 2,5 µM BAP and 2,5 µM NAA) , flavonoids (glucose 2,5 µM BAP and 2,5 µM NAA), carotenoids (sucrose, 5 µM BAP and 2,5 µM NAA), chlorophylls a (glucose 5 µM BAP and 2,5 µM NAA) and b (sucrose, 2,5 µM BAP and 2,5 µM NAA; glucose 5 µM BAP and 2,5 µM NAA) and the highest percentage of DPPH free radical inhibition (glucose 5 µM BAP and 5 µM NAA) in callus originated from cotyledonary nodes.The manipulation of explant types (leaf node, cotyledonar node, segments of epicotyl, leaf, cotyledon, hypocotyl, root), carbon source (118 mM sucrose, fructose, glucose), glutamine (0; 2,73 mM), light and dark promoted the biosynthesis of total proteins (sucrose, cotyledon, without glutamine, light), total soluble sugars (sucrose, cotyledon, leaf node, without glutamine, light), starch (sucrose, leaf node, without glutamine, light), total phenolics (sucrose, cotyledon, glutamine, light), flavonoids (sucrose, cotyledon, glutamine, light; fructose, leaf, without glutamine, light), carotenoids (fructose or glucose, leaf, glutamine, light), chlorophyll a (sucrose, cotyledon, glutamine, light; fructose, leaf, cotyledon, without glutamine, light; glucose, leaf, glutamine light); chlorophyll b (sucrose, cotyledon, glutamine, light; fructose, leaf, without glutamine, light; glucose, leaf, glutamine light); the percentage of DPPH free radical inhibition higher than 70% (sucrose, all explants types cultured either with or without glutamine, in light or dark, except for cotyledonary node (with glutamine, light), hypocotyls (with glutamine, light or without glutamine, dark).The carbon sources, glutamine, BAP and NAA influenced the calluses metabolic profiles determined through the UV-vis sacanning. The carbon sources, explants types and glutamine affected the types and concentrations of carotenoids produced by calluses, fructose promoted the biosynthesis of carotenoids types as beta-cryptoxantin and trans-betacryptoxantin which were not produced with sucrose or were detected in a few treatments with glucose, as demonstrated by the HPLC analysis. The AT-O, PAS e CBB tests indicated, respectivelly, the metacromatic reaction in the callus cell wall, the presence of ortocromatic granules, possibly phenolics and positive reactions for neutral polyssacarides, as cellulose. Few starch grains and rich protein organels were detected in the cytoplasm. The results obtained in this study showed that optimal callus culture conditions were established, through the manipulation of carbon sources, explant type, glutamine, light and dark, which increased the production of a range of metabolites of interest and could foster further investigation on biosynthesis, identification and optimization of the production of important compounds by the cell culture systems developed.
|
