Carbohydrates stimulated Amaryllidaceae alkaloids biosynthesis in Leucojum aestivum L. plants cultured in RITA® bioreactor
Agata Ptak , Emilia Morańska , Edyta Skrzypek , Marzena Warchoł , Rosella Spina , Dominique Laurain-Mattar , Magdalena Simlat
AbstractBackground: Leucojum aestivum L. is an important medicinal plant which produces Amaryllidaceaealkaloids,especiallygalanthamineandlycorine.Researchiscurrently exploring the possibility of producing these alkaloids using biotechnological methods,including invitrocultures.The biosynthesisof alkaloidsmay beaffectedby the types and concentrations of carbohydrate sources used in the medium. In the present investigation we performed such studies on in vitro cultures of L. aestivum with a view to obtaining plant material of good quality, characterized, in particular, by a high content of valuable Amaryllidaceae alkaloids. Methods: We examined the effects of various types of carbohydrate sources— sucrose, glucose, fructose and maltose—at different concentrations (30, 60 and 90 g/L)—on the quality of L. aestivum plants grown in the RITA® bioreactor. The plants’ quality was assessed by their biomass increments, as well by as analysing photosynthetic pigments, endogenous sugar, phenolics and Amaryllidaceae alkaloid content. We also investigated the effect of sugars on the activity of the antioxidant enzymes catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). Results: The highest biomass increments were observed in plants cultivated in the medium containing 90 g/L sucrose. The highest CAT activity was noted in cultures growing in the medium supplemented with 90 g/L maltose, while the highest POD activity was observed in the presence of 90 g/L fructose and 60 g/L maltose. No differences in SOD activity were observed. Moreover, the sugars did not affect the contents of chlorophyll a and carotenoids, whereas the highest amount of chlorophyll b was recorded in plants growing in the medium with 60 g/L maltose. No statistically signiﬁcant differences were observed in the contents of endogenous sugars and phenolicsin anyin vitroconditions. However,the additionof sugar hada decisive effect on the biosynthesis of the Amaryllidaceae alkaloids. The highest distributionofalkaloidsoccurredinplantsculturedinthemediumcontaining60g/L sucrose. Six Amaryllidaceae alkaloids were detected in the plant tissue. The addition of 30 g/L fructose in the medium resulted in the accumulation of ﬁve alkaloids, including ismine, which was not identiﬁed in other analysed tissues. The highest concentration of galanthamine was observed in plants cultured in the presence of 30 g/L fructose and 60 g/L sucrose (39.2 and 37.5 µg/g of dry weight (DW), respectively). The plants grown in the medium containing 60 g/L sucrose exhibited the highest lycorine content (1048 µg/g of DW). Conclusions: The type and concentration of sugar used in the medium have an essential inﬂuence on the biosynthesis of Amaryllidaceae alkaloids in L. aestivum plants cultured in a RITA® bioreactor. The results point to an interesting approach for commercial production of galanthamine and lycorine.
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