Carbohydrates stimulated Amaryllidaceae alkaloids biosynthesis inLeucojum aestivumL. plants cultured in RITA^®bioreactor

Abstract

Background Leucojum aestivum L. is an important medicinal plant which produces Amaryllidaceae alkaloids, especially galanthamine and lycorine. Research is currently exploring the possibility of producing these alkaloids using biotechnological methods, including in vitro cultures. The biosynthesis of alkaloids may be affected by 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 phenolics in any in vitro conditions. However, the addition of sugar had a decisive effect on the biosynthesis of the Amaryllidaceae alkaloids. The highest distribution of alkaloids occurred in plants cultured in the medium containing 60 g/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.

Molecular Identification of Endophytic Bacteria in Leucojum aestivum In Vitro Culture, NMR-Based Metabolomics Study and LC-MS Analysis Leading to Potential Amaryllidaceae Alkaloid Production

“…A negative correlation can be observed between the presence of lycorine and the presence of amino acids (alanine and threonine) and sugars (α-glucose and β-glucose). Also lycorine biosynthesis decreased by the addition of various concentrations of glucose in the culture medium of L. aestivum plants [ 31 ]. Using qNMR, in a single step of analysis, the quantification of the primary and specialized metabolites is realized and a global overview of our three biological samples (in vivo bulb, in vitro bulblets and isolated endophyte) has been presented.…”

“…The alkaloid tazettine (C 18 H 21 NO 4 [M + H] + 332.1486) was identified in bulbs of different varieties of Narcissus tazetta [ 50 ], Galanthus nivalis [ 51 ] but not in L. aestivum . However, tazettine was detected in this endophytic extract of Bacillus and in plant tissue culture of L. aestivum [ 31 ]. In the literature, bacterial endophytes are recognized as a rich and diverse source of alkaloids [ 52 ] such as camptothecin [ 53 ].…”

“…The pH was adjusted to 5.5. Bulblets were regenerated after transferring plants obtained from somatic embryos successively to the MS solid medium containing 5 µM zeatin during 16 weeks, then to the MS solid medium without growth regulators and supplemented with 6% w/v sucrose [ 31 ]. Cultures were placed in a growth chamber and maintained at 25 ± 2 °C under a 16/8-h (light/dark) photoperiod (Tungsram lamp, 40 WF, 90 µmol m −2 s −1 ), and subcultured once every 4 weeks under the same conditions.…”

Recent Progress in Amaryllidaceae Biotechnology

“…Recently published research showed that the most appropriate approach to optimization of the composition of nutrient media for maximum biosynthesis of plant secondary metabolites by in vitro systems is the statistical optimization of the main nutrient components. However, the latest results presented by Ptak and co-workers [ 80 ] are evidence that it is expedient to perform a single-factor optimization of the influence of various independent variables on the yield of target alkaloids and on the basis of the results received to create an experimental matrix for the full-factor statistical optimization. In this way, the most appropriate type of carbon source [ 80 ], growth regulators [ 56 , 81 ], as well as biosynthetic precursors [ 82 ] can be identified and these independent variables can subsequently be included in the experimental matrix of multifactorial optimization.…”

“…However, the latest results presented by Ptak and co-workers [ 80 ] are evidence that it is expedient to perform a single-factor optimization of the influence of various independent variables on the yield of target alkaloids and on the basis of the results received to create an experimental matrix for the full-factor statistical optimization. In this way, the most appropriate type of carbon source [ 80 ], growth regulators [ 56 , 81 ], as well as biosynthetic precursors [ 82 ] can be identified and these independent variables can subsequently be included in the experimental matrix of multifactorial optimization. In our opinion, elicitation is one of the most effective approaches to optimizing the biosynthesis of Amaryllidaceae alkaloids and it is a mandatory step in a future technology for their production [ 32 ].…”