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Leucojum aestivum is known for its ability to biosynthesize alkaloids with therapeutic properties, among which galanthamine used for the treatment of Alzheimer's disease. New sources of this alkaloid are still being explored. In this study, a novel strain PLV of endophytic bacterium Paenibacillus lautus was isolated from in vitro L. aestivum plants. We report the whole genome sequence of that strain and its capacity to produce alkaloids and growth regulators. The effect of elicitation with autoclaved bacteria on the production of alkaloids was examined. Ten alkaloids were identified in bacteria extracts: galanthamine, lycorine, ismine, lycoramine, haemanthamine, tazettine, galanthine, homolycorine, 1,2-dihydrochlidanthine, and hippeastrine. The mean contents of galanthamine and lycorine were 37.51 µg/g of dry weight (DW) and 129.93 µg/g of DW, respectively. Moreover, isolated P. lautus strain synthesized: indole-3-acetic acid, t-zeatin, c-zeatin, kinetin, gibberellin A 1 , abscisic acid, salicylic acid, benzoic acid. In vitro elicitation of cultures with P. lautus increased dry biomass, stimulated galanthamine and lycorine production, contributed to 8,9-desmethylenebis (oxy)-7,9 dimethoxy-crinan biosynthesis, change pigments content, and antioxidant enzymes activities. Our findings for the first time point out that galanthamine can be synthesized by an microorganism. Moreover isolated strain can be used as a new elictor of Amaryllidaceae alkaloids biosynthesis., (© 2022. The Author(s).)

Light-emitting diodes (LEDs) have emerged as efficient light sources for promoting in vitro plant growth and primary and secondary metabolite biosynthesis. This study investigated the effects of blue, red, and white-red LED lights on plant biomass growth, photosynthetic pigments, soluble sugars, phenolic compounds, the production of Amaryllidaceae alkaloids, and the activities of antioxidant enzymes in Leucojum aestivum L. cultures. A white fluorescent light was used as a control. The plants that were grown under white-red and red light showed the highest fresh biomass increments. The blue light stimulated chlorophyll a, carotenoid, and flavonoid production. The white-red and blue lights were favourable for phenolic acid biosynthesis. Chlorogenic, p-hydroxybenzoic, caffeic, syringic, p-coumaric, ferulic, sinapic, and benzoic acids were identified in plant materials, with ferulic acid dominating. The blue light had a significant beneficial effect both on galanthamine (4.67 µg/g of dry weight (DW)) and lycorine (115 µg/g DW) biosynthesis. Red light treatment increased catalase and superoxide dismutase activities, and high catalase activity was also observed in plants treated with white-red and blue light. This is the first report to provide evidence of the effects of LED light on the biosynthesis of phenolic acid and Amaryllidaceae alkaloids in L. aestivum cultures, which is of pharmacological importance and can propose new strategies for their production. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In vitro cultures of Leucojum aestivum are considered as an alternative for the production of galanthamine, which is used for the symptomatic treatment of Alzheimer's disease. We studied the effects of auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (picloram), 3,6-dichloro- o-anisic acid (dicamba) at concentrations of 25 and 50 µM on the induction of embryogenic callus and its capacity to induce somatic embryogenesis and alkaloid accumulation. The embryogenic response of the explants was from 30% for 25 µM of dicamba to 100% for picloram (for both 25 and 50 µM). 2,4-D (50 µM) stimulated greater callus proliferation and somatic embryo induction as compared to the other auxins. Polyethylene glycol (PEG) stimulated somatic embryo maturation. Callus grown on media containing 50 µM of auxins produced fewer phenolic compounds as compared with callus grown on media containing 25 µM of auxins. GC-MS analyses showed seven alkaloids in the in vivo bulbs and two to four in callus culture. Galanthamine was detected in callus cultivated with 2,4-D (25, 50 µM), picloram (25 µM), and dicamba (50 µM). Other alkaloids, trisphaeridine, tazettine, and 11-hydroxyvittatine were accumulated only in callus growing on medium with picloram (50 µM). [ABSTRACT FROM AUTHOR]

• Amaryllidaceae alkaloid biosynthesis in Leucojum aestivum plants was investigated. • Melatonin and NaCl were added to the culture medium. • The melatonin (10 μM) added to the culture medium led to maximum plant biomass. • Melatonin enhanced the galanthamine and lycorine accumulation in plants. • The highest distribution of alkaloids was observed with melatonin. Leucojum aestivum L. is a highly demanding pharmaceutical plant which is a natural source of pharmacologically active Amaryllidaceae alkaloids, especially galanthamine and lycorine. To establish an efficient method for the production of these alkaloids Leucojum aestivum plants derived from somatic embryos were subjected to various concentrations of melatonin (N -acetyl-5-methoxy tryptamine), NaCl and melatonin combined with NaCl. The maximum L. aestivum plant biomass was obtained when 10 μM of melatonin was applied to the medium. These plants were characterised by normal growth and did not reveal signs of browning. Enrichment of the medium with NaCl caused a reduction in plant biomass, plant pigment contents (chlorophyll a , chlorophyll b and carotenoids) and an increase in the activities of antioxidant enzymes catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). The addition of melatonin to the medium containing NaCl neutralised the negative effect of salt stress. Moreover, the stimulating effect of melatonin on Amaryllidaceae alkaloids biosynthesis was observed. In plants grown in the medium enriched with melatonin the galanthamine content was, on average, 58.6 times higher, and the lycorine content was 1.5 times higher as compared with the control. In vitro plants obtained from the medium supplemented with melatonin also showed the highest distribution of alkaloids. It is noteworthy that only the plants grown in the medium with melatonin showed the presence of tazettine and chlidanthine, 1,2-dihydro-. This study presents a promising biotechnological approach for the commercial production of alkaloids, including galanthamine and lycorine. [ABSTRACT FROM AUTHOR]