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Melatonin (MEL) can act as a plant growth regulator and biostimulator in stressful situations. Using MEL in seed pretreatment also affects the future growth of plants. Therefore, this research investigated the effects of MEL on seed germination and seedling growth under NaCl in in vitro conditions. The additional effects of MEL on the accumulation of steviol glycosides (SGs) and on the expression of appropriate genes were also studied. Five μM of MEL was the best concentration for seed germination, while 20 μM exerted a positive impact on the biomass of stevia plantlets. NaCl significantly decreased seed germination, but MEL alleviated this effect when seeds were germinated in 50 mM of NaCl. Under salinity, the values of almost all morphological traits decreased as MEL concentration increased. The highest amounts of stevioside and rebaudioside A (Reb A) were observed as a result of treating seeds with 5 and 20 μM of MEL, respectively. When adding NaCl, positive impacts of MEL on the accumulation of both SGs were also observed. Expression analyses of the genes involved in SGs biosynthesis was explored in seeds and leaves, and the transcripts of key enzymes occurred in both the tissues. However, quantitative polymerase chain reaction (qPCR) analysis showed that all tested genes were upregulated in younger leaves, contrary to older ones. Also in younger, rather than older, leaves SG gene expression varied according to MEL concentration. This study, therefore, presents the promising potential of MEL for improving stevia seed germination under salinity conditions and for enhancing the production of SGs in stevia plants.

This study aimed to examine the influence of the addition of a precursor (phenylalanine) on the accumulation of secondary metabolites in agitated shoot cultures of Ruta graveolens. Cultures were grown on Linsmaier and Skoog (LS) medium, with plant growth regulators (0.1 mg/L α-naphthaleneacetic acid—NAA—and 0.1 mg/L 6-benzylaminopurine—BAP). Phenylalanine was added to the cultures at a concentration of 1.25 g/L after 4 and 5 weeks of growth cycles. Biomass was collected after 2, 4, and 7 days of precursor addition. Both control and experimental cultures had the same secondary metabolites accumulated. Using the HPLC method, linear furanocoumarins (bergapten, isoimperatorin, isopimpinellin, psoralen, and xanthotoxin), furoquinoline alkaloids (γ-fagarine, 7-isopentenyloxy-γ-fagarine, and skimmianine), and catechin were detected and quantified in the methanolic extracts. In turn, phenolic acids, such as gallic acid, protocatechuic acid, p-hydroxybenzoic acid, syringic acid, p-coumaric acid, and ferulic acid were detected in hydrolysates. The production of phenolic acids and catechin (1.5-fold) was significantly increased by the addition of precursor, while there was no significant effect on the production of coumarins and alkaloids. The highest total content of phenolic acids (109 mg/100 g DW) was obtained on the second day of phenylalanine addition (the fourth week of growth cycles). The dominant phenolic compounds were p-coumaric acid (maximum content 64.3 mg/100 g DW) and ferulic acid (maximum content 35.6 mg/100 g DW). In the case of catechins, the highest total content (66 mg/100 g DW) was obtained on the third day of precursor addition (the fourth week of growth cycles). This study is the first to document the effect of feeding the culture medium with phenylalanine on the accumulation of bioactive metabolites in in vitro cultures of R. graveolens.

Ruta corsica is a rare and endemic plant native to Corsica. Due to its limited distribution and the priority to preserve natural sites, has been insufficiently studied. In vitro cultures provide an opportunity to research R. corsica under controlled conditions. In the present study, in vitro cultures of R. corsica were conducted in PlantformTM bioreactors. The study aimed to assess the effects of growth cycle length (5 and 6 weeks) and different concentrations of plant growth regulators (NAA and BAP) at 0.1/0.1, 0.1/0.5, 0.5/0.5, 0.5/1.0, and 1.0/1.0 mg/L on biomass growth and secondary metabolite accumulation. HPLC analysis identified compounds in the furanocoumarin and furoquinoline alkaloid groups, with furanocoumarins being the primary secondary metabolites (maximum total content: 1571.5 mg/100 g DW). Among them, xanthotoxin, psoralen, and bergapten were dominant, with maximum concentrations of 588.1, 426.6, and 325.2 mg/100 g DW, respectively. The maximum total content of furoquinoline alkaloids was 661 mg/100 g DW, with γ-fagarine as the primary metabolite, reaching 448 mg/100 g DW. The optimal conditions for secondary metabolite accumulation in R. corsica cultures were a 5-week growth cycle and the LS 0.1/0.1 medium variant.

The influence of light quality and cytokinin content in media on growth, development, photosynthetic pigments and secondary metabolite content of Myrtus communis L. was evaluated in an in vitro culture. Various treatments with light emitting diodes (LEDs): 100% blue (B), a mix of 70% red and 30% blue (RB) and 100% red were applied and compared with a traditional fluorescent lamp as control. Axillary shoots were incubated on Murashige and Skoog medium with 30 g dm−3 sucrose, 0.5% BioAgar, 0.5 μM 1-naphthaleneacetic acid and different concentrations of 6-benzyladenine (BA): 1, 2.5 and 5 µM. Cultures were maintained for 6 weeks in 23/21 ± 1 °C (day/night), 80% relative humidity and 16/8 h photoperiod; photosynthetic photon flux density (PPFD) was 35 µmol m−2 s−1 in all treatments. Light spectra and BA content in media affected biometrical and phytochemical M. communis properties. Red LEDs and 5 µM BA resulted in the highest multiplication rate. The highest shoots were obtained under red LEDs, but with the lowest concentration of cytokinin in media. Fresh weight was greatest on LEDs containing blue light in the spectrum (B and RB); moreover, 5 µM BA increased dry weight. Photosynthetic pigment levels were lower under LED light compared to control lamps. Phenolic acids and flavonoids were identified in M. communis leaf extracts. Myricetin was the major constituent with highest concentration under red LEDs and highest BA level.

Stevia rebaudiana Bertoni possesses various medicinal and food industrial applications. This study is the first to explore the effect of the cytokinins meta -Topolin ( m T; 6-(3-hydroxybenzylamino) purine), zeatin, kinetin, and BAP (6-benzylaminopurine) at concentrations of 0 (control), 5, 10, and 15 µM on shoot multiplication, as well as stevioside, rebaudioside A, phenolic acid, and flavonoid content in bioreactor cultures. The highest number of shoots (23.4 per explant) was obtained in the medium containing 5 μM of m T. However, 15 μM of m T was superior for fresh biomass production and dry biomass accumulation. Reversed-phase (RP)-HPLC analysis showed a beneficial effect of 5 μM m T on stevioside (11.43 mg/g dry weight [DW]) and rebaudioside A (10.74 mg/g DW) biosynthesis. In all conditions, the ratio of rebaudioside A/stevioside ranged from 0.75 to 1.12. The phenolic acids chlorogenic, neochlorogenic, isochlorogenic A, and rosmarinic were confirmed in the stevia extracts, as were the flavonoids isoquercetin, and quercitrin. The highest accumulations of chlorogenic and neochlorogenic acids and flavonoids were observed in shoot tissues derived from 5 µM m T, whereas 5 µM of BAP stimulated biosynthesis of chlorogenic, isochlorogenic A, and rosmarinic acids. This is the first report on the use of m T-cytokinin showing high potential in stevia cultures.

The selection of plant-based substrates for mushroom cultivation is a key factor influencing their growth and metabolism. The aim of this study was to demonstrate, in an innovative approach, differences in the content of biologically active compounds, bioelements, and antioxidant properties of Hericium erinaceus (Bull.) Pers. cultivated on various plant-based substrates derived from waste materials, specifically hemp straw and beech sawdust. Another objective was to compare various extraction methods in terms of their impact on the concentration of these compounds. Elemental analysis was performed using the TXRF method, while bioactive constituents were determined using the DAD/UV RP-HPLC technique. The plant-based substrate and extraction method influenced the levels of obtained metabolites. Dual extraction with moderate ethanol concentrations was most effective for isolating key bioactive compounds from H. erinaceus—notably ergothioneine, lovastatin, L-phenylalanine, and ergosterol—while antioxidant activity did not correlate with the concentration of the solvent used. Although dual extracts enhanced certain antioxidants and metabolites, whole fruiting bodies contained higher levels of bioelements. Overall, fruiting bodies grown on beech sawdust had greater amounts of most bioactive compounds compared to those cultivated on hemp straw, emphasizing that both substrate choice and extraction method critically influence the mushroom’s bioactive profile and its potential health benefits.

Pleurotus eryngii (DC:Fr.) Quel. is a cultivated mushroom of high culinary value and medicinal properties. Mycelium of P. eryngii is characterized by the ability of effective bio-elements absorption from growth media so it could be biofortified with trace elements with a functional activity in the human body. In this study, the ability of P. eryngii mycelia from in vitro cultures as well as fruiting bodies were investigated in terms of their effectiveness in zinc and selenium accumulation. The effect of Se and Zn biofortification on productivity, chemical compounds, and bio-elements content of P. eryngii was determined as well. To enhance Se and Zn content in P. eryngii fruiting bodies and mycelia, substrates were supplemented with sodium selenite, at a concentration of 50 mg L−1, zinc sulfate, and zinc hydro-aspartate at a concentration of 87.2 and 100.0 mg L−1, respectively. Mentioned Zn concentrations contained the same amount of zinc(II) ions, namely 20 mg L−1. The content of organic compounds include phenolic compounds and lovastatin, which were determined by a high-performance liquid chromatography with diode-array detector (HPLC-DAD) and reverse phase high-performance liquid chromatography (RP-HPLC) method with UV detection. The ability of P. eryngii to accumulate zinc and selenium from the culture medium was demonstrated. The degree of accumulation of zinc turned out to be different depending on the type of salt used. The present study also showed that conducting mycelium of P. eryngii in in vitro culture, with a higher content of zinc ions, can result in obtaining the materials with better antioxidant ability. The results of this study can be used to develop the composition of growing media, which ensures the production of biomass with the desired composition of elements.

Light-emitting diode (LED) lamps are efficient elicitors of secondary metabolites. To investigate the influence of LED light on steviol glycosides (SGs) and phenolic compounds biosynthesis, stevia shoots were cultured under the following LED lights: white–WL, blue–B, red–R, 70% red and 30% blue–RB, 50% UV, 35% red and 15% blue–RBUV, 50% green, 35% red and 15% blue–RBG, 50% yellow, 35% red and 15% blue–RBY, 50% far-red, 35% red and 15% blue–RBFR and white fluorescent light (WFl, control). RBG light stimulated shoots’ biomass production. RBFR had a beneficial impact on stevioside biosynthesis (1.62 mg/g dry weight, DW), while RBUV favoured the production of rebaudioside A (3.15 mg/g DW). Neochlorogenic, chlorogenic, caffeic, 4-feruloylquinic, isochlorogenic A, rosmarinic acids and the flavonoid quercitrin were identified in the obtained material. A stimulatory effect of RBFR and RBUV on the biosynthesis of phenolic compounds was noted. LED light also influenced stomata appearance, stomata density, photosynthetic pigments, soluble sugar content and antioxidant enzyme activities in stevia shoots. This is the first report to provide evidence of the stimulating effect of LED light on biomass yield, SGs production and phenolic compounds in stevia shoot cultures.

Fungal mycelium cultures are an alternative to natural sources in order to obtain valuable research materials. They also enable constant control and adaptation of the process, thereby leading to increased biomass growth and accumulation of bioactive metabolites. The present study aims to assess the biosynthetic potential of mycelial cultures of six Ganoderma species: G. adspersum, G. applanatum, G. carnosum, G. lucidum, G. pfeifferi, and G. resinaceum. The presence of phenolic acids, amino acids, indole compounds, sterols, and kojic acid in biomass extracts was determined by HPLC. The antioxidant and cytotoxic activities of the extracts and their effects on the inhibition of selected enzymes (tyrosinase and acetylcholinesterase) were also evaluated. The total content of phenolic acids in the extracts ranged from 5.8 (G. carnosum) to 114.07 mg/100 g dry weight (d.w.) (G. pfeifferi). The total content of indole compounds in the extracts ranged from 3.03 (G. carnosum) to 11.56 mg/100 g d.w. (G. lucidum) and that of ergosterol ranged from 28.15 (G. applanatum) to 74.78 mg/100 g d.w. (G. adspersum). Kojic acid was found in the extracts of G. applanatum and G. lucidum. The tested extracts showed significant antioxidant activity. The results suggest that the analyzed mycelial cultures are promising candidates for the development of new dietary supplements or pharmaceutical preparations.

Background and Aims Viola tricolor L. (heartsease, wild pansy) is a valuable medicinal plant obtained for pharmaceutical purposes by cultivation. Given that the species is usually strongly colonized by arbuscular mycorrhizal fungi (AMF), we tested in a pot experiment whether these microorganisms were able to influence V. tricolor mass, vitality, and the concentrations of selected elements, phenolic acids and flavonoids. Methods The following treatments were prepared: (1) control: sterile soil without AMF inoculation; (2) Rhizophagus irregularis BEG144; (3) Funneliformis mosseae BEG12; and (4) both isolates. Using a combination of physiological, phytochemical and biophysical methods, we evaluated the effects of these AMF on the performance of heartsease. Results The intensity of mycorrhizal colonization and arbuscule formation was higher when F. mosseae and R. irregularis were introduced separately than when both isolates were present. None of the AMF treatments had an impact either on V. tricolor vitality as expressed by photosynthetic performance index (PI) or on its shoot and root mass. However, in general, a negative correlation was found between the extent of mycorrhizal colonization and shoot mass. We found AMF species specificities in their influence on element, phenolic acid and flavonoid concentrations. Viola tricolor showed no response to F. mosseae. The plants inoculated with R. irregularis had higher concentrations of P, Zn, Mg, and Ca, as well as p-hydroxybenzoic acid and rutin, in comparison to control. Dual AMF species inoculation increased concentrations of Cu, Mg and rutin. Conclusions The enhanced production of secondary metabolites in V. tricolor shoots may be due to improved mineral nutrition by AMF and/or a result of general plant defense reaction to fungal colonization. The tendency towards biomass decrease in AMF treatments could be explained by the allocation of plant carbon both to the maintenance of symbionts and enhanced production of secondary compounds.