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Melanoma is a malignant neoplasm of melanocytes in the skin, and its occurrence is increasing annually. Plant-based products contain active compounds with low toxicity and are accessible alternatives for melanoma cancer treatment. The biotechnology approach for obtaining plant-based products provides continuity and allows the high-yield production of phytochemically uniform biomass. The callus biomass of Eryngium planum L. and Lychnis flos-cuculi L. was induced on Murashige and Skoog (MS) medium supplemented with growth regulators. A combination of 3.0 mg/L of 3,6-dichloro-2-methoxybenzoic acid (dicamba) and 0.3 mg/L of 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea—(thidiazuron) was used to obtain E. planum callus. Meanwhile, the callus of L. flos-cuculi was cultivated on MS medium with 2.0 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D). Methanolic extracts (EpME and LFcME), including 40% MeOH fractions (Ep40MF and LFc40MF) and 80% MeOH fractions (Ep80MF and LFc80MF), of E. planum and L. flos-cuculi cell biomass were prepared. Their cytotoxicity activity was assessed in human fibroblast cells (MRC-5) and human melanoma cells (MeWo) by direct cell counting and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Qualitative analyses using thin-layer chromatography and UPLC-HRMS/MS chromatograms showed the presence of phenolic acids and saponins within the extracts and fractions of both cell biomasses. LFc80MF and Ep80MF showed the strongest toxicity against the MeWo cell line, with IC50 values of 47 ± 0.5 and 52 ± 4 μg/mL after 72 h of treatment. EpME and LFcME had IC50 values of 103 ± 4 and 147 ± 4 µg/mL, respectively. On the other hand, Ep40MF and LFc40MF were less toxic against the MeWo cell line compared to the extracts and 80% MeOH fractions, with IC50 values of 145 ± 10 and 172 ± 7 µg/mL. This study suggests that the obtained extracts and fractions of E. planum and L. flos-cuculi cell biomass potentially possess significant cytotoxic activity against MeWo cells, which work in a time and dose-dependent manner. Although the extracts and 80% MeOH fractions were more potent, the 40% MeOH was shown to be more selective against the MeWo than the control MRC-5 cells. [ABSTRACT FROM AUTHOR]

Sedum telephium is a succulent plant used in traditional medicine, particularly in Italy, for its efficacy in treating localized inflammation such as burns, warts, and wounds. Fresh leaves or freshly obtained derivatives are directly applied to the injuries for these purposes. However, challenges such as the lack of microbiologically controlled materials and product standardization prompted the exploration of more controlled biotechnological alternatives, utilizing in vitro plant cell cultures of S. telephium. In the present study, we used HPLC-DAD analysis to reveal a characteristic flavonol profile in juices from in vivo leaves and in vitro materials mainly characterized by several kaempferol and quercetin derivatives. The leaf juice exhibited the highest content in total flavonol and kaempferol derivatives, whereas juice from callus grown in medium with hormones and callus suspensions showed elevated levels of quercetin derivatives. The in vitro anti-inflammatory and wound-healing assays evidenced the great potential of callus and suspension cultures in dampening inflammation and fostering wound closure, suggesting quercetin may have a pivotal role in biological activities. [ABSTRACT FROM AUTHOR]

Anthocyanins are water-soluble pigments found in plants. They exist in various colors, including red, purple, and blue, and are utilized as natural colorants in the food and cosmetics industries. The pharmaceutical industry uses anthocyanins as therapeutic compounds because they have several medicinal qualities, including anti-obesity, anti-cancer, antidiabetic, neuroprotective, and cardioprotective effects. Anthocyanins are conventionally procured from colored fruits and vegetables and are utilized in the food, pharmaceutical, and cosmetic industries. However, the composition and concentration of anthocyanins from natural sources vary quantitively and qualitatively; therefore, plant cell and organ cultures have been explored for many decades to understand the production of these valuable compounds. A great deal of research has been carried out on plant cell cultures using varied methods, such as the selection of suitable cell lines, medium optimization, optimization culture conditions, precursor feeding, and elicitation for the production of anthocyanin pigments. In addition, metabolic engineering technologies have been applied for the hyperaccumulation of these compounds in varied plants, including tobacco and arabidopsis. In this review, we describe various strategies applied in plant cell and organ cultures for the production of anthocyanins. [ABSTRACT FROM AUTHOR]

Natural extracts of Hypericum perforatum L. are widely used in the pharmaceutical industry due to their antiviral, antioxidant, antibacterial, antidiabetic, and antidepressant activities. Plant biotechnological methods are gaining more attention as efficient and biosustainable approaches for the production of high value compounds by using plant callus cultures in vitro. The aim of this research was to evaluate the influence of phytohormones on the formation of Hypericum perforatum L. callus culture, to explore antioxidant and antibacterial activities, and to determine the amounts of phytochemicals in the analyzed extracts. Callus cultures were induced on Murashige and Skoog basal medium supplemented with phytohormones (auxins, cytokinins, and salicylic acid), and containing newly synthesized compound N-(1,3-dioxoisoindolin-2-yl)-3-((4-methoxyphenyl)amino)propanamide. The antioxidant activities of callus cultures were evaluated using DPPH and FRAP assays and the enzyme antioxidants (superoxide dismutase, catalase, and ascorbate peroxidase) were evaluated. The antibacterial activity of plant extracts was screened against Escherichia coli and Bacillus subtilis bacteria by the diffusion agar method. There was a significant positive correlation between total phenolic content and high antioxidant efficiency in Hypericum perforatum L. callus cultures, which were formed on Murashige and Skoog medium supplemented with auxin, cytokinin, and salicylic acid (100 µM). Our results have demonstrated that callus cultures generated on MS medium supplemented with N-(1,3-dioxoisoindolin-2-yl)-3-((4-methoxyphenyl)amino)propanamide and cytokinin BAP (0.5 mg L−1) exhibited improved antioxidant activities for the first time. [ABSTRACT FROM AUTHOR]

Modern plant biotechnology often faces the problem of obtaining a stable and powerful vector for gene overexpression. It is known that introns carry different regulatory elements whose effects on transgene expression have been poorly studied. To study the effect of an intron on transgene expression, the stilbene synthase 11 (VaSTS11) gene of grapevine Vitis amurensis Rupr. was selected and overexpressed in grapevine callus cell cultures and several plant generations of Arabidopsis thaliana as two forms, intronless VaSTS11c and intron-containing VaSTS11d. The STS genes play an important role in the biosynthesis of stilbenes, valuable plant secondary metabolites. VaSTS11d contained two exons and one intron, while VaSTS11c contained only two exons, which corresponded to the mature transcript. It has been shown that the intron-containing VaSTS11d was better expressed in several generations of transgenic A. thaliana than VaSTS11c and also exhibited a lower level of cytosine methylation. As a result, the content of stilbenes in the VaSTS11d-transgenic plants was much higher than in the VaSTS11c-transgenic plants. Similarly, the best efficiency in increasing the content of stilbenes was also observed in grapevine cell cultures overexpressing the intron-containing VaSTS11d transcript. Thus, the results indicate that an intron sequence with regulatory elements can have a strong positive effect on both transgene expression level and its biological functions in plants and plant cell cultures. [ABSTRACT FROM AUTHOR]

The demand for metallic nanoparticles synthesized using green methods has increased due to their various therapeutic and clinical applications, and plant biotechnology may be a potential resource facilitating sustainable methods of AgNPs synthesis. In this study, we evaluate the capacity of extracts from Randia aculeata cell suspension culture (CSC) in the synthesis of AgNPs at different pH values, and their activity against pathogenic bacteria and cancer cells was evaluated. Using aqueous CSC extracts, AgNPs were synthesized with 10% (w/v) of fresh biomass and AgNO3 (1 mM) at a ratio of 1:1 for 24 h of incubation and constant agitation. UV-vis analysis showed a high concentration of AgNPs as the pH increased, and TEM analysis showed polydisperse nanoparticles with sizes from 10 to 90 nm. Moreover, CSC extracts produce reducing agents such as phenolic compounds (162.2 ± 27.9 mg gallic acid equivalent/100 g biomass) and flavonoids (122.07 ± 8.2 mg quercetin equivalent/100 g biomass). Notably, AgNPs had strong activity against E. coli, S. pyogenes, P. aeruginosa, S. aureus, and S. typhimurium, mainly with AgNPs at pH 6 (MIC: 1.6 to 3.9 µg/mL). AgNPs at pH 6 and 10 had a high antiproliferative effect on cancer cells (IC50 < 5.7 µg/mL). Therefore, the use of cell suspension cultures may be a sustainable option for the green synthesis of AgNPs. [ABSTRACT FROM AUTHOR]

Stilbenes are plant defense compounds known to rapidly accumulate in grapevine and some other plant species in response to microbial infection and several abiotic stresses. Stilbenes have attracted considerable attention due to valuable biological effects with multi-spectrum therapeutic application. However, there is a lack of information on natural signaling pathways and transcription factors regulating stilbene biosynthesis. It has been previously shown that MYB R2R3 transcription factor genes VaMyb40 and VaMyb60 were up-regulated in cell cultures of wild-growing grapevine Vitis amurensis Rupr. in response to UV irradiation. In this study, the effects of VaMyb40 or VaMyb60 overexpression in cell cultures of V. amurensis on their capability to produce stilbenes were investigated. Overexpression of the VaMyb60 gene led to a considerable increase in the content of stilbenes in three independently transformed transgenic lines in 5.9–13.9 times, while overexpression of the VaMyb40 gene also increased the content of stilbenes, although to a lesser extent (in 3.4–4.0 times) in comparison with stilbene levels in the empty vector-transformed calli. Stilbene content and stilbene production in the VaMyb60-transgenic calli reached 18.8 mg/g of dry weight (DW) and 150.8 mg/L, respectively. Using HPLC analysis, we detected eight individual stilbenes: t-resveratrol diglucoside, t-piceid, t-resveratrol, ε-viniferin, δ-viniferin, cis-resveratrol, cis-piceid, t-piceatannol. T-resveratrol prevailed over other stilbenoid compounds (53.1–89.5% of all stilbenes) in the VaMyb-overexpressing cell cultures. Moreover, the VaMyb40- and VaMyb60-transformed calli were capable of producing anthocyanins up to 0.035 mg/g DW, while the control calli did not produce anthocyanins. These findings show that the VaMyb40 and VaMyb60 genes positively regulate the stilbene biosynthesis as strong positive transcription regulators and can be used in biotechnological applications for stilbene production or high-quality viticulture and winemaking. [ABSTRACT FROM AUTHOR]

Plant cell cultures as valuable tools for the production of specific metabolites can be greatly improved by the application of elicitors including cyclodextrins (CDs) for enhancing the yields of the desired plant compounds. Here the effects of 2,6-dimethyl-ß-cyclodextrins (DIMEB) on the production of carotenoids and quinones from Artemisia annua L. cell suspension cultures were investigated. The addition of 50 mM DIMEB induced an early increase of intracellular carotenoid and quinone contents, which could be observed to a higher extent for lutein (10-fold), Q9 (3-fold) and Q10 (2.5-fold). Real Time PCR analysis revealed that the expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) gene in DIMEB treated cell cultures after three days was 2.5-fold higher than in untreated samples, thus suggesting that the DIMEB induced increase of carotenoids and quinones could be due to the induction of the plastidial isoprenoid biosynthetic route. In addition, the DIMEB treatment induced an enhanced release of carotenoids and quinones into the culture medium of A. annua cell suspension cultures possibly due to the ability of CDs to form inclusion complexes with hydrophobic molecules. [ABSTRACT FROM AUTHOR]

Fluoroaromatic compounds are used as agrochemicals and released into environment as pollutants. Glycosylation of 2-, 3-, and 4-fluorophenols using plant cell cultures of Nicotiana tabacum was investigated to elucidate their potential to metabolize these compounds. Cultured N. tabacum cells converted 2-fluorophenol into its β-glucoside (60%) and β-gentiobioside (10%). 4-Fluorophenol was also glycosylated to its β-glucoside (32%) and β-gentiobioside (6%) by N. tabacum cells. On the other hand, N. tabacum glycosylated 3-fluorophenol to β-glucoside (17%). [ABSTRACT FROM AUTHOR]

Stilbenes are plant phenolics known to rapidly accumulate in grapevine and other plants in response to injury or pathogen attack and to exhibit a great variety of healing beneficial effects. It has previously been shown that several calmodulin-like protein (CML) genes were highly up-regulated in cell cultures of wild-growing grapevine Vitis amurensis Rupr. in response to stilbene-modulating conditions, such as stress hormones, UV-C, and stilbene precursors. Both CML functions and stilbene biosynthesis regulation are still poorly understood. In this study, we investigated the effect of overexpression of five VaCML genes on stilbene and biomass accumulation in the transformed cell cultures of V. amurensis. We obtained 16 transgenic cell lines transformed with the VaCML52, VaCML65, VaCML86, VaCML93, and VaCML95 genes (3–4 independent lines per gene) under the control of the double CaMV 35S promoter. HPLC-MS analysis showed that overexpression of the VaCML65 led to a considerable and consistent increase in the content of stilbenes of 3.8–23.7 times in all transformed lines in comparison with the control calli, while biomass accumulation was not affected. Transformation of the V. amurensis cells with other analyzed VaCML genes did not lead to a consistent and considerable effect on stilbene biosynthesis in the cell lines. The results indicate that the VaCML65 gene is implicated in the signaling pathway regulating stilbene biosynthesis as a strong positive regulator and can be useful in viticulture and winemaking for obtaining grape cultivars with a high content of stilbenes and stress resistance. [ABSTRACT FROM AUTHOR]

Cultivated cardoon (Cynara cardunculus var. altilis L.) is a promising candidate species for the development of plant cell cultures suitable for large-scale biomass production and recovery of nutraceuticals. We set up a protocol for Agrobacterium tumefaciens-mediated transformation, which can be used for the improvement of cardoon cell cultures in a frame of biorefinery. As high lignin content determines lower saccharification yields for the biomass, we opted for a biotechnological approach, with the purpose of reducing lignin content; we generated transgenic lines overexpressing the Arabidopsis thaliana MYB4 transcription factor, a known repressor of lignin/flavonoid biosynthesis. Here, we report a comprehensive characterization, including metabolic and transcriptomic analyses of AtMYB4 overexpression cardoon lines, in comparison to wild type, underlining favorable traits for their use in biorefinery. Among these, the improved accessibility of the lignocellulosic biomass to degrading enzymes due to depletion of lignin content, the unexpected increased growth rates, and the valuable nutraceutical profiles, in particular for hydroxycinnamic/caffeoylquinic and fatty acids profiles. [ABSTRACT FROM AUTHOR]

Cannabis sativa L. is one of the most-studied species for its phytochemistry due to the abundance of secondary metabolites, including cannabinoids, terpenes and phenolic compounds. In the last decade, fiber-type hemp varieties have received interest for the production of many specialized secondary metabolites derived from the phenylpropanoid pathway. The interest in these molecules is due to their antioxidant activity. Since secondary metabolite synthesis occurs at a very low level in plants, the aim of this study was to develop a strategy to increase the production of such compounds and to elucidate the biochemical pathways involved. Therefore, cell suspensions of industrial hemp (C. sativa L. var. Futura) were produced, and an advantageous elicitation strategy (methyl jasmonate, MeJA) in combination with precursor feeding (tyrosine, Tyr) was developed. The activity and expression of phenylalanine ammonia-lyase (PAL) and tyrosine aminotransferase (TAT) increased upon treatment. Through 1H-NMR analyses, some aromatic compounds were identified, including, for the first time, 4-hydroxyphenylpyruvate (4-HPP) in addition to tyrosol. The 4-day MeJA+Tyr elicited samples showed a 51% increase in the in vitro assay (2,2-diphenyl-1-picrylhydrazyl, DPPH) radical scavenging activity relative to the control and a 80% increase in the cellular antioxidant activity estimated on an ex vivo model of human erythrocytes. Our results outline the active metabolic pathways and the antioxidant properties of hemp cell extracts under the effect of specific elicitors. [ABSTRACT FROM AUTHOR]

Many medicinal plant species are currently threatened in their natural habitats because of the growing demand for phytochemicals worldwide. A sustainable alternative for the production of bioactive plant compounds are plant biofactories based on cell cultures and organs. In addition, plant extracts from biofactories have significant advantages over those obtained from plants, since they are free of contamination by microorganisms, herbicides and pesticides, and they provide more stable levels of active ingredients. In this context, we report the establishment of Satureja khuzistanica cell cultures able to produce high amounts of rosmarinic acid (RA). The production of this phytopharmaceutical was increased when the cultures were elicited with coronatine and scaled up to a benchtop bioreactor. S. khuzistanica extracts enriched in RA were found to reduce the viability of cancer cell lines, increasing the sub-G0/G1 cell population and the activity of caspase-8 in MCF-7 cells, which suggest that S. khuzistanica extracts can induce apoptosis of MCF-7 cells through activation of the extrinsic pathway. In addition, our findings indicate that other compounds in S. khuzistanica extracts may act synergistically to potentiate the anticancer activity of RA. [ABSTRACT FROM AUTHOR]

High temperatures are a significant stress factor for plants. In fact, many biochemical reactions involved in growth and development are sensitive to temperature. In particular, heat stress (HS) represents a severe issue for plant productivity and strategies to obtain high yields under this condition are important goals in agriculture. While selenium (Se) is a nutrient for humans and animals, its role as a plant micronutrient is still questioned. Se can prevent several abiotic stresses (drought, heat, UV, salinity, heavy metals), but the action mechanisms are poorly understood. Se seems to regulate reactive oxygen species (ROS) and to inhibit heavy metals transport. In addition, it has been demonstrated that Se is essential for a correct integrity of cell membranes and chloroplasts, especially the photosynthetic apparatus. Previous results showed that in tobacco (Nicotiana tabacum cv. Bright-Yellow 2) cultures HS (5 min at 50 °C) induced cell death with apoptotic features, accompanied by oxidative stress and changes in the levels of stress-related proteins. In this work we investigated the effect of Se on the responses induced by HS. The obtained results show that Se markedly reduces the effects of HS on cell vitality, cytoplasmic shrinkage, superoxide anion production, membrane lipids peroxidation, activity of caspase-3-like proteases, and the levels of some stress-related proteins (Hsp90, BiP, 14-3-3s, cytochrome c). [ABSTRACT FROM AUTHOR]