Callus Cultures Research Articles

Tailoring enhanced production and identification of isoflavones in the callus cultures of Pueraria thomsonii Benth and its model verification using response surface methodology (RSM): a combined in vitro and statistical optimization

BackgroundScientifically, isoflavones from Pueraria thomsonii Benth possess diverse pharmacological activities and have been used to treat various diseases. In vitro propagation of callus has contributed to the reliability for large-scale production of target compounds. However, the factors affecting the biosynthesis of major isoflavones daidzin, puerarin and daidzein in the callus culture of P. thomsonii are still not known. Therefore, we aimed to enhance the in vitro production of daidzin, puerarin and daidzein by optimizing three independent factors such as temperature, NAA and 6-BA concentrations.ResultsOur findings showed that the optimal concentrations for in vitro biomass production and efficient synthesis of puerarin, daidzin and daidzein were found to be 0.158%, 0.463% and 0.057%, respectively. In addition, the HPLC fingerprint with chemo-metrics analysis was constructed by linear regression of the puerarin, daidzin and daidzein which was found to be in the range of 1.0–36.0, 5.0–72.0 and 1.0–15.0 mg/mL and the LODs and LOQs were found to be 0.15, 0.52, 0.35 and 0.28, 1.50, 0.50 mg/mL for puerarin, daidzin and daidzein, respectively. Surprisingly, our results were also in agreement with the concentration obtained from the model verification for optimal and efficient production of puerarin, daidzin and daidzein which was found to be 0.162%, 0.458% and 0.049%, respectively.ConclusionsIn summary, our present investigation provides new insights that could facilitate the enhanced production of valuable isoflavones in P. thomsonii using plant cell cultures treated with appropriate elicitor combinations and temperature. As far as the authors are concerned, this is the first report on production of daidzin, puerarin and daidzein at higher yield at laboratory level for a wide range of applications in future food, medicinal and pharmaceutical companies.

In Vitro Callus Induction in Tacca (Tacca chantrieri Andre) Leaf Explants on Murashige and Skoog Media with Different Concentrations of Sucrose

Tacca chantrieri belongs to the family Taccaceae has black flowers and has a long filiform that looks like a bat. T. chantrieri contains phytochemicals in the form of spritosol saponins used as traditional medicine by the people of China and Thailand. The amount of land clearing, forest exploitation and habitat destruction resulted in a reduction in the number of T. chantrieri, so T. chantrieri was propagated to maintain its sustainability. One way that can be used is the in vitro culture technique, namely callus culture. Callus culture is an early stage of in vitro culture technique where this stage aims to produce and multiply callus cells. The purpose of the study is were to determine the effect of different sucrose concentrations on callus induction from T. chantrieri leaf explants and determine the best sucrose concentrations for callus culture from T. chantrieri leaf explants on Murashige and Skoog (MS) media. This study used a single factor completely randomized design (CRD), namely sucrose concentrations 0 g/l, 10 g/l, 20 g/l, 30 g/l, 40 g/l, 50 g/l with five replications. The results of this study showed that the administrations of sucrose with different concentrations on MS media had an effect on increasing callus induction in Tacca chantrieri leaves. The best sucrose concertations for callus induction of Tacca chantrieri leaves was the addition of 40 g/l sucrose at 20 days after planting, 60 % callus formation percentage, callus formed in the form of compact callus and produce yellow-white callus. Keywords: Tacca chantrieri, sucrose, callus, Murashige and Skoog.

Methyl Jasmonate Activates the 2C Methyl-D-erithrytol 2,4-cyclodiphosphate Synthase Gene and Stimulates Tanshinone Accumulation in Salvia miltiorrhiza Solid Callus Cultures.

The present study characterizes the 5′ regulatory region of the SmMEC gene. The isolated fragment is 1559 bp long and consists of a promoter, 5′UTR and 31 nucleotide 5′ fragments of the CDS region. In silico bioinformatic analysis found that the promoter region contains repetitions of many potential cis-active elements. Cis-active elements associated with the response to methyl jasmonate (MeJa) were identified in the SmMEC gene promoter. Co-expression studies combined with earlier transcriptomic research suggest the significant role of MeJa in SmMEC gene regulation. These findings were in line with the results of the RT-PCR test showing SmMEC gene expression induction after 72 h of MeJa treatment. Biphasic total tanshinone accumulation was observed following treatment of S. miltiorrhiza solid callus cultures with 50–500 μM methyl jasmonate, with peaks observed after 10–20 and 50–60 days. An early peak of total tanshinone concentration (0.08%) occurred after 20 days of 100 μM MeJa induction, and a second, much lower one, was observed after 50 days of 50 μM MeJa stimulation (0.04%). The dominant tanshinones were cryptotanshinone (CT) and dihydrotanshinone (DHT). To better understand the inducing effect of MeJa treatment on tanshinone biosynthesis, a search was performed for methyl jasmonate-responsive cis-active motifs in the available sequences of gene proximal promoters associated with terpenoid precursor biosynthesis. The results indicate that MeJa has the potential to induce a significant proportion of the presented genes, which is in line with available transcriptomic and RT-PCR data.

Cytobacts: Abundant and Diverse Vertically Seed-Transmitted Cultivation-Recalcitrant Intracellular Bacteria Ubiquitous to Vascular Plants

We have recently described ‘Cytobacts’ as abundant intracellular endophytic bacteria inhabiting live plant cells based on the observations with callus and cell suspension cultures of grapevine and other plant species with the origin ascribable to field explants. In this study, we investigated the prevalence of such cytoplasmic bacterial associations in field plants across different taxa, their cultivability, and the extent of taxonomic diversity and explored the possibility of their embryo-mediated vertical transmission. Over 100 genera of field plants were surveyed for ‘Cytobacts’ through bright-field live-cell imaging as per our previous experience using fresh tissue sections from surface-sterilized shoot-tissues with parallel cultivation-based assessments. This revealed widespread cellular bacterial associations visualized as copious motile micro-particles in the cytoplasm with no or sparse colony forming units (CFU) from the tissue-homogenates indicating their general non-cultivability. Based on the ease of detection and the abundance of ‘Cytobacts’ in fresh tissue sections, the surveyed plants were empirically classified into three groups: (i) motile bacteria detected instantly in most cells; (ii) motility not so widely observed, but seen in some cells; and (iii) only occasional motile units observed, but abundant non-motile bacterial cells present. Microscopy versus 16S-rRNA V3–V4 amplicon profiling on shoot-tip tissues of four representative plants—tomato, watermelon, periwinkle, and maize—showed high bacterial abundance and taxonomic diversity (11–15 phyla) with the dominance of Proteobacteria followed by Firmicutes/Actinobacteria, and several other phyla in minor shares. The low CFU/absence of bacterial CFU from the tissue homogenates on standard bacteriological media endorsed their cultivation-recalcitrance. Intracellular bacterial colonization implied that the associated organisms are able to transmit vertically to the next generation through the seed-embryos. Microscopy and 16S-rRNA V3–V4 amplicon/metagenome profiling of mature embryos excised from fresh watermelon seeds revealed heavy embryo colonization by diverse bacteria with sparse or no CFU. Observations with grapevine fresh fruit-derived seeds and seed-embryos endorsed the vertical transmission by diverse cultivation-recalcitrant endophytic bacteria (CREB). By and large, Proteobacteria formed the major phylum in fresh seed-embryos with varying shares of diverse phyla. Thus, we document ‘Cytobacts’ comprising diverse and vertically transmissible CREBs as a ubiquitous phenomenon in vascular plants.

Stimulation of phenolic compounds accumulation and antioxidant activity in in vitro culture of Salvia tebesana Bunge in response to nano-TiO2 and methyl jasmonate elicitors

In the present study, we first attempted to achieve an efficient procedure for optimizing callogenesis from apical meristem and leaf explants of Salvia tebesana on MS media containing different concentrations of BAP alone and in combination with 2,4-D. Then, the inducing effect of nano-TiO2 (10, 60, and 120 mg L−1) and methyl jasmonate (50, 100, and 200 µM), as abiotic elicitors were studied on the enhancement of phenolic compounds, rosmarinic acid, and some individual flavonoids as well as antioxidant capacity of callus. According to the results, the highest callogenesis rate (100 and 93.33, respectively) and DW (0.55 ± 0.03 and 0.36 ± 0.02 g, respectively) per responsive explant were achieved from apical meristem on MS media containing "BAP1 + 2,4-D1" mg L−1 and from leaf explant on the medium supplemented with "BAP0.5 + 2,4-D1" mg L−1. The elicitation with 10 and 60 mg L−1 nano-TiO2 (respectively for apical meristem and leaf), and 50 µM MeJa could significantly promote the production of predominant phenolic derivatives in S. tebesana calli, where the highest content of total phenolics, O-diphenols, phenolic acid, flavonoid, flavone and flavonol, proanthocyanidin was recorded. As well as results declared that the increasing of rosmarinic acid was more correlated with nano-TiO2 treatment than MeJa elicitation. Moreover, the highest content of apigenin (0.33 ± 0.02 μg g−1 DW) was detected after MeJa-elicitation (50 µM), while the maximum level of quercetin (2.62 ± 0.09 μg g−1 DW) and rutin (13.79 ± 0.87 μg g−1 DW) were obtained after exposure to 60 mg L−1 nano-TiO2, both from leaf-derived calli. While a significant positive correlation was recorded between antioxidant assays (DPPH and FRAP) and phenolic derivatives of treated calli; a very strong correlation occurred between the content of rosmarinic acid of apical meristem-derived calli and DPPH and FRAP values (r2 = − 0.921 and r2 = − 0.913, P < 0.01 respectively). Our results showed that the combination of in vitro culture and elicitation would be a good technique to successfully produce and enhance the content of pharmacologically valuable metabolites in S. tebesana. Nano-TiO2 and methyl jasmonate elicitors in a concentration-dependent manner increased the phenolic compounds, specific flavonoids, and antioxidant properties in the callus culture of Salvia tebesana Bunge.

Enhanced Accumulation of Biologically Active Coumarin and Furanocoumarins in Callus Culture and Field-grown Plants of Ruta chalepensis Through LED Light-treatment.

Ruta chalepensis, a medicinal plant, produces biologically active coumarins (CRs) and furanocoumarins (FCRs). However, their yield is quite low in cultivated plants. In this work, the influence of light-emitting diodes (LEDs) was investigated on the accumulation of CRs and FCRs in the callus cultures and field-grown plants of R. chalepensis. Among the various tested wavelengths of LED lights, maximum accumulation of CR and FCRs was recorded under blue LED treatment in both the callus cultures as well as field-grown plants when compared with respective controls treated with white LED. Metabolite analyses of LED-treated field-grown plants showed that highest concentrations of CR (umbelliferone, 2.8-fold), and FCRs (psoralen, 2.3-fold; xanthotoxin, 3.8-fold and bergapten, 1.16-fold) were accumulated upon blue LED-treatment for 6 days. CR and FCRs contents were also analyzed in the blue LED- and red LED-treated invitro callus tissue. Upon blue LED-treatment, callus accumulated significantly high levels of umbelliferone (48.6 ± 1.2 µg g-1 DW), psoralen (370.12 ± 10.6 µg g-1 DW) and xanthotoxin (10.16 ± 0.48 µg g-1 DW). These findings imply that blue LED-treatment is a viable option as a noninvasive and low-cost elicitation technology for the enhanced production of biologically active CR and FCRs in field-grown plants and callus cultures of R. chalepensis.

Secondary metabolites of six Siberian and Crimean Armillaria species and their in vitro phytotoxicity to pine, larch and poplar

Basidiomycetes Armillaria infect deciduous, coniferous and fruit trees, causing enormous economic damage. The role of secondary metabolites (tricyclic sesquiterpene aryl esters - melleolides) in the life cycle and pathogenesis of Armillaria is under active investigation. To date, not all species of Armillaria have been tested for the biosynthesis of melleolides. We investigated the secondary metabolite profiles of six root-pathogenic species of the genus Armillaria (A. borealis Marxmüller & Korhonen, A. cepistipes Velenovský, A. gallica Marxm, A. mellea (Vahl) P. Kummer, A. sinapina Bérubé & Dessur, A. ostoyae (Romagn.) Herink) distributed in Siberia (South Krasnoyarsk Krai, Republic of Tyva, Republic of Khakassia, Taimyr Peninsula), Russian Far East (Sikhote-Alin) and Crimea (Krymsky National Park, Chatyr-Dag Mountain Lower Plateau). A total of 15 compounds were identified in the metabolome profile. Two compounds (melleolide D and melledonal C) are synthesized by all investigated strains irrespective of their geographic location and host plant. The maximum spectrum of melleolides (7-8 compounds) was found in isolates of A. borealis, A. gallica, A. sinapina, A. ostoyae. In submerged culture, the maximum accumulation of melleolides varied from 2 up to 239 mg l-1. A mixture of melleolide D and melledonal C (1:1) synthesized by the most productive strain A. mellea Cr2-17 was first found to have a phytotoxic action on the growth parameters of the callus culture Populus balsamifera and 10-day-old conifer seedlings. A 0.5% concentration of melleolides caused a credible decrease of P. balsamifera callus raw biomass; a decrease of the viability of Larix sibirica and, which is especially significant, Pinus sylvestris seedlings; inhibition of stem and root growth processes; dechromation of foliage; loss of turgor. The occurrence of a broad range of melleolides in the metabolome profile and two common compounds in all investigated strains, with a phytotoxic action at their sufficiently high concentration, enables considering the synthesis of melleolides by Armillaria fungi as one of the possible mechanisms of their pathogenicity efficiently realized in strains characterized by overproduction of melleolides under natural conditions.

Plant cell cultures: An enzymatic tool for polyphenolic and flavonoid transformations

BackgroundIn the pharmaceutical sector, tissue culture techniques for large-scale production of natural chemicals can be a less expensive alternative to large-scale synthesis. Although recent biotransformation research have used plant cell cultures to target a wide range of bioactive compounds, more compiled information and synopses are needed to better understand metabolic pathways and improve biotransformation efficiencies. PurposeThis report reviews the biochemical transformation of phenolic natural products by plant cell cultures in order to identify potential novel biotechnological approaches for ensuring more homogeneous and stable phenolic production year-round under controlled environmental conditions. MethodsArticles on the use of plant cell culture for polyphenolic and flavonoid transformations (1988 – 2021) were retrieved from SciFinder, PubMed, Scopus, and Web of Science through electronic and manual search in English. Following that, the authors chose the required papers based on the criteria they defined. The following keywords were used for the online search: biotransformation, Plant cell cultures, flavonoids, phenolics, and pharmaceutical products. ResultsThe initial search found a total of 96 articles. However, only 70 of them were selected as they met the inclusion criteria defined by the authors. The analysis of these studies revealed that plant tissue culture is applicable for the large-scale production of plant secondary metabolites including the phenolics, which have high therapeutic value. ConclusionPlant tissue cultures could be employed as an efficient technique for producing secondary metabolites including phenolics. Phenolics possess a wide range of therapeutic benefits, as anti-oxidant, anti-cancer, and anti-inflammatory properties. Callus culture, suspension cultures, transformation, and other procedures have been used to improve the synthesis of phenolics. Their production on a large scale is now achievable. More breakthroughs will lead to newer insights and, without a doubt, to a new era of phenolics-based pharmacological agents for the treatment of a variety of infectious and degenerative disorders.

Biotechnology for propagation and secondary metabolite production in Bacopa monnieri.

Bacopa monnieri (L.) Wettst. or water hyssop commonly known as "Brahmi" is a small, creeping, succulent herb from the Plantaginaceae family. It is popularly employed in Ayurvedic medicine as a nerve tonic to improve memory and cognition. Of late, this plant has been reported extensively for its pharmacologically active phyto-constituents. The main phytochemicals are brahmine, alkaloids, herpestine, and saponins. The saponins include bacoside A, bacoside B, and betulic acid. Investigation into the pharmacological effect of this plant has thrived lately, encouraging its neuroprotective and memory supporting capacity among others. Besides, it possesses many other therapeutic activities like antimicrobial, antioxidant, anti-inflammatory, gastroprotective properties, etc. Because of its multipurpose therapeutic potential, it is overexploited owing to the prioritization of natural remedies over conventional ones, which compels us to conserve them. B. monnieri is confronting the danger of extinction from its natural habitat as it is a major cultivated medico-botanical and seed propagation is restricted due to less seed availability and viability. The ever-increasing demand for the plant can be dealt with mass propagation through plant tissue culture strategy. Micropropagation utilizing axillary meristems as well as de novo organogenesis have been widely investigated in this plant which has also been explored for its conservation and production of different types of secondary metabolites. Diverse in vitro methods such as organogenesis, cell suspension, and callus cultures have been accounted for with the aim of production and/or enhancement of bacosides. Direct shoot-organogenesis was initiated in excised leaf and internodal explants without any exogenous plant growth regulator(s) (PGRs), and the induction rate was improved when exogenous cytokinins and other supplements were used. Moreover, biotechnological toolkits like Agrobacterium-mediated transformation and the use of mutagens have been reported. Besides, the molecular marker-based studies demonstrated the clonal fidelity among the natural and in vitro generated plantlets also elucidating the inherent diversity among the natural populations. Agrobacterium-mediated transformation system was mostly employed to optimize bacoside biosynthesis and heterologous expression of other genes. The present review aims at depicting the recent research outcomes of in vitro studies performed on B. monnieri which include root and shoot organogenesis, callus induction, somatic embryogenesis, production of secondary metabolites by in vitro propagation, acclimatization of the in vitro raised plantlets, genetic transformation, and molecular marker-based studies of clonal fidelity. KEY POINTS: • Critical and up to date records on in vitro propagation of Bacopa monnieri • In vitro propagation and elicitation of secondary metabolites from B. monnieri • Molecular markers and transgenic studies in B. monnieri.

Biomass Accumulation of Gynostemma Pentaphyllum (Thunb.) Makino in Cell Suspension Cultures inhibiting Human Cancer Cell Growth

Gynostemma pentaphyllum (Thunb.) Makino (GpM) is a medicinal plant in traditional medicine throughout Asia for the treatment of several diseases including cancer. GpM plant cell suspension cultures provide a time and cost effective well-controlled means promising a high-yielding biomass production of pharmaceutical compounds. The purpose of the current work is to investigate the effect of GpM cell suspension cultures on human cancer cell lines growth. The biomass was produced by cell suspension culture of GpM callus into 250 mL Erlenmeyer flask containing 50 mL of liquid medium culture. Gypenosides in GpM were confirmed by HPLC. Pharmacological activities of GpM extract were tested on human cancer cell line (HepG2, Huh7, A549 and HL-60) using Sulforhodamine B (SRB) cytotoxicity assay and Tetrazolium (MTT) assay. We successfully produced 5.485 ± 0.223 g GpM fresh biomass per 250 mL Erlenmeyer flask after 18 days culture. Total gypenosides and Rb1 in dry cell suspension were 48.844 ± 3.933 mg/g and 0.041 ± 0.004 mg/g. The crude extract from GpM cell suspension cultures exhibited significant cancerous cell growth inhibition in a dose dependent manner. From the MTT assay and SRB cytotoxicity assay, it is obvious that GpM cell suspension culture extract at 200 μg/mL significantly inhibited the growth of multiple human cancer cells including hepatoma cell lines (HepG2, Huh7), lung carcinoma cell line (A549) and leukemia cell line (HL-60). Anti-cancer cell proliferation properties of GpM cell suspension culture were significantly higher than those of natural plants’ extracts. In this framework, GpM in cell suspension cultures was found to inhibit the proliferation of several human cancer cells. Biomass accumulation of GpM in cell suspension cultures may contribute to the development of efficient strategies for plant-derived anticancer compound production.

Lipopolysaccharide and flagellin of Azospirillum brasilense Sp7 influence callus morphogenesis and plant regeneration in wheat.

In vitro somatic callus culturing is used widely in plant biotechnology, but its effectiveness depends largely on the donor plant genotype. Bacteria or components of their cells are rarely used to activate morphogenesis. In this work, inoculation of explants from immature wheat (Triticum aestivum L.) embryos with a suspension of living cells of the bacterium Azospirillum brasilense Sp7 resulted in callus death after 7days of growth, in contrast to explant treatment with a suspension of heat-killed whole cells of Sp7. The experiments used two wheat lines, LRht-B1a and LRht-B1c, which differ in morphogenic activity. Growing calluses with the lipopolysaccharide of A. brasilense Sp7 increased the yield of regenerated plants 2- to 3.5-fold in both lines. This increase was through the activation of regenerant formation from morphogenic calluses. We have demonstrated for the first time the effects of bacterial flagellin on plant tissue culture. The polar-flagellum flagellin of A. brasilense Sp7 leveled the genotypic differences in the morphogenic ability of callus tissue. Specifically, it increased the yield of morphogenic calluses in the weakly morphogenic line LRht-B1a to the yield value in the highly morphogenic line LRht-B1c but lowered the yield of regenerants in the highly morphogenic line LRht-B1c to the yield value in the weakly morphogenic line LRht-B1a. Thus, bacterial lipopolysaccharides and flagellins can be used to regulate the formation of morphogenic calluses and regenerants in plant tissue culturing in vitro.

Establishment of callus and cell suspension cultures of Ariocarpus retusus (Scheidweiler)

Ariocarpus retusus is an endemic species of Mexico subject to special protection by the Mexican government due to its vulnerability to habitat disturbance. Previous studies have reported secondary metabolites in A. retusus with interesting biological activity. However, the use of these secondary metabolites has limitations. The aim of this study was to establish protocols for obtaining friable callus and cell suspension cultures that allow the subsequent development of strategies for obtaining secondary metabolites. Seedlings were obtained from seeds which were exposed to different concentrations of auxins and cytokinins to induce callogenesis. The callus generated was subcultured in Murashige and Skoog (MS) medium with modifications in the content of ammonium nitrate and potassium nitrate, and incubated in different photoperiod conditions (16:8 h light: dark and continuous darkness). Likewise, cell suspensions were established and characterized by friable callus. The MS medium supplemented with 2 mg/L 2,4-D, 2 mg/L BAP, and 3 mg/L KIN allowed the development of vigorous callus. Callus friability was enhanced by decreasing ammonium nitrate (410 mg/L) and increasing potassium nitrate (2850 mg/L). The absence of light during incubation induced friable callus. The addition of myo-inositol (3 mg/L) significantly (p = 0.000) influenced the increase in biomass of cell suspensions.

Investigation of the chemical composition of cell cultures of DIOSCOREA CAUCASICA in connection with the production of BAS sources with cardioprotective potential

Dioscorea Caucasica is a source of various biologically active substances (BAS), e.g. saponins and polyphenols, which are known for their anti-atherosclerotic action. However, this species is in the Red List of many Russian regions. Biotechnological methods of production of callus, suspension, and root cultures in vitro can solve this problem. The research objective was to select the optimal BAS extraction parameters. The study featured in vitro cell cultures, grown according to standard methods, and extracts, obtained from these cultures using various organic solvents and extraction parameters, i.e. temperature, the ratio of raw material vs. extractant, time, etc. The best yield of the extract from callus cultures was observed when methanol was applied in a ratio of 1:10 at 40°C for 60 min. As for suspension cultures, the greatest yield was provided by isopropanol in a ratio of 1:10 at 40°C for 30 min. In case of root cultures, the most effective combination was that of isopropanol in a ratio of 1:10 at 40°C for 60 min. The root culture proved the source of the highest BAS content, namely caffeic acid, rutin, mangiferin, quercetin, and apigenin. The content of rutin was 13 and 22 times higher than that of callus and suspension cultures. TLC, HPLC, and NMR procedures demonstrated that the isopropanol extract contained such saponins as glucopyranoside, rhamnopyranoside, deltoside, protodioscin, spirosthenol A, and spirosthenol B. These substances are known for their antiatherosclerotic properties. Therefore, in v itro cell cultures of Dioscorea Caucasica can be used as raw materials for various pharmaceutical purposes, as well as in functional foods.

Effect of Flame Treatment and Radiofrequency Electromagnetic Radiations on phenolic content in in vitro cultures of Ipomoea batatas

In vitro grown callus cultures of Ipomoea batatas were exposed to flame treatment and electromagnetic radiations generated by mobile phone. The cultured tissues responded to the treatments as evidenced by the significant reduction of phenolic contents compared to controls. Even though the growth of the tissues was normal, there was a change in the phenolic content of the tissues. There exhibited not much significant variation among the treatments regarding the growth rate. The morphology and texture of the callus also remained the same. It has been concluded that like animal cells, plant cells also respond to non-ionizing radiations like electromagnetic radiation emitted by mobile phones.

Effect of UV-B Radiation on Flavonoids and Phenols Accumulation in Tempisque (Sideroxylon capiri Pittier) Callus.

Tempisque (Sideroxylon capiri Pittier) is classified as a threatened species and has been reported with a high content of phenols and flavonoids in the leaves. The use of abiotic elicitors such as radiation has been reported due to the changes it produces in the metabolism of plants by activating their defense mechanisms and increasing the biosynthesis of bioactive compounds with antioxidant capacity such as phenols and flavonoids. Therefore, the aim of this work was to evaluate the effect of UV-B radiation on growth parameters and the synthesis of bioactive compounds in in vitro culture of tempisque callus. For the callus induction, we used thidiazuron (TDZ) and 2,4-dichlorophenoxyacetic acid (2,4-D) at 0, 0.5 and 1 mg/L. Calluses were exposed to UV-B radiation (0, 1, 2, 3 and 4 h/day) for two and four weeks. The highest callus formation index was obtained with TDZ and 2,4-D at 1 mg/mL. The greatest increase in the concentration of phenols and flavonoids was detected in the fourth week with 4 h of exposure per day. The highest concentrations of quercetin (230 µg/g dry weight), kaempferol (235 µg/g dry weight) and gallic acid (240 µg/g dry weight) were found in callus obtained from leaves explants.

Impacts of heavy metal, high temperature, and UV radiation exposures on Bellis perennis L. (common daisy): Comparison of phenolic constituents and antioxidant potential (enzymatic and non-enzymatic)

Bellis perennis L., common daisy, is a beneficial medicinal plant used in folk medicine to treat skin diseases, wounds, sore throats, common colds, inflammation, and rheumatism. The objective of this study was to determine the effect of 4 different abiotic stress applications [mercury chloride (HgCl2), copper sulfate (CuSO4), high temperature (45 °C) and ultraviolet radiation (UV)] on individual phenolic constituents, non-enzymatic antioxidant activities (free radical scavenging activity, total phenol-flavonoid content, and proline level), and enzymatic antioxidant activities [superoxide dismutase (SOD) and catalases (CAT)] in common daisy callus cultures. Quantification of the 21 phenolic substances was performed using LC-ESI-MS/MS analysis and all applied stress applications increased specific phenolic constituents comparing with control (no exposure to any stress). Heavy metal stresses (HgCl2 and CuSO4) and high temperature application enhanced the quantity of the selected phenolic substances more than UV-radiation stress. The most noticeable increased quantities were observed with high temperature stress in p-coumaric acid (51.74-fold), and with CuSO4 stress in luteolin (17.76-fold) and apigenin (15.62-fold). Chlorogenic acid was the most prevalent phenolic compound in all tested callus extract including control. Although the first highest total phenol content was obtained with HgCl2 exposure and then followed by high temperature stress, the first highest total flavonoid content was obtained with high temperature stress and then UV radiation. The antioxidant capacity was significantly enhanced (2.11 fold increase) with CuSO4 stress application. Stress-related enzymatic antioxidant activity (SOD and CAT) was elevated the most with HgCl2 stress. Similarly, the highest proline content was obtained with HgCl2 treatment. These findings showed the ability of the phenolic compounds to protect common daisy against harmful heavy metals, high temperature and UV-radiation stresses. The adaptation of the callus under abiotic stress conditions developed here may be useful for the production of optimized phenolic compounds of common daisy and the exploitation of other medicinal species of interest.

Fruit derived callus and cell suspension culture as promising alternative sources for mogrosides production in Siraitia grosvenorii (Swingle) C. Jeffrey: a zero-caloric natural sweetener

The fruit of Siraitia grosvenorii (Luo Han Guo) is commercially consumed for its zero-calorie natural sweetener compounds known as mogrosides. The present work aimed to establish a cell culture platform from the fruit mesocarp and provide an alternative source for mogrosides production in S. grosvenorii. Bavistin at 1.0% with mercuric chloride at 0.1% were effective concentrations for explants sterilization. The maximum frequency of callus induction was observed in mesocarp (95.78%) and stem (97%) explants cultured on Murashige and Skoog (MS) medium supplemented with thidiazuron (0.5 mg/L) and picloram (2.5 mg/L). The maximum proliferation response of suspended cells in suspension culture was observed in MS medium fortified with thidiazuron (0.3 mg/L) and indole-3-butyric acid (0.3 mg/L). Mogroside V (2.96 mg/g DW), 11-oxo-mogroside V (0.66 mg/g DW), siamenoside I (0.26 mg/g DW), and mogroside IIIE (0.08 mg/g DW) were quantified in mesocarp callus using UPLC–PDA–ESI-Q-TOF-MS technique. A comparable content of mogroside V (1.76 mg/g DW) and 11-oxo-mogroside V (0.68 mg/g DW) was also quantified in cell suspension derived from mesocarp callus with enhanced cell biomass (FW; 184.58 g/L). Therefore, the in vitro culture derived from fruit mesocarp was shown to be a potential source for mogrosides production.

Positive Response of Hyoscyamus pusillus Callus Cultures to Exogenous Melatonin on Biochemical Traits and Secondary Metabolites under Drought Conditions

There is increased attention from specialists toward producing natural compounds from plant tissues through the combined use of elicitors. The present study investigated the effects of melatonin treatment in normal and drought stress conditions for sustainable production of biomass and secondary antioxidant metabolites in a Hyoscyamus pusillus four-month-old callus cultures. For this purpose, we used multiple concentrations of melatonin (0, 0.5, 1.0, 1.5, and 2.0 mg l−1) to assess its ability to improve growth, physiological, and biochemical properties of H. pusillus. There was two drought stress levels (0 and 30 g l−1) of polyethylene glycol (PEG). It was harvested in 28 days. The results showed a significant decrease with an increase in the concentration of PEG treatment in growth and physiological traits compared to the results of those samples when treated with melatonin. Results showed an increase in oxidative stress in tissue treated with PEG due to a significant increase in the content of hydrogen peroxide (H2O2) and malondialdehyde (MDA). The decreased oxidative stress was associated with an increased antioxidant enzyme activity superoxide dismutase (SOD), and catalase (CAT) in the samples was treated by melatonin, which resulted in increased membrane stability index (MSI) and enhanced growth traits under the PEG treatment compared to the control. By reducing the activity of phenylalanine ammonia-lyase (PAL) and peroxidase (POX), moisture tightening increased the production of phenolic compounds (PC) and flavonoid compounds (FC) in callus cultures, and high concentrations of melatonin were combined with them to improve their production. Therefore, it can be asserted that a moderate treatment with melatonin is more suitable under water stress conditions to produce secondary compounds from H. pusillus.

The regulating effect of light on the content of flavan-3-ols and derivatives of hydroxybenzoic acids in the callus culture of the tea plant, Camellia sinensis L

The objective was the study of the regulating effect of light on the composition and contents of phenolic compounds in the callus culture of the tea plant (Camellia sinensis) grown for 28 days in the light (16-h photoperiod) or in the dark. For the analyses of phenolic compounds, an ultra-performance liquid chromatography in combination with a photodiode array detector and a high-resolution Q Exactive Orbitrap mass spectrometry (UPLC-PDA-HRMS/MS) was used. Nineteen phenolic compounds were characterized. The main phenolic compounds were flavan-3-ols (mono- and oligomers of flavan-3-ols) and derivatives of hydroxybenzoic acids (mono- and digalloyl glucoses, hexosides of 2,5- and 2,3-dihydroxybenzoic and salicylic acids). It was shown that the growth of the tea callus in the light or in the dark does not affect the composition of phenolic compounds, but differently affects the content of flavan-3-ols and derivatives of hydroxybenzoic acids. In the callus grown in the light, the content of flavan-3-ols was about 2-fold higher than in the dark. On the contrary, the content of derivatives of hydroxybenzoic acids in the light was at average 3-fold lower. The regulating role of light in the biosynthesis of phenolic compounds, as well as light control of the distribution of carbon flux between the branches of the shikimate pathway, which are associated with the biosynthesis of flavan-3-ols and hydroxybenzoic acids, are discussed.

A novel spray bioreactor for the proliferation of plant callus; Hyoscyamus niger and Arnebia pulchra.

Unlike plant cell suspension culture, the proliferation of callus in bioreactors has received inadequate attention. The magnificent potential of plant callus becomes more appreciated as the research unfolds and promises interesting applications including the production of valuable metabolites, therapeutic antibodies, bioactive extracts with regenerating effects, and the generation of genetically improved plants. Issues such as the lack of 3D-access of the cells to the nutrients, using an interfering gelling substance as the support matrix, and the changes in the medium formulation during the growth phase were discouraging factors for extending research on this topic. Considering the existing drawbacks, a novel open-flow spray bioreactor (OFSB) was configured to circumvent the associated problems with the solid cell culture and promote the applicability of plant callus culture via improving the feeding strategy. Applying similar subculture conditions, the proliferation of Arnebia pulchra and Hyoscyamus niger calli as the examples of two important plant families (Boraginaceae and Solanaceae) was studied in the OFSB in comparison with similar calli that grew in Petri dishes and jars. A. pulchra and H. niger calli obtained the weight gains of (%87.3 and %106.7) in the Petri dishes, (%208.7 and %226) in the jars, and (%288.6 and %320.0) in OFSB, respectively, while no significant changes were observed in the productivity indices of the examined calli. The simple design of OFSB bypasses most of the notorious problems associated with solid plant callus culture. OFSB technical features allow the bioreactor to be used for growth optimization of various types of plant calli in a cost-effective manner.