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Main conclusion: Long-term cultured calli may experience a biosynthetic shift due to the IAA-dependent expression of the rolA gene, which also affects ROS metabolism. The “hairy root” syndrome is caused by the root-inducing Ri-plasmid of Rhizobium rhizogenes, also known as Agrobacterium rhizogenes. The Ri-plasmid contains genes known as rol genes or root oncogenic loci, which promote root development. The important implications of the rolA gene from the T-DNA include reduced plant size, resistance to infections, and the activation of specialised metabolism. Nevertheless, rolA does not belong to the plast gene group because its function is still uncertain. Recent investigations have shown two important effects of the rolA gene. First, the production of secondary metabolites has changed in long-term cultivated rolA-transgenic calli of Rubia cordifolia L. Second, the expression of both the rolA and rolB genes has a strong auxin-dependent antagonistic effect on reactive oxygen species (ROS) homeostasis. In this work, we attempted to elucidate two rolA gene phenomena: what caused the secondary metabolism of long-term cultured calli to change? How does the individual expression of the rolA gene affect ROS homeostasis? We analysed SNPs in the 5′ untranslated region and coding region of the rolA gene. These mutations do not affect the known essential amino acids of the RolA proteins. Notably, in the promoter of the rolA gene, an ACTTTA motif for auxin-mediated transcription factors was identified. Using two separate cell cultures, we demonstrated the strong auxin dependence of rolA gene expression. The expression of genes involved in ROS metabolism decreased in response to an auxin-mediated increase in rolA gene expression. Two assumptions can be made. The long-term cultivation of calli may cause changes in the hormonal state of the culture over time, which may modulate the action of the RolA protein. Moreover, auxin-dependent expression of the rolA gene led to a decrease in ROS metabolism. It can be assumed that the antagonistic interaction between rolA and rolB prevents strong rolB-induced auxin sensitivity and oxidative bursts to balance the cell state. [ABSTRACT FROM AUTHOR]

The Persian walnut, or English walnut, is one of the most prized walnuts, popular for its timber, medicinal properties, oil, and nuts. Due to its high demand across the globe, walnut cultivation has exponentially grown over the years. Despite having the appropriate climate, the state of Uttarakhand still does not cultivate walnut on a large scale. The alternative to growing walnuts with better quality and high production is through in vitro propagation. The present study was conducted to propagate walnut varieties growing in the Uttarakhand region through direct and indirect organogenesis. The DKW medium proved to be the most effective for nodal explant culture, while the MS medium yielded superior results for callus induction. We also optimized the media by varying the liquid/semisolid medium and plant growth regulator combinations. The genotype from higher altitudes of Uttarakhand was found to respond better to the in vitro treatments. The findings can prove useful in propagating good-quality walnut varieties in Uttarakhand on a large scale. [ABSTRACT FROM AUTHOR]

AbstractThe coding sequence of human basic fibroblast growth factor (hbFGF) was optimised for expression in rice. An expression cassette was constructed by fusing the PCR-amplified RAmy3D promoter, along with its 5’UTR, 3’UTR, and terminator sequences, to the codon-optimised hbFGF sequence. This cassette was inserted into the pCAMBIA1304 shuttle vector, which also contained the RAmy3D signal peptide. Agrobacterium tumefaciens strain LBA 4404 was used to transform rice callus. Among the transformed lines, the callus expressing the highest level of bFGF (38.1 mg/kg fresh weight) was identified via ELISA and selected for establishing a cell suspension culture. Expression and secretion of the recombinant bFGF into the culture medium were observed three days after incubating the transgenic rice cells in sucrose-free medium. The presence of recombinant bFGF was confirmed through Western blot and SDS-PAGE analyses. Furthermore, the rice-derived bFGF effectively stimulated the proliferation of NIH/3T3 cells, demonstrating a comparable biological activity to that of commercial bFGF. [ABSTRACT FROM AUTHOR]

The cryopreservation of Anthurium andraeanum germplasm resources is extremely important for the production and selection of new varieties. At present, the cryopreservation procedure for the callus of A. andraeanum has not been established. In this study, the leaves of A. andraeanum were used as explants to culture the callus. The cryopreservation procedure of the callus by vitrification was initially established by using the orthogonal experimental method of four factors and three levels in the preculture, loading, and dehydration steps. Furthermore, the vitrification-based cryopreservation was optimized by changing the preculture temperature and loading solution and adding exogenous substances to the plant vitrification solution (PVS2). In this procedure, the callus was precultured at 25 °C for 2 d, and loaded in 50% PVS2 at 25 °C for 60 min. The callus was dehydrated with PVS2 containing 0.08 mM reduced glutathione (GSH) at 0 °C for 60 min. After rapid-cooling in liquid nitrogen for 1 h, it was rapid-warming in a water bath at 40 °C for 90 s and unloaded for 30 min. After 1 d of recovery, the cell relative survival rate of the cryopreserved callus was 64.60%. The results provide a valuable basic and effective method for the long-term conservation of A. andraeanum germplasm resources. [ABSTRACT FROM AUTHOR]

The method of callus culture is a convenient tool for assessing of intracellular changes occurring in response to adverse effects, which reduces the experimental time and laboratory resources. Factors of intracellular resistance of coniferous trees to low positive temperatures have not been studied enough. The study of the sustainability mechanisms of the economically important species and forest-forming tree species, Scots pine, is relevant for the research region. The objective of the research was to assess the suitability of callus culture obtained from Scots pine (Pinus sylvestris L.) buds for studying the factors of bud cell resistance to low positive temperatures. Callus cultures obtained on bud explants were exposed to +4 or +1 °C for 7 days, after which the growth rate of the cultures, cell viability, and oxidative stress indicators (the content of hydrogen peroxide and superoxide anion; the activity of peroxidase and lipid peroxidation) were assessed. Before the exposure, callus cultures differed in growth rate, viability and in the development of oxidative stress, which is probably due to the genetic differences in the trees from which the buds were obtained, and is consistent with the data of other studies. Low positive temperatures suppress the growth of cultures, which indicates the restructuring of cell metabolism in the direction of protective activity. After exposure to low positive temperatures, all cultures were characterized by an increase in lipid peroxidation activity, indicating the development of oxidative stress. The absence of negative changes in cell viability and of an increasing in the content of hydrogen peroxide in all studied cultures indicates the ability of cells to withstand unfavorable changes. An increase in peroxidase activity found in all cultures indicates the activation of protective mechanisms. An increase in the content of superoxide anion may indicate the development of protective signaling. The study showed the ability of pine bud callus culture cells to withstand the used cold exposure, which makes the callus culture at researched conditions a useful test system for studying the mechanisms of cold resistance of Scots pine buds.

ABSTRACT Nanoparticles (NPs) are well‐known biostimulants in plant biotechnology, utilised to enhance the physical properties of plants and exhibit positive effects on them. The important key role is the most suitable type, effective dose and size of NP to be used in plant tissue culture systems. In this study, various concentrations of silver nanoparticles (AgNPs; 0, 25, 50, 75 and 100 μg L−1) were tested as elicitors in callus culture with the aim of enhancing secondary metabolite production in lemon balm (Melissa officinalis L.). According to the results obtained, callus formation rates have shown an increase in all applications compared to the control group. The highest callus formation, weight and diameter were observed in 50 μg L−1 application. In this application, the callus structure was compact and its colour was green. However, the aromatic compounds, neral and geranial increased significantly in 25 μg L−1 application. The maximum increase in phenolic compounds such as caffeic acid, chlorogenic acid, proto‐catechic acid, hesperidin and p‐coumaric acid was observed in the 75 μg L−1 AgNP and the highest increase in rosmarinic acid compound was determined in the 50 μg L−1 application. The study found that AgNP applications are an effective method for increasing the production of secondary metabolites in medicinal and aromatic plants, such as lemon balm, in vitro. [ABSTRACT FROM AUTHOR]

In vitro tissue culture can be an alternative method for endangered species propagation, biodiversity conservation and secondary metabolite studies. Paratecoma peroba (Record) Kuhlm. (Bignoniaceae) is an endemic and endangered Brazilian species. This work aimed to establish in vitro morphogenesis and callus induction and to perform a phytochemical analysis of P. peroba callus extract. Higher seed germination (43%) was obtained in Wood Plant Medium culture without activated charcoal (AC). Combination of 5 µM benzyladenine + 10 µM gibberellic acid, without AC, resulted in a higher number of shoots (2 shoots/explant). A callus culture was stabilised from zygotic embryos using 2,4-dichlorophenoxyacetic acid. A callus methanolic extract was used for phytochemical analysis. The isolated substance was identified as tiliroside (kaempferol 3-O-β-D-(6"-O-E-p-coumaroyl)-glucopyranoside) by NMR and quantified in callus and leaf extracts by HPLC. This study adds to the chemical knowledge of this species and it is the first report of a flavonol in Paratecoma. [ABSTRACT FROM AUTHOR]

Under a water deficit, the protective proteins known as dehydrins (DHNs) prevent nonspecific interactions in protein and membrane structures and their damage, in addition to playing an antioxidant role. The DHNs of a widespread xerophytic species Scots pine (Pinus sylvestris L.) have been poorly studied, and their role in resistance to water deficits has not been revealed. In this paper, we have expanded the list of DHNs that accumulate in the cells of Scots pine under the conditions of water deficits and revealed their relationship with the effects of water deficits. In this investigation, callus cultures of branches and buds of Scots pine were used. A weak water deficit was created by adding polyethylene glycol to the culture medium. Under the conditions of a water deficit, the activity of catalase and peroxidase enzymes increased in the callus cultures. A moderate decrease in the total water content was correlated with a decrease in the growth rate of the callus cultures, as well as with an increase in the activity of lipid peroxidation. The accumulation of Mr 72, 38, and 27 kDa DHNs occurred in the callus cultures of buds, and the accumulation of Mr 72 and 27 kDa DHNs positively correlated with the lipid peroxidation activity. An increase in the content of DHNs was observed in cultures that differed in origin, growth indicators, and biochemical parameters, indicating the universality of this reaction. Thus, previously undescribed DHNs were identified, the accumulation of which is caused by water deficiency and is associated with manifestations of oxidative stress in the kidney cells of Scots pine. [ABSTRACT FROM AUTHOR]

This study explores the in vitro cultivation of Chiliadenus montanus (Vhal.) Brullo (Asteraceae), focusing on callus multiplication, in vitro seed germination, phenolic compound production, and antioxidant activity. Callus induction was optimized, followed by multiplication using Murashige and Skoog (MS) media supplemented with 1.0 mg·L⁻¹ 2,4-Dichlorophenoxyacetic acid (2,4-D) and 2.0 mg·L⁻¹ 6-Benzylaminopurine (BAP). The highest in vitro germination rate of C. montanus seeds (11.6 ± 2.22%) was achieved using half-strength MS media supplemented with 0.5 mg·L⁻¹ gibberellic acid (GA₃) and 1.0 mg·L⁻¹ BAP. Methanol extracts from wild and in vitro samples were analyzed for Terpinen-4-ol, Eucalyptol (1,8-Cineole), and total phenolic content. In vitro microshoots exhibited an elevated Terpinen-4-ol concentration (0.01 ± 0.003 mg/g) compared to wild plants, while the concentrations of Eucalyptol (0.06 ± 0.001 mg/g) were similar in both microshoots and wild plants. Phenolic compound analysis revealed maximum levels in wild plants (30.67 ± 2.82 gallic acid equivalents [GAE]), followed by microshoots (22.81 ± 0.65 GAE), and the lowest in callus (6.37 ± 0.27 GAE). Antioxidant properties, evaluated via the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, indicated superior radical scavenging in wild plants (Inhibitory Concentration 50 [IC50] 32.13 ± 0.83 μg/ml) compared to greenhouse plants (IC50 221.04 ± 1.34 μg/ml). C. montanus emerges as a potential natural antioxidant source. In conclusion, an effective in vitro production system for phenolic compounds in C. montanus was established, offering a sustainable alternative to wild plant harvesting. The study highlights the potential benefits of C. montanus as a reservoir of bioactive substances and emphasizes the importance of in vitro cultivation for sustainable resource utilization. [ABSTRACT FROM AUTHOR]

Background: Recent interest in plant-derived exosome-like nanoparticles (ENs) has surged due to their therapeutic potential, which includes antioxidant, anti-inflammatory, and anticancer activities. These properties are attributed to their cargo of bioactive metabolites and other endogenous molecules. However, the properties of ENs isolated from plant cell cultures remain less explored. Methods: In this investigation, grape callus-derived ENs (GCENs) were isolated using differential ultracentrifugation techniques. Structural analysis through electron microscopy, nanoparticle tracking analysis, and western blotting confirmed that GCENs qualify as exosome-like nanovesicles. Results: These GCENs contained significant amounts of microRNAs and proteins characteristic of plant-derived ENs, as well as trans-δ-viniferin, a notable stilbenoid known for its health-promoting properties. Functional assays revealed that the GCENs reduced the viability of the triple-negative breast cancer cell line MDA-MB-231 in a dose-dependent manner. Moreover, the GCENs exhibited negligible effects on the viability of normal human embryonic kidney (HEK) 293 cells, indicating selective cytotoxicity. Notably, treatment with these GCENs led to cell cycle arrest in the G1 phase and triggered apoptosis in the MDA-MB-231 cell line. Conclusions: Overall, this study underscores the potential of grape callus-derived nanovectors as natural carriers of stilbenoids and proposes their application as a novel and effective approach in the management of cancer. [ABSTRACT FROM AUTHOR]

Ajuga L., a member of the Lamiaceae family, is one of the most popular species due to its medicinal, decorative, and pharmacological properties. One of them is Ajuga vestita BOISS. which is endangered and placed in the "Endangered (EN)" category. In the present study, using in vitro tissue culture techniques, Ajuga vestita BOISS. seeds were germinated in hormone-free 1/4 MS medium, and callus were regenerated from leaf explants of germinated seeds in 1/1 MS medium containing BAP (benzylaminopurine) and Kin (kinetin). It was aimed at comparatively examining the fatty acid content of Ajuga vestita BOISS. callus. When the fatty acid content of the callus obtained from the control (hormone-free), BAP, and Kin media were examined, major saturated fatty acid amounts were palmitic acid in all three extracts (59.137%, 38.836%, and 24.295%) for control, BAP, and Kin respectively. The major unsaturated fatty acid amounts were octadecanoic acid (21.097%, 32.283%) for control and BAP, respectively, and linoleic acid (21.209%) for Kin considering that high amounts of unsaturated fatty acids make the plants more reliable in terms of health, the higher amount of unsaturated fatty acid content determined in the Kin extract in our study compared to the control will shed light on future studies in this sense that can be done with tissue culture techniques. [ABSTRACT FROM AUTHOR]

Plants contain many useful substances; however, their availability and uniformity are constrained. Controlled cell culture is attracting attention as a method for identifying useful substances with better quality control than conventional plant extraction techniques. Artificial skin is widely used as an alternative to animal testing to evaluate the safety and efficacy of cosmetic materials. The Damnacanthus major Siebold & Zucc is an evergreen shrub of the madder family, and the anthraquinone series substances present in its roots have antioxidant and whitening effects, implying its potential to be used in cosmetics. In this study, the antioxidant, moisturizing, and phototoxic properties of the ethanol extract of its callus were assessed using an artificial skin alternative. It showed a distinct antioxidant effect over 600 μg/mL and a moisturizing effect over 125 μg/mL. Phospho-extracellular signal-regulated kinase 1/2 and cAMP response element-binding protein were found to be upregulated in the artificial skin due to the moisturizing mechanism, and phospho-NFκB repressing factor was observed to be upregulated in human epidermal keratinocytes (HaCaT cells) due to the antioxidant mechanism. Additionally, analysis of the artificial skin revealed no phototoxicity up to 1 mg/mL. The results of this study demonstrate that the ethanol extract of Damnacanthus major Siebold & Zucc can be used as a cosmetic material. [ABSTRACT FROM AUTHOR]

Bursera fagaroides, popularly used in México, possesses bioactive lignans. These compounds are low in the bark, and its extraction endangers the life of the trees. The aim of the present investigation was to search for alternative sources of cytotoxic compounds in B. fagaroides prepared as leaves and in vitro callus cultures. The friable callus of B. fagaroides was established using a combination of plant growth regulators: 4 mgL−1 of 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mgL−1 Naphthaleneacetic Acid (NAA) and 1 mgL−1 Zeatin. The maximum cell growth was at day 28 with a specific growth rate of μ = 0.059 days−1 and duplication time td = 11.8 days. HPLC quantification of the dichloromethane callus biomass extract showed that Scopoletin, with a concentration of 10.7 µg g−1 dry weight, was the main compound inducible as a phytoalexin by the addition of high concentrations of 2,4-D, as well as by the absence of nutrients in the culture medium. In this same extract, the compounds γ-sitosterol and stigmasterol were also identified by GC-MS analysis. Open column chromatography was used to separate and identify yatein, acetyl podophyllotoxin and 7′,8′-dehydropodophyllotoxin in the leaves of the wild plant. Cytotoxic activity on four cancer cell lines was tested, with PC-3 prostate carcinoma (IC50 of 12.6 ± 4.6 µgmL−1) being the most sensitive to the wild-type plant extract and HeLa cervical carcinoma (IC50 of 72 ± 5 µgmL−1) being the most sensitive to the callus culture extract. [ABSTRACT FROM AUTHOR]

The morphological, cytophysiological, and biochemical (formation of phenolic compounds) characteristics of the population of callus cultures cells of lemon balm (Melissaofficinalis L.), a valuable medicinal and essential oil plant, were studied. Callus cultures were obtained from explants of hypocotyls and cotyledons of seedlings in vitro and were grown for more than 1.5 years (19 cultivation passages). The increase in callus weight did not differ significantly during the first seven passages; however, with further subcultivation, the intensity of crop growth (callus weight at the end of the growing cycle) increased. The maximum increase in callus was observed in passages 17–19: the growth indices of calli initiated from cotyledons and hypocotyls reached 13.7 and 11.5, respectively, which is 3.0–3.4 times higher than in the first cycles of crop cultivation. The obtained data indicate the possibility of long-term subcultivation of callus cultures of lemon balm cells, during which autoselection of cells occurs based on growth intensity. The dynamics of callus growth, density and viability of the cell population, and the ratio of various cell types in the culture growth cycle were determined for cell culture M. officinalis for the first time. The duration of the main phases of cell population growth has been established: lag phase from 1 to 6 days; growth acceleration phase from 6 to 10 days. The exponential growth phase took place from 10 to 14 days and was characterized by a high specific growth rate µ = 0.21 days–1 From days 14 to 20, fixed phase of slowing down the growth of the culture (µ = 0.05 days–1), which gave way to a phase of linear growth (20–30 days, µ = 0.08 days–1) and stationary phase (30–40 days of the growth cycle). Thus, a “stepwise” character of culture growth has been established, which may be due to the presence in the culture of subpopulations of cells with different growth rates. During the initial screening, flavonoids and phenolcarboxylic acids were identified in calli of leaf origin in quantities comparable to leaves of intact plants, which indicates preservation of in vitro cells ability to form secondary metabolites and the prospects for further research in this direction. [ABSTRACT FROM AUTHOR]

Callus cultures of the Iranian medicinal plant Salvia atropatana were initiated from three-week-old seedlings on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) and various cytokinins. Although all tested hormonal variants of the medium and explant enabled callus induction, the most promising growth was noted for N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU)-induced calli. Three lines obtained on this medium (cotyledon line-CL, hypocotyl line—HL, and root line—RL) were preselected for further studies. Phenolic compounds in the callus tissues were identified using UPLC–MS (ultra-performance liquid chromatography–mass spectrometry) and quantified with HPLC (high-performance liquid chromatography). All lines exhibited intensive growth and contained twelve phenolic acid derivatives, with rosmarinic acid predominating. The cotyledon-derived callus line displayed the highest growth index values and polyphenol content; this was exposed to different light-emitting diodes (LED) for improving biomass accumulation and secondary metabolite yield. Under LED treatments, all callus lines exhibited enhanced RA and total phenolic content compared to fluorescent light, with the highest levels observed for white (48.5–50.2 mg/g dry weight) and blue (51.4–53.9 mg/g dry weight) LEDs. The selected callus demonstrated strong antioxidant potential in vitro based on the 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) tests. Our findings confirm that the S. atropatana callus system is suitable for enhanced rosmarinic acid production; the selected optimized culture provide high-quality plant-derived products. [ABSTRACT FROM AUTHOR]

This study explored how plant growth regulators (PGRs) impact callus induction, biomass accumulation, phytochemical synthesis, and the antioxidant and anti-aging potential of Alcea rosea L. callus cultures. Our findings revealed that the combination of 1 µM NAA and 0.1 µM TDZ leads to 100% callus induction frequency and the highest biomass accumulation, with a fresh weight of 267 g/L and a dry weight of 16.5 g/L. Maximum total phenolic (719.19 mg GAE/L) and total flavonoid (166.34 mg QE/L) production were observed in callus cultures treated with 1 µM NAA. High-performance liquid chromatography identified 9 anthocyanin compounds, with the highest total anthocyanin production (265.65 µM DW) recorded with a PGR concentration of 1 µM NAA + 0.1 µM TDZ. Antioxidant activities varied across different PGR concentrations, with notable values including DPPH-FRSA of 83.1%, ORAC value of 1089.13 µM TEAC, and maximum reduction in ROS/RNS levels by 87.56%. Anti-aging activities also varied, with tyrosinase inhibition peaking at 79.12%, vesperlysine-like AGEs’ inhibition at 68.32%, and pentosidine-like AGEs’ inhibition at 56.42%. This study introduces a novel approach that addresses traditional extraction method limitations by examining the impact of PGRs on callus culture initiation and secondary metabolite production, alongside antioxidative and anti-aging characteristics. Such methodologies potentially add to our understanding of A. rosea L.‘s therapeutic potential and contribute to sustainable techniques for producing bioactive compounds.Key message: This study explores the impact of plant growth regulators on Alcea rosea callus cultures, identifying optimal conditions for maximum anthocyanin production and highlighting their antioxidant and anti-aging potential, crucial for commercial anthocyanin resources. [ABSTRACT FROM AUTHOR]

This study investigates the potential of biologically synthesized silver nanoparticles (AgNPs) with Maerua crassifolia leaf extracts to enhance the production of bioactive compounds in Ruta chalepensis callus, alongside evaluating biomass growth, phytochemical content, antioxidant enzymes, and activities. AgNPs were characterized using transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), and UV-visible spectroscopy (UV), then introduced at various concentrations (0, 7.5, 15, 30, and 60 mg/L) into Murashige and Skoog (MS) media supplemented with 10 µM 2,4-Dichlorophenoxyacetic acid (2,4-D) and 1.0 µM 6-benzyladenine (BA). Notably, the incorporation of 30 mg/L of AgNPs into the MS medium resulted in the highest callus fresh weight (FW: 4.13 g) and callus dry weight (DW: 0.298 g) compared to both the control and other AgNPs treatments. Furthermore, this concentration exhibited elevated levels of protein (24.95 mg/ml), phenolic (90.58 mg GAE/g DW), tannin (18.57 mg TAE/g DW), flavonoid (70.97 mg QE/g DW), and flavonol (27.5 mg RE/g DW). Additionally, the enhanced antioxidant enzyme activities, including catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX) (18.81, 5.08, and 9.48 U/mg protein, respectively) were also observed at the same treatment. The 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS: 88.03%) assay and the 2,2-diphenylpicrylhydrazyl (DPPH: 85.25%) assay also showed increased activity at 30 mg/L of AgNPs, while the ferric reducing antioxidant power (FRAP) assay indicated maximum activity (33.82 mg AAE/g DW) at 60 mg/L of AgNPs. Moreover, the application of AgNPs at 30 mg/L resulted in of higher concentrations of quercetin (119.15 µg/g DW), rutin (192.96 µg/g DW), and hesperidin (219.87 µg/g DW). In conclusion, the results indicate that incorporating green AgNPs has a positive influence on the phyto-biochemical profile of R. chalepensis callus cultures, leading to enhanced levels of antioxidants and phytochemicals. Key message: Biologically synthesized AgNPs from Maerua crassifolia enhance bioactive compound synthesis in R. chalepensis callus, characterized using advanced techniques, positively impacting biomass growth, phytochemical content, antioxidant enzymes, and activities. [ABSTRACT FROM AUTHOR]

A procedure for plant regeneration from cell suspension cultures through somatic embryogenesis is described for Sapindus trifoliatus, a commercially and medicinally important tree. Callus was induced from leaf disc on agar-solidified MS medium with 5.0 mg l−1 2, 4-D and 0.01 mg l−1 Kin. Embryogenic cell suspension cultures were established by placing leaf-derived friable calli in PGR-free full-strength MS liquid medium with 3% sucrose. The growth of cell suspension culture was significantly affected by the strength of the MS mineral solution and L-glutamine. Plating of the suspension on semisolid MS medium resulted in the formation of globular structures. These embryogenic globular structures differentiated into secondary globular structures or somatic embryos on a semisolid MS medium. The differentiation of globular structures and different stages of somatic embryos (from globular to cotyledonary) was enhanced by the addition of 200 mg l−1 L-glutamine in the medium. Sucrose at relatively high concentrations (5%) or ABA (0.01 mg l−1) promoted somatic embryo maturation. The highest percentage (about 90%) of germination of somatic embryo and plantlet conversion was achieved on a half-strength MS medium containing 2% sucrose. The plants were hardened and established in soil with a 90% survival rateKey message: The present study demonstrates the establishment of suspension culture from leaf-derived callus in Sapindus trifoliatus and plant regeneration via somatic embryogenesis [ABSTRACT FROM AUTHOR]

The goal of the research was to develop a technology for introducing Salicornia europaea L.into in vitro culture. Methods of cultivating meristems and callus cultures were studied. To cultivate meristems, the tip of the apical bud were used as an explant. Fragments of stems and leaves were used toobtain callus tissue. To study the influence of various factors on germination, seeds were soaked in sterile tap water and in solutions of gibberellin, cytokinin, auxin and NaCl, and were also subjected to cold stratification (independently and with subsequent placement in Knop’s agarized medium). Salicornia seeds were sterilized with various antiseptics: 70% alcohol, 10% aqueous solution of sodium hypochlorite («Belizna»), amoxicillinand 3% hydrogen peroxide. The ability of the culture to form callus was studied in MS medium. As a result, it was determined that the highest germination of seeds was observed in Knop medium after treating theseeds with a suspension of green algae of the Scenedesmus genus, as well as after preliminary cold stratification, and slightly less after treating the seeds with a solution of NaCl and gibberellic acid. The most effective method of seed sterilization turned out to be treatment with alcohol followed by treatment with sodium hypochlorite. A comparative analysis of seed germination in filter paper in Petri dishes, Knop agar medium, Murashige and Skoog (hormone-free) was carried out. The ability of S. europaea L. to form callusin MS medium with phytohormones was assessed. Conclusion. To improve germination, it is recommended to subject the seeds to cold stratification. To obtain aseptic explants quickly, it is recommended to germinate the seeds in the Knop nutrient medium, having previously sterilized them with alcohol, then with sodiumhypochlorite, followed by washing with distilled water. The most suitable type of microclonal propagationfor S. europaea L. is the production of callus tissue followed by induction of organogenesis or embryogenesis.

Abstract Background Plants are considered the primary source of many principal bioactive compounds that have been utilized in a wide range of applications including the pharmaceutical and biotechnological industries. Therefore, there is an imperative need to modulate the production of natural bioactive components. The present study aimed to determine the importance of dried and pulverized date palm seeds (DPS) as a natural elicitor for the synthesis of secondary metabolites in Lotus arabicus L. Results The presence of various antioxidant compounds, simple sugars, amino acids, fatty acids and reasonable mineral contents was distinct in the phytochemical characterization of DPS. The major components detected in DPS analysis were the 5-(hydroxymethyl) furfural and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyranone. The induced callus of L. arabicus (seven weeks old) was supplemented with DPS at different concentrations (0, 2, 4, 8 and 10 g/l) in culture media. Treatment with 8 g/l DPS induced the highest antioxidant capacity, ascorbic acid content and secondary metabolites (total phenolics and flavonoids) in the produced callus. Stress biomarkers (hydrogen peroxide and malondialdehyde) were found in the control ranges except at 10 g/l DPS. The expression patterns of key genes involoved in secondary metabolism modulation, such as phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavonol synthase (FLS) and deoxyxylulose phosphate reductoisomerase (DXR), were triggered after DPS treatments. Moreover, the quantitative profiling of phenolic and flavonoid compounds showed that supplementation with DPS, especially at 8 g/l, led to pronounced increases in most of the measured compounds. Conclusion The marked upregulation of eliciting-responsive genes and overproduction of secondary metabolites provide molecular-based evidence for intensifying the principal pathways of phenylpropanoid, flavonoid and terpenoid biosynthesis. Overall, the present in vitro study highlights the stimulating capacity of DPS utilization to improve the bioactive components of L. arabicus at the physiological and molecular levels, enhancing its potential as a medicinal herb.

Abstract Background The study seeks to investigate the therapeutic potential of Terminalia arjuna callus in addressing atherosclerosis. In order to get maximum beneficial phytoconstituents from Terminalia arjuna, it is recommended to harvest the bark from Arjuna trees that are at least 15 years old and a gap of minimum 2 years should be kept before harvesting bark from the same plant. The callus culture technique was employed to expedite the process. The callus culture extract was subsequently converted into a nanosuspension with the aim of improving the efficacy of its phytoconstituents. It was then subjected to a comprehensive series of in vitro and in vivo evaluations to ascertain its potential for treatment of atherosclerosis. Results Liquid chromatography–mass spectrometry analysis of the callus extract confirmed the presence of flavonoids and terpenoids, known for their antioxidant and anti-inflammatory activities. Some terpenoids were even absent in Arjuna tree naturally. TEM images validated successful entrapment of the extract within the nanoparticles. In vitro analysis for antilipase and antioxidant assay confirmed the antiatherosclerotic potential of the extract. In vivo tests on rat blood serum demonstrated a significant reduction in total cholesterol, low-density lipoprotein, triglycerides, high-density lipoprotein, and very low-density lipoprotein. Histopathological analysis of rat aortas showed additional confirmation of antiatherosclerotic action. Conclusion In conclusion, the study highlights the potential of nanosuspension derived from Terminalia arjuna callus extract as a comprehensive therapeutic strategy for atherosclerosis treatment. The research highlights antioxidant, anti-inflammatory, and antiatherosclerotic properties of the callus, hinting at its viability as a potential treatment for atherosclerosis. This interdisciplinary investigation emphasizes the promising role of traditional medicinal plants within modern medical paradigms.

The cryopreservation of Anthurium andraeanum germplasm resources is extremely important for the production and selection of new varieties. At present, the cryopreservation procedure for the callus of A. andraeanum has not been established. In this study, the leaves of A. andraeanum were used as explants to culture the callus. The cryopreservation procedure of the callus by vitrification was initially established by using the orthogonal experimental method of four factors and three levels in the preculture, loading, and dehydration steps. Furthermore, the vitrification-based cryopreservation was optimized by changing the preculture temperature and loading solution and adding exogenous substances to the plant vitrification solution (PVS2). In this procedure, the callus was precultured at 25 °C for 2 d, and loaded in 50% PVS2 at 25 °C for 60 min. The callus was dehydrated with PVS2 containing 0.08 mM reduced glutathione (GSH) at 0 °C for 60 min. After rapid-cooling in liquid nitrogen for 1 h, it was rapid-warming in a water bath at 40 °C for 90 s and unloaded for 30 min. After 1 d of recovery, the cell relative survival rate of the cryopreserved callus was 64.60%. The results provide a valuable basic and effective method for the long-term conservation of A. andraeanum germplasm resources.

Licorice root is traditionally used in medicine due to its content of saponins and flavonoids. Licorice leaves are not used as pharmacopoeial raw materials, although their chemical composition and biological activity have been studied recently, which makes it possible to evaluate the possibilities of using this raw material as a medicinal one. Active harvesting of licorice can jeopardize its natural populations, so urgent tasks are to cultivate cells of this plant in systems in vitro and to study the composition of cell culture metabolites. In our study, the material for obtaining a callus culture of licorice leaves was plants from the collection of the Botanical Garden of the Ural Branch of the Russian Academy of Sciences. To select optimal conditions for growing calli, nine combinations of phytohormones were compared. The best growth of calli was found on Murashige–Skoog medium with a combination of phytohormones 1 mg/L BAP and 10 mg/L NAA. Under these conditions, flavonoids accumulated in callus in quantities comparable to their content in intact leaves and roots. The content of phenolic compounds was comparable to their amount in the roots. Ethanol extracts obtained from callus culture had pronounced antioxidant activity comparable to extracts from the intact plant and standards of rutin, gallic, and ascorbic acids. When assessing the effect of extracts on animal cell cultures in the MTT test, it was shown that all obtained extracts increased the metabolic activity of both normal human cells and the HeLa line. At the same time, the extract obtained from the leaves showed the maximum effect, while that from the callus showed the minimum and differed slightly from the root extract. Thus, calli from leaf explants can be considered as a new raw material for obtaining dietary supplements with antioxidant activity. [ABSTRACT FROM AUTHOR]

Flavonoids have numerous functions in plants, one of which is growth regulation. During the study, callus cultures were obtained from different structural elements—root, hypocotyl, shoot base, and cotyledons—of 5- and 14-day-old Lychnis chalcedony plants (Lychnis chalcedonica L.); the growth rates of the corresponding callus tissues and their accumulation of flavonoids (Fl) were studied. The total Fl content in the leaves of different tiers of vegetative plants was determined spectrophotometrically. The HPLC method was used to analyze the content of three Fls: rutin (R), quercetin (QCT), and dihydroquercetin (DHQ) in the roots, leaves, and inflorescences of flowering plants cultivated on soddy-podzolic soil of the Tomsk oblast (Russia). The growth and total content of phenolic compounds and three Fls in callus and suspension cultures obtained from root explants of young Lychnis plants were studied. The growth characteristics and organ specificity in the accumulation of individual Fl were established. The dependence of the physiological state of leaves and cell cultures on the Fl content is shown. On day 14, the suspension culture (passage ten) obtained from root callus (passage 92) had similar R and DHQ contents to the original line of callus culture and a lower level of QCT. DHQ was predominantly present in the roots of flowering plants, while R was present in the inflorescences and upper leaves. A tendency for R to decrease in a number of organs has been established: inflorescence ≥ leaves > root. The QCT distribution shows an inverse R dependence. The content of DHQ in leaves was 1.9 times less (P < 0.05) than in the inflorescence. The level of this Fl in the roots was many times higher (P < 0.05) than in aboveground organs. Following an increase in the oxidative status of the mature leaf of vegetative Lychnis plants relative to the young leaf, the osmotic and antioxidant systems, including proline and the total level of Fl, were activated. At the same time, callus (passage six), obtained from younger tissues of the hypocotyl of 5-day-old seedlings, had trace amounts of the studied Fl, while callus formed from more mature tissues of the shoot base of 14-day-old seedlings was characterized by a higher level of different Fl. Based on the obtained data, it can be assumed that Fl is involved in the regulation of the growth of leaves and cell cultures due to their antioxidant or regulatory properties. [ABSTRACT FROM AUTHOR]

Under a water deficit, the protective proteins known as dehydrins (DHNs) prevent nonspecific interactions in protein and membrane structures and their damage, in addition to playing an antioxidant role. The DHNs of a widespread xerophytic species Scots pine (Pinus sylvestris L.) have been poorly studied, and their role in resistance to water deficits has not been revealed. In this paper, we have expanded the list of DHNs that accumulate in the cells of Scots pine under the conditions of water deficits and revealed their relationship with the effects of water deficits. In this investigation, callus cultures of branches and buds of Scots pine were used. A weak water deficit was created by adding polyethylene glycol to the culture medium. Under the conditions of a water deficit, the activity of catalase and peroxidase enzymes increased in the callus cultures. A moderate decrease in the total water content was correlated with a decrease in the growth rate of the callus cultures, as well as with an increase in the activity of lipid peroxidation. The accumulation of Mr 72, 38, and 27 kDa DHNs occurred in the callus cultures of buds, and the accumulation of Mr 72 and 27 kDa DHNs positively correlated with the lipid peroxidation activity. An increase in the content of DHNs was observed in cultures that differed in origin, growth indicators, and biochemical parameters, indicating the universality of this reaction. Thus, previously undescribed DHNs were identified, the accumulation of which is caused by water deficiency and is associated with manifestations of oxidative stress in the kidney cells of Scots pine.

Background: Recent interest in plant-derived exosome-like nanoparticles (ENs) has surged due to their therapeutic potential, which includes antioxidant, anti-inflammatory, and anticancer activities. These properties are attributed to their cargo of bioactive metabolites and other endogenous molecules. However, the properties of ENs isolated from plant cell cultures remain less explored. Methods: In this investigation, grape callus-derived ENs (GCENs) were isolated using differential ultracentrifugation techniques. Structural analysis through electron microscopy, nanoparticle tracking analysis, and western blotting confirmed that GCENs qualify as exosome-like nanovesicles. Results: These GCENs contained significant amounts of microRNAs and proteins characteristic of plant-derived ENs, as well as trans-δ-viniferin, a notable stilbenoid known for its health-promoting properties. Functional assays revealed that the GCENs reduced the viability of the triple-negative breast cancer cell line MDA-MB-231 in a dose-dependent manner. Moreover, the GCENs exhibited negligible effects on the viability of normal human embryonic kidney (HEK) 293 cells, indicating selective cytotoxicity. Notably, treatment with these GCENs led to cell cycle arrest in the G1 phase and triggered apoptosis in the MDA-MB-231 cell line. Conclusions: Overall, this study underscores the potential of grape callus-derived nanovectors as natural carriers of stilbenoids and proposes their application as a novel and effective approach in the management of cancer.

The study aimed to enhance quercetin production in radish by optimizing Agrobacterium tumefaciens-mediated in-planta transformation. This protocol involved infecting radish seed embryo axis with A. tumefaciens EHA105 strain carrying the 35S::AtMYB12. Radish seeds were infected with the Agrobacterium suspension (0.8 OD600) for 30 min, followed by sonication for 60 s and vacuum infiltration for 90 s at 100 mm Hg. A 3-day co-cultivation in Murashige and Skoog medium with 150 μM acetosyringone yielded a transformation efficiency of 59.6% and a transgenic callus induction rate of 32.3%. Transgenic plant and callus lines were confirmed by GUS histochemical assay, PCR, and qRT-PCR. The transgenic lines showed an increased expression of flavonoid pathway genes (AtMYB12, CHS, F3H, and FLS) and antioxidant genes (GPX, APX, CAT, and SOD) compared to WT plants. Overexpression of AtMYB12 in transgenic callus increased enzyme activity of phenylalanine ammonia lyase, catalase, and ascorbate peroxidase. In half-strength MS medium with 116.8 mM sucrose, the highest growth index (7.63) was achieved after 20 days. In AtMYB12 overexpressed callus lines, phenolic content (357.31 mg g−1 dry weight), flavonoid content (463 mg g−1 dry weight), and quercetin content (48.24 mg g−1 dry weight) increased significantly by 9.41-fold. Micro-wounding, sonication, and vacuum infiltration improved in-planta transformation in radishes. These high-quercetin-content transgenic callus lines hold promise as valuable sources of flavonoids.

The viability, productivity and survival of higher plants under the adverse factors influence are largely determined by the functional activity of the antioxidant system. The aim of our work was to investigate changes in formation of high-molecular (superoxide dismutase and peroxidase) and low-molecular (phenolics, including flavanols and proanthocyanidins) antioxidants in callus culture of Camellia sinensis under influence of phenolic precursors (L-phenylalanine—3 mM, trans-cinnamic acid—1 mM, naringenin—0.5 mM). According to the data obtained, the effect of precursors on tea callus cultures did not lead to significant increasing of superoxide dismutase and peroxidase activity in most cases. However, it led to the increased accumulation of the total phenolics content, as well as flavanols and proanthocyanidins contents. For C. sinensis callus cultures, the most promising regulator of phenolic compounds was L-phenylalanine, in the presence of which its content increased almost twice. Thus, the exogenous effect of various precursors is possible to use for the targeted regulation of certain phenolics classes accumulation in plant cells. [ABSTRACT FROM AUTHOR]

Betalains are water soluble nitrogenous pigments produce by plants under the Caryophyllales order and has been favoured as a natural colourant in food and pharmaceutical industries due to its high stability towards pH and temperature over a wide range of food. There is a constant search for alternative source and technique for betalain production to meet the growing demand as conventional extraction method requires high quantity of plant material. Thus, this study sought to examine the potential of producing betalain through callus culture of a natural betalain bearing plant, Gomphrena globosa by inducing callus cultures from different explants using different plant growth regulators (PGRs). Subsequently, the effect of different elicitors in enhancing the betalain production was also evaluated. Callus induction from different explants showed that the percentage of callus induction from the leaf and hypocotyl explants (84.00–100.00%) was significantly higher than seeds (53.33%). A combination of 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) with 1.0 mg/L 6-benzylaminopurine (BAP) were found to be effective in producing coloured callus with relatively high biomass in full strength MS medium. Elicitation with tyrosine was the most effective in enhancing the betacyanin content (red-violet pigments) followed by salicylic acid. The highest betacyanin content, 0.371 ± 0.035 mg/mg FW of callus was obtained when 100 μM of tyrosine was supplied. Copper sulphate was found to be effective in increasing the callus size but not the betalain content. The callus size was about 13-fold bigger in MS medium supplemented with 25 μM copper sulphate compared to medium without elicitors. This is the first study reporting an optimised protocol in the induction of callus containing betalain from G. globosa using different types of explants and PGRs. In addition, tyrosine can be used as a suitable elicitor to further enhance the betalain production which provides an alternative source of betalain for the commercial production of natural colorants.Key message: Callus can be induced from the seeds, hypocotys and leaves explants of Gomphrena globosa, 0.5 mg/L of 2,4-D and 1.0 mg/L BAP were effective in producing coloured callus with high biomass, Tyrosine was found to be effective in enhancing the betalains content in the callus. [ABSTRACT FROM AUTHOR]

Noncommunicable diseases are characterized by an oxidative stress and inflammation. The search for new natural molecules that counteract these effects, is relevant for developing of new preventive and therapeutic strategies. This work evaluated the antioxidant and anti-inflammatory capacity of extracts obtained from in vitro cultures of Mammillaria candida and Turbinicarpus laui. Control callus and callus irradiated with UV light, as well as, control suspensions and suspensions elicited with chitosan were employed. The antioxidant activity was evaluated as reduction of ferric ion and by measuring the decrease in lipid peroxidation. The in vitro anti-inflammatory activity was analyzed in polymorphonuclear cells stimulated with lipopolysaccharide. For the in vivo experiments, Wistar rats were injected with carrageenan in the pad foot to induce inflammation. In both assays the anti-inflammatory effect was measured as a reduction in tumoral necrosis factor alfa. The extracts of M. candida and T. laui presented superior antioxidant activity compared to vitamin E, and the lipid peroxidation was lower when using extracts compared to indomethacin. The tumoral necrosis factor production in vitro decreased by 49% in the group treated with indomethacin and T. laui extracts, and 92% in the cells exposed to the M. candida extracts. In the in vivo assays, the indomethacin reduced 20% this cytokine, while the M. candida extracts decreased by 45%. In conclusion, the ethanolic extracts from M. candida and T. laui in vitro cultures contain secondary metabolites that could be an alternative source of new drugs with important antioxidant and anti-inflammatory activities.Key Message: The paper describes the antioxidant and anti-inflammatory activity of hydroalcoholic extracts obtained from Mammillaria candida and Turbinicarpus laui in vitro cultures, using in vitro and in vivo models. [ABSTRACT FROM AUTHOR]

Polygala sibirica L. is a rare and endangered plant species that is listed in the Red Book of the Republic of Sakha (Yakutia) under category “3 c” as a species with a narrow ecological limitation to limestone rock outcrops. For the first time, the phytochemical profile of the above-ground phytomass of P. sibirica growing in Central Yakutia was studied, and in vitro culture induction was performed. Using HPLC-MS/MS and tandem mass spectrometry methods, 74 compounds were allegedly identified in methanol extracts of the above-ground phytomass of the P. sibirica, of which 40 compounds were identified for the first time within the genus Polygala L., including 22 polyphenolic compounds: flavones (isoformononetin, syringetin, apigenin 7-O-glucoside, etc.), flavonols (herbacetin, myricetin, etc.), flavan-3-ols (afselequin, epicatechin, etc.), flavanones (hesperitin, eriocitrin), phenolic acid, and anthocyanins. A primary callus culture of P. sibirica was based on leaf explants of intact plants with the study of the dynamics of growth of raw and dried cell biomass. On the basis of callus tissue, indirect morphogenesis with abundant shoot formation is initiated. Thus, the basis has been laid for the subsequent propagation of the rare plant P. sibirica for the purpose of reintroduction in the conditions of the Yakut Botanical Garden. [ABSTRACT FROM AUTHOR]

Pistachio (Pistacia vera L) is one of the oldest cultivated plants in the world. Its fruits are rich in protein, minerals, carbohydrates, fiber, and vitamins. In addition, the demand for these plants is increasing due to the fact that they are very tasty and nutritious. On the other hand, pistachio cultivation is quite difficult. In addition, many problems are encountered in germination with seeds or reproduction with cuttings. These situations necessitate the development of different in vitro tissue culture protocols. In this study, callus culture optimization protocol was developed by using seeds of three different pistachio cultivars. Murashige and Skoog (MS) medium was supplemented with different concentrations of NAA, IAA, 2,4 D and BAP. When callus size (1,776 cm), callus weight (0.908 g) and embryogenic callus regenerations (27.94%) were considered, it was found that the best variety was Tekin. Again, in the evaluation made according to these factors, it was determined that the best improvement was in the MS medium containing 3 mg/L BAP and 1 mg/L 2,4D. The contamination rate detected throughout the studies ranged from 7.65% to 12.91%. [ABSTRACT FROM AUTHOR]

• Induction of polyploidy using colchicine in Andrographis paniculata (Burm.f.) Nees. • Polyploid Andrographis paniculata was confirmed by using flowcytometry analysis. • First time report on the in vitro callus culture from leaf explants of colchiploid Andrographis paniculata. • 160-fold increased andrographolide was quantified from colchiploid calluses than diploids using HPLC. An in vitro method to enhance the andrographolide production in Andrographis paniculata was developed using colchiploidy. Andrographis paniculata (Burm.f.) Wall. ex. Nees. is an annual herbaceous diploid medicinal plant with 2n=50, belonging to the Acanthaceae family. Andrographolide is the chief secondary metabolite present in plant and has significant commercial value due to its pharmacological properties. The objective of this study was to investigate the in vitro impact of colchiploids on the production of andrographolide. Additionally, we aimed to compare this effect in calli that were in vitro -raised from colchiploid plants with those derived from diploid plants. Colchiploidy was induced with seedlings of A. paniculata treated with 0.1% (w/v) colchicine for 12 h under dark conditions. Callus culture of Andrographis paniculata established from colchiploid leaf explants on 0.8% agar gelled Murashige and Skoog (MS) medium fortified with 5.0 µM 2,4- D + 2.3 µM Kinetin and 3% sucrose. A significant increase in andrographolide production (2.4%) was achieved in the colchiploid calluses compared to the diploid calluses (0.015%) quantified by high-performance liquid chromatography (HPLC) analysis. Plants with higher amounts of physiologically active secondary metabolites arise as a result of polyploidy. This method of andrographolide production can be used to harvest andrographolide on a large scale. [ABSTRACT FROM AUTHOR]

The rapid development of nanotechnologies has raised great interest in the use of engineered nanoparticles (NPs) and their release in the environment. The interaction between nanoparticles and crops is currently a topical problem, as nanoparticles are capable to enter the soil, plant cells and to translocate in plants. MicroRNA (miRNA) is an endogenous small RNA with a significant regulatory role in plants, cleaving or repressing the translation of a particular mRNA in response to multiple biotic and abiotic factors. In this work, the expression of miR159c in Linum usitatissimum L. calli cultures caused by various concentrations of Fe3O4 nanoparticles was studied. The obtained results showed an increase in miR159c expression with a decrease in the concentration of Fe3O4 nanoparticles. The results obtained are important in explaining the potential of nanoparticle utilisation to provide plant immunity responses. [ABSTRACT FROM AUTHOR]

Stilbenes, including resveratrol, are prominent bioactive compounds in peanuts widely recognized for their health benefits. However, limited research has focused on the production of resveratrol and its derivatives piceatannol and piceid from Arachis hypogaea. This research aims to study the production of these stilbenes using in vitro systems. Callus cultures have been established but only limited amounts of stilbenes were produced. Biotic elicitors, namely fungal culture filtrate (CF), mycelial extract (ME) and volatiles (VOCs) derived from three fungi (Serendipita indica , Fusarium solani and Alternaria alternata) were investigated in callus cultures. Based on Principal Components Analysis, 5 % v/v CF from A. alternata resulted in a significant increase in piceid production after 8 h. Similarly, 5 % v/v CF from S. indica after 16 h exhibited a notable enhancement in resveratrol and piceatannol levels. Enzyme activity of PAL and TAL was also promoted. Subsequent investigations involved establishing a cell suspension culture using CF from A. alternata and S. indica. In contrast to the callus culture, the levels of resveratrol remained comparatively lower. These results point towards a new method for piceid production using callus culture of peanuts, as well as a potential opportunity to obtain resveratrol and piceatannol via fungal elicitation. [Display omitted] • Piceid was produced in peanut callus culture without the need for elicitation. • Culture filtrate from Alternaria alternata increased piceid production. • Culture filtrate from Serendipita indica resulted in a higher amount of resveratrol and piceatannol. • Culture filtrate from Serendipita indica successfully led to extracellular resveratrol production in peanut cell suspension. • Callus culture has proved to be a more favorable biotechnological approach to the production of natural stilbenes than cell suspensions. [ABSTRACT FROM AUTHOR]

The study was carried out to compare the productivity and stability of regenerant lines of spring oat and their donor husky (Tubinsky st. (3 lines), Sayan (3), and Kazyr (1)) and naked (Tyumensky golozerny (3 lines) and Golets (2)) varieties. Field tests were carried out in the forest-steppe zone of the Krasnoyarsk krai on leached chernozem. Criteria used for the line stability evaluation were the following: superiority index Pi determining the degree of a sample deviation from the maximum yield in each of the environments (years); S(1) index characterizing the predictability of a genotype response to growing conditions; S(3) index, aimed at identifying highly productive and highly stable genotypes; and NP(2) index, intended for selection of genotypes with the optimal productivity/stability ratio. The majority of regenerants showed the yield and 1000 kernel weight parameters to be close to the donor varieties. Only three lines of the Tubinsky variety surpassed the donor in terms of the average three-year productivity by 1.78 (SR6-Tub.), 2.45 (SR11-Tub.), and 2.95 (SR15-Tub.) t/ha. Note that the 1000-kernel weight in the SR6-Tub. and ZR4-Golets lines was lower than that of the original variety by 1.9 and 1.4 g, respectively. A 1000 kernel weight in the ZR15-Kazyr line in 2017 was inferior to that in the donor variety by 1.4 g. In terms of the yield stability indices Pi and S(1), the ranks of nine out of 12 studied lines exceeded that of initial donor varieties by 5.7 on average. The similar result was observed in four (Pi higher by 2.25 rank units) and eight (S(1) higher by 6.6 rank units) regenerant lines. In relation to the S(3) index, six out of twelve lines surpassed their donors by 7 rank units on average; in terms of the 1000 kernel weight, nine lines exceeded their donors by 5.3 rank units. The rank determined for the NP(2) index exceeded that of donor varieties by 5.5 in four lines in terms of yield and by 6.2 in ten lines in terms of a 1000 kernel weight. [ABSTRACT FROM AUTHOR]

The current study was carried out in plant tissue culture lab., in center of desert studies/ University of Anbar, to determine the possibility of inducing callus from the cotyledon of Helianthus annuus L. by using different concentrations of growth regulator 2,4-D (1.0, 2.0 and 3.0 mg L-1) with other concentrations of growth regulator Kintein (0 and 0.25, 0.5 and 0.75 mg L-1). NaOCl solution was used to sterilize the seeds at four concentrations of 0, 2, 4 and 6% and for three periods of 5, 10 and 15 min to obtain sterile seedlings. The results showed that the concentration of 6.0% of NaOCl exceeded when treating the seeds for 10 min by achieving the highest percentage for eliminating contamination and the highest mean for obtaining sterile seedlings, which reached 100% and 90%, respectively. As for callus induction, the excised explant of sterile seedlings was cultured in MS media supplied with the growth regulators in the study for 35 days. The results showed that the combination of 0.25×1.0 mg L-1 achieved the highest response to callus formation, reached 100%, and the minimum time for the start of callus induction was 5.29 days. In comparison, the combination of 0.25×2.0 mg L-1 showed the minimum period for the completion of callus formation was 25.14 days. In contrast, the combination of 0.75×2.0 mg L-1 showed the highest fresh and dry weight values of 767.0 and 53.0 mg, respectively.

Ephedra is the most widely and largest widespread genus in the Gnetopsida, a subgroup of the gymnosperms. This research was done to find out how gamma radiation affected the in vitro growth of callus and suspension cultures. In addition, the effect of gamma rays (γ-rays) on the ephedrine and pseudoephedrine concentrations was evaluated through HPLC analysis. The cell suspension and callus cultures were formed on Murashige and Skoog's basal medium (MS) supplemented with 0.5 mg/l kinetin (Kn), and 3 mg/l of 2, 4-dichlorophenoxy acetic acid (2,4-D) and exposed to variable gamma radiation doses (0, 5, 10, 15, 20, 25 and 30 Gray). Highly significant differences in both fresh and dry callus weights were recorded due to the effect of gamma rays (γ-rays). The findings demonstrated that in the cell suspension and callus cultures, 15 Gray achieved the highest fresh and dry weights when compared to the control. Similarly, the highest concentrations of ephedrine and pseudoephedrine were found in suspension and callus cultures compared to control at 15 Gray.

Bursera fagaroides, popularly used in México, possesses bioactive lignans. These compounds are low in the bark, and its extraction endangers the life of the trees. The aim of the present investigation was to search for alternative sources of cytotoxic compounds in B. fagaroides prepared as leaves and in vitro callus cultures. The friable callus of B. fagaroides was established using a combination of plant growth regulators: 4 mgL−1 of 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mgL−1 Naphthaleneacetic Acid (NAA) and 1 mgL−1 Zeatin. The maximum cell growth was at day 28 with a specific growth rate of μ = 0.059 days−1 and duplication time td = 11.8 days. HPLC quantification of the dichloromethane callus biomass extract showed that Scopoletin, with a concentration of 10.7 µg g−1 dry weight, was the main compound inducible as a phytoalexin by the addition of high concentrations of 2,4-D, as well as by the absence of nutrients in the culture medium. In this same extract, the compounds γ-sitosterol and stigmasterol were also identified by GC-MS analysis. Open column chromatography was used to separate and identify yatein, acetyl podophyllotoxin and 7′,8′-dehydropodophyllotoxin in the leaves of the wild plant. Cytotoxic activity on four cancer cell lines was tested, with PC-3 prostate carcinoma (IC50 of 12.6 ± 4.6 µgmL−1) being the most sensitive to the wild-type plant extract and HeLa cervical carcinoma (IC50 of 72 ± 5 µgmL−1) being the most sensitive to the callus culture extract.

Callus cultures of the Iranian medicinal plant Salvia atropatana were initiated from three-week-old seedlings on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) and various cytokinins. Although all tested hormonal variants of the medium and explant enabled callus induction, the most promising growth was noted for N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU)-induced calli. Three lines obtained on this medium (cotyledon line-CL, hypocotyl line—HL, and root line—RL) were preselected for further studies. Phenolic compounds in the callus tissues were identified using UPLC–MS (ultra-performance liquid chromatography–mass spectrometry) and quantified with HPLC (high-performance liquid chromatography). All lines exhibited intensive growth and contained twelve phenolic acid derivatives, with rosmarinic acid predominating. The cotyledon-derived callus line displayed the highest growth index values and polyphenol content; this was exposed to different light-emitting diodes (LED) for improving biomass accumulation and secondary metabolite yield. Under LED treatments, all callus lines exhibited enhanced RA and total phenolic content compared to fluorescent light, with the highest levels observed for white (48.5–50.2 mg/g dry weight) and blue (51.4–53.9 mg/g dry weight) LEDs. The selected callus demonstrated strong antioxidant potential in vitro based on the 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) tests. Our findings confirm that the S. atropatana callus system is suitable for enhanced rosmarinic acid production; the selected optimized culture provide high-quality plant-derived products.

The application of cassava starch in the biopolymers industry has been growing significantly due to its low cost, good oxygen barrier in the dry state, and biodegradability. Plant culture technology is an alternative to traditional propagation as it does not require large areas for production, has a higher rate of multiplication, and produces disease-free plants. However, the application of cassava callus culture for starch production is limited. This study focuses on identifying the significant culture parameters for a maximum Rayong cultivar cassava callus growth and evaluation of its starch content. Cassava stems petiole and leaf explants were cultured on MS medium containing different combinations of 2,4-D (8, 12, and 15 mg/L) and BAP (1, 3, and 5 mg/L) under three light conditions (0, 16, and 24 h). The screening of the most influential parameter was done using the 2-level Factorial Design in Design Expert v13 by analyzing the frequency of callus formation. All leaf explant turned brown with no callus induction. The highest frequency of callus formation derived from stem petiole explant was achieved by the combination of 8 mg/L 2,4-D and 1 mg/L BAP under the light condition (75%) followed by 8 mg/L 2,4 D + 1 mg/L BAP under the dark condition (50%). Based on the ANOVA analysis, the individual supply of 2,4-D and BAP respectively, have a negative effect on callus formation while the combination of 2,4-D and BAP has a positive effect. Light supply did not significantly affect cassava callus formation. The amount of starch in the cassava callus was then investigated using an iodine test which yielded 0.21% of the total weight of the callus (0.0101g). The amount of starch is relatively low considering that the callus was not grown under the optimum condition for starch production. The findings of this study open prospects for future research in cassava cultures in favor of starch production. ABSTRAK: Penggunaan kanji ubi kayu dalam industri biopolimer telah meningkat secara mendadak disebabkan oleh faktor kosnya yang rendah, rintangan oksigen yang baik dan sifat keterbiodegradasi. Teknologi kultur tisu tumbuhan merupakan alternatif kepada pembiakan secara tradisional yang mana teknologi ini tidak memerlukan kawasan penanaman yang luas, penghasilan yang lebih singkat dan menghasilkan tumbuhan bebas penyakit. Walau bagaimanapun, kajian mengenai penggunaan kanji dari kultur ubi kayu masih belum meluas. Kajian ini bertujuan mengenal pasti parameter penting dalam pertumbuhan maksimum kalus kultivar ubi kayu Rayong dan kandungan kanjinya. Eksplan dari tangkai petiol dan daun ubi kayu dikulturkan dalam medium MS yang mengandungi pelbagai kombinasi hormon tumbuhan 2,4-D (8, 12 dan 15 mg/L) dan BAP (1, 3 dan 5 mg/L) di bawah tiga tempoh masa pencahayaan (0, 16 dan 24 jam). Saringan parameter paling berpengaruh dilakukan menerusi reka bentuk faktorial 2-peringkat perisian Design Expert v13 melalui analisa frekuensi pembentukan kalus. Semua eksplan daun telah bertukar dari hijau ke perang dan tidak menunjukkan induksi kalus. Bagi eksplan batang daun, frekuensi pembentukan kalus tertinggi diperoleh dari kombinasi 8 mg/L 2,4-D dan 1 mg/L BAP di bawah pencahayaan 24 jam (75%) diikuti dengan 8 mg/L 2,4 D + 1 mg/L BAP di bawah pencahayaan malap (50%). Berdasarkan analisis ANOVA, medium 2,4-D dan BAP masing-masing menunjukkan kesan negatif kepada pembentukan kalus, sementara kombinasi 2,4-D dan BAP memberikan kesan positif. Dapatan ini menunjukkan bahawa pencahayaan tidak signifikan terhadap pembentukan kalus kultur ubi kayu. Kandungan kanji di dalam kalus ubi kayu kemudiannya diuji dengan larutan iodin, dan menunjukkan dapatan sebanyak 0.21% dari berat kalus (0.0101 g). Secara relatif, nilai ini adalah rendah memandangkan eksperimen ini bukan dihasilkan di bawah keadaan optimum bagi pembentukan kanji. Hasil kajian ini membuka peluang kepada kajian-kajian lain di masa hadapan dalam penghasilan kanji dari kultur tisu ubi kayu.

Over recent years, extracellular vesicles (EVs), commonly termed exosomes, have gained prominence for their potential as natural nanocarriers. It has now been recognized that plants also secrete EVs. Despite this discovery, knowledge about EV biogenesis in plant cell cultures remains limited. In our study, we have isolated and meticulously characterized EVs from the callus culture of the model plant, Arabidopsis thaliana. Our findings indicate that the abundance of EVs in calli was less than that in the plant's apoplastic fluid. This difference was associated with the transcriptional downregulation of the endosomal sorting complex required for transport (ESCRT) genes in the calli cells. While salicylic acid increased the expression of ESCRT components, it did not enhance EV production. Notably, EVs from calli contained proteins essential for cell wall biogenesis and defense mechanisms, as well as microRNAs consistent with those found in intact plants. This suggests that plant cell cultures could serve as a feasible source of EVs that reflect the characteristics of the parent plant species. However, further research is essential to determine the optimal conditions for efficient EV production in these cultured cells. [ABSTRACT FROM AUTHOR]

Direct and indirect shoot regeneration in Vanilla odorata and indirect for V. pompona are described for the first time. Both species produce fragrant, vanillin-containing seed pods and are closely related to the commercially cultivated V. planifolia. Stem nodal segments cultured on Murashige and Skoog medium containing 2.0 mg l−1 6-benzyl aminopurine (BAP) and 1.0 mg l−1 indole butyric acid (IBA) showed > 75% bud sprouting. Media containing 1.0 mg l−1 BAP or thidiazuron (TDZ) in combination with 0.5–1.0 mg l−1 IBA produced approximately eight shoots/explant. In callus induction experiments, we found that shoot tips from in vitro plantlets were more responsive than leaf and nodal explants with the highest callus induction (47% in V. odorata and 40% in V. pompona) when cultured in the dark on medium containing 2.0 mg l−1 naphthalene acetic acid (NAA) and 1.0 mg l−1 BAP. Calluses transferred to basal medium containing 3.0 mg l−1 BAP plus 1.0 mg l−1 NAA and grown under a 16 h photoperiod produced an average of 4.1 and 3.5 shoots per callus segment for V. odorata and V. pompona, respectively, eight weeks after culture initiation. Histological analysis revealed the presence of calcium oxalate raphides, as confirmed by Raman microscopy, in callus cultured under both dark and light conditions. Over 90% of the plantlets derived from axillary buds exhibited spontaneous root formation on multiplication media; similar to that observed when shoots originating from callus were cultured on plant growth regulator-free medium. During the subsequent greenhouse acclimatization phase, which took place 12 weeks after the final subculture, these plantlets achieved a survival rate of 95%. The results of the present study will be helpful for further large-scale mass propagation efforts of V. odorata and V. pompona and other biotechnological applications such as protoplast isolation. [ABSTRACT FROM AUTHOR]