Search Results (487)

Adult trees of pedunculate oak (Quercus robur L.) are recalcitrant to vegetative propagation. In this study, we investigated the micropropagation of five oak genotypes corresponding to trees aged 60–800 years in a liquid medium. We used commercial RITA bioreactors to study the influence of the explant type, the culture medium, shoot support and number of immersions. Variables evaluated included the number of normal and hyperhydric shoots, shoot length, multiplication coefficient and number of rootable shoots per explant. All genotypes could be cultured in temporary immersion. Basal stem sections attached to callus grew better than apical sections and developed less hyperhydricity. For long-term cultivation, Gresshoff and Doy medium was the best of the three media evaluated. All genotypes produced vigorous shoots suitable for rooting and acclimation. This is the first protocol to proliferate adult oak trees in bioreactors, representing significant progress towards large-scale propagation of this and other related species. Full article

This study evaluated biomass accumulation and phenolic compound production in seven Hypericum species (H. androsaemum, H. calycinum, H. hirsutum, H. kalmianum, H. olympicum, H. perforatum, and H. triquetrifolium) cultivated in vitro under varying growth regulator treatments and culture periods. Shoots were grown on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) or meta-topoline (mT) and analyzed after 40 and 60 days. MS medium supplemented with 0.2 mg/L BA was the most effective condition for promoting biomass across all species, with shoot fresh weight increasing significantly at 60 days, particularly in H. olympicum, H. perforatum, and H. triquetrifolium. High-performance liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS) identified 13 phenolic compounds, including flavonols, hydroxycinnamic acids, anthocyanins, phloroglucinols, and naphthodianthrones. Phenolic profiles were species-specific and influenced by culture period. H. kalmianum accumulated the highest total phenolic content (37.6 mg/g DW), while H. olympicum was the top producer of hypericin and pseudohypericin. These results highlight the crucial role of culture conditions in regulating both biomass and phytochemical production and provide a promising approach for producing bioactive metabolites in Hypericum species through in vitro systems. Full article

Rye regeneration in anther cultures is problematic and affected by albino plants. DNA methylation changes linked to Cu²⁺ ions in the induction medium affect reprogramming microspores from gametophytic to sporophytic path. Alternations in S-adenosyl-L-methionine (SAM), glutathione (GSH), or β-glucans and changes in DNA methylation in regenerants obtained under different in vitro culture conditions suggest a crucial role of biochemical pathways. Thus, understanding epigenetic and biochemical changes arising from the action of Cu²⁺ and Zn²⁺ that participate in enzymatic complexes may stimulate progress in rye doubled haploid plant regeneration. The Methylation-Sensitive Amplified Fragment Length Polymorphism approach was implemented to identify markers related to DNA methylation and sequence changes following the quantification of variation types, including symmetric and asymmetric sequence contexts. Reverse-Phase High-Pressure Liquid Chromatography (RP-HPLC) connected with mass spectrometry was utilized to determine SAM, GSH, and glutathione disulfide, as well as phytohormones, and RP-HPLC with a fluorescence detector to study polyamines changes originating in rye regenerants due to Cu²⁺ or Zn²⁺ presence in the induction medium. Multivariate and regression analysis revealed that regenerants derived from two lines treated with Cu²⁺ and those treated with Zn²⁺ formed distinct groups based on DNA sequence and methylation markers. Zn²⁺ treated and control samples formed separate groups. Also, Cu²⁺ discriminated between controls and treated samples, but the separation was less apparent. Principal coordinate analysis explained 85% of the total variance based on sequence variation and 69% of the variance based on DNA methylation changes. Significant differences in DNA methylation characteristics were confirmed, with demethylation in the CG context explaining up to 89% of the variance across genotypes. Biochemical profiles also demonstrated differences between controls and treated samples. The changes had different effects on green and albino plant regeneration efficiency, with cadaverine (Cad) and SAM affecting regeneration parameters the most. Analyses of the enzymes depend on the Cu²⁺ or Zn²⁺ ions and are implemented in the synthesis of Cad, or SAM, which showed that some of them could be candidates for genome editing. Alternatively, manipulating SAM, GSH, and Cad may improve green plant regeneration efficiency in rye. Full article

Our attempts to obtain a new mushroom-derived immunostimulatory preparation containing organically bound selenium and zinc have focused on the interactions between selenites and zinc(II) in liquid culture media and their effects on transport into the mushroom cell. Previously, we found that, even if Zn²⁺ and SeO₃²⁻ concentrations in the liquid medium are not high enough to precipitate ZnSeO₃, the accumulation of selenium in the presence of zinc, and zinc in the presence of selenites, significantly dropped. This effect was more dependent on the molar ratio of ions in the medium than on the concentration values. We hypothesized that the formation of zinc–selenite soluble complexes with charges depending on the ion concentration ratio in the aquatic environment affects the first stage of ion transport into the fungal cell—biosorption. To verify this, we found the zinc–selenite molar ratio at which the complexes of the highest stability are formed, examined the influence of the molar ratio of ions in the medium on the concentration of Zn and Se in the mushroom cell wall, and investigated the correlation between the concentration of selenites not bound in complex compounds and the Se concentration in the cell wall. The results indicate that the molar fraction of Zn(II) in a liquid medium in the range of 0.5–0.6 promotes the formation of the most stable complexes. At the same time, it significantly reduces the percentage of free selenites in the medium and most strongly inhibits the biosorption process of both zinc and selenium. Full article

Raspberry (Rubus idaeus L.) micropropagation is an alternative for obtaining plantlets with high genetic and phytosanitary quality. The objective of this study was to establish a protocol for the micropropagation of raspberry (Rubus idaeus L.) using the temporary immersion bioreactor, under intermittent immersion periods and different culture medium volumes. The effect of the liquid medium using the TIB and semisolid was evaluated. Different immersion frequencies and culture medium volumes per explant were evaluated in the TIB. In all treatments, the number of shoots per explant, shoot length, number of leaves per explant, percentage of hyperhydricity, and chlorophyll and β-carotene content at multiplication stage were evaluated. The generated shoots, without a root system, were transferred to the acclimatization stage. The results show that the TIB with an immersion frequency of 2 min every 8 h and a volume of 25 mL of culture medium per explant had the best developmental parameters, with 5.75 shoots per explant, a shoot length of 3.44 cm, and 2% hyperhydricity. The highest chlorophyll and β-carotene content was observed in the TIB at different immersion frequencies of 4, 8 and 12 h, with 25 and 50 mL per explant. Survival percentages higher than 96% were observed in all methods evaluated. In conclusion, the evaluated immersion system is an efficient alternative for R. idaeus micropropagation, without using a rooting stage. Full article

Pancreatic adenocarcinoma remains one of the deadliest cancers, with limited treatment options and high chemoresistance. Traditional 2D cell cultures fail to accurately replicate the tumor architecture. Our study introduces three-dimensional (3D) pancreatic adenocarcinoma spheroid models using magnetic aggregation of pancreatic cancer cells and immortalized fibroblasts in either liquid culture medium or embedded in hydrogels. The spheroids’ growth was characterized using optical imaging, while viability was assessed using ATP quantification and flow cytometry. Results demonstrated successful spheroid formation and growth. Further analysis suggested that on one hand, culture in liquid medium and ATP-based viability assessment are practical for initial experiments. On the other hand, hydrogel culture and flow cytometry, although being more resource- and labor-intensive, provided both a more reproducible and detailed viability analysis. Full article

Background/Objectives: More than 33 billion chickens are industrially raised for meat and egg production globally and vaccinated against Marek’s disease virus (MDV). The antigenically related herpesvirus of turkey (HVT) is used as a live-attenuated vaccine, commonly provided as a recombinant vector to protect chickens against additional unrelated pathogens. Because HVT replicates in a strictly cell-associated fashion to low levels of infectious units, adherent primary chicken or duck embryo fibroblasts are infected, dislodged from the cultivation surface and distributed as cryocultures in liquid nitrogen to the site of application. Although viable cells are complex products, application of infected cells in ovo confers protection even in presence of maternal antibodies. Methods/Results: The aim of our study was to determine whether a continuous cell line in a scalable cultivation format can be used for production of HVT-based vaccines. The AGE1.CR cell line (from Muscovy duck) was found to be highly permissive in adherent cultures. Propagation in suspension, however, initially gave very low yields. The induction of cell-to-cell contacts in carrier-independent suspensions and a metabolic shock improved titers to levels suitable for vaccine production (>10⁵ infectious units/mL after infection with multiplicity of 0.001). Conclusions: Production of HVT is challenging to scale to large volumes and the reliance on embryonated eggs from biosecure facilities is complex. We demonstrate that a cell-associated HVT vector can be propagated in a carrier-independent suspension culture of AGE1.CR cells in chemically defined medium. The fed-batch production is independent of primary cells and animal-derived material and can be scaled to large volumes. Full article

Fatty acids (FAs) play a crucial role in human physiology, including energy production and serving as signaling molecules. However, a dysregulation in their balance can lead to multiple disorders, such as obesity and metabolic syndrome. These pathological conditions alter the balance between the heart’s energetic substrates, promoting an increased reliance on FAs and decreased cardiac efficiency. A therapeutic application of a non-psychotropic phytocannabinoid, cannabigerol (CBG), seems to be a promising target since it interacts with different receptors and ion channels, including cannabinoid receptors—CB₁ and CB₂, α2 adrenoceptor, or 5-hydroxytryptamine receptor. Therefore, in the current study, we evaluated a concentration-dependent effect of CBG (2.5 µM, 5 µM, and 10 µM) on H9c2 cardiomyocytes in lipid overload conditions. Gas–liquid chromatography and Western blotting techniques were used to determine the cellular lipid content and the level of selected proteins involved in FA metabolism, glucose transport, and the insulin signaling pathway. The glucose uptake assay was performed using a colorimetric method. Eighteen-hour CBG treatment in the highest concentration (10 µM) significantly diminished the accumulation of diacylglycerols (DAGs) and the saturation status of this lipid fraction. Moreover, the same concentration of CBG markedly decreased the level of FA transporters, namely fatty acid translocase (CD36) and plasma membrane fatty acid-binding protein (FABPpm), in the presence of palmitate (PA) in the culture medium. The results of our experiment suggest that CBG can significantly modulate lipid storage and composition in cardiomyocytes, thereby protecting against lipid-induced cellular dysfunction. Full article

Castanea mollissima Bl. is rich in nutrition and strong in stress resistance, and has nutritional, economic, and ecological values. A protoplast is impactful in somatic fusion and germplasm creation. Here, we propose an effective scheme for the construction of an embryonic suspension cell, protoplast isolation, and fusion. Studies have shown that when 1.0 g yellow loose embryonic callus was inoculated into MS + 1.5 mg∙L⁻¹ 6-BA + 0.2 mg∙L⁻¹ NAA + 0.5 mg∙L⁻¹ 2, 4-D liquid medium, a stable suspension cell line can be obtained. After further culturing for 2–4 days, protoplast isolation was performed. First, single-factor screening was conducted on the four enzymes, and then a two-factor random block was further set up to screen the enzyme combinations based on the results. We found that 1.0%cellulase R-10 + 0.5%pectolase Y-23 led to the highest protoplast yield (9.27 × 10⁶/g FW) and the highest activity (92.49%). Furthermore, the protoplast yield could be increased to 9.47 × 10⁶/g FW by adding 0.4 M mannitol and shaking for 8 h. The protoplasts were purified by centrifuging at 40× g for 4 min and then mixed with 30% PEG 6000 at a volume ratio of 1.5:1 for 25 min. The fusion rate could reach 70.00%. This study laid a foundation for the creation of new germplasm by Castanea mollissima Bl. Full article

The Orchidaceae family is of significant decorative, pharmaceutical, alimentary, and cultural importance worldwide. This family is very vulnerable due to illegal looting, habitat destruction, and climate change. The development of new hybrids helps meet the demand for specimens that possess outstanding appearance, fragrance, and resistance characteristics and may reduce illegal looting. The objective of this research was to investigate the in vitro propagation of the hybrid Rhyncattleanthe Queen Bee JLA 1 (Rth. Queen Bee JLA 1). Shoot induction was performed with germinated seedlings that were 1 cm in length on semi-solid MS medium with different 6-Benzylaminopurine (BAP), 1-Naphthaleneacetic acid (NAA), 3-Indoleacetic acid (IAA), and 3-indolebutyric acid (IBA) concentrations. Micropropagation was conducted using a temporary immersion system (TIS), a liquid continuous immersion system (CIS), and a conventional semi-solid system (SSS). Afterwards, all regenerated seedlings underwent an acclimatization stage. The highest numbers of shoots (7.04) and leaves (14.28) were obtained with the combination of 1.5 mg L⁻¹ BAP and 0.4 mg L⁻¹ NAA, while the addition of 0.4 mg L⁻¹ IBA in combination with 1.5 mg L⁻¹ BAP enhanced the length of stems (2.12 cm) and leaves (1.88 cm). TIS produced the highest number of shoots (15.68), leaves (22.92), stem length (5.94 cm), and number of leaves (3.50) in seedlings analyzed. The combination of growth regulators BAP and NAA together with the temporary immersion system influenced both the development of the vitroplants and their vegetative development after acclimatization of the hybrid Rth. Queen Bee JLA1 orchid. Full article

The tissue regenerative potential of the liquid component of mesenchymal stem cells has gained significant attention. Stem cells from human exfoliated deciduous teeth-conditioned medium (SHED-CM), which is often extracted during orthodontic treatment, promotes bone regeneration. However, further investigation is warranted to determine which component of SHED-CM affects bone regeneration. Therefore, we focused on the extracellular vesicles contained in SHED-CM (SHED-EVs) and aimed to study their effects on osteoblasts. SHED-EVs were isolated using a pellet-down EV extraction kit and identified using transmission electron microscopy and NanoSight. SHED-EVs were added to human calvarial osteoblasts (HCOs), and cell proliferation and migration ability were examined with Incucyte^® and BrdU. Alkaline phosphatase (ALP) expression was confirmed using real-time PCR and ALP quantification. The bone differentiation potential was examined using Alizarin Red S (ARS) staining. SHED-EVs promoted proliferation and migration of HCOs. Real-time PCR and ALP quantification results demonstrated that HCOs cultured with SHED-EVs exhibited increased ALP expression. ARS staining revealed that SHED-EVs promoted bone differentiation of HCOs. These results suggest that SHED-EVs promote cell proliferation and migration and bone regeneration of osteoblasts, highlighting their potential in the development of bone regeneration therapies. Full article

Chitosan nanoparticles (CsNPs) are biocompatible, biodegradable, and non-toxic natural polymers at low concentrations with diverse applications in in vitro plant tissue culture. This study aims to evaluate the effect of CsNPs during in vitro multiplication of sugarcane (Saccharum spp.) using temporary immersion bioreactors. CsNPs were evaluated at concentrations of 0, 25, 50, 100, and 200 mg L⁻¹ in Murashige and Skoog liquid culture medium. After four weeks of culture, response percentage, the number of shoots per explant, shoot length, number of leaves per explant, dry matter, chlorophyll content, β-carotene content, lipid peroxidation, phenolic content, hydrogen peroxide content, and antioxidant capacity were evaluated. The results showed that the highest response percentages were obtained in the treatments with 0, 25, and 50 mg L⁻¹ CsNPs, whereas the lowest response percentages were obtained in the treatments with 100 and 200 mg L⁻¹ CsNPs. Concentrations of 25 and 50 mg L⁻¹ CsNPs promoted cell growth and differentiation, whereas 100 and 200 mg L⁻¹ CsNPs inhibited it. Chlorophyll content increased by 25 and 50 mg L-1 CsNPs, whereas β-carotene content increased by 100 and 200 mg L⁻¹ CsNPs. Lipid peroxidation and antioxidant capacity increased with increasing CsNP concentrations. The phenolic content increased by 100 mg L⁻¹ CsNPs, whereas the hydrogen peroxide content decreased with increasing CsNP concentrations. In conclusion, CsNPs are an alternative for stimulating tissue growth and differentiation during the in vitro multiplication of sugarcane. Full article

Saponaria officinalis L., a plant rich in saponins, has long been used as a natural surfactant. It has traditionally been used for its cleansing and anti-inflammatory properties in the treatment of various skin conditions, including eczema, psoriasis, and acne. In this study, we investigated the potential of S. officinalis callus (SC), mass-produced via plant tissue culture, as a novel exfoliating cosmetic ingredient. The callus was induced using Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-D, and the resulting extract (SCE) was analyzed via high-performance liquid chromatography (HPLC), confirming the presence of saponarin—a bioactive compound with known anti-inflammatory properties. In vitro assays demonstrated that SCE significantly suppressed nitric oxide production and reduced the expression of pro-inflammatory mediators, including iNOS, COX-2, TNF-α, IL-1β, and IL-6, in LPS-stimulated RAW264.7 macrophages. The foaming ability and stability of SC and SCE were also comparable to commercial surfactants. Clinical studies further supported the material’s cosmetic potential: a skin patch test in 30 volunteers revealed no signs of irritation (mean score: 0.28), while a desquamation index assessment in 21 participants showed a significant reduction of 44.07%, confirming its exfoliating efficacy. Taken together, these results suggest that the SC scrub is a safe, eco-friendly, and sustainable alternative to synthetic exfoliating agents, offering functional and industrial advantages for cosmetic applications. Full article

Objective: Secretory factors, termed the secretome, in the conditioned medium (CM) from dental mesenchymal stem cells (MSCs) have shown anti-inflammatory, anti-apoptotic, and tissue regenerative potential. This cell-free product could be further developed by preconditioning cells with various biochemical agents, which lead to a change in secretome and CM profiles. Among the favorable candidates for CM production, metformin as an anti-diabetic medication is currently considered a potential agent for dental hard tissue and periodontal regeneration. Here, we aimed to assess the composition of CM from periodontal ligament stem cells (PDLSCs) grown in metformin-preconditioned media (Met-CM) compared to normal PDLSC-CM and assess the ability of Met-CM to recover the function of inflamed PDLSCs. Methods: Met-CM and normal CM were collected from PDLSCs grown with or without 50 µM metformin, respectively, under healthy culture conditions. Mass spectrometry and liquid chromatography–tandem mass spectrometry (LC–MS/MS) were performed to comparatively evaluate the proteomic profiles in PDLSC-CM versus Met-CM. We then treated the PDLSC cultures with lipopolysaccharide (LPS) from Porphyromonas gingivalis to induce inflammation and evaluated the osteogenic/cementogenic differentiation in the presence of Met-CM or normal PDLSC-CM by assessing alkaline phosphatase activity, intracellular calcium levels, and mRNA expression of osteogenic and cementogenic factors, including RUNX2, OCN, OSX, and CEMP-1. Subsequently, we performed RNA sequencing to identify transcriptomic changes in the treated cells. Results: We identified 202 differentially expressed proteins, 175 of which were significant, in Met-CM versus normal PDLSC-CM. Among the analyzed groups, the top three protein classes were protein-binding activity modulator, cytoskeletal protein, and extracellular matrix (ECM) protein. Treatment of PDLSCs with LPS significantly attenuated ALP activity, [Ca²⁺]_i, and the mRNA expression levels of RUNX2, OCN, OSX, and CEMP-1, whereas treatment with Met-CM alone markedly enhanced PDLSC differentiation activity compared with the control. Moreover, osteogenic/cementogenic differentiation of the LPS-treated PDLSCs was recovered through incubation in Met-CM. Transcriptomic analysis identified 511 and 3591 differentially expressed genes in the control versus Met-CM and LPS versus LPS + Met-CM groups, respectively. The enrichment of biological processes includes positive regulation of DNA-templated transcription and skeletal system morphogenesis in the control versus Met-CM comparison, as well as positive regulation of transcription from the RNA polymerase II promoter and negative regulation of the apoptotic process in the LPS versus LPS + Met-CM comparison. Molecular function analysis demonstrated the enrichment of protein-binding terms among the DEGs from each comparison. Conclusions: Metformin preconditioning enhanced the recovery effect of PDLSC-CM on LPS-induced inflamed PDLSCs. These findings suggest that metformin preconditioning could represent a practical formula for PDLSC-secretome, which may contribute to the development of future cell-free periodontal regenerative strategies. Full article

This study aims to optimize the bioconversion of palm kernel cake (PKC) by Pleurotus ostreatus to improve fungal biomass production, lignocellulolytic enzyme expression, and the nutritional value of the substrate as ruminant feed. Three inorganic nitrogen sources (ammonium sulfate, ammonium nitrate, and urea) were evaluated for fungal biomass production using a central composite design (CCD) in liquid fermentations. The formulated culture medium (18.72 g/L glucose and 0.39 g/L urea) effectively yielded better fungal biomass production (8 g/L). Based on these results, an extreme vertex design, mixtures with oil palm by-products (PK, hull, and fiber) supplemented with urea, were formulated, finding that PKC stimulated the highest biomass production and laccase enzyme activity in P. ostreatus. The transcriptome of P. ostreatus was obtained, and the chemical composition of the fermented PKC was determined. Transcriptomic analysis revealed the frequency of five key domains with carbohydrate-activated enzyme (CAZy) function: GH3, GH18, CBM1, AA1, and AA5, with activities on lignocellulose. In the fermented PKC, lignin was reduced by 46.9%, and protein was increased by 69.8%. In conclusion, these results show that urea is efficient in the bioconversion of PKC with P. ostreatus as a supplement for ruminants. Full article