Search Results (522)

Plant-derived extracellular vesicles (PDEVs) are emerging as promising bioactive materials for biomedical and dermatological applications. In this study, we isolated and characterized exosome-like vesicles derived from Rosa damascena callus culture medium (RSC-EXO) and evaluated their molecular features and biological activities in skin-related cellular models. Nanoparticle tracking analysis and cryo-electron microscopy showed that RSC-EXO exhibited a nanoscale size distribution and spherical morphology. Western blotting confirmed enrichment of the plant EV-associated markers PEN1 and TET8. RSC-EXO were efficiently internalized by human dermal fibroblasts and showed markedly improved biocompatibility compared with crude conditioned medium (RSC-CM). Functionally, RSC-EXO significantly increased collagen synthesis and showed a trend toward enhanced wound closure in fibroblasts. In addition, RSC-EXO reduced melanin production in α-MSH-stimulated B16F10 melanoma cells and suppressed the secretion of pro-inflammatory cytokines, including IL-1α, IL-6, and TNF-α, in LPS-stimulated RAW 264.7 macrophages. Proteomic analysis revealed a distinct cargo enriched in stress-, defense-, and metabolism-related proteins, while small RNA sequencing identified a heterogeneous small RNA population containing a limited fraction of miRNA-sized reads. Collectively, these findings suggest that RSC-EXO represents a biologically active plant-derived vesicle population with regenerative and anti-inflammatory activity observed in vitro in skin-related cellular models and support its potential as a promising platform for future cosmeceutical and dermatological applications. Full article

Since the status of C. jambhiri is “rare” and C. aurantifolia is endowed with folk medicinal properties, our study aimed at producing true-to-type seedlings for further conservation by using PGRs, ferrous sulfate, and casein hydrolysate in MS medium to induce in vitro germination and “callus” formation from seeds of C. jambhiri Lush. It also focused on evaluation of suitable species and the best type of explant for organogenesis. The present study was undertaken to develop an efficient micropropagation protocol for C. jambhiri Lush. and C. aurantifolia. The frequency of callus induction increased to its maximum when 2.0 mg/L 2,4-D and 1.0 mg/L picloram were added individually. The calli derived from 2,4-D exhibited maximum regeneration potentiality. In addition, sucrose (30 g/L), dextrose (60 g/L), and coconut water (10 mL/L and 15 mL/L) also enhanced callus induction. Regarding heavy metals, 100 ppm of Fe₂SO₄ exhibited maximum germination percentage (84.33%) from seeds of C. jambhiri Lush. However, maximum callus induction (50.00%) was induced from the seeds of C. aurantifolia incubated in Fe₂SO₄ (400 ppm). The maximum number of shoots per callus was produced (5.13) with the addition of 200 mg/L casein hydrolysate in the callus-induction medium. Pearson correlation analysis revealed a positive and significant association of the number of plantlets/calluses with shoot length and regeneration percentage, respectively. It was phenotypically observed that the tissue culture traits performed better from explants derived from C. jambhiri Lush. than C. aurantifolia when subjected to varying concentrations of PGRs, carbon sources, organic adjuvants, and heavy metals, respectively. Full article

Agave guiengola Gentry is a microendemic ornamental species native to Oaxaca, México, with restricted distribution and threatened by human activities, placing it at risk of extinction. Two culture media were evaluated: Murashige and Skoog (MS) medium and modified MS medium, together with medium consistency (semisolid and liquid) and plant growth regulators (PGRs): 6-benzylaminopurine (BA) (5.0 mg L⁻¹) for MS media and a combination of kinetin (KIN) and 3-indoleacetic acid (IAA) (5.0–9.0 and 0.1–1.0 mg L⁻¹ respectively) for modified media. Shoot clusters were grown in two consecutive 30-day phases, with transfer to opposite consistency after the first month of cultivation (first phase). Growth, hyperhydricity, rooting and callus formation were evaluated. Treatments using modified formulation + KIN + IAA under both consistencies had the greatest growth (5.5 and 5.0 cm²). Hyperhydricity was more frequent in liquid MS media (up to 100%), while adjusted MS medium had lower incidence with or without PGRs (0.0% and 6.6%). Transfer from liquid-to-semisolid medium reduced hyperhydricity (10.47%) compared with the reverse (31.35%). Rooting occurred frequently with modified formulation media. Callogenesis occurred mainly using MS + BA media (up to 93.2%), associated with adventitious shoot formation. A synergistic biphasic effect is proposed using semisolid MS + BA followed by modified MS liquid + KIN + IAA, optimizing in vitro growth for conservation. Full article

In vitro cultured plant calli, induced through dedifferentiation, are colonized by diverse endophytes. Most of these endophytes, being substantially inherited from the mother plant and highly dependent on the host’s internal ecological niche, are termed host-dependent endophytes (HDEs). Due to their close association with their hosts, HDEs exhibit heritable characteristics. However, our current understanding of plant HDEs and their effects on the host plant is limited. In this study, we characterized the composition and potential functions of the endophytic microbiota in grapevine calli derived from different varieties and organs corresponding to Cabernet Sauvignon berry flesh (CF), Rose Honey berry flesh (RF), and Rose Honey shoot tip (RS) using high-throughput sequencing and bioinformatics. Our results showed that the genotype and organotype of the explant did not affect the alpha diversity of endophytes in callus, but were associated with differences in beta diversity and community structure of the endophytic microbiota. Different types of grapevines calli inherited distinct endophytes from their mother plants, whereas sharing a conservative core endophytic microbiota consisting of a small number of amplicon sequence variants (ASVs) with high relative abundances (bacteria: 38 ASVs ranging from 79 to 92%; fungi: 9 ASVs ranging from 32 to 58). Prediction analyses using revealed conserved functional traits of the endophytic microbiota across callus types, including a core suite of bacterial adaptive phenotypes, stable central metabolism dominated by oxidative phosphorylation, and uniformly structured fungal communities dominated by saprotrophs and pathotrophs, while consistently containing yeast-form fungi. Although minor variations such as elevated trait abundance in the CF group were noted, no statistically significant functional divergence was observed, demonstrating that the endophytic microbiota of grapevine callus maintains a conserved functional profile across different types. Collectively, this study provides a methodological framework for investigating plant HDEs and offers new insights into host-endophyte interactions at the cellular level. Full article

Protein synthesis is a crucial biosynthetic process in all organisms, including plants. The integrity of the translational machinery, especially ribosomes, can be compromised during rapid cell division in ontogenesis or in response to environmental stress. In this study, Northern blotting was employed to analyze total RNA from various angiosperms, focusing on small 5′- and 3′-terminal 18S rRNA fragments. Stem-loop array RT-PCR was employed to map the cleavage sites within the target regions. Severe stress, such as extreme drought, induced the accumulation of three distinct 18S rRNA fragments across diverse angiosperm taxa, indicating that this phenomenon is likely universal. In rapidly dividing cells, such as those found in in vitro callus cultures and germinating wheat embryos, high levels of discrete 5′-terminal fragments were observed, while 3′-terminal fragments were absent. The stem-loop array RT-PCR mapping identified specific sites of 18S rRNA strand breaks. Structural annotation of the 3D model of the plant 40S subunit revealed spatial clustering of these sites in proximity to the RPS6 binding region. Notably, wheat cultivars that are tolerant to osmotic stress exhibited significantly higher levels of 18S rRNA fragmentation than sensitive cultivars. This suggests a regulatory mechanism rather than a mere byproduct of apoptotic-like regulated cell death. Additionally, fragmented ribosomes were gradually eliminated during embryo maturation, indicating a process of programmed functional ribophagy. Our findings suggest that a potential inability of plant tissues to selectively retain functional ribosomes might contribute to a decline in generative potential. Monitoring the integrity of the translational machinery could improve breeding efficiency and aid in preserving long-term stored germplasm. Full article

Efficient propagation of Hydrangea arborescens is essential for the stable production of high-quality plantlets. However, propagation via stem cuttings is often limited by environmental conditions and inconsistent rooting. This study aimed to identify an effective in vitro culture medium by integrating quantitative growth traits with image-based quality analysis. Seven culture media (M1–M7), consisting of Murashige and Skoog (MS), McCown Woody Plant (McCown), and Gamborg B5 basal media supplemented with different plant growth regulator combinations, were evaluated based on shoot number, root number, plant height, and fresh weights, and plantlet quality was assessed using Green area, ExG (excess green index), and a composite z-score. Significant differences were observed among treatments. M5 and M7 produced the highest shoot numbers, and M7 showed the greatest fresh weight. Image-based analysis indicated that M2 and M7 exhibited the highest overall quality, whereas M3 showed the lowest performance. Basal media types did not significantly affect plantlet quality, whereas hormone treatments enhanced both shoot multiplication and callus formation. A positive association was observed between callus formation rate and shoot number (Spearman’s ρ = 0.74, p < 0.001). Overall, M7 (Gamborg B5 medium supplemented with 30 g∙L⁻¹ sucrose, 1.5 mg∙L⁻¹ BA, and 0.25 g∙L⁻¹ gelrite) provided a balanced combination of high propagation efficiency and plantlet quality, and these findings contribute to the efficient production of high-quality planting materials. Full article

This study investigated the interactive effects of the culture system and carbon source on growth, shoot proliferation, and mineral nutrition dynamics in the in vitro propagation of chestnut. Explants of the ‘Akyüz’ cultivar were used in the Woody Plant Medium. Both plant tissues and culture media were analyzed for Fe, Cu, Mn, Zn, and Mg concentrations. Morphological parameters, nutrient accumulation, and depletion patterns were evaluated. The results demonstrated that the liquid culture system supplemented with sucrose significantly enhanced plant growth, chlorophyll content, callus development, and shoot multiplication. Sucrose treatments promoted higher accumulation of Fe, Cu, Zn, and Mg in plant tissues, whereas glucose treatments resulted in significantly higher Mn accumulation. Correlation and principal component analysis revealed strong positive relationships between growth parameters and Fe, Mg, Cu, and Zn, whereas Mn exhibited significant negative correlations. Among the machine learning models, Support Vector Regression showed the highest predictive performance for plant length (R² = 0.74) and SPAD (R² = 0.87). Nutrient depletion analysis showed substantial reductions in mineral concentrations in all treatments after four weeks. Overall, the combination of liquid culture systems with sucrose provides optimal conditions for chestnut micropropagation by promoting favorable nutrient interactions and minimizing antagonistic effects. Full article

Plants produce diverse metabolites with potential benefits for human health. However, the metabolomes of plant callus cultures—cell cultures analogous to stem cells—remain poorly characterized in terms of their functional relevance. This study aimed to systematically profile and functionally annotate metabolites from diverse plant callus cultures to better understand their potential biological activities and applications. We profiled the metabolomes of six plant calli: Acacia concinna (Shikakai), Daucus carota (carrot), Hibiscus sabdariffa (hibiscus), Linum usitatissimum (flax), Ocimum sanctum (tulsi), and the Nicotiana tabacum Bright-Yellow 2 (BY-2) cell line. To facilitate functional interpretation, we developed Metabolite2Function (M2F), a pipeline that annotates metabolites with biological functions using scientific literature and large language modeling. Untargeted metabolomics identified 177 metabolites, revealing clustering patterns independent of genetic relationships, culture age, or growth rate. Tulsi and carrot calli exhibited enrichment in metabolites relative to the tobacco reference line, whereas flax and hibiscus were comparatively depleted. Most metabolites varied across at least four calli, and 10% were unique to a single species. Using M2F, we annotated 87 metabolites with beneficial activities, including antioxidant, anti-glycation, anti-inflammatory, and anti-senescence functions, as well as skin-related effects such as collagen production and brightening. Notably, antioxidant and anti-senescence metabolite levels correlated with corresponding biological activities in human cells. Plant callus cultures generate distinct and functionally diverse bioactive metabolomes. M2F provides a scalable framework for systematic functional annotation relevant to human health and cosmetic applications. Full article

2,4-Dichlorophenoxyacetic acid (2,4-D) is a vital exogenous auxin for the induction and proliferation of litchi embryogenic callus. At present, its molecular regulation mechanism remains unclear. In this study, transcriptome sequencing samples were selected based on different cell growth phenotypes observed in ‘Feizixiao’ litchi embryogenic callus cultured in liquid medium with or without 2,4-D. By integrating transcriptome profiling with weighted gene co-expression network analysis (WGCNA), we identified key genes and signaling pathways dynamically responsive to 2,4-D concentration changes. We identified 558 commonly differentially expressed genes (DEGs), of which 117 were up-regulated and 387 were down-regulated; functional enrichment analysis revealed significant enrichment in the “plant hormone signal transduction” and “phenylpropanoid biosynthesis” pathways. In the former pathway, genes such as AUX28, GH3.17, GH3.6, and ARR5 were up-regulated; in the latter, by comparison, β-glucosidase 47 and Peroxidase 61 exhibited increased expression levels induced by 2,4-D. Furthermore, among these DEGs, 57 transcription factors belonged to 24 families. Notably, VRN1, FEZ, and DOF5.4 were significantly and rapidly induced by 2,4-D. WGCNA results demonstrated a significant positive correlation between the yellow module and 2,4-D treatment. Small heat shock protein (sHSP) genes constituted the core hub genes in the yellow module. Through Venn analysis of DEGs and key modules, 38 cross-genes were identified, of which non-specific lipid-transfer protein-like genes (nsLTP) were found to be specifically up-regulated without 2,4-D. The transcription factors and genes identified work in synergy to ensure the formation and sustained proliferation of embryogenic callus by precisely regulating the dynamic balance of auxin and cytokinin within cells and maintaining the stability of cell structure. Our findings provide a crucial theoretical foundation for understanding the molecular mechanism of 2,4-D in regulating litchi embryogenic callus proliferation. Full article

Background: Konjac (Amorphophallus spp.) is an economically important crop valued for the glucomannan content in its corms. Currently, the konjac industry faces germplasm degeneration due to long-term asexual propagation. Developing tissue culture and genetic transformation techniques is essential for its genetic improvement. The WUSCHEL-related homeobox (WOX) transcription factors are critical regulators of somatic embryogenesis and stem cell maintenance in plants. Methods: In this study, we performed genome-wide identification and characterization of WOX genes in the A. konjac reference genome. Furthermore, comparative transcriptomic analyses and functional verification were conducted in A. bulbifer. Results: A total of 12 AkWOX genes were identified in A. konjac, and their structural features were documented. Comparative transcriptomic analysis of A. bulbifer revealed that AbWOX genes were differentially expressed between embryogenic calli (EC) and non-embryogenic calli (nEC). Notably, AbWOX2 was significantly upregulated in EC. Overexpression of AbWOX2 significantly promoted callus proliferation and shoot regeneration in A. bulbifer. Furthermore, AbWOX2-overexpressing lines exhibited a 5.3-fold increase in genetic transformation efficiency (from 5.12% to 27.31%) compared to the control. Conclusions: We characterized the diverse expression patterns of the WOX gene family in Amorphophallus. Crucially, we identified specific individual members—most notably the markedly upregulated AbWOX2—that function as pivotal drivers of somatic embryogenesis and serve as promising candidates for enhancing regeneration and genetic engineering efficiency in Amorphophallus species. Full article

While L-tryptophan is a precursor of plant growth regulators, its effects on secondary metabolism, amino acid profile and cell wall organization in flax callus remain underexplored. This study aimed to optimize flax callus shaken cultures and evaluate the impact of L-tryptophan (0.1 mM and 1 mM) on structural properties of plant cell walls in tested callus using Fourier transform infrared spectroscopy. The impact of L-tryptophan on callus proliferation and metabolism was also determined, because amino acids (among them L-tryptophan) can promote the growth of callus. The results showed that 1 mM L-tryptophan is an effective elicitor, which stimulates flax callus to accumulate larger amounts of bioactive compounds, especially carotenoids and polyphenols, than control callus cultured without L-tryptophan. A lower concentration of L-tryptophan (0.1 mM) slightly improved the level of determined secondary metabolites (except flavonoids). The effect of L-tryptophan on polymers in plant cell walls was investigated. The data confirm that the plant cell wall is a dynamic structure, capable of remodelling in response to growth conditions and external agents. L-tryptophan (0.1 and 1 mM) reduced cellulose levels and induced structural changes in cellulose compared to the untreated control. The structural analyses also suggested a decrease in lignin level and increase in pectin amounts in flax callus after tryptophan addition in comparison to control callus. The results may reflect the relationship between tryptophan and auxins (which are derived from tryptophan) and confirm the role of these metabolites in shaping the structure of the plant cell wall. In fact, an increase in tryptophan level was confirmed in flax callus in tested experimental conditions (supplementation of cultures with both doses of L-tryptophan). These findings have practical significance, because L-tryptophan is also used as a fertilizer or component of fertilizers in plant cultivation. Full article

The global challenge of feeding a growing population while minimizing environmental impacts necessitates novel food production systems. Plant cellular agriculture offers a sustainable alternative for producing food ingredients; however, its commercial viability is hindered by the high costs and regulatory hurdles associated with conventional reagent-grade culture media. In this study, we developed a novel, cost-effective, and food-grade basal culture medium, FG-N6CI, for rice cellular agriculture. By replacing reagent-grade basal-medium components of the N6CI medium with food-grade alternatives, specifically by substituting chemical reagents with yeast extract, kelp powder, manganese yeast, and a boron supplement, we formulated a food-grade basal nutrient composition while retaining reagent-grade phytohormones. Rice (Oryza sativa L. ‘Taichung 65’) callus cultured on FG-N6CI medium exhibited significantly higher fresh weight (7.1 g) than the conventional N6CI medium (5.8 g) after 35 days (p < 0.05). Gene expression analysis showed no significant differences between the expression of OsHDA710 and OsTIR1, suggesting that FG-N6CI supports normal cellular proliferation and signaling similar to the standard medium. Economically, the cost of FG-N6CI medium was reduced by approximately 72% compared with that of the commercial reagent-grade mixture (219 JPY/L vs. 795 JPY/L). These results demonstrate that FG-N6CI is an economically competitive basal medium for scaling-up plant cellular agriculture. Full article

A simple method for the efficient high-throughput transformation of hybrid poplar (Populus tremula x alba clone 717 1B) is described. Factors considered in developing the method included the ease and efficiency of preparing large numbers of explants for transformation, and selection of culture media that enhanced cell and tissue growth while promoting shoot regeneration competence. We found that petiole explants from in vitro-cultured plantlets can be easily collected and prepared for transformation and regenerate shoots comparable to stem or leaf explants. Culturing petiole explants on Driver Kuniyuki Walnut (DKW) medium resulted in significantly greater tissue growth compared to Murashige Skoog (MS) medium. Moreover, the inclusion of low concentrations of thidiazuron (TDZ) in callus-inducing media (CIM) significantly enhanced shoot regeneration competence of cultured petiole explants. As a consequence, the combination of petiole explants cultured on DKW medium along with 2.2 ug/L TDZ during the callus induction phase resulted in rapid and efficient transformation of this hybrid poplar genotype. When applied to genomic approaches such as activation tagging or CRISPR-Cas9 and Cas12a gene editing, we obtained transformation efficiencies ranging between 70% and 90%. The described protocol provides a simple and efficient method that is easily scalable for high-throughput approaches, which could facilitate genome-wide methods for the rapid and efficient production of transformed hybrid poplars. Full article

Verbascum hasbenlii Aytaç & H. Duman is a narrowly distributed endemic species native to Çanakkale, Türkiye. This study aimed to establish and optimize callus induction and cell suspension culture systems for V. hasbenlii. Leaf explants obtained from 10-week-old seed-derived in vitro plants were cultured on six Murashige and Skoog (MS) media containing different combinations of α-naphthaleneacetic acid (NAA; 0.5 or 1.0 mg/L) and 6-Benzylaminopurine (BAP; 0.5, 1, 2 or 3 mg/L). After four weeks, callus induction was achieved in all treatments (96–100%), although significant differences were observed in explant browning, callus biomass, diameter, and morphology. The medium supplemented with 0.5 mg/L BAP + 0.5 mg/L NAA produced the highest callus biomass (1.245 g) and diameter (5.06 mm), while maintaining low explant browning and a compact-friable texture suitable for suspension culture establishment. Cell suspension cultures exhibited a typical growth pattern with lag, exponential, and stationary phases. On day 9, cultures showed increased growth parameters, including packed cell volume (PCV: 7.50%), fresh weight (FW: 0.0580 g), and dry weight (DW: 0.0052 g), with relatively high cell viability (80.72%). Biomass accumulation reached maximum levels between days 18–21, while cell viability decreased to 66.82%. These findings provide an optimized in vitro culture system for future studies on secondary metabolite production in V. hasbenlii. Full article

Anther-derived somatic embryogenesis is a valuable approach in rubber tree (Hevea brasiliensis) breeding; however, its effectiveness is highly influenced by the developmental stage of the microspores. The present investigation focused on male flower buds of the cultivar Reyan 73397 at successive developmental stages to examine the relationship between visible bud characteristics and internal microspore development, assess how microspore developmental stage affects callus induction and somatic embryo formation, and identify the stage with the greatest embryogenic potential. Cytological observations distinguished six well-defined phases of microspore development, spanning from microspore mother cells to fully mature pollen grains, each reliably linked to particular bud diameters, coloration, and anther morphology. Anthers corresponding to each developmental phase were cultured in vitro, and their ability to initiate callus and produce somatic embryos was systematically evaluated. Anthers containing uninucleate microspores exhibited the highest rates of both callus formation and somatic embryogenesis, with the early-uninucleate stage showing the strongest response. This stage consistently matched flower buds measuring 1.42–1.57 mm in transverse diameter and displaying a green to yellowish-green appearance. In contrast, anthers collected at the microspore mother cell and tetrad stages did not produce embryogenic responses. Histological evidence has indicated that both callus and somatic embryos originate from diploid somatic tissues of the anther wall, particularly connective parenchyma cells, rather than from microspores themselves. Based on these findings, a rapid, non-destructive selection method integrating bud diameter, bud color, and sieve-based size separation was developed to identify responsive explants efficiently. Overall, this study defines the optimal developmental window for anther culture in rubber trees, verifies the somatic origin of embryogenic tissues, and provides a practical morphological and cytological basis for improving anther culture efficiency in rubber tree breeding programs. Full article