Search Results (335)

Background/Objectives: Phyllostachys edulis is the most widely distributed and economically important bamboo species in China. However, the genetic transformation in P. edulis is still limited by a long regeneration cycle and low regeneration and transformation efficiency. Carbon nanotube-based delivery systems in plants have the advantages of simplicity, rapidity and low cost. Moreover, morphogenetic regulators BBM (BABY BOOM) and WUS (WUSCHEL) play significant roles in plant regeneration. Methods: Here, immature zygotic embryos were used to induce P. edulis callus, and using single-walled carbon nanotubes (SWNTs)-based delivery technology, PeBBM2, PeWUS-DNA (with introns) and PeWUS-cDNA (without introns) were introduced to P. edulis callus either individually or in combination. Conclusions: The results showed that the 0.9–1.0 mm (long axis) embryos exhibited the lowest contamination rate and the highest induction efficiency. Moreover, the results indicated that the co-transformation of PeBBM2-PeWUS more effectively boosted the growth area of the callus. However, only the PeBBM2-overexpression callus could form shoots. Compared with the wild type, the PeBBM2-overexpression lines showed reduced expression of AGL15 and increased expression of IAA30 and YUC. In conclusion, these findings suggested that SWNTs-mediated DNA delivery is a potential strategy for the genetic transformation of P. edulis callus. Additionally, the findings indicate‌ that the PeBBM2 and PeWUS genes can accelerate callus enlargement in P. edulis, whereas PeBBM2 might play a more important role in shoot formation. This study provides a basis for developing a genetic transformation system for plants based on SWNTs-mediated DNA delivery and morphogenetic regulators. 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

Lilium longiflorum is a diploid lily species valued for its tolerance to humid–hot environments and pleasant fragrance. However, its poor cold hardiness and low bulb-forming capacity limit its cultivation. To overcome these deficiencies, autotetraploids were previously generated in our laboratory via somatic doubling. In order to expand the reproductive efficiency of the two, this study optimized in vitro regeneration and bulblet enlargement protocols. We analyzed the effects of various plant growth regulators and sucrose concentrations, alongside the expression of genes related to carbohydrate metabolism and hormone signaling. Results revealed divergent regenerative pathways: diploids favored direct organogenesis (optimal medium: MS + 30 g/L sucrose + 0.5 mg/L 6-BA + 0.2 mg/L NAA + 1.0 mg/L glyphosate), whereas tetraploids thrived via a TDZ-induced callus pathway (1/2 MS + 30 g/L sucrose + 1.0 mg/L NAA + 0.2 mg/L TDZ). During bulblet enlargement, diploids were predominantly regulated by IBA and prone to proliferation (optimal enlargement medium: MS + 60 g/L sucrose + 2.0 mg/L IBA), while tetraploids were sucrose-sensitive and prioritized single-bulb hypertrophy (MS + 60 g/L sucrose + 0.5 mg/L IBA + 0.1 mg/L 6-BA + 0.1 mg/L CPPU). qRT-PCR indicated that LlAGPS1, LlGBSSI, LlSWEET15, LlMYC2, and LlSAUR32 were highly expressed in tetraploids during rapid enlargement (24–36 d), suggesting a role in bulb hypertrophy, whereas upregulated LlSUS4 and LlCWIN3 in diploids correlated with proliferation. The study provides a practical technical reference for the industrialized propagation of high-quality L.longiflorum bulbs and provide a theoretical foundation for understanding ploidy-dependent development in Lilium. Full article

Drought stress critically impacts plant growth and productivity. The bZIP transcription factor family is crucial for abiotic stress responses, yet its role in larch drought tolerance remains unclear. This study identified 19 bZIP genes in Larix kaempferi (Lamb.) Carr. and characterized LkbZIP4. Bioinformatics analysis classified it into the A subgroup. Subcellular localization and yeast two-hybrid assays confirmed that it is a nucleus-localized transactivator. Expression pattern analysis revealed that LkbZIP4 was highly specifically expressed in roots and was significantly induced by drought stress. A series of transgenic overexpression lines was successfully established through Agrobacterium tumefaciens-mediated method, using embryogenic callus of hybrid larch (L. kaempferi × L. gmelinii). Under 7% PEG-induced drought stress, LkbZIP4-overexpressing transgenic calli displayed enhanced drought tolerance relative to wild-type. This was evidenced by better growth, higher biomass, and reduced membrane damage, indicated by lower malondialdehyde content and relative electrolyte leakage. Meanwhile, these transgenic calli accumulated higher levels of osmoregulatory substances, including proline and soluble sugars, along with enhanced activities of antioxidant enzymes including superoxide dismutase and peroxidase. Our results indicate that LkbZIP4 functions to promote drought tolerance in larch, likely through the enhancement of osmotic adjustment and oxidative defense mechanisms. Full article

To overcome the seasonal constraints of explant availability and facilitate genetic improvement in Catalpa ovata, this study established a dual-pathway in vitro regeneration system (encompassing adventitious shoot organogenesis and somatic embryogenesis) using mature zygotic embryos. We systematically evaluated the synergistic effects of maternal genotypes, plant growth regulators (PGRs), basal media, and the histone deacetylase inhibitor Trichostatin A (TSA). Genotype screening revealed significant divergence in regenerative potential, with the half-sib family 32F17 exhibiting superior responsiveness (84.7% callus induction). A high cytokinin-to-auxin ratio (ZA3 medium) optimally drove direct shoot organogenesis. For adventitious shoot proliferation, the addition of TDZ significantly improved the multiplication coefficient (up to 2.99 on ZB4 medium), although a physiological trade-off with shoot elongation was observed. In parallel, the application of 10 µM TSA significantly enhanced somatic embryogenesis from embryogenic calli, effectively alleviating the inhibitory constraints of exogenous PGRs. For rhizogenesis, the DKW basal medium proved superior to half-strength MS, with the ZE3 treatment (0.1 mg·L⁻¹ NAA + 0.1 mg·L⁻¹ IBA) yielding the highest rooting frequency (69.6%) and robust root architecture. Notably, while somatic embryo conversion remained recalcitrant, plantlets derived exclusively from the adventitious shoot organogenesis pathway were successfully acclimatized ex vitro. These transplanted plantlets exhibited consistently high survival rates (83.1–84.4%) across all tested genotypes, effectively overcoming the initial genotype-dependent recalcitrance. Collectively, this optimized protocol provides a reliable technical platform for the large-scale clonal propagation and biotechnological breeding of C. ovata. 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

Salt stress severely restricts grape (Vitis vinifera L.) production. Serine/arginine-rich (SR) proteins, as a class of RNA-binding proteins, play important roles in plant growth, development and stress responses. However, the function and regulatory mechanism of VvSR34a in grape salt tolerance remain unclear. In this study, grape callus and cutting seedlings were used as materials to explore the role and molecular mechanism of VvSR34a in grape salt stress response. The results showed that, under 100 mM NaCl treatment, the relative level of VvSR34a in grape callus exhibited a ‘first increase and then decrease’ pattern, reaching a peak at 2 h, and the gene was localized in the nucleus. Transgenic experiments confirmed that the overexpression of VvSR34a significantly enhanced salt tolerance in grape callus and cuttings, as evidenced by better growth status, higher chlorophyll content and root activity, as well as lower electrolyte leakage and malondialdehyde (MDA) content under salt stress. In contrast, the silencing of VvSR34a significantly increased salt sensitivity in grapes. Y2H and LCI assays verified that VvSR34a physically interacts with VvCOP9. VvCOP9 may play a negative regulatory role in the salt stress response of the grapevine, and through the loss of the high salt-tolerant phenotype in the VvSR34a/VvCOP9-RNAi lines, it demonstrated that VvCOP9 is genetically upstream of VvSR34a. Furthermore, the ubiquitination and degradation assay demonstrated that VvCOP9 can significantly promote the degradation of VvSR34a. RNA-seq analysis showed that a total of 2834 differentially expressed genes and 202 alternative splicing events were detected in VvSR34a overexpression lines. These differentially expressed genes were significantly enriched in ATPase activity, redox and hormone signaling pathways. This study demonstrates that VvSR34a positively regulates salt tolerance in grapes, providing an important theoretical basis for molecular breeding of salt-tolerant grapevines. 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

Papaya (Carica papaya L.) is a highly cross-pollinated crop that exhibits considerable genetic variability when propagated through seeds, resulting in non-true-to-type progeny. Therefore, the development of an efficient in vitro regeneration system is essential for large-scale clonal propagation of elite cultivars. In the present study, a highly efficient and reproducible somatic embryogenesis protocol was developed for C. papaya var. TNAU Papaya CO 8 using immature zygotic embryos as explants. This study provides the first comprehensive comparative evaluation of three basal media, viz., Murashige and Skoog Medium, N6 Medium, and Woody Plant Medium, for somatic embryogenesis and plant regeneration in this variety, along with the optimization of polyamine-enriched media for enhanced plantlet recovery. The embryogenic potential of explants was assessed across different stages, including callus induction, somatic embryo development, plant regeneration, shoot elongation, rooting, and acclimatization. Maximum callus induction (81.96%) was observed on half-strength MS medium supplemented with 2,4-Dichlorophenoxyacetic acid under dark conditions, followed by ½ N6 (63.00%) and ½ WPM (58.02%). Somatic embryo initiation was highest on ½ MS medium containing 2.0 mgL⁻¹ 2,4-D (77.82%). Somatic embryos developed through distinct globular, heart, torpedo, and cotyledonary stages. Embryo maturation was significantly enhanced on MS medium supplemented with abscisic acid, polyethylene glycol, benzylaminopurine, and proline. The highest plantlet regeneration (85.02%) was achieved on MS medium enriched with putrescine, whereas comparatively lower regeneration was recorded on N6 (75.99%) and WPM (57.97%). Shoot elongation was significantly improved by supplementation with gibberellic acid (1.0 mgL⁻¹). Root induction was optimal on half-strength MS medium containing Indole-3-butyric acid, 1-Naphthaleneacetic acid, phloroglucinol, and activated charcoal, resulting in well-developed roots. Regenerated plantlets were successfully acclimatized in a cocopeat–vermicompost substrate with a survival rate of 74.01%. The optimized protocol provides a reliable and efficient system for large-scale clonal propagation and offers promising applications in genetic transformation and commercial production of papaya var. TNAU papaya CO 8. Full article

Growth Regulating Factors (GRFs) are plant-specific transcription factors that play crucial roles in regulating growth and development throughout the plant life cycle. A total of 34 Gossypium hirsutum GRF family genes were identified at the genome-wide level, which were unevenly distributed on 19 chromosomes, and were predicted to be mainly localized in the nucleus and plasma membrane. The number of GRF family genes varied greatly among different species, and they were categorized into four subfamilies (I–IV) according to their phylogenetic relationships. The G. hirsutum GRF genes possessed specific highly conserved structural domains, Trp-Arg-Cys motif (WRC) and Gln, Leu, Gln motif (QLQ), and structural analysis of the genes revealed that they contained 1–23 exons, and most of them contained UTRs. Intraspecies covariance analysis revealed that the GRF genes expanded in G. hirsutum by segmental duplication. The promoter region of the G. hirsutum GRF gene contained a large number of adversity stress response elements, as well as a small number of hormone response elements and growth and development-related response elements. Transcriptome data showed that the expression of G. hirsutum GRF genes was significantly higher in leaves than in other tissues, and some GRF genes responded to a variety of abiotic stresses. Additionally, transcriptomic sequencing revealed significantly higher expression levels of GhGRFs (e.g., GhGRF13/14/18) in embryonic callus (EC) compared to non-embryonic callus (NEC). This differential expression was validated by RT-qPCR, which confirmed that GhGRF13/14/16/20 were significantly upregulated in EC relative to NEC. These findings provide valuable candidate genes and molecular insights for improving G. hirsutum regeneration efficiency and yield-related traits through genetic manipulation, thereby accelerating the molecular breeding of elite G. hirsutum varieties. Full article

Callogenesis is a fundamental step in plant biotechnology and tissue culture, providing the basis for multiple scientific and practical applications. In this study, the impact on callogenesis of different plant growth regulators was studied on Cannabis sativa L. (a non-commercial genotype of hemp), with the objective of identifying the most suitable combination for the establishment of vigorously growing, friable calli. Forty-nine media combinations were evaluated using four PGRs: two auxins (2,4-dichlorophenoxyacetic acid, naphthaleneacetic acid) and two cytokinins (6-benzylaminopurine, kinetin). Parameters such as percentage of callus induction, proliferation, colour, texture, and growth area were assessed. Three media were identified for further spectrophotometric assays and targeted gene expression analysis: the first containing 2,4-dichlorophenoxyacetic acid 1.5 µM and benzylaminopurine 1.5 µM, the second with 2,4-dichlorophenoxyacetic acid 1.5 µM and kinetin 1.5 µM and the third supplemented with 2,4-dichlorophenoxyacetic acid 4.5 µM and kinetin 1.5 µM. The last medium proved to be superior in terms of vigour, friability and phenolic content and showed increased expression of genes involved in the early steps of the phenylpropanoid pathway. These findings highlight the central role of auxin–cytokinin interactions in regulating both callus formation and secondary metabolism. The optimised medium opens the way to subsequent biotechnological applications relying on the cultivation of plant cell suspension cultures. Full article