Search Results (1,393)

Cycloheptylprodigiosin is a promising anticancer candidate that induces cancer cell death accompanied by severe Golgi stress. Although the soybean oil-based optimized MB2216 medium produced a total prodiginine titer approximately three times that of the basal MB2216 medium, the overall production level remained limited. In addition, a substantial fraction of the pigments partitioned into floating oil droplets, hindering efficient recovery by simple centrifugation. In this study, a novel medium was rationally formulated based on genomic insights derived from homology analysis of conserved biosynthetic genes involved in cycloheptylprodigiosin production in Spartinivicinus ruber MCCC 1K03745^T. Sequential optimization through single-factor experiments, full factorial designs, steepest ascent experiments and response surface methodology identified an optimal medium consisting of peptone (5 g/L), yeast extract (1 g/L), peanut meal (7.611 g/L), and L-Proline (0.695 g/L) prepared in seawater at pH 7.6. Under the optimized conditions, the total prodiginine titer reached 53.33 mg/L, which was 11.37 times that of the basal MB2216 medium and 3.29 times that of the soybean oil-based MB2216 medium. Moreover, the pigment-associated biomass could be efficiently recovered by centrifugation. This study provides a genomics-informed strategy for improving prodiginine production in S. ruber and facilitates downstream pigment recovery. Full article

Aeromonas hydrophila is a major seafood-borne pathogen capable of persisting under preservative-associated stress by entering a viable but non-culturable (VBNC) state, thereby evading culture-based detection. Here, untargeted metabolomics was applied as the primary analytical approach to elucidate metabolic reprogramming during VBNC formation under seafood-relevant preservation conditions. Cells were incubated at 4 °C for 30 days in sodium benzoate-supplemented saline, comparing 0.85% NaCl (culturable condition) and 4% NaCl (VBNC-inducing condition), with sampling every 6 days. Under 4% NaCl with sodium benzoate, culturability declined from 6.18 log CFU/mL at day 0 to undetectable levels by day 30, while cell viability was retained, confirming VBNC induction. UHPLC–ESI–QTOF–MS profiling detected over 893 intracellular metabolic features, of which 518 metabolites were significantly altered between VBNC and culturable states at day 30. Principal component analysis revealed clear, time-dependent metabolic divergence, with the VBNC trajectory explaining 34.4% (PC1) and 11.5% (PC2) of total variance. Pathway enrichment analysis demonstrated significant remodeling of alanine, aspartate and glutamate metabolism (8/28 hits, FDR = 5.7 × 10⁻⁴); arginine biosynthesis (5/14 hits, FDR = 5.44 × 10⁻³); purine metabolism (10/70 hits, FDR = 8.34 × 10⁻³); and pyrimidine metabolism (7/39 hits, FDR = 1.35 × 10⁻²), indicating nitrogen conservation and metabolic downshifting. A robust biomarker panel, including depleted cyclic AMP, aminoadipic acid, hypotaurine, O6-CM-dG, and betaine, and enriched urocanic acid, pipecolic acid, proline, azelaic acid, and orcinol perfectly discriminated VBNC from culturable cells. These findings demonstrate that sodium benzoate-based preservation can induce a metabolically reprogrammed VBNC state in A. hydrophila, highlighting a hidden food safety risk beyond culture-based assessment. Full article

Milk fat synthesis in dairy cows is a complex process affected by ruminal fermentation, systemic metabolism, and mammary gland activity. To explore the metabolic interplay across these systems, a multi-tissue metabolomics approach (rumen fluid, plasma, and milk) using ultra-high-performance liquid chromatography–mass spectrometry was used to identify metabolic differences between Chinese Holstein cows with high (H-MF, 5.82 ± 0.41%) and low (L-MF, 3.60 ± 0.12%) milk fat content under the same diet. The bovine mammary epithelial cells (BMECs) were also cultured to evaluate the impact of a key metabolite, malic acid (MA), on lipid metabolism. Our findings reveal distinct metabolic profiles across rumen fluid, plasma, and milk, with 96, 109, and 79 differential metabolites, respectively, between the L-MF and H-MF groups. In rumen fluid, H-MF cows showed higher levels of lauric acid and succinic acid, linked to fatty acid biosynthesis, while the L-MF cows had elevated citraconic and orotic acids, associated with amino acid metabolism and liver stress. Plasma from the H-MF cows contained higher β-hydroxybutyric acid, methionine sulfoxide, and phosphatidylcholine, supporting lipogenesis, whereas L-MF plasma showed increased 3-hydroxy-L-proline, indicating tissue catabolism. In milk, the L-MF cows had higher MA, while the H-MF cows exhibited elevated L-carnitine, linked to fatty acid β-oxidation. Metabolite trend analysis during rumen fluid–plasma–milk showed that 211 metabolites were classified into 8 profiles. Profile 1 had the largest number of metabolites whose levels were down-regulated from rumen to plasma and enriched in lipid metabolism. Profile 3 (mainly related to amino acid metabolism) and profile 4 (mainly related to energy metabolism) exhibited opposite trends from plasma to milk. In vitro, 200 μM of MA reduced the triglyceride content in BMECs and down-regulated lipogenic genes and their protein expression levels (fatty acid synthase, stearoyl-CoA desaturase and sterol regulatory element binding protein 1). These results highlight how rumen fluid, plasma, and milk metabolites collectively influence milk fat synthesis, with MA acting as a key regulator of lipid metabolism in mammary epithelial cells. Full article

Objectives: Allogeneic stem cell transplantation (HSCT) is curative in acute myeloid leukemia (AML) but is limited by relapse and non-relapse mortality (NRM). Metabolomic prognostic value is unclear. We assessed whether plasma metabolite profiles at diagnosis, pre-transplant, and post-transplant are associated with overall survival (OS) and cause-specific mortality. Methods: We retrospectively analyzed plasma metabolites from 63 AML patients undergoing HSCT (263 samples). Results: Higher levels of valine (hazard ratio [HR] 24.454), citrulline (HR 20.478), 5-oxoproline (HR 11.766), and glutamine (HR 8.701) associated with higher NRM, while inosine diphosphate (HR 0.091) and pyridoxamine-5′-phosphate (HR 0.313) associated with lower NRM. For relapse-related mortality (RRM), higher levels of phenylalanine (HR 26.585), leucine/isoleucine (HR 10.755), indolepyruvate (HR 7.676), and creatinine (HR 13.874) were associated with higher RRM, while trans-4-hydroxy-L-proline (HR 0.101) was associated with lower RRM. Higher post-transplant ornithine (HR 0.063), 3-sulfocatechol (HR 0.590), and indole-3-acetate (HR 0.359) were associated with improved OS. Mixed-effects modelling identified lower dehydroascorbate and citrate in relapsed patients, with dehydroascorbate remaining significant after false discovery rate adjustment. Conclusions: Metabolomic profiling nominated candidate metabolites for validation in larger prospective studies and elucidated mechanistic pathways, potentially informing novel interventions or risk-adapted monitoring strategies in HSCT. Full article

The increasing demand for different value-added products from natural seaweeds requires a sustainable cultivation method for the regular supply of biomass and to safeguard the natural ecosystem from overexploitation. This study evaluated laboratory acclimatization of the green seaweed Acrosiphonia orientalis (DGR: 2.71 ± 0.21%; GPP: 12.55 ± 0.1 mg O₂ L⁻¹ day⁻¹), followed by a comparative evaluation of its physicochemical and biochemical characteristics, metabolite profile, and antiproliferative activity compared with naturally harvested seaweed. Metabolite profiling identified 47 compounds exhibiting differential accumulation patterns, with the natural specimens enriched in omega-3 polyunsaturated fatty acids, including docosahexaenoic acid, and the laboratory-acclimatized specimens exhibited elevated arachidonic acid levels. Amino acid profiling revealed higher concentrations of essential and non-essential amino acids in the natural specimens, with prominent levels of phenylalanine and aspartic acid, while the lab-acclimatized specimens were enriched in isoleucine, methionine, proline, and cysteine. The lab-acclimatized specimens exhibited significantly enhanced water absorption (WSC: 6 ± 0.25 mL/g DW; WHC: 2.68 ± 0.11 g/g DW) and higher total sugar (47.11 ± 0.52% Glc eq. DW) and phenolic contents (51.28 ± 0.54 mg GAE g⁻¹ extract), while the natural specimens had a superior oil-holding capacity (OHC: 1.8 ± 0.12 g/g DW); higher total flavonoid (123.62 ± 2.97 mg Q g⁻¹ extract), protein (5.11 ± 0.36 µg BSA eq/mg DW), and chlorophyll contents (8.82 ± 0.58 mg/L); and higher antioxidant activities (ABTS-EC₅₀: 67.33 ± 0.97 μg/mL extract). The mineral analysis revealed distinct elemental profiles, with enrichment of sodium, magnesium, and calcium in the lab-acclimatized specimens and a more favorable Na/K ratio (0.14 vs. 0.78) in the natural specimens. Of note, extracts from both seaweeds exhibited significant dose-dependent antiproliferative activity against HeLa cervical cancer cells (Wild EC₅₀: 118.63 ± 14.14 µg/mL extract; lab EC₅₀: 153.35 ± 10.18 µg/mL extract), suppressed colony formation in soft agar assays, induced nuclear condensation (based on Hoechst staining), and modulated the expression of key oncogenes (upregulating NDRG1, TP53, and CASP3 and downregulating BCL2, MYC, and CCND1). Collectively, this study provides an approach to acclimatize A. orientalis that may be utilized for developing a cultivation method. Moreover, this green seaweed has a great potential to be used for nutraceutical and functional food applications. Full article

Harsh environments and climate change hamper industrial hemp productivity. Under stress conditions, uniform germination and vigorous seedlings are key to sustaining crop establishment and performance. Trichoderma spp. are beneficial micromycetes, able to colonize plant roots and promote plant development even under abiotic stress conditions. Thus, the seed treatment with specifically selected Trichoderma isolates could be a useful strategy to enhance hemp seed germination and plantlet growth. In this view, a preliminary screening was performed with ‘Eletta campana’ cv. Nine out of 20 Trichoderma isolates enhanced the radicle growth (+66–111%); most of them resulted in good root colonization, but only four isolates significantly enhanced the shoot DW (+18–22%). Three isolates were selected for a pot experiment, compared to T. afroharzianum T22, to evaluate the effect on plant growth, root architecture, accumulation of photosynthetic pigments and stress-related compounds, and variation in antioxidant activity in 20-day-old plantlets. T. afroharzianum OR4 significantly promoted plantlet growth (+9% shoot DW and +11% leaf DW). The seed treatment had a low impact on the other variables studied, except in the case of foliar proline content, a marker of stress tolerance, that was greatly increased with T. afroharzianum T22 and T. atrobrunneum X44 (+32% and +17% DW). Full article

Faba bean-fed crispy tilapia represents a commercially valuable aquaculture product, renowned for its exceptional muscle firmness. However, the dynamic changes in muscle metabolite profiles during the tilapia crisping process remain largely unelucidated. In this study, proton nuclear magnetic resonance spectroscopy (¹H-NMR) combined with multivariate statistical analysis was employed to characterize and compare the muscle metabolomes of tilapia subjected to different crispness grades (CD0, CD2, CD4). A total of 11 differential metabolites were successfully identified, among which glycine, threonine, and trans-4-hydroxy-L-proline were demonstrated to be potential crispness-related biomarkers. Specifically, as the crispness grade increased from 0 to 4, the muscle contents of these key metabolites exhibited a consistent downward trend: glycine decreased significantly from 19.86 mM to 7.15 mM, threonine from 1.21 mM to 0.58 mM, and trans-4-hydroxy-L-proline from 2.25 mM to 0.89 mM. Subsequent metabolic pathway enrichment analysis further revealed that the glycine-serine-threonine metabolic pathway represented the most significantly perturbed pathway associated with the crisping process. Collectively, our findings demonstrate that faba bean-based feeding regimens enhance tilapia muscle crispness by orchestrating metabolite signatures involved in collagen biosynthesis and lipid metabolism. These results not only provide novel insights into the intrinsic molecular mechanisms underlying tilapia crisping but also establish a solid theoretical framework for the precise quality control and standardized production of high-quality crispy tilapia. Full article

Foliar nanofertilization is increasingly being explored as a strategy to enhance crop nutritional quality; however, dose-dependent physiological and metabolic responses remain insufficiently defined. This study evaluated the effects of conventional NPK (20:20:20) and nano-formulated NPK combined with zinc (3 and 5 g/L) on the mineral composition, bioactive compounds, antioxidant capacity, and metabolic profile of sweet pepper (Capsicum annuum L., cv. ‘Dora’) grown under controlled conditions. Physicochemical characterization of the nanofertilizer by dynamic light scattering and transmission electron microscopy confirmed nanoscale primary particle size and revealed concentration-dependent aggregation behavior at higher Zn levels. Significant differences (p < 0.05) were observed among treatments in macro- and microelement content, total phenolics, flavonoids, carotenoids, ascorbic acid, and antioxidant activity. The application of nano NPK combined with 3 g/L Zn resulted in the highest accumulation of total phenolics, flavonoids, and vitamin C, accompanied by enhanced antioxidant capacity, suggesting stimulation of secondary metabolism. In contrast, the higher Zn concentration (5 g/L) further increased carotenoid content but was associated with elevated proline levels, indicating the onset of physiological stress. Multivariate analyses (PCA and ROC) supported dose-dependent metabolic modulation and confirmed that combinations of selected metabolites contributed to clearer differentiation between fertilization regimes. Overall, the results highlight the existence of an optimal nano-zinc application range that enhances fruit functional quality while avoiding stress-related metabolic imbalance, emphasizing the importance of physicochemical stability in nano-enabled fertilization strategies. While this study focused on a single sweet pepper cultivar, future research should explore other pepper species to evaluate whether similar dose-dependent nano Zn effects are observed. Full article

The homogeneous nature of L-proline organocatalysts restricts their application in aldol condensation due to poor recyclability and stability. Herein, L-proline was heterogenized by ionic intercalation into Mg–Al layered double hydroxides (LDHs), yielding a series of proline-intercalated catalysts with tunable layer structures. Co-precipitation and memory-effect reconstruction strategies were employed to regulate interlayer spacing and proline loading. The resulting catalysts exhibited efficient performance in the aldol condensation of furfural with ketones under mild conditions. The reconstructed catalyst re-Mg₄Al₁P achieved a furfural conversion of 88.67% and a total product yield of 85.54% at room temperature, with product selectivity exceeding 95%. Structural characterizations confirmed that proline was stabilized within the LDH interlayers via R–COO—Mg electrostatic interaction while preserving the secondary amine active site. Mechanistic analysis indicated that the reaction proceeded through enamine- or enol-mediated pathways depending on water content, while the layered LDH framework imposed geometric confinement that suppressed side reactions. Catalyst deactivation in aqueous systems was mainly attributed to proline leaching rather than structural collapse. Full article

Selenium (Se) influences plant growth, yet its molecular regulation of amino acid metabolism in oat seedlings remains unclear. Through transcriptomic and metabolomic analyses, this study identified three major affected pathways: tryptophan metabolism (16 differentially expressed genes [DEGs], 13 differentially expressed metabolites [DEMs]), glycine, serine, and threonine metabolism (19 DEGs, 10 DEMs), and arginine and proline metabolism (24 DEGs, 13 DEMs). The T0.02 treatment (0.02 g/kg Na₂SeO₃) precisely regulates metabolism by selectively upregulating dimethylglycine in the glycine, serine, and threonine pathway and activating key genes (PRODH2, amiE2, AMD2) in the arginine–proline pathway, thereby promoting the growth of oat seedlings. The T0.1 treatment (0.1 g/kg Na₂SeO₃), promoted the accumulation of glycerate and threonine by upregulating the expression of two key genes (HPR3, ItaE1) related to glycine, serine, and threonine metabolism. Simultaneously, it enhanced the accumulation of L-ornithine, putrescine, 4-guanidinobutyric acid, and γ-aminobutyric acid through the upregulation of four key genes (ARG, ODC1, amiE1, and ALDH3) associated with arginine and proline metabolism. Additionally, the upregulation of key genes (ALDH2, 5-HTP) involved in tryptophan metabolism facilitated the accumulation of 5-methoxyindoleacetic acid and serotonin. This study primarily reveals the accumulation patterns of amino acid metabolites in oat seedlings subjected to selenium treatment and identifies key genes and metabolic pathways involved in the molecular response process. Furthermore, the research preliminarily elucidates potential regulatory nodes through which selenium treatment enhances amino acid accumulation, providing significant insights for understanding the comprehensive effects of selenium treatment on the stress resistance mechanisms of oat seedlings. Full article

This study aimed to investigate the changes in the meat quality characteristics of Duolang sheep using rumen metagenomic and muscle metabolomic analyses across different age groups. A total of 24 three-month-old male Duolang sheep were selected and reared, and samples of longissimus thoracis muscle and rumen contents were collected at 4, 6, and 8 months of age to evaluate meat quality, metabolites, rumen metagenome, and volatile fatty acids (VFAs). The results indicated that the lightness (L*_45min) and yellowness (b*_45min) of the longissimus thoracis muscle at 45 min post-slaughter were significantly higher at 4 and 6 months than at 8 months of age (p < 0.05). In terms of ruminal VFAs, butyrate concentration was significantly higher at 6 months than at 4 months (p < 0.05), and valerate concentration exhibited a quadratic relationship with age (p = 0.02). With increasing age, the relative abundances of Prevotella and Fibrobacter increased, whereas those of Methanobrevibacter and Bacteroides decreased (p < 0.05), leading to shifts in functional pathways related to amino acid, lipid, and carbohydrate and energy metabolism. Untargeted metabolomics revealed that muscle betaine and inosine peaked at 4 months of age, whereas L-arginine, L-proline, and inosinic acid were most abundant at 6 months of age (p < 0.05). Correlation analysis revealed that the b*_45min was positively associated with ruminal concentrations of propionate, butyrate, and valerate, as well as with the relative abundances of key Selenomonadales taxa (p < 0.05). Inosinic acid exhibited a positive correlation with the abundance of the genus Sodaliphilus and ruminal butyrate concentration (p < 0.05), while Sodaliphilus abundance was negatively correlated with inosine (p < 0.05). In summary, this study demonstrates that age-related variations in the meat quality of Duolang sheep are closely associated with rumen microbial ecology and muscle metabolites, offering novel insights into the molecular mechanisms underlying meat quality formation and identifying potential biomarkers. Full article

The global prevalence of obesity continues to rise, posing serious risks to human health largely because obesity itself leads to metabolic disorders of carbohydrate and lipids. Currently, effective and healthy interventions for lowering blood glucose, reducing blood lipids, and promoting weight loss remain limited due to the complexity of obesity development. Lactobacillus plantarum (GDMCC 1.140) was shown to promote catabolic processes and reduce hepatic lipid accumulation in largemouth bass fed with high-starch feed (HSF) in our previous study; however, molecular mechanisms underlying the function of this probiotic remain unclear. Here, we evaluated the effects of L-carnosine, one of metabolites produced by Lactobacillus plantarum, on carbohydrate and lipid metabolisms in an obesity model of zebrafish, which was induced by HSF. Histopathological analyses of livers from different groups indicated that a dietary supplement with L-carnosine can alleviate hepatic impairment and reduce lipid accumulation in the hepatocytes of obese zebrafish. Transcriptomic analyses revealed that L-carnosine supplementation can reverse the expression of about 70 HSF-induced genes, mainly gene-specific transcription regulators and metabolite interconversion enzymes. Furthermore, approximately 250 HSF-inhibited genes were found to be up-regulated by L-carnosine, reaching levels comparable to those in normal-starch feed (NSF) zebrafish. These genes, targeted by L-carnosine and inhibited by HSF, are highly enriched in GO terms such as lipid metabolic process, small molecule metabolic process, and cellular response to chemical stimulus, with monocarboxylic acid metabolic process, modified amino acid metabolic process and aldehyde metabolic process following, and in KEGG pathways of carbohydrate, lipid and amino acid metabolisms, such as pentose and glucuronate interconversions, glycolysis/gluconeogenesis, glycerolipid metabolism, pentose phosphate pathways, fatty acid degradation, beta-alanine metabolism and arginine and proline metabolism. These findings provide functional and molecular evidence that L-carnosine can ameliorate HSF-induced disorders of carbohydrate and lipid metabolisms. Full article

Saline–alkali land represents an important reserve of arable resources in China, and exploiting its agricultural potential is crucial for ensuring food security. In maize (Zea mays L.), which is moderately sensitive to salt stress, proline serves as a key osmoprotectant, and Δ¹-pyrroline-5-carboxylate synthetase (P5CS), the rate-limiting enzyme in its biosynthesis, plays a vital role in plant stress responses. In this study, the maize ZmP5CS gene family was systematically identified and characterized through comprehensive bioinformatics analyses. Four ZmP5CS homologs were identified, most of which were predicted to localize to chloroplasts. Phylogenetic analysis classified these genes into four major clades. Among them, ZmP5CS4 (GRMZM2G028535) expression was significantly upregulated under salt stress. Association analysis using a natural population of 278 inbred lines revealed that nine SNPs significantly associated with relative P5CS enzyme activity were located within ZmP5CS4. Haplotype analysis further identified a superior haplotype, HapA, carried by 14 inbred lines. Under salt stress, lines carried by HapA exhibited higher P5CS enzyme activity, greater proline accumulation, lower standard evaluation scores, and slightly enhanced salt tolerance compared to lines carried by HapB. Functional validation via transgenic approaches demonstrated that ZmP5CS4 overexpression significantly increased proline content and plant survival under salt stress, whereas knockout of this gene led to heightened salt sensitivity. Collectively, this study elucidates the structure and function of the maize ZmP5CS gene family, establishes the critical role of ZmP5CS4 in the salt stress response, and provides both a theoretical foundation and a candidate gene resource for improving salt tolerance in maize breeding programs. Full article

Background/Objectives: NAC transcription factors are key regulators of stress responses, yet their roles in Narcissus tazetta L. var. chinensis remain uncharacterized. This study aimed to isolate and functionally analyze NtNACa, a NAC gene from the ‘Yunxiang’ narcissus variety, to evaluate its potential in enhancing abiotic stress tolerance. Methods: NtNACa was cloned and its expression pattern under heat, salt, and ABA treatments was assessed via qRT-PCR. Subcellular localization was determined using GFP fusion in tobacco. NtNACa was overexpressed in Arabidopsis thaliana through floral dip transformation, and transgenic lines were subjected to NaCl, ABA, and drought stress assays. Results: The results showed that NtNACa has high homology with monocot NAC family members and possesses typical NAC transcription factor features. Further analyses revealed that NtNACa localizes to the nucleus, and tissue-specific expression analysis indicated that it is highly expressed in leaves, followed by roots and bulbs. The transcriptional expression of NtNACa is differentially regulated in response to 100 mM NaCl, 100 μM ABA, and 50 °C temperature stress. Overexpression of NtNACa in A. thaliana produced transgenic lines with significantly higher germination rates under ABA and NaCl treatments. Soil-grown transgenic A. thaliana plants overexpressing NtNACa showed markedly increased drought stress. Moreover, NtNACa confers drought resilience by coordinately suppressing oxidative damage (via reduced O₂⁻· production rate and MDA accumulation and elevated AtCAT2 expression), enhancing osmotic adjustment (through AtP5CR-mediated proline biosynthesis), and activating core stress-signaling components such as AtRD29A and AtSnRK2.4. Conclusions: Taken together, these results indicate that heterologous overexpression of NtNACa from ‘Yunxiang’ (N. tazetta) confers enhanced drought and salt tolerance in A. thaliana. 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