{"id":61023,"date":"2025-10-08T10:44:09","date_gmt":"2025-10-08T10:44:09","guid":{"rendered":"https:\/\/www.newsbeep.com\/il\/61023\/"},"modified":"2025-10-08T10:44:09","modified_gmt":"2025-10-08T10:44:09","slug":"synergistic-effects-of-plant-growth-regulators-and-fe%e2%82%83o%e2%82%84-nanoparticles-on-in-vitro-organogenesis-and-bioactive-compound-production-in-hypericum-perforatum-bmc-plant-biology","status":"publish","type":"post","link":"https:\/\/www.newsbeep.com\/il\/61023\/","title":{"rendered":"Synergistic effects of plant growth regulators and fe\u2083o\u2084 nanoparticles on in vitro organogenesis and bioactive compound production in hypericum perforatum | BMC Plant Biology"},"content":{"rendered":"<p>Fe\u2083O\u2084 and PGRs elevate plant growth<\/p>\n<p>Several treatments including 2,4-D (0.5 to 2 mg\/L), kinetin (0.5 to 1 mg\/L), and the Fe\u2083O\u2084 nanoparticles (1 to 4 mg\/L) produced reductions in plant height when compared to the control treatment. The treatments that had the greatest reductions in height were 2,4-D 0.5 mg\/L\u2009+\u2009kinetin 0.5 mg\/L\u2009+\u2009Fe\u2083O\u2084-NP 1 mg\/L, 2,4-D 0.5 mg\/L\u2009+\u2009kinetin 1 mg\/L\u2009+\u2009Fe\u2083O\u2084-NP 1 mg\/L, 2,4-D 1 mg\/L\u2009+\u2009kinetin 0.5 mg\/L\u2009+\u2009Fe\u2083O\u2084-NP (1, 2, and 4 mg\/L), and 2,4-D 2 mg\/L\u2009+\u2009kinetin 0.5 mg\/L\u2009+\u2009Fe\u2083O\u2084-NP 4 mg\/L (Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig1\" rel=\"nofollow noopener\" target=\"_blank\">1<\/a>a). The decrease in plant growth seen in certain treatments when compared to the control is in line with other research showing that hormone abnormalities or improper concentrations of nanoparticles can have a negative impact on plant development [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 15\" title=\"Tripathi D, Singh M, Pandey-Rai S. Crosstalk of nanoparticles and phytohormones regulate plant growth and metabolism under abiotic and biotic stress. Plant Stress. 2022;6:100107.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR15\" id=\"ref-link-section-d126582938e687\" rel=\"nofollow noopener\" target=\"_blank\">15<\/a>, <a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 16\" title=\"Karabulut F. The impact of nanoparticles on plant growth, development, and stress tolerance through regulating phytohormones. Nanoparticles in plant biotic stress management. Springer; 2024. pp. 87\u2013105.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR16\" id=\"ref-link-section-d126582938e690\" rel=\"nofollow noopener\" target=\"_blank\">16<\/a>]. The combination of 2 mg\/L 2,4-D, 1 mg\/L kinetin, and 4 mg\/L Fe\u2083O\u2084-NP increased plant height by 59.6% compared to the control, representing the highest increase observed (Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig1\" rel=\"nofollow noopener\" target=\"_blank\">1<\/a>a). Iron nanoparticles serve as a source of micronutrients and increase plant metabolism by increasing the activity of enzymes involved in cellular respiration and photosynthesis [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 17\" title=\"Pradeep M, Saxena M, Mondal D, Franklin G. Do nanoparticles delivered to roots affect plant secondary metabolism? A comprehensive analysis in float seedling cultures of Hypericum perforatum L. Chemosphere. 2024;356:141789.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR17\" id=\"ref-link-section-d126582938e696\" rel=\"nofollow noopener\" target=\"_blank\">17<\/a>, <a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 18\" title=\"Tombuloglu G, Tombuloglu H, Slimani Y, Almessiere MA, Baykal A, Bostancioglu SM, Kirat G, Ercan I. Effects of foliar iron oxide nanoparticles (Fe3O4) application on photosynthetic parameters, distribution of mineral elements, magnetic behaviour, and photosynthetic genes in tomato (Solanum lycopersicum var. cerasiforme) plants. Plant Physiol Biochem. 2024;210:108616.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR18\" id=\"ref-link-section-d126582938e700\" rel=\"nofollow noopener\" target=\"_blank\">18<\/a>]. In addition, growth hormones including kin and 2,4-D can promote root and shoot development by affecting cell division and differentiation [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 6\" title=\"Shasmita, Behera S, Mishra P, Samal M, Mohapatra D, Monalisa K, Naik SK. Recent advances in tissue culture and secondary metabolite production in Hypericum perforatum L. Plant Cell Tissue Organ Cult (PCTOC). 2023;154(1):13\u201328.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR6\" id=\"ref-link-section-d126582938e703\" rel=\"nofollow noopener\" target=\"_blank\">6<\/a>, <a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 19\" title=\"Abdollahpoor M, Kalantari S, Azizi M, Saadat YA. In vitro shoot proliferation of Hypericum perforatum L. through indirect and direct plant regeneration. J Med Plants By-products. 2017;1:81\u20139.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR19\" id=\"ref-link-section-d126582938e706\" rel=\"nofollow noopener\" target=\"_blank\">19<\/a>]. Ali and Alwani Bahm [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 20\" title=\"Ali NH, Alwani BAHM. The effect of 2, 4-D and Kinetin on Trigonella foenum-graecum L. callus using hypocotyl segments. 2021.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR20\" id=\"ref-link-section-d126582938e709\" rel=\"nofollow noopener\" target=\"_blank\">20<\/a>] analyzed the impact of a combination of 4 mg\/L 2,4-D and 4 mg\/L kin on the Trigonella foenum-graecum L. and noted a statistically significant increase in plant height along with biomass accumulation. In another investigation, Muthu et al. [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 21\" title=\"Muthu RK, Shanmugasundaram G, Karuppan T, Balakrishnan J, Doss G, Shanmugam K. Combined supplementation of 2, 4-D and Kinetin elevates callus induction from cotyledon segments of different soybean cultivars [Glycine Max (L.) MERRIL]. Plant Archives (09725210) 2023, 23(2).\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR21\" id=\"ref-link-section-d126582938e712\" rel=\"nofollow noopener\" target=\"_blank\">21<\/a>] applied 2,4-D at 5 \u00b5M mg\/L and kin at 10 \u00b5M on Glycine max (soybean) seedlings and documented better root and shoot growth than controls. Likewise, Ullah et al. [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 22\" title=\"Ullah J, Gul A, Khan I, Shehzad J, Kausar R, Ahmed MS, Batool S, Hasan M, Ghorbanpour M, Mustafa G. Green synthesized iron oxide nanoparticles as a potential regulator of callus growth, plant physiology, antioxidative and microbial contamination in Oryza sativa L. BMC Plant Biol 2024, 24939(1).\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR22\" id=\"ref-link-section-d126582938e715\" rel=\"nofollow noopener\" target=\"_blank\">22<\/a>] treated Oryza sativa L., with 15 ppm FeO-NPs alone, and observed increases in plant height.<\/p>\n<p>Fig. 1<a class=\"c-article-section__figure-link\" data-test=\"img-link\" data-track=\"click\" data-track-label=\"image\" data-track-action=\"view figure\" href=\"https:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6\/figures\/1\" rel=\"nofollow noopener\" target=\"_blank\"><img decoding=\"async\" aria-describedby=\"Fig1\" src=\"https:\/\/www.newsbeep.com\/il\/wp-content\/uploads\/2025\/10\/12870_2025_7409_Fig1_HTML.png\" alt=\"figure 1\" loading=\"lazy\" width=\"685\" height=\"442\"\/><\/a><\/p>\n<p>Effect of treatment compounds, Kin, 2,4-D and Fe\u2083O\u2084-NP on Hypericum perforatum L.: (a) plant height, (b) root length, (c) shoot number, and (d) callus FW. Data represent mean\u2009\u00b1\u2009SE (n\u2009=\u20093). Different letters indicate significant differences between treatments based on LSD test (P\u2009\u2264\u20090.05)<\/p>\n<p>Root length and shoot number boost with PGRs and Fe\u2083O\u2084-NP<\/p>\n<p>The significant increase in root length in the combined treatment of 2 mg\/L 2,4-D, 1 mg\/L kin, and Fe\u2083O\u2084-NP at concentrations of 1 and 2 mg\/L (114.04 and 112.06% compared to the control, respectively) indicates the synergistic effect of these compounds on root growth (Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig1\" rel=\"nofollow noopener\" target=\"_blank\">1<\/a>b). The combined treatment of PGRs and Fe\u2083O\u2084-NP had a positive effect on increasing the number of shoots, although this effect was not observed in all treatments. It is noteworthy that the treatment containing 1 mg\/L 2,4-D, 1 mg\/L kin and 4 mg\/L Fe\u2083O\u2084-NP caused a 180% increase in the number of shoots compared to the control (Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig1\" rel=\"nofollow noopener\" target=\"_blank\">1<\/a>c). Previous studies have shown that using growth regulators like 2,4-D and kin together can positively influence root differentiation and growth [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" title=\"Fan Y, Tang Z, Wei J, Yu X, Guo H, Li T, Guo H, Zhang L, Fan Y, Zhang C. Dynamic transcriptome analysis reveals complex regulatory pathway underlying induction and dose effect by different exogenous auxin IAA and 2, 4-D during in vitro embryogenic redifferentiation in cotton. Front Plant Sci. 2022;13:931105.\" href=\"#ref-CR23\" id=\"ref-link-section-d126582938e774\">23<\/a>,<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" title=\"Mondal M, Das S, Chandra I. Effect of individual plant growth regulators on modulation of secondary metabolites production in an important medicinal plant Gloriosa Superba L. Plant Cell Tissue Organ Cult (PCTOC). 2024;156(1):28.\" href=\"#ref-CR24\" id=\"ref-link-section-d126582938e774_1\">24<\/a>,<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 25\" title=\"Habibah NA, Yuniastuti A, Nugrahaning N, Safitri S, Puspitasari ADS. Effects of Low-Dose kinetin, 2, 4-D and monochromatic light conditions on flavonoid content in callus culture of Dioscorea esculenta. Trends Sci. 2024;21(2):7218\u20137218.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR25\" id=\"ref-link-section-d126582938e777\" rel=\"nofollow noopener\" target=\"_blank\">25<\/a>]. Additionally, Fe\u2083O\u2084-NP boosts nitrogen metabolism and enhances photosynthetic efficiency thanks to their large specific surface area and their ability to slowly release iron ions [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 26\" title=\"Feng Y, Kreslavski VD, Shmarev AN, Ivanov AA, Zharmukhamedov SK, Kosobryukhov A, Yu M, Allakhverdiev SI, Shabala S. Effects of iron oxide nanoparticles (Fe3O4) on growth, photosynthesis, antioxidant activity and distribution of mineral elements in wheat (Triticum aestivum) Plants. Plants 2022, 11(14):1894.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR26\" id=\"ref-link-section-d126582938e780\" rel=\"nofollow noopener\" target=\"_blank\">26<\/a>]. Using iron in its nano form is a much an effective way to boost root growth since plant cells absorb it better [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 27\" title=\"Rizwan M, Ali S, Ali B, Adrees M, Arshad M, Hussain A, ur Rehman MZ, Waris AA. Zinc and iron oxide nanoparticles improved the plant growth and reduced the oxidative stress and cadmium concentration in wheat. Chemosphere. 2019;214:269\u201377.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR27\" id=\"ref-link-section-d126582938e784\" rel=\"nofollow noopener\" target=\"_blank\">27<\/a>]. Iron plays a crucial role in the makeup of enzymes that are vital for plant growth, and without it, root development can really suffer [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 28\" title=\"Zhang X, Zhang D, Sun W, Wang T. The adaptive mechanism of plants to iron deficiency via iron uptake, transport, and homeostasis. Int J Mol Sci. 2019;20(10):2424.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR28\" id=\"ref-link-section-d126582938e787\" rel=\"nofollow noopener\" target=\"_blank\">28<\/a>]. The present investigation matches the findings of Tombuloglu et al. [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 18\" title=\"Tombuloglu G, Tombuloglu H, Slimani Y, Almessiere MA, Baykal A, Bostancioglu SM, Kirat G, Ercan I. Effects of foliar iron oxide nanoparticles (Fe3O4) application on photosynthetic parameters, distribution of mineral elements, magnetic behaviour, and photosynthetic genes in tomato (Solanum lycopersicum var. cerasiforme) plants. Plant Physiol Biochem. 2024;210:108616.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR18\" id=\"ref-link-section-d126582938e790\" rel=\"nofollow noopener\" target=\"_blank\">18<\/a>], since the study showed that iron nanoparticles combined with cytokinins produced enhanced root system growth and more shoot emergence in tomato seedlings. The research by Yousaf et al. [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 29\" title=\"Yousaf N, Ishfaq M, Qureshi HA, Saleem A, Yang H, Sardar MF, Zou C. Characterization of root and foliar-applied iron oxide nanoparticles (\u03b1-Fe2O3, \u03b3-Fe2O3, Fe3O4, and bulk Fe3O4) in improving maize (Zea Mays L) performance. Nanomaterials. 2023;13(23):3036.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR29\" id=\"ref-link-section-d126582938e793\" rel=\"nofollow noopener\" target=\"_blank\">29<\/a>], demonstrated that Fe\u2083O\u2084-NP together with growth regulators promoted higher biomass growth and improved root development in corn plants. In Oryza sativa L., nanogreen iron oxide (FeO-NPs) with sizes of 2\u20137.5 nm effectively enhanced tissue culture performance. Supplementation of MS media with 15 ppm FeO-NPs significantly reduced callus contamination by 80%. FeCl\u2083-based FeO-NPs promoted rapid callus induction (72%) and regeneration (43%), whereas FeSO\u2084-based FeO-NPs led to greater callus biomass (516%), diameter (300%), shoot number (200%), and root formation (114%) [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 22\" title=\"Ullah J, Gul A, Khan I, Shehzad J, Kausar R, Ahmed MS, Batool S, Hasan M, Ghorbanpour M, Mustafa G. Green synthesized iron oxide nanoparticles as a potential regulator of callus growth, plant physiology, antioxidative and microbial contamination in Oryza sativa L. BMC Plant Biol 2024, 24939(1).\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR22\" id=\"ref-link-section-d126582938e799\" rel=\"nofollow noopener\" target=\"_blank\">22<\/a>].<\/p>\n<p>Boost in callus weight by PGRs and fe\u2083o\u2084 nano<\/p>\n<p>The combined application of plant growth regulators (PGRs) and Fe\u2083O\u2084-NP increased the callus FW compared to the control treatment (Fig. 1\u2009d). Specifically, plants using the combination of 1 mg\/L 2,4-D, 1 mg\/L kin and 2 mg\/L Fe\u2083O\u2084-NP and treatment of 2 mg\/L 2,4-D, 1.5 mg\/L kin and 4 mg\/L Fe\u2083O\u2084-NP resulted in 198.28% callus FW gain compared to control. 2,4-D is a synthetic auxin that enhances cell division [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 30\" title=\"Muselikova K, Mouralova K. Synthetic auxin herbicide 2, 4-D and its influence on a model BY-2 suspension. Mol Biol Rep. 2024;51(1):444.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR30\" id=\"ref-link-section-d126582938e810\" rel=\"nofollow noopener\" target=\"_blank\">30<\/a>], while kin, is a cytokinin that promotes cell division in meristematic zones and delays cell senescence [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 31\" title=\"Mok MC. Cytokinins and plant development\u2014an overview. Cytokinins 2019:155\u201366.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR31\" id=\"ref-link-section-d126582938e813\" rel=\"nofollow noopener\" target=\"_blank\">31<\/a>]. Fe\u2083O\u2084-NP presence can enhance enzyme activities (e.g., POD, CAT) by improving cellular iron uptake and related metabolic processes [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 32\" title=\"Kumar M, Gupta G, Muhammed NP, Varghese T, Srivastava PP, Bhushan S, Shukla SP, Krishna G, Gupta S. Toxicity ameliorative effect of vitamin E against super-paramagnetic iron oxide nanoparticles on haemato-immunological responses, antioxidant capacity, oxidative stress, and metabolic enzymes activity during exposure and recovery in Labeo Rohita fingerlings. Aquacult Int. 2022;30(4):1711\u201339.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR32\" id=\"ref-link-section-d126582938e816\" rel=\"nofollow noopener\" target=\"_blank\">32<\/a>]. Iron nanoparticles stimulate callus development through their small size and high surface-to-volume ratio by improving cell wall permeability [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 33\" title=\"Holghoomi R, Colagar AH. Applications of biocompatible nanoparticles in plant biotechnology for enhanced secondary metabolite biosynthesis. Inorg Chem Commun 2024:112753.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR33\" id=\"ref-link-section-d126582938e819\" rel=\"nofollow noopener\" target=\"_blank\">33<\/a>]. Research evidence from different experiments demonstrates the same conclusion; the combination of iron nanoparticles with plant growth regulators effectively promoted callus expansion in Catharanthus roseus according to Sepehri et al. [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 34\" title=\"Sepehri B, Tohidi-Moghadam H-R, Ghooshchi F, Oveysi M, Kasraie P. Evaluation of metallic nanoparticles and plant growth regulators affecting Catharanthus roseus L. Performance under Water-Deficit stress. Int J Hortic Sci Technol 2025, 12(3).\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR34\" id=\"ref-link-section-d126582938e822\" rel=\"nofollow noopener\" target=\"_blank\">34<\/a>], Iron nanoparticles served to improve wheat tissue growth through their effects on photosynthesis and chlorophyll production according to Feng et al. [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 26\" title=\"Feng Y, Kreslavski VD, Shmarev AN, Ivanov AA, Zharmukhamedov SK, Kosobryukhov A, Yu M, Allakhverdiev SI, Shabala S. Effects of iron oxide nanoparticles (Fe3O4) on growth, photosynthesis, antioxidant activity and distribution of mineral elements in wheat (Triticum aestivum) Plants. Plants 2022, 11(14):1894.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR26\" id=\"ref-link-section-d126582938e826\" rel=\"nofollow noopener\" target=\"_blank\">26<\/a>]. The study by Zahmatkesh et al. [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 35\" title=\"Zahmatkesh H, Azizi E, Kermani M, Rahbarian R. Hypericum Perforatum L. Journal of Advanced Pharmacy Education &amp; Research| Jan-Mar 2020, 10(S1).\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR35\" id=\"ref-link-section-d126582938e829\" rel=\"nofollow noopener\" target=\"_blank\">35<\/a>], demonstrated that combining 2,4-D (1 mg\/L) with kin (0.5 mg\/L) produced significant increases in H. perforatum callus fresh weight and dry weight. The experiment by Shasmita et al. [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 6\" title=\"Shasmita, Behera S, Mishra P, Samal M, Mohapatra D, Monalisa K, Naik SK. Recent advances in tissue culture and secondary metabolite production in Hypericum perforatum L. Plant Cell Tissue Organ Cult (PCTOC). 2023;154(1):13\u201328.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR6\" id=\"ref-link-section-d126582938e832\" rel=\"nofollow noopener\" target=\"_blank\">6<\/a>] demonstrated that 2,4-D alone produced successful callus formation in H. perforatum while adding kin to the mixture produced better callus fresh weight results and enhanced hypericin production. The application of low-concentration Fe\u2083O\u2084-NP combined with PGRs in H. perforatum resulted in significant improvements in callus weight together with colorimetry measurements and enzymatic activity according to Kralova and Jampilek [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 36\" title=\"Kralova K, Jampilek J. Responses of medicinal and aromatic plants to engineered nanoparticles. Appl Sci. 2021;11(4):1813.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR36\" id=\"ref-link-section-d126582938e835\" rel=\"nofollow noopener\" target=\"_blank\">36<\/a>]. They attributed this phenomenon to stimulation of cellular metabolism via increased iron availability.<\/p>\n<p>Occurrence of black spots on leaves<\/p>\n<p>Most treatments of either PGRs and Fe\u2083O\u2084-NPs did not induce the formation of black nodules on leaves from regenerated shoots and there were no nodules in any of the treatments with 2,4-D and the concentrations of 0.5 or 1 mg\/L, kinetin and the concentrations of 0.5 or 1 mg\/L, and in the applications of Fe\u2083O\u2084-NPs at 1, 2 or 4 mg\/L. There was a slight (but not statistically significant) increase in nodule development for a few combinations at higher concentrations of kinetin and\/or Fe\u2083O\u2084-NPs, e.g., 2,4-D (0.5\u20132 mg\/L) with kinetin (1\u20131.5 mg\/L) and Fe\u2083O\u2084-NPs (2\u20134 mg\/L). These results are illustrated in Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig2\" rel=\"nofollow noopener\" target=\"_blank\">2<\/a>. Black nodules are either hypericin rich glands that are both natural and desirable. In our study, the black nodules were primarily associated with tissue browning a suggesting to be favourable hypericin accumulation. Treatments with 2,4-D, kinetin, and Fe\u2083O\u2084 nanoparticles resulted in greater callus growth, less tissue darkening and increased chlorophyll content, which suggests improved metabolic function and potential reduction in oxidative damage [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 37\" title=\"Ali S, Baloch AM. Overview of sustainable plant growth and differentiation and the role of hormones in controlling growth and development of plants under various stresses. Recent Pat Food Nutr Agric. 2020;11(2):105\u201314.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR37\" id=\"ref-link-section-d126582938e849\" rel=\"nofollow noopener\" target=\"_blank\">37<\/a>]. The investigation assessed the influence of kin with 2,4-D and Fe\u2083O\u2084-NP on H. perforatum L. callus growth and development (Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig3\" rel=\"nofollow noopener\" target=\"_blank\">3<\/a>a\u2013g). Prior research has shown that Fe\u2083O\u2084 nanoparticles have antifungal and antibacterial activity [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 38\" title=\"Ashraf H, Batool T, Anjum T, Illyas A, Li G, Naseem S, Riaz S. Antifungal potential of green synthesized magnetite nanoparticles black Coffee\u2013Magnetite nanoparticles against wilt infection by ameliorating enzymatic activity and gene expression in Solanum lycopersicum L. Front Microbiol. 2022;13:754292.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR38\" id=\"ref-link-section-d126582938e855\" rel=\"nofollow noopener\" target=\"_blank\">38<\/a>, <a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 39\" title=\"Sasani M, Fataei E, Safari R, Nasehi F, Mosayebi M. Antimicrobial potentials of iron oxide and silver nanoparticles Green-Synthesized in fusarium Solani. J Chem Health Risks 2023, 13(1).\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR39\" id=\"ref-link-section-d126582938e858\" rel=\"nofollow noopener\" target=\"_blank\">39<\/a>] and in combination with plant growth regulators, can stimulate plant defense mechanisms and mitigate cellular stress indicators [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 18\" title=\"Tombuloglu G, Tombuloglu H, Slimani Y, Almessiere MA, Baykal A, Bostancioglu SM, Kirat G, Ercan I. Effects of foliar iron oxide nanoparticles (Fe3O4) application on photosynthetic parameters, distribution of mineral elements, magnetic behaviour, and photosynthetic genes in tomato (Solanum lycopersicum var. cerasiforme) plants. Plant Physiol Biochem. 2024;210:108616.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR18\" id=\"ref-link-section-d126582938e862\" rel=\"nofollow noopener\" target=\"_blank\">18<\/a>]. Although the biosynthesis of secondary metabolites is genetically regulated, their actual production is strongly influenced by environmental conditions. Metabolic and morphogenetic changes can alter the levels of secondary metabolites by modulating the expression of specific plant genes in response to environmental factors. In addition, elicitors may trigger the activation of new genes that stimulate enzymes and, consequently, various biosynthetic pathways, leading to the formation of secondary metabolites. For example, polyketide synthase (PKS) plays a central role in the biosynthesis of hypericin and hyperforin. Similarly, phenylalanine ammonia lyase (PAL) is a key enzyme whose upregulation increases the accumulation of phenolic compounds. Under mild stress conditions, these precursors can be converted into anti-inflammatory metabolites such as hypericin, thereby enhancing the plant\u2019s resistance to stress.Pretto (2006) reported that in Hypericum perforatum cell suspension cultures, treatment with 250 \u00b5M jasmonic acid induced higher levels of hypericin compared to lower concentrations (50 or 100 \u00b5M). Moreover, another study demonstrated that reduced shading, which increases sunlight exposure and consequently daytime heat in natural habitats of H. perforatum, also contributed to higher hypericin production.Based on these findings, the present study examined Fe nanoparticles as potential elicitors in tissue culture. The results indicated that these nanoparticles could activate genes involved in hesperidin biosynthesis by enzymatic stimulation, leading to an increased accumulation of this compound (Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig2\" rel=\"nofollow noopener\" target=\"_blank\">2<\/a>). Therefore, compared with plants grown under natural conditions, it can be suggested that when mild stresses are combined with the activation of biosynthetic genes for secondary metabolites, the production of such compounds may also be enhanced in vivo.<\/p>\n<p>Fig. 2<a class=\"c-article-section__figure-link\" data-test=\"img-link\" data-track=\"click\" data-track-label=\"image\" data-track-action=\"view figure\" href=\"https:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6\/figures\/2\" rel=\"nofollow noopener\" target=\"_blank\"><img decoding=\"async\" aria-describedby=\"Fig2\" src=\"https:\/\/www.newsbeep.com\/il\/wp-content\/uploads\/2025\/10\/12870_2025_7409_Fig2_HTML.png\" alt=\"figure 2\" loading=\"lazy\" width=\"685\" height=\"447\"\/><\/a><\/p>\n<p>Effect of treatment compounds, Kin, 2,4-D and Fe\u2083O\u2084-NP on Hypericum perforatum L.: NO. of black\/leaves. Data represent mean\u2009\u00b1\u2009SE (n\u2009=\u20093). Different letters indicate significant differences between treatments based on LSD test (P\u2009\u2264\u20090.05)<\/p>\n<p>Fig. 3<a class=\"c-article-section__figure-link\" data-test=\"img-link\" data-track=\"click\" data-track-label=\"image\" data-track-action=\"view figure\" href=\"https:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6\/figures\/3\" rel=\"nofollow noopener\" target=\"_blank\"><img decoding=\"async\" aria-describedby=\"Fig3\" src=\"https:\/\/www.newsbeep.com\/il\/wp-content\/uploads\/2025\/10\/12870_2025_7409_Fig3_HTML.png\" alt=\"figure 3\" loading=\"lazy\" width=\"685\" height=\"266\"\/><\/a><\/p>\n<p>Effect of treatment compounds, Kin, 2,4-D and Fe\u2083O\u2084-NP on Hypericum perforatum L.: (a) callus induction, (b) callus growth, (c) callus formation, (d) shoot induction (e) shoot frowth (f) seedling formation (g) intact plant, (f) plant acclimatization. Data represent mean\u2009\u00b1\u2009SE (n\u2009=\u20093). Different letters indicate significant differences between treatments based on LSD test (P\u2009\u2264\u20090.05)<\/p>\n<p>Kin, 2,4-D &amp; Fe3O4 nano: callus and rooting percentage<\/p>\n<p>In this research study the percentage of callus and root formation, from the combined treatments of kinetin(Kin), 2,4-D, and Fe\u2083O\u2084 nanoparticles (Fe\u2083O\u2084-NP), was significantly increased from the control (Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig4\" rel=\"nofollow noopener\" target=\"_blank\">4<\/a>a, b). Specifically, the treatment of 2,4-D at 0.5 mg\/L, kinetin at 1 mg\/L, and Fe\u2083O\u2084-NP at 4 mg\/L resulted in a 62.26% increase in callus formation, while the treatment of 2,4-D at 1 mg\/L, kinetin at 1.5 mg\/L, and Fe\u2083O\u2084-NP at 1 mg\/L resulted in a 71.66% increase in root formation, when compared to the control (Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig4\" rel=\"nofollow noopener\" target=\"_blank\">4<\/a>a, b). The effects of these compounds on medicinal plants have been demonstrated through various studies. Also, the research performed by Rastegaran et al. [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 40\" title=\"Rastegaran MM, Hassanpour H, Ziyadi H. Synthesized Fe3O4 nanoparticles induced antioxidant activity and total phenolic and flavonoid content in Matricaria Chamomilla seedlings. Iran J Plant Physiol. 2022;12(1):4003\u201311.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR40\" id=\"ref-link-section-d126582938e962\" rel=\"nofollow noopener\" target=\"_blank\">40<\/a>] demonstrated that Fe\u2083O\u2084-NP enhances callus development alongside root formation in chamomile (Matricaria chamomilla) through better iron supply and improved metabolic enzyme performance. Another study by Amiri et al. [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 41\" title=\"Amiri M-B, Jahan M, Rezvani-Moghaddam P. Enhancing phytopharmacological compounds and traits of Persian borage (Echium amoenum fisch. &amp; Mey.) by applying organic soil amendments. Compost Sci Utilization. 2025;32(1\u20132):21\u201334.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR41\" id=\"ref-link-section-d126582938e965\" rel=\"nofollow noopener\" target=\"_blank\">41<\/a>] demonstrated how borage (Echium amoenum) growth hormones combined with iron nanoparticles enhanced root production while also improving callus quality. Certainly, phytohormones like auxins and cytokinins are critical for in vitro caulogenesis and morphogenesis, as reported by several studies that revealed detrimental effects of excess or prolonged application on plant health [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" title=\"Sabagh A, Islam MS, Hossain A, Iqbal MA, Mubeen M, Waleed M, Reginato M, Battaglia M, Ahmed S, Rehman A. Phytohormones as growth regulators during abiotic stress tolerance in plants. Front Agron. 2022;4:765068.\" href=\"#ref-CR42\" id=\"ref-link-section-d126582938e968\">42<\/a>,<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" title=\"Asghar S, Ghori N, Hyat F, Li Y, Chen C. Use of auxin and cytokinin for somatic embryogenesis in plant: a story from competence towards completion. Plant Growth Regul. 2023;99(3):413\u201328.\" href=\"#ref-CR43\" id=\"ref-link-section-d126582938e968_1\">43<\/a>,<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 44\" title=\"Sosnowski J, Truba M, Vasileva V. The impact of auxin and cytokinin on the growth and development of selected crops. Agriculture. 2023;13(3):724.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR44\" id=\"ref-link-section-d126582938e972\" rel=\"nofollow noopener\" target=\"_blank\">44<\/a>]. In Study Arain et al. [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 45\" title=\"Arain S, Kaloi G, Ahmad S, Rajput M, Mari A, Al-Qahtani W, Saleh I, Zomot N, Kiani B, Abro A. The effect of Dichlorophenoxyacetic acid (2, 4-d) concentrations on callus induction in sugarcane (Saccharum officinarum). Appl Ecol Environ Res. 2024;22(5):4951\u201360.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR45\" id=\"ref-link-section-d126582938e975\" rel=\"nofollow noopener\" target=\"_blank\">45<\/a>], excessive treatment with 2,4-D in Saccharum officinarum caused somaclonal changes, oxidative stress, and abnormal proliferative callus growth. Similarly, kinetin and principally other cytokinins used at high concentrations may alter endogenous hormone balances, lead to rooting difficulties [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 46\" title=\"Jing W, Qi D, Jing W, Qiao W. Response of root morphology, physiology and endogenous hormones in maize (Zea Mays L.) to potassium deficiency. J Integr Agric. 2016;15(4):785\u201394.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR46\" id=\"ref-link-section-d126582938e978\" rel=\"nofollow noopener\" target=\"_blank\">46<\/a>]. These complications suggest that while exogenous hormones play an important role in morphogenesis, concentration and combination are critical in reducing stress and developmental variations [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 47\" title=\"Kosakivska IV, Vedenicheva NP, Babenko LM, Voytenko LV, Romanenko KO, Vasyuk VA. Exogenous phytohormones in the regulation of growth and development of cereals under abiotic stresses. Mol Biol Rep. 2022;49(1):617\u201328.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR47\" id=\"ref-link-section-d126582938e981\" rel=\"nofollow noopener\" target=\"_blank\">47<\/a>, <a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 48\" title=\"Vongnhay V, Shukla MR, Ayyanath M-M, Sriskantharajah K, Saxena PK. In vitro morphogenesis of tobacco: modulation of endogenous growth regulators by Tulsi (Holy Basil). Plants. 2024;13(14):2002.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR48\" id=\"ref-link-section-d126582938e984\" rel=\"nofollow noopener\" target=\"_blank\">48<\/a>]. In a report on bell pepper, it was determined that the highest average proliferation for callus was recorded at a concentration of 6.0 ml\/liter of MgNPs in MS culture medium, reaching 6.1 g\/m2 [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 49\" title=\"Hadi SM, Rashed MA. The effect of MgNPs synthesized by using green method on the produc-tion and proliferation of Capisum annuum callus culture in vitro. J Kerbala Agricultural Sci. 2023;10(3):59\u201378.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR49\" id=\"ref-link-section-d126582938e990\" rel=\"nofollow noopener\" target=\"_blank\">49<\/a>].<\/p>\n<p>Fig. 4<a class=\"c-article-section__figure-link\" data-test=\"img-link\" data-track=\"click\" data-track-label=\"image\" data-track-action=\"view figure\" href=\"https:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6\/figures\/4\" rel=\"nofollow noopener\" target=\"_blank\"><img decoding=\"async\" aria-describedby=\"Fig4\" src=\"https:\/\/www.newsbeep.com\/il\/wp-content\/uploads\/2025\/10\/12870_2025_7409_Fig4_HTML.png\" alt=\"figure 4\" loading=\"lazy\" width=\"685\" height=\"453\"\/><\/a><\/p>\n<p>Effect of treatment compounds, Kin, 2,4-D and Fe\u2083O\u2084-NP on Hypericum perforatum L.: (a) callus percentage, (b) rooting percentage, (c) regeneration rate, and (d) hypericin in plant. Data represent mean\u2009\u00b1\u2009SE (n\u2009=\u20093). Different letters indicate significant differences between treatments based on LSD test (P\u2009\u2264\u20090.05)<\/p>\n<p>Regeneration rate: increase with Kin, 2,4-D &amp; Fe3O4 Nano combination<\/p>\n<p>The treatment combinations generally had a positive effect on the percentage of regeneration rate, but in some cases this effect was reduced. It is noteworthy that the treatment of 2,4-D 1 mg\/L, kin 1.5 mg\/L and different concentrations have independent significantly increased the regeneration rate by 50.85% compared to the control (Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig4\" rel=\"nofollow noopener\" target=\"_blank\">4<\/a>c). These findings highlight that specific nano-hormonal combinations can act synergistically to enhance growth and metabolite production, an approach that can benefit medicinal plant tissue culture. Research by Palmer and Keller [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 50\" title=\"Palmer CD, Keller W. Plant regeneration from petal explants of Hypericum perforatum L. Plant Cell Tissue Organ Cult (PCTOC). 2011;105:129\u201334.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR50\" id=\"ref-link-section-d126582938e1036\" rel=\"nofollow noopener\" target=\"_blank\">50<\/a>] showed that 2,4-D treatment independently triggered callus formation in H. perforatum but kin addition significantly enhanced both callus development and regeneration. The improved hormonal equilibrium together with cell division stimulation generated this effect. The research Bal et al. [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 51\" title=\"Bal A, \u00d6zen H\u00c7, Tural B, Erta\u015f E. The effects of different concentrations of foliar applied chitosan, iron oxide and chitosan-coated iron oxide nanoparticles on the secondary metabolites of Hypericum triquetrifolium turra. During full bloom. Kahramanmara\u015f S\u00fct\u00e7\u00fc \u0130mam \u00dcniversitesi Tar\u0131m Ve Do\u011fa Dergisi. 2022;25(4):811\u20138.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR51\" id=\"ref-link-section-d126582938e1039\" rel=\"nofollow noopener\" target=\"_blank\">51<\/a>] discovered that Fe\u2083O\u2084-NP treatment independently strengthened antioxidant enzyme function to improve root growth in H. perforatum. Culture medium containing 0.5 mg\/L, 1 mg\/L, and 1.5 mg\/L of Fe3O4 NPs increased the regeneration rate of flax plants [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 52\" title=\"Kokina I, Mickevi\u010da I, Jahundovi\u010da I, Ogurcovs A, Krasovska M, Jerma\u013conoka M, Mihailova I, Tamanis E, Gerbreders V. Plant explants grown on medium supplemented with Fe3O4 nanoparticles have a significant increase in embryogenesis. J Nanomaterials 2017, 2017(1):4587147.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR52\" id=\"ref-link-section-d126582938e1047\" rel=\"nofollow noopener\" target=\"_blank\">52<\/a>]. Using the three applications of Kin, 2,4-D, and Fe\u2083O\u2084-NPs resulted in a high rate of callus and root formation, but in vitro regeneration using these hormones has the potential for adverse events that need to be considered. For example, using 2,4-D or kinetin at either prolonged or high packet volumes may result in somaclonal variation, oxidative stress, abnormal morphology of callus, or tissue vitrification. Therefore, a high regeneration rate isn\u2019t necessarily a predictor of genetic fidelity. Any regenerated plants should be evaluated for homogeneity and similarity to an in vivo plant using molecular markers (e.g., RAPD, ISSR, AFLP) or cytogenetic analyses [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" title=\"Majumder S, Igamberdiev AU, Debnath SC. Somaclonal variation and clonal fidelity in commercial micropropagation: challenges and perspectives. Agronomy. 2025;15(6):1489.\" href=\"#ref-CR53\" id=\"ref-link-section-d126582938e1050\">53<\/a>,<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" title=\"Borah R, Kumaria S, Choudhury H. Micropropagation and confirmation of genetic homogeneity in Magnolia Punduana (Hook. F. &amp; Thomson) Figlar\u2014a threatened endemic plant: Borah et al. Vitro Cell Dev Biology-Plant. 2025;61(2):306\u201320.\" href=\"#ref-CR54\" id=\"ref-link-section-d126582938e1050_1\">54<\/a>,<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 55\" title=\"Linacero R, Ballesteros I. Genetic basis of Somaclonal variation. Somaclonal variation: basic and practical aspects. Springer; 2024. pp. 1\u201320.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR55\" id=\"ref-link-section-d126582938e1053\" rel=\"nofollow noopener\" target=\"_blank\">55<\/a>].<\/p>\n<p>Powerful boost of hypericin via 2,4-D, Kinetin &amp; Fe3O4 nano<\/p>\n<p>The improvement in the amount of hypericin in the plant was particularly significant with the treatment of 2,4-D 0.5 mg\/L, kin 1 mg\/L and Fe\u2083O\u2084-NP 4 mg\/L, 66.66% increase compared to the control plants (Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig4\" rel=\"nofollow noopener\" target=\"_blank\">4<\/a>d). Also, the treatments of 2,4-D 0.5 mg\/L, kin 2 mg\/L and Fe\u2083O\u2084-NP 4 mg\/L and 2,4-D 2 mg\/L, kin 1.5 mg\/L and Fe\u2083O\u2084-NP 2 mg\/L showed a 39.39% increase in hypericin compared to the control, respectively, and these two treatments did not differ significantly from each other (Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig4\" rel=\"nofollow noopener\" target=\"_blank\">4<\/a>d). The active component hypericin forms part of the healing process in H. perforatum while growing production improves the economic value and medical benefits of the product [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 1\" title=\"Kwiecie\u0144 I, Nicosia N, Ekiert H. Cultivation of hypericum perforatum (St. John\u2019s Wort) and biotechnological approaches for improvement of plant Raw material quality. Med Plants: Domestication Biotechnol Reg Importance. 2021;253:291.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR1\" id=\"ref-link-section-d126582938e1075\" rel=\"nofollow noopener\" target=\"_blank\">1<\/a>]. In the work of Al-Khayri et al. [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 56\" title=\"Al-Khayri JM, Narasimha SW, Vennapusa AR, Nagella P, Shehata WF, Al-Mssallem MQ. Biotechnological approaches for the production of hypericin and other important metabolites from the genus hypericum. Plant Cell Tissue Organ Cult (PCTOC). 2024;156(3):100.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR56\" id=\"ref-link-section-d126582938e1078\" rel=\"nofollow noopener\" target=\"_blank\">56<\/a>] H. perforatum callus cultures synthesized more hypericin in the presence of 2,4-D and kin applied simultaneously and the maximum hypericin content was reached at a lower concentration of 2,4-D (0.5\u20131 mg\/L) combined with kin (1\u20132 mg\/L). Aminizadeh et al. [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 57\" title=\"Aminizadeh M, RIAHI MA. MOHAMMADI M: Nano-Metal oxides induced Sulforaphane production and peroxidase activity in seedlings of Lepidium Draba. (Brassicaceae). 2016.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR57\" id=\"ref-link-section-d126582938e1081\" rel=\"nofollow noopener\" target=\"_blank\">57<\/a>] showed that Fe\u2083O\u2084-NP applied to Lepidium sativum led to increased activity of antioxidant enzymes and higher production of phenolic compounds. The mode of the effects of H. perforatum on the enhancement of hypericin content is comparable to Fe\u2083O\u2084-NP for their increases of activity of antioxidant enzymes and production of phenolic compounds shown in Lepidium sativum plant [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 57\" title=\"Aminizadeh M, RIAHI MA. MOHAMMADI M: Nano-Metal oxides induced Sulforaphane production and peroxidase activity in seedlings of Lepidium Draba. (Brassicaceae). 2016.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR57\" id=\"ref-link-section-d126582938e1085\" rel=\"nofollow noopener\" target=\"_blank\">57<\/a>]. The researchers Abed et al. [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 58\" title=\"Abed A, Majeed D, Ismail E, Al-Jibouri A, Bader S. Alkaloid accumulation in Catharanthus roseus L. in vitro cuiture enhanced via ag and TiO 2 nanoparticles. SABRAO J Breed Genet 2023, 55(4).\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR58\" id=\"ref-link-section-d126582938e1088\" rel=\"nofollow noopener\" target=\"_blank\">58<\/a>] reported that application of iron nanoparticles along with 2,4-D and BAP on Catharanthus roseus plants improved production of alkaloids. However, the results are also indicative of the fact that not only nanoparticles interact with PGRs and thereby enhance the availability of the minerals, but they also enhance the tissue redox status ultimately enhancing the alkaloids biosynthesis.<\/p>\n<p>Treatment-Induced changes in phenol and flavonoid levels<\/p>\n<p>The increase in phenolic content by the treatments 2,4-D 0.5 mg\/L\u2009+\u2009Kin 1 mg\/L\u2009+\u2009Fe\u2083O\u2084-NP 4 mg\/L, 2,4-D 0.5 mg\/L\u2009+\u2009Kin 2 mg\/L\u2009+\u2009Fe\u2083O\u2084-NP 1 mg\/L, 2,4-D 1 mg\/L\u2009+\u2009Kin 1 mg\/L\u2009+\u2009Fe\u2083O\u2084-NP 4 mg\/L, 2,4-D 1 mg\/L\u2009+\u2009Kin 1.5 mg\/L\u2009+\u2009Fe\u2083O\u2084-NP 1 mg\/L, 2,4-D 2 mg\/L\u2009+\u2009Kin 1 mg\/L\u2009+\u2009Fe\u2083O\u2084-NP 2 mg\/L, 2,4-D 2 mg\/L\u2009+\u2009Kin 1 mg\/L\u2009+\u2009Fe\u2083O\u2084-NP 4 mg\/L, and 2,4-D 2 mg\/L\u2009+\u2009Kin 1.5 mg\/L\u2009+\u2009Fe\u2083O\u2084-NP 1 mg\/L was 72.21%, 75.38%, 80.14%, 64.28%, 64.25%, 64.27%, and 75%, respectively, compared to the control (Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig5\" rel=\"nofollow noopener\" target=\"_blank\">5<\/a>a). Chakraborty et al. [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 59\" title=\"Chakraborty N, Banerjee D, Ghosh M, Pradhan P, Gupta NS, Acharya K, Banerjee M. Influence of plant growth regulators on callus mediated regeneration and secondary metabolites synthesis in Withania somnifera (L.) Dunal. Physiol Mol Biology Plants. 2013;19:117\u201325.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR59\" id=\"ref-link-section-d126582938e1102\" rel=\"nofollow noopener\" target=\"_blank\">59<\/a>] found that the use of 2,4-D in Withania somnifera during in vitro cultivation improved the synthesis of phenolic compounds and other secondary metabolites as well as quality of callus. Also, Emamverdian et al. [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 60\" title=\"Emamverdian A, Khalofah A, Pehlivan N, Li Y. Enhanced cd tolerance in bamboo: synergistic effects of Nano-Hydroxyapatite and Fe3O4 nanoparticles on reactive oxygen species scavenging, cd detoxification, and water balance. Agronomy. 2025;15(2):386.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR60\" id=\"ref-link-section-d126582938e1105\" rel=\"nofollow noopener\" target=\"_blank\">60<\/a>] found that applying Fe\u2083O\u2084-NP along with growth regulators like kin increased the phenol content and activity of POD and CAT enzymes that is related to enhancement of the plant defense status in stress conditions. The combined application of 2,4-D and kin increased the contents of total phenols, flavonoids and the activity of antioxidant enzymes in the in vitro cultures of Ocimum basilicum L [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 61\" title=\"Topdemir A, Buran A. Determination of antioxidant activity and phenolic and flavonoid content of Ocimum Basilicum L. callus cultures obtained by different plant growth regulators. Acta Scientiarum Polonorum Hortorum Cultus. 2023;22(2):133\u201349.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR61\" id=\"ref-link-section-d126582938e1108\" rel=\"nofollow noopener\" target=\"_blank\">61<\/a>]. Triggered phenol production was also observed in in vitro culture medium of Artemisia annua L. upon Fe\u2083O\u2084-NP and kin treatment [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 62\" title=\"Ayoobi A, Saboora A, Asgarani E, Efferth T. Iron oxide nanoparticles (Fe3O4-NPs) elicited Artemisia annua L. in vitro, toward enhancing Artemisinin production through overexpression of key genes in the terpenoids biosynthetic pathway and induction of oxidative stress. Plant Cell Tissue Organ Cult (PCTOC). 2024;156(3):85.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR62\" id=\"ref-link-section-d126582938e1111\" rel=\"nofollow noopener\" target=\"_blank\">62<\/a>]. Synergistic application of kin and Fe\u2083O\u2084-NP enhanced the content of phenolic and flavonoid metabolites in emon balm (Melissa officinalis L.). Moreover, the co-administration of Fe\u2083O\u2084-NP and kin in Thymus vulgaris L. enhanced the total phenol production and reduced oxidative stress [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 63\" title=\"Kamyab A, Samsampour D, Ahmadinasab N, Bagheri A. The combination of nanoparticles and endophytes boosts thyme (Thymus vulgaris L.) resistance to drought stress by elevating levels of phenolic compounds, flavonoids, and essential oils. Chem Biol Technol Agric. 2024;11(1):163.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR63\" id=\"ref-link-section-d126582938e1115\" rel=\"nofollow noopener\" target=\"_blank\">63<\/a>]. In a study, the synergy of a nitrophenol-based biostimulant nanocomplex caused an increase in phenols and flavonoids in tomatoes [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 64\" title=\"Tavallali V, Darvishzadeh MD. Synergistic effects of Fe nanocomplex and nitrophenolate-based biostimulant on growth and physiological performance of tomato seedlings. BMC Plant Biol. 2025;25(1):905.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR64\" id=\"ref-link-section-d126582938e1118\" rel=\"nofollow noopener\" target=\"_blank\">64<\/a>].<\/p>\n<p>Fig. 5<a class=\"c-article-section__figure-link\" data-test=\"img-link\" data-track=\"click\" data-track-label=\"image\" data-track-action=\"view figure\" href=\"https:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6\/figures\/5\" rel=\"nofollow noopener\" target=\"_blank\"><img decoding=\"async\" aria-describedby=\"Fig5\" src=\"https:\/\/www.newsbeep.com\/il\/wp-content\/uploads\/2025\/10\/12870_2025_7409_Fig5_HTML.png\" alt=\"figure 5\" loading=\"lazy\" width=\"685\" height=\"218\"\/><\/a><\/p>\n<p>Effect of treatment compounds, Kin, 2,4-D and Fe\u2083O\u2084-NP on Hypericum perforatum L.: (a) phenol, and (b) flavonoid. Data represent mean\u2009\u00b1\u2009SE (n\u2009=\u20093). Different letters indicate significant differences between treatments based on LSD test (P\u2009\u2264\u20090.05)<\/p>\n<p>Evaluation of secondary metabolites<\/p>\n<p>The synthesis of important secondary metabolites in Hypericum perforatum was significantly enhanced by the joint addition of 2,4-D, kin, and Fe\u2083O\u2084-NP. The most abundant compounds were hypericin and hyperforin (24.6\u2009\u00b1\u20090.027 and 18.2\u2009\u00b1\u20090.001 (Table <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"table anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Tab1\" rel=\"nofollow noopener\" target=\"_blank\">1<\/a>), respectively indicating more effective therapeutic usefulness. Identified compounds accounted for (93.36%) was also affected by other bioactive molecules including \u00df-patchoulene, hexadecanol and naphthalene. High data precision (r\u2009=\u20090.96; CV\u2009=\u20094.22%) confirmed the reliability of these discoveries. This work with potential use in pharmaceutical and agricultural sectors indicates the synergistic effects of PGRs with Fe\u2083O\u2084-NP to enhance the phytochemical contents in H. perforatum (Table <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"table anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Tab1\" rel=\"nofollow noopener\" target=\"_blank\">1<\/a>). Figure <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig6\" rel=\"nofollow noopener\" target=\"_blank\">6<\/a> represents the GC graph of treatment compounds 2,4- D 1 mg\/L, Kin 1 mg\/L, Fe\u2083O\u2084-NP 4 mg\/L. In a study of St. John\u2019s wort (H. perforatum L.), the compounds hypericin, pseudohypericin, hyperoside, rutin, quercitrin, and hyperforin were obtained [<a data-track=\"click\" data-track-action=\"reference anchor\" data-track-label=\"link\" data-test=\"citation-ref\" aria-label=\"Reference 65\" title=\"Smelcerovic A, Spiteller M, Zuehlke S. Comparison of methods for the exhaustive extraction of hypericins, flavonoids, and Hyperforin from Hypericum perforatum L. J Agric Food Chem. 2006;54(7):2750\u20133.\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#ref-CR65\" id=\"ref-link-section-d126582938e1173\" rel=\"nofollow noopener\" target=\"_blank\">65<\/a>].<\/p>\n<p>Table 1 Evaluation of secondary compounds in the best treatment combination including 2,4-D 1 mg\/l, Kinetin 1 mg\/l, and Fe\u2083O\u2084-NP 4 mg\/lFig. 6<a class=\"c-article-section__figure-link\" data-test=\"img-link\" data-track=\"click\" data-track-label=\"image\" data-track-action=\"view figure\" href=\"https:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6\/figures\/6\" rel=\"nofollow noopener\" target=\"_blank\"><img decoding=\"async\" aria-describedby=\"Fig6\" src=\"https:\/\/www.newsbeep.com\/il\/wp-content\/uploads\/2025\/10\/12870_2025_7409_Fig6_HTML.png\" alt=\"figure 6\" loading=\"lazy\" width=\"685\" height=\"240\"\/><\/a><\/p>\n<p>GC Chromatograph of the treatment compounds 2,4-D 1 mg\/L, kinetin 1 mg\/L, and Fe\u2083O\u2084-NP 4 mg\/L<\/p>\n<p>Multivariate trait analysis (HCA and PCA)<\/p>\n<p>The results show statistically significant positive correlations in treatments with 2,4-D, kin, Fe\u2083O\u2084-NP and all growth parameters and secondary metabolites measured. The treatment of 2,4-D 0.5 mg\/L\u2009+\u2009Kin 2 mg\/L\u2009+\u2009Fe\u2083O\u2084-NP 4 mg\/L showed significantly higher hypericin content and rooting percentage and regeneration (Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig7\" rel=\"nofollow noopener\" target=\"_blank\">7<\/a>a), which indicated the strong stimulatory effect 2,4-D, kin and Fe\u2083O\u2084-NP on growth and metabolite production. Likewise, 2,4-D 1 mg\/L\u2009+\u2009Kin 1.5 mg\/L\u2009+\u2009Fe\u2083O\u2084-NP 1 mg\/L improved regeneration and rooting rates, thereby providing support for its role in organ development and cell regeneration (Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig7\" rel=\"nofollow noopener\" target=\"_blank\">7<\/a>a). The number of shoots was significantly higher with 2,4-D 1 mg\/L\u2009+\u2009Kin 1.5 mg\/L\u2009+\u2009Fe\u2083O\u2084-NP at 2 and 4 mg\/L, indicating alterations in branching and structural growth. Additionally, hypericin production showed a positive relationship with 2,4-D 2 mg\/L\u2009+\u2009Kin 1.5 mg\/L\u2009+\u2009Fe\u2083O\u2084-NP mg\/L and 2,4-D 0.5 mg\/L\u2009+\u2009Kin 1\u2009+\u2009Fe\u2083O\u2084-NP 4 mg\/L supportive of their influence on metabolite pathways (Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig7\" rel=\"nofollow noopener\" target=\"_blank\">7<\/a>a). Similarly, root length and plant height had positive relationships with particular treatments indicating improved uptake producing greater overall plant growth.<\/p>\n<p>Fig. 7<a class=\"c-article-section__figure-link\" data-test=\"img-link\" data-track=\"click\" data-track-label=\"image\" data-track-action=\"view figure\" href=\"https:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6\/figures\/7\" rel=\"nofollow noopener\" target=\"_blank\"><img decoding=\"async\" aria-describedby=\"Fig7\" src=\"https:\/\/www.newsbeep.com\/il\/wp-content\/uploads\/2025\/10\/12870_2025_7409_Fig7_HTML.png\" alt=\"figure 7\" loading=\"lazy\" width=\"685\" height=\"1235\"\/><\/a><\/p>\n<p>Effect of treatment compounds, Kin, 2,4-D and Fe\u2083O\u2084-NP on Hypericum perforatum L. (a) Hierarchical clustering analysis (HCA) of Pearson\u2019s correlation coefficient (r) values of variable traits, where the color scale that indicates r coefficient values (r\u2009=\u20091.5 to \u22121.5) indicates positive (red) and negative (blue) correlations. (b) Principal component analysis (PCA). (c) Pearson correlation coefficient scatter plot. Pearson correlation coefficient was used to reflect the linear-related degrees of two variables. It\u2019s range is \u22121 to 1.1 depicts totally positive correlated; \u22121 depicts totally negative correlated; 0 depicts no linear correlation<\/p>\n<p>In this PCA analysis, treatments with 2,4-D (1, 0.5, and 2 mg\/L), plus kin (0.5, 1, 1.5, and 2 mg\/L), and Fe\u2083O\u2084-NP at different concentrations (1, 2, and 4 mg\/L) had positive and significant correlations with different growth parameters and secondary metabolites (Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig7\" rel=\"nofollow noopener\" target=\"_blank\">7<\/a>b). In particular, the treatment combinations of 2,4-D 1 mg\/L with Kin 1 and 1.5 mg\/L and Fe\u2083O\u2084-NP 2, 4 and 1 mg\/L; 2,4-D 0.5 mg\/L with Kin 1 and 2 mg\/L and Fe\u2083O\u2084-NP 2 and 4 mg\/L increased callus fresh weight, hypericin content, callus formation percentage, number of shoots and phenolic and flavonoid contents and decreased of black nodule accumulation. Conversely, the treatment combinations of 2,4-D 2 mg\/L with Kin 1 mg\/L and Fe\u2083O\u2084-NP 1, 2 and 4 mg\/L and 2,4-D 1 mg\/L with Kin 1 mg\/L and 2,4-D 2\u2009+\u2009Kin 1.5\u2009+\u2009NP 1 mg\/L had positive and significant correlations with root length, rooting percentage, rooting percentage and plant height (Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig7\" rel=\"nofollow noopener\" target=\"_blank\">7<\/a>b). The findings highlight the different concentrations of treatment compounds have an independent effect on enhanced growth parameters and increased production of active metabolites and they combine as a synergistic formulation that can be beneficial for enhanced tissue culture and medicinal plant production.<\/p>\n<p>Pearson correlation analysis<\/p>\n<p>Pearson correlation analysis revealed a positive and significant association between hypericin contents and most of the morphological and biochemical traits. Most notably, the highest correlation coefficient observed was hypericin and black nodule formation (r\u2009=\u20090.85) and callus formation percentage (r\u2009=\u20090.82) which both signify the roles of hypericin in tissue differentiation and morphogenic processes (Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig7\" rel=\"nofollow noopener\" target=\"_blank\">7<\/a>c). A significant correlation was also found for hypericin and rooting (r\u2009=\u20090.71), regeneration (r\u2009=\u20090.74), height (r\u2009=\u20090.67), root length (r\u2009=\u20090.65) and number of shoots (r\u2009=\u20090.66), which signifies simultaneous effects on plant growth. Biochemical data showed a strong correlation with phenolic compounds (r\u2009=\u20090.76) and flavonoids (r\u2009=\u20090.77), and may suggest a relationship between hypericin metabolism and the production of antioxidant compounds (Fig. <a data-track=\"click\" data-track-label=\"link\" data-track-action=\"figure anchor\" href=\"http:\/\/bmcplantbiol.biomedcentral.com\/articles\/10.1186\/s12870-025-07409-6#Fig7\" rel=\"nofollow noopener\" target=\"_blank\">7<\/a>c). This suggests that hypericin accumulation is positively linked with overall secondary metabolite production and antioxidant capacity in the cultures.<\/p>\n","protected":false},"excerpt":{"rendered":"Fe\u2083O\u2084 and PGRs elevate plant growth Several treatments including 2,4-D (0.5 to 2 mg\/L), kinetin (0.5 to 1&hellip;\n","protected":false},"author":2,"featured_media":61024,"comment_status":"","ping_status":"","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[7],"tags":[4386,45105,45104,45106,85,45107,46,45103,6825,141,45108,25737],"class_list":{"0":"post-61023","1":"post","2":"type-post","3":"status-publish","4":"format-standard","5":"has-post-thumbnail","7":"category-science","8":"tag-agriculture","9":"tag-callus-induction","10":"tag-feo-nanoparticles","11":"tag-hypericin","12":"tag-il","13":"tag-in-vitro-regeneration","14":"tag-israel","15":"tag-plant-growth-regulators-pgrs","16":"tag-plant-sciences","17":"tag-science","18":"tag-secondary-metabolites","19":"tag-tree-biology"},"_links":{"self":[{"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/posts\/61023","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/comments?post=61023"}],"version-history":[{"count":0,"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/posts\/61023\/revisions"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/media\/61024"}],"wp:attachment":[{"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/media?parent=61023"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/categories?post=61023"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.newsbeep.com\/il\/wp-json\/wp\/v2\/tags?post=61023"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}