In this study, the stimulatory effects of the combined treatment of plant growth regulators (2,4-D and kinetin) and iron oxide nanoparticles (Fe₃O₄-NPs) on in vitro morphogenesis and secondary metabolite production in *Hypericum perforatum* L. were investigated. The optimized treatment [2,4-D (0.5 mg/L) + kinetin (2 mg/L) + Fe₃O₄-NPs (4 mg/L)] significantly improved the plant growth parameters: plant height increased by 59.6%, root length by 114.0%, bud number by 180.0%, and callus fresh weight by 198.3% compared with the control group. This combined treatment also enhanced the regeneration efficiency (50.85%) and increased the hypericin content by 66.6%. GC-MS analysis revealed high contents of hyperoside, β-patholene, and cetyl alcohol, accounting for 93.36% of the total peak area, while the contents of total phenolics and flavonoids increased by as much as 80.1%. These results indicate that plant growth regulators (PGRs) and Fe₃O₄ nanoparticles (Fe₃O₄-NPs) exert a synergistic effect by stimulating organogenesis and accumulation of bioactive compounds, which represents a promising strategy for the biotechnological improvement of medicinal plants.
St. John’s wort (Hypericum perforatum L.), also known as St. John’s wort, is a perennial herbaceous plant of the family Hypericaceae that has economic value.[1] Its potential bioactive components include natural tannins, xanthones, phloroglucinol, naphthalenedianthrone (hyperin and pseudohyperin), flavonoids, phenolic acids, and essential oils.[2,3,4] St. John’s wort can be propagated by traditional methods; however, the seasonality of traditional methods, low seed germination, and susceptibility to diseases limit its potential for large-scale cultivation and continuous formation of secondary metabolites.[1,5,6]
Thus, in vitro tissue culture is considered an effective method for rapid plant propagation, conservation of germplasm resources, and increased yield of medicinal compounds [7, 8]. Plant growth regulators (PGRs) play a crucial role in regulating morphogenesis and are necessary for the in vitro cultivation of callus and whole organisms. Optimization of their concentrations and combinations is crucial for the successful completion of these developmental processes [9]. Therefore, understanding the appropriate composition and concentration of regulators is important for improving the growth and regenerative capacity of St. John’s wort (H. perforatum) [10].
Iron oxide nanoparticles (Fe₃O₄) are a class of nanoparticles that have been or are being developed for tissue culture. Fe₃O₄ has significant magnetic properties, good biocompatibility, and the ability to promote plant growth and reduce environmental stress, so it has attracted considerable attention in tissue culture designs. Potential applications of these nanoparticles may include optimizing in vitro culture to promote cell division, improve nutrient uptake, and activate antioxidant enzymes [11].
Although nanoparticles have shown good promoting effects on plant growth, studies on the combined application of Fe₃O₄ nanoparticles and optimized plant growth regulators in *H. perforatum* remain scarce. To fill this knowledge gap, this study evaluated the effects of their combined effects on in vitro morphogenesis and secondary metabolite production to provide new insights for improving the characteristics of medicinal plants. Therefore, this study has two objectives: (1) optimize the concentration of plant growth regulators to effectively promote callus formation, shoot regeneration, and rooting in vitro; and (2) evaluate the effects of Fe₃O₄ nanoparticles on growth parameters in vitro. Future plans include evaluating the survival rate of regenerated plants during acclimatization (in vitro). It is expected that the results of this study will significantly improve the micropropagation efficiency of *H. perforatum*, thereby contributing to the sustainable use and biotechnological applications of this important medicinal plant.
In this study, we obtained leaf explants from field-grown annual St. John’s wort plants (mother plants). These explants were used to optimize in vitro culture conditions. Before culturing, the leaves were thoroughly rinsed under running distilled water for several minutes. The explant surfaces were then disinfected by immersion in 70% ethanol for 30 seconds, followed by immersion in a 1.5% sodium hypochlorite (NaOCl) solution containing a few drops of Tween 20 for 10 minutes. Finally, the explants were rinsed three times with sterile distilled water before transferring to the next culture medium.
Over the next four weeks, shoot regeneration parameters were measured, including regeneration rate, shoot number per explant, and shoot length. When regenerated shoots reached a length of at least 2 cm, they were transferred to a rooting medium consisting of half-strength MS medium, 0.5 mg/L indolebutyric acid (IBA), and 0.3% guar gum. Rooting culture continued for three weeks, during which time rooting rate, root number, and root length were measured. Each treatment was repeated three times, with 10 explants cultured per replicate, yielding approximately 30 explants per treatment.
Plant height was measured in centimeters (cm) using a ruler, from the base of the plant to the tip of the tallest leaf. Root length was measured in millimeters (mm) immediately after carefully removing the seedlings and removing the growing medium. The number of buds per explant was counted directly on each plant. The number of black spots on the leaves, known as nodules, was measured visually. These black nodules are believed to be glands containing hypericin, or oxidative spots, and are used as a physiological indicator of the plant’s response to treatment. After removing all the growing medium, the fresh weight of the seedlings was measured using an electronic scale with an accuracy of milligrams (mg).
The method for calculating the rate of callus formation is as follows: after culturing explants in a medium containing various growth regulators (kinases, 2,4-D, and Fe3O4) for four weeks, the number of explants capable of forming callus is counted. The formula for calculating the rate of callus formation is as follows:
Each treatment was repeated three times, with at least 10 explants examined in each repetition.
The regeneration rate reflects the proportion of callus tissue that successfully completes the bud differentiation process after the callus formation stage. This indicator demonstrates the ability of callus tissue to transform into differentiated tissue and grow into new plant organs.
The rooting coefficient is the ratio of the number of branches capable of rooting to the total number of branches. This indicator reflects the success of the rooting stage, which is crucial in micropropagation and plant propagation, as good rooting helps seedlings better survive in growing conditions.
Hypericin compounds were extracted with 90% methanol. Fifty mg of dried plant material was added to 1 ml of methanol and sonicated for 20 min at 30 kHz in an ultrasonic cleaner (model A5120-3YJ) at room temperature in the dark. After sonication, the sample was centrifuged at 6000 rpm for 15 min. The supernatant was collected, and the absorbance of hypericin was measured at 592 nm using a Plus-3000 S spectrophotometer according to the method described by Conceiçao et al. [14].
Most treatments with plant growth regulators (PGRs) and iron oxide nanoparticles (Fe₃O₄-NPs) did not induce black nodule formation on regenerated shoot leaves. No nodules were observed in any of the treatments with 0.5 or 1 mg/L 2,4-D, 0.5 or 1 mg/L kinetin, or 1, 2, or 4 mg/L iron oxide nanoparticles. A few combinations showed a slight increase in nodule development (but not statistically significant) at higher concentrations of kinetin and/or iron oxide nanoparticles, such as the combination of 2,4-D (0.5–2 mg/L) with kinetin (1–1.5 mg/L) and iron oxide nanoparticles (2–4 mg/L). These results are shown in Figure 2. Black nodules represent hypericin-rich glands, both naturally occurring and beneficial. In this study, black nodules were mainly associated with browning of tissues, indicating a favorable environment for hypericin accumulation. Treatment with 2,4-D, kinetin, and Fe₃O₄ nanoparticles promoted callus growth, reduced browning, and increased chlorophyll content, suggesting improved metabolic function and potential reduction of oxidative damage [37]. This study evaluated the effects of kinetin in combination with 2,4-D and Fe₃O₄ nanoparticles on the growth and development of St. John’s wort callus (Fig. 3a–g). Previous studies have shown that Fe₃O₄ nanoparticles have antifungal and antimicrobial activities [38, 39] and, when used in combination with plant growth regulators, can stimulate plant defense mechanisms and reduce cellular stress indices [18]. Although the biosynthesis of secondary metabolites is genetically regulated, their actual yield is highly dependent on environmental conditions. Metabolic and morphological changes can influence secondary metabolite levels by regulating the expression of specific plant genes and responding to environmental factors. Furthermore, inducers can trigger the activation of new genes, which in turn stimulate enzymatic activity, ultimately activating multiple biosynthetic pathways and leading to the formation of secondary metabolites. Furthermore, another study showed that reducing shading increases sunlight exposure, thereby raising daytime temperatures in the natural habitat of *Hypericum perforatum*, which also contributes to increased hypericin yield. Based on these data, this study investigated the role of iron nanoparticles as potential inducers in tissue culture. The results showed that these nanoparticles can activate genes involved in hesperidin biosynthesis through enzymatic stimulation, leading to increased accumulation of this compound (Fig. 2). Therefore, compared to plants growing under natural conditions, it can be argued that the production of such compounds in vivo can also be enhanced when moderate stress is combined with the activation of genes involved in the biosynthesis of secondary metabolites. Combination treatments generally have a positive effect on the regeneration rate, but in some cases, this effect is weakened. Notably, treatment with 1 mg/L 2,4-D, 1.5 mg/L kinase, and different concentrations could independently and significantly increase the regeneration rate by 50.85% compared with the control group (Fig. 4c). These results suggest that specific combinations of nanohormones can act synergistically to promote plant growth and metabolite production, which is of great significance for tissue culture of medicinal plants. Palmer and Keller [50] showed that 2,4-D treatment could independently induce callus formation in St. perforatum, while the addition of kinase significantly enhanced callus formation and regeneration. This effect was due to the improvement of hormonal balance and stimulation of cell division. Bal et al. [51] found that Fe₃O₄-NP treatment could independently enhance the function of antioxidant enzymes, thereby promoting root growth in St. perforatum. Culture media containing Fe₃O₄ nanoparticles at concentrations of 0.5 mg/L, 1 mg/L, and 1.5 mg/L improved the regeneration rate of flax plants [52]. The use of kinetin, 2,4-dichlorobenzothiazolinone, and Fe₃O₄ nanoparticles significantly improved the callus and root formation rates, however, the potential side effects of using these hormones for in vitro regeneration need to be considered. For example, long-term or high-concentration use of 2,4-dichlorobenzothiazolinone or kinetin may result in somatic clonal variation, oxidative stress, abnormal callus morphology, or vitrification. Therefore, a high regeneration rate does not necessarily predict genetic stability. All regenerated plants should be assessed using molecular markers (e.g. RAPD, ISSR, AFLP) or cytogenetic analysis to determine their homogeneity and similarity to in vivo plants [53,54,55].
This study demonstrated for the first time that the combined use of plant growth regulators (2,4-D and kinetin) with Fe₃O₄ nanoparticles can enhance morphogenesis and the accumulation of key bioactive metabolites (including hypericin and hyperoside) in *Hypericum perforatum*. The optimized treatment regimen (1 mg/L 2,4-D + 1 mg/L kinetin + 4 mg/L Fe₃O₄-NPs) not only maximized callus formation, organogenesis, and secondary metabolite yield but also demonstrated a mild inducing effect, potentially improving the plant’s stress tolerance and medicinal value. The combination of nanotechnology and plant tissue culture provides a sustainable and efficient platform for large-scale in vitro production of medicinal compounds. These results pave the way for industrial applications and future research into molecular mechanisms, dosage optimization and genetic precision, thereby linking fundamental research on medicinal plants with practical biotechnology.
Post time: Dec-12-2025