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Catharanthus roseus (L.) G. Don (Apocynaceae) is a highly important medicinal plant and a valuable source of diverse pharmacologically active compounds particularly anticancer terpenoid indole alkaloids (TIAs), such as catharanthine (CAT), vinblastine (VBL) and vincristine (VCR). This study investigated the impact of various plant growth regulator (PGR) combinations on callus induction and production of CAT, VBL and VCR. Leaf and stem explants were cultured on media containing seven combinations of auxins and cytokinins, and evaluated for callus induction frequency, biomass accumulation, and alkaloid content after eight weeks. Stem explants exhibited superior responsiveness, achieving 100% callus induction under most PGR treatments, whereas the combination of GA₃ and BAP failed to induce callogenesis in both explant types. HPLC analysis revealed that the combination of 1.5 mg/L 2,4-D and 1.5 mg/L kinetin (Kin) in leaf-derived callus yielded the highest CAT and VBL concentrations (5.01 µg/gFW and 1.96 µg/gFW; respectively). However, VCR notable enhanced in the stem- derived callus grown on a medium containing a combination of 1.0 mg/L each of BAP and NAA (4.71 µg/gFW) and leaf-derived callus grown in a medium containing 1.5 mg/L each of BAP and 2,4-D (2.79 µg/gFW) as compared with the controls (leaves and stems from in vivo grown plants, 1.51 µg/gFW and 0.38 µg/gFW; respectively). Gene expression analysis revealed that the combined application of 1.0 mg/L BAP and 1.0 mg/L NAA enhanced the transcript levels of peroxidase 1 (PRX 1), secologanin synthase ( SLS ), and strictosidine synthase ( STR ) in stem-derived callus. The upregulation of these genes was positively correlated with increased VCR accumulation. In contrast, the expression levels of octadecanoid-derivative Responsive Catharanthus AP2-domain protein 3 ( ORCA3 ) and desacetoxyvindoline 4-hydroxylase ( D4H ) were reduced. These findings highlight the importance of tailored hormonal treatments for optimizing in vitro biomass production and secondary metabolite synthesis in C. roseus , offering insights into sustainable alkaloid production for pharmaceutical applications. This study addresses the link between tailored plant growth regulator regimes and the coordinated activation of late-pathway genes, resulting in the selective enhancement of individual TIAs in C. roseus callus cultures, thereby providing a novel molecular–biotechnological strategy for targeted alkaloid production.

Catharanthus roseus (L.) G. Don is a plant belonging to the genus Catharanthus of the Apocynaceae family. It contains more than one hundred alkaloids, of which some exhibit significant pharmacological activities. Chitooligosaccharides are the only basic aminooligosaccharides with positively charged cations in nature, which can regulate plant growth and antioxidant properties. In this study, the leaves of Catharanthus roseus were sprayed with chitooligosaccharides of different molecular weights (1 kDa, 2 kDa, 3 kDa) and different concentrations (0.01 μg/mL, 0.1 μg/mL, 1 μg/mL and 10 μg/mL). The fresh weights of its root, stem and leaf were all improved after chitooligosaccharides treatments. More importantly, the chitooligosaccharides elicitor strongly stimulated the accumulation of vindoline and catharanthine in the leaves, especially with the treatment of 0.1 μg/mL 3 kDa chitooligosaccharides, the contents of them were increased by 60.68% and 141.54%, respectively. Furthermore, as the defensive responses, antioxidant enzymes activities (catalase, glutathione reductase, ascorbate peroxidase, peroxidase and superoxide dismutase) were enhanced under chitooligosaccharides treatments. To further elucidate the underlying mechanism, qRT-PCR was used to investigate the genes expression levels of secologanin synthase (SLS), strictosidine synthase (STR), strictosidine glucosidase (SGD), tabersonine 16-hydroxylase (T16H), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT), peroxidase 1 (PRX1) and octadecanoid-responsive Catharanthus AP2-domain protein 3 (ORCA3). All the genes were significantly up-regulated after chitooligosaccharides treatments, and the transcription abundance of ORCA3, SLS, STR, DAT and PRX1 reached a maximal level with 0.1 μg/mL 3 kDa chitooligosaccharides treatment. All these results suggest that spraying Catharanthus roseus leaves with chitooligosaccharides, especially 0.1 μg/mL of 3 kDa chitooligosaccharides, may effectively improve the pharmaceutical value of Catharanthus roseus.

Indoor farming offers optimal conditions for high and stable ajmalicine and serpentine yields in Catharanthus roseus , enabling precise control over environmental stimuli such as light and biostimulants. This study aimed to evaluate, for the first time, the combined effects of LED light spectrum and a biostimulant containing arbuscular mycorrhizal fungi on ajmalicine and serpentine production in indoor-grown C. roseus . Following a 60 days pre-treatment under white LEDs, plants received eight treatments: white (W, control), red (R), blue (B), red-blue (RB, R:B = 6:1) LED light, and their combinations with the biostimulant (BS). Samples were collected before treatments (T0) and at harvest (T1, 92 days after pre-treatment). R and RB significantly increased aerial fresh weight compared to W and B, with no significant differences in plant height relative to W. Although no mycorrhizal colonization occurred, BS significantly increased plant height, shoot fresh weight, total dry weight, leaf serpentine concentration, and total serpentine yield (+ 34.4%) compared to the untreated condition. When combined, R and BS had a synergistic effect on ajmalicine, resulting in its highest concentration and yield. Under this treatment, ajmalicine concentration and yield were significantly higher than in all other treatments (+ 144.3% and + 138.0% compared to the W control, respectively), except for the yields under B and RB with BS, which were comparable to those under R with BS. From T0 to T1, total serpentine yield increased by 2277.1%, while ajmalicine yield rose by 716.0%. In conclusion, the combined application of R and BS, followed by harvest after a 92 days treatment period, is the most effective strategy to optimize ajmalicine and serpentine production while ensuring vigorous yet size-controlled plant growth.

Carbon-based nanomaterials (CBNs) have great potential as a powerful tool to improve plant productivity. Here, we investigated the biological effects of graphene and carbon nanotubes (CNTs) on fiber-producing species (cotton, Gossypium hirsutum ) and ornamental species (vinca, Catharanthus roseus ). The exposure of seeds to CNTs or graphene led to the activation of early seed germination in Catharanthus and overall higher germination in cotton and Catharanthus seeds. The application of CBNs resulted in higher root and shoot growth of young seedlings of both tested species. Cultivation of Catharanthus plants in soil supplemented with CBNs resulted in the stimulation of plant reproductive system by inducing early flower development along with higher flower production. Catharanthus plants cultivated in CNTs or graphene supplemented soil accelerated total flower production by 37 and 58%, respectively. Additionally, CBNs reduced the toxic effects caused by NaCl. Long-term application of CBNs to crops cultivated under salt stress conditions improved the desired phenotypical traits of Catharanthus (higher flower number and leaf number) and cotton (increased fiber biomass) compared to untreated plants of both species cultivated at the same stress condition. The drought stress experiments revealed that introduction of CBNs to matured Catharanthus plant increased the plant survival with no symptoms of leaf wilting as compared to untreated Catharanthus growing in water deficit conditions.

DELLAs are key regulators of plant growth and development, negatively regulating gibberellic acid (GA) signaling and positively regulating light and jasmonate signaling and juvenile leaf development. In the presence of GA, GID1s bind to DELLAs and signal for their degradation. Here, we identified and characterized DELLA and GID1 genes in Catharanthus roseus , the natural source of chemotherapy drugs vinblastine and vincristine. We hypothesized that CrDELLAs positively regulate vindoline biosynthesis, a precursor of vinblastine and vincristine, accumulating in light- and jasmonate-exposed young leaves. To explore this hypothesis, we silenced CrDELLA or CrGID1 genes using virus-induced gene silencing. CrDELLA -silenced plants were elongated while CrGID1 -silenced plants were dwarfed, consistent with their roles in GA-mediated growth. In the first experiment, CrDELLA -silencing significantly decreased vindoline pathway gene expression while CrGID1 -silencing significantly increased vindoline, catharanthine, ajmalicine, and serpentine accumulation. However, subsequent experiments found little to no effect. C. roseus seedlings treated with paclobutrazol, an inhibitor of GA biosynthesis shown to increase DELLA protein stability, also provided some evidence for CrDELLA’s positive role in regulating vindoline pathway gene expression. Finally, overexpressed stabilized, N-terminal truncated CrDELLAs in C. roseus seedlings yielded significant increases in vindoline pathway promoter activity ( NMT , D4H ). Overall, these experiments provide weak to moderate evidence for CrDELLAs positively regulating vindoline biosynthesis. Future experiments with transgenic approaches could strengthen the evidence and clarify this relationship. Activation of the vindoline pathway with stabilized CrDELLAs could increase the production of critical chemotherapeutics, vinblastine and vincristine. Key message Transient silencing of CrDELLA s and CrGID1 s, overexpression of truncated CrDELLA s, and application of the gibberellic acid-inhibitor paclobutrazol provide weak to moderate evidence that CrDELLAs activate vindoline biosynthesis in Catharanthus roseus .

As a member of the Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) protein kinase subfamily, FERONIA (FER) has emerged as a versatile player regulating multifaceted functions in growth and development, as well as responses to environmental factors and pathogens. With the concerted efforts of researchers, the molecular mechanism underlying FER-dependent signaling has been gradually elucidated. A number of cellular processes regulated by FER-ligand interactions have been extensively reported, implying cell type-specific mechanisms for FER. Here, we provide a review on the roles of FER in male-female gametophyte recognition, cell elongation, hormonal signaling, stress responses, responses to fungi and bacteria, and present a brief outlook for future efforts.

In plants, organ formation and cell elongation require the constant adjustment of the dynamic and adaptable cell wall in response to environmental cues as well as internal regulators, such as light, mechanical stresses, pathogen attacks, phytohormones, and other signaling molecules. The molecular mechanisms that perceive these cues and translate them into cellular responses to maintain integrity and remodelling of the carbohydrate-rich cell wall for the coordination of cell growth are still poorly understood. In the last 3 years, the function of six membrane-localized receptor-like kinases (RLKs) belonging to the CrRLK1L family has been linked to the control of cell elongation in vegetative and reproductive development. Moreover, the presence of putative carbohydrate-binding domains in the extracellular domains of these CrRLK1Ls makes this receptor family an excellent candidate for coordinating cell growth, cell-cell communication, and constant cell wall remodelling during the plant life cycle.

Catharanthus roseus is a medicinal plant widely known for producing monoterpenoid indole alkaloids (MIAs), including therapeutic compounds such as vinblastine and vincristine, which are crucial for cancer treatment. However, the naturally low concentration of these alkaloids in plant tissues poses a significant challenge for large-scale production. This study explores the application of siderophore-producing bacteria for seed bacterization of Catharanthus roseus to enhance the production of MIAs, including vindoline, catharanthine, and vinblastine. Utilizing High-Performance Liquid Chromatography (HPLC), we observed a significant increase in the concentration of these alkaloids in bacterized plants compared to controls. FTIR spectra of treated plants showed strong correlations with standard alkaloid mixtures, confirming higher alkaloid accumulation. Our findings demonstrate that bacterial siderophores play a vital role in optimizing iron uptake, which is crucial for secondary metabolite biosynthesis. This research highlights the potential of using microbial biotechnology to improve the yield of valuable pharmaceutical compounds in medicinal plants. Enhancing the biosynthetic pathways of MIAs offers a sustainable and efficient strategy for boosting the production of key therapeutic alkaloids in Catharanthus roseus , paving the way for advanced biotechnological applications in plant-based drug production.

Catharanthus roseus produces a wide spectrum of monoterpene indole alkaloids (MIAs). MIA biosynthesis requires a tightly coordinated pathway involving more than 30 enzymatic steps that are spatio-temporally and environmentally regulated so that some MIAs specifically accumulate in restricted plant parts. The first regulatory layer involves a complex network of transcription factors from the basic Helix Loop Helix (bHLH) or AP2 families. In the present manuscript, we investigated whether an additional epigenetic layer could control the organ-, developmental-and environmental-specificity of MIA accumulation. We used Whole-Genome Bisulfite Sequencing (WGBS) together with RNA-seq to identify differentially methylated and expressed genes among nine samples reflecting different plant organs and experimental conditions. Tissue specific gene expression was associated with specific methylation signatures depending on cytosine contexts and gene parts. Some genes encoding key enzymatic steps from the MIA pathway were found to be simultaneously differentially expressed and methylated in agreement with the corresponding MIA accumulation. In addition, we found that transcription factors were strikingly concerned by DNA methylation variations. Altogether, our integrative analysis supports an epigenetic regulation of specialized metabolisms in plants and more likely targeting transcription factors which in turn may control the expression of enzyme-encoding genes.

Background Catharanthus roseus (L.) G. Donis a medicinal plant species belonging to the Apocynaceae family, which produces vinblastine and vincristine along with 100 other monoterpenoid indole alkaloids. The process of biosynthesis of C. roseus alkaloids is complex, in which many genes, enzymes, and regulators are involved. Induced mutations may be considered as a potential source for producing a higher amount of vinblastine and vincristine in this plant species. Therefore, the objective of the present study was to examine the effects of different treatments utilized on the induced genetic changes in C. roseus plants and enzyme activities. Methods and results Spermine, jasmonic acid, methyjasmonate, putrescine, and cold plasma treatments were used for seed treatments. Different molecular markers, namely inter simple sequence repeat, inter retrotransposon amplified polymorphism, and retrotransposon microsatellite amplified polymorphism were employed to reveal the induced genetic changes. Antioxidant enzyme activities were also studied. The treated plants showed genetic variability and a significant increase in antioxidant enzyme activity compared to the control plants. The putrescine treatment resulted in the highest level of activity in superoxidase. A significant positive correlation occurred between the molecular markers data and antioxidant enzyme activities in treated plants. Conclusion Our data revealed that the different phytohormones and cold plasma treatments could induce both genetic and chemical content changes in C. roseus plants.