Although Calendula officinalis has been extensively investigated for its phytochemical properties and is widely grown as both a medicinal and ornamental plant, detailed morphological and anatomical information based on wild populations from Türkiye had been lacking. Most existing descriptions derive from cultivated material, which can differ substantially from plants that have established themselves under natural ecological conditions. The research team set out to close that gap by examining plants collected directly from four wild populations in Eskişehir, a province whose steppe-influenced climate and varied habitats provide a demanding test of the species’ plasticity. One of the study’s most striking observations concerns the life cycle of these plants: the investigated populations consisted predominantly of perennial individuals, with flowering extending from spring all the way into autumn under local conditions. That prolonged flowering window is notable for a species often treated as an annual in cultivation and suggests that wild Turkish populations may harbor adaptive strategies of considerable interest to breeders and ecologists alike.

The morphological component of the study was built on fresh specimens rather than dried herbarium material alone, allowing the researchers to capture characters that are often lost or distorted in preservation. Detailed descriptions of the vegetative and reproductive organs were prepared, and the team produced original illustrations documenting the plants’ structure. Such illustrated descriptions are a cornerstone of botanical taxonomy, providing a visual standard against which future collections can be compared. For a species as variable and long-cultivated as pot marigold, having verified morphological benchmarks from documented wild populations is a valuable resource, particularly for researchers attempting to distinguish genuinely wild or naturalized plants from garden escapes.

Beneath the surface, the anatomical work revealed a suite of microscopic features characteristic of the species and, more broadly, of the Asteraceae. The researchers prepared transverse and superficial sections of roots, stems, and leaves by hand from specimens preserved in 70% ethanol, a classical technique that remains indispensable for pharmacognostic quality control. The leaves proved to be amphistomatic, meaning that stomata, the pores that regulate gas exchange, occur on both the upper and lower surfaces, and these stomata were of the anomocytic type, lacking the specialized accessory cells seen in some other stomatal configurations. The leaf mesophyll was bifacial, organized into distinct palisade and spongy layers, the standard photosynthetic architecture of many dicotyledonous plants. In the roots, the team observed a well-developed secondary structure, reflecting the perennial habit of the plants and the thickening growth that supports survival across multiple seasons.

Perhaps the most pharmacologically significant anatomical finding involved trichomes, the hair-like outgrowths that cover plant surfaces. Both glandular and non-glandular multicellular trichomes were present on the stems and leaves. Glandular trichomes are of particular interest because they are the sites where many plants synthesize and store volatile compounds, including the terpenes that dominate essential oils. Their abundance and structure are therefore directly relevant to the plant’s chemistry and to the microscopic identification of herbal drug material. In the herbal medicine trade, where adulteration and misidentification are persistent concerns, such anatomical markers serve as fingerprints that allow pharmacists and regulators to verify that a batch of dried plant material is what it claims to be.

The chemical half of the study focused on the essential oils extracted from the aerial parts of the plants by hydrodistillation, the time-honored method of steam-driven separation used throughout the flavor, fragrance, and pharmaceutical industries. The resulting oils were analyzed by gas chromatography and gas chromatography coupled with mass spectrometry, the gold-standard techniques for separating and identifying volatile compounds. Together, these analyses identified 73 constituents in the essential oils, a rich chemical inventory for a species whose volatile profile has been studied far less intensively than its better-known triterpenoids and carotenoid pigments.

The dominant compound was α-cadinol, which accounted for between 30.0 and 34.5 percent of the oil in every one of the four populations. This oxygenated sesquiterpene alcohol, part of the cadinane family of compounds, was followed in abundance by δ-cadinene at 15.2 to 18.8 percent, τ-muurolol at 6.3 to 8.2 percent, τ-cadinol at 3.3 to 5.3 percent, α-muurolene at 2.4 to 3.0 percent, and γ-cadinene at 1.9 to 4.4 percent. Taken together, oxygenated sesquiterpenes made up 51.3 to 57.2 percent of the oils, with sesquiterpene hydrocarbons contributing a further 30.7 to 33.4 percent. The consistency of this cadinol-and-cadinene-dominated chemotype across all four wild populations is chemically striking, suggesting a stable, genetically anchored oil profile rather than one driven primarily by local environmental variation. It also distinguishes these wild Turkish plants from many cultivated accessions reported in earlier literature, whose oils have often been dominated by different compound classes.

That consistency carries practical weight. Essential oils rich in oxygenated sesquiterpenes such as α-cadinol and τ-muurolol are of ongoing interest to researchers exploring antimicrobial and other biological activities, and earlier studies on Calendula oils from other regions have reported antifungal properties. By documenting both the yield-relevant anatomy and the full volatile composition of wild material, the new study gives natural-product chemists a verified starting point for selecting germplasm worth further investigation. It also feeds into a broader scientific conversation about the effects of plant age, environmental conditions, and cultivation practices on essential oil composition, factors that previous researchers have shown can substantially reshape the chemical profile of this species.

The study, which grew out of the first author’s master’s thesis at the Department of Pharmaceutical Botany of Anadolu University’s Faculty of Pharmacy and received no external funding, ultimately delivers what its authors describe as baseline information for the four investigated wild populations. In a field where wild genetic resources are increasingly recognized as a reservoir of traits lost during centuries of domestication, that baseline matters. Perennial habit, extended flowering, a distinctive cadinol-rich essential oil, and a fully documented anatomical profile together make the Eskişehir populations a potentially valuable resource for taxonomy, for pharmacognostic standardization, and for phytochemical prospecting. As demand for plant-derived medicines and natural ingredients continues to grow, careful documentation of wild populations such as these provides the verified foundation on which sustainable use must be built.

Subject of Research: Morphological, anatomical, and essential oil characterization of wild Calendula officinalis populations in Türkiye

Article Title: Integrated morphological, anatomical and essential oil characterization of four wild populations of Calendula officinalis (Asteraceae) from Eskişehir, Türkiye

Article References: Kalas, M., Demirci, B., & Küçük, S. (2026). Integrated morphological, anatomical and essential oil characterization of four wild populations of Calendula officinalis (Asteraceae) from Eskişehir, Türkiye. Plant Biosystems, 160(5), Article 285. https://doi.org/10.1007/s44473-026-00300-8

Image Credits: AI Generated

DOI: 10.1007/s44473-026-00300-8

Keywords: Calendula officinalis, Asteraceae, essential oil, α-cadinol, plant anatomy, morphology, GC-MS, sesquiterpenes, Türkiye, Eskişehir, medicinal plants, pharmacognosy

The new cell line, named Calendula Root 1, or CR1, was born from a deceptively simple procedure. Seeds of Calendula officinalis were surface-sterilized and germinated on agar plates, and the emerging rootlets were sliced into small fragments and placed on callus induction medium containing the auxin 2,4-dichlorophenoxyacetic acid. Within days, a soft white callus appeared on the root pieces, and just thirty-five days after callus formation was first observed, the researchers transferred the tissue into liquid medium to start a suspension culture. The resulting cells, mostly round and measuring between fifty and three hundred micrometers, grew quickly and homogeneously, reaching packed cell volumes of fifty to sixty percent within a week when passaged weekly, figures very similar to those of BY2 cultures grown under comparable conditions.

One of the most consequential findings of the study came from an analysis of what the cells leave behind in their growth medium. When recombinant proteins are secreted into the culture supernatant, purification becomes dramatically simpler, but secreted proteins are vulnerable to the proteases that plant cells release into the medium. In BY2 cultures, these extracellular proteases have long been a thorn in the side of protein producers. Using gelatin zymography, a technique in which gelatin is embedded in a polyacrylamide gel so that proteolytic activity appears as clear bands of degraded substrate, the team found that the CR1 supernatant showed only minimal degradation at a single position of roughly eighty kilodaltons. The BY2 supernatant analyzed in the same experiment, by contrast, displayed five distinct degradation bands between fifty and sixty-eight kilodaltons plus a weaker signal at one hundred kilodaltons. This stark difference positions CR1 as a potentially far friendlier host for secreted products.

To test the production capacity of the new line, the researchers engineered a transformation construct carrying two independent expression cassettes, both driven by the cauliflower mosaic virus 35S promoter. The first encoded DsRed, the tetrameric red fluorescent protein from the coral Discosoma, targeted to the endoplasmic reticulum. The second encoded brazzein, a small, intensely sweet protein of just 6.5 kilodaltons from the West African plant Pentadiplandra brazzeana, directed to the apoplast, the space between the cell membrane and the cell wall from which proteins can reach the culture medium. DsRed served as a visible marker, allowing the team to pick fluorescent calli with a simple cold light source and filters, while brazzein represented a genuine industrial target with no commercial antibody available for detection.

Transformation was carried out using Agrobacterium tumefaciens strain GV3101, with cocultivation performed in the dark on solid medium. Ten days after plating on selection medium containing kanamycin, the first seventy-two fluorescent calli were picked, followed by another eighty-four five days later. Notably, the researchers chose not to optimize the codon sequences of either gene, reasoning that the available codon usage data for Calendula officinalis was sparse and that analysis of the closely related sunflower revealed no truly rare codons. Some calli turned visibly pink to the naked eye, indicating very high DsRed accumulation, and these were used to initiate suspension cultures that grew homogeneously within two weeks, although growth rates varied considerably between transformation events.

Quantifying brazzein posed a methodological challenge that the team solved with an elegant two-step approach. First, they purified brazzein from the supernatant of transformation event 74 using cation exchange chromatography and ultrafiltration, creating an in-house reference standard. Top-down liquid chromatography with tandem mass spectrometry confirmed the integrity of this standard, detecting the sevenfold-charged ion at a mass-to-charge ratio of 925.69 and deconvoluting the signals to a molecular weight of 6473 daltons, exactly the correct average mass of brazzein. The standard was then quantified by tryptic digest and comparison with a commercial isotope-labeled peptide, yielding a concentration of 0.21 milligrams per milliliter. Armed with this reference, the team used top-down LC-MS/MS with external calibration to measure brazzein in the supernatants of seven transformation events.

The results were striking. Brazzein concentrations in the culture supernatants ranged from 4.1 to 24.5 micrograms per milliliter, with an average of 10.5 micrograms per milliliter. Expressed per gram of fresh cell weight, the best line produced 259 micrograms of brazzein after fifteen days, roughly two hundred times more than the 1.2 micrograms per gram previously reported for brazzein production in transgenic carrot cells. Crucially, the brazzein produced in CR1 was untagged and secreted into the medium, an important distinction because attachments to either terminus of the molecule can alter or even completely abolish its sweetness. While yeast fermentation still holds the record for brazzein titers, with reports exceeding 300 milligrams per liter, the authors emphasize that the purpose of their work was to showcase the platform rather than to maximize this particular product.

The DsRed figures were even more dramatic. The best transformation event, line 155, accumulated 1322 micrograms of DsRed per gram of fresh weight, equivalent to 34.9 percent of total soluble protein, a figure the authors note clearly exceeds production rates reported for other recombinant proteins in the literature. Even the average across the seven analyzed lines, 533 micrograms per gram, was more than twice the roughly 200 milligrams per kilogram of biomass achieved with cytosol-targeted DsRed in transiently transformed BY2 plant cell packs. Because stable transformants are generally assumed to produce less recombinant protein than transiently transformed cells, the true capacity of CR1 may be even greater than these numbers suggest. Only small amounts of DsRed, between zero and 19.9 percent, were found in the supernatants, indicating that the cultures consisted largely of intact cells at harvest.

The study also revealed intriguing biology that will require further investigation. Transformation events that produced the most DsRed tended to grow more slowly, with line 155 being the most prominent example, though the authors did not explore the causal relationship in this dataset. Moreover, the molar production ratio of DsRed to brazzein varied widely between lines, from 1:2 in event 74 to 1:0.24 in event 16, showing that high DsRed output is not a reliable predictor of output from the second cassette in the tandem construct. The researchers attribute this asymmetry to position effects and to the multiple or partial T-DNA insertions that commonly occur during Agrobacterium-mediated transformation. Encouragingly, thirty-three months after transformation, all analyzed calli and dozens of others were still alive and expressing DsRed, suggesting robust transgene stability.

The authors acknowledge one cosmetic quirk: like BY2 cultures, which accumulate the pigment phytomelanin, aging CR1 cultures turn brownish-black, a trait known in marigold and other members of the Asteraceae family. Timely subculturing kept the browning from interfering with the work. Taken together, the findings establish CR1 as a rapidly growing, easily transformed cell culture with remarkably low protease activity in its medium and exceptional recombinant protein productivity in both the cytosolic-to-ER and secretory routes. Beyond the raw numbers, the choice of marigold carries strategic weight: as a well-known therapeutic herb with a positive public image, Calendula may sidestep the customer acceptance concerns that can complicate the use of tobacco-derived lines for food-related products, while its rich secondary metabolism offers untapped possibilities for producing high-value plant natural products in bioreactors.

Subject of Research: Establishment of a marigold (Calendula officinalis) suspension cell culture for recombinant protein production

Article Title: Marigold suspension cell culture as production system for recombinant DsRed and brazzein

Article References: Buntru, M., Croon, A., Müller, J., Hanke, M., & Schinkel, H. (2026). Marigold suspension cell culture as production system for recombinant DsRed and brazzein. Plant Cell Reports, 45(10), Article 317. https://doi.org/10.1007/s00299-026-03982-8

Image Credits: AI Generated

DOI: 10.1007/s00299-026-03982-8

Keywords: plant cell culture, Calendula officinalis, recombinant proteins, DsRed, brazzein, BY2 cells, Agrobacterium tumefaciens, LC-MS/MS, protease activity, suspension culture, plant biotechnology, Fraunhofer IME