The problem arises because many important plant families, from the mint family that includes rosemary to oaks, pines and countless daisies, package their seeds inside indehiscent fruits that do not open to reveal their contents. Researchers routinely treat these fruits as seeds, both for convenience and because it seems reasonable to assume that a plant investing resources in fruit tissues would fill them with a viable embryo. Yet seedless fruits are widespread across the plant kingdom, arising from self-incompatibility, male sterility, degraded pollen cells, chromosomal irregularities during meiosis and resource shortages. In most cases they are morphologically indistinguishable from seeded fruits, so there is no easy way to sort them out before an experiment begins.

Garcia-Fayos was drawn to the issue by a puzzling feature of the rosemary literature. Reviewing studies on the species’ germination, he found that in roughly 40 percent of published experiments, germination under controlled conditions never exceeded 10 percent, and reports of germination above 50 percent were rare. More striking still, the literature was riddled with contradictions. One study concluded that cold stratification boosts rosemary germination while another found no effect. Some forestry manuals recommend stratification while others dismiss it. Darkness reduced germination in some experiments but not others, gibberellic acid produced increases, decreases or nothing at all depending on the study, and even the effects of heat, smoke and soil chemistry were reported inconsistently. Madeiras and colleagues had already suggested in 2009 that these contradictions might stem from differences in the proportion of seeded fruits used across experiments, since rosemary produces many empty fruits after self-pollination, and that proportion varies between populations and years.

To test this idea rigorously, the researcher turned to experiments first conducted in 2004 with fruits collected from four large rosemary populations, each with more than 5,000 individuals, spread across the full gradient of annual temperature and rainfall that the species occupies in eastern Iberia. In each population, fruits were gathered from 15 adult plants separated by at least two metres, air-dried, shaken free from their calyxes and stored. The design crossed three alternating temperature regimes, 5 to 15, 10 to 20 and 15 to 25 degrees Celsius, with three soil water potential levels, 0.00, minus 0.01 and minus 0.05 megapascals, simulated using polyethylene glycol solutions calibrated to each thermal regime. A separate experiment crossed temperature with a light treatment comparing the natural alternating day-night cycle with continuous darkness, achieved by wrapping Petri dishes in aluminium foil.

The scale of the seedlessness problem became apparent only at the end of the experiments, when every ungerminated fruit was opened under a binocular microscope. Of 17,184 fruits collected across the four populations, just 17.2 percent contained a seed, defined as one that completely filled the fruit’s internal space with white or creamy-white, turgid tissue. Of the 8,955 fruits actually used in the germination trials, only 1,732, or 19.3 percent, held seeds. The consequences for the headline numbers were dramatic. Calculated the conventional way, over all fruits, germination was a meagre 11.2 percent. Counted only from seeded fruits, it leapt to 57.9 percent, a four- to seven-fold increase depending on the population. The ranking of the populations themselves changed, with La Murta leading at 78.7 percent germination of seeded fruits, followed by Pobla de Vallbona, Barranc dels Horts and Puebla de San Miguel.

The statistical consequences were even more consequential than the raw percentages. Using generalised linear mixed models fitted separately for each population, first with all fruits and then with seeded fruits only, Garcia-Fayos found that the apparent effects of environmental factors shifted in every population in the temperature and water potential experiment. When all fruits were included, water stress reduced germination in three of the four populations but temperature appeared to have no effect at all. When only seeded fruits were analysed, water potential emerged as a negative factor in all four populations, and warmer temperature regimes produced a clear positive response in three of them, a signal that had been completely masked by the empty fruits. In the temperature and light experiment, darkness appeared to suppress germination in three populations when all fruits were counted, but among seeded fruits this negative effect survived in only one, Puebla de San Miguel, while temperature and light otherwise lost their apparent influence entirely.

What makes the findings particularly sobering is that the remaining differences between populations could not be confidently attributed to local adaptation. If populations from hotter, drier sites had evolved seeds that germinate better at higher temperatures or tolerate drier soils, the seeded-fruit models should have shown effect sizes tracking the environmental gradient. They did not. The positive effect of temperature was indistinguishable between the populations from the warmest and coolest sites, and the effect of water potential was similar regardless of a population’s average rainfall. This fits with earlier population genetics work on the same species, which found high genetic diversity and weak population structure across the same gradient, driven by extensive gene flow through the shrub’s prolonged flowering and its generalist insect pollinators, conditions that hinder fine-scale local adaptation.

Instead, the residual inconsistencies most likely reflect a statistical artefact: the severe loss of replicates that occurs once empty fruits are removed. In the temperature and water potential experiment, only two populations retained 25 or more seeded fruits in every treatment combination, while Pobla de Vallbona fell below that threshold in eight of nine treatment levels. Given that 50 replicates per treatment level is often considered the minimum for statistically consistent results, nearly every treatment combination in both experiments was underpowered. The problem compounds if researchers, as is customary, treat Petri dishes rather than individual fruits as replicates. In this study, 30 of 180 dishes in the water potential experiment and 5 of 120 in the light experiment showed no germination at all, and 14 and 4 of those respectively contained no seeded fruits whatsoever, making the detection of any germination physically impossible.

The remedy Garcia-Fayos proposes is twofold. First, researchers should estimate the proportion of seeded fruits in their material before the experiment and increase the number of fruits sown per treatment level accordingly, so that the effective number of seeded replicates reaches a statistically meaningful target. Second, and indispensably, every ungerminated fruit should be opened and inspected at the end of the experiment so that calculations can be corrected to include only fruits that actually contained seeds. Neither step alone is sufficient; scaling up fruit numbers without verification leaves the denominator inflated, while verification without adequate starting numbers leaves the analysis starved of power. The approach echoes recommendations long voiced in the seed science literature, which has repeatedly warned that ignoring seed viability distorts germination ecology, yet the practice remains far from universal.

The implications stretch well beyond one aromatic shrub. Indehiscent, single-seeded fruits characterise a vast swath of the world’s flora, and the same blind spot affects not only germination experiments but also studies of seed rain, seed banks, predation and dispersal, where fruits rather than seeds are what researchers actually count. Ironically, seedlessness itself can be adaptive, since retaining aborted or inbred seeds until maturity may dilute the impact of pre-dispersal seed predators that lay eggs indiscriminately, a form of botanical decoy documented in junipers, pistachios and other groups. But whatever its evolutionary benefits, the new study makes clear that for the scientists trying to read the environmental rules of germination, empty fruits are noise masquerading as data. As research on endangered and rare species, where fruits and replicates are inherently scarce, becomes ever more urgent in a changing climate, the message is blunt: open the fruits, count the seeds, or risk publishing conclusions that a single overlooked emptiness could overturn.

Subject of Research: The effect of seedless fruits on germination experiments in plants with indehiscent single-seeded fruits

Article Title: Seedless fruits mask germination response to key environmental factors

Article References: Garcia-Fayos, P. (2026). Seedless fruits mask germination response to key environmental factors. Web Ecology, 26(1), 61-70. https://doi.org/10.5194/we-26-61-2026

Image Credits: AI Generated

DOI: 10.5194/we-26-61-2026

Keywords: seed germination, seedless fruits, Salvia rosmarinus, rosemary, plant ecology, experimental design, statistical power, water potential, temperature, light, Mediterranean shrubs, seed viability

The motivation behind the work is rooted in a growing set of problems with conventional sanitizers and disinfectants. Commercial germicidal products frequently rely on ethanol, isopropyl alcohol, and chemical preservatives, ingredients that the study’s authors note can cause skin dryness, irritation, burning, discoloration, dermatitis, and protein crosslinking with repeated use. Beyond the toll on human skin, there is an ecological dimension: disinfectants washed off floors and surfaces are entering sewage systems and polluting water bodies, placing aquatic ecosystems at risk. Perhaps most concerning, recent reports indicate that microorganisms are actively developing resistance to the chemicals used in sanitizers, echoing the well-known crisis of antibiotic resistance that has made pathogenic bacterial strains one of the defining public health challenges of the era.

Plant bioactive compounds have long been proposed as greener alternatives, and the researchers point out that they are less likely to induce resistance than many synthetic disinfectants. Rosemary is already known to contain rosmarinic acid, camphor, caffeic acid, ursolic acid, betulinic acid, carnosic acid, and carnosol, while hibiscus supplies saponins, flavonoids, and tannins, substances renowned for their membrane-disrupting and antimicrobial activities. Yet the team identified a gap in the literature: although antibacterial activity of plant extracts has been studied extensively, in silico analysis of plant bioactive compounds targeting specific bacterial proteins remains comparatively rare. Their study was designed to close that gap by pairing empirical assays with mechanistic computational predictions.

Methodologically, the team sourced rosemary leaves and hibiscus petals from Mumbai, Maharashtra, India, depositing voucher specimens for reproducibility. The plant material was ground into a fine powder and subjected to Soxhlet extraction with 70 percent ethanol, after which the extracts were concentrated by rotary evaporation under vacuum at a constant 40 degrees Celsius. Antibacterial activity was then measured using the agar well diffusion method against a formidable panel of eight pathogenic strains: Escherichia coli MTCC 2412, Staphylococcus aureus MTCC 2408, Salmonella species MTCC 4415, Shigella species MTCC 1457, Streptococcus pyogenes MTCC 442, Proteus vulgaris MTCC 426, Klebsiella pneumoniae MTCC 2716, and Bacillus subtilis MTCC 2010. Hydrogen peroxide served as the positive control and sterile saline as the negative control, providing benchmarks for comparison.

The results of the diffusion assays were striking, particularly for rosemary. Rosemary extract produced zones of inhibition ranging from 17.66 to 26.33 millimeters across the tested strains, while hibiscus extract produced zones ranging from 11.33 to 18.33 millimeters. Statistical analysis using one-way ANOVA followed by Tukey’s post hoc test in GraphPad Prism, with significance set at P less than 0.05, confirmed that the differences were meaningful. For rosemary, the analysis showed highly significant variation in activity across organisms (P less than 0.0001), with S. aureus emerging as the most sensitive strain, followed by moderate activity against S. pyogenes and Bacillus, while E. coli, Salmonella, Shigella, Proteus, and K. pneumoniae showed minimal or no inhibition. Hibiscus, by contrast, showed its strongest and most consistent effect against Salmonella, Shigella, S. pyogenes, Proteus, and Bacillus, with significantly lower inhibition of E. coli, S. aureus, and K. pneumoniae.

Minimum inhibitory concentrations told a complementary story about potency. Rosemary extract was effective at concentrations between 1.5 and 12.5 milligrams per milliliter, whereas hibiscus extract required 25 to 50 milligrams per milliliter to suppress bacterial growth. These values were determined using a resazurin dye reduction assay, in which the dye changes color as metabolically active bacteria reduce it, allowing growth inhibition to be tracked visually. The team also conducted preliminary phytochemical screening: rosemary revealed the presence of alkaloids, flavonoids, steroids, tannins, and saponins, while hibiscus contained alkaloids, flavonoids, glycosides, steroids, terpenoids, tannins, and saponins, a chemical arsenal consistent with the observed antibacterial effects.

To identify the specific molecules responsible, the researchers turned to gas chromatography-mass spectrometry using a Shimadzu GCMS-QP2010 Ultra instrument. The rosemary fingerprint profile revealed compounds including carbamic acid monoammonium salt, 1,2-dimethyl-3-nitrobenzene, n-hexadecanoic acid, 3-pentadecylphenol, 1,5,9,13-tetradecatetraene, tricyclo[20.8.0.0(7,16)]triacontane,1(22),7(16)-diepoxy, and 7-hexadecenal. Hibiscus yielded n-hexadecanoic acid, cis-vaccenic acid, 3-pentadecylphenol, 1,5,9,13-tetradecatetraene, bi-1-cycloocten-1-yl, alpha-santalol, and 1,3-diphenyl-1,2-butanediol. These molecular inventories then became the input for the computational phase of the study, in which AutoDock Vina, MGL Tools, and the PyMOL visualization tool were used to dock each compound against bacterial proteins selected because they are essential for cell survival or are established antibiotic targets.

The docking results provided the study’s headline finding. Tricyclo[20.8.0.0(7,16)]triacontane,1(22),7(16)-diepoxy from rosemary achieved a binding energy of minus 11.0 kilocalories per mole against the S. aureus protein (PDB: 3VSL), forming hydrogen bonds with Thr619, Thr621, Ser448, Glu623, Ser392, and Gln524 in Chain A, and with Thr621, Thr603, Ser392, and Glu623 in Chain B. The authors note that similar interactions have been observed with cefotaxime, a third-generation cephalosporin antibiotic. Meanwhile, 1,3-diphenyl-1,2-butanediol from hibiscus scored minus 7.5 kilocalories per mole against the K. pneumoniae protein (PDB: 2OV5), interacting with residues across three separate protein chains. Remarkably, these phytochemicals matched or exceeded the binding energies of the standard antibiotic meropenem, which ranged from minus 5.3 to minus 7.6 kilocalories per mole in comparative docking. Even more intriguingly, both lead compounds were relatively minor constituents of their extracts by GC-MS abundance, a phenomenon the authors note has been observed in earlier studies of essential oils, where trace compounds can exert outsized antimicrobial influence.

Before any compound can be considered a viable ingredient, however, it must pass pharmacological scrutiny. The team ran ADMET analysis, assessing absorption, distribution, metabolism, excretion, and toxicity, using the SwissADME platform. Both tricyclo[20.8.0.0(7,16)]triacontane,1(22),7(16)-diepoxy and bi-1-cycloocten-1-yl violated one of Lipinski’s rules of drug-likeness, but were deemed acceptable candidates on the strength of their bioavailability, with a bioavailability score of 0.55 indicating good absorption and distribution. The remaining bioactive compounds passed the ADMET filters cleanly, suggesting that the chemical repertoire of these two plants is not only antibacterial in principle but plausibly compatible with formulation into real products.

The broader implications reach into green chemistry and sustainable healthcare. Because plant-based bioactive compounds kill or inhibit microbes by destabilizing microbial membranes, disrupting proteins, and interfering with metabolic activities, they can serve as natural alternatives to alcohol in sanitizer formulations, which are typically built on gel or spray bases. Unlike alcohol, which dries and irritates the skin with regular use, plant-based formulations are described as gentler on both skin and environment, and their wide-spectrum activity makes them attractive for everyday hand hygiene. With recurring pandemics and epidemics underscoring the urgency of new antimicrobials, the identification of two lead candidates with antibiotic-comparable binding affinities gives the humble rosemary sprig and hibiscus bloom a new scientific pedigree, and offers formulators a concrete, evidence-backed starting point for alcohol-free germicidal products.

Subject of Research: Antimicrobial activity and molecular docking analysis of rosemary and hibiscus bioactive compounds for germicidal product development

Article Title: Exploring antimicrobial potency and in silico analysis of rosemary and hibiscus bioactive compounds to be used in germicidal product

Article References: Shedge, V., & Sambrani, S. (2025). Exploring antimicrobial potency and in silico analysis of rosemary and hibiscus bioactive compounds to be used in germicidal product. Discover Biotechnology, 2(1), Article 20. https://doi.org/10.1007/s44340-025-00028-9

Image Credits: AI Generated

DOI: 10.1007/s44340-025-00028-9

Keywords: rosemary, hibiscus, antimicrobial activity, molecular docking, bioactive compounds, plant extracts, hand sanitizer, antibiotic resistance, ADMET analysis, GC-MS, green chemistry, Staphylococcus aureus