Essential oil chemotype diversity and environmental stability in Turkish basil (Ocimum basilicum L.) germplasm: a three-layer field evaluation across contrasting ecologies

Essential oil chemotype diversity and environmental stability in Turkish basil (Ocimum basilicum L.) germplasm: a three-layer field evaluation across contrasting ecologies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Essential oil chemotype diversity and environmental stability in Turkish basil (Ocimum basilicum L.) germplasm: a three-layer field evaluation across contrasting ecologies Adem Zorlu, İsa Telci, Mahfuz Elmastaş, Oya Kaçar, Zehra Aytaç, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9131432/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract The environmental stability of essential oil (EO) chemotype expression in basil (Ocimum basilicum L.) is critical for both industrial quality assurance and food safety, yet remains poorly characterised across the multiple dimensions of environmental variation encountered in production. This study characterises the environmental modulation of EO yield and chemotype composition in 12 basil genotypes representing four chemotypes (linalool, estragole, citral, and methyl cinnamate) using a three-layer experimental design: (i) ecological variation across three contrasting Turkish locations (Bursa, Eskişehir, Tokat) over two years, (ii) ontogenetic variation across three developmental stages at Bursa, and (iii) seasonal variation between summer and autumn harvests at Tokat (144 unique EO profiles). EO yield was highest at Bursa (0.98 mL·100 g⁻¹ dry weight), increased from pre-flowering to onset of flowering, and was greater in autumn than summer harvests. The estragole chemotype (R-10A) exhibited exceptional compositional stability across ecological environments (Layer 1 CV = 2.8%), while the citral (Layer 1 CV = 12.9%) and methyl cinnamate (20.0%) chemotypes showed moderate to variable stability. Within the linalool chemotype, R-16 was the most stable genotype (SD = 5.6%). The results indicate that chemotype stability is primarily genotype-driven and that environment × harvest-stage interactions modulate secondary metabolite accumulation patterns in a chemotype-dependent manner, with implications for cultivar selection and food safety considerations. Chemotype stability Essential oil Genotype × environment interaction Ocimum basilicum Secondary metabolite Terpene biosynthesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Sweet basil (Ocimum basilicum L., Lamiaceae) is one of the most commercially important aromatic herbs worldwide, valued for its culinary, medicinal, cosmetic, and industrial applications (Makri and Kintzios 2008 ; Dhama et al. 2021 ). The species exhibits remarkable intraspecific diversity in both morphological traits and secondary metabolite profiles (Labra et al. 2004 ; De Masi et al. 2006 ), and its botany, phytochemistry, and pharmacological properties have been comprehensively reviewed (Dhama et al. 2021 ; Azizah et al. 2023 ). Essential oil (EO) composition is the primary criterion for commercial classification of basil, as it determines both aroma characteristics and biological activity (Hussain et al. 2008 ). Several chemotype classification systems have been proposed for O. basilicum based on the dominant EO constituents. Grayer et al. ( 1996 ) distinguished five major EO profiles based on the proportions of linalool, estragole, eugenol, methyl eugenol, and geraniol. Working with Turkish landraces, Telci et al. ( 2006 ) identified seven chemotypes from 18 accessions, expanding the known chemical diversity of Anatolian basil populations. Subsequent molecular and chemometric studies of Turkish germplasm confirmed this intraspecific diversity (Giachino et al. 2014 ). More recently, multivariate approaches have become standard for chemotype classification (Kirci et al. 2025 ), enabling finer resolution of compositional variation across germplasm collections. The chemodiversity of Ocimum species has been comprehensively reviewed by Gurav et al. ( 2022 ) and Dhama et al. ( 2021 ). While the genetic component of EO composition is well established, the extent to which environmental factors modulate chemotype expression remains inadequately characterised, particularly in multi-location, multi-year field trials. The genotype × environment (G × E) interaction for EO traits has been documented in several aromatic species including Origanum vulgare (Lukas et al. 2015 ) and Salvia species (Ferrante et al. 2021 ), but comprehensive G × E studies in basil remain limited. Understanding EO chemotype stability is essential for growers, breeders, and the industry, ensuring reliable raw material quality (Pimentel et al. 2023 ). Beyond spatial (ecological) variation, temporal factors also influence EO accumulation and composition. Ontogenetic variation in terpene and phenylpropanoid accumulation has been reported in basil (Eskandarzade et al. 2024 ; Beatović et al. 2015 ) and other Lamiaceae (Iijima et al. 2004a ). EO yield generally increases from vegetative to flowering stages, while compound proportions may shift in parallel with changes in terpene synthase activity during glandular trichome maturation (Iijima et al. 2004a ). Recent studies confirm that developmental stage is a critical determinant of EO quality at harvest (Kholiya et al. 2022 ; Mkaddem Mounira et al. 2022 ). In regions where multiple harvests per season are feasible, the harvest season can alter EO yield and composition due to changing photoperiod, temperature, and precipitation (Pimentel et al. 2023 ; Corrado et al. 2020 ). A further practical dimension concerns food safety. Estragole (methyl chavicol) and methyl eugenol are phenylpropanoids classified as naturally occurring genotoxic and carcinogenic (in rodent models) substances by the European Medicines Agency (EMA 2023) and the Scientific Committee on Food (SCF 2001a, 2001b). Regulation (EC) No. 1334/2008 restricts these substances in foodstuffs and flavourings. Understanding how their proportions vary with genotype, location, and developmental stage is directly relevant to food safety risk assessment, particularly as basil is widely used as a fresh herb and flavouring agent in the food industry (Hamid et al. 2024 ; Hallmann and Ponder 2024 ). The authentication of basil products with respect to genotoxic phenylpropanoids has been recognised as an emerging food safety priority (Ríos-Rodríguez et al. 2021 ; Mahendran and Vimolmangkang 2023 ). EFSA opinions on estragole (EFSA CEF Panel 2012 ) and methyl eugenol (EFSA CEF Panel 2009 ) provide the regulatory genotoxicity framework, while basil matrix effects on estragole bioactivation have also been documented (Jeurissen et al. 2008 ). Turkey harbours considerable basil genetic diversity in locally cultivated populations known as "reyhan" (Grayer et al. 1996 ). Despite this potential, Turkish basil remains insufficiently characterised regarding EO stability across environments. To the best of our knowledge, no study has simultaneously evaluated the ecological, ontogenetic, and seasonal variation of basil EO in a multi-year, multi-location design within Turkey. The present study was designed to characterise the environmental modulation of EO yield and chemotype composition in 12 basil genotypes representing four chemotypes, using a three-layer experimental approach. Unlike standard genotype × environment interaction studies that examine EO variation across a single experimental dimension (typically location or harvest time), the three-layer design simultaneously addresses ecological, ontogenetic and seasonal variation within the same genotype panel, enabling integrated cultivar recommendations that account for all three sources of compositional instability. The three layers are: (i) ecological variation across three locations; (ii) ontogenetic variation across three developmental stages; and (iii) seasonal variation between summer and autumn harvests (Fig. 1 ). The specific objectives were to quantify G × E interactions for EO yield and major compounds, to assess chemotype stability using CV analysis, and to evaluate the implications of compositional variation for food safety. Materials and methods Plant material The plant material used in this study was derived from a basil germplasm collection established within a nationally funded research project (TÜBİTAK Project No. 111O677) based on local populations collected from different regions of Türkiye, together with a limited number of introduced genotypes. After preliminary characterisation of 70 accessions, 12 genotypes were selected based on prior chemotype characterisation (Kirci et al. 2025 ) to represent the four dominant chemotypes (linalool, estragole, citral, and methyl cinnamate) and a wide essential-oil yield range while ensuring suitability for multi-location field trials. Taxonomic identity as Ocimum basilicum L. was confirmed by Prof. Dr. İsa Telci, Department of Industrial Crops, Isparta University of Applied Sciences, Türkiye. The evaluated materials are maintained as documented accessions in the institutional medicinal and aromatic plants collection, and accession records are available for verification upon request. Accession origins, chemotype assignment and collection details are provided in Online Resource 1 (Table S2). Experimental design and trial locations Field trials were conducted at three ecologically contrasting locations in Turkey over two consecutive years (Year 1 and Year 2) (Online Resource 1, Table S3). The trial locations – Bursa (40°13′N, 28°51′E, 120 m a.s.l.), Eskişehir (39°45′N, 30°33′E, 789 m a.s.l.), and Tokat (40°19′N, 36°27′E, 594 m a.s.l.) – represent sub-Mediterranean, continental transition, and semi-arid continental climates, respectively. Growing-season (May–September) mean temperature, total precipitation, and mean relative humidity were 21.5°C, 198–425 mm, and 61–72% at Bursa; 18.1°C, 130–258 mm, and 55–67% at Eskişehir; and 18.5–20.9°C, 156–187 mm, and 50–55% at Tokat, respectively, across the two experimental years (Online Resource 1, Table S3). Soil at each site was classified as clay-loam (Bursa), silty-clay (Eskişehir), and sandy-loam (Tokat). Monthly climatic data are provided in Online Resource 1, Table S3. Seedlings were produced from seed in perlite–peat mixture (5:1, v/v) and transplanted to each location after the last spring frost. The experiment was arranged in a randomised complete block design (RCBD) with three replications. Plant spacing was 40 × 30 cm. Prior to transplanting, 25 kg ha⁻¹ pure nitrogen (half of the total 50 kg ha⁻¹) and 50 kg ha⁻¹ triple superphosphate were applied. Drip irrigation was provided as needed. Three-layer experimental structure Layer 1 – Ecological variation: All 12 genotypes were harvested at the onset of flowering at each of the three locations in both years, yielding 12 genotypes × 3 locations × 2 years = 72 EO profiles. Note: the onset-of-flowering harvest at Bursa in Layer 1 is shared with the onset-of-flowering (OF) stage in Layer 2, and the first harvest at Tokat in Layer 1 is shared with the summer harvest in Layer 3; the total number of unique EO profiles is 144, while the total number of layer-specific observations is 192. Because Layers 2 and 3 used subsets of the Layer 1 dataset, results across layers should be interpreted as complementary rather than statistically independent; ontogenetic conclusions are specific to Bursa and seasonal conclusions are specific to Tokat within the tested years. Layer 2 – Ontogenetic variation: At the Bursa location, plant material was sampled at three developmental stages in both years: (i) pre-flowering (PF, vegetative), (ii) onset of flowering (OF, 10–15% open flowers), and (iii) full flowering (FF, > 50% open flowers), yielding 72 EO profiles. Layer 3 – Seasonal variation: At the Tokat location, two harvests were taken per growing season in both years: a summer harvest (first cut, June–July) and an autumn harvest (second cut, August–September), yielding 48 EO profiles. Essential oil extraction EO was extracted from dried leaf material, consistent with established practice in multi-genotype basil EO evaluation where simultaneous processing of numerous samples precludes immediate distillation of fresh tissue (Beatović et al. 2015 ; Kholiya et al. 2022 ). Leaf samples were dried in a forced-air oven at 35°C to constant weight (approximately 5–7 days), protected from direct light; this low-temperature drying protocol minimises loss of volatile monoterpenes while enabling standardised sample preparation across locations and harvest dates. Dried leaves were stored in sealed dark-glass containers at ambient temperature until distillation, within two weeks of drying to minimise monoterpene loss. Fifteen grams of dried leaf were placed in a round-bottom flask with 300 mL distilled water (1:20, w/v) and subjected to hydrodistillation for 2 h using a Neo-Clevenger-type apparatus following the European Pharmacopoeia protocol (European Pharmacopoeia Commission 2010 ). EO yield was expressed as mL·100 g⁻¹ dry weight (DW), numerically equivalent to % v/w. EO volume was read at ambient temperature after complete phase separation in the graduated Clevenger tube. Gas chromatography and gas chromatography–mass spectrometry GC analysis was performed on a Perkin-Elmer (PerkinElmer, Waltham, MA, USA) gas chromatograph equipped with an autosampler and flame ionisation detector (FID). Samples were diluted 1:10 in acetone and separated on a BPX5 capillary column (SGE Analytical Science, Melbourne, Australia; 30 m × 0.25 mm i.d. × 0.25 µm). Peaks were integrated using PerkinElmer TotalChrom software with automatic baseline detection and manual correction where necessary; integration settings were applied uniformly across all samples. Helium carrier gas at 5 psi; injector 230°C; detector 250°C; oven programme: initial 60°C (hold 2 min), ramped at 3°C·min⁻¹ to 230°C (hold 15 min); total run time 73.7 min; split injection 5:1. Quantitative data are expressed as relative FID peak area (%). Response factor correction was not applied; accordingly, all compositional values represent relative proportions rather than absolute mass concentrations. For the dominant compounds verified against authentic standards (linalool, estragole, methyl eugenol, methyl cinnamate, eugenol, geraniol), the relative FID response approximates mass proportions within ± 5% for the monoterpene and phenylpropanoid classes studied (Amor et al. 2021 ; Šovljanski et al. 2022 ). This approach is standard in multi-genotype EO characterisation (Amor et al. 2021 ; Šovljanski et al. 2022 ; Gurkan and Hayaloglu 2023 ; Abdoul-Latif et al. 2022 ). The BPX5 column was selected for broad-spectrum separation across four diverse chemotypes; while it has limited selectivity for co-eluting phenylpropanoid isomers, this does not affect the findings given the unambiguous dominance of identified compounds in each chemotype. Co-elution of estragole with trans-anethole was checked by RI comparison (estragole RI = 1195 vs. trans-anethole RI = 1283 on BPX5) and confirmed absent in all samples. The FID quantification approach, applied consistently across all 144 unique EO profiles in the present study, does not affect between-genotype or between-environment comparisons. Gas chromatography–mass spectrometry (GC-MS) analysis used a PerkinElmer GC-MS system (PerkinElmer, Waltham, MA, USA) (electron ionisation at 70 eV), same column and temperature programme, helium at 1.3 mL·min⁻¹, transfer line 250°C. Compound identification was based on: (i) comparison with authentic reference standards (linalool, estragole, methyl eugenol, methyl cinnamate, eugenol, geraniol, and α-terpineol; Sigma-Aldrich, St. Louis, MO, USA; ≥98% purity); (ii) retention index (RI) calculation relative to a homologous n-alkane series (C₈–C₂₀); and (iii) mass spectral matching against Wiley and NIST (National Institute of Standards and Technology, Gaithersburg, MD, USA) mass spectral databases (minimum forward match score ≥ 850/1000) and published data (Adams 2017 ). Compounds present at less than 0.1% relative peak area were not included in quantitative reporting; this threshold corresponds to approximately 3× the mean injection-to-injection variability (RSD < 3%), ensuring only reliably quantifiable peaks are reported. Retention indices (RI) for all identified compounds are provided in Online Resource 1 (Table S4). Identification was assigned based on agreement between RI (typically within ± 10 units) and MS library matching; dominant compounds were additionally confirmed using authentic standards where available (Adams 2017 ; Jiang et al. 2024 ). Statistical analysis Sampling and analytical replication. At each harvest, 10–15 plants were sampled randomly from each replication block. Leaf material from each replicate block was pooled, mixed, and a 15 g subsample was taken for hydrodistillation. This pooling eliminates within-plot (plant-to-plant) variation; genotypes with heterogeneous individual-plant EO profiles would appear artificially stable from pooled samples. Consequently, the CV-based stability estimates reported here reflect between-environment variation only and may underestimate true total variation, which would include the within-plot component. Each replicate was distilled and analysed separately, yielding three independent analytical observations per genotype per environment. GC-FID analysis involved a single injection per sample; peak area reproducibility was confirmed by duplicate injection of a random subset (n = 12 samples), with a mean relative standard deviation below 3%. Mean ± standard deviation (SD) values reported in tables and figures are based on these three replicate observations within each environment–year combination, except where stated otherwise. Sample processing (drying, distillation, and GC analysis) was randomised across locations and years within each analytical batch to minimise systematic confounding of batch effects with environmental effects. Analysis of variance (ANOVA) was performed separately for each experimental layer. In Layer 1, genotype (G), location (L) and year (Yr) were included as fixed factors, and all two- and three-way interactions (G×L, G×Yr, L×Yr and G×L×Yr) were tested. Replications were nested within each location × year combination. Year was treated as a fixed effect because the two experimental years represent the specific study period rather than a random sample; thus, inference applies to Year 1–Year 2 conditions (Online Resource 1, Table S3), and extrapolation to other years should be made cautiously. Analogous models were fitted for Layers 2 and 3. Mean separation was performed using Duncan's multiple range test at P ≤ 0.01 (Eberhart and Russell 1966 ). Key conclusions were verified using Tukey's HSD (α = 0.05) with consistent outcomes (Online Resource 1, Table S1 ). EO composition data are presented as relative FID peak area proportions (%, normalised to total detected peak area). Because inference focused on single dominant compounds constituting more than 30% of total EO (linalool, estragole, citral, or methyl cinnamate), the summation constraint has minimal distorting effect on individual variances. Analyses were nevertheless repeated after arcsine square-root transformation; rankings and significance conclusions were unchanged. Untransformed values are therefore reported throughout for ease of interpretation, consistent with established practice in multi-genotype basil EO studies. Duncan's multiple range test was selected because the primary objective was genotypic discrimination within the fixed set of 12 tested genotypes, rather than control of familywise error across a large set of random comparisons. This choice is consistent with established practice in aromatic crop genotype evaluation. As a robustness check, all pairwise conclusions (e.g., Bursa vs. Eskişehir and Tokat for EO yield; R-10A estragole stability vs. R-17 citral; R-16 and R-20 linalool stability rankings) were verified using Tukey's HSD at P ≤ 0.01 and remained unchanged (i.e., no genotype pair that was significantly different under Duncan became non-significant under Tukey, confirming that the less conservative Duncan approach did not inflate Type I errors in this dataset). All analyses were performed using SAS 9.4 (PROC GLM with MEANS statement; SAS Institute, Cary, NC, USA). ANOVA F-ratios, degrees of freedom, and probability values for main effects and interactions across all three experimental layers are provided as Online Resource 1 (Table S1 ). Chemotype stability was quantified by the CV (%) of the dominant compound across three locations and two years (Layer 1 – ecological variation; n = 6 environment–year combinations per genotype), following the multi-environment crop evaluation convention (Zobel et al. 1988 ): CV < 10% = highly stable; 10–20% = moderately stable; CV ≥ 20% = variable. Results Essential oil yield Ecological variation (Layer 1) EO yield varied significantly (P ≤ 0.01) among genotypes, locations, and years, and the genotype × location interaction was significant (P ≤ 0.01), indicating differential genotypic responses to environmental conditions. The three-way G×L×Yr interaction was also significant (P ≤ 0.05), though with modest statistical power (df = 22, 120); accordingly, interpretation focuses on main effects and two-way interactions (Table 1 ; Fig. 2 ). Across all genotypes and years, Bursa produced the highest mean EO yield (0.98 mL·100 g⁻¹ DW), followed by Eskişehir (0.74) and Tokat (0.72). Year 2 produced significantly higher yields than Year 1 (0.88 vs. 0.75 mL·100 g⁻¹ DW; P ≤ 0.01). Table 1 Essential oil (EO) yield (mL·100 g⁻¹ dry weight) of 12 basil genotypes across three locations over two years (Layer 1 - ecological variation). Genotype Bursa Year 1 Bursa Year 2 Eskişehir Year 1 Eskişehir Year 2 Tokat Year 1 Tokat Year 2 Mean R-1 0.89 0.82 0.6 0.88 0.51 0.62 0.72 efg R-3k 0.67 1.37 0.82 0.73 0.66 1.11 0.89 d R-4 0.53 0.86 0.66 0.67 0.69 0.62 0.67 fg R-10A 1.58 1.61 1.13 1.04 1.03 1.16 1.26 b R-15 1.1 1.58 1.22 0.95 0.82 1.13 1.13 bc R-16 0.67 1.08 0.55 0.47 0.36 0.53 0.61 gh R-17 0.87 1.1 0.82 0.87 0.82 0.82 0.88 d R-19 1.11 1.01 0.82 0.8 0.69 0.73 0.86 de R-20 0.58 0.67 0.54 0.43 0.36 0.47 0.51 h R-23 1.29 1.71 1.23 1.17 0.91 1.67 1.33 a Y-7 0.42 0.49 0.22 0.36 0.18 0.42 0.35 i Y-15 0.73 0.7 0.32 0.4 0.44 0.51 0.52 h Among genotypes, R-23 had the highest two-year mean EO yield (1.33 mL·100 g⁻¹ DW), followed by R-10A (1.26) and R-15 (1.13), while Y-7 had the lowest (0.35). The genotype × location interaction was particularly notable for R-10A, R-15, and R-23, which showed markedly higher yields at Bursa. Genotype R-4 maintained relatively stable yields across all three sites (range: 0.53–0.86 mL·100 g⁻¹ DW), suggesting high environmental stability for yield. These findings are consistent with multi-location yield stability analyses reported by Kholiya et al. ( 2022 ) and Tursun and Telci ( 2020 ). Ontogenetic variation (Layer 2) Developmental stage had a significant effect (P ≤ 0.01) on EO yield (Table 2 ; Fig. 3 ). Pre-flowering plants produced the lowest mean yield (0.78 mL·100 g⁻¹ DW), while onset of flowering and full-flowering stages produced similar and significantly higher values (0.98 and 1.00, respectively). Most genotypes followed the general trend of increasing EO yield with advancing maturity, consistent with the role of glandular trichome development in EO accumulation (Gang et al. 2001 ), and aligning with recent studies by Mkaddem Mounira et al. ( 2022 ) and Corrado et al. ( 2020 ). However, R-10A reached its maximum yield at onset of flowering rather than full flowering (1.58–1.61 vs. 1.38–1.51 mL·100 g⁻¹ DW in Year 1 and Year 2, respectively), which may reflect the earlier peak accumulation of phenylpropanoid-dominated EOs reported in basil (Iijima et al. 2004a ). Table 2 Essential oil (EO) yield (mL·100 g⁻¹ dry weight) of 12 basil genotypes across three developmental stages at Bursa (Layer 2 - ontogenetic variation). Genotype PF Year 1 PF Year 2 OF Year 1 OF Year 2 FF Year 1 FF Year 2 CV (%) Stage mean R-1 0.69 0.73 0.89 0.82 1.02 0.77 14.7 0.82 R-3k 0.93 0.83 0.67 1.37 1 1.04 24.2 0.97 R-4 0.78 0.61 0.53 0.86 0.96 0.69 21.7 0.74 R-10A 1.02 1.27 1.58 1.61 1.51 1.38 16.0 1.40 R-15 1.32 1.2 1.1 1.58 1.71 1.61 17.4 1.42 R-16 0.74 0.49 0.67 1.08 0.73 0.76 25.7 0.74 R-17 1.02 0.53 0.87 1.1 0.94 1.09 23.0 0.92 R-19 0.94 0.67 1.11 1.01 1.13 0.73 20.7 0.93 R-20 0.44 0.53 0.58 0.67 0.69 0.57 15.9 0.58 R-23 1.18 1.01 1.29 1.71 1.53 1.76 21.3 1.41 Y-7 0.29 0.37 0.42 0.49 0.4 0.49 18.6 0.41 Y-15 0.64 0.37 0.73 0.7 1 0.38 37.3 0.64 Seasonal variation (Layer 3) Autumn harvests produced significantly higher EO yields than summer harvests in both years (0.72 vs. 0.58 mL·100 g⁻¹ DW; P ≤ 0.01; Table 3 ). The year × season interaction was significant, with the difference more pronounced in Year 2 (0.82 vs. 0.58) than in Year 1 (0.62 vs. 0.57). All genotypes produced higher EO yields in autumn, supporting the value of double-cropping strategies reported by Corrado et al. ( 2020 ) and Ciriello et al. ( 2021 ). R-23 showed the largest seasonal difference, while R-17 was relatively stable across seasons. Table 3 Essential oil (EO) yield (mL·100 g⁻¹ dry weight) of 12 basil genotypes across two harvest seasons at Tokat (Layer 3 - seasonal variation). Genotype Summer Year 1 Summer Year 2 Autumn Year 1 Autumn Year 2 Summer mean Autumn mean CV (%) R-1 0.56 0.49 0.51 0.62 0.53 0.56 10.6 R-3k 0.47 0.69 0.66 1.11 0.58 0.89 36.8 R-4 0.47 0.6 0.69 0.62 0.53 0.66 15.4 R-10A 1.02 1.02 1.03 1.16 1.02 1.09 6.5 R-15 0.78 0.73 0.82 1.13 0.76 0.97 20.9 R-16 0.36 0.36 0.37 0.53 0.36 0.45 20.6 R-17 0.82 0.8 0.82 0.82 0.81 0.82 1.2 R-19 0.62 0.49 0.69 0.73 0.55 0.71 16.7 R-20 0.2 0.33 0.36 0.47 0.27 0.41 32.7 R-23 0.96 0.76 0.91 1.67 0.86 1.29 37.7 Y-7 0.14 0.36 0.18 0.42 0.25 0.30 49.5 Y-15 0.49 0.31 0.44 0.51 0.40 0.47 20.6 Essential oil composition Chemotype overview GC-MS analysis confirmed four distinct chemotypes in the evaluated genotypes, with Layer 1 compositional summaries presented in Table 4 : (i) linalool chemotype (nine genotypes: R-1, R-3k, R-4, R-15, R-16, R-19, R-20, Y-7, Y-15; note: R-4 is classified as linalool-type based on its dominant compound but uniquely co-accumulates methyl eugenol at 9.33%) with linalool ranging from 51.7 to 71.2% (grand means across six environment–year combinations; Table 4 ); (ii) estragole chemotype (R-10A) with estragole at 93.5%; (iii) citral chemotype (R-17; citral reported as the sum of its geometric isomers neral [citral b] and geranial [citral a]) with citral (geranial + neral) at 75%; and (iv) methyl cinnamate chemotype (R-23) with methyl cinnamate at 43.1% and linalool as the major secondary constituent at 34.9%. Other notable secondary compounds included eugenol (up to 13.7% in Y-7), α-bergamotene (up to 8.37% in R-19), and δ-cadinene (up to 8.45% in R-20). This chemotypic diversity mirrors the broad chemical variation documented by Gurkan and Hayaloglu ( 2023 ) and Bajomo et al. ( 2022 ) for commercially available basil varieties, and is consistent with the volatile profiles reported by Qasem et al. ( 2023 ). Table 4 Major essential oil (EO) compound relative proportion (%, uncorrected FID peak area) in basil genotypes across three locations and two years (mean ± standard deviation (SD) across six environment-year combinations; Layer 1 - ecological variation). Chemotype Genotype Bursa Year 1 Eskişehir Year 1 Tokat Year 1 Bursa Year 2 Eskişehir Year 2 Tokat Year 2 Mean ± SD Linalool R-1 49.07 59.56 77.83 56.77 56.03 56.30 59.30 ± 9.7 R-3k 62.34 66.40 80.93 54.03 56.59 66.28 64.40 ± 9.5 R-4 50.53 34.49 88.43 48.85 53.57 55.09 55.20 ± 17.9 R-15 35.51 63.43 85.34 67.96 66.51 62.24 63.50 ± 16.1 R-16 66.21 72.86 79.89 64.54 69.75 73.72 71.20 ± 5.6 R-19 50.95 53.88 73.66 47.81 51.65 55.16 55.50 ± 9.2 R-20 75.79 69.76 82.96 62.13 59.45 73.54 70.60 ± 8.8 Y-7 43.91 68.90 60.85 44.18 44.90 50.12 52.10 ± 10.4 Y-15 50.55 53.79 61.81 50.87 46.97 46.22 51.70 ± 5.7 Estragole R-10A 91.71 94.94 98.13 92.56 92.21 91.34 93.50 ± 2.6 Citral R-17 77.31 74.42 89.54 70.57 60.12 77.77 75.00 ± 9.7 Methyl cinnamate R-23 40.45 51.96 31.31 35.45 49.55 50.05 43.10 ± 8.6 Methyl eugenol R-4† 8.97 9.14 9.52 9.67 9.28 9.41 9.33 ± 0.26 Linalool chemotype – environmental variation Linalool content in the nine linalool-type genotypes varied considerably across environments (Fig. 5 ). In Layer 1, the mean linalool content was highest at Tokat (Year 1: 76.86%, Year 2: 59.85%), followed by Eskişehir and Bursa. This inverse relationship between EO yield and linalool proportion may reflect a dilution effect, wherein higher total EO production at warmer sites is accompanied by proportionally greater accumulation of minor terpenes (Marotti et al. 1996 ), consistent with findings of Tursun and Telci ( 2020 ). Individual genotypes differed markedly in linalool stability. R-16 and R-20 maintained relatively high and stable linalool proportions across all three locations (R-16: 64.54–79.89%; R-20: 59.45–82.96%). In contrast, R-4 showed the greatest location-dependent variation (34.49–88.43%), indicating high G × E interaction for this trait. In Layer 2 (ontogenetic variation), linalool content showed a general decline from pre-flowering to full flowering (Year 1 means: 57.23%, 55.38%, 52.06%), consistent with findings of Eskandarzade et al. ( 2024 ) and Beatović et al. ( 2015 ). The phenolic composition and maturity interactions reported by Lawson et al. ( 2025 ) further support the role of developmental stage in secondary metabolite accumulation. In Layer 3 (seasonal), linalool content was generally similar between summer and autumn harvests (Fig. 4 ), suggesting strong genetic control over linalool biosynthesis. Estragole chemotype – environmental stability Genotype R-10A maintained remarkably high and stable estragole proportions across all experimental layers (Fig. 6 a). In Layer 1, estragole ranged from 91.34 to 98.13% (Year 1 vs. Year 2 values shown in Table 4 ). In Layer 2, estragole ranged from 86.8 to 94.47%. In Layer 3, estragole ranged from 88.62 to 98.72%. The Layer 1 CV was 2.8%, confirming exceptional compositional stability consistent with the strong genetic determination of the estragole chemotype reported by Telci et al. ( 2006 ) and corroborated by recent chemotyping studies (Kirci et al. 2025 ; Bajomo et al. 2022 ). Citral chemotype – environmental sensitivity The citral chemotype (R-17) showed substantial sensitivity across experimental layers, particularly in ontogenetic responses (Fig. 6 b), although its Layer 1 CV (12.9%) indicated only moderate ecological instability. Citral content ranged from 60.12 to 89.54% in Layer 1 and from 37.17 to 78.16% in Layer 2, with dramatic year-to-year fluctuation in the pre-flowering stage (Year 1: 78.16% vs. Year 2: 37.17%). The Layer 1 CV was 12.9% (moderately stable), although inclusion of ontogenetic data substantially increased overall variability. This instability may be related to the environmental sensitivity of geraniol dehydrogenase, which catalyses the oxidation of geraniol to citral (Iijima et al. 2004b ), and is consistent with the broad compositional variation observed for citral-type basil accessions by Avetisyan et al. ( 2017 ). The methyl cinnamate chemotype (R-23) exhibited variable stability at the classification threshold (Layer 1 CV = 20.0%; Fig. 6 c; Table 5 ), consistent with environmental sensitivity of methyl cinnamate accumulation in Lamiaceae (Telci et al. 2006 ). Table 5 Chemotype stability assessment based on the coefficient of variation (CV%) of the dominant compound in Layer 1 (ecological variation; three locations × two years) for basil genotypes studied. n = number of genotypes per chemotype. CV (%) calculated across three locations and two years (Layer 1 – ecological variation; n = 6 environment–year combinations per genotype). Stability thresholds following multi-environment crop evaluation convention (Zobel et al. 1988 ): CV < 10% = highly stable; 10–20% = moderately stable; CV ≥ 20% = variable. Note: CV estimates are based on n = 6 environment-year means per genotype; confidence intervals may be wide for very low CV values. Chemotype Dominant compound n CV% range Mean CV% Stability class Estragole Estragole 1 2.8 2.8 Highly stable (CV < 10%) Linalool Linalool 9 7.8–32.4 17.4 Moderate to variable Methyl cinnamate Methyl cinnamate 1 20.0 20.0 Variable (CV ≥ 20%) Citral Citral (geranial + neral) 1 12.9 12.9 Moderate (10–20%) Discussion Ecological drivers of essential oil yield The superiority of Bursa for EO yield may be partly attributed to its warmer and more humid microclimate. Growing-season mean temperature was 21.5°C at Bursa versus 18.1°C at Eskişehir and 19.7°C (two-year mean) at Tokat; relative humidity averaged 66.5% at Bursa, 61.1% at Eskişehir, and 52.2% at Tokat (Online Resource 1, Table S3), though other site-specific factors – including the well-structured clay-loam soils characteristic of the Bursa plain, longer effective sunshine duration at lower altitude (120 m), and irrigation management – cannot be excluded. Eskişehir (789 m altitude; drier, continental) and Tokat (594 m; semi-arid) represent substantially different growing conditions, and the altitude gradient of 669 m between the lowest (Bursa) and highest (Eskişehir) site likely contributes to the observed EO yield gradient through temperature and growing season effects. Temperature is a primary driver of EO biosynthesis in Lamiaceae, as monoterpene and sesquiterpene synthase activities are known to be temperature-dependent (Sangwan et al. 2001 ). It should be noted that drip irrigation was applied as needed without standardised protocols across locations; differential water supply may have contributed to the observed location effects alongside climatic differences. Hussain et al. ( 2008 ) reported similar positive correlations between environmental temperature and basil EO yield. Tursun and Telci ( 2020 ) further demonstrated that elevated CO₂ and temperature significantly modulate EO composition in purple basil. The significant genotype × location interaction confirms that cultivar selection must be location-specific for optimal EO production, as also emphasised by Rahimi et al. ( 2023 ) and Mulugeta et al. ( 2023 ) in the context of environmental stress responses. The comprehensive review by da Silva et al. ( 2021 ) further contextualises how extraction method and environmental conditions jointly determine basil EO yield and composition in food applications. The between-year variation in EO yield (Year 2 > Year 1) across all locations underscores the importance of multi-year trials for reliable cultivar recommendations. Because Year was treated as a fixed effect, the conclusions strictly apply to the conditions tested and should not be generalised beyond the two-year window without further validation. Single-year data may substantially misrepresent a genotype's productive potential, a limitation commonly encountered in aromatic crop evaluation (Tena et al. 2019 ). The consistent year effect further supports treating year as a design factor rather than a random nuisance term in the present fixed-effects model. Ontogenetic patterns and harvest timing The 0.20 mL·100 g⁻¹ DW increase in EO yield from the vegetative (0.78) to the onset of flowering stage (0.98), without a further significant increase at full flowering, provides clear harvest timing guidance. This pattern is consistent with the developmental trajectory of glandular trichomes as the primary site of EO biosynthesis and storage (Gang et al. 2001 ). Beatović et al. ( 2015 ) and Eskandarzade et al. ( 2024 ) reported similar ontogenetic patterns in Serbian and Iranian basil cultivars. The ontogenetic decline in linalool content, combined with no significant additional yield gain at full flowering, supports harvesting at onset of flowering to maximise both EO yield and linalool quality. This recommendation aligns with recent findings by Kholiya et al. ( 2022 ) and Mkaddem Mounira et al. ( 2022 ). For the estragole chemotype, the earlier yield peak at onset of flowering provides a practical distinction for production scheduling. Seasonal variation and double-cropping potential The significantly higher EO yield in autumn harvests compared to summer harvests at Tokat supports the agronomic viability of double-cropping strategies. Corrado et al. ( 2020 ) reported analogous harvest sequence effects on basil metabolic profiles, noting that successive harvests modify both yield and quality. Ciriello et al. ( 2021 ) demonstrated that the interaction between genotype and harvest sequence significantly affects phenolic acid and aroma profiles in Genovese basil, highlighting the need to optimise genotype–harvest scheduling combinations. The relatively stable EO composition across seasons in linalool-type genotypes suggests that autumn double-cropping can deliver comparable quality to the primary summer harvest, a commercially relevant finding for the basil food industry (Hamid et al. 2024 ; Hallmann and Ponder 2024 ). Chemotype stability and implications for secondary metabolite management The three-layer experimental design employed here provides a more comprehensive picture of chemotype stability than conventional single-dimension G×E studies, which typically assess only spatial variation. By simultaneously examining ecological, ontogenetic and seasonal effects, the present study reveals that compositional stability varies not only among genotypes but also among the dimensions of environmental variation tested. The CV-based stability assessment reveals a clear hierarchy of environmental sensitivity among basil chemotypes. The estragole chemotype (Layer 1 CV = 2.8%) exhibited near-absolute compositional stability, suggesting strong genetic canalization of phenylpropanoid biosynthesis with minimal environmental modulation. This aligns with Telci et al. ( 2006 ), who noted the consistency of estragole-dominant chemotypes across Turkish growing conditions, and with recent chemotyping studies (Kirci et al. 2025 ; Bajomo et al. 2022 ). In contrast, the citral chemotype (Layer 1 CV = 12.9%) showed substantial environmental sensitivity, particularly across ontogenetic stages, consistent with the known variability of citral accumulation (Iijima et al. 2004b ) and the instability of geraniol-derived compounds under fluctuating temperature and moisture regimes (Tursun and Telci 2020 ). It should be noted that the estragole, citral, and methyl cinnamate chemotypes are each represented by a single genotype (R-10A, R-17, and R-23, respectively); consequently, the stability values reported for these chemotypes reflect individual genotypic stability rather than chemotype-level generalisations. Additional genotypes within each non-linalool chemotype would be needed to confirm whether the observed stability patterns are chemotype-intrinsic. Within the linalool chemotype, R-16 (SD = 5.6%) emerged as the only highly stable linalool genotype, while R-20 (SD = 8.8%) also showed comparatively low variability, together representing the most stable pair within the linalool chemotype. In contrast, R-4 (SD = 17.9%) showed the greatest compositional instability for linalool despite stable EO yield, confirming that yield stability and compositional stability are not necessarily correlated. The SD values for linalool genotypes are reported in absolute percentage points (rather than CV%) because the mean linalool content varies substantially across genotypes, making CV-based comparison misleading. It should be noted that all conclusions drawn regarding environmental (in)stability apply within the specific set of three Turkish locations, two growing years, and management conditions tested in the present study. It is acknowledged that quantitative EO data were obtained without FID response factor correction, representing relative rather than absolute proportions. This approach is widely accepted in multi-genotype EO characterisation studies (Šovljanski et al. 2022 ; Abdoul-Latif et al. 2022 ). For the dominant compounds verified against authentic standards, the relative FID response is expected to approximate mass proportions within about ± 5% for the monoterpene and phenylpropanoid classes studied. It is acknowledged that CV-based stability, while practical and easily interpretable, has inherent limitations: it is sensitive to the mean value (genotypes with low mean proportions tend to have inflated CV%) and does not capture the pattern of instability (e.g., consistent direction vs. unpredictable fluctuation). Although the CV reported in Table 5 is based on Layer 1 (ecological variation) alone, the three locations span approximately 8° of longitude and 670 m of altitude; the inclusion of ontogenetic and seasonal layers in the broader study design shows that additional variance sources exist beyond the ecological dimension captured by CV. These considerations should be borne in mind when extrapolating the stability rankings to production environments outside the tested range. Food safety implications of estragole and methyl eugenol variation The margin of exposure (MOE) calculations presented here are illustrative only and are not intended for formal risk assessment because several assumptions were not experimentally determined (including the fresh-to-dry weight ratio). Using a nominal 5:1 fresh-to-dry ratio and typical fresh basil intake of 1–2 g per eating occasion, estimated MOEs for estragole in the estragole chemotype were generally above 10,000, whereas methyl eugenol in R-4 yielded lower MOEs. A high-consumer scenario (e.g., 10 g fresh basil per occasion) would substantially reduce MOEs; therefore, chemotype-based cultivar selection remains the most practical risk-management strategy. Regulation (EC) No. 1334/2008 maximum levels apply to estragole in the final food product as consumed, not to the raw herb or essential oil. Given the genotoxic and carcinogenic classification of these phenylpropanoids, the MOE framework should be interpreted cautiously and chemotype-based cultivar selection is recommended. Conclusions This three-layer field study – encompassing 12 genotypes, three Turkish locations, three developmental stages, two harvest seasons, and two years (144 unique EO profiles; 192 layer-specific observations) – provides the first comprehensive characterisation of environmental, ontogenetic, and seasonal modulation of essential oil chemotype expression in basil. Key findings are: (i) Bursa optimises EO yield (0.98 mL·100 g⁻¹ DW) while Tokat maximises linalool proportion; (ii) onset of flowering is the optimal harvest stage for both yield and quality; (iii) autumn harvests support double-cropping with comparable linalool quality; (iv) the estragole chemotype is exceptionally stable across ecological environments (Layer 1 CV = 2.8%) while the citral (12.9%) and methyl cinnamate (20.0%) chemotypes show moderate to variable stability; (v) R-16 is the most stable linalool genotype (SD = 5.6%). These results indicate that environment × harvest-stage interactions modulate secondary metabolite accumulation in a chemotype-dependent manner and that high-phenylpropanoid chemotypes (e.g., estragole and methyl eugenol) are primarily genotype-driven and should be managed through chemotype-based cultivar selection to support food safety-oriented cultivar deployment under EU flavouring regulations. Declarations Competing interests The authors have no relevant financial or non-financial interests to disclose. Author Contribution Adem Zorlu: Conceptualization; Methodology; Investigation; Data curation; Formal analysis; Writing – original draft. İsa Telci: Conceptualization; Supervision; Methodology; Validation; Writing – review & editing. Mahfuz Elmastaş: Chemical analysis (GC–MS/GC–FID); Methodology; Validation; Data interpretation; Writing – review & editing. Oya Kaçar: Field trials and agronomic management (Bursa); Sampling; Resources; Writing – review & editing. Zehra Aytaç: Field trials and agronomic management (Eskişehir); Sampling; Resources; Writing – review & editing. Emine Bayram: Field trial support and resources; Supervision; Writing – review & editing. All authors have read and approved the final version of the manuscript. Acknowledgements The authors thank Tokat Gaziosmanpaşa University, Bursa Uludağ University and Eskişehir Osmangazi University for providing field facilities and technical support during the trials. The authors also thank the Turkish State Meteorological Service for providing climatic data for the trial locations. This research was supported by TÜBİTAK (The Scientific and Technological Research Council of Türkiye; Project No. 111O677). Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. References Abdoul-Latif FM, Elmi A, Merito A, Nour M, Risler A, Ainane A, Bignon J, Ainane T (2022) Essential oils of O. basilicum and Ocimum americanum L. from Djibouti: chemical composition, antimicrobial and cytotoxicity evaluations. Processes 10:1785. ttps://doi.org/10.3390/pr10091785 Adams RP (2017) Identification of essential oil components by gas chromatography/mass spectrometry, 4.1 ed. Carol Stream: Allured Publishing Amor G, Sabbah M, Caputo L, Idbella M, De Feo V, Porta R, Fechtali T, Mauriello G (2021) Basil essential oil: composition, antimicrobial properties, and microencapsulation to produce active chitosan films for food packaging. Foods 10:121. ttps://doi.org/10.3390/foods10010121 Avetisyan A, Markosian A, Petrosyan M, Sahakyan N, Babayan A, Aloyan S, Trchounian A (2017) Chemical composition and some biological activities of the essential oils from basil Ocimum different cultivars. BMC Complement Altern Med 17:60. ttps://doi.org/10.1186/s12906-017-1587-5 Azizah NS, Irawan B, Kusmoro J, Safriansyah W, Farabi K, Oktavia D, Doni F, Miranti M (2023) Sweet basil (O. basilicum)—a review of its botany, phytochemistry, pharmacological activities, and biotechnological development. Plants 12:4148. ttps://doi.org/10.3390/plants12244148 Bajomo EM, Aing MS, Ford LS, Niemeyer ED (2022) Chemotyping of commercially available basil (O. basilicum) varieties: cultivar and morphotype influence phenolic acid composition and antioxidant properties. NFS J 29:1–11. ttps://doi.org/10.1016/j.nfs.2022.09.002 Beatović D, Krstić-Milošević D, Trifunović S, Šiljegović J, Glamočlija J, Ristić M, Jelačić S (2015) Chemical composition, antioxidant and antimicrobial activities of the essential oils of twelve O. basilicum cultivars grown in Serbia. Rec Nat Prod 9:62–75 Ciriello M, Formisano L, El-Nakhel C, Kyriacou MC, Soteriou GA, Pizzolongo F, Romano R, De Pascale S, Rouphael Y (2021) Genotype and successive harvests interaction affects phenolic acids and aroma profile of Genovese basil for pesto sauce production. Foods 10:278. ttps://doi.org/10.3390/foods10020278 Corrado G, Chiaiese P, Lucini L, Miras-Moreno B, Colla G, Rouphael Y (2020) Successive harvests affect yield, quality and metabolic profile of sweet basil (O. basilicum). Agronomy 10:830. ttps://doi.org/10.3390/agronomy10060830 da Silva WMF, Kringel DH, Dierings de Souza EJ, Zavareze E, Dias ARG (2021) Basil essential oil: methods of extraction, chemical composition, biological activities, and food applications. Food Bioprocess Technol 14:1947–1971. ttps://doi.org/10.1007/s11947-021-02690-3 De Masi L, Siviero P, Esposito C, Castaldo D, Siano F, Laratta B (2006) Assessment of agronomic, chemical and genetic variability in common basil (O. basilicum). Eur Food Res Technol 223:273–281. ttps://doi.org/10.1007/s00217-005-0201-0 Dhama K, Sharun K, Gugjoo MB, Tiwari R, Alagawany M, Yatoo MI, Thakur P, Iqbal HMN, Chaicumpa W, Michalak I, Elnesr SS, Farag MR (2021) A comprehensive review on chemical profile and pharmacological activities of O. basilicum. Food Rev Int. ttps://doi.org/10.1080/87559129.2021.1900230 Eberhart SA, Russell WA (1966) Stability parameters for comparing varieties. Crop Sci 6:36–40. ttps://doi.org/10.2135/cropsci1966.0011183X000600010011x EFSA CEF Panel (2009) Scientific opinion on methyleugenol in flavourings: consideration of genotoxicity data on methyleugenol and structurally related alkenylbenzenes. EFSA J 7:1016. ttps://doi.org/10.2903/j.efsa.2009.1016 EFSA CEF Panel (2012) Scientific opinion on estragole in flavourings: consideration of genotoxicity data on estragole and structurally related alkenylbenzenes methyleugenol and elemicin. EFSA J 10:2798. ttps://doi.org/10.2903/j.efsa.2012.2798 Eskandarzade P, Zare Mehrjerdi M, Gruda NS, Aliniaeifard S (2024) Phytochemical compositions and antioxidant activity of green and purple basils altered by light intensity and harvesting time. Heliyon 10:e30931. ttps://doi.org/10.1016/j.heliyon.2024.e30931 European Medicines Agency (EMA) (2023) Public statement on the use of herbal medicinal products containing estragole (Revision 1). EMA/HMPC/137212/2005 Rev 1 Corr 1. London: HMPC (originally 2005) European Pharmacopoeia Commission (2010) European Pharmacopoeia, 7th ed. Strasbourg: Council of Europe Ferrante C, Orlando G, Ferro S (2021) Essential oils from Salvia species: variation and biological activity. Nat Prod Res 35:3178–3191. ttps://doi.org/10.1080/14786419.2019.1698574 Gang DR, Wang J, Dudareva N, Nam KH, Simon JE, Lewinsohn E, Pichersky E (2001) An investigation of the storage and biosynthesis of phenylpropenes in sweet basil. Plant Physiol 125:539–555. ttps://doi.org/10.1104/pp.125.2.539 Giachino RRA, Sönmez Ç, Tonk FA, Bayram E, Yüce S, Telci I, Furan MA (2014) RAPD and essential oil characterization of Turkish basil (O. basilicum). Plant Syst Evol 300:2227–2238. ttps://doi.org/10.1007/s00606-014-1034-5 Grayer RJ, Kite GC, Goldstone FJ, Bryan SE, Paton A, Putievsky E (1996) Infraspecific taxonomy and essential oil chemotypes in sweet basil, O. basilicum. Phytochemistry 43:1033–1039. ttps://doi.org/10.1016/S0031-9422(96)00429-8 Gurav TP, Dholakia BB, Giri AP (2022) A glance at the chemodiversity of Ocimum species: trends, implications, and strategies for the quality and yield improvement of essential oil. Phytochem Rev 21:947–979. ttps://doi.org/10.1007/s11101-021-09767-z Gurkan H, Hayaloglu AA (2023) Changes in volatiles and essential oil composition of three organs of purple basil (O. basilicum) by GC-MS combined with multivariate statistical approach. Food Chem Adv 2:100292. ttps://doi.org/10.1016/j.focha.2023.100292 Hallmann E, Ponder A (2024) A long-term study on chemical compounds and their location in sweet basil leaves from organic and conventional producers. Foods 13:383. ttps://doi.org/10.3390/foods13030383 Hamid S, Fadloun Oukil N, Moussa H, Djihad N, Mróz M, Kusznierewicz B, Attia A, Djenadi K, Mahdjoub MM, Bouhenna MM, Chebrouk F (2024) Chemical and biological characterization of O. basilicum phenolic extract and essential oil derived through ultrasound and microwave-assisted extraction techniques. Food Biosci 60:104359. ttps://doi.org/10.1016/j.fbio.2024.104359 Hussain AI, Anwar F, Sherazi STH, Przybylski R (2008) Chemical composition, antioxidant and antimicrobial activities of basil (O. basilicum) essential oils depend on seasonal variations. Food Chem 108:986–995. ttps://doi.org/10.1016/j.foodchem.2007.12.010 Iijima Y, Davidovich-Rikanati R, Fridman E, Gang DR, Bar E, Lewinsohn E, Pichersky E (2004a) The biochemical and molecular basis for the divergent patterns in the biosynthesis of terpenes and phenylpropenes in the peltate glands of three cultivars of basil. Plant Physiol 136:3724–3736. ttps://doi.org/10.1104/pp.104.051318 Iijima Y, Gang DR, Fridman E, Lewinsohn E, Pichersky E (2004b) Characterization of geraniol synthase from the peltate glands of sweet basil. Plant Physiol 134:370–379. ttps://doi.org/10.1104/pp.103.032946 Jeurissen SM, Punt A, Delatour T, Rietjens IM (2008) Basil extract inhibits sulfotransferase-mediated formation of DNA adducts of the procarcinogen 1′-hydroxyestragole by rat and human liver S9 homogenates and in HepG2 cells. Food Chem Toxicol 46:2296–2302. ttps://doi.org/10.1016/j.fct.2008.03.010 Jiang R, Liu J, Liu Q, Jin Z, Zhu H, Han H, Ma X (2024) Comparative analysis of volatile components and sensory profiles of four basil varieties based on HS-SPME and SD coupled with GC-MS. Processes 12:2789. ttps://doi.org/10.3390/pr12122789 Kholiya S, Punethaa A, Chauhan A, Varsha KT, Kumar D, Upadhyaya RK (2022) Essential oil yield and composition of O. basilicum at different phenological stages, plant density and post-harvest drying methods. S Afr J Bot 151:919–925. ttps://doi.org/10.1016/j.sajb.2022.11.011 Kirci D, Tabanca N, Blythe E, Demirci B (2025) Phytochemical variations in O. basilicum cultivars: essential oil composition and multivariate chemotype differentiation. Rec Nat Prod 19:688–701 Labra M, Miele M, Ledda B, Grassi F, Mazzei M, Sala F (2004) Morphological characterization, essential oil composition and DNA genotyping of O. basilicum cultivars. Plant Sci 167:725–731. ttps://doi.org/10.1016/j.plantsci.2004.04.026 Lawson HP, Aga MF, Niemeyer ED (2025) Plant maturity differentially affects the phenolic composition and antioxidant properties of green basil (O. basilicum) cultivars. ACS Omega 10:47535–47543 Lukas B, Schmiderer C, Novak J (2015) Essential oil diversity of European Origanum vulgare L. (Lamiaceae). Phytochemistry 119:32–40. ttps://doi.org/10.1016/j.phytochem.2015.09.007 Mahendran G, Vimolmangkang S (2023) Chemical compositions, antioxidant, antimicrobial, and mosquito larvicidal activity of Ocimum americanum L. and O. basilicum leaf essential oils. BMC Complement Med Ther 23:390. ttps://doi.org/10.1186/s12906-023-04228-8 Makri O, Kintzios S (2008) Ocimum sp. (basil): botany, cultivation, pharmaceutical properties, and biotechnology. J Herbs Spices Med Plants 13:123–150. ttps://doi.org/10.1300/J044v13n03_10 Marotti M, Piccaglia R, Giovanelli E (1996) Differences in essential oil composition of basil (O. basilicum) Italian cultivars related to morphological characteristics. J Agric Food Chem 44:3926–3929. ttps://doi.org/10.1021/jf9601067 Mkaddem Mounira G, Zrig A, Ben Abdallah M, Romdhane M, Okla MK, Al-Hashimi A, Alwase YA, Madnay MM, AbdElgayed G, Asard H (2022) Essential oil composition and antioxidant and antifungal activities of two varieties of O. basilicum at two phenological stages. Agronomy 12:825. ttps://doi.org/10.3390/agronomy12040825 Mulugeta SM, Sárosi S, Radácsi P (2023) Physio-morphological trait and bioactive constituents of Ocimum species under drought stress. Ind Crops Prod 206:117607. ttps://doi.org/10.1016/j.indcrop.2023.117607 Pimentel LAG, da Silva MA, de Medeiros SDS, Luz JMQ, Sala FC (2023) Agronomic, sensory and essential oil characterization of basil (O. basilicum) accessions. Horticulturae 9:831. ttps://doi.org/10.3390/horticulturae9070831 Qasem A, Assaggaf H, Mrabti HN, Minshawi F, Rajab BS, Attar AA, Alyamani RA, Hamed M, Mrabti NN, El Baaboua A (2023) Determination of chemical composition and investigation of biological activities of O. basilicum. Molecules 28:614. ttps://doi.org/10.3390/molecules28020614 Rahimi M, Mortazavi M, Mianabadi A, Debnath S (2023) Evaluation of basil (O. basilicum) accessions under different drought conditions based on yield and physio-biochemical traits. BMC Plant Biol 23:523. ttps://doi.org/10.1186/s12870-023-04540-0 Regulation (EC) No 1334/2008 of the European Parliament and of the Council on flavourings and certain food ingredients with flavouring properties for use in and on foods. Off J Eur Union L354:34–50 (2008) Ríos-Rodríguez D, Sahi VP, Nick P (2021) Authentication of holy basil using markers relating to a toxicology-relevant compound. Eur Food Res Technol 248:13–25. ttps://doi.org/10.1007/s00217-021-03849-w Sangwan NS, Farooqi AHA, Shabih F, Sangwan RS (2001) Regulation of essential oil production in plants. Plant Growth Regul 34:3–21. ttps://doi.org/10.1023/A:1013386921596 Scientific Committee on Food (SCF) (2001a) Opinion on estragole (1-allyl-4-methoxybenzene). SCF/CS/FLAV/FLAVOUR/6 ADD2 Final. Brussels: European Commission Scientific Committee on Food (SCF) (2001b) Opinion on methyleugenol (4-allyl-1,2-dimethoxybenzene). SCF/CS/FLAV/FLAVOUR/4 ADD1 Final. Brussels: European Commission Šovljanski O, Saveljić A, Aćimović M, Šeregelj V, Pezo L, Tomić A, Ćetković G, Tešević V (2022) Biological profiling of essential oils and hydrolates of O. basilicum var. Genovese and var. Minimum originated from Serbia. Processes 10:1893. ttps://doi.org/10.3390/pr10101893 Telci I, Bayram E, Yılmaz G, Avcı B (2006) Variability in essential oil composition of Turkish basils (O. basilicum). Biochem Syst Ecol 34:489–497. ttps://doi.org/10.1016/j.bse.2006.01.009 Tena E, Goshu F, Mohamad G, Tesfa M, Tesfaye D, Seife A (2019) Genotype × environment interaction by AMMI and GGE-biplot analysis for sugar yield in sugarcane. Cogent Food Agric 5:1651925. ttps://doi.org/10.1080/23311932.2019.1651925 Tursun AO, Telci I (2020) The effects of carbon dioxide and temperature on essential oil composition of purple basil (O. basilicum). J Essent Oil Bear Plants 23:1090–1104. ttps://doi.org/10.1080/0972060X.2020.1854128 Zobel RW, Wright MJ, Gauch HG (1988) Statistical analysis of a yield trial. Agron J 80:388–393. ttps://doi.org/10.2134/agronj1988.00021962008000030002x Additional Declarations No competing interests reported. Supplementary Files OnlineResource11.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 18 Mar, 2026 Reviews received at journal 18 Mar, 2026 Reviews received at journal 16 Mar, 2026 Reviewers agreed at journal 16 Mar, 2026 Reviewers agreed at journal 16 Mar, 2026 Reviewers invited by journal 16 Mar, 2026 Editor assigned by journal 16 Mar, 2026 Submission checks completed at journal 16 Mar, 2026 First submitted to journal 15 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9131432","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":608460816,"identity":"2c717103-bcc7-4e89-a2d6-4e9b6b405029","order_by":0,"name":"Adem Zorlu","email":"","orcid":"","institution":"Sağlık Bilimleri Üniversitesi","correspondingAuthor":false,"prefix":"","firstName":"Adem","middleName":"","lastName":"Zorlu","suffix":""},{"id":608460817,"identity":"2f21aa39-dae4-4dbf-b7f3-1bb5238b8ee9","order_by":1,"name":"İsa Telci","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYBACNjBZwMDDz4wQNCCkhbEBqIZHshmhGkgk4NUF1sJgcIBYLXz8h58/+GBgJ2N8nP3Zg597/sgzsDdvk2D8cQ+3wyTSDBtnGCTzmB3mMTfseWZg2MBzrEyCIaEYjxYGw2YegwMgLWwSPAcMGBskcsyAWnC7jI3/+MfmP0Atxs3szyT/HDCwb5B/Q0ALQ45hM9DvPAbMDGbSQFsSGyR4CGiRyCmc2QP0iwTQL8YyB4yT23jSii0S0nBrke8/vuHDjwo7e/7+488evjkgZ9vPfnjjjQ82uLWgOhJOEqcBpmUUjIJRMApGAToAABc3SHXg7VWDAAAAAElFTkSuQmCC","orcid":"","institution":"Isparta University of Applied Sciences","correspondingAuthor":true,"prefix":"","firstName":"İsa","middleName":"","lastName":"Telci","suffix":""},{"id":608460819,"identity":"21c8581a-15e8-4e3b-b005-ab546fd6f31f","order_by":2,"name":"Mahfuz Elmastaş","email":"","orcid":"","institution":"Sağlık Bilimleri Üniversitesi","correspondingAuthor":false,"prefix":"","firstName":"Mahfuz","middleName":"","lastName":"Elmastaş","suffix":""},{"id":608460821,"identity":"e44ed245-1a91-4b38-af38-67a4d992e72b","order_by":3,"name":"Oya Kaçar","email":"","orcid":"","institution":"Bursa Uludağ University","correspondingAuthor":false,"prefix":"","firstName":"Oya","middleName":"","lastName":"Kaçar","suffix":""},{"id":608460823,"identity":"bd6d9293-8f97-4e4b-8936-9cb37447e4d1","order_by":4,"name":"Zehra Aytaç","email":"","orcid":"","institution":"Eskişehir Osmangazi University","correspondingAuthor":false,"prefix":"","firstName":"Zehra","middleName":"","lastName":"Aytaç","suffix":""},{"id":608460827,"identity":"4792f2ea-8d80-40d2-940c-752e91fec530","order_by":5,"name":"Emine Bayram","email":"","orcid":"","institution":"Ege University","correspondingAuthor":false,"prefix":"","firstName":"Emine","middleName":"","lastName":"Bayram","suffix":""}],"badges":[],"createdAt":"2026-03-15 23:08:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9131432/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9131432/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105004473,"identity":"5c8b4b32-cd7f-40e3-b740-a4c9ede8855d","added_by":"auto","created_at":"2026-03-19 17:56:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8188127,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic overview of the three-layer experimental design and analytical workflow used to characterise essential oil yield and chemotype composition in 12 basil (Ocimum basilicum) genotypes. Layer 1 (ecological variation): three locations (Bursa, Eskişehir, Tokat) × two years; Layer 2 (ontogenetic variation): three developmental stages (pre-flowering, onset of flowering, full flowering) at Bursa × two years; Layer 3 (seasonal variation): two harvest seasons (summer, autumn) at Tokat × two years. Fresh plant material was dried, hydrodistilled (Clevenger apparatus), and analysed by GC-FID (quantification) and GC-MS (identification). Four chemotypes were identified: linalool (nine genotypes), estragole (R-10A), citral (R-17), and methyl cinnamate (R-23). Food safety implications are discussed in the context of EU Regulation (EC) No 1334/2008\u003c/p\u003e","description":"","filename":"Fig11.png","url":"https://assets-eu.researchsquare.com/files/rs-9131432/v1/70bf459ce390b02a8c23439d.png"},{"id":105004479,"identity":"97158339-b74e-4035-9a13-c6026d17e2ea","added_by":"auto","created_at":"2026-03-19 17:56:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18874608,"visible":true,"origin":"","legend":"\u003cp\u003eEssential oil (EO) yield (mL·100 g⁻¹ dry weight (DW)) of 12 basil genotypes across three locations (genotype × location interaction; Layer 1 – ecological variation). Bars represent individual year values for each location (Bursa Year 1, Bursa Year 2, Eskişehir Year 1, Eskişehir Year 2, Tokat Year 1, Tokat Year 2). Different lowercase letters above each genotype group denote significant differences among genotype means across all six environment–year combinations (Duncan's multiple range test, P ≤ 0.01)\u003c/p\u003e","description":"","filename":"Fig21.png","url":"https://assets-eu.researchsquare.com/files/rs-9131432/v1/7cce13d741b3af254efba70e.png"},{"id":105004476,"identity":"8faa7cd3-a4d2-49f7-a5f5-44121494bbca","added_by":"auto","created_at":"2026-03-19 17:56:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":19804972,"visible":true,"origin":"","legend":"\u003cp\u003eEssential oil (EO) yield (mL·100 g⁻¹ dry weight (DW)) of 12 basil genotypes across three developmental stages at Bursa (genotype × stage interaction; Layer 2 – ontogenetic variation). PF pre-flowering (vegetative), OF onset of flowering (10–15% open flowers), FF full flowering (\u0026gt;50% open flowers), Year 1 first growing year, Year 2 second growing year. Different lowercase letters above genotype groups denote significant differences among stage means (Duncan's test, P ≤ 0.01)\u003c/p\u003e","description":"","filename":"Fig31.png","url":"https://assets-eu.researchsquare.com/files/rs-9131432/v1/8e23d34b7c3d6103d5e7784e.png"},{"id":105004480,"identity":"1ee04370-cf4f-4dd6-8973-4009cc40f04f","added_by":"auto","created_at":"2026-03-19 17:56:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":9585823,"visible":true,"origin":"","legend":"\u003cp\u003eSeasonal variation in the dominant compound relative proportion (%) for each basil chemotype at Tokat (Layer 3 – seasonal variation). a Linalool: mean of nine linalool-type genotypes (n = 9); b Genotype R-10A: estragole; c Genotype R-17: citral; d Genotype R-23: methyl cinnamate. Summer first cut (June–July), Autumn second cut (August–September), Year 1 first growing year, Year 2 second growing year. Individual Year 1 and Year 2 values are shown separately for each season (n = 2 years per season). The brackets with ns denote non-significant seasonal differences (P \u0026gt; 0.05) based on the season main effect in the year × season ANOVA. Dashed lines indicate the seasonal mean for each compound\u003c/p\u003e","description":"","filename":"Fig41.png","url":"https://assets-eu.researchsquare.com/files/rs-9131432/v1/ca27052c0697898d3cf71ee8.png"},{"id":105004475,"identity":"5c17d986-2e36-4575-a531-e13a15905f46","added_by":"auto","created_at":"2026-03-19 17:56:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":21335420,"visible":true,"origin":"","legend":"\u003cp\u003eLinalool content (%) of nine linalool-type basil genotypes across three locations and two years (Layer 1 – ecological variation). Six bars per genotype represent Bursa Year 1, Eskişehir Year 1, Tokat Year 1, Bursa Year 2, Eskişehir Year 2, and Tokat Year 2. Error bars show the mean ± standard deviation (SD) across six environment–year combinations (diamond markers)\u003c/p\u003e","description":"","filename":"Fig51.png","url":"https://assets-eu.researchsquare.com/files/rs-9131432/v1/3220eab5c40e767ed7412889.png"},{"id":105004474,"identity":"9a0b55e3-e2b3-4a12-91dd-89d342cfe874","added_by":"auto","created_at":"2026-03-19 17:56:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":9326919,"visible":true,"origin":"","legend":"\u003cp\u003eDominant compound variation in non-linalool chemotypes across three locations and two years (Layer 1 – ecological variation). a Estragole (%) in genotype R-10A (Layer 1 CV = 2.8%); b citral (geranial + neral) (%) in genotype R-17 (Layer 1 CV = 12.9%); c methyl cinnamate (%) in genotype R-23 (Layer 1 CV = 20.0%). Six bars per panel represent Bursa Year 1, Eskişehir Year 1, Tokat Year 1, Bursa Year 2, Eskişehir Year 2, and Tokat Year 2. Dashed lines indicate the overall mean for each chemotype. CV coefficient of variation across six environment–year combinations\u003c/p\u003e","description":"","filename":"Fig61.png","url":"https://assets-eu.researchsquare.com/files/rs-9131432/v1/8dd6f828139ce77203f043f1.png"},{"id":105562854,"identity":"420bfc1a-a94c-46ee-b165-9e7ebea14414","added_by":"auto","created_at":"2026-03-27 12:44:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27042657,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9131432/v1/c6570cd8-9de1-41d9-9d33-026f25b8725d.pdf"},{"id":105004477,"identity":"571f6b7d-fdff-45ad-b744-a12c61c19df3","added_by":"auto","created_at":"2026-03-19 17:56:53","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":196208,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineResource11.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9131432/v1/6a2678518549f35f4f5c1688.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Essential oil chemotype diversity and environmental stability in Turkish basil (Ocimum basilicum L.) germplasm: a three-layer field evaluation across contrasting ecologies","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSweet basil (Ocimum basilicum L., Lamiaceae) is one of the most commercially important aromatic herbs worldwide, valued for its culinary, medicinal, cosmetic, and industrial applications (Makri and Kintzios \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Dhama et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The species exhibits remarkable intraspecific diversity in both morphological traits and secondary metabolite profiles (Labra et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; De Masi et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), and its botany, phytochemistry, and pharmacological properties have been comprehensively reviewed (Dhama et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Azizah et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Essential oil (EO) composition is the primary criterion for commercial classification of basil, as it determines both aroma characteristics and biological activity (Hussain et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral chemotype classification systems have been proposed for O. basilicum based on the dominant EO constituents. Grayer et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) distinguished five major EO profiles based on the proportions of linalool, estragole, eugenol, methyl eugenol, and geraniol. Working with Turkish landraces, Telci et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) identified seven chemotypes from 18 accessions, expanding the known chemical diversity of Anatolian basil populations. Subsequent molecular and chemometric studies of Turkish germplasm confirmed this intraspecific diversity (Giachino et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). More recently, multivariate approaches have become standard for chemotype classification (Kirci et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), enabling finer resolution of compositional variation across germplasm collections. The chemodiversity of Ocimum species has been comprehensively reviewed by Gurav et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and Dhama et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile the genetic component of EO composition is well established, the extent to which environmental factors modulate chemotype expression remains inadequately characterised, particularly in multi-location, multi-year field trials. The genotype \u0026times; environment (G \u0026times; E) interaction for EO traits has been documented in several aromatic species including Origanum vulgare (Lukas et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and Salvia species (Ferrante et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), but comprehensive G \u0026times; E studies in basil remain limited. Understanding EO chemotype stability is essential for growers, breeders, and the industry, ensuring reliable raw material quality (Pimentel et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBeyond spatial (ecological) variation, temporal factors also influence EO accumulation and composition. Ontogenetic variation in terpene and phenylpropanoid accumulation has been reported in basil (Eskandarzade et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Beatović et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and other Lamiaceae (Iijima et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004a\u003c/span\u003e). EO yield generally increases from vegetative to flowering stages, while compound proportions may shift in parallel with changes in terpene synthase activity during glandular trichome maturation (Iijima et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004a\u003c/span\u003e). Recent studies confirm that developmental stage is a critical determinant of EO quality at harvest (Kholiya et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mkaddem Mounira et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In regions where multiple harvests per season are feasible, the harvest season can alter EO yield and composition due to changing photoperiod, temperature, and precipitation (Pimentel et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Corrado et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA further practical dimension concerns food safety. Estragole (methyl chavicol) and methyl eugenol are phenylpropanoids classified as naturally occurring genotoxic and carcinogenic (in rodent models) substances by the European Medicines Agency (EMA 2023) and the Scientific Committee on Food (SCF 2001a, 2001b). Regulation (EC) No. 1334/2008 restricts these substances in foodstuffs and flavourings. Understanding how their proportions vary with genotype, location, and developmental stage is directly relevant to food safety risk assessment, particularly as basil is widely used as a fresh herb and flavouring agent in the food industry (Hamid et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Hallmann and Ponder \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The authentication of basil products with respect to genotoxic phenylpropanoids has been recognised as an emerging food safety priority (R\u0026iacute;os-Rodr\u0026iacute;guez et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Mahendran and Vimolmangkang \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). EFSA opinions on estragole (EFSA CEF Panel \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and methyl eugenol (EFSA CEF Panel \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) provide the regulatory genotoxicity framework, while basil matrix effects on estragole bioactivation have also been documented (Jeurissen et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTurkey harbours considerable basil genetic diversity in locally cultivated populations known as \"reyhan\" (Grayer et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Despite this potential, Turkish basil remains insufficiently characterised regarding EO stability across environments. To the best of our knowledge, no study has simultaneously evaluated the ecological, ontogenetic, and seasonal variation of basil EO in a multi-year, multi-location design within Turkey.\u003c/p\u003e \u003cp\u003eThe present study was designed to characterise the environmental modulation of EO yield and chemotype composition in 12 basil genotypes representing four chemotypes, using a three-layer experimental approach. Unlike standard genotype \u0026times; environment interaction studies that examine EO variation across a single experimental dimension (typically location or harvest time), the three-layer design simultaneously addresses ecological, ontogenetic and seasonal variation within the same genotype panel, enabling integrated cultivar recommendations that account for all three sources of compositional instability. The three layers are: (i) ecological variation across three locations; (ii) ontogenetic variation across three developmental stages; and (iii) seasonal variation between summer and autumn harvests (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The specific objectives were to quantify G \u0026times; E interactions for EO yield and major compounds, to assess chemotype stability using CV analysis, and to evaluate the implications of compositional variation for food safety.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material\u003c/h2\u003e \u003cp\u003eThe plant material used in this study was derived from a basil germplasm collection established within a nationally funded research project (T\u0026Uuml;BİTAK Project No. 111O677) based on local populations collected from different regions of T\u0026uuml;rkiye, together with a limited number of introduced genotypes. After preliminary characterisation of 70 accessions, 12 genotypes were selected based on prior chemotype characterisation (Kirci et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) to represent the four dominant chemotypes (linalool, estragole, citral, and methyl cinnamate) and a wide essential-oil yield range while ensuring suitability for multi-location field trials. Taxonomic identity as Ocimum basilicum L. was confirmed by Prof. Dr. İsa Telci, Department of Industrial Crops, Isparta University of Applied Sciences, T\u0026uuml;rkiye. The evaluated materials are maintained as documented accessions in the institutional medicinal and aromatic plants collection, and accession records are available for verification upon request. Accession origins, chemotype assignment and collection details are provided in Online Resource 1 (Table S2).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExperimental design and trial locations\u003c/h3\u003e\n\u003cp\u003eField trials were conducted at three ecologically contrasting locations in Turkey over two consecutive years (Year 1 and Year 2) (Online Resource 1, Table S3). The trial locations \u0026ndash; Bursa (40\u0026deg;13\u0026prime;N, 28\u0026deg;51\u0026prime;E, 120 m a.s.l.), Eskişehir (39\u0026deg;45\u0026prime;N, 30\u0026deg;33\u0026prime;E, 789 m a.s.l.), and Tokat (40\u0026deg;19\u0026prime;N, 36\u0026deg;27\u0026prime;E, 594 m a.s.l.) \u0026ndash; represent sub-Mediterranean, continental transition, and semi-arid continental climates, respectively. Growing-season (May\u0026ndash;September) mean temperature, total precipitation, and mean relative humidity were 21.5\u0026deg;C, 198\u0026ndash;425 mm, and 61\u0026ndash;72% at Bursa; 18.1\u0026deg;C, 130\u0026ndash;258 mm, and 55\u0026ndash;67% at Eskişehir; and 18.5\u0026ndash;20.9\u0026deg;C, 156\u0026ndash;187 mm, and 50\u0026ndash;55% at Tokat, respectively, across the two experimental years (Online Resource 1, Table S3). Soil at each site was classified as clay-loam (Bursa), silty-clay (Eskişehir), and sandy-loam (Tokat). Monthly climatic data are provided in Online Resource 1, Table S3.\u003c/p\u003e \u003cp\u003eSeedlings were produced from seed in perlite\u0026ndash;peat mixture (5:1, v/v) and transplanted to each location after the last spring frost. The experiment was arranged in a randomised complete block design (RCBD) with three replications. Plant spacing was 40 \u0026times; 30 cm. Prior to transplanting, 25 kg ha⁻\u0026sup1; pure nitrogen (half of the total 50 kg ha⁻\u0026sup1;) and 50 kg ha⁻\u0026sup1; triple superphosphate were applied. Drip irrigation was provided as needed.\u003c/p\u003e\n\u003ch3\u003eThree-layer experimental structure\u003c/h3\u003e\n\u003cp\u003eLayer 1 \u0026ndash; Ecological variation: All 12 genotypes were harvested at the onset of flowering at each of the three locations in both years, yielding 12 genotypes \u0026times; 3 locations \u0026times; 2 years\u0026thinsp;=\u0026thinsp;72 EO profiles. Note: the onset-of-flowering harvest at Bursa in Layer 1 is shared with the onset-of-flowering (OF) stage in Layer 2, and the first harvest at Tokat in Layer 1 is shared with the summer harvest in Layer 3; the total number of unique EO profiles is 144, while the total number of layer-specific observations is 192. Because Layers 2 and 3 used subsets of the Layer 1 dataset, results across layers should be interpreted as complementary rather than statistically independent; ontogenetic conclusions are specific to Bursa and seasonal conclusions are specific to Tokat within the tested years.\u003c/p\u003e \u003cp\u003eLayer 2 \u0026ndash; Ontogenetic variation: At the Bursa location, plant material was sampled at three developmental stages in both years: (i) pre-flowering (PF, vegetative), (ii) onset of flowering (OF, 10\u0026ndash;15% open flowers), and (iii) full flowering (FF, \u0026gt;\u0026thinsp;50% open flowers), yielding 72 EO profiles.\u003c/p\u003e \u003cp\u003eLayer 3 \u0026ndash; Seasonal variation: At the Tokat location, two harvests were taken per growing season in both years: a summer harvest (first cut, June\u0026ndash;July) and an autumn harvest (second cut, August\u0026ndash;September), yielding 48 EO profiles.\u003c/p\u003e\n\u003ch3\u003eEssential oil extraction\u003c/h3\u003e\n\u003cp\u003eEO was extracted from dried leaf material, consistent with established practice in multi-genotype basil EO evaluation where simultaneous processing of numerous samples precludes immediate distillation of fresh tissue (Beatović et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kholiya et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Leaf samples were dried in a forced-air oven at 35\u0026deg;C to constant weight (approximately 5\u0026ndash;7 days), protected from direct light; this low-temperature drying protocol minimises loss of volatile monoterpenes while enabling standardised sample preparation across locations and harvest dates. Dried leaves were stored in sealed dark-glass containers at ambient temperature until distillation, within two weeks of drying to minimise monoterpene loss. Fifteen grams of dried leaf were placed in a round-bottom flask with 300 mL distilled water (1:20, w/v) and subjected to hydrodistillation for 2 h using a Neo-Clevenger-type apparatus following the European Pharmacopoeia protocol (European Pharmacopoeia Commission \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). EO yield was expressed as mL\u0026middot;100 g⁻\u0026sup1; dry weight (DW), numerically equivalent to % v/w. EO volume was read at ambient temperature after complete phase separation in the graduated Clevenger tube.\u003c/p\u003e\n\u003ch3\u003eGas chromatography and gas chromatography–mass spectrometry\u003c/h3\u003e\n\u003cp\u003eGC analysis was performed on a Perkin-Elmer (PerkinElmer, Waltham, MA, USA) gas chromatograph equipped with an autosampler and flame ionisation detector (FID). Samples were diluted 1:10 in acetone and separated on a BPX5 capillary column (SGE Analytical Science, Melbourne, Australia; 30 m \u0026times; 0.25 mm i.d. \u0026times; 0.25 \u0026micro;m). Peaks were integrated using PerkinElmer TotalChrom software with automatic baseline detection and manual correction where necessary; integration settings were applied uniformly across all samples. Helium carrier gas at 5 psi; injector 230\u0026deg;C; detector 250\u0026deg;C; oven programme: initial 60\u0026deg;C (hold 2 min), ramped at 3\u0026deg;C\u0026middot;min⁻\u0026sup1; to 230\u0026deg;C (hold 15 min); total run time 73.7 min; split injection 5:1. Quantitative data are expressed as relative FID peak area (%). Response factor correction was not applied; accordingly, all compositional values represent relative proportions rather than absolute mass concentrations. For the dominant compounds verified against authentic standards (linalool, estragole, methyl eugenol, methyl cinnamate, eugenol, geraniol), the relative FID response approximates mass proportions within \u0026plusmn;\u0026thinsp;5% for the monoterpene and phenylpropanoid classes studied (Amor et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Šovljanski et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This approach is standard in multi-genotype EO characterisation (Amor et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Šovljanski et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Gurkan and Hayaloglu \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Abdoul-Latif et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The BPX5 column was selected for broad-spectrum separation across four diverse chemotypes; while it has limited selectivity for co-eluting phenylpropanoid isomers, this does not affect the findings given the unambiguous dominance of identified compounds in each chemotype. Co-elution of estragole with trans-anethole was checked by RI comparison (estragole RI\u0026thinsp;=\u0026thinsp;1195 vs. trans-anethole RI\u0026thinsp;=\u0026thinsp;1283 on BPX5) and confirmed absent in all samples. The FID quantification approach, applied consistently across all 144 unique EO profiles in the present study, does not affect between-genotype or between-environment comparisons.\u003c/p\u003e \u003cp\u003eGas chromatography\u0026ndash;mass spectrometry (GC-MS) analysis used a PerkinElmer GC-MS system (PerkinElmer, Waltham, MA, USA) (electron ionisation at 70 eV), same column and temperature programme, helium at 1.3 mL\u0026middot;min⁻\u0026sup1;, transfer line 250\u0026deg;C. Compound identification was based on: (i) comparison with authentic reference standards (linalool, estragole, methyl eugenol, methyl cinnamate, eugenol, geraniol, and α-terpineol; Sigma-Aldrich, St. Louis, MO, USA; \u0026ge;98% purity); (ii) retention index (RI) calculation relative to a homologous n-alkane series (C₈\u0026ndash;C₂₀); and (iii) mass spectral matching against Wiley and NIST (National Institute of Standards and Technology, Gaithersburg, MD, USA) mass spectral databases (minimum forward match score\u0026thinsp;\u0026ge;\u0026thinsp;850/1000) and published data (Adams \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Compounds present at less than 0.1% relative peak area were not included in quantitative reporting; this threshold corresponds to approximately 3\u0026times; the mean injection-to-injection variability (RSD\u0026thinsp;\u0026lt;\u0026thinsp;3%), ensuring only reliably quantifiable peaks are reported. Retention indices (RI) for all identified compounds are provided in Online Resource 1 (Table S4). Identification was assigned based on agreement between RI (typically within \u0026plusmn;\u0026thinsp;10 units) and MS library matching; dominant compounds were additionally confirmed using authentic standards where available (Adams \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Jiang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eSampling and analytical replication. At each harvest, 10\u0026ndash;15 plants were sampled randomly from each replication block. Leaf material from each replicate block was pooled, mixed, and a 15 g subsample was taken for hydrodistillation. This pooling eliminates within-plot (plant-to-plant) variation; genotypes with heterogeneous individual-plant EO profiles would appear artificially stable from pooled samples. Consequently, the CV-based stability estimates reported here reflect between-environment variation only and may underestimate true total variation, which would include the within-plot component. Each replicate was distilled and analysed separately, yielding three independent analytical observations per genotype per environment. GC-FID analysis involved a single injection per sample; peak area reproducibility was confirmed by duplicate injection of a random subset (n\u0026thinsp;=\u0026thinsp;12 samples), with a mean relative standard deviation below 3%. Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) values reported in tables and figures are based on these three replicate observations within each environment\u0026ndash;year combination, except where stated otherwise. Sample processing (drying, distillation, and GC analysis) was randomised across locations and years within each analytical batch to minimise systematic confounding of batch effects with environmental effects.\u003c/p\u003e \u003cp\u003eAnalysis of variance (ANOVA) was performed separately for each experimental layer. In Layer 1, genotype (G), location (L) and year (Yr) were included as fixed factors, and all two- and three-way interactions (G\u0026times;L, G\u0026times;Yr, L\u0026times;Yr and G\u0026times;L\u0026times;Yr) were tested. Replications were nested within each location \u0026times; year combination. Year was treated as a fixed effect because the two experimental years represent the specific study period rather than a random sample; thus, inference applies to Year 1\u0026ndash;Year 2 conditions (Online Resource 1, Table S3), and extrapolation to other years should be made cautiously. Analogous models were fitted for Layers 2 and 3. Mean separation was performed using Duncan's multiple range test at P\u0026thinsp;\u0026le;\u0026thinsp;0.01 (Eberhart and Russell \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1966\u003c/span\u003e). Key conclusions were verified using Tukey's HSD (α\u0026thinsp;=\u0026thinsp;0.05) with consistent outcomes (Online Resource 1, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEO composition data are presented as relative FID peak area proportions (%, normalised to total detected peak area). Because inference focused on single dominant compounds constituting more than 30% of total EO (linalool, estragole, citral, or methyl cinnamate), the summation constraint has minimal distorting effect on individual variances. Analyses were nevertheless repeated after arcsine square-root transformation; rankings and significance conclusions were unchanged. Untransformed values are therefore reported throughout for ease of interpretation, consistent with established practice in multi-genotype basil EO studies.\u003c/p\u003e \u003cp\u003eDuncan's multiple range test was selected because the primary objective was genotypic discrimination within the fixed set of 12 tested genotypes, rather than control of familywise error across a large set of random comparisons. This choice is consistent with established practice in aromatic crop genotype evaluation. As a robustness check, all pairwise conclusions (e.g., Bursa vs. Eskişehir and Tokat for EO yield; R-10A estragole stability vs. R-17 citral; R-16 and R-20 linalool stability rankings) were verified using Tukey's HSD at P\u0026thinsp;\u0026le;\u0026thinsp;0.01 and remained unchanged (i.e., no genotype pair that was significantly different under Duncan became non-significant under Tukey, confirming that the less conservative Duncan approach did not inflate Type I errors in this dataset). All analyses were performed using SAS 9.4 (PROC GLM with MEANS statement; SAS Institute, Cary, NC, USA). ANOVA F-ratios, degrees of freedom, and probability values for main effects and interactions across all three experimental layers are provided as Online Resource 1 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eChemotype stability was quantified by the CV (%) of the dominant compound across three locations and two years (Layer 1 \u0026ndash; ecological variation; n\u0026thinsp;=\u0026thinsp;6 environment\u0026ndash;year combinations per genotype), following the multi-environment crop evaluation convention (Zobel et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1988\u003c/span\u003e): CV\u0026thinsp;\u0026lt;\u0026thinsp;10% = highly stable; 10\u0026ndash;20% = moderately stable; CV\u0026thinsp;\u0026ge;\u0026thinsp;20% = variable.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eEssential oil yield\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003eEcological variation (Layer 1)\u003c/h2\u003e \u003cp\u003eEO yield varied significantly (P\u0026thinsp;\u0026le;\u0026thinsp;0.01) among genotypes, locations, and years, and the genotype \u0026times; location interaction was significant (P\u0026thinsp;\u0026le;\u0026thinsp;0.01), indicating differential genotypic responses to environmental conditions. The three-way G\u0026times;L\u0026times;Yr interaction was also significant (P\u0026thinsp;\u0026le;\u0026thinsp;0.05), though with modest statistical power (df\u0026thinsp;=\u0026thinsp;22, 120); accordingly, interpretation focuses on main effects and two-way interactions (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Across all genotypes and years, Bursa produced the highest mean EO yield (0.98 mL\u0026middot;100 g⁻\u0026sup1; DW), followed by Eskişehir (0.74) and Tokat (0.72). Year 2 produced significantly higher yields than Year 1 (0.88 vs. 0.75 mL\u0026middot;100 g⁻\u0026sup1; DW; P\u0026thinsp;\u0026le;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEssential oil (EO) yield (mL\u0026middot;100 g⁻\u0026sup1; dry weight) of 12 basil genotypes across three locations over two years (Layer 1 - ecological variation).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBursa Year 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBursa Year 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEskişehir Year 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEskişehir Year 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTokat Year 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTokat Year 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.72 efg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-3k\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.89 d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.67 fg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-10A\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.26 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-15\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.13 bc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-16\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.61 gh\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-17\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.88 d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-19\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.86 de\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-20\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.51 h\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-23\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.33 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eY-7\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.35 i\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eY-15\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.52 h\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong genotypes, R-23 had the highest two-year mean EO yield (1.33 mL\u0026middot;100 g⁻\u0026sup1; DW), followed by R-10A (1.26) and R-15 (1.13), while Y-7 had the lowest (0.35). The genotype \u0026times; location interaction was particularly notable for R-10A, R-15, and R-23, which showed markedly higher yields at Bursa. Genotype R-4 maintained relatively stable yields across all three sites (range: 0.53\u0026ndash;0.86 mL\u0026middot;100 g⁻\u0026sup1; DW), suggesting high environmental stability for yield. These findings are consistent with multi-location yield stability analyses reported by Kholiya et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and Tursun and Telci (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eOntogenetic variation (Layer 2)\u003c/h2\u003e \u003cp\u003eDevelopmental stage had a significant effect (P\u0026thinsp;\u0026le;\u0026thinsp;0.01) on EO yield (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Pre-flowering plants produced the lowest mean yield (0.78 mL\u0026middot;100 g⁻\u0026sup1; DW), while onset of flowering and full-flowering stages produced similar and significantly higher values (0.98 and 1.00, respectively). Most genotypes followed the general trend of increasing EO yield with advancing maturity, consistent with the role of glandular trichome development in EO accumulation (Gang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), and aligning with recent studies by Mkaddem Mounira et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and Corrado et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, R-10A reached its maximum yield at onset of flowering rather than full flowering (1.58\u0026ndash;1.61 vs. 1.38\u0026ndash;1.51 mL\u0026middot;100 g⁻\u0026sup1; DW in Year 1 and Year 2, respectively), which may reflect the earlier peak accumulation of phenylpropanoid-dominated EOs reported in basil (Iijima et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004a\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEssential oil (EO) yield (mL\u0026middot;100 g⁻\u0026sup1; dry weight) of 12 basil genotypes across three developmental stages at Bursa (Layer 2 - ontogenetic variation).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePF Year 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePF Year 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOF Year 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eOF Year 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFF Year 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFF Year 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCV (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eStage mean\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e14.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-3k\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e24.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e21.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-10A\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e16.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-15\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e17.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-16\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e25.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-17\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e23.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-19\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e20.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-20\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e15.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-23\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e21.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eY-7\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e18.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eY-15\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e37.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSeasonal variation (Layer 3)\u003c/h2\u003e \u003cp\u003eAutumn harvests produced significantly higher EO yields than summer harvests in both years (0.72 vs. 0.58 mL\u0026middot;100 g⁻\u0026sup1; DW; P\u0026thinsp;\u0026le;\u0026thinsp;0.01; Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The year \u0026times; season interaction was significant, with the difference more pronounced in Year 2 (0.82 vs. 0.58) than in Year 1 (0.62 vs. 0.57). All genotypes produced higher EO yields in autumn, supporting the value of double-cropping strategies reported by Corrado et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and Ciriello et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). R-23 showed the largest seasonal difference, while R-17 was relatively stable across seasons.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEssential oil (EO) yield (mL\u0026middot;100 g⁻\u0026sup1; dry weight) of 12 basil genotypes across two harvest seasons at Tokat (Layer 3 - seasonal variation).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSummer Year 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSummer Year 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAutumn Year 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAutumn Year 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSummer mean\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAutumn mean\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCV (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e10.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-3k\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e36.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e15.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-10A\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e6.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-15\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e20.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-16\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e20.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-17\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-19\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e16.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-20\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e32.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eR-23\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e37.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eY-7\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e49.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eY-15\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e20.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEssential oil composition\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003eChemotype overview\u003c/h2\u003e \u003cp\u003eGC-MS analysis confirmed four distinct chemotypes in the evaluated genotypes, with Layer 1 compositional summaries presented in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e: (i) linalool chemotype (nine genotypes: R-1, R-3k, R-4, R-15, R-16, R-19, R-20, Y-7, Y-15; note: R-4 is classified as linalool-type based on its dominant compound but uniquely co-accumulates methyl eugenol at 9.33%) with linalool ranging from 51.7 to 71.2% (grand means across six environment\u0026ndash;year combinations; Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e); (ii) estragole chemotype (R-10A) with estragole at 93.5%; (iii) citral chemotype (R-17; citral reported as the sum of its geometric isomers neral [citral b] and geranial [citral a]) with citral (geranial\u0026thinsp;+\u0026thinsp;neral) at 75%; and (iv) methyl cinnamate chemotype (R-23) with methyl cinnamate at 43.1% and linalool as the major secondary constituent at 34.9%. Other notable secondary compounds included eugenol (up to 13.7% in Y-7), α-bergamotene (up to 8.37% in R-19), and δ-cadinene (up to 8.45% in R-20). This chemotypic diversity mirrors the broad chemical variation documented by Gurkan and Hayaloglu (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and Bajomo et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) for commercially available basil varieties, and is consistent with the volatile profiles reported by Qasem et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMajor essential oil (EO) compound relative proportion (%, uncorrected FID peak area) in basil genotypes across three locations and two years (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) across six environment-year combinations; Layer 1 - ecological variation).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChemotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBursa Year 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEskişehir Year 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTokat Year 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBursa Year 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEskişehir Year 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTokat Year 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003e\u003cb\u003eLinalool\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eR-1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e49.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e59.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e77.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e56.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e56.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e56.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e59.30\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eR-3k\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e62.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e66.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e80.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e54.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e56.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e66.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e64.40\u0026thinsp;\u0026plusmn;\u0026thinsp;9.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eR-4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e88.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e48.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e53.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e55.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e55.20\u0026thinsp;\u0026plusmn;\u0026thinsp;17.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eR-15\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e35.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e63.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e85.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e67.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e66.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e62.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e63.50\u0026thinsp;\u0026plusmn;\u0026thinsp;16.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eR-16\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e66.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e72.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e79.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e64.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e69.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e73.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e71.20\u0026thinsp;\u0026plusmn;\u0026thinsp;5.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eR-19\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e53.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e73.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e47.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e51.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e55.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e55.50\u0026thinsp;\u0026plusmn;\u0026thinsp;9.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eR-20\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e75.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e69.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e82.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e62.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e59.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e73.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e70.60\u0026thinsp;\u0026plusmn;\u0026thinsp;8.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eY-7\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e68.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e60.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e44.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e44.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e50.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e52.10\u0026thinsp;\u0026plusmn;\u0026thinsp;10.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eY-15\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e50.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e53.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e61.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e50.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e46.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e46.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e51.70\u0026thinsp;\u0026plusmn;\u0026thinsp;5.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEstragole\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eR-10A\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e91.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e98.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e92.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e92.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e91.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e93.50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCitral\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eR-17\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e77.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e74.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e89.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e60.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e77.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e75.00\u0026thinsp;\u0026plusmn;\u0026thinsp;9.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMethyl cinnamate\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eR-23\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e51.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e31.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e35.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e49.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e50.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e43.10\u0026thinsp;\u0026plusmn;\u0026thinsp;8.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMethyl eugenol\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eR-4\u0026dagger;\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e9.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e9.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e9.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e9.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eLinalool chemotype \u0026ndash; environmental variation\u003c/h2\u003e \u003cp\u003eLinalool content in the nine linalool-type genotypes varied considerably across environments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In Layer 1, the mean linalool content was highest at Tokat (Year 1: 76.86%, Year 2: 59.85%), followed by Eskişehir and Bursa. This inverse relationship between EO yield and linalool proportion may reflect a dilution effect, wherein higher total EO production at warmer sites is accompanied by proportionally greater accumulation of minor terpenes (Marotti et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1996\u003c/span\u003e), consistent with findings of Tursun and Telci (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIndividual genotypes differed markedly in linalool stability. R-16 and R-20 maintained relatively high and stable linalool proportions across all three locations (R-16: 64.54\u0026ndash;79.89%; R-20: 59.45\u0026ndash;82.96%). In contrast, R-4 showed the greatest location-dependent variation (34.49\u0026ndash;88.43%), indicating high G \u0026times; E interaction for this trait. In Layer 2 (ontogenetic variation), linalool content showed a general decline from pre-flowering to full flowering (Year 1 means: 57.23%, 55.38%, 52.06%), consistent with findings of Eskandarzade et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and Beatović et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The phenolic composition and maturity interactions reported by Lawson et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) further support the role of developmental stage in secondary metabolite accumulation. In Layer 3 (seasonal), linalool content was generally similar between summer and autumn harvests (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e), suggesting strong genetic control over linalool biosynthesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEstragole chemotype \u0026ndash; environmental stability\u003c/h2\u003e \u003cp\u003eGenotype R-10A maintained remarkably high and stable estragole proportions across all experimental layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). In Layer 1, estragole ranged from 91.34 to 98.13% (Year 1 vs. Year 2 values shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In Layer 2, estragole ranged from 86.8 to 94.47%. In Layer 3, estragole ranged from 88.62 to 98.72%. The Layer 1 CV was 2.8%, confirming exceptional compositional stability consistent with the strong genetic determination of the estragole chemotype reported by Telci et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and corroborated by recent chemotyping studies (Kirci et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Bajomo et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCitral chemotype \u0026ndash; environmental sensitivity\u003c/h2\u003e \u003cp\u003eThe citral chemotype (R-17) showed substantial sensitivity across experimental layers, particularly in ontogenetic responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), although its Layer 1 CV (12.9%) indicated only moderate ecological instability. Citral content ranged from 60.12 to 89.54% in Layer 1 and from 37.17 to 78.16% in Layer 2, with dramatic year-to-year fluctuation in the pre-flowering stage (Year 1: 78.16% vs. Year 2: 37.17%). The Layer 1 CV was 12.9% (moderately stable), although inclusion of ontogenetic data substantially increased overall variability. This instability may be related to the environmental sensitivity of geraniol dehydrogenase, which catalyses the oxidation of geraniol to citral (Iijima et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2004b\u003c/span\u003e), and is consistent with the broad compositional variation observed for citral-type basil accessions by Avetisyan et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe methyl cinnamate chemotype (R-23) exhibited variable stability at the classification threshold (Layer 1 CV\u0026thinsp;=\u0026thinsp;20.0%; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec; Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), consistent with environmental sensitivity of methyl cinnamate accumulation in Lamiaceae (Telci et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eChemotype stability assessment based on the coefficient of variation (CV%) of the dominant compound in Layer 1 (ecological variation; three locations \u0026times; two years) for basil genotypes studied.\u003c/b\u003e n\u0026thinsp;=\u0026thinsp;number of genotypes per chemotype. CV (%) calculated across three locations and two years (Layer 1 \u0026ndash; ecological variation; n\u0026thinsp;=\u0026thinsp;6 environment\u0026ndash;year combinations per genotype). Stability thresholds following multi-environment crop evaluation convention (Zobel et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1988\u003c/span\u003e): CV\u0026thinsp;\u0026lt;\u0026thinsp;10% = highly stable; 10\u0026ndash;20% = moderately stable; CV\u0026thinsp;\u0026ge;\u0026thinsp;20% = variable. Note: CV estimates are based on n\u0026thinsp;=\u0026thinsp;6 environment-year means per genotype; confidence intervals may be wide for very low CV values.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChemotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDominant compound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCV% range\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMean CV%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eStability class\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEstragole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEstragole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHighly stable (CV\u0026thinsp;\u0026lt;\u0026thinsp;10%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLinalool\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLinalool\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.8\u0026ndash;32.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e17.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eModerate to variable\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethyl cinnamate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethyl cinnamate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVariable (CV\u0026thinsp;\u0026ge;\u0026thinsp;20%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCitral\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCitral (geranial\u0026thinsp;+\u0026thinsp;neral)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eModerate (10\u0026ndash;20%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEcological drivers of essential oil yield\u003c/h2\u003e \u003cp\u003eThe superiority of Bursa for EO yield may be partly attributed to its warmer and more humid microclimate. Growing-season mean temperature was 21.5\u0026deg;C at Bursa versus 18.1\u0026deg;C at Eskişehir and 19.7\u0026deg;C (two-year mean) at Tokat; relative humidity averaged 66.5% at Bursa, 61.1% at Eskişehir, and 52.2% at Tokat (Online Resource 1, Table S3), though other site-specific factors \u0026ndash; including the well-structured clay-loam soils characteristic of the Bursa plain, longer effective sunshine duration at lower altitude (120 m), and irrigation management \u0026ndash; cannot be excluded. Eskişehir (789 m altitude; drier, continental) and Tokat (594 m; semi-arid) represent substantially different growing conditions, and the altitude gradient of 669 m between the lowest (Bursa) and highest (Eskişehir) site likely contributes to the observed EO yield gradient through temperature and growing season effects. Temperature is a primary driver of EO biosynthesis in Lamiaceae, as monoterpene and sesquiterpene synthase activities are known to be temperature-dependent (Sangwan et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). It should be noted that drip irrigation was applied as needed without standardised protocols across locations; differential water supply may have contributed to the observed location effects alongside climatic differences. Hussain et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) reported similar positive correlations between environmental temperature and basil EO yield. Tursun and Telci (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) further demonstrated that elevated CO₂ and temperature significantly modulate EO composition in purple basil. The significant genotype \u0026times; location interaction confirms that cultivar selection must be location-specific for optimal EO production, as also emphasised by Rahimi et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and Mulugeta et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) in the context of environmental stress responses. The comprehensive review by da Silva et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) further contextualises how extraction method and environmental conditions jointly determine basil EO yield and composition in food applications.\u003c/p\u003e \u003cp\u003eThe between-year variation in EO yield (Year 2\u0026thinsp;\u0026gt;\u0026thinsp;Year 1) across all locations underscores the importance of multi-year trials for reliable cultivar recommendations. Because Year was treated as a fixed effect, the conclusions strictly apply to the conditions tested and should not be generalised beyond the two-year window without further validation. Single-year data may substantially misrepresent a genotype's productive potential, a limitation commonly encountered in aromatic crop evaluation (Tena et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The consistent year effect further supports treating year as a design factor rather than a random nuisance term in the present fixed-effects model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eOntogenetic patterns and harvest timing\u003c/h2\u003e \u003cp\u003eThe 0.20 mL\u0026middot;100 g⁻\u0026sup1; DW increase in EO yield from the vegetative (0.78) to the onset of flowering stage (0.98), without a further significant increase at full flowering, provides clear harvest timing guidance. This pattern is consistent with the developmental trajectory of glandular trichomes as the primary site of EO biosynthesis and storage (Gang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Beatović et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and Eskandarzade et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) reported similar ontogenetic patterns in Serbian and Iranian basil cultivars. The ontogenetic decline in linalool content, combined with no significant additional yield gain at full flowering, supports harvesting at onset of flowering to maximise both EO yield and linalool quality. This recommendation aligns with recent findings by Kholiya et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and Mkaddem Mounira et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For the estragole chemotype, the earlier yield peak at onset of flowering provides a practical distinction for production scheduling.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eSeasonal variation and double-cropping potential\u003c/h2\u003e \u003cp\u003eThe significantly higher EO yield in autumn harvests compared to summer harvests at Tokat supports the agronomic viability of double-cropping strategies. Corrado et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) reported analogous harvest sequence effects on basil metabolic profiles, noting that successive harvests modify both yield and quality. Ciriello et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) demonstrated that the interaction between genotype and harvest sequence significantly affects phenolic acid and aroma profiles in Genovese basil, highlighting the need to optimise genotype\u0026ndash;harvest scheduling combinations. The relatively stable EO composition across seasons in linalool-type genotypes suggests that autumn double-cropping can deliver comparable quality to the primary summer harvest, a commercially relevant finding for the basil food industry (Hamid et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Hallmann and Ponder \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eChemotype stability and implications for secondary metabolite management\u003c/h2\u003e \u003cp\u003eThe three-layer experimental design employed here provides a more comprehensive picture of chemotype stability than conventional single-dimension G\u0026times;E studies, which typically assess only spatial variation. By simultaneously examining ecological, ontogenetic and seasonal effects, the present study reveals that compositional stability varies not only among genotypes but also among the dimensions of environmental variation tested. The CV-based stability assessment reveals a clear hierarchy of environmental sensitivity among basil chemotypes. The estragole chemotype (Layer 1 CV\u0026thinsp;=\u0026thinsp;2.8%) exhibited near-absolute compositional stability, suggesting strong genetic canalization of phenylpropanoid biosynthesis with minimal environmental modulation. This aligns with Telci et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), who noted the consistency of estragole-dominant chemotypes across Turkish growing conditions, and with recent chemotyping studies (Kirci et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Bajomo et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In contrast, the citral chemotype (Layer 1 CV\u0026thinsp;=\u0026thinsp;12.9%) showed substantial environmental sensitivity, particularly across ontogenetic stages, consistent with the known variability of citral accumulation (Iijima et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2004b\u003c/span\u003e) and the instability of geraniol-derived compounds under fluctuating temperature and moisture regimes (Tursun and Telci \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It should be noted that the estragole, citral, and methyl cinnamate chemotypes are each represented by a single genotype (R-10A, R-17, and R-23, respectively); consequently, the stability values reported for these chemotypes reflect individual genotypic stability rather than chemotype-level generalisations. Additional genotypes within each non-linalool chemotype would be needed to confirm whether the observed stability patterns are chemotype-intrinsic.\u003c/p\u003e \u003cp\u003eWithin the linalool chemotype, R-16 (SD\u0026thinsp;=\u0026thinsp;5.6%) emerged as the only highly stable linalool genotype, while R-20 (SD\u0026thinsp;=\u0026thinsp;8.8%) also showed comparatively low variability, together representing the most stable pair within the linalool chemotype. In contrast, R-4 (SD\u0026thinsp;=\u0026thinsp;17.9%) showed the greatest compositional instability for linalool despite stable EO yield, confirming that yield stability and compositional stability are not necessarily correlated. The SD values for linalool genotypes are reported in absolute percentage points (rather than CV%) because the mean linalool content varies substantially across genotypes, making CV-based comparison misleading. It should be noted that all conclusions drawn regarding environmental (in)stability apply within the specific set of three Turkish locations, two growing years, and management conditions tested in the present study. It is acknowledged that quantitative EO data were obtained without FID response factor correction, representing relative rather than absolute proportions. This approach is widely accepted in multi-genotype EO characterisation studies (Šovljanski et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Abdoul-Latif et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For the dominant compounds verified against authentic standards, the relative FID response is expected to approximate mass proportions within about\u0026thinsp;\u0026plusmn;\u0026thinsp;5% for the monoterpene and phenylpropanoid classes studied.\u003c/p\u003e \u003cp\u003eIt is acknowledged that CV-based stability, while practical and easily interpretable, has inherent limitations: it is sensitive to the mean value (genotypes with low mean proportions tend to have inflated CV%) and does not capture the pattern of instability (e.g., consistent direction vs. unpredictable fluctuation). Although the CV reported in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e is based on Layer 1 (ecological variation) alone, the three locations span approximately 8\u0026deg; of longitude and 670 m of altitude; the inclusion of ontogenetic and seasonal layers in the broader study design shows that additional variance sources exist beyond the ecological dimension captured by CV. These considerations should be borne in mind when extrapolating the stability rankings to production environments outside the tested range.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eFood safety implications of estragole and methyl eugenol variation\u003c/h2\u003e \u003cp\u003eThe margin of exposure (MOE) calculations presented here are illustrative only and are not intended for formal risk assessment because several assumptions were not experimentally determined (including the fresh-to-dry weight ratio). Using a nominal 5:1 fresh-to-dry ratio and typical fresh basil intake of 1\u0026ndash;2 g per eating occasion, estimated MOEs for estragole in the estragole chemotype were generally above 10,000, whereas methyl eugenol in R-4 yielded lower MOEs. A high-consumer scenario (e.g., 10 g fresh basil per occasion) would substantially reduce MOEs; therefore, chemotype-based cultivar selection remains the most practical risk-management strategy. Regulation (EC) No. 1334/2008 maximum levels apply to estragole in the final food product as consumed, not to the raw herb or essential oil.\u003c/p\u003e \u003cp\u003eGiven the genotoxic and carcinogenic classification of these phenylpropanoids, the MOE framework should be interpreted cautiously and chemotype-based cultivar selection is recommended.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis three-layer field study \u0026ndash; encompassing 12 genotypes, three Turkish locations, three developmental stages, two harvest seasons, and two years (144 unique EO profiles; 192 layer-specific observations) \u0026ndash; provides the first comprehensive characterisation of environmental, ontogenetic, and seasonal modulation of essential oil chemotype expression in basil. Key findings are: (i) Bursa optimises EO yield (0.98 mL\u0026middot;100 g⁻\u0026sup1; DW) while Tokat maximises linalool proportion; (ii) onset of flowering is the optimal harvest stage for both yield and quality; (iii) autumn harvests support double-cropping with comparable linalool quality; (iv) the estragole chemotype is exceptionally stable across ecological environments (Layer 1 CV\u0026thinsp;=\u0026thinsp;2.8%) while the citral (12.9%) and methyl cinnamate (20.0%) chemotypes show moderate to variable stability; (v) R-16 is the most stable linalool genotype (SD\u0026thinsp;=\u0026thinsp;5.6%). These results indicate that environment \u0026times; harvest-stage interactions modulate secondary metabolite accumulation in a chemotype-dependent manner and that high-phenylpropanoid chemotypes (e.g., estragole and methyl eugenol) are primarily genotype-driven and should be managed through chemotype-based cultivar selection to support food safety-oriented cultivar deployment under EU flavouring regulations.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAdem Zorlu: Conceptualization; Methodology; Investigation; Data curation; Formal analysis; Writing \u0026ndash; original draft. İsa Telci: Conceptualization; Supervision; Methodology; Validation; Writing \u0026ndash; review \u0026amp; editing. Mahfuz Elmastaş: Chemical analysis (GC\u0026ndash;MS/GC\u0026ndash;FID); Methodology; Validation; Data interpretation; Writing \u0026ndash; review \u0026amp; editing. Oya Ka\u0026ccedil;ar: Field trials and agronomic management (Bursa); Sampling; Resources; Writing \u0026ndash; review \u0026amp; editing. Zehra Ayta\u0026ccedil;: Field trials and agronomic management (Eskişehir); Sampling; Resources; Writing \u0026ndash; review \u0026amp; editing. Emine Bayram: Field trial support and resources; Supervision; Writing \u0026ndash; review \u0026amp; editing. All authors have read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors thank Tokat Gaziosmanpaşa University, Bursa Uludağ University and Eskişehir Osmangazi University for providing field facilities and technical support during the trials. The authors also thank the Turkish State Meteorological Service for providing climatic data for the trial locations. This research was supported by T\u0026Uuml;BİTAK (The Scientific and Technological Research Council of T\u0026uuml;rkiye; Project No. 111O677).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdoul-Latif FM, Elmi A, Merito A, Nour M, Risler A, Ainane A, Bignon J, Ainane T (2022) Essential oils of O. basilicum and Ocimum americanum L. from Djibouti: chemical composition, antimicrobial and cytotoxicity evaluations. Processes 10:1785. ttps://doi.org/10.3390/pr10091785\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdams RP (2017) Identification of essential oil components by gas chromatography/mass spectrometry, 4.1 ed. Carol Stream: Allured Publishing\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmor G, Sabbah M, Caputo L, Idbella M, De Feo V, Porta R, Fechtali T, Mauriello G (2021) Basil essential oil: composition, antimicrobial properties, and microencapsulation to produce active chitosan films for food packaging. Foods 10:121. ttps://doi.org/10.3390/foods10010121\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAvetisyan A, Markosian A, Petrosyan M, Sahakyan N, Babayan A, Aloyan S, Trchounian A (2017) Chemical composition and some biological activities of the essential oils from basil Ocimum different cultivars. BMC Complement Altern Med 17:60. ttps://doi.org/10.1186/s12906-017-1587-5\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAzizah NS, Irawan B, Kusmoro J, Safriansyah W, Farabi K, Oktavia D, Doni F, Miranti M (2023) Sweet basil (O. basilicum)\u0026mdash;a review of its botany, phytochemistry, pharmacological activities, and biotechnological development. Plants 12:4148. ttps://doi.org/10.3390/plants12244148\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBajomo EM, Aing MS, Ford LS, Niemeyer ED (2022) Chemotyping of commercially available basil (O. basilicum) varieties: cultivar and morphotype influence phenolic acid composition and antioxidant properties. NFS J 29:1\u0026ndash;11. ttps://doi.org/10.1016/j.nfs.2022.09.002\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeatović D, Krstić-Milošević D, Trifunović S, Šiljegović J, Glamočlija J, Ristić M, Jelačić S (2015) Chemical composition, antioxidant and antimicrobial activities of the essential oils of twelve O. basilicum cultivars grown in Serbia. Rec Nat Prod 9:62\u0026ndash;75\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCiriello M, Formisano L, El-Nakhel C, Kyriacou MC, Soteriou GA, Pizzolongo F, Romano R, De Pascale S, Rouphael Y (2021) Genotype and successive harvests interaction affects phenolic acids and aroma profile of Genovese basil for pesto sauce production. Foods 10:278. ttps://doi.org/10.3390/foods10020278\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorrado G, Chiaiese P, Lucini L, Miras-Moreno B, Colla G, Rouphael Y (2020) Successive harvests affect yield, quality and metabolic profile of sweet basil (O. basilicum). Agronomy 10:830. ttps://doi.org/10.3390/agronomy10060830\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eda Silva WMF, Kringel DH, Dierings de Souza EJ, Zavareze E, Dias ARG (2021) Basil essential oil: methods of extraction, chemical composition, biological activities, and food applications. Food Bioprocess Technol 14:1947\u0026ndash;1971. ttps://doi.org/10.1007/s11947-021-02690-3\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Masi L, Siviero P, Esposito C, Castaldo D, Siano F, Laratta B (2006) Assessment of agronomic, chemical and genetic variability in common basil (O. basilicum). Eur Food Res Technol 223:273\u0026ndash;281. ttps://doi.org/10.1007/s00217-005-0201-0\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDhama K, Sharun K, Gugjoo MB, Tiwari R, Alagawany M, Yatoo MI, Thakur P, Iqbal HMN, Chaicumpa W, Michalak I, Elnesr SS, Farag MR (2021) A comprehensive review on chemical profile and pharmacological activities of O. basilicum. Food Rev Int. ttps://doi.org/10.1080/87559129.2021.1900230\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEberhart SA, Russell WA (1966) Stability parameters for comparing varieties. Crop Sci 6:36\u0026ndash;40. ttps://doi.org/10.2135/cropsci1966.0011183X000600010011x\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEFSA CEF Panel (2009) Scientific opinion on methyleugenol in flavourings: consideration of genotoxicity data on methyleugenol and structurally related alkenylbenzenes. EFSA J 7:1016. ttps://doi.org/10.2903/j.efsa.2009.1016\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEFSA CEF Panel (2012) Scientific opinion on estragole in flavourings: consideration of genotoxicity data on estragole and structurally related alkenylbenzenes methyleugenol and elemicin. EFSA J 10:2798. ttps://doi.org/10.2903/j.efsa.2012.2798\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEskandarzade P, Zare Mehrjerdi M, Gruda NS, Aliniaeifard S (2024) Phytochemical compositions and antioxidant activity of green and purple basils altered by light intensity and harvesting time. Heliyon 10:e30931. ttps://doi.org/10.1016/j.heliyon.2024.e30931\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEuropean Medicines Agency (EMA) (2023) Public statement on the use of herbal medicinal products containing estragole (Revision 1). EMA/HMPC/137212/2005 Rev 1 Corr 1. London: HMPC (originally 2005)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEuropean Pharmacopoeia Commission (2010) European Pharmacopoeia, 7th ed. Strasbourg: Council of Europe\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerrante C, Orlando G, Ferro S (2021) Essential oils from Salvia species: variation and biological activity. Nat Prod Res 35:3178\u0026ndash;3191. ttps://doi.org/10.1080/14786419.2019.1698574\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGang DR, Wang J, Dudareva N, Nam KH, Simon JE, Lewinsohn E, Pichersky E (2001) An investigation of the storage and biosynthesis of phenylpropenes in sweet basil. Plant Physiol 125:539\u0026ndash;555. ttps://doi.org/10.1104/pp.125.2.539\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiachino RRA, S\u0026ouml;nmez \u0026Ccedil;, Tonk FA, Bayram E, Y\u0026uuml;ce S, Telci I, Furan MA (2014) RAPD and essential oil characterization of Turkish basil (O. basilicum). Plant Syst Evol 300:2227\u0026ndash;2238. ttps://doi.org/10.1007/s00606-014-1034-5\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrayer RJ, Kite GC, Goldstone FJ, Bryan SE, Paton A, Putievsky E (1996) Infraspecific taxonomy and essential oil chemotypes in sweet basil, O. basilicum. Phytochemistry 43:1033\u0026ndash;1039. ttps://doi.org/10.1016/S0031-9422(96)00429-8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGurav TP, Dholakia BB, Giri AP (2022) A glance at the chemodiversity of Ocimum species: trends, implications, and strategies for the quality and yield improvement of essential oil. Phytochem Rev 21:947\u0026ndash;979. ttps://doi.org/10.1007/s11101-021-09767-z\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGurkan H, Hayaloglu AA (2023) Changes in volatiles and essential oil composition of three organs of purple basil (O. basilicum) by GC-MS combined with multivariate statistical approach. Food Chem Adv 2:100292. ttps://doi.org/10.1016/j.focha.2023.100292\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHallmann E, Ponder A (2024) A long-term study on chemical compounds and their location in sweet basil leaves from organic and conventional producers. Foods 13:383. ttps://doi.org/10.3390/foods13030383\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamid S, Fadloun Oukil N, Moussa H, Djihad N, Mr\u0026oacute;z M, Kusznierewicz B, Attia A, Djenadi K, Mahdjoub MM, Bouhenna MM, Chebrouk F (2024) Chemical and biological characterization of O. basilicum phenolic extract and essential oil derived through ultrasound and microwave-assisted extraction techniques. Food Biosci 60:104359. ttps://doi.org/10.1016/j.fbio.2024.104359\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHussain AI, Anwar F, Sherazi STH, Przybylski R (2008) Chemical composition, antioxidant and antimicrobial activities of basil (O. basilicum) essential oils depend on seasonal variations. Food Chem 108:986\u0026ndash;995. ttps://doi.org/10.1016/j.foodchem.2007.12.010\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIijima Y, Davidovich-Rikanati R, Fridman E, Gang DR, Bar E, Lewinsohn E, Pichersky E (2004a) The biochemical and molecular basis for the divergent patterns in the biosynthesis of terpenes and phenylpropenes in the peltate glands of three cultivars of basil. Plant Physiol 136:3724\u0026ndash;3736. ttps://doi.org/10.1104/pp.104.051318\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIijima Y, Gang DR, Fridman E, Lewinsohn E, Pichersky E (2004b) Characterization of geraniol synthase from the peltate glands of sweet basil. Plant Physiol 134:370\u0026ndash;379. ttps://doi.org/10.1104/pp.103.032946\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJeurissen SM, Punt A, Delatour T, Rietjens IM (2008) Basil extract inhibits sulfotransferase-mediated formation of DNA adducts of the procarcinogen 1\u0026prime;-hydroxyestragole by rat and human liver S9 homogenates and in HepG2 cells. Food Chem Toxicol 46:2296\u0026ndash;2302. ttps://doi.org/10.1016/j.fct.2008.03.010\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang R, Liu J, Liu Q, Jin Z, Zhu H, Han H, Ma X (2024) Comparative analysis of volatile components and sensory profiles of four basil varieties based on HS-SPME and SD coupled with GC-MS. Processes 12:2789. ttps://doi.org/10.3390/pr12122789\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKholiya S, Punethaa A, Chauhan A, Varsha KT, Kumar D, Upadhyaya RK (2022) Essential oil yield and composition of O. basilicum at different phenological stages, plant density and post-harvest drying methods. S Afr J Bot 151:919\u0026ndash;925. ttps://doi.org/10.1016/j.sajb.2022.11.011\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKirci D, Tabanca N, Blythe E, Demirci B (2025) Phytochemical variations in O. basilicum cultivars: essential oil composition and multivariate chemotype differentiation. Rec Nat Prod 19:688\u0026ndash;701\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLabra M, Miele M, Ledda B, Grassi F, Mazzei M, Sala F (2004) Morphological characterization, essential oil composition and DNA genotyping of O. basilicum cultivars. Plant Sci 167:725\u0026ndash;731. ttps://doi.org/10.1016/j.plantsci.2004.04.026\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLawson HP, Aga MF, Niemeyer ED (2025) Plant maturity differentially affects the phenolic composition and antioxidant properties of green basil (O. basilicum) cultivars. ACS Omega 10:47535\u0026ndash;47543\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLukas B, Schmiderer C, Novak J (2015) Essential oil diversity of European Origanum vulgare L. (Lamiaceae). Phytochemistry 119:32\u0026ndash;40. ttps://doi.org/10.1016/j.phytochem.2015.09.007\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMahendran G, Vimolmangkang S (2023) Chemical compositions, antioxidant, antimicrobial, and mosquito larvicidal activity of Ocimum americanum L. and O. basilicum leaf essential oils. BMC Complement Med Ther 23:390. ttps://doi.org/10.1186/s12906-023-04228-8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMakri O, Kintzios S (2008) Ocimum sp. (basil): botany, cultivation, pharmaceutical properties, and biotechnology. J Herbs Spices Med Plants 13:123\u0026ndash;150. ttps://doi.org/10.1300/J044v13n03_10\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarotti M, Piccaglia R, Giovanelli E (1996) Differences in essential oil composition of basil (O. basilicum) Italian cultivars related to morphological characteristics. J Agric Food Chem 44:3926\u0026ndash;3929. ttps://doi.org/10.1021/jf9601067\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMkaddem Mounira G, Zrig A, Ben Abdallah M, Romdhane M, Okla MK, Al-Hashimi A, Alwase YA, Madnay MM, AbdElgayed G, Asard H (2022) Essential oil composition and antioxidant and antifungal activities of two varieties of O. basilicum at two phenological stages. Agronomy 12:825. ttps://doi.org/10.3390/agronomy12040825\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMulugeta SM, S\u0026aacute;rosi S, Rad\u0026aacute;csi P (2023) Physio-morphological trait and bioactive constituents of Ocimum species under drought stress. Ind Crops Prod 206:117607. ttps://doi.org/10.1016/j.indcrop.2023.117607\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePimentel LAG, da Silva MA, de Medeiros SDS, Luz JMQ, Sala FC (2023) Agronomic, sensory and essential oil characterization of basil (O. basilicum) accessions. Horticulturae 9:831. ttps://doi.org/10.3390/horticulturae9070831\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQasem A, Assaggaf H, Mrabti HN, Minshawi F, Rajab BS, Attar AA, Alyamani RA, Hamed M, Mrabti NN, El Baaboua A (2023) Determination of chemical composition and investigation of biological activities of O. basilicum. Molecules 28:614. ttps://doi.org/10.3390/molecules28020614\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahimi M, Mortazavi M, Mianabadi A, Debnath S (2023) Evaluation of basil (O. basilicum) accessions under different drought conditions based on yield and physio-biochemical traits. BMC Plant Biol 23:523. ttps://doi.org/10.1186/s12870-023-04540-0\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRegulation (EC) No 1334/2008 of the European Parliament and of the Council on flavourings and certain food ingredients with flavouring properties for use in and on foods. Off J Eur Union L354:34\u0026ndash;50 (2008)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR\u0026iacute;os-Rodr\u0026iacute;guez D, Sahi VP, Nick P (2021) Authentication of holy basil using markers relating to a toxicology-relevant compound. Eur Food Res Technol 248:13\u0026ndash;25. ttps://doi.org/10.1007/s00217-021-03849-w\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSangwan NS, Farooqi AHA, Shabih F, Sangwan RS (2001) Regulation of essential oil production in plants. Plant Growth Regul 34:3\u0026ndash;21. ttps://doi.org/10.1023/A:1013386921596\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScientific Committee on Food (SCF) (2001a) Opinion on estragole (1-allyl-4-methoxybenzene). SCF/CS/FLAV/FLAVOUR/6 ADD2 Final. Brussels: European Commission\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScientific Committee on Food (SCF) (2001b) Opinion on methyleugenol (4-allyl-1,2-dimethoxybenzene). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003eSCF/CS/FLAV/FLAVOUR/4\u003c/span\u003e\u003cspan address=\"http://SCF/CS/FLAV/FLAVOUR/4\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ADD1 Final. Brussels: European Commission\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eŠovljanski O, Saveljić A, Aćimović M, Šeregelj V, Pezo L, Tomić A, Ćetković G, Tešević V (2022) Biological profiling of essential oils and hydrolates of O. basilicum var. Genovese and var. Minimum originated from Serbia. Processes 10:1893. ttps://doi.org/10.3390/pr10101893\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTelci I, Bayram E, Yılmaz G, Avcı B (2006) Variability in essential oil composition of Turkish basils (O. basilicum). Biochem Syst Ecol 34:489\u0026ndash;497. ttps://doi.org/10.1016/j.bse.2006.01.009\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTena E, Goshu F, Mohamad G, Tesfa M, Tesfaye D, Seife A (2019) Genotype \u0026times; environment interaction by AMMI and GGE-biplot analysis for sugar yield in sugarcane. Cogent Food Agric 5:1651925. ttps://doi.org/10.1080/23311932.2019.1651925\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTursun AO, Telci I (2020) The effects of carbon dioxide and temperature on essential oil composition of purple basil (O. basilicum). J Essent Oil Bear Plants 23:1090\u0026ndash;1104. ttps://doi.org/10.1080/0972060X.2020.1854128\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZobel RW, Wright MJ, Gauch HG (1988) Statistical analysis of a yield trial. Agron J 80:388\u0026ndash;393. ttps://doi.org/10.2134/agronj1988.00021962008000030002x\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"genetic-resources-and-crop-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gres","sideBox":"Learn more about [Genetic Resources and Crop Evolution](https://www.springer.com/journal/10722)","snPcode":"10722","submissionUrl":"https://submission.nature.com/new-submission/10722/3","title":"Genetic Resources and Crop Evolution","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Chemotype stability, Essential oil, Genotype × environment interaction, Ocimum basilicum, Secondary metabolite, Terpene biosynthesis","lastPublishedDoi":"10.21203/rs.3.rs-9131432/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9131432/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe environmental stability of essential oil (EO) chemotype expression in basil (Ocimum basilicum L.) is critical for both industrial quality assurance and food safety, yet remains poorly characterised across the multiple dimensions of environmental variation encountered in production. This study characterises the environmental modulation of EO yield and chemotype composition in 12 basil genotypes representing four chemotypes (linalool, estragole, citral, and methyl cinnamate) using a three-layer experimental design: (i) ecological variation across three contrasting Turkish locations (Bursa, Eskişehir, Tokat) over two years, (ii) ontogenetic variation across three developmental stages at Bursa, and (iii) seasonal variation between summer and autumn harvests at Tokat (144 unique EO profiles). EO yield was highest at Bursa (0.98 mL\u0026middot;100 g⁻\u0026sup1; dry weight), increased from pre-flowering to onset of flowering, and was greater in autumn than summer harvests. The estragole chemotype (R-10A) exhibited exceptional compositional stability across ecological environments (Layer 1 CV\u0026thinsp;=\u0026thinsp;2.8%), while the citral (Layer 1 CV\u0026thinsp;=\u0026thinsp;12.9%) and methyl cinnamate (20.0%) chemotypes showed moderate to variable stability. Within the linalool chemotype, R-16 was the most stable genotype (SD\u0026thinsp;=\u0026thinsp;5.6%). The results indicate that chemotype stability is primarily genotype-driven and that environment \u0026times; harvest-stage interactions modulate secondary metabolite accumulation patterns in a chemotype-dependent manner, with implications for cultivar selection and food safety considerations.\u003c/p\u003e","manuscriptTitle":"Essential oil chemotype diversity and environmental stability in Turkish basil (Ocimum basilicum L.) germplasm: a three-layer field evaluation across contrasting ecologies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-19 17:56:47","doi":"10.21203/rs.3.rs-9131432/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-18T19:03:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-18T18:57:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-16T15:50:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"320433139381210655408461810690619502894","date":"2026-03-16T15:05:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"241605264409623872088914037350467309612","date":"2026-03-16T11:58:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-16T09:15:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-16T08:54:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-16T08:53:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Genetic Resources and Crop Evolution","date":"2026-03-15T22:56:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"genetic-resources-and-crop-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gres","sideBox":"Learn more about [Genetic Resources and Crop Evolution](https://www.springer.com/journal/10722)","snPcode":"10722","submissionUrl":"https://submission.nature.com/new-submission/10722/3","title":"Genetic Resources and Crop Evolution","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"508b059f-0647-49b7-91f0-da730826a0c5","owner":[],"postedDate":"March 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-16T07:11:27+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-19 17:56:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9131432","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9131432","identity":"rs-9131432","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}