The Chemical Analysis of Wild Thyme Variability for the Enhanced Production of Bioactive Compounds and Agro-Ecosystem Sustainability in the Mountains of Pistoia (Italy)

Abstract

The Pistoia Mountains exhibit a great variety of flora, particularly rich in aromatic plants, such as juniper, mint, savory, helichrysum, and thyme. Thyme is especially notable for its essential oil, typically displaying high thymol and carvacrol content. While the chemotype of thyme determined by its specific terpene composition is genetically controlled, environmental factors, plant age, and seasonality can influence terpene production. This article investigates the morpho-chemical variability of wild thyme plants collected from two different regions of the Pistoia Mountains, identifying five distinct chemotypes. The gas chromatography–mass spectrometry (GC-MS) technique was used to characterize the chemical profiles and determine the seasonal variation in terpene production, identifying spring and summer as the balsamic period, the optimal time for essential oil collection. Furthermore, high-value thyme clones were preserved through in vitro micropropagation, ensuring chemotype stability. These findings contribute to a deeper understanding of wild thyme biodiversity and provide a foundation for practical applications, including the development of value-added products like herb-infused cheeses, plant and animal disease treatments, and integrated pest management strategies in agricultural systems.

1. Introduction

Over the past two decades, agriculture has evolved significantly due to technological advancements, rising energy demands, and shifting farming practices. These changes have been largely influenced by the environmental and climatic impacts of crop production, along with a growing emphasis on sustainability, particularly through organic farming [1]. Agriculture plays a crucial role in agro-ecosystems, not only as a means of food production but also in natural resource management, landscape preservation, and the conservation of plant biodiversity [2].

However, agricultural intensification has led to the widespread use of biotechnology and chemical inputs. While these advances enhance productivity, they have also contributed to reduced crop biodiversity and increased dependence on agrochemicals [3]. The preference for high-yielding crop varieties has made agricultural systems more vulnerable to ecological shifts, while the extensive use of pesticides and fertilizers, despite their short-term benefits, has led to resistance issues, soil microbiota disruptions, and broader environmental degradation [4]. To address these challenges, more ecologically sustainable approaches are needed to maintain high production levels, while preserving resources for future generations [4,5].

In this context, the Pistoia Mountains region, renowned for its rich biodiversity, serves as a valuable reservoir of plant species, including aromatic plants, which exhibit considerable chemical and genetic variability.

Among the diverse plant species that contribute to agro-ecosystem sustainability, medicinal and aromatic plants (MAPs) stand out for their rich bioactive compounds and essential ecological roles, such as their roles in pollinator attraction, herbivore deterrence, and allelopathic interactions. They are attracting increasing interest due to their biological properties. Beyond their culinary applications, they hold significant potential in various industries, including pharmaceuticals, herbal medicine, cosmetics, and plant protection products [6,7]. As open-field crops, MAPs contribute to multifunctional and sustainable agriculture, requiring minimal energy for cultivation while offering diverse uses, from nutraceutical products and phytonutrients, to supporting land valorization [8].

The cultivation of MAPs, when carried out using an integrated and sustainable approach, can enhance biodiversity in agro-ecosystems while also aiding in the restoration of degraded and marginal lands [9]. The targeted cultivation and conservation of these plants is essential not only for preserving their natural richness and biodiversity, but also for standardizing parameters for various applications [4,9].

One of the key indicators of MAP quality is the content and composition of the derived essential oils (EOs), which are among the most potent natural pesticides [10,11].

Thyme (Thymus spp.), a member of the Lamiaceae family, is widely recognized for its EO, which is rich in bioactive compounds such as thymol and carvacrol. These compounds not only give thyme its distinctive aroma, but also provide strong antimicrobial properties, making its essential oil a valuable product for the food, cosmetics, and pharmaceutical industries [12,13,14].

The chemical composition of thyme essential oil varies significantly due to multiple factors, including genetic traits, environmental conditions, plant age, and seasonal fluctuations. These elements collectively influence the formation of different chemotypes, defined as mixtures of terpenes with a distinct composition [15,16,17,18]. The diversity of thyme chemotypes plays a significant role in enhancing pest management strategies, offering natural alternatives to synthetic pesticides [19].

Among the different species, Thymus sp. of the serpyllum group (common name, wild thyme) is a perennial, aromatic shrub endemic to the Pistoia Mountains, growing at elevations up to 1000 m above sea level. In our previous work, we characterized the Thymus serpyllum L. population through chemotype analysis, revealing considerable variability across five distinct regions of the Pistoia Mountains [18,20].

This article aims to explore the chemical diversity of wild thyme plants collected across the Mountains of Pistoia, with a specific focus on identifying stable chemotypes and their potential role in enhancing essential oil production and contributing to sustainable agro-ecosystems. Specifically in this region of Italy, despite the increasing interest in the phytochemical diversity of Thymus species, the relationship between seasonal stability of chemotypes and terpene yield remains little explored. It was hypothesized that wild thyme with different terpene profiles exhibits stable chemotypes and differential yield across seasons, which can be leveraged for targeted applications in food, agricultural, and pharmaceutical sectors. Additionally, several scientific questions were addressed, including the identification of the optimal harvesting time, commonly referred to as the balsamic period; the potential association between terpene profile and morphological traits; and the feasibility of conserving high-value aromatic chemotypes through in vitro clonal propagation.

To address these questions, a morpho-chemical characterization was conducted on wild thyme plants collected from two locations in the Pistoia Mountains. Based on their terpene composition, representative plants corresponding to distinct chemotypes were selected and subsequently propagated. This high-value collection is currently maintained both in our experimental plots and in in vitro culture [17,21].

The seasonal monitoring of terpene quantity enabled us to define the balsamic period, crucial for refining essential oil extraction strategies and to maximize both yield and oil quality [21,22]. Our results may aid the production of high-quality wild thyme EO, broadening the potential for developing products such as herb-infused cheeses and natural remedies for plant and animal diseases [23,24,25].

Furthermore, thyme’s EO has well-documented therapeutic properties; many studies on the antimicrobial and anti-inflammatory effects of thymol and carvacrol (both components of thyme essential oil) confirm their efficacy against various pathogens, including Escherichia coli, Staphylococcus aureus, and Alternaria alternata [12,18,25,26,27]. These properties not only enhance thyme’s value as a natural preservative for the food industry, but they also position it as a promising candidate for pharmaceutical applications [13,25].

This work serves as the first crucial step for future research, laying the groundwork for the study of the chemical biodiversity of Thymus serpyllum L., which holds great potential for the development of plant-based agricultural applications.

2. Materials and Methods

2.1. Collection of Wild Thyme and Its Maintenance in Pots

A total of 17 thyme samples of the Thymus serpyllum group were collected in November 2023 from the Pistoiese Apennines (Italy), specifically from two locations: Osservatorio (San Marcello Pistoiese-Piteglio, 1000 m above sea level) and Pracchia (616 m above sea level). The collected plants were analyzed through gas chromatography–mass spectrometry (GC-MS), as described in the following paragraphs. After harvesting, the plants were transferred into pots containing a mixed substrate of peat and perlite at the ratio of 70:30 and maintained in an experimental plot in Sesto Fiorentino (FI) (55 m above sea level). The 17 plants were kept for one year under shade, outdoors, and exposed to seasonal variations. They were irrigated as needed.

2.2. Morphological Analysis

The morphological analysis of wild thyme plants was conducted during the flowering period, following the guidelines of the European Cooperative Programme for Plant Genetic Resources (ECPGR) [28]. Highly discriminative descriptors were selected for the morphometric analysis. Highly discriminative descriptors were selected for the morphometric analysis.

Images of the plants, flowers, and leaves were obtained using a stereomicroscope (Zeiss Stemi 2000 C, Jena, Germany), and morphological traits were measured using ImageJ-win 32 v1.54k software. For each characteristic, at least three measurements per plant were taken.

2.3. Terpene Identification and Quantification by Gas Chromatography Coupled to Mass Spectrometry (GC-MS) Analyses

The GC-MS analysis was conducted on plant material at the time of collection and across different seasons. Specifically, wild thyme plants were analyzed over the course of one year, from autumn to summer. All chemicals and analytical-grade GC standards were obtained from Sigma-Aldrich (St. Louis, MO, USA). Inert gases (He and N₂, 99.999% purity) were supplied by the SOL Gas Company (Monza, Italy). The terpene extraction and GC-MS analysis were performed following the method described by Bellumori et al., with some modifications [29]. Approximately 0.5 g of fresh plant material from each sample was subjected to liquid extraction with 1 mL of heptane, containing a known amount of tridecane as an internal standard, in a screw-cap GC-MS glass vial. The sample was sonicated (three cycles of 15 min each) and was then agitated overnight. After centrifugation at 2057 relative centrifugal force (RCF) for 10 min, 100 μL of the supernatant was collected for GC-MS analysis.

The analysis was performed using an Agilent 7820A Gas Chromatograph (GC) (Agilent, Palo Alto, CA, USA) equipped with a Mass Selective Detector (MSD) model 5977E (Agilent, Palo Alto, CA, USA). One microliter of the heptane extract was injected into a split/splitless injector operating in splitless mode. A Gerstel MPS2 XL autosampler (Gerstel, Mülheim an der Ruhr, Germany) with a liquid injection option was used. Helium was used as the carrier gas (pressure: 33 psi; flow rate: 1.2 mL/min). The chromatographic conditions were as follows: splitless injector set at 240 °C; Agilent (Palo Alto, CA, USA) DB-Wax UI column (60 m, 0.25 mm i.d., 0.5 μm df); oven temperature program—initial hold at 40 °C for 5 min, followed by an increase of 3 °C/min to 150 °C and then 20 °C/min to 240 °C with a hold time of 10 min. The mass spectrometer operated in electron ionization mode (70 eV), scanning the m/z range of 29–330 at a rate of three scans per second [29].

Data processing was performed using the official NIST MS library (NIST14) within the MassHunter WorkStation (B.07.01 version) software (Agilent, Palo Alto, CA, USA). Peak identification was confirmed using authentic standards analyzed under the same conditions. Total Ion Current (TIC) chromatograms were recorded, and the relative concentration of each identified compound was calculated as the peak area relative to the total area of all identified peaks. To quantify individual terpenes, calibration curves were generated by injecting known concentrations of authentic standards (Sigma-Aldrich, St. Louis, MO, USA) into the GC-MS system. The terpene content was expressed as PPM (µg/g of fresh or dry weight). The absolute terpene yield per plant, intended as the sum of all the single molecules identified, was also calculated in mg, accounting for the dry biomass of the wild thyme samples. Data are the means of three determinations (SD < 5%).

2.4. In Vitro Micropropagation of Wild Thyme Plants

Wild thyme plant material was collected from the experimental plot in Sesto Fiorentino (FI) during the early growing season (April/May). The Thymus micropropagation protocol was adapted from Mendes et al., 2013 [30]. Specifically, stem segments measuring 2–3 cm in length, containing axillary buds, underwent a sterilization process. Initially, the explants were rinsed with water containing a few drops of detergent and thoroughly washed under running tap water for 30 min. Subsequently, the wild thyme segments were disinfected by immersing them in 70% (v/v) ethanol for 15 s, followed by multiple washes with sterile distilled water under aseptic conditions within a laminar airflow cabinet. A second sterilization step involved treatment with a 2% (v/v) sodium hypochlorite solution for 10 min, after which the segments were rinsed three times with sterile distilled water. To ensure sterility, the explants were first cultured in basal autoclaved Murashige and Skoog (MS) agar medium for 7–10 days [29]. Following this period, contamination-free segments were transferred to MS medium supplemented with 1 mg/L 6-benzylaminopurine (BAP), 0.25 mg/L indole-3-butyric acid (IBA), 3% (w/v) sucrose, and 6% (w/v) agar. The pH of the medium was adjusted to 5.7–5.8 before autoclaving at 121 °C for 21 min. The cultures were maintained in a growth chamber at 24 ± 2 °C under a 16 h photoperiod. Subcultures were carried out every 6–8 weeks to ensure healthy growth of the explants, with plantlets reaching an average of 4–6 cm in height.

To evaluate whether the chemotype and terpene profile remained stable over time, three representative chemotypes were chosen and tested at different time points. Namely, the chemotypes were thymol (thy3), thymol/carvacrol (thy9), and geraniol (thy14). GC-MS analyses were performed six months and one year after the in vitro establishment, as described in the previous section. The results were compared to the terpene profiles obtained from genetically identical plants grown in pots.

2.5. Statistical Analysis

Cluster analyses were performed on the base of GC-MS normalized data representative of our experimental population (N = 17), aiming to assess the similarity between individuals and the presence of groups having different expressions of terpenes in the wild thyme population investigated. The dissimilarity data matrix was built using the Manhattan distance [31]. Subsequently, hierarchical clustering was applied to the obtained distance matrix by using a single-linkage method, which merges clusters based on the minimum distance between points from different groups. Cluster outcomes were represented by a dendrogram, and the goodness of their fit was tested by using Cophenetic Correlation Coefficient (CCC), considering results of a CCC > 0.8 as accurately representing the data. Preliminary silhouette index analyses were conducted to obtain the optimal number of clusters describing the population. The subdivision into groups based on volatile chemical profiles, obtained through the cluster analysis, was used to verify if a statistically significant difference between groups exists. For this analysis, a new dissimilarity matrix was constructed using the Bray–Curtis method and the Analysis of Similarities (ANOSIM) test with permutations (n_p = 999) was conducted. The R statistics of this test were used to assess if a difference exists among the groups (where R ≈ 0 indicates little or no separation, and R ≈ 1 indicates strong group separation). The results were considered significant if the associated p value was less than 0.05 [32,33].

The morphological data of the measured parameters (N = 17, replicates ≥ 3) were plotted on a bar graph. The results were further analyzed for significant differences using the non-parametric Kruskal–Wallis test, followed by Dunn’s multiple comparison test, considering the thymol chemotype as the standard group. The results were considered statistically significant when the associated p value with test statistics was less than 0.05.

Cluster analyses and statistical analyses on the GC-MS data were performed in the R environment (R Core Team, 2024) [31]; for clustering the basic functions “dist” and “hlust” were used. For ANOSIM analysis, the “anosim” function of the vegan package (version 2.6-10) was used [34].

Morphological data were processed and analyzed in the GraphPad Prism 8.0.1 environment (GraphPad Software, Inc., La Jolla, CA, USA).

3. Results

3.1. Chemical and Morphological Characterization of Wild Thyme Plants

The 17 wild thyme samples collected in autumn 2023 from Pracchia and Osservatorio (Pistoia Apennines) were analyzed via GC-MS to determine their terpene profiles. These data were used to perform analyses which allowed for the identification of distinct chemotypes within the population. Through the cluster analyses, four distinct chemotypes were identified, as illustrated in Figure 1. Statistical group difference was detected as significant by ANOSIM testing (Supplementary Table S1).

The further evaluation of terpene expression, in terms of quantity (absolute and relative), allowed for the comparison of the different terpene levels within the groups. Plants Thy5 and Thy11 belong to the sabinene hydrate chemotype, Thy14 to the geraniol chemotype, and Thy16 is classified within the linalool chemotype. The remaining plants predominantly produce thymol as their main terpene, considered along with its precursors γ-terpinene and p-cymene.

Within the thymol-dominant group, plant Thy4 stands out due to its comparable amounts of thymol and carvacrol. Since carvacrol is a thymol isomer whose synthesis is influenced by environmental factors, Thy4 can be considered a subgroup of the thymol chemotype. As further discussed in the next section, plants Thy3, Thy9, and Thy13 will also be classified within the thymol/carvacrol chemotype subgroup, as they exhibit both terpenes as main components across different seasons.

During the flowering period, the plants were analyzed for their morphological characteristics (Figure 2).

All measured morphological parameters and their corresponding values are reported in Supplementary Table S2. The data were then examined to determine whether the plants of the different chemotypes identified showed morphological differences compared to the thymol chemotype, the most prevalent one.

Figure 3 highlights the main morphological parameters observed across the chemotypes of wild thyme. The linalool chemotype is characterized by significantly shorter calyx and corolla lengths compared to the thymol chemotype. The geraniol chemotype, on the other hand, has flowers with a significantly shorter calyx length. Finally, plants belonging to both sabinene hydrate chemotype and thymol/carvacrol chemotype display notable differences in leaf morphology, with significantly smaller leaf areas and leaf lengths.

3.2. Chemotype Stability and Optimal Harvesting Season

The GC-MS analyses were performed at the time of collection (autumn 2023) and in the following seasons once the plants had acclimatized to a different climate and altitude (Supplementary Table S3). The representative GC-MS chromatograms illustrate the peak annotated with the corresponding compound names and retention times identified (Supplementary Figure S1). The seasonal terpene analyses carried out across different seasons demonstrate that the chemotype remains stable despite physiological variations due to environmental changes. Specifically, Figure 4 presents graphs for each chemotype described in the previous section. As shown, the most represented terpene for each chemotype remains consistent across seasons, despite the plants being grown in a different environment from their place of origin.

Once the chemotype stability throughout the four seasons had been confirmed, the identification of the balsamic period was subsequently pursued. For all chemotypes, the balsamic period (expressed as the total terpene content per fresh weight) occurs in spring/summer (Figure 5A). Indeed, thymol chemotypes (including the thymol/carvacrol group) reached terpene concentrations of approximately 2300–3300 PPM/fresh weight, while the geraniol and linalool chemotypes recorded intermediate values around 1700–2100 PPM/fresh weight. The sabinene hydrate chemotype showed the lowest yield, with values not exceeding 800 PPM/fresh weight, suggesting a lower biosynthetic capacity for volatile compounds. Specifically, for these two seasons (namely spring and summer), the absolute terpene production (expressed as the terpene content per dry weight) was evaluated to investigate the total production capacity (Figure 5B). As illustrated in the graphs, differences in the yield among chemotypes can be observed. In particular, the thymol and thymol/carvacrol chemotypes maintained the highest terpene yields, exceeding 12,700 PPM/dry weight in some case. Also here, the sabinene hydrate chemotype showed a lower yield (3400 PPM/dry weight) compared to the other chemotypes.

Since spring and summer correspond to the flowering period of wild thyme, an analysis was further conducted to determine whether flowers accumulate more terpenes than other vegetative parts. As shown in Figure 5C, the overall terpene content in the flowers tends to be higher, suggesting that essential oil extraction should be performed during flowering to maximize oil yield and quality. However, an exception was observed in the linalool chemotype, where the flower exhibits a lower terpene concentration than the vegetative parts. This result aligns with the morphological observations (Figure 3A), in which linalool chemotype plants presented smaller flowers, likely resulting in reduced accumulation of floral volatiles.

3.3. In Vitro Maintenance of High-Value Wild Thyme Chemotypes

In vitro culture techniques were successfully applied to maintain selected wild thyme clones identified in the Pistoia Mountains. The morphological and chemical traits were maintained over time. The in vitro seedlings retained typical thyme foliage characteristics such as compact growth and ovate–lanceolate leaves (Figure 6A).

The comparison between in-pot and in vitro data over time shows that the terpene yield remains comparable to that of the potted plant even after one year of in vitro cultivation (Figure 6B). For example, the thymol chemotype (thy3) maintained a yield of approximately 3000 PPM/dry weight in vitro, closely matching the 2000 PPM/dry weight observed in pot-grown wild thyme. Similarly, Figure 6C confirms that the chemotype was conserved—thymol remained the major compound in thy3, geraniol in thy14, and thymol/carvacrol was the most abundant terpene in the thy9 plant.

4. Discussion

The Pistoia Mountains, characterized by their rich endemic flora and fauna, are an important reservoir of biodiversity. This natural wealth offers opportunities to develop innovative products and strategies that support various agro-ecosystem functions including food production, natural resource management, and landscape and plant biodiversity conservation, as well as the cultural, historical, and economic sustainability of rural areas [20,35].

Within this context, the Thymus serpyllum group from the Pistoia Mountains emerges as a wild, endemic aromatic plant showing high biodiversity within its populations. Our recent investigation demonstrated that natural population of wild thyme located in five areas of the Pistoia Mountains, can be subdivided into at least seven groups based on distinct aromatic profiles [18], each potentially suited for specific applications.

In the present study, we focused on a subset of this diversity, analyzing 17 plants collected from two selected areas of the Pistoia Mountains (Pracchia and Osservatorio), representing the previously described variability.

Indeed, the plants sampled were classified into four distinct chemotypes based on gas chromatography–mass spectrometry (GC-MS) findings and supported by cluster and statistical analyses. This classification aligns with prior studies emphasizing the significant role of genetic diversity and environmental factors on essential oil composition in various thyme species [36,37,38,39]. While the five chemotypes identified are representative of the broader variability described, it is important to recognize that the full chemotypic spectrum of the Thymus was not captured. This limits the generalizability of our findings and the possibility of replicating and validating them externally in independent thyme populations. Despite these limitations, the study provides a solid framework for further research. The chemical characterization and initial clonal preservation of selected chemotypes offer a valuable resource for future ecological, pharmacological, and agronomic studies.

The identification of five chemotypes (thymol, thymol/carvacrol, sabinene hydrate, geraniol, and linalool) confirms and expands upon prior findings in other Thymus species, where high intraspecific chemotypic variation has been reported. For instance, studies on T. vulgaris, T. zygis, and T. caramanicus in the Mediterranean basin and Central Europe have consistently demonstrated the presence of multiple chemotypes within localized populations, often shaped by both genetic factors and microclimatic conditions [40,41,42]. In our samples, the predominance of the thymol chemotype, characterized by thymol and its precursors γ-terpinene and p-cymene, aligns with the findings from several Mediterranean Thymus populations, where thymol is frequently observed as the dominant component [42,43]. Notably, our thymol-dominant group also includes a subgroup characterized by high carvacrol content. The presence of other less common chemotypes such as sabinene hydrate, linalool, and geraniol within the same population underscores the rich chemical polymorphism of the Pistoia wild thyme and suggests a valuable gene pool for further domestication or selection programs.

The morphological analysis of the 17 wild thyme plants suggested some trends that may correlate with their chemical profiles, even if the sample size is limited and not fully representative of the broader Thymus populations in the Pistoia Mountains. As detailed in our findings, the thymol chemotype exhibited significantly larger leaves compared to the linalool chemotype, which had smaller flowers and leaves. These observations are consistent with the earlier literature suggesting that distinct morphological traits are correlated with specific chemotypes [37,38]. Furthermore, variations in calyx length and flower size among chemotypes could influence not just esthetic attributes but also potential ecological interactions [39,44]. The smaller flowers observed in the linalool chemotype could suggest adaptations for attracting pollinators, which signals the need for further ecological studies on the symbiotic relationships between wild thyme chemotypes and their environments.

Regarding chemotype stability and optimal harvesting seasons, our study showed that wild thyme plants maintain their chemotype across different seasons, even when transferred and grown in a new environment, different from their original habitat. Despite physiological variations due to environmental changes, GC-MS analyses confirmed that the predominant terpene for each chemotype remained consistent throughout the year.

This stability is crucial for applications where consistency in essential oil composition is required, such as in pharmaceuticals, cosmetics, and food industries. Similar seasonal stability in essential oil composition has been reported in Thymus serpyllum populations in the Western Himalayas, where thymol content remained relatively stable across different seasons [45]. From an ecological perspective, different terpenes exhibit distinct properties. For instance, the linalool chemotype attracts a wide range of pollinators [46], whereas geraniol has insecticidal and repellent properties [47]. Similarly, the sabinene hydrate chemotype acts as a deterrent to herbivores [48], while the thymol and thymol/carvacrol chemotypes play a crucial role in combating pathogens [49]. These ecological roles underscore the importance of chemotype diversity in plant defense mechanisms and interactions with the environment.

From an organoleptic standpoint, different terpenes are associated with unique aromas (The Good Scents Company Information System, http://www.thegoodscentscompany.com/, accessed on 23 November 2024). In particular, among the terpenes characterizing this wild thyme subpopulation, thymol and carvacrol confer a spicy taste, while geraniol offers a floral flavor. The sabinene hydrate presents woody, citrus, and spicy notes, whereas linalool, as expected, exhibits a distinct citrus-like aroma.

With regard to the balsamic period, the results indicate that spring and summer represent the best time for harvesting most of the chemotypes, which also corresponds with the flowering phase of thyme. This observation is consistent with findings in other Thymus species and aromatic plants, where essential oil yield peaks were recorded during flowering, whereas the lowest yield was observed during the dormant season [45,50,51].

Interestingly, while all chemotypes exhibited increased terpene levels in spring and summer, a distinct pattern in the linalool chemotype was observed. In this group, flowers showed lower terpene concentrations compared to vegetative tissues. This contrasts with the general trend observed in other chemotypes and suggests a chemotype-specific regulation of volatile accumulation. Morphological observations revealed that the linalool chemotype has smaller flowers, which may correlate with a reduced number of secretory structures or a lower gland density, as previously reported in studies linking floral anatomy or development to essential oil output [52,53]. These findings suggest that, in addition to seasonal timing, specific morpho-chemotype traits must also be considered when optimizing harvesting strategies for essential oil production.

From an applied perspective, identifying spring and summer as optimal harvesting periods provides valuable guidance for local cultivators and industries interested in standardizing the quality and yield of wild thyme essential oils. However, our results also underscore the importance of tailoring harvest practices to the specific chemotype being cultivated. The linalool chemotype, despite its aromatic and potential ecological value, may require harvesting that targets vegetative traits rather than floral biomass. This nuance is critical considering the increasing interest in chemotype-specific cultivation for applications in pharmaceuticals, perfumery, and agroecology.

The observed chemotype stability across seasons suggests a strong genetic component in determining terpene profiles. This is consistent with findings in other Thymus species, where essential oil composition is known to be influenced by both genetic and environmental factors [53].

This finding strengthens the rationale for establishing chemotype-specific cultivation programs, especially for obtaining high-value compounds like thymol/carvacrol, geraniol, sabinene hydrate, or linalool, each of which has distinct organoleptic and functional properties. Our study also underscored the importance of in vitro techniques for maintaining high-value wild thyme chemotypes, particularly in the context of preventing hybridization and preserving plants with desirable chemical profiles. The successful in vitro conservation of the different chemotypes for over a year, with no significant alterations in terpene composition and quantity, demonstrates the viability of this method for long-term preservation and potential commercial propagation.

5. Conclusions

This study provides a partial characterization of the chemical and morphological variability of Thymus serpyllum populations from the Pistoia Mountains. We identified five distinct chemotypes (thymol, thymol/carvacrol, geraniol, linalool, and sabinene hydrate) with stable terpene profiles across seasons, even after transplantation to a new environment. These findings confirm that chemotype expression in our wild thyme is predominantly genetically determined, with important implications for its agricultural and industrial use.

The seasonal assessment of terpene content revealed that the balsamic period occurs during spring and summer for all chemotypes, with floral tissues contributing significantly to the overall yield. Interestingly, the linalool chemotype showed lower terpene content in flowers, correlating with its smaller floral structures. Morphological analysis further highlighted that chemotypes differ not only chemically, but also in some key leaf and floral traits, underscoring the potential for targeted selection in breeding programs.

Importantly, our in vitro conservation approach demonstrated that it is possible to maintain the specific terpene profile and terpene yield over time, offering a valuable tool for preserving high-value chemotypes and supporting biodiversity conservation efforts.

Altogether, these results directly support the study’s goal of enhancing the sustainable use of wild thyme resources. They emphasize the need to preserve local chemotypic diversity as a means to maximize essential oil quality and optimize harvest timing. Future studies should aim to unravel the genetic mechanisms driving chemotype differentiation and extend this research to other wild thyme populations across different ecological contexts to better inform cultivation and conservation strategies.