GIS Bioclimatic Profile and Seed Germination of the Endangered and Protected Cretan Endemic Plant Campanula cretica (A. DC.) D. Dietr. for Conservation and Sustainable Utilization

Abstract

This study focused on the seed germination of the local Cretan endemic Campanula cretica, an endangered and nationally protected species with ornamental value. To determine its seed germination requirements, high-resolution bioclimatic (temperature and precipitation) maps were integrated with geographic distribution data of C. cretica using Geographic Information Systems. The seed germination was tested at four constant temperatures (10, 15, 20, and 25 °C) with a photoperiod of 12 h light/12 h dark and under light/darkness and darkness at 15 °C. Pre-treatments with gibberellic acid solutions (500 and 1000 mg·L⁻¹ GA₃) and cold moist stratification at 5 °C were applied to investigate seed dormancy. Seed germination was significantly affected by the interaction of temperature and seed pre-treatments; without pre-treatment, the seeds germinated better (>85%) at 10 and 15 °C. The detected seed germination pattern matched the natural temperatures prevailing in situ during late autumn. Pre-treatments with GA₃ solutions and cold stratification first reported herein widened the seed germination range at 20 and 25 °C. The seeds germinated better in light (94.38%) than in darkness (69.38%). The results of this investigation addressed existing research gaps (GIS-derived bioclimatic profiling, effects of incubation temperature, cold stratification, GA₃, and light investigated for the first time), thus facilitating species-specific conservation efforts and enabling sustainable utilization strategies.

1. Introduction

Greece represents a significant center of evolution and diversity for members of the genus Campanula, hosting approximately 96 taxa (species and subspecies) out of the 448 Campanula taxa globally [1], of which ca. 60 are single-country endemics confined only to Greece [2,3]. This exceptional richness and uniqueness underscore the importance of Greek flora within this highly diverse and evolutionarily complex genus of Campanulaceae [2]. Many of the Greek endemic Campanula species are neglected and underutilized plants (NUPS) with potential applications across various sectors, such as the Cretan endemic C. pelviformis Lam. in the agro-alimentary and the medicinal-cosmetic sectors [4,5] or the latter as well as the Greek endemics C. incurva Aucher, C. cretica (A. DC.) D. Dietr., and C. laciniata L. in the ornamental-horticultural sector [6,7,8,9]. Such wild bellflowers provide a valuable genetic reservoir for developing new cropped cultivars and varieties [9]. Conserving and utilizing these species not only preserves Greece’s botanical heritage but also creates economic opportunities by enabling the development of value chains on local and global scales [10].

Crete, the largest island in Greece and the fifth largest in the Mediterranean Basin [11,12], is recognized as the most significant hot spot for plant endemism in the Mediterranean region [13], providing habitats for 11.5% of the Greek Campanula members among which eight taxa are local Cretan endemics and two are Greek endemics shared with other mainland or insular regions of Greece [3]. Designated by the International Union for Conservation of Nature (IUCN) as a Global Center of Plant Diversity [11], Crete’s unique biodiversity is largely attributed to its varied environmental conditions, complex topography, and long-term isolation due to troubled geological history (mountain building and tectonic movements) [11,12,14]. The dramatic terrain of this island with several mountains is peaked by three major mountain ranges—Lefka Ori, Psiloritis, and the Dikti Mountains—which serve as climatic refugia and are home to a significant number of threatened endemic species [12,13]. Among these, Lefka Ori hosts the highest concentration of endemic taxa assessed as threatened under IUCN criteria [13], as well as half of the Cretan endemic Campanula taxa [15].

Previous studies to date have investigated the effects of constant temperatures (10, 15, and 20 °C) on the germination of the local Cretan endemics Campanula hierapetrae Rech. fil., Campanula laciniata, and Campanula saxatilis L. subsp. saxatilis. The findings consistently indicate that the optimal temperature for seed germination in these single-island endemic species is 15 °C [16]. A more recent study on C. saxatilis subsp. saxatilis has assessed seed germination under four constant temperatures (10, 15, 20, and 25 °C) with a 12 h light/dark cycle. The results have revealed the highest germination percentage (85%) at 10 °C, with a slightly lower but faster germination rate observed at 15 °C [17]. Additionally, germination experiments involving five populations of the Cretan endemic bellflower C. pelviformis have demonstrated the highest germination percentages (>85%) at both 10 °C and 15 °C [8]. Collectively, these studies suggest that lower to moderate temperatures (10–15 °C) are generally favorable for the germination of Cretan endemic Campanula species. Temperature has significant effects on the induction and termination of dormancy and is considered the most influential environmental factor regulating the timing of seed germination [18,19]. By controlling germination under constant temperatures, both the range of germination temperatures and the germination season of a species can be determined [19]. In addition to the influence of temperature, the application of gibberellic acid (GA₃) has been shown to enhance germination in various Campanula species. For example, treating C. incurva seeds with 400 mg·L⁻¹ GA₃ at 21 ± 1 °C has significantly improved the seed germination percentage [6]. Similarly, germination stimulation through GA₃ pre-treatment has been observed in C. persicifolia L. (1000 mg·L⁻¹ GA₃) at alternating temperatures of 25/15 °C, C. rumeliana (Hampe) Vatke at 20/10 °C [20], and C. punctata Lam. var. punctata at both 15/6 °C and 25/15 °C [21]. Cold stratification is widely recognized as a critical pre-germination treatment for overcoming seed dormancy and enhancing germination percentages in many temperate species [22]. Previous studies have confirmed its effectiveness in various members of the genus Campanula. Notably, the sub-Balkan endemic Campanula trachelium L. subsp. athoa (Boiss. & Heldr.) Hayek has demonstrated a remarkable germination percentage of 99% at alternating temperatures of 25/15 °C after undergoing one month of cold stratification [20]. Similarly, in C. punctata var. punctata, seeds subjected to cold stratification for 4, 8, or 12 weeks, followed by four weeks of incubation at a constant 25 °C, have exhibited high germination percentages ranging from 77.0% to 87.0% [21]. These findings underscore the importance of temperature regulation, gibberellic acid application, and cold stratification as key strategies in promoting successful seed germination and seedling establishment in Campanula species, highlighting their potential utility in conservation and propagation efforts.

For the development of species-specific bioclimatic profiles, Geographic Information Systems (GIS) provide an essential tool offering valuable insights into the abiotic factors that prevail in the natural habitats of wild-growing populations of specific local endemic species. These profiles help assess whether such conditions can be replicated in human-made environments, thereby supporting ex situ conservation and cultivation efforts [17,23,24]. By defining the environmental conditions necessary for successful germination, growth, and acclimatization of specific species, bioclimatic (ecological) profiles are instrumental not only in species reintroduction and in situ conservation but also in the development of sustainable exploitation strategies [24]. In recent years, such bioclimatic (ecological) profiles have been established for several Greek endemic species of conservation concern (for example, [6,8,9,25]), underscoring their significance for ex situ conservation. Consequently, such GIS applications in plant conservation play a critical role in optimizing species management efforts [26].

One notable example of Crete’s unique flora is Campanula cretica (Campanulaceae), an impressive wildflower species that is locally endemic to Crete and primarily found at the foothills of the Lefka Ori mountain range, being classified as endangered (EN) [13] and assigned a protection status under the Greek Presidential Decree 67/1981 [3]. Beyond the biological rarity of C. cretica and its ecological role, a recent study has demonstrated that the species possesses strong antioxidant and anti-inflammatory properties [4], thus suggesting a new potential source of bioactive compounds. Additionally, the plant has considerable ornamental and horticultural value and has sparked commercial interest [10] since it is already catalogued and traded through the Internet by foreign nurseries, being available online as both living plants and seeds [11]. This uncontrolled commercialization raises concerns about potential overcollection from its natural habitats and/or its unauthorized propagation and trade in terms of the EU Regulation 511/2014 enforcing the Nagoya Protocol [10,11]. Therefore, this study specifically focused on C. cretica, aiming to develop a comprehensive and effective seed germination protocol for authorized use and correctly assign the species’ seed dormancy class. To this end, the effects of four different temperatures (10, 15, 20, and 25 °C) under alternating light/dark conditions, as well as the effects of different lighting conditions (light/darkness and darkness) at 15 °C, on seed germination behavior were investigated. Additionally, to explore seed dormancy in C. cretica, pre-treatments with gibberellic acid solutions (500 and 1000 mg·L⁻¹) and cold moist stratification at 5 °C were applied. Furthermore, a bioclimatic (ecological) profile was constructed to support both the conservation and sustainable utilization of this showy Cretan bellflower.

2. Materials and Methods

2.1. Plant Material and Value

Campanula cretica (Figure 1) is a hemicryptophyte with a restricted range, found exclusively on the island of Crete, Greece [15]. It primarily grows in the gorges located in the southern and western foothills of the Lefka Ori mountains, with a recent discovery in a gorge in eastern Crete [15]. This species thrives in the shady crevices and ledges of limestone cliffs within ravines and is occasionally found on rocky road embankments in macchie and in gaps of Castanea sativa Mill. forests [15]. Its altitude range spans from sea level to 700 m above sea level and less frequently up to 1400 m, blooming from May to July [15] and occasionally (but to a lesser extent) in autumn. This impressive local endemic plant has relatively large and narrowly bell-shaped, white to deep violet-blue flowers developed on simple and erect flowering stems up to 60 cm (often bent downward when growing on inclined rocky surfaces), which are surrounded by large and vivid green, heart-shaped basal leaves; for detailed morphology, see [15].

2.2. GIS Ecological Profiling

To establish a bioclimatic (ecological) profile for the local Cretan endemic C. cretica, it was essential to accurately depict its natural range in Crete (in total, 15 dot occurrences; see [27]). Utilizing digitized geographic data from a previous study built on a grid cell size of 8.25 × 8.25 km [28], the main distribution areas for C. cretica wild-growing populations (n = 8) were georeferenced through GIS (Geographical Information Systems). High-resolution bioclimatic maps were then employed and linked accordingly, with a pixel size of 30 arcsec and a spatial resolution of 1 km². These maps provided detailed data on the minimum, maximum, and average monthly temperatures and precipitation levels, along with 19 additional bioclimatic variables for the natural distribution areas depicting the overall range of C. cretica. The climate data, covering the period from 1970 to 2000, were obtained from the open-access WorldClim database [29].

2.3. Seed Collection and Storage

Using an authorized collection permit (YPEN/DPD/15539/845 of 24 February 2022) issued by the competent Greek authorities (domestic Ministry of Environment and Energy) and following taxonomic verification, mature fruits (capsules) of C. cretica (Figure 2) were collected prior to their natural dispersal from a wild-growing population in Crete on 27 August 2022 found in Kakopetros, Chania, Crete Island (N35.419.784, E23.740.193, 518 m above sea level).

Upon collection, the capsules were placed on filter paper and were maintained for a week under laboratory conditions. Subsequently, manual cleaning of the seeds was performed using a series of sieves with varying mesh sizes, according to the dimensions of the seeds. The taxonomically identified and manually cleaned seeds were assigned the IPEN (International Plant Exchange Network) accession number GR-BBGK-1-22,128. After thorough cleaning, the seeds were stored in glass containers at a temperature of 3–5 °C in a low-humidity environment (<15%) until the initiation of the experiments. To further ensure the conservation of the species’ genetic variability, new authorized seed collections were made in different areas in Crete during 2023–2025 (YPEN/DPD/38262/2306 of 2 August 2023, 80381/5557 of 9 August 2024, and 22236/1535 of 10 April 2025), resulting in representative seed lots stored for long-term conservation and further future experimentation.

2.4. Seed Germination Experiment

The two germination experiments for C. cretica seeds commenced in December 2022 and were conducted at the Laboratory of Floriculture, Department of Agriculture, Aristotle University of Thessaloniki (Thermi, Greece). In the first experiment, the seeds were subjected to the following different pre-treatments: (i) no treatment (control), (ii) seeds immersed in a solution of gibberellic acid (GA₃) at a concentration of 500 mg·L⁻¹ for 24 h, (iii) seeds immersed in a solution of GA₃ at a concentration of 1000 mg·L⁻¹ for 24 h, and (iv) seeds subjected to cold and moist stratification at 5 °C for 25 days. In the present study, the seeds of C. cretica were not stratified for more than 25 days, due to a pre-experiment conducted showing that germination of seeds stored for about four months was observed by the end of the fourth week of cold stratification (Subsequently, the germination response of the pre-treated seeds was evaluated across four constant temperatures, 10 °C, 15 °C, 20 °C, and 25 °C, using temperature-controlled growth chambers (CRW-500SD, Chrisagis, Athens, Greece). At each incubation temperature and for each pre-treatment, four replications were employed each with 40 individual seeds. The seeds were placed on filter paper moistened with distilled water in 9 cm sterile plastic Petri dishes. A layer of sterile, wet river sand about 1 cm thick was placed under the filter paper. The Petri dishes were randomly arranged on the shelves of the growth chambers. To maintain moisture, distilled water was added to the filter paper as needed throughout the experiment. Within the chambers, light was provided by fluorescent lamps at an intensity of 82 μmol m⁻² s⁻¹, with a photoperiod of 12 h light and 12 h dark, and relative humidity (RH) was maintained at 75 ± 1%. Germination was recorded every four days over a 36-day period, with germinated seeds being removed at each observation. Seed germination was defined by the visible emergence of the radicle from the seed coat (Figure 3).

Immediately after the completion of the germination test of control seeds of the first experiment (which lasted for 36 days), a second experiment was conducted to assess the light requirements for the germination of C. cretica seeds. Seeds of the seedlot used in the first experiment were subjected to germination at 15 °C. For each light treatment, a sample of four replications of 40 seeds each was used. The Petri dishes were placed in a chamber under either alternating light/dark conditions (12 h light/12 h dark) or continuous darkness. For the dark treatment, Petri dishes were covered with double layers of aluminum foil, which was removed only briefly for the counting of germinated seeds and then replaced. The mean germination time (MGT) for each treatment (light/dark and dark) was calculated based on the average of four replicates. The MGT for each replicate was determined using the following equation [30]:

$$\mathrm{MGT}={\displaystyle \frac{\mathsf{\Sigma }\left(\mathrm{D}\times \mathrm{n}\right)}{\mathsf{\Sigma }\mathrm{n}}}$$

(1)

where n is the number of seeds that germinated on day D, and D is the number of days counted from the onset of the experiment.

2.5. Statistical Analysis

In the first germination experiment, the experimental design employed a completely randomized factorial design in a split-plot arrangement with the factors being the incubation temperature (4 levels, 10, 15, 20, and 25 °C) and the seed pre-treatments (4 conditions, control, 500 mg·L⁻¹ GA₃, 1000 mg·L⁻¹ GA₃, and cold stratification). The incubation temperature was the main plot factor, and the treatments were the subplots. Two-way ANOVA [31] was conducted to evaluate the effects of the applied factors (temperature and pre-treatment) and their interaction on germination of C. cretica seeds, and the comparisons of the means were conducted using the least significance difference (LSD) test at significance level a = 0.05 [32]. The data were first checked for normality (Kolmogorov–Smirnov and Shapiro–Wilk test) and homogeneity of variances (Levene’s test) prior to analysis. In the second experiment, a t-test was conducted to evaluate the effects of light conditions on the germination percentage and mean germination time (days) of C. cretica seeds incubated at 15 °C. All statistical analyses were carried out using SPSS 28.0 (SPSS, Inc., Chicago, IL, USA).

3. Results

3.1. GIS-Derived Bioclimatic (Ecological) Profile

A GIS-based bioclimatic (ecological) profile was developed for C. cretica, utilizing natural occurrence records to capture the abiotic conditions prevailing in its natural habitats, particularly temperature and precipitation (Figure 4). According to historical climate data, the lowest average temperature for C. cretica was recorded in January (7.18 ± 1.74 °C) and February (7.38 ± 1.74 °C). From March to May, the average monthly temperatures gradually increased (8.85 ± 1.69 °C, 12.16 ± 1.62 °C, and 16.19 ± 1.49 °C, respectively), reaching 20.54 ± 1.44 °C at the beginning of summer (June). Notably, the highest average temperatures were observed in July (22.46 ± 1.45 °C) and August (22.21 ± 1.45 °C). Following the peak in temperatures, a gradual decrease was noted from September (19.72 ± 1.50 °C) to December (8.84 ± 1.71 °C). The bioclimatic profile of C. cretica (Figure 4) indicated that the minimum mean temperatures could reach 4.37 ± 2.20 °C in February, while the maximum mean temperatures could reach 18.14 ± 1.58 °C in July. The mean recorded diurnal range was 7.25 ± 0.15 °C, and the annual mean temperature was 14.44 ± 1.60 °C. The relatively low values of these two bioclimatic variables, combined with the absence of extreme temperatures throughout the seasons—historically not dropping below 0 °C or rising above 30 °C (minimum of Tmin = 1.09 °C in February and maximum of Tmax = 29.40 °C in July)—suggested that C. cretica wild-growing populations may thrive in relatively favorable environmental conditions in terms of plant growth, with a mild winter season in the study area.

In terms of precipitation, the bioclimatic (ecological) profiling indicated that the rainy season may extend from October to March (Figure 4), with most precipitation occurring during this period. Historically, the rainiest months in the species’ natural range were herein shown to be December, January, and February, with January standing out as the month with the highest rainfall (152.60 ± 14.16 mm). From April onward, precipitation was shown to decrease, leading to the onset of the dry season by June (7.43 ± 2.35 mm). July and August were found to be the two driest months (4.30 ± 1.78 and 4.02 ± 1.24 mm, respectively). From September onward, the precipitation patterns were reported as slightly increasing (18.62 ± 1.90 mm), and they were gradually more pronounced during October and November (77.19 ± 8.04 and 94.63 ± 5.64 mm, respectively).

3.2. Seed Germination Experiment and Seed Germination Success

3.2.1. Effects of Incubation Temperature and Different Pre-Treatments

According to the results of two-way analysis of variance, the incubation temperature and pre-treatment factors, as well as their interaction, had a significant effect (p < 0.05) on seed germination of C. cretica (

Table 1. Results of two-way ANOVA regarding the effects of temperature, pre-treatment, and their interaction on final germination percentage of Campanula cretica seeds.

Source	Sum of Squares	df	Mean Square	F	Sig. (p-Value)
Temperature	8484.15	3	2828.05	132.26	<0.001
Pre-treatment	4951.43	3	1650.48	77.19	<0.001
Temperature × pre-treatment	6346.03	9	705.11	32.99	<0.001

).

In the light of the significant effect of the interaction of temperature × pre-treatment on the percentage of germinated seeds (p < 0.05), only the latter was analyzed (single-factor effects were not analyzed). Explicitly, at 10 °C, high germination percentages (>86%) were observed across all pre-treatments (Figure 5). However, seeds pre-treated with 500 mg·L⁻¹ GA₃ exhibited a higher percentage of germinated seeds compared with those pre-treated with 1000 mg·L⁻¹ GA₃. It was noteworthy that in the cold stratification treatment, the first germinated seeds were observed on the fourth day, whereas in the other treatments, they were observed on the eighth day from the starting day of the germination experiment. Seed germination was completed by the 20th day in the 500 mg·L⁻¹ GA₃ pre-treatment and by the 24th day for the other pre-treatments.

At 15 °C, the germination percentages were very high across all treatments with no detected statistically significant differences (Figure 5). In the cold-stratified seeds, germination began before the 4th day and was completed by the 24th day from the starting day of the germination experiment. In the control seeds and in the seeds treated with 500 mg·L⁻¹ and 1000 mg·L⁻¹ GA₃, the first germinated seeds were observed on the 8th day, and their germination was completed by the 24th, 20th, and 16th days, respectively.

At 20 °C and 25 °C, the pre-treatment of seeds with the GA₃ regardless of solution concentration resulted in the highest germination percentages, with no statistically significant differences observed between them, while the lowest germination percentage was recorded in the control seeds (Figure 5). Specifically, at 25 °C, only one seed germinated out of 120 control seeds. At 20 °C, germination in the control seeds began on the eighth day from the starting day of the germination experiment. Whereas, in seeds treated with 500 and 1000 mg·L⁻¹ GA₃ or cold-stratified ones, the germination began before the 4th day and was completed by the 16th or 28th, respectively. At 25 °C, germination began on the 4th day for seeds treated with 500 and 1000 mg·L⁻¹ GA₃ or cold-stratified ones and was completed after the 24th day.

In the control, the highest germination was observed at 10 °C (90.00%) and 15 °C (85.00%), with no statistically significant differences between them. In contrast, a significant reduction in the number of germinated seeds was observed at 20 °C (56.88%) and especially at 25 °C (0.63%) (

Table 2. Effect of incubation temperature on the germination percentage of Campanula cretica seeds without pre-treatment (control) and pretreated with 500 mg·L⁻¹ GA₃, 1000 mg·L⁻¹ GA₃, and cold stratification (CS). Means ± standard deviation values are given.

Pre-Treatment	Incubation Temperature
	10 °C	15 °C	20 °C	25 °C
Control	90.00 a 1 ± 2.04	85.00 a ± 4.08	56.88 b ± 8.98	0.63 c ± 1.25
0.5 g·L−1 GA3	93.13 a ± 5.54	87.50 ab ± 4.56	89.38 ab ± 2.39	81.88 b ± 6.25
1 g·L−1 GA3	86.25 a ± 5.95	87.50 a ± 7.07	88.13 a ± 6.88	76.25 b ± 4.79
CS	90.63 a ± 4.27	85.63 a ± 5.54	74.38 b ± 5.91	43.75 c ± 6.61

¹ Means in the same row followed by the same letter are not significantly different (p > 0.05), according to the LSD test.

). In the seeds treated with 500 mg·L⁻¹ GA₃, statistically significant differences in germination percentages were observed only between 10 °C and 25 °C incubation temperature (93.13 and 81.88%, respectively). Furthermore, in the 1000 mg·L⁻¹ GA₃ pre-treatment, the germination percentages at 10 °C (86.25%), 15 °C (87.50%), and 20 °C (88.13%) did not differ significantly in statistical terms (Table 2), while a significant reduction in germination was observed at 25 °C (76.25%). Finally, in the seeds subjected to the cold stratification treatment, the highest germination percentages were observed at incubation temperatures of 10 °C and 15 °C (90.63 and 85.63%, respectively), with no statistically significant differences between them, while the increase in incubation temperature to 20 °C and 25 °C resulted in a significant reduction in the germinated seed percentages (74.38 and 43.75%, respectively) (Table 2).

3.2.2. Effects of Light Treatment

In the experiment assessing the effect of light on the germination of C. cretica seeds, statistically significant differences were observed in both the germination percentage and the mean germination time (

Table 3. Effect of light conditions on germination of Campanula cretica seeds incubated at 15 °C. Means ± standard deviation values are given.

Factor	Germination (%)	Mean Germination Time (Days)
Light/dark	94.38 a 1 ± 1.25	9.70 a ± 0.80
Dark	69.38 b ± 6.88	15.34 b ± 1.21

¹ Means in the same column followed by the same letter were not significantly different (p > 0.05). The comparisons were performed using the t-test.

). Seeds that germinated under alternating light/dark conditions exhibited a higher germination rate (94.38%) compared with those germinated in continuous darkness (69.38%). Additionally, the mean germination time for seeds in alternating light/dark conditions was 9.7 days, whereas in darkness, the mean germination time was 15.34 days.

4. Discussion

The conservation of threatened and protected local Cretan endemic plants such as the herein investigated C. cretica requires a deep understanding of species-specific propagation and acclimatization techniques. Successful conservation strategies must incorporate both in situ assessments focused on wild-growing populations and ex situ approaches with a particular focus on developing effective seed germination protocols [7,33]. Sexual propagation is critical for maintaining genetic diversity, yet seed dormancy poses a significant challenge to large-scale plant production [34,35,36], especially in Campanulaceae [25]. For this reason, in the frame of the project CON-CRETE supported by the Hellenic Foundation for Research and Innovation and the Natural Environment & Climate Change Agency), additional seed lots of Campanula cretica were collected from geographically distinct populations (e.g., Samaria gorge, Lefka Ori, and Kakopetros) and different habitats (rocky outcrops and woodland) to be stored ex situ for future targeted seed germination experiments.

The high seed germination results of non-treated seeds at 10 °C and 15 °C (90.00% and 85.00%, respectively) and the significantly decreased germination at 20 °C and 25 °C (58.75% and <1%, respectively) detected in this investigation were consistent with a previous study on C. cretica [20]. However, in the present study, fresh seeds of C. cretica were not used in the germination test. The used seeds were stored for about 4 months, and this may have affected to some extent the dormancy of the seeds. Therefore, a further germination experiment with freshly collected seeds will need to be carried out to draw more secure conclusions regarding the germination requirements of the species. Similarly, high germination percentages at 10 and 15 °C have also been observed in other Cretan endemic species of Campanula, such as C. saxatilis subsp. saxatilis [17] and C. pelviformis [8]. Comparable results were observed in Petromarula pinnata (L.) A. DC. (also Campanulaceae), with relatively high percentages (>50%) of germinated seeds at 20 °C, a dramatic decrease (<3%) at 25 °C, and optimal seed germination at 10 and 15 °C [25]. These findings also agree with an earlier study indicating that many species within the Greek Campanulaceae germinating without pre-treatments prefer low temperatures, specifically (5)10–15 °C [20]. In the natural habitats of C. cretica, such germination trends probably occur from late autumn to early winter (November and December) when monthly temperatures decreased enough to fall within the range of those required for germination, as herein indicated by GIS-based bioclimatic (ecological) profiling. During this period, the increased soil moisture resulting from higher rainfall likely created favorable conditions for seed germination, while the relatively mild winter temperatures facilitated the growth and survival of seedlings. The uneven distribution of rainfall (mainly limited during the winter months) and the high summer temperatures in the Mediterranean (Greek) ecosystems were probably strongly associated with the adaptation of the plant life cycle of Greek native Campanula members to these climatic conditions. This comprehensive profiling offered valuable insights into the in situ climatic requirements of C. cretica in the wild, thus providing a deeper understanding of the species’ biological cycle and the specific conditions met in natural habitats (sexual reproduction of wild-growing populations) or man-made settings (ex situ seed germination).

Apart from informing the selection of appropriate temperature intervals for seed gemination experiments, the development of seed germination protocols informed by GIS-bioclimatic profiles can also be perceived as a solution for creating efficient propagation protocols for threatened endemic plants and, subsequently, as an opportunity to produce large numbers of individuals in man-made settings. This leads to the establishment of ex situ collections of plants of wild origin, which offer the opportunity to be used for translocation and reinforcement of wild-growing populations in situ with the aim to preserve or restore the species genetic diversity [36,37,38], facilitation of possible commercial interest for horticulture and floriculture [10,39,40,41,42,43,44], or exploration of agro-alimentary [45,46] and pharmacognostic interest [47,48,49,50,51] in a sustainable fashion, which is not based on plant material directly sourced from the wild. The insights presented herein regarding this rare, endangered, and valuable endemic plant of Crete can be effectively harnessed for conservation strategies and may be used to support its sustainable utilization, given its recognized ornamental value [10,11] and the newly discovered medicinal properties [4]. Accordingly, these GIS-facilitated germination protocols can be employed to generate adequate plant material of C. cretica for conservation actions, as well as for pilot-scale pot or field cultivation in nurseries and/or botanical gardens. The latter end was met in the frame of the agricultural research project titled “Establishment of an operational group to promote new flower crops from rare and seldom-seen autochthonous plants of Greece for cut flower production by precision fertilization” (acronym: Greek cut-flowers). In this project, upon successful application of the seed germination protocols herein developed and the acclimatization of the produced young plants, the first pilot cultivations were established recently (2024) at the premises of the Institute of Plant Breeding and Genetic Resources of the Hellenic Agricultural Organization Demeter in Thermi, metropolitan Thessaloniki, Greece (pot cultivation), and, with the co-operation of local cut-flower producers, in another two locations in Attica, Greece (greenhouse and outdoor field cultivations).

In general, seed dormancy is a mechanism that many plants have evolved to regulate the time of seed germination, thus ensuring successful seedling establishment and growth under favorable conditions [22,52,53,54,55]. Given that genes influencing seed germination and dormancy are among those under the strongest selection pressure in plants [56], germination and dormancy mechanisms hold significant ecological importance [52]. The failure to consider these traits in population restoration planning often leads to unsuccessful plant establishment [35,57]. Understanding the species-specific seed dormancy and the respective germination requirements of C. cretica is therefore essential for both conservation and ex situ cultivation efforts. In the present study, pre-treating C. cretica seeds with GA₃ (500 mg·L⁻¹ and 1000 mg·L⁻¹) and applying cold stratification (5 °C) were implemented for the first time. These treatments significantly enhanced the germination percentages of the seeds at temperatures of 20 and 25 °C compared with the control. The increase in germination percentage following GA₃ pre-treatment has also been reported in other species of the genus Campanula. For example, the seeds of C. incurva exhibit increased germination percentages following pre-treatment with 400 mg·L⁻¹ GA₃ at 21 ± 1 °C [6]. Similarly, enhanced germination percentages after 1000 mg·L⁻¹ GA₃ pre-treatment have been reported for C. persicifolia L. at alternating temperatures 25/15 °C, for C. rumeliana (Hampe) Vatke at 20/10 °C [20], and for C. punctata var. punctata at 15/6 °C and 25/15 °C [21]. Furthermore, cold moist stratification for 25 days resulted in higher germination percentages at 20 °C and 25 °C compared with seeds of C. cretica that did not undergo cold stratification. This increase in germination percentages following cold stratification (5 °C) is consistent with findings for the eastern Asiatic C. punctata var. punctata [21] and the sub-Βalkan endemic C. trachelium L. subsp. athoa (Boiss. Heldr.) Hayek presenting 99% germination at 25/15 °C after one month of cold stratification [20].

In this study, the germination behavior observed in C. cretica seeds indicated that both cold stratification and GA₃ treatment resulted in the loss of the effect of temperature on germination, and the seeds germinated over a wider range of temperatures. At the incubation temperatures tested herein (10, 15, 20, and 25 °C), the germination of cold stratified seeds was observed in the first four days, whereas the seeds treated with GA₃, as well as the control seeds, started to germinate before the eighth day (see Figure 5). At the incubation temperature of 15 °C, the pre-treatments applied to the seeds in the present study (GA₃, cold stratification) resulted in an increase in germination speed; specifically, about 80% of seeds germinated on the eighth day from the beginning of germination experiment. According to previous studies [22], seeds are considered to exhibit physiological dormancy when cold stratification reduces the specific temperature requirement for germination or enhances the germination rate. Additionally, the application of gibberellins has been shown to be effective in breaking this type of dormancy [22]. Nevertheless, accurately classifying the type of dormancy in a species requires investigation of the pre-germination embryo development [22,58,59]. In the present study, this aspect was not examined, as measuring embryo size was not feasible due to the extremely small size of C. cretica seeds (see Figure 2). It has been reported that seeds of Campanulaceae exhibit either morphological dormancy (MD) or morphophysiological dormancy (MPD), with embryo growth occurring at temperatures of 15 °C or higher [58]. For example, seeds of C. americana L. exhibit morphological dormancy (MD) [22,58,60] as the embryo increases in size by 103% prior to germination when seeds are placed at 25/15 °C under light conditions without any dormancy-breaking treatment [60]. Similarly, in seeds of various Campanulaceae species from mountainous regions of Hawaii (USA), embryo growth ranging from 87% to 179% has been reported prior to radicle emergence, depending on the species [61]. On the other hand, the east Asiatic C. punctata Kam. var. punctata (syn. C. takesimana Nikai) demonstrates morphophysiological dormancy (MPD) [21] as its seeds possess underdeveloped embryos at dispersal and require GA₃ treatment or short-term cold stratification to break physiological dormancy. Undoubtedly, further research on C. cretica is recommended to accurately determine the specific type of seed dormancy.

Species of Campanulaceae typically produce small to very small seeds that require light for germination [20]. The findings of the present study are consistent with previous research reporting that the final germination rate of C. cretica seeds in light conditions may reach 99% at a constant temperature of 15 °C, while in darkness, it can only reach 24% [20]. Other species within the genus Campanula such as the Balkan endemics C. spatulata Sm. subsp. spatulata and subsp. spruneriana (Hampe) Hayek and the sub-Balkan species C. versicolor Andrews and C. sparsa Friv. also show higher germination rates in light compared with darkness [20]. It is noteworthy that although an earlier study has reported higher germination rates for C. pelviformis seeds under light conditions (98%) compared with continuous darkness (11%) [20], a more recent study has found no statistically significant differences in germination percentage and mean germination time between the two conditions [8]. In the latter study [8], both light treatments exhibited high germination percentages and similar MGT values (98.75% and 10.95 days under light/dark conditions and 95% and 11.32 days in dark conditions) at the incubation temperature of 15 °C. A comparable germination pattern has also been observed in the seeds of Petromarula pinata [25].

5. Conclusions

This study provides a comprehensive analysis of the abiotic environmental conditions under which the wild-growing populations of C. cretica (Cretan bellflower) have evolved, focusing on their natural distribution across diverse habitats in Crete, Greece. Utilizing GIS-derived bioclimatic (ecological) profiling, this research established crucial environmental parameters that can support both the in situ conservation efforts and ex situ cultivation of this endangered Cretan endemic species. The insights gained herein are vital for developing strategies to integrate C. cretica into either natural habitats or artificial environments, thereby facilitating both conservation purposes and its sustainable utilization in man-made settings for different purposes. Additionally, the study addressed significant gaps in the understanding of the germination requirements of C. cretica, specifically investigating the effects of incubation temperature, cold stratification, gibberellic acid (GA₃), and light exposure (pre-treatments investigated for the first time). The findings revealed optimal conditions for seed germination, which are instrumental in formulating effective propagation protocols. These protocols are essential for initiatives aimed at reinforcing natural populations or restoring degraded habitats. Moreover, the study identified the ideal incubation parameters necessary for maximizing germination success, which is a critical step toward the ex situ conservation of this species in seed banks and botanical gardens. In conclusion, the results of this research not only contribute to the preservation of C. cretica’s genetic diversity but also provide insights into its potential utilization in the ornamental-horticultural and medicinal-cosmetic sectors. In addition, this research serves ongoing efforts to support integrated conservation actions for this endangered local endemic plant species and facilitates its sustainable management as a unique but neglected and underutilized Cretan phytogenetic resource.