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Article

Seed Germination Trials and Ex Situ Conservation of Local Prioritized Endemic Plants of Crete (Greece) with Commercial Interest

by
Virginia Sarropoulou
,
Nikos Krigas
*,
Georgios Tsoktouridis
,
Eleni Maloupa
and
Katerina Grigoriadou
*
Balkan Botanic Garden of Kroussia, Institute of Plant Breeding and Genetic Resources, Hellenic Agricultural Organization Demeter (ELGO-DIMITRA), P.O. Box 60458, GR-570 01 Thessaloniki, Greece
*
Authors to whom correspondence should be addressed.
Seeds 2022, 1(4), 279-302; https://doi.org/10.3390/seeds1040024
Submission received: 15 September 2022 / Revised: 22 October 2022 / Accepted: 25 October 2022 / Published: 1 November 2022
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Abstract

:
The in vivo germination course (15, 30, 45, and 60 days) of nine threatened local endemic plants of Crete (Greece) was studied due to conservation concerns and commercial interest in economic sectors. We used seeds directly collected from the wild sown in peat (Terrahum):perlite (1:1 v/v ratio)covered with coarse-grained vermiculite in a greenhouse mist bench with a substrate temperature of 19 ± 2 °C. The onset of in vivo germination was defined by the visible radicle protrusion (6th–9th day from sowing). After 60 days, 100% germination was observed for seeds of Campanula cretica, Dianthus fruticosus subsp. creticus, and Petromarula pinnata, followed by Draba cretica (91%) and Sanguisorba cretica (86%). Average–low germination capacity was observed for seeds of Calamintha cretica (26%), Lomelosia minoana subsp. minoana (38%), and Origanum microphyllum (23%), and very low capacity was observed for Onopordum bracteatum subsp. creticum (1.5%). After keeping seeds at 4–5 °C for three months, pre-treatments were performed (immersion in 50 or 250 ppm of gibberellic acid solution for 24 h) on three taxa with low germination capacity, thus resulting to the germination acceleration of Calamintha cretica seeds and increased germination capacity of Onopordum bracteatum subsp. creticum seeds. Apart from the facilitation of future species-specific conservation actions, the study showcases that the obtained results herein may permit an upgrade in the feasibility and readiness timescale assessments for the sustainable exploitation of studied taxa in different economic sectors.

1. Introduction

In situ plant biodiversity conservation is often but should always be supported by ex situ conservation actions that involve: storing seed lots in seed banks to secure the enhanced representation of the genetic diversity of prioritized plant species; employing different propagation trials with varied methods to aid the development of species-specific propagation protocols; using acclimatization methods regarding high-cost and valuable plant stocks raised ex situ destined for future reintroduction; and reinforcing the population of targeted species in the wild habitats [1,2,3]. Nevertheless, to date severe knowledge gaps (e.g., largely unknown biological cycle in man-made settings, appropriate species-specific propagation techniques, ex situ acclimatization potential) are still extant for the majority of plant species that are of conservation concern, such as insular local endemic plants [3,4,5,6,7,8,9]. Foregoing studies on the sexual reproduction of several conservation priority plants have focused on the settings or conditions that promise a satisfactory germination of seeds, thus introducing effective propagation protocols [3,10,11,12,13,14,15]. Nonetheless, the seeds of many plant species often present varied dormancy types with innate mechanisms securing the appropriate timing of seed germination; therefore, species-specific research is required [16]. To overcome seed dormancy for conservation purposes or sustainable exploitation needs, a plethora of stratification and pre-treatment regimes have been proposed to date in the literature [17,18,19,20].
Neglected and underutilized plants (NUPs) often encompass scarcely known plant species, subspecies, or crop varieties with interesting or remarkable potential in economic sectors, but only infrequently do they refer to local endemic floristic elements of small geographical territories, namely from an island [4]. The native phytogenetic resources of given regions are globally and locally valuable, meriting both conservation and new ventures to establish value chains, and they are also used or are appreciated at local scales. However, they have never been mainstreamed and they hardly appeal to research communities, politicians, and stakeholders [4,21,22]. Given this shortcoming, the critical step for the sustainable exploitation of NUPs is the development of species-specific propagation and cultivation protocols which are both effective and affordable; upon such development, agronomical approaches and agro-processing procedures may be facilitated [23], thus unlocking the commercialization potential of NUPs and permitting value chain creation [24]. Furthermore, both evidence and storytelling regarding high-added value products of unique identity are needed for effective commercial prospecting and fascination of consumers, stakeholders, producers, wholesalers, and retailers. Hence, multidisciplinary approaches have been suggested for the sustainable exploitation of NUPs in conjunction with applied research methods, political and consumer preferences, and policy awareness [24]. The availability, development, and/or revival of knowledge on NUPs may also trigger the mitigation of extant barriers hindering the sustainable exploitation of unique resources [24].
In geographical terms, the present study was focused on the wild flora of Crete (south Aegean Archipelago) which represents an important center of endemism in Greece as well as in the Mediterranean and European contexts [25]. The flora of Crete includes at least 223 local endemic species and subspecies (taxa) that grow exclusively to this island [26], among which some have already been sustainably managed to date, which has allowed for the establishment of value chains [4]. For example, the dittany of Crete (Origanum dictamnus) and recently the Cretan mountain tea (Sideritis syriaca subsp. syriaca) have already established value chains in at least Crete and Greece, which formed after the domestication and effective crossing of all barriers for the species, starting as exclusively wild-growing species in natural habitats, ending up being threatened species in the wild, and finally being extant commercial crops cultivated for marketed products in man-made settings [4,27].
Primary dormancy and the imposition of secondary dormancy, seed photosensitivity, and the range of germination temperatures in relation to habitat, life-form, and size of seeds appear to be related to the specific adaptation strategies that the Mediterranean plants have developed among others, ensuring that seed germination is achieved under appropriate conditions that are favorable for the appearance and survival of seedlings [28]. Dormancy in species native to Mediterranean climate areas can be overcome through cold or hot stratification [29], widening the range of temperatures at which seeds germinate, alternating day/night temperatures, reducing the level of abscisic acid through the application of growth regulators such as gibberellic acid (GA3) or other chemicals such as nitrate ions (i.e., KNO3) and smoke substances, keeping seeds in dry conditions (pre-ripening or dry storage), and increasing seed photosensitivity in the light regime [30].
In the context of the plant conservation efforts of threatened local endemic plants and in the frame of sustainable exploitation strategies of Greek native NUPs, this study focused on exploring the in vivo seed germination of some perennial Cretan endemic plants (mostly rock-dwellers) that have been threatened with extinction [31]. Apart from conservation concerns, for these plants there are both extant applied research gaps and distinctive interest in various economic sectors [4,5,6]. The herein focal endemic plants of Crete which are threatened with extinction [31] include: Campanula cretica (A. DC.) D. Dietr. (Cretan bellflower), Dianthus fruticosus L. subsp. creticus (Tausch) Runemark (Cretan fruticose carnation), Draba cretica Boiss. & Heldr., Lomelosia minoana (P.H. Davis) Greuter & Burdet subsp. minoana, Calamintha cretica (L.) Lam. (Cretan calamint), Origanum microphyllum (Benth.) Vogel (Cretan marjoram), Petromarula pinnata (L.) A. DC. (Cretan rock lettuce), Sanguisorba cretica Hayek (Cretan burnet), and Onopordum bracteatum L. subsp. creticum Franco (Cretan wild artichoke). Furthermore, this study attempted to re-examine the feasibility of value chain creation and readiness timescale [4] for the sustainable exploitation of the studied Cretan endemic plants.

2. Materials and Methods

2.1. Focal Local Endemic Plants of Crete (Greece)

The selection of the focal threatened endemic plants of Crete (n = 223) [26] with interest in different economic sectors [4,5,6] incorporated several criteria, such as extant rarity in the wild, endemism, valuable properties (aromatic and/or medicinal, floricultural and/or ornamental, edible and/or flavoring value), and took also into consideration the estimated accessibility of their wild-growing populations for the acquisition of propagation material. Additionally, previous own propagation experience in related Greek native plants of similar life-forms such as annuals, biennials, and herbaceous perennials were incorporated [3,4,32,33,34,35]. The focal local endemic plants of Crete (Greece) are as follows (Table 1).
Dianthus fruticosus subsp. creticus (Carophyllaceae, Figure 1) is a range-restricted and endangered [31] chamaephyte [36]. Draba cretica (Brassicaceae) (Figure 1) is a range-restricted and critically endangered [31] dwarf hemicryptophyte [36]. Sanguisorba cretica (Rosaceae) is a range-restricted and critically endangered [31] hemicryptophyte (Figure 1) [36].
Campanula cretica (Campanulaceae, Figure 2) is a range-restricted and critically endangered [31] hemicryptophyte [36]. Petromarula pinnata (Campanulaceae) (Figure 2) is a widespread but locally endemic hemicryptophyte of Crete which has been assessed as vulnerable [31].
Calamintha cretica (Lamiaceae) (Figure 3) is a critically endangered [31] chamaephyte [36].
Lomelosia minoana subsp. minoana (Dipsacaceae, Figure 4) is a range-restricted and critically endangered [31] chamaephyte [36]. Onopordum bracteatum subsp. creticum (Asteraceae, Figure 4) is an endangered [31] and range-restricted hemicryptophyte [36].

2.2. Seed Collections

The seed collections were performed during botanical expeditions in Crete (Table 1) using a special permit of the Balkan Botanic Garden of Kroussia (BBGK) that is issued yearly by the Greek Ministry of Environment and Energy (Permits 154553/1861/13-7-2017 & 182336/879). Seeds of the targeted taxa (species and subspecies) were collected from wild-growing populations, and the collected material was promptly transferred to the facilities of the Balkan Botanic Garden of Kroussia (BBGK), Institute of Plant Breeding and Genetic Resources (IPBGR), Hellenic Agricultural Organization Demeter in Thessaloniki (Thermi) for taxonomic identification. Seed cleaning (removal of soil, foreign matter, and perishable materials) was then performed, followed by storage at 15 °C at a relative humidity of <15% for approximately 30 days. Upon taxonomic identification, all collected seed lots received a unique International Plant Exchange Network (IPEN) accession number (Table 1). The visited habitats and illustrations of the seed collections performed per Cretan endemic plant are illustrated in Figure 1, Figure 2, Figure 3 and Figure 4.

2.3. Seed Germination Trials

The first in vivo sowing of the seeds of the nine Cretan endemic taxa took place during autumn and winter (20 November 2018–20 January 2019) without any pre-treatment of the seeds or prior storage in a cold room (Table 2).
The second in vivo sowing took place during spring (6 February 2019–6 April 2019) after 80 days of storing the seeds in a cold room (4–5 °C, relative humidity <5%) for the six selected taxa (Calamintha cretica, Dianthus fruticosus subsp. creticus, Draba cretica, Onopordum bracteatum subsp. creticum, Origanum microphyllum, and Sanguisorba cretica) (Table 3). The six taxa were selected due to their low germination capacity during the first (autumn-winter) sowing and, therefore, the procedure was repeated during spring using pre-treatments to increase their germination. Two different 24 h pre-treatments took place prior to sowing in the case of Calamintha cretica seeds (0 and 50 ppm of GA3), and three pre-treatments were performed for Onopordum bracteatum subsp. creticum seeds (0, 50, and 250 ppm of GA3). The seeds of Origanum microphyllum were only pre-treated in 50 ppm of GA3 solution for 24 h prior to sowing (24 h immersion in H2O was not tested due to very low availability of seeds). For Calamintha cretica, Onopordum bracteatum subsp. Creticum, and Origanum microphyllum, the 24 h immersion of seeds in dH2O (same or equivalent to 24 h in 0 ppm of GA3 solution) served as the control treatment to the increased concentrations of GA3 (50 and 250 ppm for O. bracteatum subsp. creticum, but only 50 ppm for C. cretica and O. microphyllum); thus, 24 h in a 0 ppm GA3 solution is equivalent to 24 h in dH2O (same pre-treatment). In the other three taxa, including Dianthus fruticosus subsp. creticus, Draba cretica, and Sanguisorba cretica, pre-treatment was performed by immersing the seeds in dH2O for 24 h (Table 3). The reason behind the additional pre-treatment of Calamintha cretica, Onopordum bracteatum subsp. creticum, and Origanum microphyllum seeds with GA3 with the exception of dH2O in spring sowing was the significantly lower germination percentages that were detected during the previous autumn sowing period combined with the limited availability of seed material for each taxon.
We assessed the seed germination ability of the studied plants in different seasons of the year (Table 2 and Table 3). In all cases (autumn and spring sowing), single crates with peat substrate (Terrahum): perlite (1:1 v/v) were used, and their surface was covered with vermiculite. The crates were placed on a heated greenhouse bench (19 ± 2 °C, relative humidity 80–90%), and the time of in vivo seed germination was determined with regular measurements at 15-day intervals (15, 30, 45, and 60 days). The in vivo germination criterion in both trials was the radicle protrusion from the seed coat. In addition, the median germination time (t50) was calculated according to the formula by Farooq et al. [37] as the time needed (in days) to reach 50% of the final/maximum germination (Table 2 and Table 3). The unit measure used for “Germination onset day” was the number of days, and the onset (i.e., initiation) of germination was defined as the day when the first seed was germinated (Table 4).
The two in vivo sowing experiments (first and second), considering that they were performed at different times of the year (autumn and spring) with a different age of the seeds (i.e., different physiological state, level of dormancy, humidity, etc.) cannot be eventually compared. Seeds of the first sowing trial in autumn received no previous cold storage or 24 h pre-treatment (nine taxa), whereas seeds of the second trial in spring were kept for 80 days in a cold chamber (4–5 °C, RH <5%) (six taxa) and were pre-treated for 24 h with dH2O and/or GA3 solutions. Due to the different seed storage conditions, different pre-treatment regimes, and different number of tested taxa between the two sowing seasons, no further comparisons were made.
The in vivo seed germination procedure in either autumn or spring took place in three single seat crates of 100 seeds each, comprising a total of 300 seeds per taxon (3 replicates × 100 seeds).
After the germination of the seeds and growth of the seedlings for 2–4 months (species- or subspecies-dependent), the seedlings were transplanted into multi-hole trays filled with a substrate mixture of peat (Terrahum):perlite (1:1 v/v ratio), and were progressively filled into larger volume pots (0.33 and 1 L) with a mixture of peat (TS2, Klassmann):perlite (3:1 v/v ratio). Τhe criterion for transplanting the plants into larger volume pots was the first emergence of the root system out of the container, thus indicating symptoms of intolerance and non-symmetrical growth of the underground part compared with the above ground part.

2.4. Experimental Design and Statistical Analysis

The experimental layout was completely randomized. The data related to the in vivo seed germination experiments were analyzed by using the statistical package SPSS 17.0 (SPSS Inc., Chicago, IL, USA) and Analysis of Variance (one-way ANOVA). The descriptive data were compared using the Duncan’s multiple range test at a level of p ≤ 0.05. Statistical analysis was conducted separately for each plant taxon as well as per pre-treatment.

3. Results

3.1. Seed Germination

After a period of 60 days during the first in vivo autumn–winter sowing, 100% germination was observed in Campanula cretica, Dianthus fruticosus subsp. creticus and Petromarula pinnata (Figure 5 and Figure 6). The seeds of Draba cretica and Sanguisorba cretica also exhibited high germination rates, i.e., 91% and 86%, respectively (Figure 6). Intermediate germination capacity was evidenced in seeds of Calamintha cretica (26%), Lomelosia minoana subsp. minoana (38%), and Origanum microphyllum (23%) (Figure 7), while the germination rate of Onopordum bracteatum subsp. creticum was only 1.5% (Figure 8). The onset of germination for the nine studied Cretan plant taxa was defined by the visible growth of the radicle from the seed coat, and its appearance ranged from 6 to 9 days after sowing (Table 2, Figure 5, Figure 6, Figure 7 and Figure 8).
In the second trial, the germination of Calamintha cretica, Draba cretica, and Sanguisorba cretica seeds was completed after 30 days, after being placed in cold storage for 80 days (4–5 °C, RH < 5%) and being pre-treated in dH2O for 24 h prior to spring sowing, which exhibited the maximum germination rate. Further increase in the percentages of the germinated seeds after 30 and up to 60 days was non-significant, i.e., 89% for Sanguisorba cretica, 71–74% for Draba cretica, and 36% for Calamintha cretica. Regarding the other three Cretan endemic taxa studied in spring, the germination reached maximum competence after 15 days from sowing while no statistically significant changes were noticed in germination rate (%) in the subsequent period (15–60 days), i.e., 79–80% for Dianthus fruticosus subsp. criticus, 16–21% for Origanum microphyllum, and 18% for Onopordum bracteatum subsp. creticum. For all Cretan taxa studied in spring (n = 6), the t50 values ranged between 8.42 and 20.61 days, while the germination onset was recorded between 5 and 12 days, depending on species identity (Table 3).
Concerning the immersion of Onopordum bracteatum subsp. creticum seeds for 24 h in different concentrations of GA3 solutions (0, 50, 250 mg/L), their germination process was initiated on day 7 for the GA3-untreated seeds and on day 6 for the seeds pre-treated with GA3, regardless of concentration. The germination rates obtained ranged between 18 and 25%. In particular, an 18% germination rate was recorded throughout the 60-day period (15, 30, 45, 60 days) in the case of dH2O for 24 h (equivalent to 0 ppm GA3 for 24 h) pre-treatment; 23–24% germination rate for seeds pre-treated with 50 mg/L of GA3; and 23–25% for those imbibed for 24 h in a 250 mg/L GA3 solution prior to sowing. The t50 values ranged between 8.42 (dH2O for 24 h) and 10.41 days (50 mg/L GA3) and was 9.61 days for seeds pre-treated with 250 mg/L GA3 (Table 3).
According to Table 4 (combined overview of Table 2 and Table 3), the sowing season of the year (autumn, spring), cold and non-previous storage of seeds, 24 h seed pre-treatment prior to sowing in dH2O and/or GA3 solutions, and taxon identity appear to be important factors that can affect the germination ability of the seeds either positively, negatively, or not at all. In specific, there was an acceleration in the germination onset from 9 to 13 days after sowing, and there were higher germination percentages for Calamintha cretica seeds sown in spring after a 24 h pre-treatment with 50 ppm GA3 compared with autumn sowing under the same pre-treatment condition (24 h in 50 ppm GA3) or spring sowing after a 24 h pre-treatment in dH2O. Moreover, higher germination percentages were obtained for Dianthus fruticosus subsp. creticus and Draba cretica seeds sown in autumn compared to those sown in spring. In the case of Origanum microphyllum seeds pre-treated with 50 ppm of GA3 for 24 h, the initiation of germination (in days) occurred earlier during autumn sowing than during spring (Table 4). The germination percentage of Onopordum bracteatum subsp. creticum seeds pre-treated for 24 h in dH2O in the spring (18%) was 12-fold higher than that (1.5%) obtained in the autumn sowing, while the pre-treatment of spring-sown seeds with different concentrations of GA3 solutions (0, 50, 250 ppm) did not manage to considerably raise the germination percentages (23–25%) of the control treatment (18%) (24 h in dH2O or 0 ppm of GA3) (Table 4, Figure 9).
Ex situ conservation efforts were successfully accomplished regarding all mother plants derived ex situ from the germination trials with seeds that were directly collected from wild-growing populations in their natural habitats. Therefore, an adequate amount of plant propagation material was obtained for each of the threatened local endemic species of Crete, and subsequent horticultural experience was gained; currently, all materials are cultivated ex situ for conservation purposes and future experimentations.

4. Discussion

4.1. From Seed Germination to Ex Situ Conservation and Sustainable Exploitation

This study presents first-time in vivo germination data for nine threatened local endemic plants of Crete (Greece), for which only scarce or limited documentation has existed to date [4,5,6]. This investigation employed seeds that directly originating from wild habitats, which were collected during maturity and are illustrated for the first time herein. The research was primarily focused on the exploration of seed dormancy, the appropriate season, and the temperatures for in vivo germination, as temperature is known as the most important environmental factor controlling germination of Mediterranean plant species [38]. This adaptation allows seeds to avoid harsh environmental conditions for seedling establishment, as low water availability in Mediterranean ecosystems often forces the seeds to germinate in autumn when the rainy season starts and temperatures are cool. The germination data generated in this study may bridge extant applied research gaps for the threatened Cretan local endemics studied herein; thus, the data are critical for in situ conservation actions targeting wild-growing endemic populations, can facilitate their ex situ conservation in botanic gardens, and may also pave the way for the sustainable exploitation of species in different economic sectors [4].
Many members of the genus Campanula are currently appreciated for their ornamental flowers mainly due to their typical flower color ranging from deep violet to the very palest milky blue or even bluish white [39]. They are often sold as garden plants or pot plants and as cut flowers. Despite the numerous species of this genus, only few are currently available on the market. Wild bellflowers can represent an important genetic resource for finding new crops and varieties that can contribute towards diversifying plant and flower production in the ornamental industry [39]. Most perennial Campanula species and cultivars are propagated by conventional vegetative propagation methods to produce homogeneous populations, such as shoot division and stem rooting [40]. It is known that many Campanulaceae seeds harvested in June or July have to be sown within the following 6–8 months to obtain maximum germination rate [40]. For example, in Campanula trachelium L., low temperatures are reported to delay germination time and to reduce germination percentages, whereas other species such as C. sparsa Friv. and C. spatulata Sibth & Sm. do not seem to have their germination prevented by low temperatures [41]. Light intensity can also affect seed germination as evidenced in C. carpatica Jacq., for which there are reports that enhanced light intensity combined with either no or low-salt levels may produce up to 89% more germinated seedlings [42]. However, no such data were extant for the critically endangered Campanula cretica studied herein, thus hindering future conservation actions and sustainable exploitation strategies for this threatened species. The in vivo seed germination of Campanula cretica was completed in our study within 60 days with a 100% germination rate after collection from the wild habitats and direct sowing in autumn, with visible signs of sprouting after 9 days (t50: 30 days). Similar to our study, the application of plant growth regulators in other members of this genus such as C. glomerata L. [43] and C. carpatica [44] is not required for their seed germination. High germination percentages at 15 °C were also achieved in other local Cretan or south Greek endemic species of Campanula such as C. hierapetrae (>90%) having t50 = 11.9 days and C. laciniata L. (100%) having t50 = 4–5 days [28]. Currently, there is no indication of dormancy in C. cretica. This first-time seed germination data can possibly serve specific conservation actions if needed for C. cretica and may facilitate its sustainable exploitation in the future.
Petromarula pinnata (also Campanulaceae) is an attractive wild-growing Cretan endemic plant with ornamental value [4] and agro-alimentary potential, andit is often harvested in Crete directly from natural habitats to be used as an edible wild green [5]. The germination of the vulnerable Petromarula pinnata was completed (100%) in our study within 30 days during the autumn period with 5–15 °C mean temperatures. Other studies report similar performances for this species [45], indicating that the final germination percentage of P. pinnata seeds is higher in light conditions (61–76%) than in continuous dark (3–22%). Herein, 50% of the P. pinnata seeds germinated within the first 15 days (t50: 15 days) of the onset of germination were reported to occur after 9 days from sowing due to visible sprouting signs. It is known that fluctuating temperatures during the in vivo germination process can act as a sensing mechanism for both soil depth and vegetation cover due to direct sun exposure, and such soil temperature fluctuations can even be experienced by seeds buried at depths greater than those situated in upper soil layers [46]. Most members of Campanulaceae appear to have a light requirement for seed germination, which can be replaced by GA3 and partly by nitrates such as KNO3 [45]. Previous and current seed germination data can possibly play a role in conservation actions for P. pinnata and may further facilitate its sustainable exploitation in the future.
Calamintha cretica is a sweet-scented medicinal and aromatic plant of Crete that is marked as critically endangered [36]. Its leaves contain essential oil, with major compounds such as piperitenone, piperitone oxide, and p-menthane, thus rendering it a candidate plant for use in perfumery and in the pharmaceutical industry [47] due to piperitenone oxide being shown to have anticarcinogenic properties on human colon cancer cells [48]. In Crete, C. cretica is sometimes used as a herbal tea. This plant species could also be introduced in gardening and landscape applications as a ground cover for urban and peri-urban gardens, parks, and green roofs, which would be similar to a closely related species such as Clinopodium nepeta (K.) Kuntze [previously known as Calamintha nepeta (L.) Savi] that has been suggested for such uses [49,50]. In our study, seed germination of Calamintha cretica was completed earlier (within 30 days) and with a higher germination rate (36%) after cold storage of seeds in comparison to those that had direct sowing after collection from the natural environment. In a previous study conducted on the same species [51], Calamintha cretica seeds immediately after harvesting germinated at 100% and in a short period of time at 15 or 20 °C without any pre-treatment, and stored seeds for six months germinated equally well compared to those used directly after harvesting; seeds stored for 12 months had slightly reduced germination. Despite the lower germination percentage recorded in this study compared with previous ones (perhaps due to not fully ripened seeds harvested from the wild), the t50 value for C. cretica herein was three-fold higher for cold-stored seeds during the spring period compared with those directly sown after collection during autumn. In accordance with our findings, the germination of Calamintha cretica seeds was reported to be fastest at 20 °C [51]. The high germination without any seed pre-treatment that has been reported soon after seed harvest and for at least one year after indicates the absence of dormancy. The current in vivo seed germination data complement previous in vitro seed germination studies and can be exploited both for conservation actions and the sustainable exploitation of Calamintha cretica.
The critically endangered and sweet-scented Cretan marjoram has a noteworthy agro-alimentary potential as a spice, and it is often used in Crete as a local medicinal plant for tea making [5,6]; this is similar to its close relative Origanum majorana L., which has approved indications as a traditional herbal medicine with approved indications according to its monography of the European Medicines Agency (https://www.ema.europa.eu/en, accessed on 15 September 2022) [52]. In addition, Cretan marjoram also has interesting potential in the horticultural-ornamental sector [4]. Herein, Origanum microphyllum seeds directly sown without previous cold storage gave the highest germination rate (22.7%). On the contrary, Markaki [53] found that O. microphyllum seeds may germinate in vitro easily (100%) within 12 days at 20 °C under the influence of white light. Undoubtedly, the germination rates (16–21%) achieved after a 60-day period for O. microphyllum seeds pre-treated with cold storage and GA3 were indeed low in our study. However, there are in vitro germination data [54] for nine different Origanum species, and the germination rates (50–100%) seem to be species-dependent (when examined in petri dishes containing 1% agar alone or with 250 mg/L of GA3, or after mechanical scarification in different photoperiod regimes). Similarly to the findings of this study, a wide range of germination percentages have been reported in vitro for other Origanum species, including O. minutiflorum O. Schwartz & P. H. Davis (20%) [55], O. acutidens (Hand.-Mazz.) Iestw. [56], O. vulgare L. (54–74%) [57], O. compactum Benth. (81%) [58], O. cordifolium (Montbret & Aucher ex Benth.) Vogel (80–100%) [59], and the closely related O. majorana (71–88%) [60]. Even though the onset of germination (6th day) was early for directly sown seeds of Origanum microphyllum with non-previous storage, the time finally needed for 50% of the seeds to germinate was increased (18.14 days) in relation to the cold storage and GA3 pre-treated seeds (9 days, t50: 12 days). Consistent with our results, the germination percentage of untreated O. vulgare seeds was only 25.8%; the percentage increased to 39% after chilling treatment at 4 °C for seven days, and when soaked for 36 h in a 100 mg/L GA3 solution, their germination raised to 48%, with mean germination time ranging from 5.75 to 10 days. Thus, the results suggest that Origanum seeds have a rather combined dormancy that is both exogenous and endogenous [61]. Further research is suggested to fully master the germination potential of Origanum microphyllum prior to conservation efforts and sustainable exploitation attempts.
Many species (and numerous hybrids) of the genus Dianthus are used as ornamental plants of rocky gardens, owing to remarkably beautiful and often fragrant blossoms in spring, summer, or often deep into the autumn. Carnation plants are also often used as cut-flowers; given that the flower color of dry plants is retained, they are useful for pressed flower crafts and arrangements. From a horticultural viewpoint, species of particular significance are those that have beautiful and large flowers (preferably of different shades of pink to red) with long blossoming periods, capable of thriving on poor, acidic soils and being drought-resistant [62]. As such, carnation plants are commonly planted in parks, especially in rock gardens as the floral edges of seedbeds, or are used as covers of barren sites in settlements [63]. Some Dianthus species such as D. barbatus L., D. chinensis L., and D. plumarius L. have been easily reproduced with both seeds and cuttings [64]. Previous studies indicate that Dianthus fruticosus seeds (without reference to its geographically separated subspecies) can successfully germinate in vitro (97%) at 15 °C [65]. However, the germination rates of the endangered Dianthus fruticosus subsp. creticus in this study were notably lower (79–80%) after the cold storage of seeds (t50: 9.3 days) compared with those directly sowed after collection from the natural environment (93–100%) (t50: 10 days); the seed germination was optimum (100%) on the 30th day under autumn day-night fluctuating temperatures within a greenhouse temperature ranging between 5 °C and 15 °C. The current in vivo seed germination data may complement previous in vitro seed germination studies, and both can be exploited for conservation actions and the sustainable exploitation of Dianthus fruticosus subsp. creticus as a new and rare ornamental carnation.
In 13 different Draba species (including the studied Draba cretica), in vivo seed germination has been referred to occur in less than two weeks after sowing at 20 °C [66], which unfortunately does not have any further information available. According to the results of this study, the seed germination ability of the critically endangered high-mountain Draba cretica within a 60-day period ranged between 71% and 91% irrespective of the cold storage period or season of the year. In line with the present findings, similar or lower germination percentages were reported in other arctic species of the genus Draba [67]. The directly sown seeds of Draba cretica in autumn without previous cold storage showed better germination ability (50–91%) than the respective cold-stored ones sown during spring (60–74%). It is known that many Draba species have inferred physiological dormancy [68], and specifically D. glabella Richardson has been reported to germinate under alternating light and dark conditions following cold stratification for four weeks [69]; meanwhile, the conditional dormancy of D. verna L. seeds can be broken after ripening, as seeds seem to prefer lower temperatures and germinate in autumn temperatures in nature [70,71]. It has been postulated that D. verna seeds fall to the soil and remain there until germination in September or October as germination does not occur during the summer months due to the seeds being dormant and needing about three months to after-ripen before they can successfully germinate [70]; thus, if the seeds are covered before receiving 5–7 weeks of sunlight, they will not germinate well in the autumn, indicating that other factors affect its germination and light is the lowest influencer [72]. However, this is not the case with the high-mountain rock-dweller Draba cretica studied herein. The germination process of Draba cretica under experimentation was completed after a 30-day period, and the onset of germination was observed after 9 days from sowing in both treatments. The seed germination data generated herein can possibly serve conservation actions for Draba cretica and may further facilitate its sustainable exploitation in the future.
Members of the closely related genera Lomelosia and Scabiosa also have attractive flowers, and their seeds may germination rates from as low as 50–53% (e.g., Scabiosa triniifolia Friv. and S. africana L.) [54] to easy germination (100%), such as in S. canescens Waldst. & Kit. [73] and S. columbaria L. [74]. In different Lomelosia and Scabiosa spp. [54], the above average-to-high germination success (50–100%) is reported in in vitro seeds treated with GA3 or after mechanical scarification. The in vivo seed germination of the critically endangered Lomelosia minoana subsp. minoana in the present study was completed within 30 days and reached its maximum rate (34–38%) during autumn (t50: 10.74 days). In agreement with our findings, low germination rates (<5% or <30%) such as those recorded herein have also been reported for Lomelosia graminifolia (K). Greuter & Burdet [75] and Scabiosa atropurpurea L., thus indicating the necessity of a storage period to successfully increase the final germination (from approximately 40–50% in fresh seeds to 70–80% in stored ones) and thus suggesting innate dormancy [76]. However, Lolelosia minoana subsp. asterusica (Greuter) Greuter & Burdet seeds, another critically endangered local endemic subspecies of Crete [30], may germinate well at all temperatures tested (10, 15, 20 °C), with an optimum (100%) of 15 °C after scarification [28]. The latter introduces the need for a new series of trials and scarification techniques to be attempted in the future with Lomelosia minoana subsp. minoana seeds, which would be conducted to aim at improving their in vivo germination capacity as observed to date to aid conservation efforts and sustainable exploitation attempts.
The germination rates of the endangered Onopordum bracteatum subsp. creticum under study were significantly higher (18%) after 15 days with cold storage of seeds as compared with those directly sowed after collection from the natural environment (1.5%). On the contrary, a previous in vitro study in the widespread O. bracteatum Boiss. & Heldr. subsp. bracteatum have revealed that seeds cultured in petri dishes can exhibit increased germination percentages (77–80%) [54]. Consistent with our findings, four populations of the closely related Scotch thistle (O. acanthium L.) exhibited a range of germination responses from readily germinable to strongly dormant in both freshly collected seeds and seeds stored for 67 days at room temperature; the study pointed out that the dry-stored seeds germinated faster than the fresh ones [77]. Despite the onset of seed germination of the studied Onopordum bracteatum subsp. creticum being recorded after 6 and 7 days from sowing and undergoing non-storage and cold storage, the time needed for half of the cold storage seeds to germinate was shorter (t50: 8.42 days) compared with the non-stored ones (t50: 10.5 days), thus indicating the key role effect of cold storage on its earlier germination and higher germination ability. Similarly, it has been reported that a higher percentage of O. acanthium seeds can germinate under controlled conditions (66–89%), but a significant number across treatments will not germinate until scarified [78], thus emphasizing the importance of cold scarification prior to seed germination. Therefore, further research is suggested to fully master the germination potential of Onopordum bracteatum subsp. creticum with the aim to facilitate conservation efforts and sustainable exploitation attempts.
Members of the genus Sanguisorba (generally called burnets) are used as leafy salads and can often be propagated by seeds and division, but the germination requirements for propagation in controlled environments and commercial production are still unclear. Small burnet (Sanguisorba minor Scop.) individuals can be established from the seed [79] or by sprouting from the caudex [80], and can occasionally even be regenerated from rhizomes [81]. To date, there are available records for nine Sanguisorba taxa showing a generally high germination success (56–100%) for seeds germinated in vitro within a varied periods (7 to 298 days) under a wide range of constant or alternating temperatures [54]. In various Sanguisorba species, namely S. canadensis L., S. hakusanensis Makino, S. obtusa Maxim., S. officinalis L., and S. tenuifolia Fisch. ex Link, a treatment of cold stratification for 2–4 weeks has proven to be beneficial [66]. In the Sanguisorba cretica studies here for the first time, the germination process was completed on the 30th day and exhibited similar maximum germinations rates (86–89%) irrespective of previous cold storage. The seed germination data generated herein can possibly facilitate both conservation actions and the sustainable exploitation for Sanguisorba cretica.

4.2. Re-Evaluation of Feasibility and Readiness Timescale for Sustainable Exploitation

Ex situ conservation efforts were successfully accomplished regarding all mother plants derived ex situ from the germination trials, with seeds being directly collected from wild-growing populations in their natural habitats. Therefore, an adequate amount of plant propagation material was obtained for each of the threatened local endemic species of Crete, and horticultural experience was gained. Currently, all materials are cultivated ex situ for conservation purposes and future experimentations. The seed germination data generated in this study for the first time (Section 3.1) bridge extant applied research gaps for the studied local endemic plants of Crete, thus allowing their propagation and ex situ cultivation at pilot scales for conservation purposes. The latter application, when paired with recent publications [31], imposes the need for re-evaluating the studied Cretan endemic taxa in terms of feasibility for value chain creation and readiness timescale for sustainable exploitation in the future (Level II and III evaluations after [4]).
When the above-mentioned re-evaluation procedure is endeavored again for the attributes previously scored (see methodological scheme and scoring in Krigas et al., 2021) [4], in light of the data presented herein and/or recently published [31], the feasibility for value chain creation regarding the studied taxa is considerably increased, and the readiness timescale is remarkably upgraded in all cases. These increased scores and upgraded designations per studied taxon are summarized and outlined in Table 5. More specifically, it is shown that the estimated feasibility for sustainable exploitation regarding Lomelosia minoana subsp. minoana (30.55%), Sanguisorba cretica (33.33%), and Calamintha cretica (16.67%) are notably increased, and their respective readiness timescales can be considered “achieved”, whereas previous assessments rendered them as either “achievable in the long-term or short-term (for the first two taxa and the last one, respectively). Similarly, the increased feasibility for the sustainable exploitation of Campanula cretica by 25.00%, Draba cretica by 33.33%, and Petromarula pinnata by 16.67% result in an upgrade to their readiness timescale for value chain creation, changing from “long-term” to “short-term”; the respective evaluations for Origanum microphyllum (increase by 9.72%) and Dianthus fruticosus subsp. creticus (increase by 36.11%) can upgrade both readiness timescales for sustainable exploitation from either “medium-term” (O. microphyllum) or “indeterminable” (D. fruticosus subsp. creticus) to “short-term” (Table 5). The feasibility for the sustainable exploitation of Onopordum bracteatum subsp. creticum is increased by 15.28%, and its readiness timescale for value chain creation is upgraded from “long-term” to “medium-term” (Table 5). It should be highlighted that the differences reported herein per taxon are due to the newly available extinction risk assessments [31], the increased representation of seed storage in ex situ conservation facilities (see Table 1 and Krigas et al. [4]), and their first-time pilot ex situ cultivations (as reported in this study after research on their asexual propagation) paired with the development of species-specific seed germination protocols. These advancements result in higher individual scores per attribute and higher percentages of respective optimum possible scores, thus enabling the process of value chain creation in the near future [4].

5. Conclusions

This paper presents new data regarding the in vivo seed germination for nine local Cretan endemic plants with conservation interests (all threatened); these plants were associated with sustainable exploitation potential and an enhanced feasibility to create new value chains for unique but neglected and underutilized phytogenetic resources of Crete. To this end, it was reported herein that the seeds of Campanula cretica, Dianthus fruticosus subsp. creticus, Draba cretica, Petromarula pinnata, and Sanguisorba cretica did not appear to have dormancy as they satisfactorily germinated under the examined in vivo conditions. Refrigeration and pre-treatment with GA3 were reported to increase the in vivo germination rate in Onopordum bracteatum subsp. creticum and to accelerate the onset of germination in Calamintha cretica seeds. In this study, species-specific sexual propagation protocols through in vivo seed germination were developed for the studied taxa, thus facilitating conservation efforts as well as their sustainable exploitation; the latter may now be considered feasible in the near future or already achieved. At the same time, the data generated in this study have increased the feasibility and readiness timescale for the sustainable exploitation of these threatened, local endemic (unique), neglected, and underutilized phytogenetic resources of Crete, which considerably upgrades the possibility of paving the road toward the establishment of new and species-specific value chains in the future.

Author Contributions

Conceptualization, N.K., G.T. and K.G.; methodology, V.S., N.K., G.T. and K.G.; validation, G.T. and E.M.; formal analysis, V.S. and K.G.; investigation, V.S., N.K. and K.G.; resources, E.M.; data curation, V.S. and K.G.; writing—original draft preparation, V.S., N.K., G.T. and K.G.; writing—review and editing, V.S., N.K., G.T., E.M. and K.G.; visualization, V.S., N.K. and K.G.; supervision, G.T. and K.G.; project administration, G.T. and K.G.; funding acquisition, N.K., G.T., E.M. and K.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research has been co-financed by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH—CREATE—INNOVATE (project code: T1EDK-05380), entitled “Conservation and sustainable utilization of rare threatened endemic plants of Crete for the development of new products with innovative precision fertilization”.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data supporting the results of this study are included in the manuscript, and the datasets are available upon request.

Conflicts of Interest

The authors declare no conflict of interest.

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Seeds 01 00024 g001 550
Figure 1. Studied Cretan endemic members of Brassicaceae, Caryophyllaceae, and Rosaceae in their wild habitats (A,D,G) and illustration of collected parts (B,E,H) with mature seeds (C,F,I).
Figure 1. Studied Cretan endemic members of Brassicaceae, Caryophyllaceae, and Rosaceae in their wild habitats (A,D,G) and illustration of collected parts (B,E,H) with mature seeds (C,F,I).
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Figure 2. Studied Cretan endemic members of Campanulaceae in their wild habitats (A,D) and illustration of collected parts (B,E) with mature seeds (C,F).
Figure 2. Studied Cretan endemic members of Campanulaceae in their wild habitats (A,D) and illustration of collected parts (B,E) with mature seeds (C,F).
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Seeds 01 00024 g003a 550Seeds 01 00024 g003b 550
Figure 3. Studied Cretan endemic members of Lamiaceae in their wild habitats (A,D) and illustration of collected parts (B,E) with mature seeds (C,F).
Figure 3. Studied Cretan endemic members of Lamiaceae in their wild habitats (A,D) and illustration of collected parts (B,E) with mature seeds (C,F).
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Figure 4. Studied Cretan endemic members of Dipsacaceae and Asteraceae in their wild habitats (A,D) and illustration of collected parts (B,E) with mature seeds (C,F).
Figure 4. Studied Cretan endemic members of Dipsacaceae and Asteraceae in their wild habitats (A,D) and illustration of collected parts (B,E) with mature seeds (C,F).
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Seeds 01 00024 g005 550
Figure 5. Individual seeds (A,D), germinated seedlings (B,E), and transplanted individuals (C,F) of studied Cretan endemic members of Campanulaceae.
Figure 5. Individual seeds (A,D), germinated seedlings (B,E), and transplanted individuals (C,F) of studied Cretan endemic members of Campanulaceae.
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Seeds 01 00024 g006a 550Seeds 01 00024 g006b 550
Figure 6. Individual seeds (A,D,G), germinated seedlings (B,E,H), and transplanted individuals (C,F,I) of studied Cretan endemic members of Brassicaceae, Caryophyllaceae, and Rosaceae.
Figure 6. Individual seeds (A,D,G), germinated seedlings (B,E,H), and transplanted individuals (C,F,I) of studied Cretan endemic members of Brassicaceae, Caryophyllaceae, and Rosaceae.
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Figure 7. Individual seeds (A,D), germinated seedlings (B,E), and transplanted individuals (C,F) of Cretan endemic members of Lamiaceae.
Figure 7. Individual seeds (A,D), germinated seedlings (B,E), and transplanted individuals (C,F) of Cretan endemic members of Lamiaceae.
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Seeds 01 00024 g008a 550Seeds 01 00024 g008b 550
Figure 8. Individual seeds (A,D), germinated seedlings (B,E), and transplanted individuals (C,F) of studied Cretan endemic members of Dipsacaceae and Asteraceae.
Figure 8. Individual seeds (A,D), germinated seedlings (B,E), and transplanted individuals (C,F) of studied Cretan endemic members of Dipsacaceae and Asteraceae.
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Figure 9. Onopordum bracteatum subsp. creticum seedlings pre-treated with different GA3 concentrations (0, 50, and 250 mg/L) for 24 h after 30 and 60 days of spring sowing.
Figure 9. Onopordum bracteatum subsp. creticum seedlings pre-treated with different GA3 concentrations (0, 50, and 250 mg/L) for 24 h after 30 and 60 days of spring sowing.
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Table
Table 1. Extinction risk, protection status, and seed collection details for the studied Cretan endemic plants (ordered alphabetically according to scientific name) including the IPEN (International Plant Exchange Network) accession numbers stored and accessions used in different periods of this investigation (A: autumn; S: spring). Extinction risk assessments according to Kougioumoutzis et al. [31]: CR, critically endangered; EN, endangered; VU, vulnerable.
Table 1. Extinction risk, protection status, and seed collection details for the studied Cretan endemic plants (ordered alphabetically according to scientific name) including the IPEN (International Plant Exchange Network) accession numbers stored and accessions used in different periods of this investigation (A: autumn; S: spring). Extinction risk assessments according to Kougioumoutzis et al. [31]: CR, critically endangered; EN, endangered; VU, vulnerable.
Extinction RiskPresidential Decree 67/1981Scientific NameFamilySeed Accessions StoredIPEN Accession Number Used (Period)Collection DateCollection SitePrefectureLatitude
(North)		Longitude
(East)
EN-Campanula cretica (A. DC.) D. Dietr.Campanulaceae2GR-BBGK-1-19,147 (A)29/8/2018Samaria GorgeChania35.51353624.017463
CRYESCalamintha cretica (L.) Lam.Lamiaceae4GR-BBGK-1-19,146 (A,S)29/8/2018Samaria GorgeChania35.51353624.017463
EN-Dianthus fruticosus L. subsp. creticus (Tausch) RunemarkCaryophyllaceae5GR-BBGK-1-19,17 (A.S)29/8/2018Agios Georgios SelinarisLasithi35.28480325.540993
CR-Draba cretica Boiss. & Heldr.Brassicaceae3GR-BBGK-1-19,10 (A,S)29/8/2018SkinakasHeraklion35.2114624.89459
CR-Lomelosia minoana (P.H. Davis) Greuter & Burdet subsp. minoanaDispacaceae4GR-BBGK-1-19,16 (A)7/10/2018Ano ViannosHeraklion35.0542125.40998
EN-Onopordum bracteatum Boiss. & Heldr. subsp. creticum FrancoAsteraceae1GR-BBGK-1-19,1 (A,S)29/8/2018ZomynthosRethymno35.2425724.88549
CR-Origanum microphyllum (Benth.) VogelLamiaceae5GR-BBGK-1-
19,31 (A.S)		7/10/2018Dikti MountainLasithi35.11819125.492043
VU-Petromarula pinnata (L.) A. DC.Campanulaceae27GR-BBGK-1-19,124 (A)29/8/2018Gorge of Agia IriniChania35.299523.8346
CRYESSanguisorba cretica HayekRosaceae4GR-BBGK-1-19,154 (A,S)29/8/2018Samaria GorgeChania35.5135324.017463
Table
Table 2. In vivo seed germination procedure of nine local endemic plants of Crete without any pre-treatments during autumn.
Table 2. In vivo seed germination procedure of nine local endemic plants of Crete without any pre-treatments during autumn.
Plant TaxonDays after SowingGermination (%)t50 (Days)p-Values
Calamintha cretica152.0 b24.170.000 ***
3020.0 a
4526.0 a
6026.0 a
Campanula cretica1540 c30.000.000 ***
3050 c
4570 b
60100 a
Dianthus fruticosus subsp. creticus1593 b10.000.001 **
30100 a
45100 a
60100 a
Draba cretica1550 b12.160.000 ***
3090 a
4590 a
6091 a
Lomelosia minoana subsp. minoana1520 b10.740.009 **
3034 a
4536 a
6038 a
Onopordum bracteatum subsp. creticum151.5 a10.501.000 ns
301.5 a
451.5 a
601.5 a
Origanum microphyllum158.3 b18.140.017 *
3022.7 a
4522.7 a
6022.7 a
Petromarula pinnata1550 b15.000.000 ***
30100 a
45100 a
60100 a
Sanguisorba cretica156 b21.940.000 ***
3086 a
4586 a
6086 a
Statistical analysis was separately conducted for each taxon. Means with the same letter in the germination (%) column are not statistically significant different from each other according to the Duncan’s multiple range test at p ≤ 0.05; n.s.: p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
Table
Table 3. In vivo seed germination (%) of six local endemic plants of Crete collected from wild habitats after storage for 80 days in a cold chamber (4–5 °C, RH < 5%) and pre-treatment with dH2O for 24 h or GA3 solution for 24 h prior to spring sowing.
Table 3. In vivo seed germination (%) of six local endemic plants of Crete collected from wild habitats after storage for 80 days in a cold chamber (4–5 °C, RH < 5%) and pre-treatment with dH2O for 24 h or GA3 solution for 24 h prior to spring sowing.
Cretan Taxon/
(Pre-Treatment)		Days after SowingGermination (%)t50 (Days)p-Values
Calamintha cretica
(dH2O for 24 h)		155 b22.450.025 *
3018 a
4520 a
6022 a
Calamintha cretica
(50 ppm GA3 solution for 24 h)		1525 b9.230.044 *
3036 a
4536 a
6036 a
Dianthus fruticosus
subsp. creticus
(dH2O for 24 h)		1579 a9.300.992 ns
3080 a
4580 a
6080 a
Draba cretica
(dH2O for 24 h)		1560 b13.270.026 *
3071 a
4574 a
6074 a
Onopordum bracteatum subsp. creticum
(dH2O for 24 h)		1518 a8.421.000 ns
3018 a
4518 a
6018 a
Onopordum bracteatum subsp. creticum
(50 ppm GA3 solution for 24 h)		1523 a10.410.411 ns
3024 a
4524 a
6024 a
Onopordum bracteatum subsp. creticum
(250 ppm GA3 solution for 24 h)		1523 a9.610.323 ns
3024 a
4525 a
6025 a
Origanum microphyllum
(50 ppm GA3 solution for 24 h)		1516 a12.000.587 ns
3017 a
4517 a
6021 a
Sanquisorba cretica
(dH2O for 24 h)		3017 a20.610.000 ***
4517 a
6021 a
6089 a
Statistical analysis separately conducted for each plant taxon and pre-treatment prior to sowing. Means with the same letter in the germination (%) column (individually per taxon and per pre-treatment) are not statistically significant different from each other according to the Duncan’s multiple range test at p ≤ 0.05; ns: p > 0.05, * p ≤ 0.05, *** p ≤ 0.001.
Table
Table 4. Overview of in vivo seed germination trials of nine local endemic plants of Crete (!: first-time data) studied right after collection from wild habitats in autumn (A) without any pre-treatment and in spring (S) after 80 days storage at 4–5 °C.
Table 4. Overview of in vivo seed germination trials of nine local endemic plants of Crete (!: first-time data) studied right after collection from wild habitats in autumn (A) without any pre-treatment and in spring (S) after 80 days storage at 4–5 °C.
Germination Rate (%) every Forthright (in Days) during Autumn (A)/Spring (S)
Scientific NamePre-Treatment (Spring)Germination Onset Day
A/S		15th
A/S		30th
A/S		45th
A/S		60th
A/S
! Campanula cretica-9/-40/-50/-70/-100/-
! Calamintha cretica24 h in H2O-/12-/5 -/18-/20-/22
50 ppm GA3 (24 h)13/92/2520/3626/3626/36
! Dianthus fruticosus subsp. creticus24 h in H2O6/593/79100/80100/80100/80
! Draba cretica24 h in H2O9/950/6090/7190/7491/74
! Lomelosia minoana subsp. minoana-6/-20/-34/-36/-38/-
! Onopordum bracteatum
subsp. creticum		0 ppm GA3 (24 h in H2O)6/71.5/181.5/181.5/181.5/18
50 ppm GA3 (24 h)-/6-/23-/24-/24-/24
250 ppm GA3 (24 h)-/6-/23-/24-/25-/25
! Origanum microphyllum50 ppm GA3 (24 h)6/98.3/1622.7/1722.7/1722.7/21
Petromarula pinnata-9/-50/-100/-100/-100/-
! Sanguisorba cretica24 h in H2O13/26/1886/8986/8986/89
Table
Table 5. Overview of the general and special interest in different economic sectors with a multifaceted evaluation of the feasibility and readiness timescale for sustainable exploitation regarding the nine studied local endemic plants of Crete. The table also includes the experimentation period (A: autumn; S: spring) and maximum in vivo germination percentage achieved after trials (in parentheses), which resulted in increased individual scores, pilot ex situ cultivations, and upgrade of previous assessments (Level II and III evaluations).
Table 5. Overview of the general and special interest in different economic sectors with a multifaceted evaluation of the feasibility and readiness timescale for sustainable exploitation regarding the nine studied local endemic plants of Crete. The table also includes the experimentation period (A: autumn; S: spring) and maximum in vivo germination percentage achieved after trials (in parentheses), which resulted in increased individual scores, pilot ex situ cultivations, and upgrade of previous assessments (Level II and III evaluations).
Cretan Endemic PlantGeneral Ornamental Interest [4]* Special Ornamental Interest [4]Agro-Alimentary Interest [5]Medicinal Interest [6]Level II & III Assessments ** [4]Sowing Period (Maximum Germination Percentage Achieved)Increase in Sum of Scores (+) and Upgraded Percentage (%)
Calamintha creticaLow
(37.50%)		Below average to very high
(31.25%/43.90%/51.61%/63.24%)		High
(59.52%)		Below average
(46.30%)		69.44%/Short-termA, S (36%)+12 (86.11%)/Achieved
Campanula creticaAverage
(50.00%)		Average
(55.21%/49.09%/46.77%/51.07%)		Low
(33.33%)		Low
(33.33%)		36.11%/Long-termA (100%)+18 (61.11%)/Short-term
Dianthus fruticosus subsp. creticusLow
(36.67%)		Low
(23.96%/21.04%/25.81%/28.06%)		Low
(35.71%)		Very low(11.11%)34.72%/IndeterminableA, S (100%)+23 (66.67%)/Short-term
Draba creticaAverage
(53.33%)		Very high
(59.38%/68.83%/67.74%/85.38%)		Very low
(14.29%)		Very low
(11.11%)		36.11%/Long-termA, S (91%)+24 (69.44%)/Short-term
Lomelosia minoana subsp. minoanaAverage
(51.67%)		Very high
(62.50%/62.08%/68.28%/70.75%)		No
(0)		Very low
(20.37%)		40.28%/Long-termA (38%)+22 (70.83%)/Achieved
Onopordum bracteatum subsp. creticumLow
(34.17%)		Low to below average
(31.25%/38.70%/45.70%/47.04%)		Low
(38.10%)		Below average(46.39%)38.89%/Long-termA, S (25%)+11 (54.17%)/Medium-term
Origanum microphyllumBelow average
(44.17%)		Low to average
(35.42%/44.42%/47.31%/54.35%)		Very high
(80.95%)		High (59.26%)52.78%/Medium-termA, S (22.7%)+7 (62.50%)/Short-term
Petromarula pinnataAverage
(52.50%)		Low to average
(54.17%/45.45%/50.54%/54.15%)		Below average
(42.86%)		Low(37.04%)45.83%/Long-termA (100%)+12 (62.50%)/Short-term
Sanguisorba creticaLow
(32.50%)		Low
(31.25%/30.39%/39.25%/38.34)		Below average
(45.24%)		Low
(27.78%)		38.89%/Long-termA, S (89%)+24 (72.22%)/Achieved
* Special ornamental interest refers to pot/patio plant (1st percentage), home gardening (2nd percentage), landscaping (3rd percentage), and xeriscaping (4th percentage), modelled after Krigas et al. [4]. ** Assessments regarding the feasibility (Level II evaluation) and readiness timescale (Level III evaluation) for the sustainable exploitation of the studied taxa.
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Sarropoulou, V.; Krigas, N.; Tsoktouridis, G.; Maloupa, E.; Grigoriadou, K. Seed Germination Trials and Ex Situ Conservation of Local Prioritized Endemic Plants of Crete (Greece) with Commercial Interest. Seeds 2022, 1, 279-302. https://doi.org/10.3390/seeds1040024

AMA Style

Sarropoulou V, Krigas N, Tsoktouridis G, Maloupa E, Grigoriadou K. Seed Germination Trials and Ex Situ Conservation of Local Prioritized Endemic Plants of Crete (Greece) with Commercial Interest. Seeds. 2022; 1(4):279-302. https://doi.org/10.3390/seeds1040024

Chicago/Turabian Style

Sarropoulou, Virginia, Nikos Krigas, Georgios Tsoktouridis, Eleni Maloupa, and Katerina Grigoriadou. 2022. "Seed Germination Trials and Ex Situ Conservation of Local Prioritized Endemic Plants of Crete (Greece) with Commercial Interest" Seeds 1, no. 4: 279-302. https://doi.org/10.3390/seeds1040024

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