horticulturae
Article
GIS-Facilitated Germination of Stored Seeds from Five
Wild-Growing Populations of Campanula pelviformis Lam. and
Fertilization Effects on Growth, Nutrients, Phenol Content and
Antioxidant Potential
Ioannis Anestis 1 , Elias Pipinis 2 , Stefanos Kostas 1 , Evgenia Papaioannou 3,4 , Eleftherios Karapatzak 5 ,
Eleftherios Dariotis 5 , Parthena Tsoulpha 6 , Emmanouil Koundourakis 1 , Eleni Chatzileontari 1 ,
Georgios Tsoktouridis 5,7 , Stefanos Hatzilazarou 1, * and Nikos Krigas 5, *
1
2
3
4
5
Citation: Anestis, I.; Pipinis, E.;
Kostas, S.; Papaioannou, E.;
6
7
Karapatzak, E.; Dariotis, E.; Tsoulpha,
P.; Koundourakis, E.; Chatzileontari,
E.; Tsoktouridis, G.; et al. GISFacilitated Germination of Stored
Seeds from Five Wild-Growing
Populations of Campanula pelviformis
Lam. and Fertilization Effects on
Growth, Nutrients, Phenol Content
and Antioxidant Potential.
Horticulturae 2023, 9, 877.
https://doi.org/10.3390/
horticulturae9080877
Academic Editor: Kalliopi
Kadoglidou
Received: 5 July 2023
Revised: 26 July 2023
Accepted: 31 July 2023
Published: 2 August 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
*
Laboratory of Floriculture, School of Agriculture, Aristotle University of Thessaloniki,
54124 Thessaloniki, Greece; ianestis@agro.auth.gr (I.A.); skostas@agro.auth.gr (S.K.);
manoskoundourakis1@gmail.com (E.K.); elenichatzileontarii@gmail.com (E.C.)
Laboratory of Silviculture, School of Forestry and Natural Environment, Aristotle University of Thessaloniki,
54124 Thessaloniki, Greece; epipinis@for.auth.gr
Laboratory of Soil Science, School of Agriculture, Aristotle University of Thessaloniki,
54124 Thessaloniki, Greece; eapapaioa@agro.auth.gr
Laboratory of Forest Soil Science, School of Forestry and Natural Environment, Aristotle University of
Thessaloniki, 54124 Thessaloniki, Greece
Institute of Plant Breeding and Genetic Resources, Hellenic Agricultural Organization Demeter,
P.O. Box 60458, 57001 Thermi, Greece; ekarapatzak@gmail.com (E.K.); eleftheriosdariotis@yahoo.com (E.D.);
gtsok@elgo.gr (G.T.)
Laboratory of Forest Genetics and Plant Breeding, School of Forestry and Natural Environment,
Aristotle University, 54124 Thessaloniki, Greece; thena@for.auth.gr
Theofrastos Fertilizers, Industrial Area of Korinthos, Irinis & Filias, Ikismos Arion, Examilia,
20100 Korinthos, Greece
Correspondence: hatzilaz@agro.auth.gr (S.H.); nikoskrigas@gmail.com (N.K.)
Abstract: This study was designed to bridge extant research gaps regarding the vulnerable and
protected local endemic Campanula pelviformis, a wild edible green traditionally consumed in Crete
(Greece) with agro-alimentary and medicinal interest as well as ornamental value. The C. pelviformis
ecological profile was generated using the climate and temperature conditions prevailing in its
wild habitats through mapping of natural distribution linked with online bioclimatic databases
in geographical information systems. We tested the germination of seeds from five wild-growing
populations at four different temperatures (10, 15, 20 and 25 ◦ C) and under different light conditions
(light/dark and darkness), and we performed fertilization trails [integrated nutrient management
(INF), chemical fertilization (ChFe), control] examining morphological and physiological characteristics, above- and below-ground macro- and micronutrients and phenol contents, as well as their
antioxidant capacity. We found population and temperature effects on seed germination with their
interaction being statistically significant. Campanula pelviformis germinated better at 10 and 15 ◦ C
(>85% for all populations) with no preference on light conditions (98.75% and 95% in light and dark
conditions). The INF application increased root dry mass, chlorophyll content index and chlorophyll
fluorescence compared to other treatments and was beneficial for macro- and micronutrient concentrations in above-ground parts compared to previously studied wild-growing material, while
below-ground parts were positively impacted by both fertilization types. Total phenols and antioxidant capacity were both increased by ChFe fertilization. The data furnished herein permitted the
re-evaluation and upgrade of its sustainable exploitation potential in different economic sectors.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Keywords: endemic; GIS; integrated nutrient management; macronutrients; medicinal; micronutrients;
NUPs; ornamental; phytogenetic resources; sustainable exploitation; wild edible greens
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Horticulturae 2023, 9, 877. https://doi.org/10.3390/horticulturae9080877
https://www.mdpi.com/journal/horticulturae
Horticulturae 2023, 9, 877
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1. Introduction
Campanula spp. (Campanulaceae) are widely recognized plant species for their ornamental value, and many of them are frequently used as ornamental plants around
the world [1]. In fact, Campanula glomerata L., Campanula medium L., Campanula cochleariifolia Lam. are used as garden plants; Campanula isophylla Moretti, Campanula carpatica
Jacq., Campanula rotundifolia L., Campanula pyramidalis L. are used as potted plants, while
C. medium, Campanula persicifolia L., C. pyramidalis, and C. glomerata are used for cut flowers.
To date, there is an ongoing quest for new Campanula species with interesting or promising
ornamental potential in this sector [1,2].
Apart from the horticultural value, species within the genus Campanula are also being
exploited for agro-alimentary purposes in different regions [3]. For example, local Spanish
residents use raw roots of C. rapunculus L. in salads for their nutritional benefits, and
Italians collect the same plant using its raw roots or aerial parts in salads [3,4]. Furthermore,
Campanula pelviformis Lam. has been reported as a wild green traditionally consumed as
food in the eastern part of Crete [5,6]. In fact, the phytochemical profile of C. pelviformis
has been recently analyzed, and several isolated compounds have been found to possess
pharmaceutical properties, thus rendering it as an emerging medicinal plant [6]. These findings render C. pelviformis as a trustworthy source of diverse phytonutrients and bioactive
compounds [6].
The genus Campanula comprises 448 taxa (species and subspecies) worldwide (https:
//powo.science.kew.org/, accessed on 19 April 2023), most of which are perennials (occasionally annuals or biennials), often thriving in rocky areas, slopes, or grounds; many
Campanula members are often range-restricted or endemic plants in different regions, thus
associated with high biological value [7]. In Greece, there are 95 wild-growing Campanula
taxa, and most of them are exclusively found in crevices of limestone cliffs growing as
chasmophytes [8]. Of these, 11 taxa are to be found in Crete, with 8 being local single-island
endemics [8,9]. C. pelviformis is included as a local endemic plant in the extraordinary plant
diversity of Crete [9], which involves numerous single-island endemic taxa that are still
neglected and underutilized plants (NUPs) [2,10–12]. Previous studies have shown that
the exploitation of such unique phytogenetic resources holds great potential for different
economic sectors, such as in agriculture, medicinal-cosmetics, and ornamental horticulture [1,2,11–14]. Furthermore, conservation efforts have been increased to date to achieve
effective protection for local endemic NUPs, especially for those threatened with extinction [2]. Significant information on both the economic and biological value of threatened
local endemic NUPs has been gathered through species-specific studies [2,11–13], and
basic research gaps related to their propagation and ex-situ cultivation protocols have been
addressed during the last decade [2,15–29].
Applied research to develop appropriate propagation methods for local endemic
NUPs aiming to re-enforce threatened populations in the wild has primarily been driven by
their appreciated biological rarity and priceless ecological value [2,10]. However, this has
also been often coupled by potential economic benefits associated with their sustainable
utilization and concomitant exploitation [2,11,12]. The effectiveness of these two research
directions can be facilitated by information on the environmental conditions associated
with targeted NUPs reflecting their adaptation in natural habitats and their ecological preferences [2,15–20,22–26,28]. Such data can be utilized to adjust or improve their propagation
conditions, for example, through seed propagation [30], which is the most economical
and efficient propagation method for many plant species [31]. Furthermore, for the ex
situ conservation of targeted NUPs, and particularly for threatened species originating
from the challenging terrain of the Mediterranean basin, it is crucial to achieve successful
propagation and cultivation allowing for the production of plant stocks [2,32]. Various
cultural practices have been employed in the past with reasonable ex situ cultivation and
acclimatization results in terms of morphological and physiological traits, thus leading
to the conclusion that ex situ cultivation can contribute significantly to the production
of high-quality plants [33]. The application of fertilizers during the propagation stage
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in the nursery has been shown to increase the nutrient reserves in propagated plants for
population restoration efforts, thus rendering fertilization as an efficient method leading to
enhanced establishment success in their natural habitat [34].
Species-wise, this study is focused on Campanula pelviformis, a vulnerable [35] and
range-restricted local endemic plant of eastern Crete, which is protected by the Greek
Presidential Decree 67/1981, although it is traditionally consumed as a wild edible green [6],
thus meriting conservation concern. Given that many species from genus Campanula are
already used for ornamental and ethnobotanical purposes [1,3,4,6], C. pelviformis also takes
advantage of its sustainable exploitation, since high demand of such unique resources
may lead to excessive collection in the wild, risking the degradation of genetic pools
and local biodiversity. Context-wise, this study was designed to bridge extant research
gaps regarding the studied focal species. The aims of this research were to: (i) define the
optimal temperature and light conditions required for the successful seed germination of
C. pelviformis, examining seed responses of different wild-growing populations from low
and semi-mountainous areas of Crete; (ii) identify the preferred ecological conditions of
C. pelviformis based on its natural distribution; (iii) compare the impact chemical fertilizers
and integrated nutrient management on the growth of seedlings and estimate their nutrient
content; (iv) assess the overall phenolic content and antioxidant capacity of its aerial parts;
and in the light of all the aforementioned, (v) reassess the sustainable utilization potential
of C. pelviformis in the horticultural-ornamental, agro-alimentary and medicinal sectors.
2. Materials and Methods
2.1. Plant Characteristics
Campanula pelviformis (Figure 1) is a hemicryptophyte with a restricted range occurring
only in the eastern part of the island of Crete, Greece [8]. This local endemic species
thrives in rocky slopes and ledges, stony dry places or walls in villages and rocky road
embankments, spanning an elevation range from sea level to 850 m above sea level [8].
Currently, it is considered as Vulnerable taxon [35] and a protected plant species due to its
inclusion in the Greek Presidential Decree 67/1981 (https://portal.cybertaxonomy.org/
flora-greece/cdm_dataportal/taxon/332301ee-177e-4f9f-95b9-62fcea5e9714, accessed 29
June 2023).
2.2. GIS Ecological Profiling
The natural occurrence points of C. pelviformis derived from a previously published
study [36]. To create the ecological profile in geographical information systems (GIS) for
this species, historical (1970–2000) climate and precipitation data as well as similar values
regarding 19 bioclimatic variables (minimum, maximum, and average values for every
month) with a pixel size 30 sec and spatial resolution of 1 km2 , were obtained from the
WorldClim database (https://www.worldclim.org/data/worldclim21.html, accessed on
15 June 2023) and were linked with the raster file containing its natural distribution points
in Crete (n = 16).
2.3. Seed Collection and Storage
Mature seeds from approximately five to ten wild-growing individuals of five wildgrowing populations thriving in different altitudes were collected by hand during the
summer of 2019 and the summer of 2020, prior dispersal (Table 1). As C. pelviformis is both
a protected and threatened species, the seeds were collected after applying for a special
permission, and the collection permits (182336/879 of 16 May 2019 and 64886/2959 of 6 July
2020) were issued by the Greek Ministry of Environment and Energy. Upon collection,
the collected capsules of each population were spread out on filter papers in laboratory
conditions and left for a week to dry out. Then, the seeds were cleaned manually from
capsule and/or stem remnants, and they were dry-stored in glass containers at 3–5 ◦ C
before experimentation.
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(A)
(B)
(C)
(D)
Figure 1. Plant individual of Campanula pelviformis as chasmophyte on vertical rocky crevices of Kalo
Chorio (Lasithi, Greece) in full flowering (A), ripe fruits collected in Mt Thrypti (B), seed germination
in Petri dishes (C) and individual germinated seeds (D).
Table 1. Geographical details about the seed collections performed and IPEN (International Plant
Exchange Network) accession numbers of Campanula pelviformis seed lots collected from lowland and
semi-mountainous areas of Eastern Crete,ffGreece.
IPEN Accession
Number
Collection Date
Collection Site in
Lasithi, Eastern
Crete
Latidude (North)
Longitude (East)
Altitude (m)
GR-BBGK-1-20,365
GR-BBGK-1-19,1056
26 July 2020
6 August 2019
After Kalo Chorio
Istron
35.1242
35.1248
25.7490
25.7444
45
25
GR-BBGK-1-19,1194
29 July 2019
Mochlos
area
35.1725
25.9051
105
GR-BBGK-1-19,1187A
14 July 2019
Thrypti
northern
outskirts
35.0931
25.8639
835
GR-BBGK-1-19,1187B
14 July 2019
Central Thrypti
35.0903
25.8658
870
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2.4. Germination Tests
The germination experiments were conducted in April 2022 at the Laboratory of
Floriculture, School of Agriculture, Aristotle University of Thessaloniki (Thermi, Greece).
Prior to experimentation, measurements of seeds’ weight, length and width were conducted
for each population. The weights of 1000 seeds for each population were measured using
a laboratory scale with four decimals, and photos from 30 seeds per population were
taken for calculating their length and width using the ImageJ software (ImageJ, U. S.
National Institutes of Health, Bethesda, MD, USA). Based on the above measurements,
the seeds from lowland populations used in the experiments had a 1000-seed weight of
0.099 g for GR-BBGK-1-20,365, 0.07 g for GR-BBGK-1-19,1056 and 0.042 g for GR-BBGK1-19,1194, whereas both semi-mountainous populations had a lower 1000-seed weight of
0.02 g in both cases. The average length/width of the seeds from lowland populations
ranged from 0.615/0.283 mm (GR-BBGK-1-19,1194) to 0.691/0.358 mm (GR-BBGK-1-20,365).
In comparison, the semi-mountainous populations showed an average length/width of
0.592/0.284 mm (GR-BBGK-1-19,1187A) and 0.583/0.280 mm (GR-BBGK-1-19,1187B).
The temperature experiments employed seeds from five distinct populations originating from various altitudes, as described above, and evaluated their seed responses
to four constant temperatures of 10, 15, 20 and 25 ◦ C within four different temperaturecontrolled growth chambers. For each temperature treatment, we tested four replications of
20 seeds (n = 4) placed in 9 cm sterile plastic Petri dishes with two filter papers moistened
with distilled water. All Petri dishes were randomly placed on the shelves of the growth
chambers with the photoperiod adjusted at 12 h light/12 h dark. During the experimental
period, all filter papers were kept regularly moist. Every five days for a period of 45 days,
all germinated seeds were totaled and removed. The criterion used for determining seed
germination of an individual seed was the evident emergence of radicle protrusion (at least
2 mm) from the seed coat.
After completion of the temperature-related germination experiments, new experiments were carried out to investigate the light requirements for C. pelviformis seed germination. The population GR-BBGK-1-19,1194 with the highest gemination percentages and
the temperature of 15 ◦ C for seed germination were selected for these experiments. The
same experimental design/protocol was followed, and Petri dishes were chambered under
alternating light/dark (12 h light/ 12 h dark photoperiod) and continuous dark conditions.
For the treatment in the dark, each Petri dish was covered with aluminum foil, which was
opened only for less than a few minutes to allow scoring for seed germination and was
immediately reclosed. For each treatment (light/dark, dark), the mean germination time
(MGT) was calculated as the average of four replications consisted of 20 seeds each. The
MGT was calculated for each replication per treatment according to the following equation:
MGT =
Σ( D ∗ n)
Σn
where n is the number of seeds which germinated on day D, and D is the number of days
counted from the beginning of the test [37].
2.5. Plant Production
The seeds of C. pelviformis GR-BBGK-1-19,1056 (Table 1) were used for a pilot germination experiment performed in December 2020 aiming at plant production. Specifically,
three germinated seeds were placed in each plastic pot (6 × 6 × 6.5 cm) filled with a 3:1
ratio (v/v) substrate of sphagnum peat moss (TS2, Klasmann-Deilmann GmbH, Geeste,
Germany) and perlite (Perloflor Hellas, Volos, Greece); the latter substrate was carefully
topped with river sand, and all the containers were placed randomly on a greenhouse
bench. After seedling emergence and the fully developed cotyledons, the seedlings were
thinned so that one seedling remained per pot. In total, there were 36 pots with one seedling
each. The potted seedlings were periodically irrigated to maintain the proper moisture
conditions for further growth until developing a potent root system. Three months after
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sowing (in the middle of March 2021), all seedlings were carefully transplanted into larger
pots (8.5 × 8.5 × 9.5 cm). The substrate of the larger pots was soil, sphagnum peat moss
(TS2, Klasmann-Deilmann GmbH, Geeste, Germany) and perlite (Perloflor Hellas, Volos,
Greece) in a ratio of 4:5:1 (v/v). To assess the soil fertility before the substrate preparation,
a soil sample of approximately 1.5 kg was chemically and mechanically analyzed (Table 2).
Table 2. Chemical and physical properties of the soil used for Campanula pelviformis seedling development.
Chemical Analysis
Mechanical Analysis
pH
Organic
Matter (%)
Soluble
Salts
(mS/cm)
CaCO3
(%)
Sand
(%)
Silt
(%)
Clay
(%)
8.12
0.36
0.35
5.50
56.00
28.00
16.00
Macronutrient concentrations (ppm)
N-NO3
P
K
Mg
Ca
8.00
8.00
104.00
842.00
>2000
Micronutrient concentrations (ppm)
Fe
Zn
Mn
Cu
4.7
2.00
7.06
0.77
After transplantation, a total of 36 seedlings were randomly divided into 3 groups of
12 plants each (n = 12) to employ 3 different fertilization treatments: seedling group with
integrated nutrient management (INM) applied; a second group with chemical fertilization
(ChFe) applied; and a third group with no applied fertilization (control). Both types of
fertilizers were foliar spray applications using a low-pressure electric sprayer system;
the plants were sprayed with the INM and ChFe treatment solutions until saturation,
and the control group was sprayed only with tap-water. The INM fertilization consisted
of a nutrient solution of THEOCOPPER at 7 mL/L, THEOCAL at 1.5 g/L, THEOFAST
at 5 mL/L (THEOFRASTOS fertilizers, Korinthos, Greece), 10-47-10 (AGRI.FE.M. LTD
Fertilizers, Athens, Attica, Greece) at 3.2 g/L, K2 SO4 (0-0-52, AGRI.FE.M. LTD Fertilizers,
Greece) at 2.07 g/L, micronutrients (Plex Mix, AGRI.FE.M. LTD Fertilizers, Athens, Attica,
Greece) at 1.5 mL/L and MgSO4 (Mg 25.6%, AGRI.FE.M. LTD Fertilizers, Greece) at
0.6 g/L [21,27]. For the conventional fertilization (ChFe), the nutrient solution consisted of
NH4 NO3 (34,4-0-0, Neofert® , Neochim PLC, Dimitrovgrad, Bulgaria) at 2.7 g/L, Ca(NO3 )2
(NITROCAL, Agrohimiki, Patras, Greece) at 1.7 g/L, 10-47-10 at 3.2 g/L, K2 SO4 (0-0-52) at
2.27 g/L, micronutrients Plex Mix at 1.5 mL/L and MgSO4 (Mg 25.6%) at 0.6 g/L [21,27].
Both fertilization treatments obtained the same units of macronutrients at ratio of 15-15-15,
Mg at 1.5%, Ca at 4.6% and the same amounts of micronutrients. The INM treatment
consisted of an additional amount of copper at 1% w/v, polysaccharides, organic nitrogen
(at urea and amino acid form) and a foliar biostimulator (THEOFAST) compared to the
ChFe treatment. The application of fertilization treatments was performed every week
from late March 2021 until the middle of June 2021, and the plants were irrigated every
2–3 days depending on ambient temperature conditions. The plants were grown inside an
unheated greenhouse of the Laboratory of Floriculture, School of Agriculture, located in
the farm of Aristotle University of Thessaloniki (Thermi, Greece).
2.6. Morphological and Physiological Measurements of Seedlings
At the end of June, we evaluated the effect of fertilization treatment on morphological
and physiological variables of C. pelviformis. In each plant individual per treatment, we
measured the main shoot height (SH) using a metal ruler, root collar diameter (RCD) using
a digital calliper and the shoot number. In addition, we measured the root dry biomass
(RDB) and the above-ground dry biomass (AGDB) in four randomly sampled plants per
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treatment. All dry weights were defined after drying of the materials in the oven at 74 ◦ C
for 48 h.
Along with the above-mentioned, gas exchange parameters, chlorophyll content and
fluorescence were determined in the fertilized and control seedlings of C. pelviformis. A
chlorophyll meter CCM 200 (Opti-sciences, Tyngsboro, MA, USA) was used to calculate
the chlorophyll content index (CCI) based on the ratio of transmittance measurement
at 660 and 940 nm [38]. The chlorophyll fluorescence parameter Fv/Fm was measured
by OS30p+ Rapid Plant Stress Screening Device (Opti-sciences, Tyngsboro, MA, USA).
Generally, Fv/Fm is the ratio of variable (v) to maximum (m) fluorescence after darkadaptation, thus representing the maximum photochemical efficiency of photosystem
II, which is commonly used for detection of various stress conditions [39]. For each
fertilization treatment, 13 measurements with CCM200 and 10 measurements with OS30p+
were performed on fully expanded young leaves of the C. pelviformis plant individuals.
2.7. Plant Tissue Analyses of Seedlings
After drying in the oven at 74 ◦ C for 48 h, the tissue samples of the C. pelviformis plant
individuals were finely ground to pass a 40-mesh sieve. More precisely, for each treatment
the above-ground part (leaves and shoots) and the below-ground part (roots) of the dried
plants were separately grounded to record the respective tissue nutrient concentration.
Three samples of fine powder were formed for each plant part (either above-ground or
below-ground), which corresponded to the three fertilization treatments (ChFe, INM,
control) examined. Subsequently, from each fine powdered and homogenized sample,
three subsamples of ca. 0.25 g each were randomly taken (n = 3), and each subsample was
disorganized by the method of wet oxidation until a transparent solution was obtained
using a triple acid mixture of H2 SO4 , HNO3 and HClO4 in a ratio of 5:1:1 at 80 ◦ C [40].
The solutions resulting from the digested samples were analyzed colorimetrically for the
total P (phosphorus) determination according to the molybdenum blue method by using
a Shimadzu spectrophotometer model UV-1201V [41]. The potassium (K), calcium (Ca),
magnesium (Mg), sodium (Na), copper (Cu), iron (Fe), zinc (Zn) and manganese (Mn)
concentrations were assessed by atomic absorption spectroscopy (Perkin-Elmer Analyst
300), while total nitrogen (N) was determined by the Kjeldahl method [42].
2.8. Total Phenol Content and Antioxidant Capacity
Fresh leaves from C. pelviformis plant individuals were selected per fertilization treatment at the end of the experiment and were grounded to powder by liquid nitrogen. The
amount of 0.1 g of lyophilized leaves was extracted by adding 1 mL methanol, was vortexed
for 20 min and was centrifuged at 10,000× g at room temperature for 5 min. The upper
liquid portion was separated from the solid residue, was transferred to a new vial, and was
stored under −20 ◦ C until analyzed.
We employed the Folin–Ciocalteu method [43] for the determination of total phenol
content (TPC) in samples. Each sample (0.5 mL) was diluted in MeOH (1:50 v/v) with the
addition of 2.5 mL of the Folin–Ciocalteu solution (1:10 v/v). Two (2) mL of Na2 CO3 (7.5%
w/v) solution were also added after 6 min, and the whole mixture was vortexed. Finally,
the prepared sample mixtures were placed in a water bath for 5 min at 50 ◦ C (LabTech
Digital Water Bath, Gurgaon, India). Analyses of all samples were conducted by recording
their absorbance at 760 nm by a Helios Alpha spectrophotometer (Thermo Spectronic,
Cambridge, UK). We used a gallic acid calibration curve (100, 50, 25, 12.5 and 0 g/mL) to
calculate the TPC as mg of gallic acid equivalents per g of fresh weight (mg GAE/g FW).
We employed the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method [43] for the determination of antioxidant capacity (AC). In 0.2 mL of samples dissolved in 80% v/v MeOH
(1:20 v/v), the amount of 2.8 mL 4% w/v DPPH was added, vortexed and incubated in
darkness for 30 min at room temperature. The AC of the samples was recorded at 517 nm
in a spectrophotometer, was compared to an ascorbic acid calibration curve (0.1, 0.05, 0.025,
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0.01 and 0 g/mL), thus was expressed in mg of ascorbic acid equivalents per g of fresh
weight (mg AAE/g FW).
The final readings of TPC and AC determination of each C. pelviformis sample represented the mean of three biological replications, and they were shown as mean ± standard
error. Analysis of variation (ANOVA) was used for comparisons of means using SPSS 27
(IBM Corp., Armonk, NY, USA) and Duncan’s multiple range test at p < 0.05.
2.9. Statistical Analysis
In the germination experiment, the experimentation employed a completely randomised design with two factors (different populations, and incubation temperature). The
germination data at 25 ◦ C was not further analysed since none of the seeds germinated
for populations GR-BBGK-1-19,1194; GR-BBGK-1-19,1187A; GR-BBGK-1-19,1187B, or the
percentages of the germinated seeds were very low, i.e., <6% in populations GR-BBGK-120,365 and GR-BBGK-1-19,1056. Therefore, in the statistical analysis, the levels of the factors
were: five for the populations and three for the incubation temperature (5 × 3 factorial
design). The data were analysed using ANOVA in the frame of the procedure general linear
model (GLM) [44], and the means were compared using the LSD test at a significance level
p ≤ 0.05. In the remaining statistical analyses, the comparisons of the means were made
using the Duncan’s test at a significance level p ≤ 0.05 [45]. All statistical analyses were
performed with SPSS 21.0 (SPSS, Inc., IBM Corp., Armonk, NY, USA).
3. Results
3.1. Seed Germination Success
3.1.1. Effects of Temperature Treatments
The population (p = 0.002) and temperature (p = 0.000) factors as well as their interaction (p = 0.000) had a significant effect on seed germination of C. pelviformis.
The most successful germination was achieved at 10 and 15 ◦ C for populations GRBBGK-1-19,1194 and GR-BBGK-1-19,1187A (97.5% and 92.5%, respectively), while populations GR-BBGK-1-20,365 (96.25%) and GR-BBGK-1-19,1187B (95%) exhibited higher
germination percentage at 15 and 10 ◦ C, respectively, than at 20 ◦ C (Figure 2). For population GR-BBGK-1-19,1056, temperature had no statistically significant impact on seed
germination (Figure 2).
Regarding the germination speed of C. pelviformis seeds, it was observed that in
all populations the seeds germinated faster at 15 ◦ C in comparison with the rest of the
temperature treatments (Figure 2). In fact, population GR-BBGK-1-20,365, GR-BBGK-119,1056 and GR-BBGK-1-19,1187B recorded higher rates of germination (>60%) on the
10th day (Figure 2). Furthermore, the germination of seeds at 20 ◦ C was quite gradual
compared to the rest of the examined temperatures for all populations (Figure 2).
The temperature level of 10 ◦ C had a significant impact on seed germination of
C. pelviformis, with seeds of GR-BBGK-1-19,1194 population exhibiting higher germination
percentage compared to those of population GR-BBGK-1-19,1056 (Table 3). At 15 ◦ C, the
seeds of the lowland populations GR-BBGK-1-20,365 and GR-BBGK-1-19,1194 achieved
higher germination percentages than those of the semi-mountainous populations GR-BBGK1-19,1187A and GR-BBGK-1-19,1187B (Table 3). The temperature level of 20 ◦ C notably
reduced all seed germination percentages of the examined populations, particularly in GRBBGK-1-19,1187A and GR-BBGK-1-19,1194. However, in the same temperature the seeds of
the lowland population GR-BBGK-1-19,1056 germinated at their highest percentage and
those of population GR-BBGK-1-19,1187B still showed 81.25% germination (see Table 3).
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Population GR-BBGK-1-20,365
100
a1
ab
b
ns2
80
60
40
40
40
20
20
20
0
5
10
15
20 25 30
Time (days)
35
40
0
45
a
a
80
60
0
Population GR-BBGK-1-19,1194
100
60
0
5
10
15
20
25
Time (days)
30
35
40
0
45
b
0
5
10
15
20
25
Time (days)
30
35
40
45
(A)
Population GR-BBGK-1-19,1187A
100
a1
Population GR-BBGK-1-19,1187B
100
a
80
Germination (%)
Germination (%)
80
Population GR-BBGK-1-19,1056
100
a
ab
b
80
b
60
60
40
40
20
20
0
0
0
5
10
15
20
25
Time (days)
30
35
40
45
0
5
10
15
20
25
Time (days)
30
35
40
45
(B)
Figure 2. Cumulative germination percentage diagrams of Campanula pelviformis seeds of the five populations ((A): lowland populations; (B): semi-mountainous
populations, see Table 1) incubated at 10 (•●), 15 (■) and 20 (□). The comparisons were made using the LSD test. 1 —In each population, means were statistically
ff
tt
ff
different at p < 0.05 when not sharing a common letter (n = 4); ns2 —non-significant differences (n = 4).
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Table 3. Effect of Campanula pelviformis populations from lowland and semi-mountainous areas
on the seed germination percentage for each incubation temperature with high seed germination.
Means ± standard deviation values are given.
Population
Area
10 ◦ C
Incubation Temperatures
15 ◦ C
20 ◦ C
96.25 ± 4.79 a
91.25 ± 2.50 ab
96.25 ± 4.79 a
86.25 ± 4.79 b
95.00 ± 4.08 a
73.75 ± 8.54 cd
85.00 ± 4.08 b
87.50 ± 5.00 b
70.00 ± 10.80 d
81.25 ± 8.54 bc
1
Lowland
GR-BBGK-1-20,365
GR-BBGK-1-19,1056
GR-BBGK-1-19,1194
90.00 ± 4.08 ab
88.75 ± 4.79 b
97.50 ± 2.89 a
Semimountainous
GR-BBGK-1-19,1187A
GR-BBGK-1-19,1187B
92.50 ± 2.89 ab
95.00 ± 4.08 ab
1
Values in the same column followed by the same letter are not significantly different (p > 0.05, n = 4) according
to LSD test.
3.1.2. Effects of Light Treatments
No statistically significant results were observed in terms of mean germination percentage and mean germination time (MGT) between C. pelviformis seeds germinated under
alternating light/dark and continuous dark conditions. Germination percentages were
high for both treatments (98.75% in light/dark and 95% in dark) (Table 4), and the examined seeds showed similar behaviour regarding MGT under different light conditions
(10.95 days in light/dark and 11.32 days in dark conditions).
Table 4. Effect of light conditions on germination of Campanula pelviformis population GR-BBGK-119,1194 seeds. Means and standard deviation values are given.
Germination Percentage (%)
Light/dark
Dark
98.75 ± 2.50 a
1
95.00 ± 4.08 a
Mean Germination Time (Days)
10.95 ± 0.14 a
11.32 ± 0.53 a
1
In each column, means followed by the same letter are not significantly different at p < 0.05, n = 4. The
comparisons were made using T-test.
3.1.3. Ecological Profiling
To better understand the biological circle of C. pelviformis, an ecological profile was
created in GIS depicting the temperature and precipitation needs of the wild-growing
populations (Supplementary Material Table S1).
The results of the temperature-related attributes showed that C. pelviformis populations
are naturally adapted to a relatively warm winter season (mean temperature of coldest
quarter = 11.12 ± 0.87 ◦ C; minimum temperature of coldest month = 7.63 ± 0.86 ◦ C),
with the lowest average temperatures recorded in January (10.77 ± 0.89 ◦ C) and February (10.69 ± 0.86 ◦ C). The average temperature is shown to rise gradually from March
(11.96 ± 0.84 ◦ C) until June (22.71 ± 0.70 ◦ C). Onwards in mid-summer (July–August), the
average temperature steadily rises to 26 ◦ C. After the summer season, the average temperature decreases from 22.72 ± 0.79 ◦ C in September to 12.25 ± 0.88 ◦ C in December (winter
season). The aforementioned factors, combined with the absence of extreme temperatures
in its natural distribution sites (Tmin of Tmin = 5.71 ◦ C in February, Tmax of Tmax = 30.29 ◦ C
in July) and the low mean diurnal range (6.86 ± 0.13 ◦ C), may indicate that C. pelviformis is
naturally adapted in relatively warm environments with regards to temperature.
Historical precipitation data depicted a seasonality of rainfall in the natural range of
C. pelviformis. Specifically, high precipitation was recorded from December (120.09 ± 11.06 mm)
until February (98.31 ± 8.99 mm), with the highest precipitation values recorded in January
(134.03 ± 12.20 mm). From March (75.16 ± 7.74 mm) onwards, the mean of precipitation
was shown to decrease until May (16.90 ± 2.75 mm), leading to summer season, which is
the driest period with scarce precipitation (4.54 ± 1.22 mm in June, 1.68 ± 0.60 mm in July
and 2.00 ± 0.52 mm in August). After summer, the mean of precipitation was shown to
rise again from 17.42 ± 1.20 mm in September to 72.80 ± 4.80 mm in November, initiating
the wet season.
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3.2. Seedlings’ Growth
The parameters main shoot height (p = 0.085), root collar diameter (p = 0.827), number
of shoots (p = 0.146), and above-ground dry biomass (p = 0.086) were not affected by the
applications of fertilizers (Table 5), whereas fertilizing had impact on root dry biomass
(p = 0.005), chlorophyll content index (p = 0.000), and chlorophyll fluorescence (p = 0.017) of
C. pelviformis plant individuals (Table 5). As far as the root dry biomass is concerned, control
plants showed significantly lower values compared to INM fertilized plants, whereas no
significant difference was observed between plants fertilized with INM and ChFe (Table 5).
The non-fertilized plants recorded the lowest values of chlorophyll content, and the plants
fertilized with INM had the highest values (Table 5). The chlorophyll fluorescence of
fertilized plants regardless of the type of fertilizer was significantly higher compared to
that of the non-fertilized plants of C. pelviformis (Table 5).
Table 5. Effect of fertilization (INM: integrated nutrient management; ChFe: chemical fertilization) on morphological and physiological characteristics of Campanula pelviformis plant individuals.
Means ± standard deviation values are provided.
Attribute (Unit)
Control
INM
ChFe
Shoot height (cm)
Root collar diameter (mm)
Number of shoots
Root dry biomass (g)
Above ground dry biomass (g)
Chlorophyll content index (CCI)
Chlorophyll fluorescence (Fv /Fm )
18.58 ± 2.23 a *
8.94 ± 1.27 a
11.58 ± 1.73 a
1.00 ± 0.12 b
1.89 ± 0.29 a
17.72 ± 2.96 c
0.748 ± 0.006 b
17.75 ± 2.30 a
9.27 ± 1.51 a
11.08 ± 2.74 a
1.72 ± 0.41 a
1.85 ± 0.30 a
50.59 ± 7.66 a
0.763 ± 0.022 a
19.92 ± 2.43 a
9.28 ± 1.80 a
13.25 ± 3.49 a
1.39 ± 0.42 ab
2.32 ± 0.26 a
29.94 ± 7.44 b
0.768 ± 0.009 a
* Values in the same row followed by the same letter are not significantly different (p > 0.05, n = 12) according to
Duncan’s test.
3.3. Leaf Nutrient Concentration
Macronutrient concentrations in leaves and shoots of C. pelviformis (Table 6) were generally affected by fertilization (p = 0.000 for N; p = 0.002 for P; p = 0.000 for K; p = 0.000 for
Ca; p = 0.000 for magnesium; p = 0.000 for Na). The macronutrient content of C. pelviformis
leaves and shoots showed a trend for increased concentration of N, K, Ca, Mg and Na in
plants treated with INM compared to unfertilized plants (control) or ChFe, while P was
detected in the highest concentration in the leaves and shoots of untreated plants (Table 6).
Table 6. Macronutrient concentrations in above-ground (leaves and shoots) and below-ground (roots)
parts of cultivated Campanula pelviformis plant individuals in greenhouse with regards to fertilization
(INM: integrated nutrient management; ChFe: chemical fertilization; Control: absence of INM or
CHFe, only water).
Treatment
Nitrogen
(N) (%)
Phosphorus Potassium
(P) (mg/g)
(K) (mg/g)
Calcium
(Ca)
(mg/g)
Magnesium
(Mg)
(mg/g)
Sodium
(Na)
(mg/g)
4.88 b
5.52 a
4.79 b
8.35 c
14.63 a
11.06 b
1.93 c
2.37 a
2.17 b
3.04 a
3.28 a
2.98 a
Above-ground part (leaves, stems)
Control
INM
ChFe
1.25 c *
2.12 a
1.65 b
3.20 a
2.99 b
3.01 b
26.39 b
30.17 a
25.45 c
15.18 c
18.85 a
16.19 b
Below-ground part (roots)
Control
INM
ChFe
1.11 c
2.06 a
1.34 b
1.23 a
0.87 b
1.24 a
5.27 a
4.41 b
5.27 a
2.48 b
3.43 a
2.30 b
* In the same column, the means are statistically different at p < 0.05, n = 3 when not sharing a common letter. The
comparisons were made using the Duncan’s test.
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In the roots of the plant, fertilization affected all macronutrient concentrations (p = 0.000
for N; p = 0.004 for Pl; p = 0.000 for K; p = 0.000 for Ca; p = 0.005 for Mg) except Na (p = 0.198).
INM fertilization showed increased contents of N, Ca and Mg compared to ChFe and
control plants, while the application of INF decreased P and K compared to the other two
treatments.
The fertilization affected the concentration of micronutrients (Table 7) in the aboveground parts (p = 0.000 for Fe; p = 0.008 for Mn; p = 0.000 for Zn; p = 0.000 for Cu) of
cultivated C. pelviformis. A remarkable 10-fold increase was detected in the concentration
of Cu in plants treated with INM compared to control plants (Table 7). A high increase was
also noticed in the concentration of Zn, but no statistical differences were found regarding
Mn between the plants treated with INM and ChFe. The concentration of Fe was increased
in ChFe plants compared to the other two treatments.
Table 7. Micronutrient concentration in above-ground (leaves and shoots) and below-ground (roots)
parts of Campanula pelviformis plant individuals with regards to fertilization (INM: integrated nutrient
management; ChFe: chemical fertilization; Control: only water).
Treatment
Iron (Fe) (ppm)
Manganese
(Mn) (ppm)
Zinc (Zn) (ppm)
Copper (Cu)
(ppm)
Above-ground part (leaves, stems)
Control
INM
ChFe
633.08 c *
721.71 b
797.25 a
52.71 b
64.84 a
66.94 a
12.22 c
19.80 a
15.43 b
15.65 b
152.79 a
2.96 c
17.00 a
17.14 a
15.06 a
13.77 b
22.68 a
14.30 b
Below-ground part (root)
Control
INM
ChFe
553.60 a
452.77 a
527.01 a
27.05 a
20.52 b
26.47 a
* In the same column, the means are statistically different at p < 0.05, n = 3 when not sharing a common letter. The
comparisons were made using the Duncan’s test.
Similarly, root micronutrients were also affected by INF fertilization (p = 0.052 for
Fe; p = 0.025 for Mn; p = 0.001 for Cu; p = 0.525 for Zn). INF fertilization increased the
concentration of Cu, while ChFe and control plants had higher concentration for Mn
(Table 7).
3.4. Total Phenol Content and Antioxidant Capacity
The presence of total phenols was detected in all samples of C. pelviformis, but it was
more prominent and significantly higher in the fertilized samples, especially those treated
with ChFe (Table 8). The same trend was observed for the AC of the samples, where the
one with the most potent antioxidant capacity was again the samples from fertilized plants
with ChFe.
Table 8. Effect of fertilization (INM: integrated nutrient management; ChFe: chemical fertilization;
Control: only water) on the concentration of total phenols and antioxidant capacity of Campanula
pelviformis young leaves, expressed in mg gallic acid equivalents (GAE) and ascorbic acid equivalents
(AAE) per g of fresh (FW), respectively.
Treatment
Total Phenols
(mg GAE/g FW)
Antioxidant Capacity
(mg AAE/g FW)
Control
INM
ChFe
0.993 ± 0.037 b *
1.022 ± 0.009 b
1.195 ± 0.021 a
0.094 ± 0.0004 c
0.102 ± 0.0008 b
0.106 ± 0.0006 a
* Values are means ± standard error of the mean. Means followed by a different letter are statistically significantly
different (p < 0.05, n = 3).
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4. Discussion
4.1. Seed Germination Protocols
The results of this study suggested a strong temperature effect (p = 0.0001) in the
germination of C. pelviformis seeds in line with previous studies examining other members
of the genus Campanula [32]. The highest percentages (>85%) of seed germination were
observed at 10 and 15 ◦ C, while high rates were even recorded at 20 ◦ C (>70%). Similar
results have been recently reported for another chasmophytic local endemic plants of
Crete, namely Campanula saxatilis L. subsp. saxatilis [43]. Previous studies have indicated
that various taxa of the genus Campanula can germinate even at 25 ◦ C under specific
pretreatment methods [32], i.e., 99% for Campanula merxmuelleri Phitos, 91% for Campanula
pangea Hatvig, and 80% for C. glomerata. However, no pretreatments were tested herein for
C. pelviformis, and at this temperature, C. pelviformis seed germination was quite low and
was not included in the analysis.
The investigation herein attempted, for the first time, to examine the germination of
seeds collected from different populations of C. pelviformis from lowlands (three populations) and semi-mountainous areas (two populations) of Eastern Crete, Greece. This study
reports, for the first time, on the combined influence of altitude and temperature in seed
germination behavior of C. pelviformis as recorded for the different populations examined.
Although individual effects were not considered herein, this study provides evidence on
variation in seed germination of lowland C. pelviformis populations; for example, the seeds
of population GR-BBGK-1-19,1194 achieved higher germination percentage compared to
population GR-BBGK-1-19,1056 at 10 ◦ C, while the latter showed its highest seed germination at 20 ◦ C. Some variation was also detected between the two semi-mountainous
populations of C. pelviformis examined herein from Mt. Thrypti (the second highest mountain in the Lasithi region of Crete), resulting in different germination percentages but similar
decrease rates. In general, it is well established that differences may exist in the germination characteristics (rate and percentage of germination) of a taxon’s populations [30].
Previous studies have indicated that other Balkan endemic Campanula species may have
quite different germination responses related to temperatures depending on the altitude of
original collections; for example: (i) 10%, 11.1%, and 61.1% at 5, 15 and 25 ◦ C, respectively,
in C. spatulata Sm. seeds from intermediate (900 m) altitudes compared to 0%, 21.1% and
2.2% at 5, 15 and 25 ◦ C, respectively, in seeds collected from high (1850 m) altitudes [46]:
(ii) 0%, 64.4% and 65.6% at 5, 15 and 25 ◦ C, respectively, in C. lingulata Waldst. & Kit.
seeds collected from lowlands (450 m) compared to 0%, 54.4% and 30% at 5, 15 and 25 ◦ C,
respectively, for seeds originating from plants of intermediate (850 m) altitudes; and (iii) 0%,
56.7% and 16.7% at 5, 15 and 25 ◦ C, respectively, for seeds of plants from higher (1300 m)
altitudes as well as 0%, 36.7% and 4.4% at 5, 15 and 25 ◦ C, respectively, for seeds of subalpine altitudes (1700 m) close to timberline [46]. It should be noted, however, that the
above-mentioned examples refer to rather common and widespread in Greece Campanula
members which are Balkan endemics, with broad ecological adaptations and different
habitat preferences [46]. As such, these findings should not be directly compared with
other range-restricted chasmophytic Campanula members with narrow niche confined in
small geographical territories such as C. pelviformis studied herein (single-island endemic
of Crete). Undoubtedly, it would be useful to conduct further experimentation in freshly
collected seeds to draw safer conclusions in relation to the germination requirements of the
species in concern and in fact this presents another research line that we will soon follow.
Regarding the light requirements for C. pelviformis seed germination, the findings
herein suggest that this taxon has no specific preference, as high percentages of germination
were achieved in both light treatments (98.75% in alternating light/dark conditions and
95% in darkness), thus aligning with previous studies found in grey literature (AntonidakiGiatromanolaki as cited by Koutsouvoulou et al. [32]). On the other hand, the seeds of
several Campanula taxa clearly exhibit a preference for light for their germination [32],
i.e., C. andrewsii A. DC. subsp. andrewsii (93% in light and 26% in darkenss), C. cretica (A.
DC.) A. Dietr. (99% in light and 24% in darkness), C. spatulata subsp. spruneriana (Hampe)
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Hayek (90% in light 60% in darkness), C. spatulata subsp. spatulata (92% in light and 63% in
darkness), and C. versicolor Andrews (85% in light and 38% in darkness).
Based on the available data, it appears that the seeds of C. pelviformis germinate
best at temperatures of 10 and 15 ◦ C. Consequently, its natural germination in the wild
habitats is expected to occur between late autumn and early winter, where the mean
monthly air temperature may range between 10 and 15 ◦ C as indicated by the GIS-derived
ecological profile. During this period, the increased soil moisture due to high amount of
precipitation is probably expected to create the ideal conditions for the germination of
seeds (Supplementary Material Table S1). Furthermore, this timing of seed germination
probably allows the growth of seedlings through winter to develop into fresh rosettes of
C. pelviformis by February when local inhabitants of Crete traditionally collect this wild
edible green from the wild [6].
C. pelviformis is widely used in the eastern part of Crete (Lasithi), and young rosettes
of leaves are sourced directly from the wild habitats at the end of winter or early in spring
together with other wild edible plants. This represents a very localized old tradition in
the Lasithi area; for example, C. pelviformis is not mentioned as an edible wild green by
Pieroni et al. [47] in their study in central Crete, although it occurs in Heraklion prefecture [8]. This wild gathering of C. pelviformis is mainly being performed to prepare the
typical local culinary preparation called “tsigarolachana” (meaning to stir in olive oil).
The “tsigarolachana” can be served in everyday life with other basic food ingredients
(fish, other seafood, or meat), can be cooked in casserole with all the latter and/or with
eggs and other common vegetables. Ultimately, “tsigarolachana” is also the basic filling
(together with other wild greens) for pot pies with handmade dough [6]. The daily use and
consumption of wild ‘chórta’ (or hórta) including C. pelviformis is linked with the traditional
Mediterranean (Cretan) diet, which is associated with high adult life expectancy (longest
in the world) and decreased rates of coronary heart disease, certain types of cancer, and
other chronic diseases [47]. Among other wild greens, C. pelviformis is considered as a
medicinal plant of high nutritional value [6]. In the Lasithi area, there are many regular
open markets selling wild herbs and chórta in bunches, especially at the end of winter and
beginning of spring, at very high prices equivalent to those of high-quality meat. It should
be noted, however, that the high demand of such localized products even at local scales
can trigger over-collection from range-restricted wild populations, thus jeopardizing local
genetic pools of the local Cretan endemic C. pelviformis. Therefore, the ex situ propagation and cultivation with fertilization regimes as presented herein, for the first time, may
offer a sustainable exploitation strategy able to satisfy any future demand regarding the
traditionally consumed nutritious rosettes of C. pelviformis.
4.2. Fertilization and Morphological-Physiological Traits
Previous studies have indicated that fertilization applications can enhance the plant
photosynthetic rate [48]. The findings herein suggested that the photosynthetic rate such
as chlorophyll content index and chlorophyll fluorescence had positive impact from the
INF treatment. Similar outcomes had been observed in other rare local endemic species
such as the Tunisian endemics Teucrium luteum (Mill.) Degen subsp. gabesianum (S. Puech)
Greuter or Marrubium aschersonii Magnus, where fertilization treatments led to an increased
photosynthetic rate [22,28]. Additionally, another three Cretan endemic species namely
Lomelosia minoana (P. H. Davis) Greuter & Burdet subsp. minoana and subsp. asterusica
(Greuter) Greuter & Burdet, and Eryngium ternatum Poir. were affected in the same direction regarding these physiological characteristics from the application of fertilization
regimes [43]. More specifically, the INF treatment that resulted in this study in increased
root dry biomass in C. pelviformis in a similar pattern to the observations related with
M. aschersonii, while L. minoana subsp. asterusica and subsp. minoana showed a negative
impact following the INF treatment [28].
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4.3. Nutrient Content and Absorption
The effect of fertilization on ex situ cultivated C. pelviformis plants is the first study
to date, describing the content and the absorption of macro- and micronutrients. Both
INM and ChFe fertilization schemes resulted positively regarding most of the measured
parameters compared to the control. The INM fertilization showed statistically significant
higher values in chlorophyll content index (CCI) and root dry biomass (RDB) compared to
the ChFe and control; however, ChFe resulted in higher values for the rest of the parameters
measured, but these were not statistically significant (Table 5).
Tissue analysis of the above-ground parts (leaves, stems) indicated a trend of the INM
fertilization to lead to a higher nutrient content and absorption for N, K, Ca, Mg, Na, Zn
and Cu compared to the ChFe and control; however, ChFe and control plants resulted in
higher values only for Fe and P, respectively (Tables 6 and 7).
Tissue analysis for the below-ground part (roots) indicated that INM fertilization
showed higher nutrient content and absorption for N, Ca, Mg, and Cu compared to the
ChFe and control; however, both ChFe and control resulted in higher values for P, K, and
Mn compared to INM (Tables 6 and 7).
Measurements conducted on the leaves of other local endemic plants of Crete revealed
that L. minoana subsp. minoana benefited from the INF application in terms of all macronutrients examined except for Na [43]. E. ternatum showed benefits in terms of N and K,
while L. minoana subsp. asterusica exhibited high N and P levels. Additionally, in terms of
micronutrient measurements in plant leaves of these taxa, the INF fertilization resulted in
increased Cu levels for L. minoana subsp. minoana, Mn and Cu levels for E. ternatum, as well
as in all micronutrients except Mn for L. minoana subsp. asterusica [43]. It is well known that
Cu is an important micronutrient in photosynthesis and in carbon metabolism [49,50], but
it can also be toxic to plants in excess quantities [51]. In the current study, it is worth noting
a 10-fold increase and 58.6–64.7% higher amounts of Cu through INM foliar application
for the tissue analysis of above-ground parts and below-ground parts of C. pelviformis,
respectively, as compared to the ChFe and control plants. The latter means that Cu moves
systemically in plant tissues and, moreover, is translocated downstream to the root system
of C. pelviformis plants, without showing any toxicity effects. Since Cu plays an important
role in controlling fungal diseases in plants [52], the high amounts of Cu in C. pelviformis
leaves, stems and roots probably act as protective agent against fungal pathogens. Undoubtedly, more experiments are needed to investigate the beneficial properties of Cu
absorption in different developmental stages of C. pelviformis prior to further conclusions.
By analyzing the concentrations of nutrients in different plant organs, valuable insights
are obtained that enhance the understanding of plant health and ecological adaptation,
as well as specific needs in terms of fertilization [53]. At the same time, identifying
nutrient deficiencies in plants can contribute significantly to the optimization of precision
agricultural technology, forming appropriate strategies for applying nutrients to plants
through fertilization [54,55]. This approach ensures both economic and environmental
benefits which are very important aspects of modern agriculture [56].
This study also showcased the beneficial effect of fertilization and the ease of responsiveness of the cultivated germplasm to fertilization, and it can provide evidence on the
nutritional superiority of the cultivated germplasm. The latter, if sustainably exploited and
managed, reveals a noteworthy economic potential for new product development with
high added value which can be marketed in an exclusive way to address the commercial
demand for C. pelviformis rosettes at least at local (Crete) or national (Greece) scales [2]. In
the light of the above-mentioned, INF fertilization appears to offer promising prospects for
enhancing cultivation practices for C. pelviformis when preparing nursery stocks, either for
population re-introduction purposes or sustainable exploitation strategies.
4.4. Total Phenol Content, Antioxidant Capacity and Fertilization Protocols
The study herein provides, for the first time, an insight on the macro- and micronutrient
profiles of the ex situ cultivated C. pelviformis and on the effect of fertilization regimes in
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TPC and AC of C. pelviformis. The presence of total phenols was evidenced in all samples
of ex situ cultivated C. pelviformis regardless of fertilization, but they were prominent and
significantly higher in fertilized samples, especially those treated with ChFe. The same
trend was observed for the AC of the cultivated samples of C. pelviformis.
Considering other Cretan endemic plant species examined to date, the ChFe application has been shown to have a positive impact on both total phenols and antioxidant
capacity for L. minoana subsp. minoana and E. ternatum, similar to the patterns observed
in C. pelviformis, while total phenol content is reported to increase in plants without any
fertilization application; antioxidant capacity is reported as negatively affected by the INF
application for L. minoana subsp. asterusica [43]. Previous research on other Campanula
species has shown that the presence of total phenolics and significant antioxidant activity
is a fact. For instance, previous studies [57] demonstrated strong antioxidant activity in
methanol extracts of whole plants of C. alliariifolia Willd. Similarly, other studies [58] investigated, in a comparative way, the antioxidant activity of 15 Campanula species from
Palestine, concluding that C. sulphurea Boiss. and C. sidoniensis Boiss. & C.I.Blanche show
comparatively the best DPPH radical scavenging activity. In a separate study, the phytochemical analysis of C. latifolia L. has reported very high levels of DPPH radical scavenging
activity in both the aerial parts and roots of the species [59]; however, comparatively lower
concentrations of total phenols have been detected in the aerial parts (0.053 mg Gallic
acid/g extract) and root extracts (0.037 mg Gallic acid/g extract) of C. latifolia compared to
the concentrations found in fresh leaves of C. pelviformis in the present study.
4.5. Re-Evaluation of Feasibility and Readiness Timescale for Sustainable Exploitation
The data furnished in this study may facilitate future conservation efforts and population re-introduction actions in the wild habitats and also pave the way for the sustainable
utilization of C. pelviformis. In this sense, the amount of data furnished in this study shed
light on the urgent need to re-evaluate the feasibility and readiness timescale of value chain
establishment for the species of concern. The medicinal potential (Level I evaluation) of
C. pelviformis has increased considerably to above average to high potential (68.2%) due to
newly furnished data on isolated phytochemical compounds with medicinal properties [6],
coupled with evidence on its nutritive value in terms of macro- and micronutrients (both
Tsiftsoglou et al. [6] and this study), thus rendering C. pelviformis as a noteworthy medicinal
plant of agro-alimentary interest.
Furthermore, the feasibility of value chain establishment for C. pelviformis (Level II
evaluation in Krigas et al. [2]) was improved from 43.06% [2] to 66.67% after re-evaluation
of related attributes in light of newly furnished data. This upgrade affected the readiness
timescale for its sustainable exploitation (Level III evaluation) from achievable in long-term
to achievable in medium-term (see Table 9). This re-evaluation was based on the change
of threat status for C. pelvifromis (Vulnerable according to Kougioumtzis et al. [35]), which
resulted in a score of four compared to zero; the successful seed germination, resulting in a
score of six compared to zero; the definition of its cultivation needs in terms of watering
and fertilization (score six from four), resulting in full cultivation protocols (score of six
instead of two); and the documentation of its extant commercial prices as wild edible greens
gathered from the wild [6].
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Table 9. Overview of the multifaceted evaluation regarding Campanula pelviformis (see Krigas et al.
[2] which is revisited herein in light of the data furnished in this study. Previous assessments and
upgrades of individual scores after the data presented herein are discerned with right arrow (→).
General
Ornamental
Interest [2]
* Special
Ornamental
Interest [2]
AgroAlimentary Interest
[11]
Medicinal
Interest [12]
Sustainable Exploitation
Upgrades (Level II and III
Assessments **)
Below average
Average
Below average
Low → Above average
to high
43.06% → 66.67%
42.86%
38.89% → 68.52%
Achievable in long-term →
in medium-term
41.67%
53.13%/8.05%/52.15%/59.29%
* 1st percentage: as pot/patio plant; 2nd percentage: for home gardening; 3rd percentage: for landscaping; 4th
percentage: for xeriscaping after Krigas et al. [2]. ** Assessments regarding the feasibility (Level II evaluation)
and readiness timescale (Level III evaluation) for the sustainable exploitation as estimated in [2].
5. Conclusions
This study provided for the first time comprehensive information on the abiotic
environmental conditions of the C. pelviformis based on its distribution in natural habitats
of Crete, Greece. The GIS ecological profile developed offers valuable insights into the
successful ex situ conservation and cultivation of this vulnerable local endemic species,
as well as guidance for its adaptation in anthropogenic environments for conservation
purposes. The results of the seed trials clearly demonstrated the effective seed germination
of C. pelviformis achieved herein for the first time, thus marking a significant step towards
its in situ conservation and its potential sustainable utilization for noteworthy ornamental,
agro-alimentary and medicinal properties. The experimental cultivation achieved in this
study for the first time provided valuable insights into the beneficial effects of different
types of fertilization in morphological-physiological characteristics, in phytochemicals
such as total phenols and antioxidant capacity as well as in terms of increased contents
of macro- and micro-nutrients contents as documented in the above-ground compared
to wild-growing plants as well as in the below-ground part of C. pelviniformis which
was studied herein for the first time. All the data furnished in this multidisciplinary
investigation built to bridge extant applied research gaps was finally exploited to provide
an update on the feasibility and readiness timescale for the sustainable exploitation of
C. pelviformis, thus further facilitating the creation of a new value chain for the ex situ
cultivated C. pelviformis. The latter is expected to alleviate the collection pressure exerted to
the traditionally harvested wild-growing populations of this remarkable yet vulnerable
local endemic plant species of Crete.
Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/horticulturae9080877/s1, Table S1: Ecological profile across the
natural distribution range of Campanula pelviformis wild-growing populations in Crete, Greece.
Author Contributions: Conceptualization, S.H., N.K., I.A. and G.T.; data curation, N.K., I.A., S.K., E.P.
(Elias Pipinis), E.P. (Evgenia Papaioannou), E.D. and G.T.; formal analysis, I.A., E.P. (Elias Pipinis), E.P.
(Evgenia Papaioannou), N.K., S.K. and S.H.; investigation, I.A., N.K., S.H., E.D., E.P. (Elias Pipinis),
E.P. (Evgenia Papaioannou), E.K. (Eleftherios Karapatzak), S.K., P.T., E.K. (Emmanouil Koundourakis),
E.C. and G.T.; methodology, I.A., N.K., S.H., E.P. (Elias Pipinis), E.P. (Evgenia Papaioannou), S.K.,
E.D., E.K. (Eleftherios Karapatzak), P.T., E.K. (Emmanouil Koundourakis), E.C. and G.T.; project
administration, S.H., N.K. and G.T.; resources, N.K., S.H. and S.K.; software, I.A.; supervision, N.K.,
S.H. and G.T.; validation, N.K., I.A., E.K. (Eleftherios Karapatzak), E.D. and G.T.; visualization,
I.A., E.P. (Elias Pipinis) and N.K.; writing—original draft, I.A., E.P. (Elias Pipinis), S.H., N.K., E.K.
(Eleftherios Karapatzak) and G.T.; writing—review and editing, all authors. All authors have read
and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: All data supporting the results of this study are included in the
manuscript, and the datasets are available upon request.
Horticulturae 2023, 9, 877
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Acknowledgments: The fieldwork of N.K., G.T., E.D. and I.A. has been partially supported by the
MULTI-VAL-END project (2018–2022) entitled “Multifaceted Valorisation of single-country Endemic
plants of Crete, Greece, Tunisia and Rif, Morocco for sustainable exploitation in the agro-alimentary,
horticultural-ornamental and medicinal-cosmetic sectors” (ARIMNet2 2017 Transnational Joint Call).
Conflicts of Interest: The authors declare no conflict of interest.
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