Asexual Propagation of Campanula pelviformis and Petromarula pinnata—Two Local Endemic Plants of Crete with Multipurpose Crop Potential

Abstract

The urgency for sustainable propagation methods is increasing as anthropogenic disturbances threaten global biodiversity and plant productivity. This study focused on the cutting propagation of Campanula pelviformis and Petromarula pinnata, two local endemic species of Crete (Greece) with known multipurpose crop potential. For C. pelviformis, the effects of cutting type and indole-3-butyric acid (IBA) treatments applied with or without the commercially available and environmentally friendly fertilizer Theocopper and population were investigated. For P. pinnata, the effects of cutting type and IBA treatments were examined. Root dry mass was assessed four weeks after propagation onset, while rooting success, root number and length were measured in the third week to evaluate overall rooting performance. Vegetative propagation of P. pinnata was straightforwardly achieved across cutting types (>90% rooting success), and superior root traits were observed in young shoot cuttings. In C. pelviformis, high rooting success (90%) was recorded in four of the six treatments using adventitious shoot cuttings, while the combined application of 1% Theocopper and IBA efficiently enhanced both rooting percentages and root traits in young shoot cuttings and sub-apical stem sections. The results further indicated a population effect on rooting success and traits among cutting types. The data produced may guide conservation purposes and/or sustainable utilization of these neglected species as novel multipurpose crops, especially in landscaping design with native plants.

1. Introduction

Human-induced environmental disturbances such as global warming, climate change, and industrial pollution pose challenges to biodiversity, plant health, and productivity [1,2]. The intensive utilization of plant genetic resources for various applications has increased globally and locally, imposing the need to develop new, sustainable cultivation methods and novel crops [3,4,5,6], responding to increased consumer and market demands [7].

Recent research highlights the potential of several neglected and underutilized plant species (NUPs) in the agro-alimentary sector but also in the medicinal–cosmetic sector, which is on an ongoing quest for new bioactive plant-derived compounds, or the horticultural–ornamental sector seeking native species for landscaping design; these sectors are important in global economic domains [8,9,10,11] and across spatial scales, requiring constantly novel valuable products [12,13,14,15,16]. Native plant germplasm, particularly NUPs of the Mediterranean basin, are often associated with local endemism, ancient ethnobotanic heritage and traditional practices, ancestral breeding efforts based on wild crop relatives, and often include NUPs with documented nutritional or pharmaceutical [8,9] and ornamental value [10,17,18,19]. In this way, the Mediterranean biodiversity and the biocultural heritage in the Mediterranean region constitute an immense source tank to address modern challenges related to biodiversity, plant health, and productivity [8,9,10,20]. To this end, new research lines and focus are channeled towards developing applied initiatives for novel crops and new value chains’ creation. To achieve the latter, efficient propagation methods should first be established to ensure availability of certified and sustainably produced selected propagation material of NUPs, and such endeavors are conducted worldwide [8].

Among the numerous NUPs of the Mediterranean Basin, two members of the Campanulaceae family, namely Campanula pelviformis Lam. (protected by the Greek Presidential Decree 67/1981) and Petromarula pinnata (L.) A. DC. emerge as new and promising multipurpose crop species [21]. Both species are local endemics of Crete Island (Greece) and are well-documented for their promising potential across various economic sectors, exhibiting ornamental value suitable for potted or patio plants as well as for landscaping applications in the horticultural industry [8,21]. Evidence of existing consumer interest in these species from an ornamental perspective has already been demonstrated through uncontrolled electronic trade [22]. Furthermore, these species contain bioactive phytochemicals for medicinal–cosmetic purposes [9,23,24] and long-standing nutritional value and benefits as edible wild greens consumed traditionally in Crete [10,21,23,24]. Notably, nine secondary metabolites with medicinal value have been recently isolated from the aerial parts of C. pelviformis [23], and both species contain substantial levels of nutritional compounds, while P. pinnata exhibits notable antioxidant and bactericidal activities in addition [21,23,24]. These findings highlight the need for ex situ conservation strategies in support of sustainable utilization of these Cretan local endemic NUPs (single-island endemics). This can be initiated via a particular set of actions that include the development and application of both sexual and asexual propagation methods [21].

Sexual propagation of C. pelviformis and P. pinnata has been extensively studied to date, including germination tests on both freshly collected and stored seeds [25]. Additionally, seed traits such as length, width, and mass of freshly collected seeds from these taxa have been analyzed [25,26]. While sexual propagation promotes genetic diversity among seedlings, large-scale seedling production for conservation or sustainable exploitation can be cost-prohibitive and labor-intensive due to the very small size of seeds of Campanulaceae species [25,27], requiring specialized handling and/or seed dormancy release after maturation. Interestingly though, there is no previous experience or published records regarding the asexual propagation of these multipurpose Cretan NUPs [21], and, therefore, this extant research gap should be bridged to allow sustainable utilization initiatives.

From an agronomic perspective, asexual propagation may ensure the uniformity of favorable plant traits inherent in the studied species and can bypass biological issues encountered in sexual propagation such as low seed viability [28,29]; upon successful establishment, high-quality plants can be raised ex situ in large quantities, compensating for the generally increased cost of production and delayed mass production compared to sexual propagation [28]. Propagation-wise, different parts of plants can be used as cuttings such as shoots, roots, and leaves, while the type of cutting, the age of parent plants, the environmental conditions, the seasonal timing (both in terms of collection and establishment), and the application of plant growth regulators are critical factors that influence rooting success of cuttings [28,30]. The application of exogenous plant growth regulators is considered a key factor for the rooting of cuttings, with the use of indole-3-butyric acid (IBA) being regarded as the most reliable rooting enhancer [31]. Although IBA is commonly used for mass production via vegetative propagation, increasing interest has also arisen in recent years regarding the synergistic action of IBA with other substances and environmentally friendly compounds such as organic biostimulators, or natural rooting hormones like coconut oil [32,33,34,35,36,37,38].

This study aimed to develop effective asexual propagation protocols for the local Cretan endemic NUPs belonging to Campanulaceae, namely P. pinnata and C. pelviformis. In absence of previous experience for the focal Cretan NUPs, this investigation first aimed to define whether these species can generate appropriate cutting material with considerable rooting capacity under ex situ man-made settings and whether a successful propagation protocol can be developed via conventional and alternative methods; such clonal propagation protocols should be envisaged as a complement to, rather than a replacement for, conservation strategies, offering the possibility for raising uniform stocks of specific genotypes or facilitating sustainable cultivation efforts. To identify the most effective combinations for rooting success of cuttings, the evaluation for C. pelviformis focused on the effects of population, cutting type, and IBA treatment applied with or without a commercial and environmentally friendly fertilizer with biostimulant activity, as well as on the physical characteristics of the produced roots. A similar approach was followed for P. pinnata, which included the evaluation of IBA and cutting type, using the same rooting parameters.

2. Materials and Methods

2.1. Establishment of Mother Stock Plants from Seedlings

Mature capsules with seeds of C. pelviformis and P. pinnata were collected from the wild in July 2022, during targeted botanical expeditions in various locations of Crete, Greece, using a special permit from the Greek Ministry of Environment and Energy (5539/845 of 24 February 2022) (

Table 1. IPEN (International Plant Exchange Network) accession numbers and geographical details of collected seeds of the studied Campanulaceae species from different areas of Crete Island, Greece.

Species	IPEN Accession Number	Collection Site and Cretan Region	Latitude (North)	Longitude (East)	Altitude (m)
Campanula pelviformis	GR-1-BBGK-22,70	Istron, Lasithi	35.1248	25.7485	43
	GR-1-BBGK-22,84	Tsigouni Gorge, Lasithi	35.1528	25.9102	224
	GR-1-BBGK-22,73	Thrypti, Lasithi	35.0931	25.8640	834
Petromarula pinnata	GR-1-BBGK-22,68	Agia Eirini Gorge, Herakleion	35.2819	25.1650	132

). The seed collection was carried out manually, choosing 10 individual plants per population of the focal NUPs. The mixed collected materials were transferred to the laboratory, allowing them to dry naturally in shade at room temperature for 10 days. Seeds were then manually cleaned from each of the dried materials, placed in 125 mL glass bottles (Sigma-Aldrich, St. Louis, MI, USA), and stored in a fridge (model: CRW500SD, Chrisagis, Athens, Greece) until experimentation.

On 3 November 2023, a total of 250 seeds per population were sown in propagation trays filled with peat (TS1, Klassmann-Deilmann GmbH, Geeste, Germany) and vermiculite (RHP, ’s-Gravenzande, The Netherlands) at 3:1 v/v (Figure 1). The trays were placed in a growth chamber set at 15 °C, based on the results of previous seed germination studies [26] (Figure 1A). Seedlings were irrigated using tap water manually every four days. After two months, when more than 100 seedlings per population had reached approximately 2 cm in height, the trays were moved to a greenhouse bench at the Institute of Plant Breeding and Genetic Resources (IPBGR), Hellenic Agricultural Organization-Demeter, where they were transplanted into 0.33 L pots containing a mixture of peat (TS2, Klassmann-Deilmann GmbH, Geeste, Germany) and perlite (Geoflor, Perlite Hellas S.A., Volos, Greece) (3:1, v/v). In the greenhouse, the pots were irrigated automatically three times a day for two min each via sprinkler at a rate of 50 L per h. On 3 June 2024, following seedling establishment, 50 plants per population were transplanted into 1.6 L pots containing a mixture of peat TS2 and perlite (3:1, v/v), and irrigation was continued using the same automatic system. On 4 November 2024, following the growth pattern of C. pelviformis plants, 50 plants were selected phenotypically based on plant health, vigor, and optimal growth and were transplanted into 3 L pots filled with a substrate composed of peat TS2, perlite, and soil (4:2:1, v/v) (Figure 1B,C) to enable further growth and shoot development and produce adequate cutting material for experimentation.

For P. pinnata GR-1-BBGK-22,68, three-year-old mother stock plants were used for harvesting of different cuttings (Table 1); these mother plants were previously established from seeds [25] under similar ex situ conditions with those of C. pelviformis.

2.2. Asexual Propagation Experiments

The propagation experiments for C. pelviformis included three types of cuttings and were commenced in two stages based on the developmental patterns of the mother plants. Once the produced seedlings formed multiple new adventitious shoots consisting of 4–6 cm stem segments in mid-January 2025, they were excised from the upper part of the shoot, each containing the apical meristem (Figure 2A). Leaves were thinned to 2–3 fully developed leaves per cutting. Consecutively, when mother stock plants began producing young shoots in late March, two additional types of cuttings were collected, particularly young shoot cuttings 4–6 cm in length bearing the apical meristem (Figure 2B) and sub-apical stem sections bearing two axillary buds (Figure 2C). The leaves of each cutting were thinned to one to two leaves per cutting.

For P. pinnata, propagation trials began in mid-April 2025, coinciding with the development of newly emerged stems on mother plants. Collected cuttings included fresh young shoots containing the apical meristem (4–6 cm) (Figure 3A), as well as sub-apical stem sections taken from lower parts of the stem with two axillary buds (Figure 3A). Similarly, each cutting was thinned to one or two remaining leaves to ensure a total leaf area of one fully developed leaf.

For C. pelviformis, the IBA application (Indole-3-butyric acid 98%, Thermo Fisher Scientific Inc., Leicestershire, UK) was evaluated on the three collected cutting types either alone and/or in combination with a commercially available and environmentally friendly alternative fertilizer containing nitrogen, potassium, copper, and amino acids (THEOCOPPER, Theofrastos fertilizers, Korinthos, Greece, Supplementary Table S1). Six treatments were evaluated: (a) control—cuttings were immersed for 10 s in a solution prepared with water in 50% ethanol and placed directly into propagation trays immediately after excision from the stock mother plant to maintain turgor; (b) treatment of 1000 mg × L⁻¹ IBA—cuttings were immersed for 10 s in a 1000 mg × L⁻¹ IBA solution prepared in 50% ethanol; (c) treatment of 2000 mg × L⁻¹ IBA—cuttings were immersed for 10 s in a 2000 mg × L⁻¹ IBA solution prepared in 50% ethanol; (d) 1% Theocopper—cuttings were immersed for 10 min in 1% Theocopper solution prepared with deionized water (for details, see [39]); (e) Treatment of 1000 mg × L⁻¹ IBA + 1% Theocopper—Cuttings were first immersed for 10 min in 1% Theocopper in deionized water and then for 10 s in a 1000 mg × L⁻¹ IBA in 50% ethanol; and (f) treatment of 2000 mg × L⁻¹ IBA + 1% Theocopper—cuttings were first immersed for 10 min in 1% Theocopper in deionized water and then for 10 s in a 2000 mg × L⁻¹ IBA in 50% ethanol. In total, 24 cuttings per population and cutting type were evaluated across the six treatments. For P. pinnata, the first three treatments described above were evaluated on two types of collected cuttings due to limited availability of plant material.

Rooting conditions entailed propagation trays filled with perlite, peat moss (Terrahum, Klasmann-Deilmann, Gmbh, Germany), peat (3:1, v/v) under an intermittent automated benchtop fog system inside a greenhouse at ambient temperature conditions with a relative humidity of 70–85%.

2.3. Measurements Conducted on the Asexual Propagation Experiments

In the third week, data were collected on rooting success, number and length of the produced roots by cutting type. By the fourth week, the endpoint of the experiment was set when several treatments of C. pelviformis had reached 100% rooting. A similar methodological approach was applied to P. pinnata, as it exhibited a comparable response pattern. At the end of the experiments and after the measurements of root parameters, the produced roots from eight rooted cuttings were carefully removed from each treatment using a scalpel, placed in paper bags, and then dried in an oven at 65 °C for 72 h. After drying, root dry mass was determined using a four-digit analytical balance.

Consecutively, 16 of the remaining rooted cuttings from the three most effective treatments in terms of rooting percentages per population and cutting type for C. pelviformis and for each cutting type for P. pinnata were transplanted into 0.33 L pots containing a substrate mixture of peat TS2 and perlite (3:1 v/v) and were placed in a greenhouse equipped with an automated irrigation system. Two weeks later, the surviving plants were recorded to estimate the frequencies of the successfully acclimatized plants.

2.4. Statistical Analysis

Completely randomized factorial experiments with a split-plot arrangement were used for both studied species. For C. pelviformis, population (3 populations) was the main plot factor, treatment (6 levels) was the sub-plot factor, and cutting type (3 types) was assigned as a sub-sub-plot factor (as a within-unit factor nested within mother plants). For P. pinnata, treatment (3 treatments) was the main plot factor, with cutting type (2 types) designated as the sub-plot factor. The dataset was analyzed within the methodological framework of Mixed Linear Models with the ANOVA method [40,41]. The LSD criterion was used for the comparison of mean values. Since treatments’ combinations resulted in different rooting success, the parameters of root number, root length, and root dry mass had unequal replications. For the application of the LSD criterion, the harmonic mean of the number of replications per combined treatment was used (detailed data are presented in the Supplementary Tables S5, S7, S9, S11 and S13). In addition, since the experiment was unbalanced, Type III Sum of Squares was used in all ANOVA models.

For rooting success, the 24 cuttings were randomly allocated into three different trays of eight cuttings each, and the rooting percentage (%) was estimated from eight cuttings using binary data (0 for no rooting and 1 for rooted cuttings). This process resulted in three replications of rooting success per combined treatment. Count data were collected for the number of roots produced for each cutting (24 cuttings per treatment). The mean length of roots produced per cutting was used for each of the 24 cuttings per treatment. The same procedure was followed for root dry mass; however, only eight (or fewer, when necessary) randomly selected cuttings per treatment were used for the statistical analysis of dry mass measurements. For C. pelviformis, the number, length, and dry mass of produced roots were square root transformed, while untransformed raw data were used for rooting success. For P. pinnata, only the dry mass of produced roots was square root transformed, while root number and length were analyzed without transformation. These transformations aimed to achieve normality and homoscedasticity of the models’ residuals.

Normality and homoscedasticity were assessed using the residuals of linear models. Particularly, normality was evaluated using histograms, boxplots, median values of residuals (should be near 0), and coefficients of skewness and kurtosis, as well as the Kolmogorov–Smirnov test (p > 0.05). The homoscedasticity was checked through residuals-versus-model predicted values and Spearman’s rho correlations between the predicted values estimated by the model and the absolute values of the residuals. These assumptions were not significantly violated. All the above-mentioned procedures were done using the IBM SPSS Statistics v.27.0 (SPSS, Inc., IBM Corp., Armonk, NY, USA). In all hypothesis-testing procedures, the significance level was preset at a = 0.05 (p ≤ 0.05).

3. Results

3.1. Asexual Propagation of Campanula pelviformis

The individual effects of population, treatment, and cutting type were statistically significant for all examined parameters in C. pelviformis; additionally, most of the two-way and the three-way interactions were also statistically significant. Exceptions (not statistically significant) included the population X cutting type interaction for root number (p = 0.111) and the treatment X cutting type interaction for root dry mass (p = 0.471,

Table 2. Significance (p ≤ 0.05) and partial eta squared of individual factors and interactions thereof examined for mean rooting percentage, root number per cutting, root length and root dry mass in Campanula pelviformis cuttings.

Source of Variation	Root Percentages (%)		Root Number		Root Length (cm)		Root Dry Mass (mg)
	p-Values	Partial Eta Squared	p-Values	Partial Eta Squared	p-Values	Partial Eta Squared	p-Values	Partial Eta Squared
Population	<0.001	0.926	<0.001	0.073	<0.001	0.395	<0.001	0.574
Treatment	<0.001	0.953	<0.001	0.333	<0.001	0.094	<0.001	0.391
Population × treatment	<0.001	0.747	<0.001	0.071	<0.001	0.125	<0.001	0.234
Cutting type	<0.001	0.822	<0.001	0.101	<0.001	0.031	<0.001	0.066
Population × cutting type	<0.001	0.439	0.111	0.015	<0.001	0.062	<0.001	0.086
Treatment × cutting type	<0.001	0.564	<0.001	0.13	<0.001	0.185	0.471	0.041
Population × treatment × cutting type	<0.001	0.474	<0.001	0.223	<0.001	0.298	<0.001	0.231

).

All parameters except root percentages were square root transformed before analysis; partial eta squared is an effect size index. Moreover, high values of partial eta squared were observed in population (η² = 0.926) and treatment (η² = 0.953) on root percentages, treatment (η² = 0.333), and the three-way interaction (η² = 0.223) on root number, population (η² = 0.395) and the three-way interaction (η² = 0.298) on root length, and population (η² = 0.574) and treatment (η² = 0.391) on root dry mass. Detailed descriptive statistics and the ANOVA results are provided in the Supplementary Materials (Supplementary Tables S2–S9).

Adventitious shoot cuttings exhibited comparably the highest rooting frequencies across all treatments (>66%). High rooting success (>94%) was significantly enhanced by increasing concentrations of IBA, while similar values were observed by the combined application of IBA and 1% Theocopper (Figure 4). Moderate rooting percentages were observed in young shoot cuttings (44–57%), and relatively low rooting percentages were observed in sub-apical stem sections (14–28%) under control and 1% Theocopper treatments (Figure 4). Rooting was increased under 1000 and 2000 mg × L⁻¹ IBA treatments for both cutting types (>75%), reaching a maximum in young shoot cuttings treated with 2000 mg × L⁻¹ IBA (97%). The combined application of IBA and 1% Theocopper resulted in 57–61% rooting success for sub-apical stem sections.

Across populations (Figure 4), cuttings derived from adventitious shoots generally exhibited the highest rooting percentages (>84%), with adventitious shoot cuttings of population GR-1-BBGK-22,84 demonstrating the highest percentages (90.3 ± 3.6%). In addition, young shoot cuttings of C. pelviformis population GR-1-BBGK-22,84 and population GR-1-BBGK-22,73 showed similarly high rooting percentages (79–83%), while sub-apical stem sections consistently showed the lowest percentages across populations (<64%).

According to Figure 5, control and 1% Theocopper treatments had a limited effect on root production, with adventitious shoot cuttings producing the highest number of roots under both treatments (10.6 ± 1.0 and 14.5 ± 1.0 roots, respectively). In contrast, the ap-plication of IBA had a strong impact in root production with adventitious shoot cuttings presenting the highest number of roots at 1000 mg × L⁻¹ IBA (28.8 ± 1.7 roots), whereas treatment with 2000 mg × L⁻¹ IBA increased root production across all three cutting types, with young shoot cuttings and sub-apical stem sections exhibiting the highest values (32.8 ± 2.0 and 31.0 ± 1.9 roots, respectively). Similarly, the combined treatments increased root production for all cutting types compared to control and 1% Theocopper treatments (>21.8 roots). The highest root production was observed in adventitious shoot and young shoot cuttings treated with 1% Theocopper with 1000 mg × L⁻¹ IBA (29.0 ± 2.0 and 28.4 ± 2.0 roots, respectively) and in young shoot cuttings treated with 1% Theocopper and 2000 mg × L⁻¹ IBA (28.9 ± 2.1 roots).

Based on the observed population effects (Figure 5), adventitious shoots and young shoot cuttings produced higher numbers of roots, particularly in C. pelviformis population GR-1-BBGK-22,73 (26.3 ± 1.6 and 24.2 ± 1.4 roots, respectively) and population GR-1-BBGK-22,84 (23.4 ± 1.1 and 27.4 ± 1.9 roots, respectively). Sub-apical stem sections also exhibited relatively high mean root number in these populations (20–23 roots), whereas all cutting types showed the lowest root number in population GR-1-BBGK-22,70.

As shown in Figure 6, control, 1% Theocopper, and 1000 mg × L⁻¹ IBA treatments had a limited effect on root length, particularly in young shoot cuttings (0.7 ± 0.04, 0.5 ± 0.03, 0.7 ± 0.03 cm, respectively) and sub-apical stem sections (0.4 ± 0.03, 0.4 ± 0.09, 0.6 ± 0.03 cm, respectively). However, treatment with 1000 mg × L⁻¹ IBA significantly increased root length in adventitious shoot cuttings (1.1 ± 0.09 cm). Moreover, application of the higher IBA concentration had a strong effect on root length, with young shoot cuttings and sub-apical stem sections producing the longest roots (1.0 ± 0.04 and 1.0 ± 0.05 cm, respectively). Similarly, combined treatments increased root length in young shoot cuttings (0.9 ± 0.06 cm at T + 1000 mg × L⁻¹ IBA and 0.8 ± 0.03 cm at T + 2000 mg × L⁻¹ IBA) and in sub-apical stem sections (1.1 ± 0.1 and 0.8 ± 0.1 cm, respectively) compared with control and 1% Theocopper treatments.

According to Figure 7, all examined treatments affected the dry mass of produced roots compared with the control treatment (3.1–4.6 mg across all cutting types). Treatment with 1000 mg × L⁻¹ IBA increased root dry mass in all cutting types (8.4–9.2 mg), and the same was detected after combined application with 1% Theocopper (7.9–9.3 mg). Notably, the highest root dry mass was observed in young shoot cuttings that were treated with 1% Theocopper combined with 1000 and 2000 mg × L⁻¹ IBA (10.5 ± 1.9 and 12.6 ± 1.9 mg, respectively).

Young shoot cuttings and sub-apical stem sections derived from C. pelviformis population GR-1-BBGK-22,84 produced the highest dry mass values (12.3 ± 1.5 and 10.5 ± 1.6 mg, respectively). Adventitious shoot cuttings from this population, as well as population GR-1-BBGK-22,73 (Figure 7) also showed relatively increased dry mass (7.5 ± 0.9 and 8.8 ± 0.7 mg, respectively), while the remaining cutting types from GR-1-BBGK-22,73 and all cuttings from GR-1-BBGK-22,70 produced comparably lower dry mass values (>6.8 ± 0.6 mg, Figure 7).

All cutting types derived from C. pelviformis population GR-1-BBGK-22,84 (Figure 8B) produced longer roots, particularly adventitious shoot cuttings (1.2 ± 0.05 cm). Adventitious shoot cuttings from population GR-1-BBGK-22,73 (Figure 8A) also exhibited increased root length (0.9 ± 0.04 cm), whereas the remaining cutting types from both populations GR-1-BBGK-22,73 and GR-1-BBGK-22,70 produced shorter roots (>0.7 ± 0.03 cm).

The survival rates of the rooted cuttings by the 6th week after experimentation reached 93.8% for adventitious shoots and young shoot cuttings of C. pelviformis populations GR-1-BBGK-22,84 and GR-1-BBGK-22,73, while the corresponding cuttings of population GR-1-BBGK-22,70 were 87.5%. In addition, survival rates of 87.5% were recorded for all sub-apical cuttings across the examined populations of C. pelviformis.

3.2. Asexual Propagation of Petromarula pinnata

In P. pinnata GR-1-BBGK-22,68, the effects of the two studied factors (IBA treatment and cutting type) were statistically significant for root number (p < 0.001), root length (p < 0.001) and root dry mass (p = 0.007 for IBA treatment, and p < 0.001 for cutting type), while their interaction was significant for root number (p = 0.005) and root dry mass (p = 0.044,

Table 3. Significance (p ≤ 0.05) and partial eta squared of the examined factors and their interaction per cutting, root length and root dry mass in Petromarula pinnata GR-1-BBGK-22,68.

Source of Variation	Root Number 1		Root Length 1		Root Dry Mass 2
	p-Values	Partial Eta Squared	p-Values	Partial Eta Squared	p-Values	Partial Eta Squared
Treatment	<0.001	0.597	<0.001	0.268	0.007	0.373
Cutting type	<0.001	0.794	<0.001	0.484	<0.001	0.692
Treatment × cutting type	0.005	0.147	0.121	0.062	0.044	0.257

¹ Denotes parameters without transformation, and ² denotes parameters with a squared-root transformation; partial eta squared is an effect size index.

). Details on the descriptive statistics and the ANOVA results are provided in the Supplementary Materials (Supplementary Tables S10–S15). High values of partial eta squared were observed in cutting type (η² = 0.794) and treatment (η² = 0.597) on root number, cutting type (η² = 0.484) and treatment (η² = 0.268) on root length, and cutting type (η² = 0.692) and treatment (η² = 0.373) on root dry mass.

Rooting success for P. pinnata GR-1-BBGK-22,68 cuttings was 100% across all treatments in young shoot cuttings, whereas in sub-apical stem sections reached 91.7% in the control, 95.83% at 1000 mg × L⁻¹ IBA treatment, and 100% at 2000 mg × L⁻¹ IBA treatment.

Root number increased significantly with the application of IBA (91.3 ± 5.9 roots at 1000 mg × L⁻¹ IBA and 85.4 ± 4.8 roots at 2000 mg × L⁻¹ IBA) compared with the control treatment (49.1 ± 5.0 roots,Figure 9). In addition, young shoot cuttings produced approximately twice as many roots as sub-apical stem sections (103.0 ± 3.6 vs. 46.8 ± 3.2 roots, respectively).

The application of IBA at both concentrations increased root length (>1.6 ± 0.06 cm) compared with the control (1.3 ± 0.09 cm) (Figure 10). Regarding cutting type, young shoot cuttings produced longer roots than those of sub-apical stem sections (1.8 ± 0.05 cm and 1.3 ± 0.05 cm, respectively).

The application of IBA increased the dry mass of the produced roots (>19 mg), with higher IBA concentration resulting in further increases (23.0 ± 2.9 mg) compared to the control (14.2 ± 3.0 mg) (Figure 11 and Figure 12). Young shoot cuttings produced almost twice as high dry root mass (26.3 ± 1.7 mg) as sub-apical stem sections (11.4 ± 2.0 mg) (Figure 11B and Figure 12).

After six weeks, both cutting types of P. pinnata exhibited a survival rate of 93.8%.

4. Discussion

The development of successful vegetative propagation protocols either for conservation purposes and/or sustainable exploitation of NUPs can provide a reliable and cost-effective approach, ensuring genetic uniformity, maintenance of selected genotypes, and rapid production of plant material [28]. Over the past decade, significant efforts for the development of ex situ vegetative propagation protocols via cuttings have been conducted for several herbaceous NUPs [21,42,43,44,45,46]. Although detailed research on sexual propagation via seeds has already been conducted for the focal NUPs of the current study, resulting in comprehensive protocols indicating appropriate temperatures (10 and 15 °C) and no preference in light conditions for increased seed germination (>80–85% for both species) [25,26], unfortunately, no previous studies have yet addressed their asexual propagation [21]. In this context, the present investigation developed effective asexual propagation protocols via cuttings for the focal NUPs. The results demonstrated a rooting performance (>90%) across IBA treatments with or without 1% Theocopper for adventitious shoot cuttings and with 2000 mg × L⁻¹ IBA for young shoot cuttings in C. pelviformis, as well as across all treatments and cutting types examined in P. pinnata under man-made settings, which are commercially accepted according to current standards [31]. Petromarula pinnata exhibited a straightforward rooting capacity, as untreated and treated cuttings resulted in very high rooting success (>90%) for both examined cutting types. In C. pelviformis, high rooting success was observed primarily in adventitious shoot cuttings under four of the six treatments (>93%). Vegetative propagation studies focusing on cuttings of different Campanulaceae species that are endemic to Greece are scarce in the literature; however, similar studies in the Eurasiatic Campanula rapunculoides L. and the Balkan endemic Campanula portenschlagiana Schult. have recorded high rooting capacity (72% and 85–100%, respectively) [47,48,49]. Comparable results employing IBA application have been reported for other herbaceous perennial Greek endemic species with conservation priority belonging to different families. In these cases, adventitious shoot cuttings of the local endemics of Crete Carlina diae (Rech.f.) Meusel & Kästner (Asteraceae), Limonium chersonesum Erben & Brullo (Plumbaginaceae), Erysimum krendlii Polatschek (Brassicaceae), and Dianthus ingoldbyi Turrill (Caryophyllaceae) have also exhibited high rooting capacity [42,44,45,46]. The same applies to another local endemic plant of Paxi Islands of the Ionian Archipelago, namely Centaurea paxorum Phitos & T. Georgiadis (Asteraceae) [45].

The results of the current study showed that the highest rooting success was observed in both adventitious and young shoot cuttings treated with 2000 mg × L⁻¹ IBA for C. pelviformis. This response may be partly attributed to higher concentrations of exogenous auxin interacting with endogenous indole-3-acetic acid (IAA), thereby potentially promoting the cambium differentiation and enhancing further rooting induction in both cutting types [30,50,51]. Similar results have been reported for other herbaceous perennial Greek endemic NUPs such as the local Cretan endemic Lomelosia minoana (P.H.Davis) Greuter & Burdet subsp. minoana (Dipsacaceae) [43] or the local endemic of Naxos Island (Cyclades, Aegean Archipelago) Erysimum naxense Snogerup (Brassicaceae) [45]. Moreover, the application of 2000 mg × L⁻¹ IBA in stem cuttings has been reported to increase rooting percentages in the ornamental herbaceous perennial Paeonia ‘Yang Fei Chu Yu’ (86.7%), as well as in the eastern Mediterranean medicinal and aromatic plant Salvia tomentosa Mill. (Lamiaceae) when treated with 1500 mg × L⁻¹ IBA (79.5%), while the application of IBA powder resulted in 100% rooting in the Greek endemic herbaceous perennial Nepeta camphorata Boiss. & Heldr. (Lamiaceae) [52,53,54].

Although improvements in root-related attributes following the application of IBA (without using biostimulants) have been previously reported in studies on herbaceous Greek endemic perennial plants [42,44,46] or on various woody species originating from other regions of the world (e.g., [34,55]), literature reports remain scarce regarding the environmentally friendly alternative fertilizer applied in this study as a rooting enhancer (1% Theocopper). A previous study using added amino acids such as L-glutamate and L-tryptophan to the nutrient solution of C. portenschlagiana cuttings under an aeroponic system has shown increased root capacity in terms of number of rooted explants by 40 to 100% for two genotypes, as well as increased root length, while enhanced root biomass has been recorded for one of the two genotypes. Such amino acids may contribute indirectly to root development, through nitrogen remobilization in the case of glutamate or via IAA biosynthesis in the case of tryptophan [56,57,58]. Among the IBA-supplemented treatments that outperformed the non-IBA treatments in the current study, the most significant enhancement of the measured rooting traits (root number, length, and dry mass) was observed in 2000 mg × L⁻¹ IBA and its combined application with the alternative fertilizer applied as a rooting enhancer (1% Theocopper). These effects were particularly pronounced in sub-apical stem sections and young shoot cuttings of C. pelviformis. Moreover, our results showed that root dry mass was further increased with higher IBA concentrations in P. pinnata. Notably, a similar effect of 1% Theocopper combined with 1000/2000 mg × L⁻¹ IBA on root dry mass has been recently reported for another herbaceous perennial Greek subendemic species, namely Helichrysum orientale (L.) Gaertn. (Asteraceae) [39]; in the latter study, all treatments of 1% Theocopper combined with three concentrations of IBA (1000/2000/4000 mg × L⁻¹) are reported to result in the production of statistically significant high amounts of root dry mass [39]. In general, a higher root volume in cuttings has been correlated in the literature with root surface area and the quality of the produced plants in other herbaceous ornamental perennials such as Oenothera lindheimeri (Engelm. & A.Gray) W.L.Wagner & Hoch (Siskiyou Pink’ gaura; Onograceae), Pixie Star’ dianthus (Dianthus alpinus L.; Caryophyllaceae), Salvia yangii B.T.Drew (Perovskia, previously known as Perovskia atriplicifolia Benth.; Lamiaceae), and ‘Mainacht’ salvia (Salvia × sylvestris L.) [59].

The current results indicated that superior root characteristics were achieved from apical cuttings in both studied species. In contrast, vegetative propagation of C. rapunculoides in the absence of any plant growth regulator shows a higher rooting capacity of basal cuttings (72%) compared to tip cuttings (14–17%), thus implying that the possible use of plant growth regulators may further enhance the efficiency of this propagation method [47]. Moreover, the combined effect of the alternative fertilizer applied in the current study as a rooting enhancer with IBA showed the capacity to increase the measured root characteristics in sub-apical cutting types. The application of the fertilizer as a rooting enhancer with IBA may have provided further availability of nutritive elements (e.g., nitrogen and potassium) to the cuttings along with IBA. As suggested by other studies in cultivated commercial roses [60], increased nitrogen availability during cutting propagation may be positively correlated with increased root number along with enhancement of IBA uptake and carbohydrate availability coming from the photosynthesizing leaves of the cutting. Although the precise mechanism of the observed enhancement of rooting characteristics of the sub-apical stem sections via the use of 1% Theocopper remains unclear from the current data, this enhancement in the studied Cretan NUPs may translate into higher propagation success. Moreover, the effective use of both sub-apical and apical cuttings in commercial propagation could improve the overall protocol efficiency by increasing the availability of suitable plant material for propagation and subsequent cultivation of these multipurpose NUPs.

In general, genotypic variation in rooting success among cutting types has been reported in previous studies for woody species such as Santalum austrocaledonicum Vieill. (Santalaceae) [61] and Colutea istria Mill. (Fabaceae) [62], shrubby species like Rosa damascena Mill. [63] or cultivated mother stock plants of different varieties of the herbaceous ornamental perennials C. portenschlagiana and Salvia greggii A.Gray (Lamiaceae) [48,64]. Vegetation propagation trials using cuttings from two genotypes of endemic C. portenschlagiana have revealed important differences between genotypes in terms of root-related traits, including rooting success, number of roots, root length, and fresh and dry weight. Such findings, combined with hormonal content analysis, have shown higher concentrations of IBA and IAA and lower concentrations of abscisic acid (ABA) in the genotype with higher root capacity [48,65]. The latter confirms the established role of auxins and ABA in adventitious root development, while increased flavonoid levels detected in the same genotype may also affect root efficiency [48,65]. A population effect has also been reported in previous seed germination studies regarding C. pelviformis and P. pinnata, with different populations showing varied patterns [25,26]. In this study, variation in all examined rooting metrics was observed among populations of C. pelviformis, thus indicating a possible population effect. Although the superior rooting capacity of adventitious shoot cuttings in C. pelviformis was consistent across the different populations investigated herein (>84%), young shoot cuttings and sub-apical stem sections showed variable rooting success among populations (34–83%). It is generally known that intraspecific genotype variation can significantly affect the success of vegetative propagation and that different genotypes can exhibit notable differences in their physiological responses such as varied endogenous hormone levels, carbohydrate allocation ratios, or varied responses of the same cutting type to the same rooting conditions [66]. In this context, it is notable that such intraspecific genotypic patterns were also detected in the current study for C. pelviformis, and further studies are needed to define genotypic differences in terms of specific physiological responses. Compared to other populations or genotypes examined in the present study, the high rooting success of C. pelviformis population GR-1-BBGK-22,84, with improved root characteristics in terms of number, length, and mass across all tested cutting types, was further enhanced via the combined effect of IBA and 1% Theocopper. This observation may imply a positive combined effect of IBA and 1% Theocopper on adventitious root growth for this population, resulting in faster exploitation of the substrate and enhanced survival of the rooted cuttings.

Taken altogether, the asexual propagation of both studied species can be regarded as a straightforward, rapid, and reliable propagation method, suitable for producing plant material intended for landscaping and gardening purposes or for agro-alimentary or medicinal–cosmetic cultivation contexts [67]. The asexual propagation of P. pinnata via young shoot cuttings can be readily achieved even without the use of rooting enhancers; however, their application can further improve root quality. In contrast, although C. pelviformis cuttings also exhibited high rooting success, the combined use of hormonal and non-hormonal and environmentally friendly rooting enhancers containing nutrients appears beneficial for achieving better root traits and increasing successful plant establishment.

Compared to seed germination and seedling development, the vegetative propagation of C. pelviformis through cuttings may shorten the time needed to first flowering, thus representing a notable advantage for this species in terms of ornamental value. In the present study, first flowering of the biennial (or short-lived perennial) C. pelviformis [68] was observed in all asexually propagated populations during their ex situ assessment, thus highlighting the noteworthy potential of this propagation method. Specifically, mother plants of C. pelviformis developed ex situ from seedlings required more than 13 months to reach an immature flowering stage, whereas cuttings in this study initiated their flowering within only four to five weeks under vegetative propagation conditions. This indicates a clear advantage for the rapid mass production of flowering individuals compared to sexual propagation and seedling development.

In contrast to C. pelviformis and as expected due to the perennial habit of P. pinnata [68], this species exhibited a dissimilar response in ex situ conditions, with most of the donor mother plants developed from seedlings requiring at least three years to reach their first flowering. The cuttings in this study that were excised from mother plants at the immature first flowering stage were not observed to commence to bloom under vegetative propagation conditions within the four to six weeks’ timeframe in which they were monitored. However, even though it cannot be concluded from the current data that the rooted cuttings commence to immediate bloom, the observed fact is that the cuttings reach ex situ a well-developed stage (approximately 10 cm in height with fully developed leaves and vascular system) by the sixth week after onset, while the corresponding timeframe for seedlings to reach the same stage needs at least 12 weeks under similar environmental conditions [25]. Therefore, the vegetative propagation of P. pinnata through cuttings may represent a practical approach for the rapid production of established plants. Although the present study did not assess subsequent growth performance, time to flowering, or directly compare cutting-derived plants with seedlings, the successful rooting and establishment observed suggest that this propagation method could have practical advantages for large-scale plant production. Further studies are needed to evaluate these potential benefits. Although P. pinnata in wild habitats is considered as a perennial species [68], some foreign nurseries consider that plants of P. pinnata cultivated in a pilot way outside the species’ native range (Crete) can be monocarpic at their grounds (e.g., in UK, see https://cotswoldgardenflowers.co.uk/products/petromarula-pinnata, accessed on 13 April 2026), and often that they begin to flower within two years (e.g., https://plantlust.com/plants/12922/petromarula-pinnata/, accessed on 13 April 2026). However, there is no scientific ex situ monitoring of the lifespan of P. pinnata, and therefore the monocarpic nature of this species or its exact lifespan has not yet been scientifically documented. The latter also raises questions about how cuttings of this species might respond if taken at later developmental stages, closer to the end of the species’ lifespan. Therefore, relevant further research is proposed in this direction to determine the lifespan of P. pinnata in both natural habitats and ex situ conditions.

5. Conclusions

This study provided the first evidence that the local Cretan endemic NUPs Campanula pelviformis and Petromarula pinnata (Campanulaceae) can produce cuttings of different types possessing significant rooting capacity, suggesting their suitability for nursery propagation under controlled conditions. Although mother plant productivity, propagation costs, scalability, or long-term post-acclimatization performance was not substantiated herein, this study showed that asexual propagation by cuttings of C. pelviformis may be a straightforward procedure for the rapid mass production of flowering individuals destined for ornamental use and landscape applications compared to sexual propagation and seedlings’ development. In this study, we proposed an ex situ asexual propagation protocol for P. pinnata using the conventional rooting hormone IBA; although its cuttings may root readily even without treatment, young shoot cuttings should be markedly preferred. For C. pelviformis, high rooting success and superior root quality were achieved through specific combinations of population, cutting type, and hormone treatment. Notably, the combination of 1% Theocopper with IBA enhanced rooting success in certain treatments, thus highlighting the potential use of environmentally friendly fertilizers that act as biostimulants in applied vegetative propagation of the focal NUPs and possibly other herbaceous perennials. Overall, the findings of the current study establish a solid baseline for future genotype conservation efforts and pave the way for the sustainable cultivation and potential commercial exploitation of populations of C. pelviformis and P. pinnata as novel multipurpose crops with strong local identity shaped by endemism (single-island endemics) and promising value across agro-alimentary, medicinal–cosmetic, and ornamental domains, especially in landscaping design with native plants.