Enhancing the Propagation and Cultivation Framework of Greek Rosa canina L. Germplasm via Sustainable Management Techniques

Abstract

The current study aimed to enhance the sustainable utilization framework of the underutilized Greek native Rosa canina L. (rosehip) germplasm as a potential novel crop that can deliver high-quality products with minimum environmental impact. The first part of the work includes asexual propagation trials on cuttings of two Greek R. canina genotypes, assessing the potential of various alternative rooting enhancers to the conventionally used indole-3-butyric acid (IBA), as affected by rooting substrate and cutting type. The propagation results showed commercially acceptable (>50%) rooting rates for 2500 ppm IBA, coconut water, rooting gel and aloe vera treatments and similar rooting attributes of root number and length, providing evidence for the feasibility of using alternative rooting enhancers. The second part of the work presents the results of an ongoing ex situ cultivation trial assessing the potential of a diversified organic fertilization regime against conventional fertilization on fruit size and yield, coupled with macro- and micro-nutrient concentration, in the leaves of four Greek R. canina genotypes. The results showed a genotype-specific response in rosehip fruit size and yield to fertilization, with the organic regime showing comparable results to the conventional fertilization. In addition, diverse patterns, depending on the element, of macro- and micro-nutrient content were measured in the leaves in both fertilization regimes, which were, however, genotype-dependent. Overall, the current study reports for the first time the potential of alternative rooting enhancers for commercial R. canina propagation coupled with the application of organic fertilization as a means of establishing a diversified cultivation protocol for underutilized R. canina germplasm. The current results can be employed to further facilitate a value chain creation for Greek rosehips as a raw material for use in the agro-alimentary and medicinal–cosmetic sectors.

1. Introduction

The primary production sector across Europe and beyond faces unprecedented challenges nowadays relating to climatic change, increased consumer demand and the latest energy crisis, on top of already increased production costs [1,2,3]. Additional structural challenges currently faced by the EU fruit and vegetable sector have been proposed to be related to weak organization, a lack of adequate innovation and an unremarkable response to consumer needs [4]. At the same time, further environmental challenges relating to environmental adaptation of crops to climatic change, biodiversity conservation and food insecurity put additional pressure on the plant-based primary sector [5,6]. Options for novel, highly nutritive crops with diverse uses that meet contemporary consumer demands and can deliver high-quality products with minimum environmental impacts and costs are becoming imperative for the sustainability of current and future production systems. Such novel crop alternatives can be either discovered or re-discovered through the utilization and sustainable exploitation of wild-occurring species that are otherwise neglected or underutilized (recently termed neglected and underutilized plant species (NUPs) elsewhere) which have been given considerable scientific recognition in recent years [7,8,9,10,11]. Within the category of NUPs fall several regional wild-growing plant species, with profuse advantages relating to environmental resilience, diversified crop quality and high nutraceutical potential [9,12].

Such an example of a local NUP germplasm with documented agronomical and nutraceutical potential is Greek Rosa canina (dogrose) [13,14]. Species-wise, dogrose Rosa canina (Rosaceae) is a small forest tree or shrub that bears hermaphroditic epigynous flowers that produce a type of pseudo-fruits, called rosehips. Across the relatively widespread distribution range of the species in Greece, R. canina trees flower from late spring to early summer and produce ripe rosehips from late summer to mid-autumn [15]. Rosehips are widely known and valued for their superior nutraceutical properties as a result of their documented high content of ascorbic acid and antioxidants, among other things [16]. Indicatively, the antioxidant activity of rosehips has been extensively documented in a diverse array of plant-based nutritional and pharmaceutical studies [13,17,18,19,20]. The documented value of rosehips has brought about the significant economic potential of the species both worldwide and regionwide in the Greek horticultural sector, establishing a high exploitation feasibility and an already achieved readiness timescale for commercial agronomic exploitation [14]. Indicatively, traditional and contemporary uses of rosehips in Greece and elsewhere include culinary and beverage uses [13,14]. Additional studies have highlighted the versatility of R. canina providing data for further use in species enrichment for degraded forest mitigation programs, enhancing biodiversity and soil erosion protection [21,22]. R. canina has been shown to enhance the soil’s organic matter, reduce the risk of erosion and enhance soil fertility in forest landscapes [22]. Furthermore, rosehip residues have been shown to have multilateral potential [23], including use for biogas production [24]. As such, it is believed herein that multifaceted uses of rosehips in sectors like landscape management, agro-alimentary and medicinal–cosmetic are warranted.

Based on the above-mentioned versatile potential of rosehips, an integrated domestication framework for the sustainable exploitation of Greek R. canina has recently been proposed, implementing molecular authentication coupled with propagation and phytochemical evaluation of the fruits of wild-occurring material [13]. Consequently, the establishment of selected genotypes under experimental cultivation was further evaluated [14]. The results of the above work refurbished the exploitation feasibility of Greek native R. canina germplasm, highlighting at the same time the current research gaps and potential limitations.

Considering the domestication steps for Greek R. canina, as are depicted above, asexual propagation and cultivation establishment have been considered herein the two pivotal steps for further research on enhancing the exploitation framework of this germplasm. In addition, the evaluation of novel environmentally friendly or alternative management methods and inputs for achieving successful propagation and cultivation has also been considered worthy of research, especially when considering the contemporary challenges of environmentally sustainable production systems under a changing climate [5,6].

Asexual propagation via cuttings in commercial terms has long been conducted via the application of artificial hormonal substances to enhance rooting, with indole-3-butyric acid (IBA) being the main substance used [25]. The same holds true for the asexual propagation of native plant material for domestication purposes [26,27], including R. canina [13,28]. The use of alternative rooting enhancers to the conventionally used IBA during propagation is a subject that has been given research attention recently [29,30], with encouraging evidence for small tree species like Cornus spp. and the Rosa sp. [31,32]. Indicative examples are coconut water, which includes crucial phytohormones that support the plant’s maintenance and development [33], along with various rooting gels [29]. Coconut water is a very versatile substance that, in terms of the current study, contains natural phytohormones of most known groups, including the natural auxin indole-3-acetic acid (IAA) [34]. A further example of natural plant extracts that contain natural auxin substances is aloe vera gel [35]. Apart from substances with rooting enhancing properties, the use of arbuscular mycorrhizal fungi (AMF) as alternative rooting enhancers in asexual plant propagation has also been evaluated and discussed in the past [36], taking advantage of the natural properties of arbuscular mycorrhizal fungi in enhancing plant growth and development or acting as natural bio-fertilizers [37].

Trial cultivation efforts of small tree or shrub NUP germplasm in Mediterranean environments that also explore the fertilization effects or requirements have thus far shown potential to facilitate integrated sustainable utilization frameworks [14,38]. The cultivation of NUPs that are tree species inevitably falls into the principles of orchard management. According to these principles, organic management mainly considers diversification in terms of lower inputs or inputs with a lesser environmental impact for fertilization, crop protection and/or water management, as opposed to conventional management, where traditionally no such limitations apply [39,40]. A large body of evidence from the literature supports the environmental sustainability of organic tree crop management compared to conventional systems as part of an integrated or diversified management system [41,42,43,44].

Prompted by the above-described multifaceted premise and considering the previously proposed exploitation framework for Greek R. canina [13,14], this study firstly investigated whether alternative rooting enhancers in combination with arbuscular mycorrhizal fungi against the conventionally used indole-3-butyric acid (IBA) could be used for asexual propagation with the cuttings of two different R. canina genotypes; secondly, this study investigated the effects of a diversified organic fertilization regime against conventional fertilization and no fertilization on the fruit yield, fruit size and macro- and micro-nutrient content in the leaves during the trial cultivation of four distinct Greek R. canina genotypes. The pivotal goal of the study was to enhance the integrated cultivation framework of underutilized Greek R. canina germplasm as a potential novel crop that can deliver high-quality products with minimum environmental impact.

2. Materials and Methods

2.1. Greek Rosa canina Germplasm

In succession with previous research efforts undertaken at the Institute of Plant Breeding and Genetic Resources (IPBGR), Hellenic Agricultural Organization DIMITRA, Thessaloniki, Greece (ELGO-DIMITRA), concerning the domestication and sustainable utilization of Greek R. canina germplasm, the current study utilized cultivated R. canina germplasm that has been previously molecularly authenticated, phytochemically evaluated, asexually propagated, ex situ adapted and established under experimental cultivation as part of a research scheme for evaluating the sustainable exploitation potential of the germplasm [13,14]. Four distinct Greek R. canina genotypes have been used herein, consisting of 3-year-old plants that were already established in a pilot orchard-type field cultivation trial (Figure 1) [14]. The genotypes used had previously been allocated unique IPEN (International Plant Exchange Network) accession numbers by the IPBGR, ELGO-DIMITRA, as follows: GR-1-BBGK-19,191, GR-1-BBGK-19,193, GR-1-BBGK-19,635 and GR-1-BBGK-19,674 [13].

2.2. Asexual Propagation

Between the asexual propagation experiments conducted, five alternative rooting enhancers to the conventionally used IBA rooting hormone were tested, which were: (i) Rizobac (a root growth stimulator which contains beneficial soil bacteria in a total population of 1 × 10¹¹ cfu (colony forming units) (Humofert, Athens, Greece)); (ii) Coconut water 75% (containing water, coconut water (5.3%) (coconut cream, water), rice (3.3%), calcium neutral phosphate, stabilizers (guar gum, gellan gum, xanthan gum), sea salt, flavorings, vitamins (B12, D2)); (iii) BIOSHELL ZFE biostimulator (0.2% w/v) (Agroecosystem, Thessaloniki, Greece); (iv) Aloe vera inner leaf natural gel (99.7%) (Forever Living LLC, Athens, Greece) and (v) Vitax organic rooting gel containing natural plant extracts (Vitax, Leicester, UK). The rooting enhancers were applied undiluted based on the manufacturer’s instructions for each product. In addition, the effects of the enrichment of the rooting substrate with externally applied mycorrhizal fungi, consisting of a commercial mix of the Funneliformis mosseae and Rhizoglomus irregulare strains (CLICK NATURE, Italpollina S.p.A., Rivoli, Italy), coupled with the pre-treatment of cuttings with a mycorrhizal inoculator (Glomus spp., Traver, Geenea Med. Ltd., Larnaca, Cyprus) before the application of the rooting enhancer, were also tested in separate experiments.

Three propagation experiments were conducted on the Greek R. canina genotype GR-1-BBGK-19,674 and two propagation experiments on genotype GR-1-BBGK-19,635 under a completely randomized design, which were replicated twice for each experiment during three propagation periods, namely summer 2022, autumn 2022 and summer 2023. In the summer of 2022, the experimentation kicked off with genotype GR-1-BBGK-19,674. Two cutting types were tested, which were 10–12 cm stem sections of primary soft wood from the tip of the stem bearing the stem’s apical meristem (apical cuttings) and internode sections forming the lower parts of the stem (sub-apical cuttings). Each cutting type was treated with six rooting enhancer treatments (which included a control treatment, BIOSHELL ZFE (0.2% w/v), Rizobac, coconut water 75%, 2500 ppm IBA and 0.25% IBA powder), resulting in 12 treatments in total with 30 replicate cuttings each. Consecutively, based on the results of the summer 2022 experiment, in the autumn of 2022, two identical experiments were conducted on the two separate Greek R. canina genotypes mentioned above, where the effects of mycorrhizal enrichment in the substrate were evaluated via the application of two substrate types (peat:perlite (1:3 v/v) and Mycorrhizae-enriched peat:perlite (1:3 v/v), according to the manufacturer’s instructions), each with five rooting enhancer treatments (control, Vitax rooting gel, Rizobac, coconut water 75% and 2500 ppm IBA), resulting in 10 treatments in total and 20 replicate apical cuttings in each treatment for each experiment.

Consecutively, in the summer of 2023, for genotype GR-1-BBGK-19,674, four rooting enhancers were evaluated (control, aloe vera gel (99.7%), Vitax rooting gel and 2500 ppm IBA) with 20 replicate apical cuttings each. At the same time, based on the results of the autumn 2022 experiment for genotype GR-1-BBGK-19,635, in the summer of 2023, the effects of mycorrhizal pre-treatment of the apical cuttings prior to the rooting enhancer treatment were tested via the application of five rooting enhancers (control, coconut water (75%), 2500 ppm IBA, Rizobac and rooting gel) with or without mycorrhizal pre-treatment with ready-made mycorrhizal inoculator solution, resulting in 10 treatments in total of 20 replicate cuttings each.

The “quick-dip method” was used for the application of the rooting enhancer, where the basal tip of each cutting is dipped into the enhancer solution for 5–7 s. The pre-treatment was applied by dipping the basal tip of the cuttings into a 1% v/v mycorrhizal inoculator solution prior to the rooting enhancer dip. The parameters measured were the rooting percentage, average root length and number of roots per cutting. The rooting took place for 30 days under intermittent misting with a relative humidity of >85% in a plastic greenhouse with propagation trays filled with peat (Klasmann, KTS 1)–perlite (1:3 v/v) as the main substrate, unless otherwise stated, across all propagation experiments (Figure 1).

2.3. Experimental Cultivation of Four Rosa canina Genotypes under Distinct Fertilization Regimes

The trial cultivation setup was established since March 2020 when 8-month-old ex situ adapted plants of the four Greek R. canina genotypes studied herein were planted under mainstream orchard establishment techniques. These genotypes were previously selected for cultivation based on a multifaceted evaluation [13,14]. The site of the cultivation setup has been previously described in [14,38]. The cultivation trial consisted of 15 individuals for each genotype planted in rows/plots of 5 plants each, where a separate fertilization regime was applied in each row (5 plants) according to a completely randomized design (Figure 1 and Figure S1). The established plants were drip-irrigated on a weekly basis at a rate of 1.92 L/h throughout the 2022 growing season from April to October.

The implemented fertilization regimes were as follows: no fertilization (control) (NF); conventional crop fertilization (CF) and organic crop fertilization (OF). The CF and OF protocols were adjusted based on the soil analysis and were applied throughout the 2022 growing season monthly, similarly to the previous growing seasons of 2020 and 2021 (see Table S1). The commercial inorganic fertilizers that are typically used in orchard husbandry were employed for the CF regime. The applied fertilization regime consisted of a soil application of 200 g/plant (21% N, 17% P₂O₅, 0.15% Zn and 4% S), a foliar spray application (Mo 1% w/v, B 15% w/v and Zn 30% w/v), a soil application of 200 g/plant (N 13.7%, K₂O 46.3%), a second foliar spray application (2% w/w total N, 4% w/w P₂O₅, 0.2% w/w K₂O, 0.6% w/w CaO, 0.09% w/w MgO and 0.1% w/w S) and a final foliar spray application of 230 g/L CaO.

Similarly, the applied OF regime consisted of a soil application of 50 g/plant organic fertilizer (organic water-soluble N 11% w/w, organic C 40% w/w, total amino acids 69.2% w/w) with additional 4 g/plant organic fertilizer (organic and humic compounds: 68–78%, 5% Ν, 3% Ρ₂O₅, 3–5% CaO, 0.7–1.0% MgO, 1.2 Fe and trace elements), a foliar spray application (Mo 1% w/v, B 15% w/v, Zn 30% w/v), a soil application of 50 g/plant organic fertilizer (organic water-soluble N 11% w/w, organic C 40% w/w, total amino acids 69.2% w/w) with additional 50 g/plant organic fertilizer (30% K₂O, 10% MgO, 42.5% SO₃), a second foliar spray application (2% w/w total N, 4% w/w P₂O₅, 0.2% w/w K₂O, 0.6% w/w CaO, 0.09% w/w MgO, 0.1% w/w S) and a final foliar spray application of 230 g/L CaO (Table S1).

The growth and productivity of the trees was monitored on a long-term basis throughout the period of the cultivation trial since planting (2020–2022) [14]. Building upon the previously collected data, in the current study, the rosehip fruit size growth patterns and final yield, coupled with the concentrations of macro- and micro-elements in leaves, were measured across the 2022 growing season, which was the 3rd year since field establishment and the 2nd year since plants had entered the fruit production stage.

2.4. Determination of Rosehip Fruit Size Growth Patterns and Yield of Cultivated Greek Rosa canina Genotypes

Throughout the 2022 growing season, the fruit growth curve was assessed by measuring the fruit length (from the pedicel base to the tip of the fruit), as well as the fruit height and the width in terms of perpendicular and horizontal diameter (mm), respectively, using a digital caliber (Figure 1). The measurements were conducted at 7 time points following full bloom (days after anthesis—DAA) on a 50-fruit sample, which was averaged per replicate tree on 3 replicate trees for every fertilization regime in every genotype. The fruit size was then determined using the formula:

$$V({\mathrm{c}\mathrm{m}}^3)=\frac{\left(\left(4÷3\right)\times \pi \times \left(\left(L\times W1\times W2\right)÷8\right)\right)}{1000}$$

where L is the measured length (mm) and W1 and W2 are the measured widths in terms of the perpendicular and horizontal diameter, respectively (mm).

The fresh weight of the harvested rosehip fruits was determined in grams per tree (experimental replicate) for all harvested fruits with the use of a digital scale.

2.5. Determination of Macro- and Micro-Elements in the Leaves of Cultivated Greek Rosa canina Genotypes

The concentration of mineral elements (P, K, Ca, Mg, B, Mn, Zn, Fe, Cu) in the Rosa canina leaves was determined using an inductively coupled plasma optical emission spectrometry (ICP–OES) system (Avio 220 Max, PerkinElmer, Waltham, MA, USA) after each sample’s incineration at 550 °C for 6 h and following ash dissolution in 6 N HCl [45]. Three biological replicates were conducted for each treatment and the results were expressed in dry weight. The total nitrogen (N) was determined following the Kjeldahl method using a VAPODEST 50 s system (Gerhardt, Königswinter, Germany). The results were expressed in N%.

2.6. Statistical Analysis

In all the propagation experiments, an analysis of variance (GLM MANOVA) was performed according to a completely randomized design to assess the overall treatment effects on the rooting attributes measured (root number, average root length, rooting). Following the results of the GLM MANOVA, the data were split according to the experimental design in each experiment and were analyzed separately to dissect the specific treatment effects for each applied factor, and differences between means were determined via Duncan’s multiple range post hoc test (p < 0.05) in each case as appropriate. Similarly, for the cultivation data of fruit size, an analysis of variance was performed to assess the overall genotype and fertilization treatment effects on fruit size, coupled with discreet analysis for each measurement date to pinpoint any specific treatment effects on fruit growth during the cropping period, and means were compared via Duncan’s multiple range test (p < 0.05).

For the final fruit yield data (fruit weight), an analysis of variance was conducted to determine the genotype and fertilization effects on the fruit weight, and mean comparison was conducted via Duncan’s multiple range test (p < 0.05). Similarly, for the leaf macro- and micro-element content data, an analysis of variance was conducted to determine the genotype and fertilization effects on each measured element, followed by mean comparison via Duncan’s multiple range test (p < 0.05)

The statistical software used for all analyses was IBM-SPSS v.21 and the graphs were formulated using Microsoft Excel 365.

3. Results

3.1. Asexual Propagation of Greek Rosa canina Germplasm

In the propagation experiment of summer 2022 on genotype GR-1-BBGK-19,674, the 2500 ppm IBA and coconut water showed the highest rooting frequencies among the enhancers tested, which was more profound in apical cuttings with 80% and 60% rooting, respectively, whereas BIOSHELL ZFE showed the lowest rooting in the apical cuttings and among the lowest in the sub-apical cuttings with 13.3% and 16.6% rooting, respectively (

Table 1. Overview of the rooting results for Greek Rosa canina genotype GR-1-BBGK-19,674 for the experiment conducted in the summer of 2022. The table presents the measured rooting attributes of root number, average root length of the rooted cuttings and the corresponding rooting frequencies (±SEM, p < 0.05) for each cutting type and rooting enhancer treatment. Cuttings in both experiments were leafy soft wood sections of primary stem growth either from the tip of the stem bearing the stem’s apical meristem (apical cuttings) or internode sections with two lateral buds (sub-apical cuttings).

GR-1-BBGK-19,674
Rooting Enhancer	Root Number		Average Root Length (mm)		% Rooting
	Apical Cuttings	Sub-Apical Cuttings	Apical Cuttings	Sub-Apical Cuttings	Apical Cuttings	Sub-Apical Cuttings
Control	5.80 (±0.90) abc	4.83 (±0.81) c	4.156 (±0.387) ab	3.415 (±0.253) bc	50.0 (±9.2) bc	40.0 (±9.1) bcde
IBA (2500 ppm)	8.21 (±0.47) a	8.20 (±1.12) a	3.786 (±0.250) bc	3.928 (±0.292) ab	80.0 (±7.4) a	50.0 (±9.2) bc
BIOSHELL ZFE (0.2% w/v)	6.00 (±1.47) abc	4.00 (±1.48) c	2.154 (±0.819) c	3.757 (±0.797) bc	13.3(±6.3) e	16.6 (±6.9) de
Coconut water (75%)	4.33 (±0.65) c	6.50 (±1.06) abc	5.346 (±0.685) a	4.156 (±0.387) ab	60.0 (±9.1) ab	26.6 (±8.2) cde
IBA 0.25%	4.46 (±0.52) c	7.89 (±1.29) ab	3.672 (±0.412) bc	3.641 (±0.360) bc	43.3 (±9.2) bcd	30.0 (±8.5) cde
Rizobac	6.85 (±1.13) abc	6.00 (±0.84) abc	3.141 (±0.378) bc	3.287 (±0.288) bc	43.3 (±9.2) bcd	33.3 (±8.7) bcde

Values within both columns of apical and sub-apical cuttings for each measured attribute that do not share the same letter are significantly different (Duncan’s MRT, p < 0.05). The analysis was conducted for each measured attribute separately. Cuttings in both experiments were leafy soft wood sections of primary stem growth, either from the tip of the stem bearing the stem’s apical meristem (apical cuttings) or internode sections with two lateral buds (sub-apical cuttings).

, p < 0.05). The applied rooting enhancers had a significant effect on the root number in the rooted cuttings, with 2500 ppm IBA showing a similar root production to BIOSHELL ZFE and Rizobac but a higher root production than coconut water and 0.25% IBA powder in the apical cuttings (Table 1, p < 0.05). In the sub-apical cuttings, 2500 ppm IBA showed a higher root production only than BIOSHELL ZFE (Table 1, p < 0.05). Concerning the length of the produced roots, in the apical cuttings, coconut water had the longest roots, similar to the control, whereas in the sub-apical cuttings, no significant effects of the rooting enhancers on root length were observed after 30 days of rooting (Table 1, p < 0.05).

Following the initial propagation results of genotype GR-1-BBGK-19,674, during the following experiment in autumn 2022, BIOSHELL ZFE was replaced with Vitax rooting gel under a new experimental design using only apical cuttings and two substrate treatments, exploring the effects of mycorrhizal enrichment and introducing a further genotype into the experimentation, GR-1-BBGK-19,635. Substrate enrichment with mycorrhizal fungi did not seem to affect the root production and length in the rooted cuttings for either of the two genotypes tested (

Table 2. Overview of the rooting results for Greek Rosa canina genotypes GR-1-BBGK-19,674 and GR-1-BBGK-19,635 for the two experiments conducted in autumn of 2022. The table presents the measured rooting attributes of root number, average root length of the rooted cuttings and the corresponding rooting frequencies (±SEM, p < 0.05) for each substrate type and rooting enhancer treatment.

GR-1-BBGK-19,674
Rooting Enhancer	Root Number		Average Root Length (mm)		% Rooting
	1 Peat:3 Perlite	1 Peat:3 Perlite + Mycorrhizae	1 Peat:3 Perlite	1 Peat:3 Perlite + Mycorrhizae	1 Peat:3 Perlite	1 Peat:3 Perlite + Mycorrhizae
Control	4.00 (±1.30) ab	4.29 (±1.50) ab	1.429 (±0.714) a	1.619 (±0.415) a	25.0 (±9.9) bcd	35.0 (±10.9) bc
Coconut water (75%)	3.50 (±0.77) ab	2.50 (±0.50) ab	1.125 (±0.217) a	0.958 (±0.208) a	40.0 (±11.2) bc	10.0 (±6.8) d
IBA (2500 ppm)	5.63 (±0.89) ab	6.60 (±0.79) a	1.521 (±0.163) a	1.642 (±0.243) a	80.0 (±9.1) a	50.0 (±11.4) b
Rizobac		1.67 (±0.66) b		0.733 (±0.145) a	00.0 (±0.0) e	15.0 (±8.1) cd
Rooting gel	4.09 (±0.73) ab	2.75 (±0.85) ab	0.740 (±0.113) a	1.104 (±0.490) a	50.0 (±11.4) b	20.0 (±9.1) cd
GR-1-BBGK-19,635
Control	2.00 (±0.26) bc	2.89 (±0.53) bc	0.487 (±0.029) d	0.855 (±0.195) cd	33.0 (±9.8) a	38.0 (±10.1) a
IBA (2500 ppm)	4.46 (±0.61) a	3.47 (±0.44) ab	2.423 (±0.285) ab	2.833 (±0.362) a	54.0 (±10.4) a	63.0 (±10.1) a
Coconut water (75%)	2.50 (±0.45) bc	3.10 (±0.37) abc	1.293 (±0.204) cd	1.660 (±0.373) bc	67.0 (±9.8) a	42.0 (±10.3) a
Rizobac	2.25 (±0.52) bc	2.62 (±0.40) bc	1.275 (±0.293) cd	0.838 (±0.179) cd	50.0 (±10.4) a	54.0 (±10.4) a
Rooting gel	2.58 (±0.60) bc	1.78 (±0.40) c	1.450 (±0.245) cd	1.288 (±0.598) cd	50.0 (±10.4) a	38.0 (±10.1) a

Values within both columns of substrate type for each genotype and measured attribute that do not share the same letter are significantly different (Duncan’s MRT, p < 0.05). The analysis was conducted for each measured attribute and genotype separately. The substrate types were a mixture of 1 peat:3 perlite v/v and the same mixture inoculated with mycorrhizal fungi. The ACN of each genotype is given in bold for each part of the table.

, p < 0.05). In genotype GR-1-BBGK-19,674, 2500 ppm IBA showed higher rooting percentages in the substrate without mycorrhizal enrichment, with 80% rooting, which was also the highest among the applied rooting enhancers for the above substrate, followed by rooting gel with 50% rooting and coconut water with 40% rooting (Table 2, p < 0.05). At the same time, the respective rooting frequencies in the mycorrhizal-enriched substrate were lower with 50%, 20% and 10% rooting in the 2500 ppm IBA, rooting gel and coconut water, respectively (Table 2, p < 0.05). Rizobac in GR-1-BBGK-19,674 managed to root only in the mycorrhizal-enriched substrate with 15% rooting (Table 2). In genotype GR-1-BBGK-19,635, on the other hand, mycorrhizal enrichment in the substrate did not affect the rooting percentages for all rooting enhancers applied apart from the control, showing >50% rooting in the substrate without mycorrhizal enrichment (Table 2).

Following the propagation results of autumn 2022, in the summer of 2023, for genotype GR-1-BBGK-19,674, substrate without mycorrhizal enrichment was used testing a further roοting enhancer, which was aloe vera gel, against the previously highest rooting treatments of 2500 ppm IBA and rooting gel. The results showed a similar root production and length among the rooting enhancers tested, coupled with similar rooting frequencies, with 2500 ppm IBA showing 68% rooting, rooting gel showing 50% rooting and aloe vera showing 45% rooting (

Table 3. Overview of the rooting results for Greek Rosa canina genotype GR-1-BBGK-19,674 for the experiment conducted in the summer of 2023. The table presents the measured rooting attributes of root number, average root length of the rooted cuttings and the corresponding rooting frequencies (±SEM, p < 0.05) for each rooting enhancer treatment applied.

GR-1-BBGK-19,674
Rooting Enhancer	Root Number	Average Root Length (mm)	% Rooting
Aloe vera 99.7%	1.83 (±0.20) a	5.753 (±0.731) a	45.0 (±8.0) a
Control	1.72 (±0.15) a	6.155 (±0.556) a	45.0 (±8.0) a
IBA (2500 ppm)	1.78 (±0.13) a	7.029 (±0.523) a	68.0 (±7.5) a
Rooting gel	1.70 (±0.08) a	6.129 (±0.320) a	50.0 (±4.0) a

Values within each column for each measured attribute that do not share the same letter are significantly different (Duncan’s MRT, p < 0.05).

).

Concurrently, for genotype GR-1-BBGK-19,635, the effects of mycorrhizal fungi as a pre-treatment on cuttings before the application of the rooting enhancer was tested in the summer of 2023 under the same rooting enhancers applied previously for this genotype (

Table 4. Overview of the rooting results for Greek Rosa canina genotype GR-1-BBGK-19,635 for the experiment conducted in summer 2023. The table presents the measured rooting attributes of root number, average root length of the rooted cuttings and the corresponding rooting frequencies (±SEM, p < 0.05) for each rooting enhancer treatment with or without pre-treatment.

GR-1-BBGK-19,635
Rooting Enhancer	Root Number		Average Root Length (mm)		% Rooting
	No Pre-Treatment	Mycorrhizal Pre-Treatment	No Pre-Treatment	Mycorrhizal Pre-Treatment	No Pre-Treatment	Mycorrhizal Pre-Treatment
Control	2.30 (±0.22) bc	2.88 (±0.37) abc	5.062 (±0.479) b	5.044 (±0.600) b	100 (±0.0) a	85.0 (±8.2) ab
Coconut water (75%)	1.94 (±0.24) c	2.47 (±0.17) bc	7.177 (±0.588) a	6.639 (±0.382) ab	90.0 (±6.9) ab	85.0 (±8.2) ab
IBA (2500 ppm)	3.00 (±0.33) ab	3.47 (±0.34) a	5.641 (±1.218) ab	6.182 (±0.356) ab	55.0 (±11.4) c	75.0 (±9.9) abc
Rizobac	2.59 (±0.21) abc	2.54 (±0.31) abc	6.786 (±0.629) ab	6.028 (±0.769) ab	85.0 (±8.2) ab	70.0 (±10.5) bc
Rooting gel	2.93 (±0.42) ab	2.58 (±0.24) abc	5.622 (±0.733) ab	6.947 (±0.506) ab	70.0 (±10.5) bc	95.0 (±5.0) ab

Values within both columns of pre-treatment for each measured attribute that do not share the same letter are significantly different (Duncan’s MRT, p < 0.05). The analysis was conducted for each measured attribute separately. Pre-treatment refers to dipping the basal tip of the cuttings into a 1% v/v mycorrhizal inoculator solution prior to the rooting enhancer dip.

). Negligible effects of mycorrhizal pre-treatment of the cuttings were observed on the root number and length, which ranged from 1.94 in coconut water without pre-treatment to 3.47 in 2500 ppm IBA with pre-treatment in the root number, and from 5.044 mm in the control with pre-treatment to 7.177 mm in coconut water without pre-treatment in the root length after 30 days (Table 4). Concerning the rooting rates, they were found similar among the rooting enhancers with mycorrhizal pre-treatment and all above 70% rooting, whereas in the cuttings without pre-treatment, the control, coconut water and Rizobac performed better than 2500 ppm IBA, with 100%, 90% and 85% compared to 55% rooting, respectively (Table 4, p < 0.05).

3.2. Rosehip Development Patterns and Yield of Greek Rosa canina Genotypes as Affected by Fertilization

The fertilization regime effects on the rosehip size were evident towards the later stages of fruit set across the four studied genotypes (Figure 2, p < 0.05). The genotype GR-1-BBGK-19,674 showed a larger rosehip size under conventional and organic fertilization compared to the control at 35, 72 and 91 DAA, but the differences were diminished between all treatments at 107 DAA (Figure 2, p < 0.05). The genotypes GR-1-BBGK-19,635 and GR-1-BBGK-19,191, on the other hand, showed a significantly larger rosehip size at full maturity (>100 DAA) under conventional and organic fertilization compared to the control, and the genotype GR-1-BBGK-19,193 showed a significantly larger rosehip size at full maturity under organic fertilization (Figure 2, p < 0.05).

The fertilization regime significantly affected the final rosehip yield in genotype GR-1-BBGK-19,674, with organic fertilization outweighing the rest (Figure 3, p < 0.05). In addition, the rosehip yield varied significantly among genotypes with the genotype GR-1-BBGK-19,191 being differentiated from the rest by presenting the lowest rosehip fruit yield per plant across all three fertilization treatments (Figure 3, p < 0.05). The average fresh rosehip yield ranged from 172.41 g per plant with conventional fertilization in genotype GR-1-BBGK-19,191 to 1251.44 g per plant with organic fertilization in genotype GR-1-BBGK-19,674 (Figure 3, p < 0.05).

3.3. Inorganic Element Content in Leaves of Greek Rosa canina Genotypes as Affected by Fertilization

The effects of the fertilization regimes on the macro-element and micro-element concentration in the leaves of cultivated germplasm of Greek R. canina were found to be genotype-dependent as they varied in magnitude among genotypes and among elements within each genotype (

Table 5. Concentrations of macro-elements measured in leaves of cultivated germplasm of four Greek Rosa canina genotypes (GR-1-BBGK-19,191, GR-1-BBGK-19,193, GR-1-BBGK-19,635 and GR-1-BBGK-19,674) that were established in the pilot cultivation trial under conventional fertilization, organic fertilization and control (no fertilization), expressed as mg content g⁻¹ DW ± SD, n = 3.

Genotype	Fertilization Regime	N	P	K	Ca	Mg
GR-1-BBGK-19,191	Control	29.79 ± 0.27 ab	3.32 ± 0.16 a	12.63 ± 0.59 a	13.79 ± 0.84 ab	3.35 ± 0.12 bcd
	Conventional	31.98 ± 2.17 a	2.70 ± 0.11 bcd	12.81 ± 0.66 a	11.20 ± 0.39 d	3.33 ± 0.10 bcd
	Organic	26.20 ± 1.65 cd	2.78 ± 0.19 bc	12.31 ± 0.96 a	13.75 ± 1.12 ab	3.35 ± 0.30 bcd
GR-1-BBGK-19,674	Control	25.80 ± 0.63 cd	2.83 ± 0.16 bc	10.75 ± 0.98 bc	13.53 ± 1.38 ab	3.29 ± 0.32 cd
	Conventional	23.00 ± 2.05 e	2.16 ± 0.06 e	10.30 ± 0.32 bc	13.29 ± 0.53 bc	2.96 ± 0.07 d
	Organic	25.98 ± 0.55 cd	2.94 ± 0.17 bc	10.84 ± 0.59 b	13.93 ± 1.18 ab	3.20 ± 0.22 d
GR-1-BBGK-19,635	Control	29.07 ± 0.20 b	2.61 ± 0.13 cd	9.77 ± 0.49 cd	10.74 ± 0.71 d	3.70 ± 0.27 abc
	Conventional	30.40 ± 1.77 ab	2.75 ± 0.11 bc	9.04 ± 0.27 d	11.38 ± 0.35 d	3.78 ± 0.16 ab
	Organic	28.84 ± 1.97 b	2.70 ± 0.02 bcd	10.70 ± 0.29 bc	11.36 ± 1.36 d	4.01 ± 0.49 a
GR-1-BBGK-19,193	Control	23.97 ± 0.94 de	2.48 ± 0.15 d	8.95 ± 0.52 d	15.15 ± 0.89 a	3.88 ± 0.20 a
	Conventional	27.89 ± 0.73 bc	2.75 ± 0.02 bc	12.51 ± 0.10 a	11.94 ± 0.05 cd	3.40 ± 0.01 bcd
	Organic	25.36 ± 1.03 de	2.82 ± 0.07 bc	11.11 ± 0.38 b	13.90 ± 0.28 ab	3.68 ± 0.10 abc

Means that do not share the same letter within each element column are significantly different (Duncan’s multiple range test, p < 0.05).

and

Table 6. Concentrations of micro-elements measured in leaves of cultivated germplasm of four Greek Rosa canina genotypes (GR-1-BBGK-19,191, GR-1-BBGK-19,193, GR-1-BBGK-19,635 and GR-1-BBGK-19,674) that were established in the pilot cultivation trial under conventional fertilization, organic fertilization and control (no fertilization) expressed as ppm ± SD, n = 3.

Genotype	Fertilization Regime	Cu	Fe	Zn	Mn	B
GR-1-BBGK-19,191	Control	5.412 ± 0.280 c	29.956 ± 0.257 de	16.136 ± 0.853 cd	147.900 ± 2.685 b	76.113 ± 0.771 ef
	Conventional	8.245 ± 1.020 ab	31.070 ± 1.153 cd	19.510 ± 0.711 a	98.930 ± 2.259 g	68.736 ± 2.136 g
	Organic	7.125 ± 0.112 b	28.926 ± 0.291 e	14.840 ± 0.180 de	107.266 ± 2.478 f	79.993 ± 1.256 de
GR-1-BBGK-19,674	Control	7.340 ± 0.362 b	31.170 ± 1.549 cd	18.120 ± 0.848 b	155.866 ± 4.952 a	99.656 ± 4.280 a
	Conventional	7.868 ± 0.220 ab	38.613 ± 0.127 a	14.716 ± 0.355 e	139.633 ± 2.936 c	90.820 ± 1.935 c
	Organic	8.413 ± 0.334 a	36.870 ± 0.888 a	17.966 ± 0.524 b	120.400 ± 1.824 de	94.570 ± 1.003 bc
GR-1-BBGK-19,635	Control	8.501 ± 0.259 a	37.746 ± 0.661 a	15.990 ± 0.347 cde	76.546 ± 1.540 i	83.086 ± 2.108 d
	Conventional	7.240 ± 0.320 b	31.150 ± 0.815 cd	16.180 ± 0.475 cd	78.736 ± 2.654 i	96.693 ± 3.489 ab
	Organic	5.907 ± 0.762 c	32.246 ± 1.017 bc	16.283 ± 0.365 c	92.670 ± 0.800 h	78.126 ± 0.138 ef
GR-1-BBGK-19,193	Control	5.933 ± 0.257 c	32.213 ± 2.706 bc	12.400 ± 1.545 f	146.233 ± 8.450 b	91.363 ± 5.171 c
	Conventional	8.649 ± 0.116 a	34.066 ± 0.539 b	15.910 ± 0.970 cde	114.466 ± 0.776 e	71.980 ± 0.386 fg
	Organic	7.215 ± 0.137 b	38.590 ± 0.919 a	15.153 ± 0.730 cde	121.900 ± 4.194 d	83.303 ± 0.785 d

Means that do not share the same letter within each element column are significantly different (Duncan’s multiple range test, p < 0.05).

). The concentration of nitrogen (N) in the leaves was found to be higher under conventional fertilization compared to organic in the genotypes GR-1-BBGK-19,191 and GR-1-BBGK-19,193, whereas the opposite effect was observed in GR-1-BBGK-19,674 with conventional fertilization showing a lower N content than organic (2.3% g⁻¹ DW and 2.6% g⁻¹ DW, respectively), with the remaining genotype GR-1-BBGK-19,635 not being affected by fertilization in terms of the N content (Table 5, p < 0.05). Similarly, the phosphorus (P) concentration increased in the leaves of genotype GR-1-BBGK-19,674 under organic fertilization compared to conventional (0.29% g⁻¹ DW and 0.21% g⁻¹ DW, respectively, Table 5, p < 0.05). Potassium (K), on the other hand, demonstrated a higher leaf content under organic fertilization in genotype GR-1-BBGK-19,635 with 1.07% g⁻¹ DW and under conventional fertilization in genotype GR-1-BBGK-19,193 with 1.25 g⁻¹ DW (Table 5, p < 0.05). The calcium (Ca) content was found to be decreased under conventional fertilization in GR-1-BBGK-19,191 compared to organic (1.12% g⁻¹ DW and 1.37% g⁻¹ DW, respectively), with no further observed differences among the two fertilization regimes applied (Table 5, p < 0.05). Similarly, negligible effects were observed in Mg content in the leaves of cultivated R. canina.

As far as the concentration of micro-elements measured in the leaves of cultivated R. canina germplasm is concerned, conventional fertilization increased the content of Fe and Zn while the leaf content of Mn and B was found to be lower with conventional fertilization compared to organic in genotype GR-1-BBGK-19,191 (Table 6, p < 0.05). In genotype GR-1-BBGK-19,674, organic fertilization showed a higher leaf content of Zn and lower leaf content of Mn compared to conventional fertilization (Table 6, p < 0.05). Comparatively, in genotype GR-1-BBGK-19,635, organic fertilization showed a higher Mn leaf content and lower B leaf content compared to conventional fertilization (Table 6, p < 0.05). In genotype GR-1-BBGK-19,193, organic fertilization presented a higher leaf content of Fe, Mn and B and lower leaf content of Cu compared to conventional fertilization (Table 6, p < 0.05).

4. Discussion

4.1. Asexual Propagation of Greek Rosa canina Germplasm

Within the current work, the use of alternative rooting enhancers to the conventionally used IBA has been evaluated for the first time on Greek R. canina germplasm. Among the rooting factors tested (rooting enhancers, cutting type, mycorrhizae), rooting enhancers appeared to exert the main influence on the rooting of R. canina cuttings in both tested genotypes. In the majority of the current experiments, the use or lack of use of mycorrhizae had little or no effect on the mean root length, root number and rooting rate. The rooting response of the cuttings of shrub species to mycorrhizae has been suggested to depend, among other things, on the type of substrate, genotype and the applied fungal strain [36,37,46]. From the current results, it is not clear whether the lack of a mycorrhizal effect observed is due to the applied substrate or the genetic make-up of the tissue, which requires further research at the biochemical level. Regarding the rooting enhancers, although IBA showed high rooting rates throughout, in many cases, similar rooting rates with no differences in the rooting attributes were measured with other rooting enhancers such as aloe vera, coconut water and rooting gel. Similar results were obtained by Shidiki et al. [29] who experimented on Vitex diversifolia and Cordia milleneii and found equal performance among IBA, aloe vera and coconut water on the rooting of cuttings. This may stem from the fact that coconut water is a composition of numerous nutrients; is considered a natural anti-inflammatory and contains naturally occurring hormones such as cytokinins, and can thus act as an alternative rooting promoter [33,34]. Aryan et al. [47] experimented on the effects of different rooting promoters on cuttings of pomegranate plants such as aloe vera in comparison with IBA and concluded no statistically significant differences in the root number or length of the rooted cuttings. Additional studies have tested several rooting hormone alternatives, including aloe vera and coconut water, which could potentially replace chemical rooting hormones, which are costly and pose a toxicity risk to humans and animals [30]. Issues like the risk or toxicity of artificial rooting hormones like IBA to humans or the environment coupled with a potential cost analysis were not addressed in the current study. However, the rooting enhancers of natural origin like coconut water and aloe vera that have been used herein and showed similar performance to IBA have been considered environmentally friendly and suitable for use in organic horticulture [30]. As such, the current study encourages the use of environmentally friendly, alternative rooting enhancers with similar performance to conventional rooting hormones such as IBA and provides an early step toward documenting the potential of the rooting enhancers tested for the asexual propagation of Greek R. canina.

4.2. Rosehip Development Patterns and Yield of Cultivated Greek Rosa canina Genotypes

The current study presents for the first-time rosehip fruit yield data from ex situ adapted Greek R. canina germplasm under cultivation conditions. The presence of external fertilization inputs seemed to enhance the fruit size in all tested genotypes but not the final cumulative yield, which was found to be genotype-dependent. Fertilization rich in N, like the conventional fertilization treatment herein, showed an improved fruit yield, affecting its quality in cultivated tree crop varieties of pears (Pyrus spp.) and almonds (Prunus amygdalus Batch) under Mediterranean conditions [48,49]. From the current data, the fact that larger fruits had a similar cumulative weight to smaller fruit, may indicate variations in crop load in terms of the response of distinct genotypes to fertilization treatments; however, from the current results, no clear conclusion can be drawn. In wild germplasm of other tree crops like Amelanchier ovalis that has been experimentally cultivated, the application of conventional fertilization increased the fruit size and fresh weight but with a higher water content compared to organic fertilization, insinuating effects on the fruit’s phytochemical profile [38]. Further research is suggested on fruits of the R. canina genotypes cultivated herein in terms of the fruit’s phytochemical profile to draw a more integrated picture on the cultivation protocol.

Albeit at an experimental scale, the similarity of the rosehip fruit production response to organic fertilization compared to conventional observed herein highlights the potential of a diversified organic management system. By taking into additional account the previously determined high feasibility and readiness timescale for the sustainable exploitation of R. canina [14], it is believed that upscaling to substantial commercial production capacities can be viable. Of course, further issues need to be addressed in horticultural terms, like investment and production costs, crop protection in man-made environments and the potential for improving the current germplasm via breeding efforts. These issues should be investigated to further facilitate the value chain creation for the current germplasm, the basis of which is proposed herein.

4.3. Inorganic Element Content in Leaves of Greek Rosa canina Genotypes as Affected by Fertilization

The concentration of macro- and micro-elements in the leaves of experimentally cultivated R. canina genotypes was determined herein under distinct fertilization regimes. The fertilization regimes were applied gradually across the growing season (from March to September) and affected the concentrations of different macro-elements across different genotypes. In micro-element leaf content, the genotypes GR-1-BBGK-19,674 and GR-1-BBGK-19,193 generally showed increased leaf micro-element content under organic fertilization. The fertilization regimes applied were rich in N throughout, coupled with P and K and additional micro-elements. As expected, the most profuse macro-element concentrated in the leaves was N throughout the experiment, followed by K and Ca, with the latter two also found in high concentrations in the soil where the trial was established. The balanced uptake by the plants of K and Ca is considered essential for fruit crops like apples [50,51]. On the other hand, Mn was comparably the most abundant micro-element measured in the leaves of all genotypes, which was not applied externally via the applied fertilization regimes, but it was found in high abundance in the trial’s soil. The external enrichment of the soil with nutrients can enhance nutrient uptake, translocation and metabolism, but it should be applied at the right time and at an appropriate magnitude [52,53].

The current results concerning the nutrient concentration in the leaves of the R. canina genotypes studied demonstrate the plants’ nutrient uptake by the soil. Nevertheless, the magnitude of nutrient uptake in terms of particular elements’ leaf concentrations appear to be genotype-dependent herein. In general, genotypic divergence in terms of nutrient uptake by the plants has been documented [54]. For instance, the genotype dependence of macro- and micro-nutrient uptake and utilization has been shown in cultivated germplasm of trees like cacao [55]. Elementwise, the enrichment of the soil with already existing elements appeared to enhance their uptake by the plants under both conventional and organic regimes in the R. canina genotypes studied.

The positive effects of external organic C amplification on plant productivity and soil fertility on tree crops are well known [56,57]. The organic fertilization enriched in organic C that was applied herein showed genotype-dependent effects on the concentrations of macro- and micro-elements in the leaves, but the productivity of the plants was similar to the conventional fertilization regime applied.

5. Conclusions

This study presents for the first time a systematized cultivation effort for Greek R. canina evaluating the application of organic fertilization complemented with asexual propagation and alternative rooting enhancers to the conventionally used IBA. The organic fertilization rich in organic C applied herein is proposed as an equivalent regime to conventional fertilization in terms of the rosehip yield and nutrient uptake by the plants. Concerning the asexual propagation of R. canina, the rooting enhancers of aloe vera, coconut water and the organic rooting gel tested herein, coupled with the use of apical cuttings, can constitute an alternative organic propagation protocol. Further research on the environmental benefits of the alternative rooting enhancers, on the fruit phytochemical profile under the cultivation of R. canina and on upscaling to commercial production volumes is also suggested. The overall study encourages a diversified organic management approach to the propagation and cultivation of Greek R. canina and strengthens the creation of a value chain for the current and future agro-alimentary and medicinal–cosmetic sectors.