In Vitro Germination, Micropropagation and Addressing the Hyperhydricity of the Balkan Native Dianthus cruentus, a Plant with High Ornamental and Xeriscaping Potential

Abstract

Dianthus cruentus Griseb. (Caryophyllaceae) is an herbaceous perennial native to Greece with a strong ornamental potential when used as a pollinator-friendly component of xeric gardens and green roofs, where it is valued for its tolerance of poor, dry soils, and its showy colorful inflorescences. Aiming to develop an efficient mass propagation protocol appropriate for the introduction of the species as a novel floricultural crop, the in vitro seed and clonal propagation of a Greek native xeric ecotype were investigated in this paper for the first time. A total of 90–100% of the seeds, after being stored in the dark at room temperature for 12 months, germinated when incubated at 10 to 25 °C after their surface sterilization and transfer in vitro. Sixty-day-old seedlings grown in vitro were then used as a source of nodal explants for the initial establishment of micropropagation cultures, more efficiently on MS medium with 0.1 mg L⁻¹ 6-benzylaminopurine (BA). In the multiplication stage, either normal or hyperhydric micro-shoots were used as explant sources, assessing the possibility of incorporating usually discarded material in the propagation procedure. Different solid media were tested, with the highest multiplication indices (5.1) recorded in an MS medium containing 0.1 mg L⁻¹ BA and 0.05 mg L⁻¹ NAA, regardless of explants’ hyperhydricity, while an MS medium containing 0.1 mg L⁻¹ BA and 12 g L⁻¹ agar proved optimal for the effective reversal of hyperhydric explants (MI: 5.2). Despite higher hyperhydricity and reaction rates being observed when hyperhydric explants were used, modifications in the multiplication medium proved to be highly effective in controlling hyperhydricity, with the highest number of normal shoots (2.4–2.6) produced in BA-containing media. Micro-shoots rooted readily in ½ MS medium (60–100%), with rooting rates and quality positively affected by the presence of 0.5 mg L⁻¹ IBA in the rooting medium and the absence of cytokinins in the multiplication one. Rooted micro-shoots were successfully acclimatized ex vitro at high rates (65–100%), their origin influencing their acclimatization and morphology. Thus, the concurrent use of normal and hyperhydric shoots in the proposed micropropagation protocol is proven to be both feasible and desirable, as it is able to significantly increase efficiency and facilitate the sustainable exploitation and dissemination of D. cruentus as a promising multivalent horticultural crop.

1. Introduction

Dianthus cruentus Griseb. (Caryophyllaceae) (Figure 1) is one of the 45 native Dianthus species of Greece, which is situated in the southern part of the wider Balkan peninsula floristic area, a well-attested regional center of diversity for this species-rich genus [1,2]. This is largely due to geological and climatic processes that gave rise to the highly fragmented landscapes of the wider Mediterranean region [3,4]. This plant species can be found growing in dry montane grassland, woodland openings, and other open habitats, on a variety of substrates and in a wide altitudinal range, from 200 to 2100 m above sea level. It is fairly common in the continental part of the country, being found from the mountains of northern Peloponnese and throughout the mainland, while its global distribution encompasses the southern and central parts of the Balkan peninsula, as well as the wider Anatolian region in western Asia. A highly variable species with multiple distinct morphs described under numerous synonyms, it is a perennial, herbaceous plant with a short woody base and compact, tussock-like growth, whose linear, glaucous-green basal leaves are borne on short stems. Flowering lasts from late April to mid-July, when numerous pink to deep reddish purple, more rarely pale red to salmon flowers appear in dense terminal capitulas subtended by erect stems 30 to 70 cm tall [5,6,7]. As with other similar species such as Dianthus carthusianorum, the multiple, showy inflorescences are desirable for their architectural, durable form and the eye-catching color of their numerous flowers [8]. At the same time, it is a hardy, drought-tolerant, and low-maintenance native plant suitable for nutrient-poor, stony sites, with its extensive fibrous root system helping it get established in unstable and shallow soils [9], while its nectar-rich flowers are attractive to locally endangered groups of pollinators such as long-tongued bees and butterflies [10]. Thus, it can be considered a promising novel floricultural crop suitable for use in urban and peri-urban gardens, extensive green roofs, xeric planting beds, and rock gardens. Recent research indicates strong potential for its possible use as a medicinal plant due to its rich phytochemical content and potent antioxidant activity [9,11].

The introduction of novel xeric ornamental crops, mostly native species, holds significant promise for horticultural and ecological applications, particularly in specialized contexts such as green roofs and irrigation-free, xeric gardens [12,13,14,15,16,17,18]. Many Dianthus species native to southern Europe are renowned for their adaptability to arid conditions and problematic soil types (e.g., rich in heavy metals) in open, exposed environments, making them ideal candidates for challenging landscaping projects in water-limited regions such as the wider Mediterranean area [19,20,21]. Moreover, their diverse array of colors and forms, usually compact growth habit, and often delightful fragrance render them highly desirable for ornamental purposes [22,23]. Dianthus cruentus, in particular, stands out as a native perennial species with considerable potential for cultivation in various regions, as already exemplified by its propagation, offering, and use by various nurseries, seed suppliers, and landscape architects all over Europe and North America [24]. At the same time, numerous other Greek native plant taxa have been assessed for their possible introduction as novel ornamental crops, one of them being the Greek endemic Dianthus fruticosus subsp. fruticosus, a shrubby, woody perennial with succulent leaves, impressive flowers, and intense drought tolerance when grown in a Mediterranean green roof setup [19,23]. Thus, underutilized Dianthus taxa and ecotypes from biodiversity hotspots around the Mediterranean can and should be sought in situ and then assessed, introduced, and integrated into modern floriculture practice, green roof systems, and other specialized applications after the successful development of reliable mass propagation protocols, offering not only aesthetic appeal but also contributing to biodiversity conservation and ecological resilience [25,26,27,28]. This holistic approach to horticultural innovation underscores the importance of incorporating native and drought-tolerant species like D. cruentus into urban landscapes, aligning with sustainable development goals and enhancing the functionality and visual appeal of built environments in the age of biodiversity and climate crisis [12,16,29,30,31].

Historically, the importance of floricultural crops such as carnations (Dianthus caryophyllus) in the horticultural industry, coupled with the escalating need for expedient and efficient cut flower production, as well as the demand for consistent and robust propagation material, has spurred extensive research efforts dating back to the late 1970s. These studies have predominantly focused on in vitro propagation methods and other biotechnological approaches for breeding this species [32,33]. Subsequently, there has been a burgeoning interest in exploring in vitro propagation techniques for other Dianthus species of significant ornamental value, such as Dianthus chinensis [34], driven by the confluence of factors including the growing market demand for novel crops, the utilization of these species in breeding programs to create new hybrid cultivars, and the need to expand the range of available floricultural products [23,33]. More recently, there has been a notable shift towards leveraging in vitro propagation methods for the propagation and conservation of a wide array of rare, threatened, or range-restricted Dianthus species, with a particular focus on species native to the floristically rich Balkan peninsula [35]. This trend underscores the pivotal role played by biotechnological advancements in addressing conservation challenges and meeting the evolving demands of an ever-diversifying horticultural sector. There is only one report found in the literature on the in vitro propagation of D. cruentus mainly focusing on in vitro rooting and ex vitro acclimation in a hydroponic system [24]. Previous studies on D. caryophyllus and D. chinensis have assessed, among others, the efficacy of various types and concentrations of PGRs, nutrient media, explant types, and environmental conditions such as temperature and light [23,32,35] while at the same time, many challenges remain, such as low reaction and multiplication rates [23], a high extent and intensity of hyperhydricity [32,35], and the apparent difficulty in extracting and sterilizing viable explants from mature ex vitro plants [24,32,33,34,35]. Each Dianthus species responds in a different way in vitro, e.g., D. pinifolius when cultured on MS medium supplemented with 0.5 mg L⁻¹ BA and 0.1 mg L⁻¹ NAA, achieved a maximum rate of regeneration (100%) and a total of 15.4 shoots per explant [35], while D. fruticosus on the same medium, using the same type and origin of explants achieved 89% regeneration and a total of 1.9 shoots per explant [23].

One of the most important aspects in the introduction of new ornamental crops is the development of a simple, efficient, and low-cost propagation protocol. The use of in vitro-grown seedlings from in situ collected seeds is the most widely used approach when assessing the propagation of native perennial species, as it minimizes the risk of contamination and establishment failure, while accelerating the establishment and the proliferation of in vitro cultures [36,37], at the same time ensuring a greater genetic diversity than other in vitro methods, making possible the selective breeding of elite genotypes with enhanced, unique traits and restricting the extent of low genetic variability due to clonality in ecological restoration plantings [38,39,40]. All species of the genus Dianthus reproduce mainly sexually, with the production and dispersal of a high number of seeds. Most species of the genus Dianthus produce non-dormant seeds, as they become capable of germinating over a range of temperatures without any pre-treatment [41,42,43], thus, being able to form only transient soil seed banks in their habitats [44]. As for all plant species originating from regions with a hot-summer climate, the effect of temperature is particularly important for the ecophysiology of the seed germination of the Mediterranean species, as the latter can be delayed or even prevented in spatial and/or temporal circumstances with unsuitable environmental conditions, and sometimes significant changes in germination are observed during dry storage of their seeds [45,46,47,48].

The phenomenon of hyperhydricity is a particularly important problem for the in vitro propagation of many horticultural crops, including most species of the genus Dianthus [49,50]. It is also a well-attested and commonly occurring problem in the micropropagation of various Mediterranean xeric perennial species such as Globularia alypum [51], Lithodora zahnii [52], Clinopodium nepeta [36], Calamintha cretica [38], and Anthyllis barba-jovis [53], consisting of phenotypic and physiological maladaptations which adversely affect the propagation and ex vitro survival of in vitro produced micro-shoots. In Dianthus caryophyllus, hyperhydricity is manifested by morphological aberrations such as undeveloped, thin cell walls, large intracellular spaces, and a non-fully developed vascular system, in addition to the presence of biochemical indicators such as elevated H₂O₂ markers and free radicals production, as well as evidence of epigenetic regulation of the expression of photosynthesis, metabolism, translation, and production of secondary metabolite genes [54,55]. In Dianthus chinensis, various intrinsic and environmental factors co-modulate and regulate the intensity and the reversibility of this complex phenomenon, the most important of which are genotypic sensitivity, ethylene concentration, the composition of the culture medium, and the conditions of gas exchange in the vessels used for the in vitro culture [56,57].

Therefore, with the incentive to introduce Dianthus cruentus as a novel xeric floricultural crop, our research focused on a promisingly adapted and morphologically unique native population of the species, with the aims of studying its seed germination ecophysiology and its micropropagation starting from seedling-derived explants. Specifically, we investigated the effect of seed-storage period and germination temperature on D. cruentus germination ability and the effect of type and concentration of plant growth regulators, nutrient medium, and agar concentration on in vitro shoot proliferation and rooting. In order to maximize the efficiency of the protocol, we concurrently assessed the viability of in vitro reversal of hyperhydricity in affected micro-shoots.

2. Materials and Methods

2.1. Plant Material

Fully ripe, intact seeds were representatively collected [58] from a small population of Dianthus cruentus situated in the western foothills of Mount Kallidromo, growing locally in dry, exposed openings of dense kermes oak-dominated macchia (Phillyreo-Querquetum cocciferaea) on stony, calcareous soil, near the old Athens-Thessaloniki national road (38°00′53.2″ N, 23°45′46.9″ E, Phthiotis county, Greece) in August 2020 and August 2021. The seeds were isolated from ready-to-dehisce fruits (capsules), which had been left to mature and dry on the mother plants until the opening of their apical teeth. Following the removal of debris and non-viable (infected, empty) seeds, the two collected seed lots were labeled and dry stored in the dark for either 1 week (0-month-old seeds) or 1 year (12-month-old seeds) in paper bags at 25 °C.

A total of 1200 seeds were used for germination studies.

For the micropropagation experiments seedlings grown in vitro were used as starting plant material.

2.2. In Vitro Germination

In August 2021, the seeds were surface sterilized by an initial 10-sec wash with 90.0% v/v ethanol solution, followed by immersion for 10 min in a 20.0% (v/v) solution of commercial bleach (4.6% w/v sodium hypochlorite), finally rinsed three times (3.0 min per rinse) with sterile double-distilled water. The surface sterilized seeds were then cultured in vitro, in plastic Petri dishes (9 cm), containing a solid (8 g L⁻¹ agar) half-strength Murashige and Skoog [59] medium (½ MS). Seed incubation was carried out in constant-condition growth chambers at five different temperatures, i.e., 5, 10, 15, 20, and 25 °C and 16-h cool white fluorescent light (37.5 μmolm⁻² s⁻¹/8-h dark photoperiod).

Germination was recorded every second day for a period up to 40 d. Germination was defined as the emergence of a radicle at least 2.0 mm long [60], and T₅₀ was defined as the time taken for cumulative germination to reach 50.0% of its maximum [42]. 120 seeds were used per treatment, consisting of six Petri dishes per treatment and 20 seeds per Petri dish. All seeds that had failed to emerge had their viability assessed using a cut-test on the 40th day; seeds with a plump, firm, and white-greenish embryo were considered viable [46].

2.3. Establishment of In Vitro Cultures

Two days after the end of the germination experiment, young D. cruentus seedlings grown in vitro were transferred to 100 cm³ glass vessels containing solid (8.0 g L⁻¹ agar) hormone-free (Hf) MS medium and covered with Magenta B-caps (Sigma-Aldrich, St. Louis, MO, USA) for further growth. Both single-node and apical explants, derived from 60-day-old seedlings, were cultured on Hf MS medium or MS medium containing either 6-benzyladenine (BA) or 2-isopentenyladenine (2iP) at 0.1 or 0.5 mg L⁻¹ (initial culture).

2.4. Multiplication Stage and Addressing of Hyperhydricity

At the multiplication stage, single-node and apical explants derived from either normal or hyperhydric micro-shoots produced at the establishment stage were subcultured on solid MS medium, either Hf or supplemented with 0.1 mg L⁻¹ BA or 2iP, with the addition of 1-naphthaleneacetic acid (NAA) at concentrations of 0.00, 0.01 or 0.05 mg L⁻¹.

The experiment was repeated in the following subculture, aiming to further reduce the extent and intensity of hyperhydricity. Single-node and apical explants of either normal or hyperhydric physiology were subcultured on four different solid media of different cytokinin type, BA or zeatin (ZEA), agar concentration, 8 or 12 mg L⁻¹, and nutrient medium, either MS or Woody Plant Medium (WPM) [61].

2.5. In Vitro Rooting and Ex Vitro Acclimatization

Micro-shoots with at least 3 visible nodes, produced by sub-culturing on MS medium with either Hf or 0.1 mg L⁻¹ BA or ZEA. During the last phase of the multiplication stage, they were transferred for rooting onto ½ MS medium containing 0.0 or 0.5 mg L⁻¹ indole-3-butyric acid (IBA).

After their successful rooting, they were rinsed under running tap water and then were transferred to containers (500 mL), on peat (pH 5.5–6.5; Klasmann-Delimann Gmbh, Geeste, Germany): perlite (particles diameter 0.1–0.5 cm, Perloflor; Isocon S.A., Athens, Greece) substrate 1:1 (v/v). The containers were covered with transparent plastic wrap (Sanitas; Sarantis S.A., Athens, Greece) to maintain humidity. They were then placed for one week in a growth chamber (25 °C, 16-h cool white, fluorescent light 37.5 μmolm⁻² s⁻¹/8-h dark photoperiod). Following this, they were uncovered and transferred to a heated glasshouse (37°58′58.0″ N, 23°42′19.2″ E) for two more weeks.

2.6. In Vitro Culture Conditions and Data Collection

In vitro cultures took place in 100 mL glass vessels sealed with Magenta B-caps (Sigma-Aldrich, St. Louis, MO, USA), with five explants installed per vessel. The cultures were maintained at 25 °C and 16-h cool white, fluorescent light 37.5 μmolm⁻² s⁻¹/8-h dark photoperiod. The media contained 30 g L⁻¹ sucrose, were solidified with 8.0 g L⁻¹ agar (M. Roumboulakis SA, Athens, Greece), and their pH was adjusted to 5.7–5.8 before the addition of the agar and autoclaving (121 °C for 20 min).

Measurements in the vitro experiments consisted of shoot formation percentage, total number of shoots per explant, number of normal (NS) and hyperhydric (HS) shoots, number of nodes per ΝS, as well as percentage of rooting, root number, and root length. All establishment, multiplication, and rooting data were collected after 30 d of culture. Acclimatization percentage was recorded 30 d after plantlets were transferred to ex vitro conditions. To obtain the proliferation potential of the cultures the multiplication index (MI) was calculated by multiplying the percentage of explants that produced shoots by the multiplication product of the mean number of shoots per responding explants and the node number per shoot.

2.7. Experimental Design and Statistical Analysis

In every stage of the micropropagation procedure, a completely randomized design was used, with the significance of the results tested by one-way analysis of variance (ANOVA). In addition, two-way ANOVA was used for the factorial study on seed age and temperature in seed germination. Similarly, a MANOVA approach was applied to study the effect of explant physiology and medium composition on the abundance and quality of micro-shoots produced in the multiplication stage, as well as in studying the interaction between multiplication and rooting media in the rooting and acclimatization stages. The data of germination, shoot formation, rooting, and acclimatization percentages were arcsine-transformed prior to statistical analysis; the means were compared by the Student’s t-test at p < 0.05 (JMP 14.0 software, SAS Institute Inc., Cary, NC, USA, 2013). The replicate number per treatment is shown in the corresponding data tables.

3. Results

3.1. In Vitro Germination

Radicle emergence was first noticed from day 2 at the three highest temperatures tested (15 to 25 °C) and from day 4 at 10 °C for both seed age treatments (0- or 12-month-old seeds), while at 5 °C germination was delayed, firstly occurring on day 6 for 12-month-old seeds and on day 14 for 0-month-old seeds. The 12-month-old seeds germinated at higher rates than did the 0-month-old seeds, especially at the two extreme temperatures (5 and 25 °C) tested in the germination experiment, where both seed lots exhibited their minimum germination rates. Apart from the lowest temperature tested, there were no pronounced differences in seed germination speed between the two seed lots, as determined through the calculation of the T₅₀ value. However, seed germination was completed earlier for 12-month-old seeds at temperatures of 10, 20, and 25 °C (8, 14, and 22 days earlier, respectively), whereas, at 5 °C and 15 °C, the difference was much smaller (2 days earlier) (

Table 1. In vitro germination of D. cruentus seeds: final germination percentage, time taken for 50% of the final germination percentage (T₅₀), and time for full germination as a function of seed age and incubation temperature.

Seed Age (Months)	Temperature (°C)	Germination (%) ± SE	T50 † (d)	Time for Full Germination (d)
0	5	18.0 d	18	32
0	10	90.0 b	4	14
0	15	98.0 a	4	8
0	20	82.0 c	4	22
0	25	15.0 d	4	30
12	5	91.0 b	14	30
12	10	98.0 a	4	6
12	15	100.0 a	4	6
12	20	99.0 a	2	6
12	25	96.0 ab	2	8
	Fseed age	***	-	-
	Ftemperature	***	-	-
	Fseed age × temperature	***	-	-
	Fone-way	***	-	-

^† (T₅₀): the days needed to reach 50% of the final germination percentage. Values followed by different lowercase letters in each column denote significant differences at the 5.0% level (Student’s t-test), ***: significant at p ≤ 0.001 (one-way ANOVA, MANOVA); n = 6, 20 seeds/Petri dish (total 120 seeds per treatment).

, Figure 2).

3.2. Establishment of In Vitro Cultures

Four media containing the cytokinins BA or 2iP at concentrations of either 0.1 or 0.5 mg L⁻¹ were used during the initial establishment of the in vitro culture from 60-day-old seedlings. Except for the medium with 0.5 mg L⁻¹ BA, which gave a low reaction rate (33%), all others showed quite high reaction rates, the highest being that with 0.5 2iP mg L⁻¹ (87%). Quite large differences were detected in the shoot formation rate among the media tested, with the highest value (55%) observed in the medium containing 0.1 mg L⁻¹ BA. The presence of 0.1 mg L⁻¹ BA resulted in the highest number of normal shoots (2.3), while its lowest value (0.7) was observed in the medium containing 0.5 mg L⁻¹ 2iP. Media with 2iP induced the development of a higher number of nodes compared to those with BA. In all the media tested, most explants developed shoots of an apparent aberrant morphology, bearing deformed, vitreous leaves, resulting in their characterization as hyperhydric. The rates of hyperhydricity were found to be high to very high in all media tested, with the lowest value obtained from the one containing 0.1 mg L⁻¹ BA. In contrast, the highest number of hyperhydric shoots per explant (4.1) was observed on the medium with 0.1 mg L⁻¹ 2iP, although with no statistically significant differences among different media. In toto, the highest multiplication index (2.76) was achieved with the use of the medium containing 0.1 mg L⁻¹ BA, its value considerably larger than those resulting from the other three establishment media tested, although with no significant differences among treatments due to high intrinsic variability (

Table 2. Establishment of initial in vitro cultures of D. cruentus on MS medium supplemented with either 6-benzyladenine (BA) or 2-isopentenyladenine (2iP) from single node explants excised from in vitro grown seedlings.

Cytokinin (mg L−1)	Reaction (%)	Shoot Formation (%) †	NS †† Number	Node Number per NS	HS ††† Number	MI ††††
BA 0.1	76.0 b	55.0 a	2.3 a	2.2 b	2.7 a	2.8 a
BA 0.5	33.0 c	16.0 b	1.8 ab	2.1 b	2.4 a	0.6 a
2iP 0.1	72.0 b	34.0 a	1.1 bc	2.6 a	4.1 a	1.0 a
2iP 0.5	87.0 a	40.0 a	0.7 c	2.9 a	3.8 a	0.8 a
Fone-way ANOVA §	***	**	***	***	NS	NS

^† At least one non-hyperhydric shoot per explant produced. ^†† NS: normal shoot (non-hyperhydric shoot); ^††† HS: visibly deformed, hyperhydric shoots; ^†††† MI: multiplication index = shoot formation (%) × mean normal shoot number per explant × mean node number. Mean values (n = 55–65) in each column followed by the same lower-case letter do not differ significantly at p ≤ 0.05 by Student’s t-test. ^§ NS, **, ***: non-significant at p ≤ 0.05, significant at p ≤ 0.01, p ≤ 0.001, respectively.

, Figure 3).

3.3. Effect of Explant Physiology on Multiplication and Addressing of Hyperhydricity

Reaction rates were found to be higher when hyperhydric shoots were used as a source of explants. Contrastingly, the corresponding shooting rates were lower than those of normal explants in most cases, with the lowest value recorded in the medium containing 0.1 mg L⁻¹ 2iP and 0.01 mg L⁻¹ NAA, and the highest in the medium with 0.1 mg L⁻¹ BA and 0.05 mg L⁻¹ NAA. In all media containing BA, the number of shoots produced per explant was significantly higher in both types of explants, while the number of nodes per normal shoot did not show significant differences. As expected, the formation of hyperhydric shoots was extremely pronounced in the hyperhydric explants, peaking with the use of a medium containing 0.1 mg L⁻¹ 2iP in the absence of NAA. In general, the use of hyperhydric shoots as explants was associated with a pronounced increase in the already high rates of hyperhydricity, concurrently with an increase in the intensity of base callusing and rooting rates, with 98% of the hyperhydric explants rooting on the medium without plant growth regulators. Overall, in both cases, the highest multiplication indexes were recorded for the media containing BA, while the lowest for the medium containing 0.1 mg L⁻¹ 2iP. The addition of NAA was found to be significant in reducing the number of hyperhydric shoots in the hyperhydric shoots-derived explants, its presence at the highest tested concentration (0.05 mg L⁻¹) leading to the achievement of an equally high multiplication index regardless of the physiological status of the explants used. Contrastingly, the presence of NAA led to a higher multiplication index only when hyperhydric explants were used, with no noticeable positive effect on normal explants (

Table 3. Effect of two different cytokinins, i.e., 6-benzyladenine (BA) and 2-isopentenyladenine (2iP) and naphthaleneacetic acid (NAA), on shoot proliferation from single node explants, excised from normal or hyper hydric micro-shoots of D. cruentus grown on MS medium supplemented with BA or 2iP at 0.1 mg L⁻¹.

Explant Physiology	Cytokinin (mg L−1)	NAA (mgL−1)	Reaction (%)	Shoot Formation (%) †	NS †† Number	Node Number per NS	HS ††† Number	MI ††††
Normal	-	-	76.0 de	58.0 cde	1.7 cd	2.4 b	2.4 ef	2.4 cde
	BA 0.1	-	85.0 b	80.0 ab	2.5 ab	2.5 ab	2.0 f	5.0 ab
	BA 0.1	0.01	85.0 b	82.0 a	2.0 abc	2.6 ab	1.5 f	4.3 ab
	BA 0.1	0.05	83.0 b	78.0 a	2.6 a	2.5 ab	1.8 f	5.1 a
	2iP 0.1	-	80.0 bc	62.0 cde	1.5 cde	2.4 ab	2.6 def	2.2 de
	2iP 0.1	0.01	72.0 e	54.0 de	1.7 cd	2.5 ab	2.2 ef	2.3 de
	2iP 0.1	0.05	76.0 cde	70.0 ab	1.8 bcd	2.6 a	2.2 ef	3.3 bcd
Hyperhydric	-		91.0 ab	69.0 abc	1.4 de	2.5 ab	4.1 cd	2.4 de
	BA 0.1	-	100.0 a	67.0 bcd	1.4 cde	2.5 ab	7.1 ab	2.3 de
	BA 0.1	0.01	100.0 a	73.0 abc	1.5 cde	2.6 ab	6.1 abc	2.9 abc
	BA 0.1	0.05	100.0 a	87.0 a	2.1 abc	2.8 a	5.6 bc	5.1 ab
	2iP 0.1	-	100.0 a	40.0 e	0.8 e	2.3 b	8.3 a	0.7 e
	2iP 0.1	0.01	87.0 bc	53.0 de	1.1 de	2.4 ab	5.8 abc	1.4 e
	2iP 0.1	0.05	100.0 a	60.0 bcd	1.2 cde	2.4 ab	4.1 cde	1.7 de
Fphysiology			***	NS	***	NS	***	*
Fcytokinin			*	***	***	NS	NS	***
FNAA			NS	*	NS	NS	**	NS
Fphys × NAA §			NS	NS	NS	NS	*	NS
Fone-way ANOVA			***	*	***	NS	***	***

^† At least one non-hyperhydric shoot per explant produced. ^†† NS: normal shoot (non-hyperhydric shoot); ^††† HS: visibly deformed, hyperhydric shoots; ^†††† MI: multiplication index = shoot formation (%) × mean normal shoot number per explant × mean node number. Mean values (n_normal = 50–60, n_hyper = 15–40) in each column followed by the same lower-case letter do not differ significantly at p ≤ 0.05 by Student’s t-test. ^§ NS, *, **, ***: non-significant at p ≤ 0.05, significant at p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, respectively.

, Figure 4).

In the subsequent multiplication subculture, assessing additional treatments for their efficiency in reducing hyperhydricity, it was again found that the reaction rate of the hyperhydric explants was higher, but the corresponding shooting rate showed very large differences among the four media tested and was affected by the physiology of the explants used, with its highest value recorded in the medium containing 0.1 mg L⁻¹ BA and 12% agar for both types of explants. Similarly, the number of normal shoots produced showed wide variation among treatments, with the maximum recorded on the same medium when hyperhydric explants were used. Again, the number of hyperhydric shoots was elevated for hyperhydrated explants, with particularly higher values for the two media containing cytokinins (BA, ZEA) with 0.8% agar and MS. Of particular interest is the vast differences noted among the multiplication indices of the different media tested when hyperhydric explants were used, with the 2 MS, 0.8% agar media containing cytokinins performing very poorly (MI: 0.4–0.8), in contrast to the more uniform results from the use of normal explants. In both cases, the medium containing 0.1 mg L⁻¹ BA and 12% agar proved to be more effective for both types of explants, especially when hyperhydric explants were used, where its highest value among all treatments (MI: 5.2) was recorded (

Table 4. Effect of growth medium, agar concentration, and cytokinin type on shoot proliferation from single node explants excised from normal or hyperhydric micro-shoots of D. cruentus grown on MS medium supplemented with BA at 0.1 mg L⁻¹.

Explant Physiology	Medium Composition (mg L−1)	Reaction (%)	Shoot Formation (%) †	NS †† Number	Node Number per NS	HS ††† Number	MI ††††
Normal	MS/8/0.1 BA	90.0 abc	77.0 a	2.0 ab	2.5 ab	1.4 de	3.8 ab
	MS/12/0.1 BA	87.0 bc	87.0 a	2.0 ab	2.3 b	0.1 e	4.0 ab
	WPM/8/0.1 BA	93.0 ab	87.0 a	1.6 b	2.3 b	0.2 e	3.2 b
	MS/8/0.1 ZEA	80.0 c	57.0 bc	1.5 b	2.4 ab	4.0 bc	2.0 bc
Hyperhydric	MS/8/0.1 BA	93.0 ab	43.0 cd	0.7 c	2.6 ab	5.3 b	0.8 c
	MS/12/0.1 BA	93.0 ab	83.0 a	2.4 a	2.6 a	2.5 cd	5.2 a
	WPM/8/0.1 BA	90.0 abc	67.0 ab	2.0 ab	2.2 b	2.1 cde	2.9 b
	MS/8/0.1 ZEA	100.0 a	30.0 d	0.5 c	2.6 ab	12.3 a	0.4 c
Fphysiology		*	**	NS	NS	***	NS
Fsubstrate		NS	***	***	NS	***	***
Fphysiology × substrate §		NS	NS	**	NS	***	*
Fone-way		NS	***	***	NS	***	***

^† At least one non-hyperhydric shoot per explant produced. ^†† NS: normal shoot (non-hyperhydric shoot); ^††† HS: visibly deformed, hyperhydric shoots; ^†††† MI: multiplication index = shoot formation (%) × mean normal shoot number per explant × mean node number. Mean values (n_normal = n_hyper = 30) in each column followed by the same lower-case letter do not differ significantly at p ≤ 0.05 by Student’s t-test. ^§ NS, *, **, ***: non-significant at p ≤ 0.05, significant at p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, respectively.

, Figure 5).

3.4. In Vitro Rooting and Ex Vitro Acclimatization

Rooting rates were consistently high, with their highest value (100%) recorded from the transfer of micro-shoots grown on hormone-free MS medium to ½ MS medium with the addition of 0.5 mg L⁻¹ IBA. The mean number of roots was positively affected by the presence of IBA in the rooting medium, with the exception of micro-shoots originating from the 0.1 mg L⁻¹ BA multiplication medium, where no differences were observed. The mean root length appeared to be significantly reduced in the IBA-containing medium, with its lowest value (2.1 cm) recorded for micro-shoots derived from the two multiplication media containing cytokinins. In these, differences were also observed in the shoot formation and callusing morphology of the rooting explants, as they showed a slightly increased number of shoots and nodes, as well as an elevated callogenesis rate, which was however more pronounced when the micro-shoots were derived from cytokinin-free substrates. The use of IBA affected the morphology of the roots, with more pronounced secondary branching, particularly in micro-shoots derived from cytokinin-free medium (

Table 5. Rooting response of micro-shoots of D. cruentus produced at the multiplication stage on three MS media, i.e., hormone-free or with either 6-benzyladenine (BA) or zeatin (ZEA) at 0.1 mg L⁻¹, as affected by the cytokinin content in the medium of origin and the presence of indole-3-butyric acid (IBA) when transferred in ½ ΜS medium.

IBA (mg L−1)	Cytokinin (mg L−1)	Rooting (%)	Root Number	Root Length (cm)	Callus (%)
0.0	0.0	90.0 ab	5.7 bc	4.0 a	53.0 bc
0.0	0.1 BA	86.0 b	4.6 cd	3.0 b	44.0 bc
0.0	0.1 ZEA	60.0 c	4.0 d	3.2 b	33.0 bc
0.5	0.0	100.0 a	7.6 a	2.6 bc	83.0 a
0.5	0.1 BA	89.0 ab	4.6 cd	2.1 c	52.0 b
0.5	0.1 ZEA	83.0 bc	6.1 b	2.1 c	44.0 bc
FIBA		*	**	***	*
Fcytokinin		***	***	**	**
FIBA × cytokinin §		NS	NS	NS	NS
Fone-way ANOVA		***	***	***	***

Mean values (n = 25–30) in each column followed by the same lower-case letter do not differ significantly at p ≤ 0.05 by Student’s t-test. ^§ NS, *, **, ***: non-significant at p ≤ 0.05, significant at p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, respectively.

, Figure 6).

Ex vitro acclimatization rates were very high (average acclimatization rate: 89%), with the exception of rooted micro-shoots derived from the hormone-free multiplication medium, especially those rooted in the medium without IBA (65%). The mean number of shoots showed slight differences between treatments, with micro-shoots derived from the medium containing 0.1 mg L⁻¹ ZEA developing slightly more shoots (1.5) when the rooting substrate did not contain IBA. Significant differences were recorded in mean shoot length, with the shortest shoots (1.2–1.5 cm) recorded in plantlets derived from the ZEA-containing medium. Smaller differences were observed in the mean number of nodes, where again plantlets derived from micro-shoots subcultured in 0.1 mg L⁻¹ ZEA and rooted in the substrate without IBA were ranked last. Finally, the morphological characteristics of the leaves varied significantly between the various treatments, especially the mean maximum length of the lamina, which showed the highest value for plantlets subcultured on medium absent of cytokinins and rooted in the presence of 0.5 mg L⁻¹ IBA (

Table 6. Acclimatization success and morphology of successfully rooted micro-shoots of D. cruentus after 30 d of their transfer to ex vitro conditions, as affected by the rooting medium and the preceding multiplication medium.

IBA in Rooting (mg L−1)	Cytokinin (mg L−1)	Acclimatization (%)	Stem Number	Stem Length (cm)	Node Number	Max Leaf Length (cm)	Max Leaf Width (mm)
0.0	0.0	65.0 b	1.1 b	2.2 b	6.3 a	4.8 c	2.6 a
0.0	0.1 BA	100.0 a	1.0 b	3.0 a	6.2 a	7.4 ab	2.6 a
0.0	0.1 ZEA	92.0 a	1.5 a	1.2 c	5.2 b	6.4 b	2.3 b
0.5	0.0	83.0 a	1.2 b	2.4 b	6.0 a	8.4 a	2.6 a
0.5	0.1 BA	100.0 a	1.3 ab	2.3 b	5.8 a	6.2 bc	2.3 b
0.5	0.1 ZEA	96.0 a	1.1 b	1.5 c	5.9 a	7.6 ab	2.6 a
FIBA		NS	NS	NS	NS	***	NS
Fcytokinin		***	NS	***	*	NS	*
FIBA × cytokinin		*	*	**	***	***	***
Fone-way		***	*	***	**	***	***

Mean values (n = 20–30) in each column followed by the same lower-case letter do not differ significantly at p ≤ 0.05 by Student’s t-test. NS, *, **, ***: non-significant at p ≤ 0.05, significant at p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, respectively.

, Figure 7).

4. Discussion

4.1. In Vitro Seed Germination

The first aim of the experimental procedure was to investigate the presence or absence of dormancy in Dianthus cruentus seeds, the determination of the cardinal temperatures for their germination, and the assessment of the effects of dry storage on their germination ecophysiology. Based on the measurements obtained in vitro, D. cruentus seeds are characterized by the absence of dormancy, as they became capable of germinating rapidly over a range of temperatures without any pre-treatment. The absence of dormancy is a common feature of most species of the genus Dianthus, while some species native to boreal and alpine habitats such as Dianthus superbus show shallow physiological dormancy, characteristics that can be linked to the need to germinate and survive in harsh, variable conditions [42,43].

Dry storage had a significant effect on seed germination, with seeds stored for 12 months germinating at significantly higher rates than those subjected to germination tests immediately after collection for most incubation temperatures. Thus, the dry after-ripening effect appears to play a particularly important role in the germination ecophysiology of the species, increasing both the temperature range of germination and the final germination rate in a manner similar to that observed for other native Mediterranean species facing a long dry summer and dispersing their seeds before the wet season, such as Clinopodium nepeta [38], Satureja thymbra [45], Periploca angustifolia [62] and Helianthemum caput-felis [63]. A similar positive effect of dry storage has been reported for other species of the genus, most notably the Sardinian endemic psammophyte Dianthus morrisianus, in which the phenomenon is an ecophysiological adaptation to protect seeds from premature germination in the harsh thermo-Mediterranean summer conditions in littoral habitats [46]. At the same time, it is reported that the phenomenon does not seem to be important for other species of the genus such as D. deltoides, D. arenarius, and D. gratianopolitanus, species originating from regions with temperate climate and frequent summer rainfall [43].

Regarding the effect of temperature, in all experiments, the highest germination values were obtained when seeds were incubated at 15 °C, while high values were also recorded for 10 °C, a range typical for plant taxa native to regions with a dry Mediterranean climate [36,37,45]. In contrast, significant differences were observed at the other temperatures, especially in freshly collected seeds, with the lowest germination values recorded for the two cardinal temperatures of 5 °C and 25 °C. T₅₀ values were particularly low for all germination temperatures tested (2 to 6 d), with the exception of 5 °C (14 to 18 d), indicating a very high germination rate, in line with most species of the genus, i.e., Dianthus fruticosus [23], D. morrisianus [46] and D. xylorrhizus [64]. Large differences were noted in the total time required for the completion of the germination procedure, with the lowest values recorded for both seed storage treatments at 15 °C (6–8 d), and the highest at 5 °C (30–32 d). At the other temperatures tested, the time period required for complete germination of fresh seeds was 8 to 16 days longer than for seeds dry-stored for one year. These results further illustrate the incidence of dry after-ripening and indicate the good adaptation of the species for rapid establishment in areas prone to periodic physiological stresses, with similar behavior to other Mediterranean species living in dry habitats with variable, highly demanding conditions such as Centranthus ruber and Silene hicesiae [65,66].

4.2. In Vitro Clonal Propagation

During the development of the current in vitro clonal propagation research for Dianthus cruentus there was no formally published research concerning any aspect of its propagation. Therefore, the investigation of its in vitro propagation was based on reports of related and morphologically and/or ecologically related species that have already been investigated, such as the Greek natives D. pinifolius and D. fruticosus [23,35]. A study concerning the in vitro micropropagation of the species was recently published, using seedlings grown from commercially available seeds as an explant source [24].

Establishing in vitro cultures from in vitro grown, seedling-derived explants of D. cruentus proved to be highly successful, as also found by Ivanovic et al. [24]. In general, the use of seedling-derived explants is indicated for the establishment of native Mediterranean plants, as juvenile tissues show increased response rates, low recalcitrance, and a lack of microbial contaminants, while the use of sexual propagules introduces higher genetic diversity, enhancing the selection of elite ornamental genotypes [37,40,51,52,67]. In the present study, the use of cytokinins BA and 2iP was initially investigated at two concentrations (0.1 and 0.5 mg L⁻¹), based on previous research on several other Dianthus species [23,35,68]. Due to practical difficulties because of the extremely short internodes and the limited availability of in vitro-grown seedlings, both nodal and apical explants were used interchangeably, as their response was similar, which was also reported by Ivanovic et al. [24]. All treatments showed high response rates at establishment, with the exception of the medium containing 0.5 mg L⁻¹ BA. The number of shoots per explant that responded was relatively low, especially in the media containing 2iP, confirming the superiority of BA in low concentrations as in other in vitro culture establishment protocols of Mediterranean native plant taxa [38,39,53,69,70,71,72].

Hyperhydricity proved to pose a significant barrier for the successful development of a high yield in vitro propagation protocol for Dianthus cruentus, noting the proliferation of a high number of micro-shoots with impaired stomatal function and decreased mechanical strength that are expected to perform poorly during their ex vitro acclimatization, similarly to other horticulturally important species of the genus such as D. caryophyllus and D. chinensis [49,55,73,74]. There are several studies establishing the connection between elevated cytokinin content and hyperhydricity, especially in Mediterranean native plant taxa including medicinal species with potential as ornamentals such as Salvia spp. [70,71], Origanum dictamnus [72], and Alkanna tinctoria [75], while in Salvia tomentosa it has been shown that other factors such as the provenance and type of explants can further influence its occurrence [70]. Various approaches have been experimentally assessed to deal with this complex phenomenon in the commercially important floricultural crop D. caryophyllus, with most of them aiming to lessen its impact by a priori controlling the growth of hyperhydric micro-shoots through modifications in the culture conditions such as bottom cooling, improved aeration, decreased relative humidity, and LED lighting, which have proven to be efficient but usually correlated to a parallel decrease in the number of non-hyperhydric micro-shoots produced [55,76,77,78]. Another approach entails the effort to revert the hyperhydric status of already produced micro-shoots through strategic inclusion of various compounds with a proven ability to discourage ethylene production and stimulate healthy growth (such as AgNO₃, Ca(NO₃)₂, silver nanoparticles, silicon and polyamines) leading to a significant increase in the efficiency and productivity of the propagation protocol thanks to the inclusion of previously discarded micro-shoots [34,56,57,79,80,81]. Subcultivation of explants from normal and hyperhydric shoots on Hf-free MS substrate offered evidence for significant differences in the response and shoot formation between the two types of explants. The use of such substrates in three cultivars of D. caryophyllus has been shown to significantly reduce the intensity of hyperhydricity or even reverse its expression, resulting in the production of normal micro-shoots [82]. It is reported that the use of hyperhydric shoots as a source of explants during micropropagation of several commercial strains of Dianthus chinensis caused a significant increase in the reaction rate and the number of normal shoots [57]. This result highlights the increased multiplication potential of the hyperhydric shoots and provides the impetus for further investigation of their possible integration in the in vitro propagation procedure.

The addition of NAA at different concentrations during the multiplication stage has been investigated in several species of the genus Dianthus, such as D. pinifolius, D. giganteiformis subsp. kladovanus and D. juniperinus subsp. bauhinorum, as well as in other Mediterranean native plant taxa, including Calamintha cretica and Anthyllis barba-jovis, with positive effects on increasing shoot formation and shoot number, as well as reducing hyperhydricity thanks to its phytohormonal balancing action as a potent auxin, reducing the deleterious effects of an excess of cytokinins [35,36,38,53,68,83]. In the first subculture, three different NAA concentrations (0.00, 0.01 or 0.05 mg L⁻¹) were assessed in the presence of BA or 2iP at 0.1 mg L⁻¹ when two types of explants, NS or HS, were used. In explants derived from normal shoots, the addition of NAA did not appear to significantly affect shoot formation in BA-containing media, while a small increase in multiplication index was recorded at the highest concentration of NAA tested when 2iP was used. Here again, significant differences were observed between the two types of cytokinin, as the use of BA was again proven to be superior to 2iP, giving more shoots and higher rates of shoot formation. Similar results were observed during the in vitro propagation of Anthyllis hermanniae, a xeric Mediterranean native, where the use of BA induced significantly higher numbers of shoots per explant, as well as higher multiplication indices compared to all other cytokinins [37]. When hyperhydric micro-shoots were used as a source of explants, increasing the concentration of NAA had a positive effect on reducing the number of hyperhydric shoots and increasing the multiplication index, especially in BA-containing media. An MS medium containing 0.1 mg L⁻¹ and 0.05 mg L⁻¹ NAA proved to be the optimal treatment in both types of explant. Ivanovic et al. [24] did not report hyperhydricity in the in vitro culture of D. cruentus, using MS medium supplemented with BA, NAA, and solidified with 0.8 g L⁻¹ agar, as we did in the present work, possibly due to the increased concentration of NAA that they used. The addition of 0.1 mg L⁻¹ NAA in the multiplication medium was also found to eliminate hyperhydricity in Clinopodium nepeta and C. cretica [36,38].

In addition to optimizing the concentration and type of PGRs in the culture medium, modifying the type and concentration of the solidifying agent and nutrient medium can help reduce hyperhydricity and increase shoot formation. In the Greek native xerophytic plants Globularia alypum [51], Lithodora zahnii [52], Clinopodium nepeta [38], and Calamintha cretica [36] increasing the concentration of agar significantly reduced the hyperhydricity rate and increased the number of normal shoots. Similarly, in herbaceous species, increasing agar concentration resulted in a reduction of hyperhydricity [84,85,86]. The agar concentration in the medium regulates the water potential, the diffusion of micronutrients, and the uptake of cytokinins by the explants [50].

In other species such as Aloe polyphylla [87,88], Salvia santolinifolia [89], and Gypsophila paniculata [90], it has been shown that the optimization of the NO₃:NH₄ ratio is crucial for controlling hyperhydricity, as this is affected by the synergistic action of cytokinin concentrations and ammonium ion levels. In the second subculture, the modification of the composition of the culture medium significantly affected the shoot formation rates and the multiplication indices. Regardless of the physiology of the explants, when BA was used as cytokinin at a concentration of 0.1 mg L⁻¹, replacing MS medium with low-ammonium WPM or increasing the agar concentration to 12 g L⁻¹ significantly reduced the number of hyperhydric shoots. For explants of hyperhydric origin, this reduction was accompanied by a significant increase in the number of normal shoots and multiplication index, especially in the medium with increased agar concentration, which proved to be the optimal treatment. ZEA is a cytokinin with particularly favorable shoot-forming activity in many species that show a reduced response at the proliferation stage, especially when they are established by explants derived from adult plants [52,91]. However, in many native Mediterranean species such as Clinopodium nepeta and Limoniastrum monopetalum, its presence in the medium is associated with the increased development of hyperhydric micro-shoots, especially at high concentrations, while its high cost is a further obstacle to its extensive use [38,92]. In the present study, the use of ZEA produced low numbers of normal shoots and the highest number of hyperhydric ones, especially when hyperhyric explants were used. Across the two subcultures, multiplication indices were a direct expression of the highly variable response of both types of explants to different media, with the highest divergence observed among hyperhydric explants, possibly due to their particular morphophysiology interacting with the tested media. As in Dianthus caryophyllus, plausible underlying physiological mechanisms explaining the highly variable extent and intensity of hyperhydricity between the various treatments include differences in levels of oxidative stress, gas exchange rates, and ethylene concentration, among others, mediated by the interactions between the explants and the different physicochemical properties of the tested multiplication media [54,55,56,57]. Rooting of in vitro-produced micro-shoots is a critical stage for micropropagation of most species, with the rapid development of a strong and abundant root system being the main objective of the stage. In order for this to be possible, the micro-shoots are transferred to special rooting media, which usually contain an auxin [37,39]. Different types and concentrations of auxins, as well as different nutrient media, have been tested to optimize root formation in species of the genus Dianthus, with significant differences between species and intraspecific genotypes. Balkan native Dianthus species such as D. pinifolius [35] and D. giganteiformis subsp. kladovanus [68] have been found to root at higher rates and to produce a greater number of roots in ½ MS substrates, in the presence of the auxins IBA or NAA at low concentrations. During the in vitro propagation of Mediterranean native ornamentals such as Cotinus coggygria [93], Muscari muscarimi [94], and Capparis orientalis [95], the content of the multiplication media can continue affecting the rooting of the micro-shoots that originated from them (carry-over effect). In all the rooting experiments carried out, the presence of IBA in the media at a concentration of 0.5 mg L⁻¹ positively affected the intensity and quality of rooting. Interestingly, D. cruentus shoots were recently reported to root at lower percentages in Hf and 0.5 mg L⁻¹ IBA full MS media (68–78%), with the highest rooting percentages recorded with the use of NAA (93–98%), quite possibly due to the use of a rich in nutrients medium [24]. The composition of the multiplication medium of provenance had also a significant effect, with the cytokinin carry-over effect stronger in ZEA-provenance micro-shoots, while the combined effect of PGRs used in both stages significantly determined the callus formation rates. The use of ½ MS medium produced very high rooting rates even when the medium did not contain IBA, confirming its suitability as an optimal rooting medium for multiple Mediterranean natives including Teucrium fruticans and Limoniastrum monopetalum, where higher rooting rates and number of roots were observed compared to the use of full MS medium [36,38,53,69,92].

It is clear that the effectiveness of all propagation methods in practice is an expression of the number of plants that can survive in ambient conditions after acclimatization to ex vitro conditions, posing a critical step that can limit the propagation potential of novel xeric ornamentals such as Greek endemic Ebenus sibthorpii [40]. At the same time, the effect of the micropropagation process on the morphological characteristics of the produced plantlets should be evaluated, with the aim of producing uniform and true-to-type plantlets that are identical to the mother plants [21]. The main factors that affect acclimatization consist of the environmental conditions in the area of acclimatization, the acclimatizing taxon’s habit and ecophysiology, cultural treatments such as fertilizing and pruning, as well as the morpho-physiological characteristics of the rooted micro-shoots [36,37,38]. As in rooting, sometimes the earlier stages of micropropagation can significantly affect the morphological characteristics of the acclimatized plantlets, a phenomenon mainly due to the continued effect of cytokinins, with reports of such observations in horticultural crops such as Zantedeschia aethiopica [96], Uniola paniculata [97], and Fragaria × ananassa [98]. Rooted micro-shoots with a Hf-free provenance acclimatized at lower rates. Significant differences were observed between the average shoot length and average number of nodes of acclimatized plantlets of different provenances, with smaller differences in leaf characteristics and a strong interaction between multiplication and rooting media. The effect of IBA on the morphology of the acclimatized plantlets was generally limited, with the exception of the maximum leaf length, contrary to the results of the acclimatization of the species in hydroponic culture, where the presence of 0.5 mg L⁻¹ IBA in the rooting medium enhanced the number of shoots and the weight of roots compared to the Hf control, an observation plausibly attributed to the significantly faster growth of plantlets in liquid media [24].

5. Conclusions

In the current study, a xeric ecotype of the Greek native Dianthus cruentus was successfully propagated in vitro both by seed and clonal propagation. Very high seed germination rates (90–100%) were achieved in seeds dry after-ripened for 12 months at room temperatures in the dark, in a temperature range of 10 to 25 °C. An efficient micropropagation protocol was developed by initiating in vitro cultures from explants excised from in vitro grown seedlings, highly suitable to make use of both normal and hyperhydric micro-shoots, thus addressing the limitations posed by hyperhydricity. It is recommended to culture both types of explants in an MS medium containing 0.1 mg L⁻¹ BA and 0.05 mg L⁻¹ NAA for shoot multiplication, while an MS medium containing 0.1 mg L⁻¹ and 12 g L⁻¹ agar is optimal for the efficient reversal of hyperhydric explants. Micro-shoots should be transferred in a half-strength MS medium with 0.5 mg L⁻¹ IBA for rooting. Ex vitro acclimatization of microplants was achieved at high percentages (85–100%) in a substrate of peat and perlite (1:1, v/v) when rooted micro-shoots were sourced from media containing PGRs. The use of normal explants was more efficient than hyperhydric ones in most media tested. Nevertheless, the use of specific media resulted in equal or even higher micro-shoot multiplication when explants were excised from hyperhydric shoots, permitting their efficient integration into the propagation procedure. Thus, the micropropagation of this particularly promising and attractive native was proved to be highly effective in all its stages and could facilitate sustainable exploitation of D. cruentus in commercial floriculture as a pollinator-friendly and drought-tolerant green-roof plant, with additional possible applications in ornamental breeding programs and pharmaceutical research. Finally, the current study offers a robust alternative management strategy capable of both addressing hyperhydricity and enhancing the efficiency of micropropagation by incorporating the hyperhydric shoots into the propagation procedure, thereby avoiding their disposal and enabling their use as a viable explants’ source. In light of the ongoing search for reliable, economic, and resourceful mass propagation methods and protocols, we propose that such an integrative approach be explored and developed further in other novel floricultural crops, especially those assessed as highly prone to hyperhydricity.