Seed germination and vegetative and in vitro propagation of Hieracium lucidum subsp. lucidum (Asteraceae), a critically endangered endemic taxon of the Sicilian flora

Introduction

As is well known, Hieracium L. s. str. (Asteraceae) is a genus of vascular plants in which diplosporous agamospermy, hybridisation and polyploidy have a fundamental role (Krahulcová, Krahulec & Chapman, 2000; Chrtek, Mráz & Sennikov, 2006). Sexuality is very rare and confined to a few diploid species, mostly distributed in southern Europe (Chrtek, Mráz & Severa, 2004; Mráz & Zdvořák, 2019). This complicated reproductive biology has given rise to a very large number of morphotypes that are hardly distinguishable from each other, and they have been described as either subspecies or at the rank of species (Mráz, Chrtek & Kirschner, 2001). Many of the taxa described are punctual endemics and require specific actions of protection.

Due to their climatic conditions, southern Italy and, in particular, Sicily are suitable for only a few Hieracium relict taxa. However, over the past 20 years, in-depth taxonomic studies of this genus have resulted in several endemic taxa with a very local distribution. Additionally, little-known taxa have been rediscovered or reported as new to Italian flora (Table 1). Several studies on cariology (Di Gristina, Domina & Geraci, 2014, 2021), seed germination (Di Gristina, Varshanidze & Domina, 2020), conservation status (Di Gristina, Raimondo & Domina, 2022) and nomenclature (Di Gristina et al., 2012) have also been conducted on these taxa.

Sicily, the largest island of the Mediterranean basin, is situated at the southern fringe of the distribution range of Hieracium. On this island, the genus is currently represented by 12 taxa, 11 of which are endemic.

Hieracium lucidum Guss. subsp. lucidum is a suffruticose chamaephyte endemic to Mt. Gallo (Fig. 1), a limestone promontory located near the city of Palermo (NW-Sicily). It is a chasmophyte strictly localised on the shady northwest cliffs, between 220 and 310 m a.s.l. These cliffs often benefit from the formation of fog due to the fresh and humid winds coming from the sea. These conditions allow the survival of other punctual endemic species, such as Anthemis ismelia Lojac., Genista gasparrinii (Guss.) C. Presl, Limonium panormitanum (Tod.) Pignatti and Pseudoscabiosa limonifolia (Vahl) Devesa. Although Mt Gallo is a regional natural reserve, it is affected almost yearly by wildfires that reach the vertical walls. The morpho-anatomical study of H. lucidum subsp. lucidum has made it possible to detect a structural organisation of leaves and stems that demonstrate a poor tolerance to the xerophilic environment of the mountain; it is markedly mesophilic, and its survival is allowed by the constant use of the non-rainfall water provided by the fog (Colombo, Melati & Princiotta, 1981). Therefore, H. lucidum subsp. lucidum is a paleoendemism, representative of the subtropical flora that occupied the Mediterranean area in the Tertiary period, which today finds refuge only on Mt. Gallo (Brullo & Brullo, 2020; Gianguzzi, Caldarella & Pasta, 2022). H lucidum subsp. lucidum is also one of the few sexual and diploid examples (Merxmüller, 1975; Brullo & Pavone, 1978; Brullo, Campo & Romano, 2004) in the genus Hieracium, and according to Pignatti (1979, 1982, 1994, 2018), it could be an ancestral taxon from which several European taxa may be derived. Although the plants grow on rocks and vertical cliffs, the recurrent presence of fire progressively reduces the size of its single population and according to Di Gristina, Raimondo & Domina (2022), H. lucidum subsp. lucidum can be classified as Critically Endangered (CR B1ab(iii,v)). Currently, the protection of the species is not provided for in any law or convention. Due to the current state of conservation, the phytogeographic relevance and its peculiar taxonomic role within Hieracium, the taxon requires immediate and special protection measures. Hence, there is a need to operate not only with fire control but also with planned population reinforcement interventions. Consequently, we initiated an extended work to develop efficient protocols for seed germination and vegetative and in vitro propagation. The protocols for seed germination and vegetative propagation for reproduction of Endangered Species of the Mediterranean area are quite developed (Magrini & Salmeri, 2022). To date, in vitro micropropagation studies aimed at species conservation are less common, and no studies are known about Hieracium taxa. To conserve genetic heritage, in vitro propagation can be used in ex situ conservation strategies. Indeed, establishing a micropropagation method for culturing that facilitates the rapid production of a substantial number of plantlets could make a significant contribution to the conservation of species. Germplasm conservation requires cooperation among a spectrum of different technologies (Falk, 1990; Fay, 1992). Micropropagation and other in vitro techniques have been widely used in conservation for the propagation of endangered species and species that present difficulties with traditional propagation techniques. Many threatened species can be quickly propagated with this technology using a minimum of plant material and reducing the impact on populations (Bengi & Yeald, 2005; Trejgell, Michalska & Tretyn, 2010; Gianguzzi et al., 2023).

The results concerning the three reproduction protocols reported in this article aimed to contribute to the conservation of this remarkable element of Sicilian flora.

Materials and Methods

Vegetative propagation test

During the winter of 2021, 13–18 cm long apical stem cuttings (Fig. 2) were collected from pot-grown H. lucidum subsp. lucidum plants in the dormant phase at the Department of Agriculture, Food and Forest Sciences of the University of Palermo. In total, 24 cuttings were collected. The cuttings were disinfected with 3% sodium hypochlorite for 5 s and then washed with sterile distilled water. The basal portion of the 12 cuttings was treated with a talc formulation of indole-3-butyric acid (IBA), and 12 control cuttings were not treated. Of the 12 cuttings treated with IBA, six were placed in a rooting medium consisting of 50% sand-perlite and 50% peat, while the remaining six were placed in a rooting medium consisting of 50% fine sand and 50% sawdust. The same was done for the control cuttings. The vegetative propagation test was carried out in a greenhouse without heating (10–20 ± 1 °C). The low number of samples studied did not allow statistical analysis to be carried out but gave an indication of the treatment to be suggested for vegetative reproduction protocols.

In vitro propagation test

The in vitro propagation test followed the following steps: collection of the plant material, sterilisation, passage in the culture medium, callus induction, shoot regeneration, rooting of the regenerated shoots and acclimatisation.

Collection of the plant material and surface sterilisation

The experiment was conducted at the micropropagation laboratory of the Department of Agricultural, Food and Forestry Sciences, at the University of Palermo. A total of 20 leaves from 10 individuals of Hieracium lucidum subsp. lucidum were collected in the field in mid-September. Initially, the leaves were kept under running water for 1 h to remove any traces of dust and stirred in water plus detergent for 30 min. Subsequently, to reduce the contamination caused by fungi and endogenous or exogenous bacteria, the leaves were sterilised under a vertical laminar flow hood in ethanol at 70% for 5 min and then immersed in a commercial solution of 15% bleach for 15 min. Finally, the leaves were rinsed in distilled water, cut into pieces of about 1 cm², and placed in Petri dishes with plant growth regulators (PGRs).

Culture media and growth condition

The sterilised leaves were placed inside Petri dishes with basal medium with vitamins (Murashige & Skoog, 1962) enriched with 30 g L⁻¹ sucrose and 7 g L⁻¹ plant agar (Duchefa, Haarlem, Netherlands) as an agent and integrated with different concentrations of plant growth regulators, as indicated in Table 2. A pH of 5.8 was reached by adding 1M NaOH or 1M HCl solution. The Petri dishes were maintained at 25 ± 1 °C in a climatic growth chamber in the dark and then subjected to a photoperiod of 16 h light under a white fluorescent lamp with a photosynthetic photon flux density (PPFD) of 35 μmol m⁻¹ s⁻¹.

Table 2:

Plant growth regulators used in the media to induce the regeneration of callus and shoots from Hieracium lucidum subsp. lucidum.

Medium	PGR
	BAP mgL−1	mT mg L−1	NAA mg L−1	2,4-D mg L−1
MS0	0	0	0	0
B1	1	0	1	0
B2	2	0	1	0
B3	1	0	0	1
B4	2	0	0	1
mT1	0	1	1	0
mT2	0	2	1	0
mT3	0	1	0	1
mT4	0	2	0	1

DOI: 10.7717/peerj.16839/table-2

Note:

MS0, control, Murashige and Skoog basal medium, hormone free; B1, B2, B3, B4, medium with 6-Benzylaminopurine (BAP), at different concentration of 1 or 2 mg L⁻¹ in combination with 1-Naphthylacetic acid (NAA) and 2,4-Dichlorophenoxyacetic acid (2.4-D) at the concentration of 1 mg L⁻¹; mT1, mT2, mT3, mT4, medium with meta-Topolin (mT) at concentration of 1 or 2 mg L⁻¹ in combination with NAA at the concentration of 1 mg L⁻¹.

Callus induction

For callus induction, leaves were added to MS basal medium with vitamins (Murashige & Skoog, 1962) enriched with 30 g L⁻¹ sucrose and 7 g L⁻¹ plant agar, and containing various concentrations and combinations of 6-benzylaminopurine (BAP), meta-Topolin (mT), 2,4-Dichlorophenoxyacetic acid (2,4-D) and Naphthylacetic acid (NAA) (Fig. 3A). An MS medium without any hormones (MS0) was used as a control. The pH range of the plant tissue culture medium is essential for the development and growth of explants (Rashid et al., 2018). In this experiment, the pH of the medium was adjusted to 5.7. A total of 56 explants were used (seven replicates with eight explants). The culture of the callus was carried out in Petri dishes of 90 mm diameter with 30 mL of medium and kept in culture for 4 weeks, after which the effect of the different PGRs on callus production was evaluated (Figs. 3B and 3C).

Shoot regeneration

To evaluate the percentage of regeneration of the shoots, the obtained callus was transferred to microbox vessels (125 × 65 × 80 mm) containing 80–100 mL of culture media (Figs. 3D and 3E). The callus was subcultured twice in fresh medium after 15 days (Fig. 3F). The regeneration of the shoots from the callus was evaluated after 7 weeks. All cultures were incubated in a growth climatic chamber at 25 ± 1 °C in the dark and then placed under a fluorescent white lamp with a PPFD of 35 μmol m⁻¹ s⁻¹ and a photoperiod of 16 h of light and 8 h of darkness.

Rooting of the regenerated shoots and acclimatisation

To promote root development and further plant growth, the regenerated shoots were cut from the callus and transferred to hormone-free MS basal medium with vitamins (Murashige & Skoog, 1962) and MS containing various concentrations of NAA and IBA (Fig. 4A and Table 3). Rooting was performed on 56 explants (seven replicates with eight explants). After 4 weeks of rooting, the microshoot percentage, root length and number of roots were counted. Plantlets with well-developed roots (Fig. 4B) were placed in Jiffy-7-Pellet® (Fig. 4C). Subsequently, once the roots escaped from the Jiffy, the plants were placed in plastic pots containing a mixture of peat and perlite (75% and 25%, respectively) (Fig. 4D). To favour hardening, the plantlets were kept at 25 ± 1 °C with 16 h of light and 8 h of darkness.

Table 3:

Plant growth regulators used in the growth media to induce the regeneration of roots from Hieracium lucidum subsp. lucidum.

Medium	PGR
	IBA mg L−1¹	NAA mg L−1
MS0	0	0
R1	0.5	0
R2	1	0
R3	0	0.5
R4	0	1

DOI: 10.7717/peerj.16839/table-3

Note:

MS0, control, hormone-free; R1, R2, medium with different concentration of Indole-3-butyric acid (IBA) at 0.50 and 1 mg L⁻¹ respectively; R3, R4, medium with concentration of Naphthaleneacetic Acid (NAA) 0.50 and 1 mg L⁻¹ respectively.

Statistical analysis of in vitro propagation tests

In this experiment, a completely randomised design was conducted to test the effect of medium on callus, shoot regeneration and rooting. A two-way analysis of variance (ANOVA) was conducted to identify statistically significant disparities and potential interactions among the PGRs (with a significance level of P ≤ 0.05) (Pereira et al., 2018). When the interaction between two factors lacked significance, a one-way ANOVA was executed, individually analyzing each factor, and determining mean distinctions through Tukey’s test. The statistical analysis was carried out utilizing SYSTAT 13 software.

Results

Discussion

The aims of this study were to develop efficient protocols for seed germination and vegetative and in vitro propagation to improve applied knowledge about the ex situ conservation of critically endangered Sicilian endemic Hieracium lucidum subsp. lucidum. For this purpose, germination assays, vegetative propagation and in vitro tests were accomplished as part of an integrated investigation.

The results obtained from the germination tests showed that seeds of H. lucidum subsp. lucidum had no dormancy and a high germination capacity (70–95%) immediately following harvest. This adaptation allows populations living in vertical cliffs to have an immediate availability of new individuals under unfavourable ecological conditions and thus greater opportunities for affirmation of the progeny (Di Gristina, Varshanidze & Domina, 2020).

Among other things, as evidenced by monitoring the population of H. lucidum subsp. lucidum in nature, its seedlings have low competitive capacity, and those emerging at the base of the cliffs are quickly overwhelmed by the surrounding vegetation and destined to perish. This last phenomenon plays a fundamental role in limiting the expansion of the species outside its primary habitat.

The highest percentage of germination (95%) was obtained at a constant temperature of 15 °C in the dark. However, high germination rates were also obtained at all tested thermoperiods, although they decreased as the temperature increased, reaching a minimum of 70% at an alternating temperature of 30/15 °C. Based on these results, H. lucidum subsp. lucidum germinates better at cool or medium constant temperatures. The germination tests conducted in GA₃ did not show substantial variations from those conducted in pure water; therefore, the seed germination of H. lucidum subsp. lucidum was not influenced by GA₃. No other data on germination are available for this taxon.

Regarding the vegetative propagation test, the total percentage of rooting cuttings obtained in the shortest time, the greater number of roots and their greater length, and the greater number of leaves emitted after rooting suggest that treatment with IBA is beneficial for rooting H. lucidum subsp. lucidum cuttings. However, a larger-scale trial is required to confirm this observation.

Vegetative propagation tests also showed that the best rooting medium consisted of 50% sand-perlite and 50% peat. Overall, the results obtained from the rooting cuttings can be defined as encouraging, considering the low productivity capacity of mature H. lucidum subsp. lucidum seeds found in nature (about 10–15 per capitulum). Vegetative reproduction could be a useful multiplication technique for the ornamental use of this taxon.

A highly efficient in vitro micropropagation protocol was developed using leaf explants. The effect of the combination of two different cytokinins, such as BAP and mT, combined with NAA and 2,4-D was examined for the regeneration of callus and shoots. Callus formation was observed in leaf explants incubated on MS basal medium with vitamins (Murashige & Skoog, 1962) and different auxin/cytokinin combinations after 4 weeks of incubation and shoot regeneration after 7 weeks. The induction rates of callus and shoots varied depending on the combination of the applied PGRsgrowth regulators; mT is an alternative to benzyladenine (BA), zeatin (ZEA), and kinetin (KIN) in plant tissue culture (Aremu et al., 2012). It has been used to increase the in vitro plant propagation efficiency in Citrus (Chiancone et al., 2015), Capparis (Gianguzzi et al., 2019) and Carthamus (Vijayakumar et al., 2017). The direction of organogenesis is regulated by the type, ratio and concentration of cytokinins and auxins (Xi et al., 2013). Cellular recognition and/or mechanism of action of cytokinins may affect the different responses to PGR combinations (Kim et al., 2001). In the present study, when mT was present in the culture medium at a concentration of 1 or 2 mg L⁻¹ in combination with 1 mg L⁻¹ 2,4-D and NAA, there was a greater production of callus compared to media in which mT was replaced with BAP. Maximum callus production was observed when 2 mg L⁻¹ mT was present in the medium in combination with 1 mg L⁻¹ 2,4-D (86%) with an average number of shoot regenerations equal to 14.43. Despite hormone-free medium stimulated callus formation on the shoots and plantlets of explants in Lilium (Kedra & Bach, 2005). In our experiments callus induction and shoot regeneration did not occur in the hormone-free medium but occurred at all combinations at different percentages and numbers of shoots. Leaf cultures of other Asteraceae, such as Adenostyles alpina subsp. macrocephala (Huter, Porta & Rigo) Dillenb. & Kadereit (Gianguzzi et al., 2023), Carlina acaulis L. (Trejgell, Dąbrowska & Tretyn, 2009), Echinacea purpurea (L.) Moench (Korach et al., 2002) and Eclipta alba (L.) Hassk. (Dhaka & Kothari, 2005), benefit from the combined effect of cytokinins and auxins, and the highest multiplication rate was achieved with supplementation with BA MS medium. The response of the auxin–cytokinin ratio in organogenic differentiation has also been documented for Bignoniaceae, Hypericaceae and Piperaceae (Lisowska & Wysokinska, 2000; Pereira et al., 2000; Pretto & Santarém, 2000).

From the obtained results, a decrease in the percentage of callus and the number of shoots per explant was observed after substitution in the medium with BAP instead of mT at the same concentration. Of the two concentrations of BAP tested in combination with NAA and 2,4-D, the 2 mg L⁻¹ BAP in combination with 1 mg L⁻¹ of 2,4-D induced a higher percentage of callus (39%) with an average number of shoots per explant of 4.86. A response percentage lower than 13% was observed in the medium containing 1 mg L⁻¹ BAP in combination with 1 mg L⁻¹ of NAA. The callus-regenerated shoots were harvested and transferred to rooting media. The response in terms of rooting percentage, number and length was evaluated after 4 weeks. The best results were recorded in the hormone-free MS basal medium and in the medium with IBA. Root regeneration is a crucial step during plant micropropagation. IBA is preferably used for in vitro root regeneration (Deklerke, Terbruyge & Marinova, 1997; Ramesh et al., 2005). In the present study, the maximum rooting percentage (79%) was obtained in MS basal medium without hormones, with 9.4 roots per explant and an average length of 1.49 cm. Among the solutions tested (IBA at 0.5, 1 mg L⁻¹ or NAA at 0.5, 1 mg L⁻¹) the highest root production occurred with the presence of IBA at a concentration of 1 mg L⁻¹. IBA is one of the most commonly used auxins to induce root formation by organogenesis. The efficacy of IBA for rooting in regenerating shoots has been reported for several species, such as Adenostyles alpina subsp. macrocephala (Gianguzzi et al., 2023) and Capparis spinosa L. (Gianguzzi, Barone & Sottile, 2020). The acclimation step was successfully performed with Jiffy-7® pellets. Subsequently, the plants were placed in pots with a mixture of peat and perlite (75% and 25%, respectively). Once hardened, the plantlets were planted in pots containing garden soil, maintained in the greenhouse for 2 months, and finally in open air, ready for reinforcement activities of the natural population.

Conclusions

The carbonate cliffs are an important refuge for plant biodiversity in the Mediterranean, as they are the only truly natural environments left in this geographical area that have been so strongly anthropised since the dawn of agriculture. All possible actions should be taken to preserve the environment and the plant species living there.

This study is the first integrated report of seed germination and vegetative and in vitro propagation for critically endangered Sicilian endemic Hieracium lucidum subsp. lucidum.

We examined the different propagation techniques of this taxon as a case study that can also be adapted to the conservation of other species that share the same conservation problems.

Seed germination does not appear to be controlled by temperature; in fact, a high germination capacity was obtained at all tested temperatures. The seeds had no dormancy, and the germination process was not influenced by giberellins.

A satisfactory vegetative reproductive capacity appeared from the number of rooted stem cuttings obtained. The application of a talc formulation of IBA to the basal portion of the cuttings improved rooting compared to untreated cuttings.

This research confirmed that leaves have great organogenic potential for shoot formation for in vitro propagation; however, the response is directly related to the combinations of growth regulators. Maximum regeneration of the callus and shoots was achieved in the medium containing 2 mg L⁻¹ of mT and 1 mg L⁻¹ of 2,4-D, and maximum regeneration of roots was induced in hormone-free MS medium or containing 1 mg L⁻¹ of IBA. In vitro micropropagation provides an efficient method for the regeneration of adventitious shoots from H. lucidum leaf explants that will be useful for species conservation.

Based on the obtained results, it can be stated that H. lucidum subsp. lucidum can be successfully propagated using one of the tested techniques reported in this study, depending on the availability of the material for reproduction, thus contributing to the conservation of this critically endangered element of the Sicilian flora. Propagation by seed allows the genetic variability of the populations, but the studied species is not able to produce fertile seeds in quantities suitable for multiplication programmes. Vegetative and in vitro reproduction can be used to support propagation by seed to have a large number of plants in a short time but do not determine genetic variability. In vitro reproduction resulted in the most effective multiplication technique, based on the number of plants produced in a relatively short time. It is essential that propagation material be collected from several individuals to preserve the highest genetic variability.

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