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

Hieracium lucidum subsp. lucidum is a critically endangered endemic taxa of the Sicilian flora. It is a relict of the Tertiary period surviving on the cliffs of Monte Gallo (NW-Sicily). This research focused on finding the best protocols for seed germination and vegetative and in vitro propagation to contribute to ex situ conservation. Seed germination tests were carried out using constant temperatures of 15 °C, 20 °C and 25 °C in continuous darkness and an alternating temperature of 30/15 °C (16 h/8 h, light/dark). The seeds had no dormancy, and a high germination capacity (70-95%) was obtained at all tested thermoperiods. The possibility of vegetative propagation of the taxon was evaluated through the rooting capacity of stem cuttings treated or not treated with indole-3-butyric acid (IBA). All cuttings were treated with IBA rooted within 2 months, while only 50% of the untreated cuttings were rooted within a longer time. An efficient protocol for rapid in vitro propagation from leaf portions was developed. The response of explants was tested on hormone-free Murashige and Skoog (MS) basal medium and MS enriched with different types of cytokinins: 6-Benzylaminopurine (BAP) and meta-Topolin (mT) in combination with naphthaleneacetic acid (NAA) and 2,4-Dichlorophenoxyacetic acid (2,4-D) at the same concentration. The combination of mT (2 mg L-1) and 2,4-D (1 mg L-1) in the medium was the most effective and showed the highest percentage of callus induction and the mean number of regenerated shoots. The maximum rate of root regeneration and the maximum number and length of roots were obtained on hormone-free MS and MS enriched with IBA at concentrations of 1 mg L-1. From the results obtained, it can be concluded that H. lucidum subsp. lucidum can be successfully propagated using one of the tested techniques, subject to the availability of the material for reproduction.

Micropropagation of Endemic Endangered Taxa of the Italian Flora: Adenostyles alpina subsp. macrocephala (Asteraceae), as a Case Study.

The conservation of endangered, rare, and endemic plant species is based on in situ and ex situ conservation strategies. When in situ conservation alone is not sufficient to guarantee the survival of the species, ex situ techniques are adopted in support. This study aimed to develop an efficient micropropagation protocol for Adenostyles by evaluating the effect of different plant growth regulators on leaf explants. Adenostyles alpina subsp. macrocephala (Asterace) is a perennial herbaceous plant endemic to Calabria (Southern Italy). The genus Adenostyles includes three species confined to the mountains of the Mediterranean and southern Europe. For callus induction, media supplemented with different concentrations of Benzylaminopurine (BAP) (0.5, 1, 2, and 3 mg L-1), Naphthaleneacetic Acid (NAA) (1 mg L-1), and 2,4-Dichlorophenoxyacetic Acid (2,4-D) (1 mg L-1) were tested. Shoot regeneration and proliferation were obtained in media supplemented with BAP (1, 2, and 3 mg L-1) and NAA (1 mg L-1). Root induction was obtained in media supplemented with IBA (0.25, 0.50, and 1 mg L-1) and NAA (0.25, 0.50, and 1 mg L-1). Statistically significant differences in callus induction and shoot regeneration were observed between the various media tested. The medium containing Murashige and Skoog (MS) supplemented with 3 mg L-1 of BAP and 1 mg L-1 of NAA showed the highest percentage of callus induction and increased shoot regeneration. The regenerated shoots showed more effective root induction in the hormone-free MS medium and in the presence of Indole-3-Butyric Acid (IBA) at concentrations of 0.25, 0.50, and 1 mg L-1. These results can be used as a basis for the preparation of a micropropagation protocol for different taxa of Adenostyles, as well as other species of Asteraceae specialized to the Mediterranean mountain habitat.

In Vitro Rooting of Capparis spinosa L. as Affected by Genotype and by the Proliferation Method Adopted During the Multiplication Phase.

The in vitro rooting of three caper (Capparis spinosa L.) selected biotypes, grown in a commercial orchard on the Sicilian island of Salina (38°33'49" N), was performed using-as base material for rooting experiments-shoot explants proceeding from two different in vitro culture systems: solid medium and liquid culture in a PlantForm bioreactor (TIS). The regenerated shoots of each accession were submitted to different auxin treatments (NAA, IBA, IAA - 1 or 2 mg L-1; NAA+IBA 0.75 and 0.25 mg L-1, respectively), supplemented with sucrose or fructose (mg L-1). The highest rooting rate in terms of root percentage (67%) was reached with the explants of the selected accession 'Sal 39' proceeding from liquid culture in PlantForm and induced in the MS medium with sucrose, as a carbon source, supplemented with NAA 0.75 mg L-1 + IBA 0.25 mg L-1, after six days in a climatic growth chamber at 25 ± 1 °C in the dark and then placed under a cool white fluorescent lamp, with a PPFD of 35 µmol m-1 s-1 and a photoperiod of 16 h. On the other hand, poor rooting rate was generally achieved under all the tested experimental conditions with the other biotypes, 'Sal 37' and 'Sal 35', demonstrating the strong role exerted by the previously adopted proliferation method and by the genotype for successful caper in vitro rooting.

In Vitro Regeneration of Capparis spinosa L. by Using a Temporary Immersion System.

Three caper (Capparis spinosa L.) biotypes grown on the Sicilian island of Salina (38°33'49″ N) were micropropagated to evaluate two different in vitro culture systems: one using the traditional solid medium, and the other based on liquid culture in a PlantForm bioreactor. PlantForm is a temporary immersion system (TIS), a new propagation method in which the shoots undergo temporary immersion in a liquid medium in order to avoid the accumulation of gas through forced ventilation. This study proposes a protocol to improve the efficiency of in vitro propagation of caper plants, while also reducing production costs, because of the elimination of the gelling agent, and manual labor, requiring limited subcultures and posing minimal contamination risks. Our results show that the caper shoots propagated in bioreactors demonstrated good adaptability and better growth rates than those grown in the conventional system. Statistically significant differences were observed between plants grown in the PlantForm liquid culture and those grown in solid medium regarding the number and length of shoots, which were further promoted by the addition of plant growth regulators (PGRs). The relative growth and real proliferation rate of the caper explants were higher when using meta-Topolin than when using 6-benzylaminopurine as a PGR. Overall, the TIS improved in vitro caper culture by promoting the proliferation, length, and vigor of the shoots.

Early embryo achievement through isolated microspore culture in Citrus clementina Hort. ex Tan., cvs. 'Monreal Rosso' and 'Nules'.

Microspore embryogenesis is a method of achieving complete homozygosity from plants. It is particularly useful for woody species, like Citrus, characterized by long juvenility, a high degree of heterozygosity and often self-incompatibility. Anther culture is currently the method of choice for microspore embryogenesis in many crops. However, isolated microspore culture is a better way to investigate the processes at the cellular, physiological, biochemical, and molecular levels as it avoids the influence of somatic anther tissue. To exploit the potential of this technique, it is important to separate the key factors affecting the process and, among them, culture medium composition and particularly the plant growth regulators and their concentration, as they can greatly enhance regeneration efficiency. To our knowledge, the ability of meta-Topolin, a naturally occurring aromatic cytokinin, to induce gametic embryogenesis in isolated microspores of Citrus has never been investigated. In this study, the effect of two concentrations of meta-Topolin instead of benzyladenine or zeatin in the culture medium was investigated in isolated microspore culture of two genotypes of Citrus. After 11 months of isolated microspore culture, for both genotypes and for all the four tested media, the microspore reprogramming and their sporophytic development was observed by the presence of multinucleated calli and microspore-derived embryos at different stages. Microsatellite analysis of parental and embryo samples was performed to determine the embryo alleles constitution of early embryos produced in all tested media, confirming their origin from microspores. To our knowledge, this is the first successful report of Citrus microspore embryogenesis with isolated microspore culture in Citrus, and in particular in Citrus clementina Hort. ex Tan, cvs. 'Monreal Rosso' and 'Nules.'