The Effect of Heat Shock on Seed Dormancy Release and Germination in Two Rare and Endangered Astragalus L. Species (Fabaceae).

Many Astragalus species exhibit seeds with physical dormancy (PY), but little is known about the ecological context of this dormancy. We focused on A. maritimus and A. verrucosus, two threatened Sardinian endemic species inside the subgenus Trimeniaeus Bunge. Fresh seeds collected from the only two respective known populations were used to investigate the effect of mechanical scarification, heat shock, and water imbibition processes on PY release and germination. PY can be overcome through mechanical scarification of the water-impermeable seed coats, while no dormancy break was detected, nor a subsequent increase in seed germination due to fire-induced heat. This suggests that fire does not trigger dormancy release and seed germination in these species. The seeds tolerate relatively high heat shock temperatures (up to 120 and 100 °C for A. verrucosus and A. maritimus, respectively), but after 120 °C for 10 min, the number of dead seeds increases in both species. These facts suggest the capacity to develop a soil seed bank that can persist after fires and delay germination until the occurrence of optimal conditions. As regards water imbibition, both Astragalus species did not show the typical triphasic pattern, as germination started without further water uptake. This study emphasizes the significance of understanding germination processes and dormancy in threatened species. In fire-prone ecosystems, PY dormancy plays a crucial role in soil seed bank persistence, and it may be selectively influenced by post-fire conditions. Understanding such adaptations provides useful insights into conservation strategies.

Greenhouse investigation on the phytoremediation potential of pioneer tree Pinus halepensis Mill. in abandoned mine site.

Tailings and mine dumps are often pollutant sources that pose serious environmental threats to surrounding areas. The use of pioneer vascular plants to extract or stabilize metals is considered among the more effective mine tailing reclamation techniques. The study aimed at evaluating the phytoremediation potential of Pinus halepensis in abandoned mine-tailing (SW-Sardinia, Italy). Plant ability to tolerate high Zn, Pb, and Cd concentration and their accumulation in roots and aerial parts were assessed at greenhouse conditions. Experiments were performed on 45 seedlings planted in different substrates (mine-tailings, mine-tailings compost-amended, and reference) and on 15 seedlings grown spontaneously in the contaminated mine site investigated with their own substrates. The phytostabilization potential of plant was evaluated through biological accumulation and translocation indexes together with plant survival and biometric parameters. The outcomes showed the adaptability of P. halepensis to grow and survive in contaminated substrates. Compost addition did not improve plant survival and growth, however, it enhanced total carbon and nitrogen contents of soil, restricted metal bioavailability, and accumulation in plant aerial parts. These findings highlight that P. halepensis may be considered for phytostabilization given the great potential to limit Zn, Pb, and Cd toxicity in plant tissues by applying compost amendment in metal contaminated mine sites.

The novelty of this study is the selection of Pinus halepensis Mill. as a proper tree species for long-term phytoremediation of multi-heavy metal mine tailing sites. This plant species not only had adaptation to the Mediterranean climate and could tolerate high temperatures and high metal concentrations, but also showed high survival and growth percentage of its roots and epigean organs in highly contaminated mine tailing. Pinus halepensis could limit metal accumulation and toxicity in the aerial part of the plant with the addition of compost amendment. This study demonstrates that compost can enhance soil properties and modify metal bioavailability. The outcomes can be beneficial for the phytostabilization project and restoration of similar Mediterranean mine sites.

Using Mediterranean Native Plants for the Phytoremediation of Mining Sites: An Overview of the Past and Present, and Perspectives for the Future.

Mining exploitation in the Mediterranean Basin has left evident scars on the environment, and poses serious risks for human health and biodiversity, especially when mine wastes are left abandoned. This review analysed the main issues of metal(loid)s pollution related to mine exploitation in the Mediterranean Basin. Here, a list of Mediterranean native plant species studied for phytoremediation is given and, considering their biological forms, vegetational types, and ecology, we categorised them into halotolerant and hydro/hygrophilous vegetation, annual and perennial meadows, garrigues and maquis, and high maquis and woods. The main conclusions of the review are as follows: (1) plant communities established on mine environments are often rich in endemic taxa which ensure a high biodiversity and landscape value, and can help in the psychophysical health of local inhabitants; (2) political and land management should take greater account of the use of native plants for the remediation of contaminated soils; (3) a multidisciplinary approach that includes, among others, studies on biochemical response to metal(loid)s as well as the application of innovative soil amendments gives better results; (4) phytoextraction applications require a detailed recovery plan that takes into consideration several issues, including the negative influence on biodiversity due to extensive use of monotypic plantations, disposal of harvested hazardous plants, and the risk of phytoextracts entering the food chain; and (5) more studies are necessary to increase knowledge and to detect suitable species-especially halophytic ones-for phytoremediation purposes.

Germination and early seedling development of Helichrysum microphyllum Cambess. subsp. tyrrhenicum Bacch., Brullo & Giusso in the presence of arsenates and arsenites.

Arsenate, As(V), and arsenite, As(III), are the most available arsenicals present in the soil solutions, in particular in mine polluted substrates, and cause several symptoms of toxicity in plants (like inhibition of seed germination and reduction of seedling development). For these reasons, seeds germination studies are essential for the design of phytoremediation activities of mine sites. Seed germination and seedling development of Helichrysum microphyllum subsp. tyrrhenicum, were evaluated at 15 °C using various concentrations of As(V) and As(III) (0-500 mg/L and 0-200 mg/L, respectively). Seeds were harvested (I) into a mine dump contaminated in As, (II) nearby this site, and (III) faraway the As contaminated area and without mine activities. Seed germination, cotyledons emergence, and seedling mortality were evaluated for 90 days. As(V) and As(III) acted differently, showing a much higher toxicity when arsenite was added than arsenate. The taxon was able to germinate, develop cotyledons, and survive under all arsenate concentrations, whereas arsenite acted on these steps already at 2.5 mg/L. Moreover, a linear decrease in cotyledons emergence was assessed with the increase of both arsenicals' concentrations, as well as a linear decrease of seedling survival under arsenite. The taxon showed great adaptability to As pollution, giving an important contribution in phytoremediation of mining sites.

Integrative Taxonomy of Armeria arenaria (Plumbaginaceae), with a Special Focus on the Putative Subspecies Endemic to the Apennines.

Three subspecies of Armeria arenaria are reported from Italy, two of which are considered endemic to the Apennines. The taxonomic value of these two taxa (A. arenaria subsp. marginata and A. arenaria subsp. apennina) is unclear. Moreover, the relationships between A. arenaria subsp. praecox and Northern Italian populations-currently ascribed to A. arenaria subsp. arenaria-have never been addressed. Accordingly, we used an integrated taxonomic approach, including morphometry, seed morpho-colorimetry, karyology, molecular systematics (psbA-trnH, trnQ-rps16, trnF-trnL, trnL-rpl32, and ITS region), and comparative niche analysis. According to our results, French-Northern Italian populations are clearly distinct from Apennine populations. In the first group, there is evidence which allows the recognition of A. arenaria s.str. (not occurring in Italy) and A. arenaria subsp. praecox. In the second group, the two putative taxa endemic to the Northern Apennines cannot be separated, so a single subspecies is here recognized: A. arenaria subsp. marginata.

Seed Morphology in Species from the Silene mollissima Aggregate (Caryophyllaceae) by Comparison with Geometric Models.

The description of seed shape by comparison with geometric models allows shape quantification, providing the means for an accurate comparison between different species or populations. Geometric models described for the lateral and dorsal views of the seeds of Silene species are applied to the quantification of the shape in the seeds belonging to twenty populations of the eleven taxa of S. mollissima aggregate. Cardioid models LM1, LM5 and LM6 adjust differentially to the lateral views of the seeds, while models DM1, DM5 and DM6 are applied to the dorsal views of the seeds. Quantification of the lateral view of seeds with LM5 results in two groups of species of different geographic origin. The seeds more resembling DM5 include S. andryalifolia, S. badaroi, S. gazulensis, S. hifacensis and S. tomentosa, i.e., the taxa with a continental distribution from southern Spain to northern Italy; in contrast, the group of seeds with lower similarity to DM5 includes those from species in northern Africa and the Mediterranean Tyrrhenian islands: S. auricolifolia, S. hicesiae, S. ichnusae, S. mollissima, S. oenotriae and S. velutina. The description of the seed shape based on geometric models contributes to investigating the relationships between related species and constitutes a promising technique for taxonomy.

Formulation and In Vitro Efficacy Assessment of Teucrium marum Extract Loading Hyalurosomes Enriched with Tween 80 and Glycerol.

The extract of Teucrium marum L. (Lamiaceae) was obtained using the aerial parts of the plant, by means of a maceration process. Verbascoside, caffeic acids derivatives and flavonols were the main components contained in the extract as detected using high-performance liquid chromatography coupled with diode array detector (HPLC-DAD) as an analytical method. The extract was successfully incorporated into hyalurosomes, which were further enriched by adding a water cosolvent (glycerol) and a surfactant (Tween 80), thus obtaining glycerohyalurosomes. Liposomes, transfersomes and glycerosomes were prepared as well and used as comparisons. All vesicles were small, as the mean diameter was never higher than ~115 nm, thus ideal for topical application and stable on storage, probably thanks to the highly negative surface charge of the vesicles (~-33 mV). The cryo-TEM images confirmed the formation of close-packed, oligolamellar and multicompartment hyalurosomes and glycerohyalurosomes in which around 95% of the used extract was retained, confirming their ability to simultaneously load a wide range of molecules having different chemical natures. Moreover, the extract, when loaded in hyalurosomes and glycerohyalurosomes was able to counteract the damages induced in the fibroblasts by hydrogen peroxide to a better extent (viability~110%) than that loaded in the other vesicles (viability~100%), and effectively promoted their proliferation and migration ensuring the healing of the wound performed in a cell monolayer (scratch assay) during 48 h of experiment. Overall in vitro results confirmed the potential of glycerohyalurosomes as delivery systems for T. marum extract for the treatment of skin lesions connected with oxidative stress.

The Endemic Vascular Flora of Sardinia: A Dynamic Checklist with an Overview of Biogeography and Conservation Status.

The vascular flora of Sardinia has been investigated for more than 250 years, with particular attention to the endemic component due to their phylogeographic and conservation interest. However, continuous changes in the floristic composition through natural processes, anthropogenic drivers or modified taxonomical attributions require constant updating. We checked all available literature, web sources, field, and unpublished data from the authors and acknowledged external experts to compile an updated checklist of vascular plants endemic to Sardinia. Life and chorological forms as well as the conservation status of the updated taxa list were reported. Sardinia hosts 341 taxa (15% of the total native flora) endemic to the Tyrrhenian Islands and other limited continental territories; 195 of these (8% of the total native flora) are exclusive to Sardinia. Asteraceae (50 taxa) and Plumbaginaceae (42 taxa) are the most representative families, while the most frequent life forms are hemicryptophytes (118 taxa) and chamaephytes (106 taxa). The global conservation status, available for 201 taxa, indicates that most endemics are under the 'Critically Endangered' (25 taxa), 'Endangered' (31 taxa), or 'Least Concern' (90 taxa) IUCN categories. This research provides an updated basis for future biosystematics, taxonomic, biogeographical, and ecological studies and in supporting more integrated and efficient policy tools.

Ex situ phytoremediation trial of Sardinian mine waste using a pioneer plant species.

The mitigation of metals contamination is currently a crucial issue for the reclamation of mine sites. Indeed, mine wastes are often disposed in open dumps and consequently pollutants are subjected to dispersion in the surrounding areas. In this study, the potential use of Helichrysum microphyllum subsp. tyrrhenicum for phytostabilization was evaluated in ex situ conditions. Ninety specimens were randomly selected and were planted in three substrates (reference substrate, mine waste materials, and mine wastes with compost). Mineralogical compositions of substrates, rhizosphere, and roots were assessed through X-ray diffraction (XRD). Zn, Pb, and Cd concentrations of substrates, rhizosphere, soil pore waters, and plant tissues were determined. The phytostabilization potential was determined through the application of biological accumulation coefficient (BAC), biological concentration factor (BCF), and translocation factor (TF). Moreover, survival and biometric parameters were assessed on plant specimens. The polluted substrates and related rhizosphere materials were mainly composed of dolomite, quartz, pyrite, and phyllosilicate. Zn was the most abundant metal in substrates, rhizosphere, and soil pore waters. XRD analysis on roots showed the presence of amorphous cellulose and quartz and Zn was the most abundant metal in plant tissues. H. microphyllum subsp. tyrrhenicum restricts the accumulation of the metals into roots limiting their translocation in aereal parts, indicating its potential use as phytostabilizer (BCF, BAC, TF < 1). Survival and growth data showed a great adaptability to different substrates, with an evident positive effect of the implementation of compost which increased the plant survival and decreased the metals uptake into roots.

Differential Interpretation of Mountain Temperatures by Endospermic Seeds of Three Endemic Species Impacts the Timing of In Situ Germination.

Predicting seed germination in the field is a critical part of anticipating the impact of climate change on the timing of wild species regeneration. We combined thermal time and soil heat sum models of seed germination for three endemic Mediterranean mountain species with endospermic seeds and morphophysiological dormancy: Aquilegia barbaricina, Paeonia corsica, and Ribes sandalioticum. Seeds were buried in the soil within the respective collection sites, both underneath and outside the tree canopy, and their growth was assessed regularly and related to soil temperatures and estimates of the thermal characteristics of the seeds. The thermal thresholds for embryo growth and seed germination of A. barbaricina assessed in previous studies under controlled conditions were used to calculate soil heat sum accumulation of this species in the field. Thermal thresholds of seed germination for P. corsica and R. sandalioticum were not previously known and were estimated for the first time in this field study, based on findings of previous works carried out under controlled conditions. Critical embryo length and maximum germination for A. barbaricina were reached in April, and in December for R. sandalioticum. Seeds of P. corsica stay dormant in the ground until the following summer, and the critical embryo length and highest germination were detected from September to December. Soil heat sum models predicted earlier germination by one month for all three species under two Intergovernmental Panel on Climate Change (IPCC) scenarios, based on the assumption that the estimated thermal thresholds will remain constant through climate changes. This phenological shift may increase the risk of mortality for young seedlings. The models developed provide important means of connecting the micro-environmental niche for in situ seed germination and the macro-environmental parameters under a global warming scenario.

Extraction, Characterization and Incorporation of Hypericum scruglii Extract in Ad Hoc Formulated Phospholipid Vesicles Designed for the Treatment of Skin Diseases Connected with Oxidative Stress.

An extract of Hypericum scruglii, an endangered endemic plant of Sardinia (Italy), was prepared and characterized. It was loaded in special phospholipid vesicles, glycerosomes, which were modified by adding maltodextrin (glucidex) and a polymer (gelatin or hyaluronan). The corresponding liposomes were also prepared and used as reference. The vesicles disclosed suitable physicochemical features for skin delivery. Indeed, their mean diameter ranged from 120 to 160 nm, they were homogeneously dispersed (polydispersity index ≤ 0.30), and their zeta potential was highly negative (~-45 mV). The vesicle dispersions maintained unchanged characteristics during 60 days of storage, were highly biocompatible, and were able to protect keratinocytes against damages due to oxidative stress induced by treating them with hydrogen peroxide. Vesicles were also capable of promoting cell proliferation and migration in vitro by means of a scratch wound assay. The results confirmed the fruitful delivery of the extract of H. scruglii in glycerosomes modified with glucidex and gelatin and their promising ability for skin protection and treatment.

Morpho-Colorimetric Characterization of the Sardinian Endemic Taxa of the Genus Anchusa L. by Seed Image Analysis.

In this work, the seed morpho-colorimetric differentiation of the Sardinian endemic species of Anchusa (Boraginaceae) was evaluated. In Sardinia, the Anchusa genus includes the following seven taxa: A. capellii, A. crispa ssp. crispa, A. crispa ssp. maritima, A. formosa, A. littorea, A. montelinasana, and A. sardoa. Seed images were acquired using a flatbed scanner and analyzed using the free software package ImageJ. A total of 74 seed morpho-colorimetric features of 2692 seed lots of seven taxa of Anchusa belonging to 17 populations were extrapolated and used to build a database of seed size, shape, and color features. The data were statistically elaborated by the stepwise linear discriminant analysis (LDA) to compare and discriminate each accession and taxon. In addition, the seed morpho-colorimetric differences among coastal and mountainous taxa were evaluated. Considering the ecological conditions, the LDA was able to discriminate among the Anchusa taxa with a correct identification of 87.4% and 90.8% of specimens for mountainous and coastal plants, respectively. Moreover, the LDA of the 17 populations of Anchusa showed a low separation among species and populations within the coastal group, highlighting how the long-distance dispersal by flotation on the sea water surface and the pollination network may influence the similarity patterns observed. In addition, a misattribution was observed for A. crispa ssp. crispa, which was misclassified as A. crispa ssp. maritima in 14.1% of cases, while A. crispa ssp. maritima was misidentified as A. crispa ssp. crispa in 21.1% of cases, highlighting a close phenotypic relationship between these two taxa. The statistical results obtained through the seed image analysis showed that the morpho-colorimetric features of the seeds provide important information about the adaptation and evolution of Anchusa taxa in Sardinia.

Does Storage under Gene Bank Conditions Affect Seed Germination and Seedling Growth? The Case of Senecio morisii (Asteraceae), a Vascular Plant Exclusive to Sardinian Water Meadows.

Understanding seed viability under long-term storage conditions provides basic and useful information to investigate the effectiveness of seed banking. Besides the germination success, seedling establishment is also an important requirement, and a decisive step to ensure plant propagation. We used comparative data of germination, seedling growth, and survival percentage between fresh and 10-years-stored seeds of Senecio morisii, a narrow endemic and vulnerable species of Sardinia (Italy), in order to evaluate if differences exist in these traits between fresh and 10-years-stored seeds. Stored seeds showed higher germination percentages than fresh ones, whereas seedling growth and survival did not present significant differences between them, except for seedling growth in plants produced from seeds germinated at 25 °C. This study allowed us to assess if seeds of S. morisii were able to germinate under controlled conditions, and if they maintained their viability and germination capacity for at least 10 years of long-term storage in the seed bank. In addition, the high seedling survival detected in both fresh and stored seeds suggests that stored seeds of S. morisii can be used to support reinforcement or reintroduction actions when fresh materials are not available.

Predicting the consequences of global warming on Gentiana lutea germination at the edge of its distributional and ecological range.

BACKGROUND: Temperature is the main environmental factor controlling seed germination; it determines both the percentage and the rate of germination. According to the Intergovernmental Panel on Climate Change, the global mean surface temperature could increase of approximately 2-4 °C by 2090-2099. As a consequence of global warming, the period of snow cover is decreasing on several mountain areas. Thermal time approach can be used to characterise the seed germination of plants and to evaluate the germination behaviour under the climate change scenarios. In this study, the effect of different cold stratification periods on seed dormancy release and germination of Gentiana lutea subsp. lutea, a taxon listed in Annex V of the Habitats Directive (92/43/EEC), was evaluated. Furthermore, the thermal requirements and the consequences of the temperature rise for seed germination of this species were estimated. In addition, a conceptual representation of the thermal time approach is presented. METHODS: Seeds of G. lutea subsp. lutea were harvested from at least 50 randomly selected plants in two representative localities of the Gennargentu massif (Sardinia). Germination tests were carried out under laboratory conditions and the responses at 5, 10, 15, 20, 25 and 30 °C were recorded. Different cold stratification pre-treatments at 1 ± 1 °C (i.e. 0, 15, 30, 60 and 90 days) were applied. Successively, the base temperature (T b) and the number of thermal units (θ, °Cd) for germination were estimated. Additionally, this study examined the consequences of an increase in temperatures based on the Representative Concentration Pathways (RPC) scenarios. RESULTS: The results indicated that from 0 to 30 days of cold stratification, the germination was null or very low. After 60 and 90 days of cold stratification the seed dormancy was removed; however, 25 and 30 °C negatively affected the germination capacity of non-dormant seeds. Seeds cold-stratified for 90 days showed a lower T b than those stratified for 60 days. However, 60 and 90 days of cold stratification did not cause great variations in the thermal time units. Analysing the RPC scenarios, we detected that the number of days useful for dormancy release of seeds of G. lutea may be less than 30 days, a condition that does not permit an effective dormancy release. CONCLUSIONS: We conclude that seeds of G. lutea need at least 60 days of cold stratification to remove dormancy and promote the germination. The thermal time model developed in this work allowed us to identify the thermal threshold requirements of seed germination of this species, increasing the knowledge of a plant threatened by global warming. Our results emphasise the need for further studies aiming at a better characterisation of germination efficiency, especially for species that require cold stratification. This would improve the knowledge on the germination mechanisms of adaptation to different future global warming conditions.

Thermal niche for in situ seed germination by Mediterranean mountain streams: model prediction and validation for Rhamnus persicifolia seeds.

BACKGROUND AND AIMS: Mediterranean mountain species face exacting ecological conditions of rainy, cold winters and arid, hot summers, which affect seed germination phenology. In this study, a soil heat sum model was used to predict field emergence of Rhamnus persicifolia, an endemic tree species living at the edge of mountain streams of central eastern Sardinia. METHODS: Seeds were incubated in the light at a range of temperatures (10-25 and 25/10 °C) after different periods (up to 3 months) of cold stratification at 5 °C. Base temperatures (Tb), and thermal times for 50 % germination (θ50) were calculated. Seeds were also buried in the soil in two natural populations (Rio Correboi and Rio Olai), both underneath and outside the tree canopy, and exhumed at regular intervals. Soil temperatures were recorded using data loggers and soil heat sum (°Cd) was calculated on the basis of the estimated Tb and soil temperatures. KEY RESULTS: Cold stratification released physiological dormancy (PD), increasing final germination and widening the range of germination temperatures, indicative of a Type 2 non-deep PD. Tb was reduced from 10·5 °C for non-stratified seeds to 2·7 °C for seeds cold stratified for 3 months. The best thermal time model was obtained by fitting probit germination against log °Cd. θ50 was 2·6 log °Cd for untreated seeds and 2·17-2·19 log °Cd for stratified seeds. When θ50 values were integrated with soil heat sum estimates, field emergence was predicted from March to April and confirmed through field observations. CONCLUSIONS: Tb and θ50 values facilitated model development of the thermal niche for in situ germination of R. persicifolia. These experimental approaches may be applied to model the natural regeneration patterns of other species growing on Mediterranean mountain waterways and of physiologically dormant species, with overwintering cold stratification requirement and spring germination.