6 Seed-based restoration and conservation

6.1 Addressing seed needs for reforestation in the western United States

Aghai, M.M.1, Kildisheva, O.A.2

1Silvaseed Company, USA; 2The Nature Conservancy, USA

Climate change has increased the frequency and intensity of disturbance regimes in the Western United States, negatively impacting native conifer forests. High intensity wildfires are driving the loss of critical tree populations from which wild-type seed can be sourced for restoration. In parallel, there are many regional initiatives calling for reforestation en masse as a pathway to carbon sequestration and stabilization of these habitats. Although a mature forest management industry exists in the region, its seed collection and processing capability is limited and largely reliant on genotypes derived from orchards and tree improvement programs developed for timber production. Wild seed collection is both an art and a science, requiring knowledgeable practitioners to survey for mast and rely on a detailed understanding of myriad conifer species’ seed biology to develop a collection strategy. Today skilled seed collection practitioners are few, as academic or professional institutions have not maintained training programs to support this sector in much of the United States. To address non-industrial reforestation needs and establish genetically resilient and structurally diverse forests, significant innovation and investment in scaling up responsible and genetically appropriate seed collection efforts from wild populations is needed. This presentation will provide an overview of the current practices of seed collection, cleaning and banking, as well as highlight examples of scaling up collection efforts to meet new reforestation needs in the Western United States. We will feature Silvaseed - a 130 year old historic regional seed provider - as an example of a legacy operation that is innovating processes and products to meet seed needs for ecologically relevant reforestation in the era of climate change.

6.2 MedGermDB: building a seed germination database of Mediterranean plants

Cruz-Tejada, D.M.1, Fernández-Pascual, E.2, Mattana, E.3, Carta, A1.

1University of Pisa, Italy; 2University of Oviedo, Spain; 3Royal Botanic Gardens, Kew, UK

Seed-based restoration is an effective tool to address biodiversity loss as a consequence of climate change. However, knowledge of regeneration traits is still very limited compared to that of vegetative plant traits. Therefore, if we advance in the understanding of the functional ecology of regeneration traits, we can improve the efficiency of projects aimed at biodiversity conservation. In the Mediterranean basin, climatic dynamics are even more challenging when it comes to regeneration from seeds under a warming climate. Seed germination will be likely affected by warmer winters as it is limited by “typical” Mediterranean cold signals and the release of dormancy is reduced in those species that usually germinate in spring. In addition, hotter and drier summers will compromise the survival of plants by affecting their seedling establishment phase. Taking into account these predicted scenarios, we will identify native species from the Mediterranean region that are pre-adapted to these expected climatic changes and can resist extreme environmental conditions during their regeneration stage. We will use the species from the EUNIS Mediterranean habitats to compile a database that will include both regenerative primary data and vegetative traits, as well as information on their potential interest related to their contribution to the associated ecosystem services for private companies and public bodies involved in ecological restoration (e.g. species with horticultural, gardening or pollinator interest). We will then carry out a meta-analysis of regenerative primary data that will allow us to identify species that resist extreme environmental conditions. In conclusion, the gathered data will provide information based on regenerative traits that can be used in effective seed-based conservation strategies in the Mediterranean basin.

6.3 Greening Iberian olive groves with native seeds

Jiménez-Alfaro, B.1, Gálvez, C.2

1University of Oviedo, Spain; 2Semillas Silvestres S.L., Spain

Agricultural intensification of Mediterranean woody crops (vineyards, olive groves and fruit trees) has dramatically changed traditional landscapes that were relatively sustainable before the twentieth century. Current agroecological practices suggest the need to move towards the establishment of herbaceous ground covers. However, we do not know which species are suitable for restoration, and how seeds of these species can be produced on large scales. Here, we present an overview of major challenges for greening olive groves in southern Iberia and recent initiatives that combine applied research with seed production in Andalusia, Spain. We developed a comprehensive framework for selecting regionally adapted species for restoration that also emphasizes considerations for seed production. A major finding was that research on ground covers for regenerative agriculture has largely overlooked native species at the expense of commercial and ill-suited varieties. Our assessment of native annuals showed that 85% of the evaluated grasses and forbs exhibit a suite of ecological and production traits that can be tailored to meet the requirements of farmers, seed producers and environmental agencies. Moreover, seed experiments conducted with natural populations of suitable species allowed us to describe the seed ecological niche of preferential grasses and forbs to predict the natural regeneration of established populations. The contribution of scientific data and the trends in agroecological practices have strengthened the production of native seeds by specialized producers and the early growth of a seed market for greening Spanish olive groves. Together with other initiatives and trials conducted on experimental olive groves, it seems we are facing a change of paradigm in the sustainability of these agroecosystems. Nevertheless, we are still far from a healthy seed market coupled with national-based policies on native seed production.

6.4 Selecting native seeds for the revegetation of coastal dunes altered by Carpobrotus species

Alonso-Simón, A.1; Magrini, S.1; Canini, F.1; Attorre, F.2; Fabrini, G.2; Zucconi, L.1

1Tuscia University, Italy; 2Sapienza University, Italy

The RIVALSA project aims for an integrated plant and fungal approach to the revegetation of coastal dunes altered by the invasive alien species Carpobrotus edulis and C. acinaciformis, introduced in Mediterranean Europe to stabilize sandy areas and for ornamental purposes. In addition to their ability to grow and spread, their litter has an allelopathic effect, reducing germination and establishment rates of native species. Among the objectives of the project, here we focus on the evaluation of seed germination in the presence of allelopathic substances, to select a pool of wild plant species whose seeds can tolerate the presence of such substances in the soil. Seeds of 9 species were collected in invaded or non-invaded dunes of Latium (central Tyrrhenian Italy). To evaluate the allelopathic effect, three different leaf extracts were prepared for both Carpobrotus species using water, 70% ethanol, and acetone as solvents. Germination tests were performed using the optimal protocol for each species, with increasing concentrations of the extracts (0, 0.3, 0.66, 1 and 2 mg/ml). Preliminary results showed different sensitivity of seeds from various species to the Carpobrotus extracts. For example, Anthemis maritima showed a higher germination rate in the control (64%) than in the presence of the water extract of C. acinaciformis (37% at the highest concentration). Besides, a comparison of populations of species growing in non-invaded or invaded areas, such as Centaurea sphaerocephala, revealed that the latter plants were less or not sensitive to allelopathic extracts (96% vs 89% for the control and the highest concentration, respectively), demonstrating how plants adapt to chemicals from the invader. Thus, it may be possible to identify seeds from native species not inhibited by Carpobrotus extracts and, together with a selected fungal consortium, use them for the recovery of the native dune vegetation after the eradication of Carpobrotus plants.

6.5 Germination protocol for herbarium-stored seeds to recover extinct plants

Albani Rocchetti, G.1, Brancaleoni, L.2, Caneva, G.1, Davis, C.3, D’Agostino, M.1, Fabrini, G.4, Godefroid, S.5,6,7, Iberite, M.4, Mayer, A.1, Mondoni, A.8, Abeli, T.1

1University Roma Tre, Italy; 2University of Ferrara, Italy; 3Harvard University, USA; 4La Sapienza University, Italy; 5Meise Botanic Garden, Belgium; 6Wallonia-Brussels Federation, Belgium; 7Brussels Free University, Belgium; 8University of Pavia, Italy

Herbarium specimens have been proposed to be valuable sources of viable diaspores (pollens, spores and seeds), which can provide native germplasm for recovering extinct plant species and their genetic variation lost. Diaspores taken from herbarium specimens or similar collections can remain viable for decades, and therefore may have potential for recovering extinct species (de-extinction s.l.). Although the lack of documented experiments of plant de-extinction using herbarium material, some attempts with locally extirpated species demonstrate the feasibility of this approach, which is supported by a general consensus of conservationists and curators. Indeed, a protocol for herbarium specimens’ seeds germination was not developed so far. Already existent protocols and guidelines, like germination standard protocols developed by the MSB (Millennium Seed Bank Partnership and Royal Botanical Garden, Kew) can be applied. Our research is dedicated to find germination protocols for trying plant de-extinction from seeds preserved in herbarium specimens. To this aim, we chose 26 species congeneric to worldwide extinct species, collecting more than 20,000 seeds from circa 400 herbarium specimens held in 8 Institutions (BR, RO, FI, PAV, FER, MSNM, MSPC, and Roma Tre University). Criteria for seed collection were specimens’ age, seed ripening, number of specimens, abundance of seeds per specimens, and ad hoc curators’ guidelines. In addition, we tested freshly harvested seeds to countercheck. Several germination protocols are tested, including different pre-treatments, sterilization techniques, priming and germination conditions (e.g. temperature, light cycle, substrate). To date, we obtained more than 200 germinations of seeds taken from herbaria, from the 19th century until 2016, and we expect more germinations from the ongoing tests. This project represents the first documented and systematic development of germination protocols for old seeds from herbarium collections for conservation purpose, which lays the basis for the next attempt to the first herbarium-based seed plant de-extinction.

6.6 Fit for the future: alpine plant responses to climatic stress after 20 years of seed bank conservation

White, F.J.1, Porro, F.1, Rosbakh, S.2, Mondoni, A.1

1University of Pavia, Italy; 2University of Regensburg, Germany

Climate warming is occurring in alpine environments faster than the global average, making cold-adapted alpine specialists particularly vulnerable to population decline and extinction. Ex situ seed conservation is of growing importance but little is known about how plants from genebank stored seeds will respond to climatic changes. We grew plants from seeds of the artic-alpine specialist Viscaria alpina, collected from the same location at different time points and held under seed bank conditions over the last 20 years. The site has experienced a warming trend of 0.3 °C per decade, with large interannual variation in temperature and precipitation. For six accessions (three old and three recent) we exposed juvenile plants to the stresses of heat and/or drought for one month. Plant biomass and leaf traits (leaf area, specific leaf area, leaf dry matter content) were measured as proxies of fitness under the different treatments. Interestingly, the temporal origin of the seeds did not have a significant effect on plant traits. However, we found differences in the stress response that were linked to inter-annual variation of the climate at the collection site. Specifically, plants from seeds produced in wet/cold years had significantly lower biomass and smaller leaf area when exposed to heat/dry and heat/wet stresses than those from seeds produced in drier/warmer years. As a result, these latter plants showed increased fitness when exposed to similar stress experienced by the mother plant. Our results demonstrate that 20 years of storage produces viable plants of V. alpina, but it may be important to collect seeds frequently as those produced in different years respond differently to stress. Using temporally separated accessions to study climate change and stress response, we highlight a lack of contemporary evolution but observe environmentally induced plastic responses that may have important implications for V. alpina survival under climate change.

6.7 Frost resistance of pedunculate oak seeds and the implications for their storage

Chmielarz, P.1; Suszka, J.1; Wawrzyniak, M.K.1

1Institute of Dendrology, Polish Academy of Sciences, Poland

Effective seed storage is extremely problematic for short-lived pedunculate oak (Quercus robur L.) seeds, categorized as recalcitrant. We examined the impact of seeds cold acclimation to -7 °C as well as decreasing of seeds moisture content on their frost resistance and further seedlings growth. Simultaneously, we investigated the effect of acorns thermotherapy (the pathogen elimination procedure, 40°C/3h) and storage temperature of acorn in terms of the production of reactive oxygen species (ROS) in their embryonic axes. Results show that fresh pedunculate oak seed can survive freezing temperature down to -10°C and produce good quality seedlings. Temperature around -11° to -13°C is near-lethal to seeds and significantly reduces their viability. Overall, desiccation does not increase their frost resistance, thus acorns should be stored in a highly hydrated state. Thermotherapy did not increase the level of three types of ROS (H2O2; O2-• and •OH). The temporal heat stress of thermotherapy (40°C/3h) resulted in slightly reduced levels of H2O2, indicating activation of the antioxidant systems in acorn preparation for storage. The effect of constant storage temperatures (-3, -5, -7 °C) and their combinations (cold acclimation: -3 → -5 °C or -3 → -5 →-7 °C) on ROS levels and seed viability showed that the highest ROS levels were detected in acorns stored at -7 °C, whereas three-step cold acclimation was beneficial for reducing ROS levels. Thermotherapy and cold acclimation of acorns can improve their viability after storage by decreasing ROS levels.

6.8 Inter-population variability in seed germination, reproductive ecology and genetic structure of a rare chasmophyte (Campanula raineri, Campanulaceae): insights for conservation purposes

Villa, S.1, Magoga, G.1, Iannella, M.2, Di Musciano, M.2, Montagna, M.3, Pierce, S.1

1University of Milan, Italy; 2University of L’Aquila, Italy; 3University of Napoli, Italy

Campanula raineri Perp. is a stenoendemic chasmophyte of the Italian Prealps. A multidisciplinary approach combining seed germination tests and other reproductive ecology traits (e.g. pollen quality, the trade-off between sexual and vegetative reproduction) across populations, population genomics and past-to-future Species Distribution Models (SDMs) will highlight potential threats for this species and support conservation strategies involving plant material produced from seed. Scattered populations at different elevations across the entire distributional range of C. raineri provide data and material (e.g. seeds for in vitro germination tests and pollen for quality assessment) for the analyses. Population structure is currently being defined using a 2b-RAD genotyping approach followed by bioinformatics analyses of SNPs. SDMs are being elaborated to combine presence/absence data with ecological information (bioclimatic variables, elevation and substrate). Seed germination rate and pollen viability exhibited strong positive relationships with elevation, while flower production decreased slightly. Initial results regarding population structure suggest extensive outbreeding across populations and scarce nucleotide diversity within populations. This genetic uniformity hinders the species ability to cope environmental changes, making it particularly threated. Preliminary models combining only species occurrence localities and bioclimatic variables correctly predict the current distribution area of C. raineri, and are being integrated with elevation and substrate information and extended in time to describe past and future potential distributions. Preliminary genomics results suggest the need of conservation actions for C. raineri. Projections of future species distribution will identify populations at most risk of climate change, and suitable areas for survival and colonization. In vitro production of plants can easily provide material for population reinforcements or reintroductions, even transferring material between populations -suggested by the high gene flow naturally occurring between populations-, taking into account that high-altitude populations produce less but higher-quality flowers.

6.9 Seed ecology of candidate species for restoration of humid forest in New Caledonia: the case of Koniambo Massif

Fogliani, B.1, Martini, A.2, Brescia, F.2, Ititiaty, Y.2

1University of New Caledonia, New Caledonia; 2ARBOREAL, New Caledonian Agronomic Institute, New Caledonia

New Caledonia has the important mission to reconcile its exceptional biodiversity with the rapid development of human activities. One of the most important threats is habitat fragmentation especially due to mining exploration on ultramafic soils. Over the past 40 years, revegetation has been developed to reduce such impacts but ecological restoration concept capability and concepts have only been developed in the past 15 years. It involves knowledge of ecological succession and the study of seed ecology for restoration. According to the obligation of KNS mining company, as part of compensation, we investigated on how to accelerate the evolution of para- (MPF) and pre-forest (PF) towards a dense humid forest (F). We first identified forest zones to be studied and reinforced on their edge on the Koniambo massif. We then realized species inventories in the forest and in surrounding vegetation to evaluate species composition. This step permitted to reinforce our database dealing with 41 life-traits such as the reproductive type, the adult height, the type of fruit, the size and weight of seeds, the dispersion type, the germination and dormancy types. It also led to a list of candidate species that can participate to the acceleration due to their presence in all type of vegetation from MPF to F, but also their capacity to close the vegetation and to attract dispersers. From this list, seeds were harvested and studied regarding their structure, germination, dormancy breaking, and conservation capacities in relation with their environment. This also allowed producing plantlets we used for a plantation of 1 hectare both in MPF and PF. An evaluation of success according to the planted vegetation and species was made. An analysis of all these data will be presented and will led to recommendations to provide to managers for implementing ecological restoration projects in forest environment.

6.10 Dormancy release and germination of selected forest species under controlled conditions

Wawrzyniak, M.K.1, Suszka, J.1, Chmielarz, P.1

1Institute of Dendrology, Polish Academy of Sciences, Poland

To maintain the ambitious plan for wood production and forest conservation, adopted under the 2015 Paris Agreement, effective seedling production is crucial. However, the most economically important forest species in Europe produce dormant seeds, which can affect the final seedlings outcome. Species like European hornbeam (Carpinus betulus L.), mountain ash (Sorbus aucuparia L.), Norway maple (Acer platanoides L.), or common yew (Taxus baccata L.), can differ in their dormancy level and easily induce secondary dormancy. In the case of European hornbeam, viable but non-germinated seeds can consist of ca. 20% of sowed seeds. As a result, not all viable seeds can germinate and eventually converse to seedling. This can be even more important in the case of valuable seeds stored in genebanks used for restoration purposes. The common practice of Autumn sowing in nurseries can result in erratic and unpredictable germination due to the risk of predation and weather conditions during winter and early spring. The stratification and germination under controlled conditions allows for minimizing losses and sowing the seeds to the soil at the optimal time. To delay germination already stratified seed, it can be stored below 0°C for a short time which is commonly used in beech (Fagus sylvatica L.), common ash (Fraxinus excelsior L.), yew and many other species. In this way, it is possible to sow seeds that are fully prepared in advance in the nursery, to avoid their premature germination and minimize the risk of secondary dormancy and unify germination time. In a laboratory, good practice for seed germination capacity test in many forest tree species test is applying temperature cycles of 3°/20°C at 16/8 h intervals, which limits the risk of induction of secondary dormancy and/or allows dormancy release in those seeds in batch, with have its deeper level. Better understanding of environmental control of dormancy release and germination in many forest species allow to produce higher amounts of quality seedling vital in any reforestation programs.

6.11 Understanding germination processes can enhance restoration: a role for the woody endocarp of Morella parvifolia (Benth.) Parra-Os from the south Andes of Ecuador

Crespo, A.1, Inga, D.1, Pérez, H.E.2

1University of Azuay, Ecuador; 2University of Florida, USA

The Andes of Ecuador are a highly diverse yet threatened biodiversity hotspot. The urgency to recover native biodiversity here is growing, as is the need for knowledge to better inform conservation efforts. We present novel information on temporal patterns of imbibition and germination for endocarps and seeds of Morella parviflora (Myricaceae), a native Tropical Andean shrub ideal for initial stages of restoration. Endocarps and seeds extracted from fresh fruits of M. parvifolia did not express physical or physiological dormancy. Imbibition was completed within 12 hours. However, imbibition rate was about 1.2-fold greater in seeds compared to endocarps. Germination occurred readily at ambient temperatures after 7 (seeds) and 14 (endocarps) days. Interestingly, although seeds tended to germinate more rapidly, endocarps displayed 2.0-fold higher final germination compared to seeds. Seeds displayed high levels of fungal contamination by day 30. Alternatively, contamination did not exceed 1% for endocarps throughout the 60-day germination trial. Fruit storage for 4 or 8 months had a significant effect on imbibition and germination. Imbibition was relatively unchanged in endocarps, but germination lag increased. In seeds, imbibition was reduced while germination lag increased after 8 months of storage. Germination did not exceed 26% for any type of unit after 4 to 8 months of storage. Fresh endocarps contained about 11 mg of residual wax. However, residual wax increased 2.6-fold by 8 months of storage. This may contribute to an osmo-regulatory mechanism in drupes and when combined with protection against pathogen attack may support a functional role for the endocarp. We suggest that large-scale propagation of M. parvifolia is feasible when using mature, fresh drupes processed within 72 hours of collection. Processing should include the complete removal of the waxy flesh to enhance imbibition. Extracting seeds from endocarps, however, is not recommended for large scale propagation.

6.12 Seed treatments improve plant recruitment in dryland environment

Kildisheva, O.A.1, Merritt, D.J.2,3, Best, C.4, Bakker, J.D.5, Erickson, T.E.2,3

1The Nature Conservancy, USA; 2Kings Park Science, Australia; 3University of Western Australia, Australia; 4Australian Grains Genebank, Australia; 5University of Washington, USA

The restoration of terrestrial ecosystems is a recognized global priority. While large-scale restoration efforts rely on direct seeding, plant recruitment from seed is most severely limited during the germination and emergence phases of plant development. Seed treatments may promote plant establishment in degraded landscapes, particularly in drylands where severe disturbance and moisture limitations critically curtail natural recovery. The goal of this study was to determine whether seed treatments would increase seedling recruitment of a keystone Australian endemic species (Triodia pungens) in a harsh post-mining restoration scenario. We evaluated seeds treatments (removal of floral appendages, priming in a karrikinolide [KAR1] solution, and seed coating) in their effectiveness to relieve physiological seed dormancy and to promote plant recruitment. We also examined the germination, emergence, and morphological traits of seedlings exposed to a range of moisture conditions typical of the Pilbara Bioregion of northwestern Australia. Seed dormancy and low soil moisture limited seedling recruitment. Combining floret removal with KAR1 priming increased germination up to 67% compared to untreated seeds (7%) or untreated seeds retained in florets (<1%). Seed coating reduced emergence slightly, but priming seeds prior to coating diminished the negative effects of coating and in some cases improved seedling growth in certain moisture conditions. Our study indicates that removing key germination restrictions through seed treatment (floret removal and KAR1 priming) prior to sowing can enable higher recruitment in moisture-limited conditions and may contribute to greater recruitment of difficult to establish native plant species in highly degraded dryland environments.

6.13 Seed biopriming and pelleting with indigenous microbes for restoring native plants in Australian ecosystems

Muñoz-Rojas, M.1,2, Machado de Lima, N.2; Dadzie, F.2; Ooi, M.K.J.2; Merritt, D.J.3,4; Erickson, T. E.3,4

1University of Seville, Spain; 2University of New South Wales, Australia; 3Kings Park Science, Australia; 4University of Western Australia, Australia

Terrestrial ecosystems worldwide are being seriously affected by degradation processes. Developing cost-effective large-scale solutions to restore these landscapes is crucial to preserve biodiversity and achieve ecosystem functionality and sustainability. In seed-based revegetation, enhancing seed germination and seedling establishment is essential to ensure restoration success. Indigenous soil bacteria, including cyanobacteria from soil biocrusts, have shown promise as bio-fertilizers given their ability to promote seedling growth of native plants. However, these bio-inoculants have not yet been fully exploited in seed-based ecological restoration. In this presentation, we will showcase our most recent research on (i) the effects of bio-priming seeds with indigenous bacteria and cyanobacteria on plant growth of native plants used in ecosystem restoration and (ii) the development of novel methodologies for targeted delivery of functional bacteria and cyanobacteria (i.e., via seed bio-encapsulation in extruded pellets). Our results have shown that both bacteria and cyanobacteria promote germination and seedling growth of native Australian plants, including hummock grasses, Acacia trees and shrubs. We also found that microbial inoculation can increase the levels of soil carbon and microbial diversity and promote seedling growth of native plants under abiotic stress conditions, i.e., salinity and drought. These microbial-based approaches are now ready to be applied in large-scale ecosystem restoration programs.

6.14 Enhancing direct seeding with unmanned aerial vehicles, operational capacity, and seed technology in the western US and beyond

Aghai, M.M.1

1DroneSeed Co., USA

DroneSeed is a Seattle-based startup developing software, hardware, and infrastructure for land surveying, and aerial seed deployment in forests and rangelands. Wildfire and other large-scale ecosystem disturbances are increasing in frequency and severity. Constraints to post-disturbance revegetation include accessibility to remote areas, difficulty with precise distribution of limited seed supply at scale, biotic and abiotic conditions (e.g. invasive species, seed predation, and moisture availability), and associated costs (e.g. labor). DroneSeed provides post-disturbance recovery programs that include surveying and distribution of native seed that has been manufactured into a vessel to deliver substrates and amendments to protect seed and promote germination. DroneSeed is trialing this technology for public, private, and philanthropic land management organizations throughout the US, with hundreds of hectares of trials underway in New Zealand and Canada as well. The presentation will provide an overview of the company’s technology and innovations to revegetation processes, introduce select projects, and highlight the research and development supporting our data-driven approach. We will focus on efforts from 2018 to 2022 to collect greenhouse and field data on the efficacy of seed enablement technology in direct seeding post-fire and post-harvest landscapes across several biomes and ecotypes. We will present the seedling establishment results of project sites with conifers, grasses and forbs, as well as tropical hardwoods. We will share next steps and how this seeding technology can play a role in restoration and address challenges we still face while building towards scalable solutions for ecosystem restoration.

6.15 The project Life SEEDFORCE, an integrated approach for plant translocation

Fois, M.1, Bacchetta, G.1, Buhagiar, J.2, Casolo, V.3, Ceriani, R.4, Cristaudo, A.5, Cuena-Lombraña, A.1, Dessì, L.1, Di Martino, L.6, Dixon, L.7, Fabrini, G.8, Magrini, S.9, Mariotti, M.10, Meloni, F.1, Podda, L.1, Porceddu, M.1, Ravnjak B.11, Salmeri, C.12, Sarigu, M.1, Villani, M.13, Bonomi, C.14

1University of Cagliari, Italy; 2University of Malta, Malta; 3University of Udine, Italy; 4Monte Barro Natural Park, Italy; 5University of Catania, Italy; 6Maiella National Park, Italy; 7Mediterranean National Botanical Conservatory of Porquerolles, France; 8La Sapienza University, Italy; 9University of Tuscia, Italy; 10University of Genoa, Italy; 11University of Ljubljana, Slovenia; 12University of Palermo, Italy; 13University of Padua, Italy; 14Trento Science Museum, Italy

Developing successful plant conservation projects is a challenging task and requires an inclusive and multi-disciplinary approach, putting together diverse expertise to address all critical issues for a successful outcome. An integrated in-situ and ex-situ approach can yield numerous benefits and offer opportunities to showcase to the public the actual plants that need protection, building participation and support from the local community. The Life+ SEEDFORCE project aims at improving the conservation status of 29 plant species from Italy, Malta, Slovenia and France, reported in bad conditions according to art. 17 of the 92/43/EEC Habitats Directive. The project includes best practice preparatory actions to assess the genetic make-up of the target species and populations, analyse the current species climatic envelope and project possible future scenarios, taking into account trophic decencies in target species. The outcome of such preparatory actions will be used to prepare the propagation mix best adapted for each site to be use for reintroduction and population reinforcement. Threats to each species will be first identified and removed. The genetic make-up and the ecological niche will support the identification of the appropriate donor populations and the development of joint protocols for species propagation and population reinforcements or re-establishments in extinct sites. A series of engagement activities have been devised to gain public support and to involve farmers that in most cases can influence the long-term conservation of many plant species in secondary habitats. The transnational partnership, covering all the natural distribution range of the selected species will allow a fruitful exchange of experience to better investigate the plant biology, aiming at understanding differences in species conservation status and management among neighboring countries.

6.16 The impact of ozone treatment on seed germination and pathogen infections in wheat, barley, triticale and rapeseeds

Hlásná Čepková, P.1, Palicová, J.1, Kubinová, Š.2, Dejneka, A.2, Čurn, V.3, Papoušková, L.1

1Crop Research Institute, Czech Republic; 2Czech Academy of Science, Czech Republic; 3University of South Bohemia, Czech Republic

Seeds of agriculture crops can be contaminated by fungal species that affect the germination rate of seeds and the final yield of crops. Seed treatment plays an important role in sustainable agriculture and therefore alternatives to chemical seed treatment are being sought. Physical seed treatment of seeds could be one of environmentally friendly technology. The objective of this study was to assess the efficacy of O3 treatment on seeds germination (germination rate, root and germ length) and fungi infection in four agriculture crops – spring wheat (cultivar Registana), winter barley (cv. KWS Meridian), winter triticale (cv. Tulus) and spring rapeseed (cv. Ovace). The seeds were divided into control groups (no O3 gas) and treated groups (3 ppm), which were exposed for 5, 10 and 60 min (maximum length treatment). The ozone-treated samples of both variants (5 and 10 min) were vigorous, and very healthy. However, the initial development of the ozone treated seeds was slower compared to the control. After seven days of germination, the ozone treated samples showed values comparable to or higher than the control, the response was quite individual according to the crop species tested. Interestingly, the application of a 60 min ozone dose did not affect germination parameters. The control seeds (no O3 gas) and ozone treated seeds (5 and 10 min) of four crops were cultivated on potato dextrose agar to monitor microscopic fungi. Statistical analysis proved important differences between tested crops and between the ozone exposure times on the seeds. The highest number of CFU (colony forming units) of microscopic fungi was detected in non-treated samples (no O3 gas) of all crops. The highest effect of ozone treatment was observed in barley and wheat samples treated for 10 min. The most frequent genera of cultivated fungi were Penicillium, Aspergillus and Alternaria.

6.17 Seed lipid thermal fingerprints of Mediterranean terrestrial orchids in relation to their ecology and ex situ conservation

Magrini, S.1, Pritchard, H.W.2,3, Ballesteros, D.2,4

1Tuscia University, Italy; 2Royal Botanic Gardens, Kew, UK; 3Kunming Institute of Botany, China; 4University of Valencia, Spain

Ex-situ seed storage offers a potential solution to mitigate the extinction risk of threatened plants. However, dry seed longevity under conventional seedbank conditions (-20°C) is known to be extremely variable, with some families tending to be relatively short-lived, like Orchidaceae. Seed lipids have been thought to be a determinant of seed ageing with lipid composition impacting differing susceptibility to oxidation and variation in thermal behavior: e.g. unsaturated fatty acids are more prone to oxidation during ageing at high temperatures but they seem to provide larger thermal stability (than saturated fatty acids) during -20°C storage. Understanding the balance of lipid composition and thermal characteristics of their melting and crystallization behavior could prove a useful tool for identifying longer-term cold storage problems in lipid-rich seeds, helping to inform storage practices. Terrestrial orchid seeds are orthodox in storage behavior and have lipid as the main storage compound. Only very few data are available on the thermal properties of orchid seeds. We have used differential scanning calorimetry to explore potential links between poor storage performance, lipid composition and lipid thermal fingerprints of seeds of 15 Mediterranean terrestrial orchids. We have determined the temperature spread and complexity of the lipid melt, the peak temperature and enthalpy of the main peak, and some aspects of the short-term crystallization kinetics. We aimed to test the hypothesis that Mediterranean orchid seeds, as typical for temperate environments, have polyunsaturated fatty acids with melting temperatures around -30°C as the main storage lipids. We wanted to test whether species presenting short lifespans present lipid compositions that tend to show multiple melting events at -20°C and high temperature melting ends, as typical in the saturated fatty acids present in many tropical seeds. Data are interpreted in relation to the ecology of the species and the risk of lipid crystallization during cold storage.

6.18 Ex situ conservation guidelines for Australia’s national plant treasures

Martyn Yenson, A.J.1,2, Guja, L.K.3,4, Commander, L.E.1,5 on behalf of the Germplasm Guidelines steering committee

1Australian Network for Plant Conservation, Australia; 2Australian Botanic Garden, Australia; 3National Seed Bank, Australian National Botanic Gardens, Australia; 4Center for Australian National Biodiversity Research, Australia; 5University of Western Australia, Australia

The Australian Network for Plant Conservation recently released the third edition of Plant Germplasm Conservation in Australia: strategies and guidelines for developing, managing and utilizing ex situ collections. Known as the Australian Germplasm Guidelines, this publication is technical and practical, setting a benchmark for ex situ conservation of wild species into the future. The Germplasm Guidelines distils research on seed biology and ecology into practical actions for effective seed banking at various scales, with a workflow to address each step of acquiring, maintaining and utilizing genetically representative collections. An understanding of seed ecology informs sections of the Guidelines dealing with seed germination, seed storage behaviour and seed longevity in storage. The need to capture and utilize ‘special’ types of germplasm (material from ferns, mosses, liverworts), or taxa with ‘special’ life history stages or growing requirements (terrestrial orchids with mycorrhizal associations, carnivorous and parasitic plants) requires an understanding of species ecology. Ex situ collections such as those maintained by the Australian Seed Bank Partnership also enable ecological research to be conducted on difficult-to-collect, rare or threatened species. The Germplasm Guidelines complements the ‘Guidelines for the Translocation of Threatened Plants in Australia 3rd edition’ and the ‘Florabank Guidelines for native seed collection and use 2nd edition’, published in 2018 and 2021 respectively. This trio of publications provides guidance on how to conserve and restore the biodiverse flora of Australia in the face of multiple threats. The approaches, techniques and technologies included in the Guidelines are broadly applicable to plants across the globe. These Guidelines were updated with funding from The Ian Potter Foundation, in collaboration with the Australian Seed Bank Partnership and the assistance of the restoration and agriculture sectors, botanic gardens, CSIRO and universities. Many contributors play a role in ecological restoration, seed conservation, ecological research, or related fields.

6.19 Best-practice guidelines for threatened plant translocation, native seed use, and plant germplasm conservation

Commander, L.E.1,2, Martyn Yenson, A.J.1,3, Guja, L.K.4,5, Fernance, C.1

1Australian Network for Plant Conservation, Australia; 2University of Western Australia, Australia; 3Australian Botanic Garden, Australia; 4National Seed Bank, Australian National Botanic Gardens, Australia; 5Center for Australian National Biodiversity Research, Australia

Over the past few years, the Australian Network for Plant Conservation (ANPC) has led the revision and publication of the following three best-practice guidelines: (1) Guidelines for the Translocation of Threatened Plants in Australia; (2) Florabank Guidelines for native seed collection and use; (3) Plant Germplasm Conservation in Australia for ex situ conservation. These guidelines can be used separately or together to inform plant conservation and restoration in Australia and across the world. All guidelines support action towards restoring functional, self-sustaining plant populations by providing guidance on different aspects of this process. Each one brings together current literature and best practice to create an accessible, evidence-based handbook. They also link readers into international guidelines, with relatable Australian examples. The Germplasm Guidelines and Translocation Guidelines feature case studies that capture collaborative efforts and showcase innovative methods. All three guidelines are available for free download from the ANPC website https://www.anpc.asn.au/product-category/publications/.

6.20 Ex situ conservation experiences for seven threatened taxa: the case of the Life FLORANET project for the conservation of plant biodiversity in the Apennines (Italy)

Di Martino, L.1, Conti, F.2, Bartolucci, F.2, Di Musciano, M.3, Frattaroli, A.R.3, Di Santo, M.1, Di Cecco, V.1

1Maiella National Park, Italy; 2University of Camerino, Italy; 3University of L’Aquila, Italy

The Life Floranet project has just ended, with the main objective of protecting and improving the conservation status of seven plant species of EU importance (Annexes II-IV to Directive 92/43/EEC) within the Natura 2000 areas of the Central Apennines (Italy): Maiella National Park, National Park of Abruzzo, Lazio and Molise and Sirente-Velino Regional Park. The selected species investigated in this project are: Cypripedium calceolus L., Adonis distorta Ten., Androsace mathildae Levier, Iris marsica I. Ricci & Colas., Astragalus aquilanus Anzal., Klasea lycopifolia (Vill.) Á. Löve & D. Löve, and Jacobaea vulgaris subsp. gotlandica (Neuman) B. Nord. In situ and ex situ conservation strategies have been adopted, as well as reducing the tourist impact, awareness campaigns among stakeholders. These strategies are essential to carry out concrete actions for effective species conservation. Specifically, for each species, we have performed: seed collection, in vitro propagation, seed reproduction, long-term conservation, in situ repopulation and creation of new populations. The project involved collecting seeds using ENSCONET protocols and thousands of seeds were obtained which were processed at the Maiella germplasm bank in the Plant Biodiversity Conservation Center. Part of the seeds were stored at -20 ° C (with duplicates at KEW’s Millennium Seed Bank) and part was used to carry out germination tests. From these tests, four germination protocols were developed with very high percentages, and seedlings from all target species were obtained. These seedlings through the nurseries of the Maiella National Park were made to grow and were used for some restocking interventions in nature. In this context, the Floranet project plays a crucial role in implementing conservation strategies and monitoring endemic or rare species, reducing their extinction risk. These actions strengthen the conservation role of the ex situ structures through an integrated conservation strategy combining in situ and ex situ activities.

6.21 Success of native hay-seed mix for dry grassland restoration: do not neglect the role of soil seed bank

Vuerich, M.1, Fabro, M.2, Petrussa, E.1, Braidot, E.1, Boscutti, F.1

1University of Udine, Italy; 2ERSA, Italy

Ecological restoration has been proved to be an effective strategy to contrast biodiversity loss due to land use change. Among terrestrial ecosystems, dry grasslands represent biodiversity hotspot strongly threatened by soil consumption. Passive grassland restoration, based on spontaneous succession, is highly unpredictable and, in some cases, unsuitable to re-establish native communities. The use of spontaneous seeds (hay seed) and or/ commercial seed mix might contribute to boost early restoration stages by triggering native plant colonization. A crucial stage for the success of any restoration action and for determining community composition is the seeds germination. In addition, the soil seedbank of restoration sites can exert main effect on the final plant assembly, even if it is often neglected. We designed a controlled environment experiment in which commercial grassland seed-mix and spontaneous hay seeds collected in local dry grassland were sown on commercial soil (seedbank less) and agricultural soil (with seedbank) with two seed density. We hypothesized soil, seed mix type and density to interplay in determining community diversity and composition. Commercial seed provided greater species abundance and rapid containment of seedbank species. The abundance of the seedbank ruderal species greatly contributed to the total biodiversity in the case of hay seeds. The number of individuals of species not belonging to the seedbank has always been higher if commercial seed mix was sown; in the case of hay seeds this parameter was favored by agricultural soil probably due to the substrate or to positive interaction with seedbank species. The composition of functional groups (i.e. monocot vs dicot and life span) and species assembly were also influenced by the soil and seed mix type. Soil seedbank effect in determining the success of site restoration when operating seed sowing should not be overlooked, alternative management actions aimed at curb seedbank germination should be, hence, considered.

6.22 Ecology and physiology of coastal dune plant seeds as a model for their conservation

Hernández-Mendoza, V.1, Álvarez-Espino, R.1, Mendoza-González, G.2

1Yucatán Scientific Research Center, Mexico; 2Sisal-Yucatán Campus of the National Autonomous University of Mexico, Mexico

Coastal dune vegetation is adapted to tolerate extreme environments of high salinity, high temperature, low precipitation, low nutrients, and burial on accreting beaches. This vegetation provides important ecosystem services, such as erosion prevention and control, protection against hurricanes, biodiversity refuge, and provision of forest resources. Nevertheless, most coastal dune ecosystems are endangered due to habitat degradation resulting from the exploitation of natural resources for housing, tourism, agriculture and industry, and other factors such as invasive species, sea level rise, and climate change. In Mexico, these ecosystems are undervalued and among the less studied, estimating more than 50% of their 800,000 hectares have been lost. Studies on the use of native pioneer species have shown these represent a potential for successful dune stabilization, but more studies on dune management in restoration projects are required. Studies of physiology, ecology, and conservation of native seeds are needed in restoration projects to recover the health of these degraded ecosystems. To be successful, restoration programs must consider the establishment of different stages in their protocols, before and during revegetation, choosing optimal collection sites, properly collecting seeds, describing dormancy and germination cues to improve the production of native plant materials, and storing viable seeds in adequate conditions for each species, functioning as a gene bank. Gene banks are an ex situ conservation option for storing dried seeds from wild populations at low temperatures. However, not all seeds can be stored in traditional seed bank conditions, so it is necessary to study each species physiology to propose better conservation options. The present work evaluates the ecological and physiological behavior of coastal dune seeds in order to find the adequate conditions of ex situ conservation by analyzing the germinative responses, dormancy break and desiccation tolerance of the main pioneer coastal dune species in two Southeast Mexican beaches.

6.23 Seed priming to optimize germination in Arthrocnemum Moq.: a prime halophyte candidate for agricultural and biotechnological uses

Ramírez, E.1, Hernández-Apaolaza, L.1, Sánchez-Gavilán, I.1 and de la Fuente, V.1

1Autonomous University of Madrid, Spain

Seed germination and seedling growth constitute the first stage of a plant’s life cycle for crop establishment. Arthrocnemum Moq. is a halophyte of the subfamily Salicornioideae (Amaranthaceae), which could be recognized in the foreseeable future as an emerging candidate in applied biosaline agricultural programs, mainly due to the large biomass it represents in coastal and inland saltmarshes, in addition to its interesting nutritional and pharmacological properties. However, to ensure their subsequent use as a crop, it is necessary to optimize their germination through appropriate seed priming treatments. The main goal of this work was to seek the optimization of Arthrocnemum germination process using different pretreatments (exposure to high concentrations of salt in the dark and its subsequent transferred to distilled water separately and together with the combination of pH, salinity, and iron conditions). The experiments were tested on six samples of two different species: A. meridionale (from Tunisia) and A. macrostachyum (from Spain). Salinity priming of seeds for 15 days in darkness improved germination percentages by almost 25% at 600 mM NaCl, in both Tunisian and Spanish species. However, keeping seeds at different salt concentrations for 30 days produced higher improvement percentages at lower concentrations in A. meridionale (100-200 mM NaCl), while in A. macrostachyum the highest improvement percentages were obtained at 600 mM NaCl (percentage improvement of 47%). When the dark time period is reduced to 5 days at higher salt concentrations, the greater germination percentages were reached in all the samples at the concentration of 800 mM NaCl, increasing the improvement of germination between 17% and 50%. Finally, the conditions of pH = 7, pretreatment in darkness at 800 mM NaCl and 400 µM or iron, turned out to be an effective medium for seed germination.

6.24 FLEURS LOCALES, a project on the value chain of native seed production in south-western Europe

Galvez-Ramírez, C.1, López-Almeida, G.1, López-Camacho, A.1, Medrán-Viñas, A.1

1Semillas Silvestres S.L., Spain

Demand for native seeds with certified geographic and genetic traits is on the rise, with increasing necessities for ecological restoration, sustainable landscaping and promotion of useful biodiversity in agroecosystems. Each country is meeting this demand according to its own administrative constraints and social context, but every European state has seen the emergence of a nascent native seed industry. In southwestern Europe the industry faces technical limitations and lack of experience, making it necessary to adopt and share quality standard for an ethical native seed production, guaranteeing genetic origins and allowing for an organized development of the sector. The European project INTERREG-SUDOE FLEURS LOCALES is analyzing the value chain of native seed production in Southwestern Europe, with the goal of describing the current situation of the industry, its limitations and future possibilities. Project participants come from three states (France, Portugal, Spain) and different interested parties (producers, professional users, politicians, farmer unions, regional and state administrations). During the projects lifespan (2020-23) work is undergoing in several territories to experiment with common work practices, with different parties implementing local restoration actions on woody orchards, meadows or areas affected by wildfires.

6.25 Reducing seed losses in commercial seed production through understanding of the genetic mechanisms controlling seed shattering

Kourmpetli, S.1

1Cranfield University, UK

Seed loss due to unfavorable weather conditions close to the time of harvest or lack of uniform seed maturation poses a great challenge for the commercial seed production of several crops. Non-shattering phenotypes have been selected through domestication in crops such as rice and wheat, and the genetic mechanisms controlling this trait have been widely investigated. However, in less studied plants, such as parsnip, where no reference genome sequence is available and genetic resources are limited, understanding how seed shattering is controlled is crucial in order to develop strategies for the reduction of seed losses in a commercial seed production setting. We have developed a method for the assessment of seed shattering in parsnip and we screened a population of 33 genetically diverse parsnip lines for their ability to retain the majority of seed on the plant after commercial seed maturation. We have observed a great variation between these genotypes and identified lines with extreme phenotypes that will be used for further QTL mapping. In addition, we are using a reverse genetics approach to identify orthologues of known key regulators of seed shattering and study their function in parsnip. Our work will help to identify and develop markers of reduced seed shattering in parsnip that could be incorporated in breeding programs to reduce seed losses.