Societal Impact Statement Ex situ seed conservation is an essential tool in our efforts to conserve wild and cultivated plant diversity, as mentioned in the Global Strategy for Plant Conservation and the Sustainable Development Goals. While there are established standards which seed banks follow, it is important that we continue to evaluate protocols and optimize processes. Here, we encourage seed banks to re-evaluate their seed drying protocols. There is increasing evidence that two-stage drying, an initial active drying phase followed by equilibrium drying to the target moisture content for storage, increases the subsequent longevity of seeds which will result in improved conservation outcomes. Summary Billions of seeds are stored around the world in seed banks-either conservation seed banks preserving species diversity or genebanks conserving primarily intra-species agrobiodiversity. As well as providing long-term conservation, many of these seed banks offer samples of seeds for use in restoration programs, breeding and other research activities. Most seed banks follow international standards to manage collections, with specific standards concerning each step in the conservation cycle. One of the standards describes the conditions under which seeds should be dried before packing and storage: Seeds should be dried to equilibrium in a controlled environment of 5 degrees C-20 degrees C and 10%-25% relative humidity. These conditions theoretically mean that seeds will be close to an optimum moisture content for the maintenance of viability during storage. However, considerable gains in subsequent longevity can potentially be achieved, particularly for seeds that are harvested before maturation drying is completed in planta, if a two-stage drying process is adopted. The first, brief active drying phase should be at 30 degrees C-45 degrees C and 20%-70% RH and should be followed by slower final drying towards equilibrium in a cool, dry environment (15 degrees C +/- 5 degrees C with 15% +/- 5% RH). We encourage seed banks to consider this two-stage drying approach, even before the genebank standards are revised. Conservation research is important for improving current best practices to ensure the effective safeguarding of species and agricultural biodiversity.

Coastal dune plants provide critical ecosystem and cultural services, which are severely threatened by ongoing climate change and sea-level rise. Coastal habitats account for a disproportionate extent of land on islands, emphasizing the importance of these ecosystems for island biodiversity. We investigated salinity tolerance in a diverse pool of 19 native and invasive plant species from Hawaiʻi’s coastal dunes for insights into their vulnerability to sea-level rise. Salinity tolerance was investigated experimentally in seedling and juveniles by treating plants with artificial seawater for 3 weeks, followed by a 2-week freshwater recovery period. For mechanistic insights, stomatal conductance and leaf chlorophyll content were measured before, during, and after seawater treatment. Salinity tolerance was highly variable among species. The least tolerant species experienced 100

For many decades, seed germination data have been modelled by probit analysis. In particular, it is the basis of the seed viability equation used, in the first instance, to describe the decline in germination of seeds in storage, but then also the rate of the decline, depending on seed moisture content and the temperature of storage. The underlying assumption of a probit model is that the response follows a normal distribution, in this case, loss of the ability to germinate over time. Probit analysis also takes into account the binomial error associated with germination data. Many statistical packages have probit analysis as an option within the generalized linear modelling framework; here, we present code for applying probit analysis in the free software, R. Codes are provided for fitting a single survival curve, for a single seed lot stored in a constant storage environment; for fitting multiple survival curves and evaluating the effect of constraining parameters for the different seed lots; and lastly, to model the moisture relations of seed longevity. The code bases provided could also be used in pollen and fern/bryophyte spore longevity modelling.

The Hawaiian archipelago’s sole native member of the Capparaceae plant family, the endemic maiapilo ( Capparis sandwichiana ), is a species of conservation importance with poor natural regeneration. This study identifies important seed characteristics, such as its seedcoat water permeability, embryo development, and time to germination, to determine dormancy classification. To test for water-permeable coats, seeds were mechanically scarified and imbibed in tap water over a 48-hour time period. During imbibition, scarified and nonscarified seed masses increased at similar rates. The same seeds were then tested for germination with daily alternating regimens of 12 hours of light/dark and temperatures of 25/15 °C. The nonscarified seeds germinated at a significantly higher rate than scarified treatments, indicating the presence of water-permeable seedcoats to rule out physical dormancy (PY). To test for physiological dormancy, gibberellic acid was used in germination tests. Caper seeds were sown without (control) and with (500, 1000 ppm) gibberellic acid (GA 3 ) treatments to help determine dormancy classification. Mean germination for each replicate ranged between 10% (500 ppm GA 3 ) and 60% (1000 ppm GA 3 ). There was no significant difference with final germination or overall time (days) to reach 25% total germination ( T 25 ) among treatments and controls. Because 1) seedcoats were permeable to water, 2) embryos were fully developed, and 3) germination took longer than 30 days, Capparis sandwichiana seeds most likely have nondeep physiological dormancy. These results will benefit managers and growers with a better understanding of seed characteristics for dormancy alleviation and successful germination of this threatened endemic species.

Seed (gene)banking is an effective way to conserve cultivated and wild plant diversity. However, long-term funding is not always consistently sufficient, and there is a need to both strengthen the effectiveness of genebank operations and maximize cost efficiency. One way to control the cost of maintaining a germplasm collection is to optimize the quantity of seeds per accession that is placed into storage, depending on the expected length of time a seed lot will remain above the viability threshold, expected rates of use for distribution and viability testing and on the requirement to ensure a reserve. Here, we express this as an equation, which can be applied to cultivated species and adjusted to different scenarios, but also to inform decisions about use of accessions of wild species where the number of seeds available is limited, a common scenario for wild-species conservation seed banks. For many crop genebanks, given the expected longevity of seeds, it would be worthwhile to increase the number of seeds produced and processed for storage. This would also help to diminish the risk of genetic drift due to frequent cycles of regeneration but would have implications in terms of how accessions are regenerated, in particular, how many plants are used for regeneration and the size of storage facilities. The equation we present can also be rearranged and used to plan how to allocate seeds for testing and use when the number of seeds available is limited. This may have particular relevance for species conservation seed banks.

Ferns are an important part of many ecosystems, especially on islands, but are often overlooked in conservation priorities. Fern propagation can be challenging and requires signifi-cant time and resources, especially for rare ferns with limited propagation history. Some fern spores can survive for decades in the soil bank and some dry adapted ferns may store well at room temperatures for several years. Herbaria have been proposed as a source of viable propagules of rare plants for propagation. We explored herbarium specimens as a source of viable spores for conservation of rare and endangered Hawaiian ferns. We examined 156 herbarium specimens for the presence of spores and sampled spores from 50 of them spanning four decades. Germination of spores occurred in 24 specimens representing eight of nine included species within the 15-month experiment. However, gametophytes from spores from only five specimens (10%) representing three species (33%) produced sporophytes during the experiment. While fresh spores provide higher germination success, sporophytes developed from specimens of Pellaea ternifolia collected in 1984, 1989, and 2018, as well as from a specimen of Cyrtomium caryotideum from 2019 and a Doryopteris decora specimen from 2020. This result confirms that herbaria can be a source of dry adapted spores of rare species or populations that may be extinct or otherwise difficult to source. Increased attention on rare fern propagation techniques is critical to successful conservation efforts from both fresh and dried collections such as herbarium specimens.

Premise:Genebanks must maintain viable seeds for decades. Seeds that germinate are clearly alive, but some seeds, often from wild populations, do not germinate because they are dormant, empty, aged, or damaged (D.E.A.D.). This work evaluates the effects of D.E.A.D. factors on genebanked seeds using a unique dataset to improve genebanking practices and standards for ex situ conservation of seed collections. Methods:Seeds from over 100 species were recently collected from the same populations as seeds that were genebanked decades ago. Germination proportion and speed were measured after applying various temperature, chemical, or seed coat abrasion treatments. Viability was further tested using vital staining of samples with a low germination proportion. Proportions of dormant, empty, aged, and damaged seeds were compared between seed cohorts. Results:Germination proportion and speed varied among samples, and cues to stimulate germination of dormant seeds were identified for individual species, leading to a positive correlation between viability metrics of germination and vital staining. Empty seeds primarily contribute to low germination in this study. Aging, indicated by lower and slower germination, was evident in several of the stored samples, compared to those that had been recently harvested. Discussion:This unique approach demonstrates the feasibility of genebanking seeds from diverse endangered plant species using freezer storage. Genebanking methods that are more relevant for crop seeds need to be modified when applied to seeds from wild populations because the sample sizes tend to be small and the seeds tend to germinate slowly and asynchronously.

Premise:Embryo excision is an effective, under-described means of promoting germination in the sunflower family and may help to ensure the survival of endangered taxa or lineages with limited seed availability. Methods and Results:We describe and illustrate a detailed method of embryo excision used successfully to stimulate germination in a diversity of composites and that requires minimal materials and expense, using Layia platyglossa as an example. We show how this procedure greatly increases germination compared to control treatments in Madia elegans, a close relative of Hawaiian silverswords that exhibits physiological dormancy. Conclusions:This technique can be learned quickly and is highly effective. Embryo excision can aid conservation efforts dependent on minimal seed resources by enhancing germination and allowing evaluation of seed quality before or after storage, as well as synchronizing seedling development, thereby allowing for refinement of ex situ seed bank conditions and efficient use of horticultural resources.

Context Seed banking is the most efficient and cost-effective method of preserving plant germplasm, but not all species can be conserved by conventional practices. Further, restoration of plant populations from seeds often is hindered by a lack of a priori knowledge of seed dormancy and germination requirements. Aims Our objective was to determine seed desiccation, initial freezing tolerance and the dormancy-breaking and germination requirements of Coprosma kauensis, a dioecious shrub endemic to Kauaʻi Island in Hawaiʻi. Methods The effects of temperature and gibberellic acid (GA3) on germination of fresh seeds were tested in light/dark at 15/5, 25/15 and 20/10°C with and without treatment with GA3. To test effects of desiccation and initial freezing on germination, seeds were dried to 15% relative humidity at 15°C and then stored at −20°C. Key results Regardless of incubation temperature and treatment (or not) with GA3, final germination ranged from 78 to 88%; however, time to 50% germination decreased at 20/10°C for seeds treated with 500 ppm GA3. There was no significant difference in final germination percentage between freshly harvested seeds and those desiccated, nor of those desiccated and then subsequently frozen at −20°C. Conclusions Fresh seeds of C. kauensis exhibit a low degree of nondeep physiological dormancy. They can germinate over a range of temperatures, but germination speed is increased by GA3. Fresh seeds are desiccation and freeze tolerant (after 6 months storage). Implications This vulnerable Kauaʻi endemic species easily can be propagated from seeds. Future studies should investigate long-term longevity at various storage temperatures.

Plants are essential to life on earth. Plants provide a wide range of essential ecosystem services. Importantly, they are also of immense cultural and spiritual value to our species. Plant conservation has changed in one fundamental way over the last decade. The stakes are higher and so the risks of failure are extraordinarily high. Human activity is directly eroding the world's ecological foundations with plants a key asset for society. The rates of plant extinction are not slowing. In this chapter we broadly review many facets of plant conservation and discuss the current state and future outlook of the topic.

The plant species Sesbania tomentosa (‘ōhai; Fabaceae) is endemic to the Hawaiian Islands, federally listed as endangered in the USA and has been proposed for categorization as Vulnerable on the IUCN Red List. In 2021, c. 12,000 seeds from 12 seed lots collected during 1990–1992 from across the Hawaiian Islands were discovered in ambient herbarium conditions (55% relative humidity (RH) at 20 °C). International gene bank standards suggest drying seeds in equilibrium with 15% RH and stored at −18 °C. To investigate seed viability, we mechanically scarified then sowed 15 seeds from each accession at daily alternating regimes of 12 h light and 12 h dark at temperatures of 25/15 °C, respectively. Germination was observed after 7 days and ended after 34 days. Mean final germination was 88.9 ± SD 0.1% (range 73–100%). Each seed lot was accessioned into the National Tropical Botanical Garden's Seed Bank and Laboratory. In seeds with a water-impermeable seed coat (i.e. physical dormancy), such as S. tomentosa, seeds can desorb but not absorb water. Therefore, if the seeds were initially dried, although exposed to high RH for up to 32 years, seed equilibrium RH may have remained low, which may in part explain the observed high germinability. This study holds significance for managers who are working to conserve this endangered Hawaiian species and suggests that even suboptimal conditions may still yield highly viable seeds several decades into the future.

Background and aims – Gardenia species are ecologically, culturally, and economically significant but the three native species of Gardenia in Hawai‘i are assessed as Critically Endangered. Seed banking is the most cost effective and efficient means of conserving plant material ex situ. To better understand the conservation physiology of Hawaiian and South Pacific Gardenia spp. and support their conservation, we asked 1) How do seeds respond to different temperatures and light and dark regimes? 2) What class of dormancy, if any, do seeds exhibit? 3) How does seed germinability respond over time in a seed bank? and 4) What is the conservation status and level of ex situ representation of Gardenia globally? Material and methods – To answer these questions, we used 19 accessions of fresh seeds and seeds stored for varying periods of time in the National Tropical Botanical Garden’s Conservation Seed Bank and Laboratory of Hawaiian (G. brighamii, G. remyi), New Caledonian (G. aubryi, G. oudiepe), and Tahitian (G. taitensis) species. Seeds were incubated at varying temperatures and in light, and in dark. Key results – We found that (1) seeds of all species tested germinated slowly and only at higher temperatures in the light and dark, (2) seeds have non-deep physiological dormancy, (3) seeds of the Hawaiian species are short lived at conventional seed bank conditions, and (4) only 40% of Gardenia spp. are represented in ex situ facilities, and 66% of the species have not been evaluated for the IUCN Red List. Conclusion – Seeds of Hawaiian Gardenia spp. are short lived in storage. Since seeds germinate in darkness, they are unlikely to form a persistent soil seedbank. Although seeds of all species tested are physiologically dormant, they can be easily propagated from seed at warmer temperatures, giving some hope to the conservation and restoration of the Critically Endangered Hawaiian species. Since our dataset was limited by a lack of continuous viability monitoring, we emphasize the need for initial germination testing and ongoing viability tests to better understand seed longevity. Lastly, we discuss the ecological relevance of our results in the context of the Hawaiian archipelago.

BACKGROUND AND AIMS:Islands, with their long coastlines and increased vulnerability to sea level rise, offer compelling opportunities to investigate the salinity tolerance of coastal plants. Seeds are generally more vulnerable than other plant stages to increased stressors. The aim of this study was to characterize salinity tolerance during germination across a diverse pool of 21 species from 14 plant families found in coastal communities throughout the Hawaiian Islands in order to increase our general understanding of coastal plant ecology for conservation and restoration. METHODS:Seeds of each species were exposed to unfiltered/untreated seawater (35 ppt total salinity) and two salinity treatments (10 and 20 ppt) in which the seawater was diluted with distilled water, and germination percent and timing were compared to seeds in a distilled water control. Non-germinated seeds were then tested for recovery germination. We quantified and compared germination percent, time and recovery among species and across salinity levels and tested for heterogeneity related to seed size, dormancy class, habit and threatened status. KEY RESULTS:Although salinity tolerance varied considerably among species, salinity exposure generally reduced and delayed germination. The greatest effects were detected at higher salinity levels. Recovery germination overall was higher for seeds that had been exposed to higher salinity. None of the factors we explored emerged as predictors of salinity tolerance except seed mass, which tended to enhance germination at higher salinity. CONCLUSIONS:Species responses to salinity exposure indicate high vulnerability of coastal systems to increased salinity stress, and variability among species could lead to shifts in community assembly and composition under sea level rise. These results can help guide coastal ecosystem conservation and restoration management decisions in the face of climate change.

Moisture content determination is one of the most important and common assessments made on seeds. It contributes to the estimation of the value of a seed lot in commercial settings and the physiological status of seeds in research and conservation. The most common method for determining seed moisture content is by measuring the change in weight of a sample after drying at a constant temperature for a set period of time. This, however, renders the seeds unusable for other purposes, such as viability testing. Here, we review various methods for assessing the moisture status of seeds, including non-destructive methods. Controlling the amount of water in seeds is just as important as being able to measure the amount of water in seeds. Perhaps most notably, orthodox seeds are normally dried to low moisture content to maintain viability and vigour for longer, with the seeds either stored in breathable containers in a dry environment or sealed in air-tight containers such that they cannot reabsorb water. However, and especially in seed research, we are sometimes interested in having seeds at an intermediate or high moisture content. Hence, in this review we also discuss various ways to adjust the moisture content of seeds.

Societal Impact Statement Trees are an important part of many ecosystems. The Global Tree Assessment data can be used to focus conservation and restoration efforts for the circa 30% of tree species that are threatened worldwide. The conservation status for the tree flora of Limahuli Valley on Kaua'i Island and a restoration plan for 11 endangered tree species is evaluated in the Global Tree Assessment framework. Lessons learned from Limahuli Valley, one of the most biodiverse valleys in the Hawaiian Islands, provide recommendations for developing long‐term sustainable restoration projects. Summary Based on conservation status assessments of the world's circa 60,000 trees, the Global Tree Assessment (GTA) report revealed that 30% (17,500) of known tree species are currently at risk of extinction. This study aims to evaluate the conservation status for the tree flora of Limahuli Valley and a restoration plan for 11 endangered tree species, in the Global Tree Assessment framework. Of the 117 tree taxa found in Limahuli Valley, 83 (71%) have been assessed for the International Union for the Conservation of Nature (IUCN) Red List and 90% of the assessed tree taxa are threatened. However, only 19 (21%) of these are federally listed and nine tree taxa were not found under their current name or at all in the GlobalTree Portal, suggesting a need for additional curation as well as a conservation status assessment gap. Progress has been made in Limahuli Valley on most restoration goals suggested by the GTA framework, but challenges remain related to both access to material for restoration, mitigation of threats, and understanding correlates of survivorship. Furthermore, trees only constitute about 40% of the flora in Limahuli Valley, and it is important to also consider nontrees including ferns and allies to strive for restoration of an ecosystem as well as the targeted species.

Eryngium sparganophyllum is a perennial endemic to ciénegas in warm North American deserts. Only four populations remain, yet little scientific knowledge exists to guide its conservation. Based on a spatially balanced sampling design, we quantified plant density, individual size, and reproductive potential of two populations in the U.S. We also assessed conditions influencing germination in the lab. The population in a warmer climate had a larger population size, higher short-term population growth rate, hosted larger individuals, and showed a negative correlation between plant size and plant density. In both populations, plant density was positively correlated with shallow soil moisture and probability of flowering was positively correlated with plant size. Seeds only germinated in the spring, and responded to interactions between temperature, light exposure and population location. We hypothesize that 1) recruitment of E. sparganophyllum may be episodic, relying on a rare niche opportunity of reliable shallow soil moisture and low competition; and 2) adult survival of the species may be limited by interspecific density dependence and climatic extremes. The conservation of this species depends on ensuring high adult survival and promoting recruitment, which can be accomplished through actively protecting extant populations and exploring restoration and introduction possibilities.

Understanding the relative longevity of different seed lots, perhaps of different species or genotypes, but also following production under different environments or using different cultivation methods, or following different post-harvest treatments, is relevant to anyone concerned with the retention of seed lot viability and vigour during storage. However, different scientists over the years have used different conditions to assess seed lot longevity, as well as different variables as the measure of 'longevity.' Here, we give some of the backgrounds to how two standard protocols, with an open and closed system respectively, were derived, and explain why we consider p(50), defined as the time during storage when seed lot viability, as measured through a germination test, has declined to 50%, is a suitable longevity trait parameter.