Seed Bank Conditions Research Articles

Gardenia (Rubiaceae) seed conservation physiology with emphasis on rare Hawaiian species

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.

Fit for the future? Alpine plant responses to climatic stress over two decades of seed bank storage

Climate warming is occurring in high mountain areas faster than the global average, making cold-adapted alpine specialists vulnerable. Ex situ seed conservation is a solution, but little is known about how plants from stored seeds will cope with the altered climate. 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. During this time the site has warmed by 0.3 °C per decade and it has been drier than average. For three old (‘ancestor’) and three recent (‘descendant’) accessions, we exposed juvenile plants to temperature and/or soil moisture stress for one month. To determine plant fitness we measured vegetative traits (above ground biomass, specific leaf area), reproductive performance (flower number, stalk height) and seed traits (number, mass). Descendants had lower above ground biomass than ancestors and produced heavier seeds. In response to drought, compared to the control, ancestors had lower biomass whereas descendants did not, though the actual biomass did not vary between ancestors and descendants. Ancestors had higher seed production under drought than descendants, but the variation was large. We show plant traits in an alpine specialist have changed over 20 years, with plants originating from recent accessions apparently better adapted to drought. These findings indicate that alpine species have adjusted to the changed climate, implying older seed bank stored seeds may be unsuitable for use in plant conservation. Seed banks should therefore increase the frequency of seed collection.

Can alpine plant species "bank" on conservation?: Using artificial aging to understand seed longevity.

PremiseTo conserve native plants, many institutions are turning toward ex‐situ conservation methods, such as storage in seed banks; however, not all seeds are able to survive in seed bank conditions, or may not in the long term. Experimental aging has shown that alpine species lose viability more quickly than low‐elevation species. Furthermore, the germination requirements for rare species are largely unknown, but are a necessary first step in understanding storage behavior and viability decline.MethodsFive alpine species were subjected to germination and accelerated aging experiments to understand their longevity in storage. For the accelerated aging experiment, the seeds were rehydrated in a dark incubator and subsequently placed in a drying oven. Following the aging process, the seeds were placed into previously determined germination conditions.ResultsAll species had p 50 values of <13.7 days, which is the threshold to consider a species short lived. These results suggest that we cannot haphazardly store seeds and assume that all species will survive for decades.DiscussionAccelerated aging experiments are not a perfect measure of seed longevity, and true longevity needs to be empirically determined. However, this experimental method allows us to predict which species may be short lived and whether alternative ex‐situ conservation methods might be needed beyond conventional seed banking.

Does oxygen affect ageing mechanisms of Pinus densiflora seeds? A matter of cytoplasmic physical state.

During desiccation, the cytoplasm of orthodox seeds solidifies into an intracellular glass with highly restricted diffusion and molecular mobility. Temperature and water content govern seed ageing rates, while oxygen (O2) can promote deteriorative reactions. However, whether the cytoplasmic physical state affects involvement of O2 in seed ageing remains unresolved. We aged Pinus densiflora seeds by controlled deterioration (CD) at 45 °C and distinct relative humidity (RH), resulting in cells with a glassy (11% and 30% RH) or fluid (60% and 80% RH) cytoplasm. Hypoxic conditions (0.4% O2) during CD delayed seed deterioration, lipid peroxidation, and decline of antioxidants (glutathione, α-tocopherol, and γ-tocopherol), but only when the cytoplasm was glassy. In contrast, when the cytoplasm was fluid, seeds deteriorated at the same rate regardless of O2 availability, while being associated with limited lipid peroxidation, detoxification of lipid peroxide products, substantial loss of glutathione, and resumption of glutathione synthesis. Changes in metabolite profiles provided evidence of other O2-independent enzymatic reactions in a fluid cytoplasm, including aldo-keto reductase and glutamate decarboxylase activities. Biochemical profiles of seeds stored under seed bank conditions resembled those obtained after CD regimes that maintained a glassy cytoplasm. Overall, O2 contributed more to seed ageing when the cytoplasm was glassy, rather than fluid.

Editorial: Seed Science and Technology. Volume 49 Issue 1 (2021)

At the time of writing, in the UK and Northwest Europe the wood anemone (Anemone nemorosa L., Ranunculaceae) is in full flower, as Spring gets under way. This common herbaceous perennial of woodland and hedgerows is a possibly somewhat extreme example of the challenges facing conservationists striving to preserve seeds of their native species, in all their diversity, ex situ in conventional seed banks. Its seeds will survive drying, but only after the achenes, still green when shed, are dispersed from the parent plant. Ali et al. (2007) observed an initial increase in desiccation tolerance, followed by a decline; and the proportion of seeds surviving desiccation and their increase in subsequent longevity coincided with the growth and development of their embryos ex planta. However, developmental arrest of the embryo was not required for the acquisition of desiccation tolerance, and continued growth and development of the embryo resulted in loss of desiccation tolerance, analogous to that seen in orthodox (sensu Roberts, 1973) seeds upon radicle emergence. Consequently, the window of desiccation tolerance and maximum potential longevity is comparatively short; and in nature occurs when seeds are on or close to the soil surface. Though the seeds can be air-dried with care and stored at sub-zero temperatures, high initial viabilities are difficult to achieve and their storage lives are short (ten years or less under conventional seed bank conditions); throwing doubt on the role of conventional seed banking as a means of long, or even medium-term ex situ conservation for this species. Furthermore, while A. nemorosa seeds, like those of many species from the Ranunculaceae, might be described as having morphological or morphophysiological dormancy (Baskin and Baskin, 2014), the observed lack of developmental arrest is somewhat at odds with most definitions of dormancy.

Exploring seed longevity of UK native trees: implications for ex situ conservation

AbstractUK trees require increased conservation efforts due to sparse and fragmented populations. Ex situ conservation, including seed banking, can be used to better manage these issues. We conducted accelerated ageing tests on seeds of 22 UK native woody species, in order to assess their likely longevity and optimize their conservation in a seed bank. Germination at four ageing time points was determined to construct survival curves, and it was concluded that multiple samples within a species showed comparable responses for most species tested, except for Fraxinus excelsior. Of all species studied, one could be classified as very short-lived, four as short-lived and 17 as medium, with none exceeding the medium category. The most important finding of this manuscript is that although some taxonomic trends were observed, the results indicate the need for caution when making broad conclusions on potential seed storage life at a species, genus or family level. Longevity predictions were compared to actual performance of older collections held in long-term storage at the Millennium Seed Bank, Kew. Although most collections remain high in viability in storage after more than 20 years, for short-lived species at least, there is some indication that accelerated ageing predicts longevity in seed bank conditions. For species with reduced potential longevity, such as Fagus sylvatica and Ulmus glabra, additional storage options are recommended for long-term gene banking.

Germination behaviour of forbs native to the southwestern United States following exposure to short-term seed bank conditions (−20°C)

AbstractSeeds are increasingly being stored in seed banks for ex situ conservation, food security, and restoration reserves. Although it is hypothesized that seeds of many species can survive the dry, freezer conditions (−20°C) of seed banks for many years, shelf life is unknown for many species. In particular, changes to dormancy and germination requirements following freezer storage have not yet been studied extensively. To improve our understanding of how seed dormancy and germination respond to seed banking conditions, we evaluated seed of six restoration-priority species in the southwestern United States, which is a region of increasing restoration need. Germination tendencies of seeds that were and were not exposed to a −20°C treatment for 4–6 months were evaluated under 22 cold-moist stratification and incubation treatments to broadly assess changes to dormancy and germination requirements and speed. Direct or indirect (via interactions with stratification and/or incubation treatments) differences in dormancy breaking and germination were observed in seeds for four of the six species studied. Specifically, storage temperature accounted for differences in the final germination proportion, germination during stratification, and/or rate of germination in five of the six species. Notably, seeds of the one species that exhibit a combination of physiological and physical dormancy showed significant differences in all germination measures. However, while significant differences were found between seeds exposed to short-term seed bank storage and those that were not, these differences were small and may not be biologically meaningful, indicating that seed banking these species should not change how they are used for restoration or reintroduction purposes.

Seed germination ecology of Viola calaminaria, an endangered metallophyte with a narrow distribution

AbstractViola calaminaria is an endangered metallophyte endemic to a small area close to the border between Belgium, Germany and the Netherlands, where it grows on rock outcrops rich in heavy metals (zinc, lead and cadmium). Because V. calaminaria reproduces mainly by seeds, it is of crucial importance to understand its germination requirements. Germination percentage and speed at constant (11–25°C) and alternating (23/09°C) temperatures were investigated in five large populations. Germination percentage was positively correlated to seed weight. Germination was low (&lt;25%) at 11 and 16°C, intermediate (around 65%) between 20 and 25°C and the highest (93%) at the alternating temperature regime (23/09°C). V. calaminaria is a slow germinator requiring 41 days on average to germinate at 23/09°C and considerably more at 20 to 25°C (105 days on average). Our results also highlighted that the species is desiccation tolerant and can therefore be safely conserved under standard seed bank conditions.

Wheat seed ageing viewed through the cellular redox environment and changes in pH

To elucidate biochemical mechanisms leading to seed deterioration, we studied 23 wheat genotypes after exposure to seed bank storage for 6–16 years compared to controlled deterioration (CD) at 45 °C and 14 (CD14) and 18% (CD18) moisture content (MC) for up to 32 days. Under two seed bank storage conditions, seed viability was maintained in cold storage (CS) at 0 °C and 9% seed MC, but significantly decreased in ambient storage (AS) at 20 °C and 9% MC. Under AS and CS, organic free radicals, most likely semiquinones, accumulated, detected by electron paramagnetic resonance, while the antioxidant glutathione (GSH) was partly lost and partly converted to glutathione disulphide (GSSG), detected by HPLC. Under AS the glutathione half-cell reduction potential (EGSSG/2GSH) shifted towards more oxidising conditions, from −186 to −141 mV. In seeds exposed to CD14 or CD18, no accumulation of organic free radicals was observed, GSH and seed viability declined within 32 and 7 days, respectively, GSSG hardly changed (CD14) or decreased (CD18) and EGSSG/2GSH shifted to −116 mV. The pH of extracts prepared from seeds subjected to CS, AS and CD14 decreased with viability, and remained high under CD18. Across all treatments, EGSSG/2GSH correlated significantly with seed viability (r = 0.8, p<.001). Data are discussed with a view that the cytoplasm is in a glassy state in CS and AS, but during the CD treatments, underwent transition to a liquid state. We suggest that enzymes can be active during CD but not under the seed bank conditions tested. However, upon CD, enzyme-based repair processes were apparently outweighed by deteriorative reactions. We conclude that seed ageing by CD and under seed bank conditions are accompanied by different biochemical reactions.

Conservation of Garcinia imberti Bourd. through seeds

Garcinia imberti seeds were collected during 2015-2017 from Shangili, Cheenikkala and Bonaccord evergreen forests of Agasthyamala Biosphere Reserve, the only abode of this endangered endemic species. Germinability of seeds were analysed through decoating, Gibberellic acid (GA3) and light inductive pre-treatments on fresh (62.8 % moisture content; MC) and desiccated (fast; 23.3% MC and slow; 30.5 % MC) seeds. The seed germination with impermeable coat (0.7-1.2 mm) was restricted which on decoating got enhanced. Application of GA3 along with exposure to light breaked dormancy within 4-6 days compared to non-treated seeds that took 238-254 days to germinate. Stored seeds behaviour revealed that seed moisture content and rate of germination were negatively correlated. Seed storage was found to be more efficient only up to 80 days at controlled seed banking conditions (20 ± 20C, 20 % relative humidity; RH). Both fast and slow desiccated seeds stored for 60 days in seed bank conditions exhibited 50.4 and 43.4 % of germination compared 39.4% germination of non-desiccated seeds. Hence fast desiccated and decoated G. imberti seeds pre-treated with GA3 on subsequent exposure to light alleviated dormancy. For seed banking, fast desiccated seeds with MC in between 40-20% are found to be promising.

Survival and growth of embryo axes of temperate trees after two decades of cryo-storage

Liquid nitrogen (LN) storage is recommended for conserving plants with seeds for which standard seed banking is not feasible, but there are few empirical reports confirming its long-term effectiveness. In this study we evaluated in vitro viability and ex vitro growth of embryo axes of a species that is short-lived in seed bank conditions (Juglans nigra) and two recalcitrant seeded species (Aesculus hippocastanum and A. glabra) that were stored 11–23 years in LN. Viability of J. nigra axes did not decrease significantly after 23 years and produced normal-appearing plantlets. Similarly, viability of A. hippocastanum axes did not decrease after 23 years. However, A. glabra axes showed a significant decline in viability after 23 years, from 80% when freshly harvested to 33%. These results demonstrate that LN storage can preserve embryo axes of J. nigra and Aesculus sp. for over two decades, providing a workable conservation tool for these and similar species.

Seed banking of terrestrial orchids: evaluation of seed quality in Anacamptis following 4‐year dry storage

Good-quality dry seeds of some orchids have the potential to survive for decades under conventional seed bank conditions, but further research is needed to fill existing gaps in knowledge regarding seed behaviour under long-term dry storage. The objectives of this study were to evaluate germination ability on two asymbiotic culture media with different nitrogen source; to assess seed desiccation tolerance needed for the storage at sub-zero temperatures; and to study the effects of dry storage at low temperature. Asymbiotic seed germination tests of four Anacamptis species were carried out to evaluate the effects of different culture media, dehydration and dry storage on germination ability. Viability of 4-year-stored seeds was assessed by means of the tetrazolium test. Generalised linear model (GLM) analysis detected significant effects (P<0.01) of the species, medium and storage time on total germination, while dehydration did not significantly affect it. Except for A.palustris, germination percentage was minimum after 1-month storage and increased with longer storage periods. Tetrazolium viability tests detected high percentages of viable seed (>90%) following 4-year storage in three out of four species. Seeds of the four Anacamptis species proved to be desiccation tolerant and have orthodox storage behaviour. The consequence of these findings is of interest to practical conservation approaches for orchids in seed-banking. The results highlight the importance of multiple assessments of seed quality, both viability and germination, to understand seed storage behaviour.

Changes in tocochromanols and glutathione reveal differences in the mechanisms of seed ageing under seedbank conditions and controlled deterioration in barley

“Orthodox” seeds are desiccation tolerant, enabling dry storage for agricultural and conservational purposes. Protocols of “controlled deterioration” (CD) using elevated temperatures and seed water contents (WCs) are frequently used to study mechanisms of seed ageing. However, the relevance of using CD to understand how seeds age at lower water contents and temperatures has been disputed. Here, comparisons in antioxidant changes are drawn between barley seed stored for up to 15 years in a seedbank with seeds aged using CD (45 °C, 75% RH; WC = 13% FW) under normoxia (21%) or elevated (75%) O2. Regardless of ageing method, glutathione (GSH) levels decreased concomitantly with an oxidative shift in the glutathione redox state (EGSSG/2GSH) as viability was lost. An earlier oxidative shift in EGSSG/2GSH and more rapid loss of seed vigour was found in seeds aged by CD under 75% O2, compared to 21% O2, but longevity was not shortened by elevated O2. Seedbank-aged seeds contained more glutathione disulphide (GSSG) levels, leaked more electrolytes upon imbibition and contained less tocochromanols than non-stored seeds. In contrast, in response to CD, levels of GSSG fell, electrolyte leakage did not increase and tocochromanol levels increased (21% O2), or remained stable (75% O2). In a further set of 24 barley genotypes stored for up to 15 years at 20 °C (WC = 11% FW) and 0 °C (WC = 8% FW), tocochromanol levels positively (r = 0.42) correlated with total germination, which ranged between 1-78% (20 °C) and 65-97% (0 °C). Of all tocochromanol species, α-tocopherol correlated most with total germination (r = 0.49) and γ-tocotrienol least (r = 0.08). It is concluded that CD is not a perfect model for understanding mechanisms of ageing that occur under seedbank conditions.

Physiology of Desiccation-Sensitive (Recalcitrant) Seeds and the Implications for Cryopreservation

Desiccation-tolerant (orthodox) seeds can survive in the dry state for considerable periods and so can be stored successfully at low water content and subzero temperatures. Orthodox seeds possess a variety of protective processes and mechanisms that confer desiccation tolerance, important among them being metabolic shutdown and intracellular dedifferentiation. In contrast, desiccation-sensitive (recalcitrant) seeds cannot tolerate water loss and so cannot be stored using conventional seed bank conditions. Particularly with respect to storage, recalcitrant seeds do not undergo intracellular dedifferentiation nor any significant metabolic shutdown. Embryos of recalcitrant seeds remain metabolically active, with little or no reduction in extent of the extensive intracellular membranes. At the water content at which they are shed, with the ongoing metabolism in recalcitrant seeds, developmental phenomena grade imperceptibly into those associated with germination. Thus, recalcitrant seeds can be stored intact ...

Germination capacity and seed storage behaviour of threatened metallophytes from the Katanga copper belt (D.R.Congo): implications for ex situ conservation

Background and aims – Plant species adapted to metalliferous soil are of high conservation value, and actions for preserving these species (some of them are endemics) are urgent given the threat of mining activities. In the framework of an integrated conservation programme of cuprophytes (plants that tolerate a soil with a high level of copper) in Katanga (D. R. Congo), this study aims at: (1) providing new data on species whose germination has never been studied so far; (2) gaining new insight into the storage behaviour of these species; (3) discussing implications for ex situ conservation of these highly threatened species. Methods – Germination tests were conducted on fresh seeds of nineteen species. These tests were repeated after 6, 12 and 24 months of storage in dry-cold conditions. Key results – Most species kept or increased their germination capacity after 2 years storage in dry-cold conditions. Nine species showed a slight decrease in their viability (from 100% to &gt; 80%) after 2 years storage in dry-cold conditions. The present study gives evidence that at least six of the 19 studied species are desiccation-tolerant (orthodox). Among these, two are strict endemics, Haumaniastrum robertii and Faroa malaissei, and two are broad endemics, Diplolophium marthozianum and Gladiolus robiliartianus . This means that ex situ seed banking of these species could form a useful part of a more comprehensive conservation strategy. Only two species have been identified as desiccation-sensitive (recalcitrant), i.e.inappropriate for conservation in standard seed bank conditions. An orthodox behaviour has not been ruled out for the other species tested, but their response was less clear and needs further investigation.

Laboratory germination and seed bank storage of Ranunculus peltatus subsp. baudotii seeds from the Tuscan Archipelago

Germination requirements and suitability for ex situ conservation were investigated for seeds from the only population of Ranunculus peltatus Schrank subsp. baudotii (Godron) Meikle ex C.D.K. Cook (Ranunculaceae) from the Tuscan Archipelago (Italy). A 2 × 2 × 3 factorial experiment investigated the interactive effects of light, alternating temperatures and mean temperature on the germination of seeds sown on agar. Logistic regression analysis showed significant effects of both temperature (P < 0.0001) and light (P < 0.05). Alternating temperatures improved germination but only at mean temperatures of 20 and 15°C. The results indicate that seeds of R. baudotii exhibit morphological dormancy and a phenological adaptation to seasonal ponds. Drying led to a significant reduction in germination in seeds collected in both 2009 and 2010 (P < 0.05); the reduction was larger for 2009. The better quality of the 2010 seed was probably due to a post-harvest treatment that enabled continued maturation of the seeds. Storage of seeds for one year under seed bank conditions (-20°C) did not reduce viability. Seeds of R. baudotii possess orthodox storage behaviour and are therefore suited to ex situ conservation in seed banks; however, further research is needed to confirm their long term storage prospects.

Plant regeneration in Arachis stenosperma Krapov. and W. C. Gregory from roots and calluses derived from leaflets of in vitro plants

Seed explants of A. stenosperma were cultured on MS medium supplemented with 6-benzylaminopurine with the aim of rescuing nonviable accessions stored in seed bank conditions. The regeneration potential of leaf explants from in vitro plants derived from embryonic axes was studied by using whole leaflets and leaflet segments. Explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzylaminopurine and naphthalene acetic acid. Indirect organogenesis was observed in response to 6-benzylaminopurine, either alone or in association with naphthalene acetic acid, in both explant types. Media supplemented with naphthalene acetic acid as the sole growth regulator induced rhizogenesis in whole leaflets and leaflet segments, with subsequent shoot production directly from the roots.