Identification of the rice Rc gene as a main regulator of seed survival under dry storage conditions.

Seed deterioration during storage results in poor germination, reduced vigour, and non-uniform seedling emergence. The aging rate depends on storage conditions and genetic factors. This study aims to identify these genetic factors determining the longevity of rice (Oryza sativa L.) seeds stored under experimental aging conditions mimicking long-term dry storage. Genetic variation for tolerance to aging was studied in 300 Indica rice accessions by storing dry seeds under an elevated partial pressure of oxygen (EPPO) condition. A genome-wide association analysis identified 11 unique genomic regions for all measured germination parameters after aging, differing from those previously identified in rice under humid experimental aging conditions. The significant single nucleotide polymorphism in the most prominent region was located within the Rc gene, encoding a basic helix-loop-helix transcription factor. Storage experiments using near-isogenic rice lines (SD7-1D (Rc) and SD7-1d (rc) with the same allelic variation confirmed the role of the wildtype Rc gene, providing stronger tolerance to dry EPPO aging. In the seed pericarp, a functional Rc gene results in accumulation of proanthocyanidins, an important sub-class of flavonoids having strong antioxidant activity, which may explain the variation in tolerance to dry EPPO aging.

Experimental rice seed aging under elevated oxygen pressure: Methodology and mechanism.

Seed aging during storage results in loss of vigor and germination ability due to the accumulation of damage by oxidation reactions. Experimental aging tests, for instance to study genetic variation, aim to mimic natural aging in a shorter timeframe. As the oxidation rate is increased by elevating the temperature, moisture, and oxygen levels, this study aimed to (1) investigate the effect of experimental rice seed aging by an elevated partial pressure of oxygen (EPPO), (2) elucidate the mechanism of dry-EPPO aging and (3) compare aging under dry-EPPO conditions to aging under traditional moist-controlled deterioration (CD) conditions and to long-term ambient storage. Dry seeds from 20 diverse rice accessions were experimentally aged under EPPO (200 times higher oxygen levels), at 50% relative humidity (RH), along with storage under high-pressure nitrogen gas and ambient conditions as controls. While no decline in germination was observed with ambient storage, there was significant aging of the rice seeds under EPPO storage, with considerable variation in the aging rate among the accessions, with an average decline toward 50% survival obtained after around 21 days in EPPO storage and total loss of germination after 56 days. Storage under high-pressure nitrogen gas resulted in a small but significant decline, by an average of 5% germination after 56 days. In a second experiment, seven rice seed lots were stored under EPPO as compared to a moist-CD test and two different long-term ambient storage conditions, i.e., conditioned warehouse seed storage (CWSS) and traditional rice seed storage (TRSS). Untargeted metabolomics (with identification of lipid and volatile compounds profiles) showed a relatively high increase in levels of oxidized lipids and related volatiles under all four storage conditions. These compounds had a high negative correlation with seed viability, indicating oxidation as a main deteriorating process during seed aging. Correlation analysis indicated that EPPO storage at 50% RH is more related to aging under TRSS at 60% and CD-aging at 75% ERH rather than CWSS at 40% ERH. In conclusion, aging rice seeds under EPPO conditions is a suitable experimental aging method for analyzing variation among seed lots or genotypes for longevity under storage.

Seed Moisture Isotherms, Sorption Models, and Longevity.

Seed moisture sorption isotherms show the equilibrium relationship between water content and equilibrium relative humidity (eRH) when seeds are either losing water from a hydrated state (desorption isotherm) or gaining water from a dry state (adsorption isotherm). They have been used in food science to predict the stability of different products and to optimize drying and/or processing. Isotherms have also been applied to understand the physiological processes occurring in viable seeds and how sorption properties differ in relation to, for example, developmental maturity, degree of desiccation tolerance, or dormancy status. In this review, we describe how sorption isotherms can help us understand how the longevity of viable seeds depends upon how they are dried and the conditions under which they are stored. We describe different ways in which isotherms can be determined, how the data are modeled using various theoretical and non-theoretical equations, and how they can be interpreted in relation to storage stability.

A Performance Management System for Long-Term Germplasm Conservation in CGIAR Genebanks: Aiming for Quality, Efficiency and Improvement.

UN Sustainable Development Goal 2 Target 2.5 focuses on the conservation of genetic diversity in soundly managed genebanks. In examining the term "soundly managed", it becomes quickly evident that there is much more to long-term conservation than placing samples of seeds or other germplasm in long-term conservation conditions. There are several important factors that determine whether germplasm samples will remain viable in storage for long periods of time. To manage these factors efficiently and effectively, genebanks require sound data and quality management systems. The CGIAR Genebank Platform, coordinated by the Crop Trust, put in place a number of mechanisms that enabled effective online reporting, performance management, quality management, audit and external review and validation. These mechanisms do not conform to the usual monitoring systems put in place for research programs and have only been possible thanks to the flexibility of CGIAR in recognising that the genebanks were exceptional. As a result, in the past 10 years, CGIAR genebanks have significantly improved their performance and the conservation status of collections.

Variation in Seed Metabolites between Two Indica Rice Accessions Differing in Seed Longevity.

For a better understanding of germination after seed storage, metabolite profiling was conducted using hybrid triple quadrupole time-of-flight (QTOF) mass spectrometry. After moisture content (MC) equilibration, seeds of "WAS170" (short-lived) and "IR65483" (long-lived) were stored at 10.9% MC and 45 °C. Samples for metabolite analysis were taken after 0 and 20 days of storage. Among 288 metabolites, two flavonoids (kaempferide and quercetin-3-arabinoside), one amino acid (S-sulfocysteine) and one sugar (D-glucose) increased in "IR65483" seeds after storage but were not detected in "WAS170" seeds. Based on the genome sequence database, we identified clear allelic differences with non-synonymous mutations on the six flavonol synthase genes regulating the accumulation of kaempferol- and quercetin-metabolites. On the other hand, two metabolites (thiamine monophosphate and harmaline) increased in short-lived seeds after storage; these metabolites could be potential biochemical indicators of seed deterioration.

Why Seed Physiology Is Important for Genebanking.

Genebank management is a field in its own right; it is multifaceted, requiring a diverse set of skills and knowledge. Seed physiology is one area that is critical to the successful operation of seed genebanks, requiring understanding of seed quality during development and maturation, seed dormancy and germination, and seed longevity in storage of the target species. Careful management of the workflow between these activities, as seeds move from harvest to storage, and the recording and management of all relevant associated data, is key to ensuring the effective conservation of plant genetic resources. This review will discuss various aspects of seed physiology that genebank managers should be aware of, to ensure appropriate decisions are made about the handling and management of their seed collections.

Temporal patterns of seed germination in early spring-flowering temperate woodland geophytes are modified by warming.

BACKGROUND AND AIMS: Understorey species in temperate deciduous woodlands such as wild daffodil (Narcissus pseudonarcissus) and common snowdrop (Galanthus nivalis) have complex dormancy: seeds that are shed in late spring require warm summer temperatures for embryo elongation and dormancy alleviation, but then cooler temperatures for germination in autumn. As seasons warm and tree canopies alter, how will different seasonal temperature sequences affect these complex dormancy responses? METHODS: The effect of different sequences of warmer (+5 °C), current or cooler (-5 °C) seasons (summer to spring) on seed germination patterns over seven successive seasons were investigated, with all sequences combined factorially to determine the consequences of differential seasonal temperature change for the temporal pattern of germination (and so seedling recruitment). KEY RESULTS: Little (<1 %, G. nivalis) or no (N. pseudonarcissus) seed germination occurred during the first summer in any treatment. Germination of N. pseudonarcissus in the first autumn was considerable and greatest at the average (15 °C) temperature, irrespective of the preceding summer temperature; germination was also substantial in winter after a warmer autumn. Germination in G. nivalis was greatest in the warmest first autumn and influenced by preceding summer temperature (average > warmer > cooler); the majority of seeds that germinated over the whole study did so during the two autumns but also in year 2's cooler summer after a warm spring. CONCLUSIONS: Warmer autumns and winters delay first autumn germination of N. pseudonarcissus to winter but advance it in G. nivalis; overall, warming will deplete the soil seed bank of these species, making annual seed influx increasingly important for recruitment and persistence. This study provides a comprehensive account of the effects of temperature changes in different seasons on seed germination in these early spring-flowering geophytes and consequently informs how these and other temperate woodland species with complex seed dormancy may respond to future climate change.

Variation in seed longevity among diverse Indica rice varieties.

BACKGROUND AND AIMS: Understanding variation in seed longevity, especially within closely related germplasm, will lead to better understanding of the molecular basis of this trait, which is particularly important for seed genebanks, but is also relevant to anyone handling seeds. We therefore set out to determine the relative seed longevity of diverse Indica rice accessions through storage experiments. Since antioxidants are purported to play a role in seed storability, the antioxidant activity and phenolic content of caryopses were determined. METHODS: Seeds of 299 Indica rice accessions harvested at 31, 38 and 45 d after heading (DAH) between March and May 2015 and differing in harvest moisture content (MC) were subsequently stored at 10.9 % MC and 45 °C. Samples were taken at regular intervals and sown for germination. Germination data were subjected to probit analysis and the resulting parameters that describe the loss of viability during storage were used for genome-wide association (GWA) analysis. KEY RESULTS: The seed longevity parameters, Ki [initial viability in normal equivalent deviates (NED)], -σ-1 (σ is the time for viability to fall by 1 NED in experimental storage) and p50 [time for viability to fall to 50 % (0 NED)], varied considerably across the 299 Indica accessions. Seed longevity tended to increase as harvest MC decreased and to decrease as harvest MC increased. Eight major loci associated with seed longevity parameters were identified through GWA analysis. The favourable haplotypes on chromosomes 1, 3, 4, 9 and 11 enhanced p50 by ratios of 0.22-1.86. CONCLUSIONS: This is the first study to describe the extent of variation in σ within a species' variety group. A priori candidate genes selected based on rice genome annotation and gene network ontology databases suggested that the mechanisms conferring high seed longevity might be related to DNA repair and transcription, sugar metabolism, reactive oxygen species scavenging and embryonic/root development.

Seed longevity phenotyping: recommendations on research methodology.

The length of time for which seed lots maintain their viability and vigour in storage has become a trait of interest for rice and other crop and model species. However, different research groups have taken different approaches to measuring this trait, including storing seeds under 'natural ageing', accelerated ageing, and controlled deterioration test conditions. There has also been a tendency to use only a single germination test result to assess the relative longevity of different genotypes. Here, we explain why we have taken a different approach in a genome-wide association study of seed longevity in Oryza sativa (Indica Group), and offer suggestions as to how, in the future, this trait should be assessed to make it possible to develop varieties with improved seed longevity, depending on the target storage environment.

Further Evidence That the Genebank Standards for Drying Orthodox Seeds May Not Be Optimal for Subsequent Seed Longevity.

Maximizing seed longevity is important for genebanks to efficiently manage their accessions, reducing the frequency of costly regeneration cycles and the loss of genetic integrity. Research on rice seeds has shown that subsequent longevity in air-dry storage can be improved by drying seeds, which are metabolically active at harvest (moisture contents above a critical value close to 16.5%), for an initial period at a higher temperature (40°C-60°C) than that currently recommended by the current genebank standards (5°C-20°C). The aim of this study was to test whether similar benefits could be achieved in two legume species-cowpea and soya bean-by drying freshly harvested seeds, from two separate harvests, at 40°C and 35% relative humidity, for up to 8 days before equilibrium drying in a drying room (17°C and 15% relative humidity). Improvements in longevity were observed in three of the four accessions of soya bean, with the greatest improvement generally occurring after the maximum duration (8 days) at the higher temperature. However, of the five accessions of cowpea, only seeds of TVu-9698 and TVu-13209 from the first harvest, and of TVu-13193 from the second harvest, showed an improvement in longevity compared with drying following the standard protocol. A negative effect of high-temperature drying was also observed in one accession of cowpea, TVu-11980, but only in seeds harvested later in the season, 13 weeks after planting. This research not only provides evidence of the potential benefits of drying orthodox seeds at an alternative, higher, temperature instead of at the conventional lower temperature, before long-term storage, but also raises awareness of how genebanks can improve the management of their accessions.

Rethinking the approach to viability monitoring in seed genebanks.

Seed viability monitoring, usually through a germination test, is a key aspect of genebank management; a low viability result triggers the regeneration of an accession in order to ensure that the genetic diversity of the accession is conserved and available for distribution. However, regular viability monitoring of large collections is costly in terms of seeds, labour and other resources. Genebanks differ in how they conduct their viability monitoring and how they collect, manage and store the data that are generated. In this article, we propose alternatives to the current norm of conducting an initial germination test soon after arrival at the genebank and then testing after regular, set storage intervals, as recommended in the Food and Agriculture Organization's Genebank Standards for Plant Genetic Resources for Food and Agriculture. We use real data from the International Rice Genebank (held at the International Rice Research Institute) to illustrate some of the issues regarding the accuracy and reliability of germination test results, in particular when they are used to predict the longevity of a seed lot in storage and to set viability monitoring intervals. We suggest the use of seed storage experiments on samples of seeds to identify which seed lots from a particular crop season to test first. We also give advice on the use of sequential testing schemes potentially to reduce the number of seeds used for viability testing; the use of tolerance tables to identify unlikely results when samples are subdivided into replicates; and what data to include in a genebank management database to improve the management of seed collections.

Resilience of rice (Oryza spp.) pollen germination and tube growth to temperature stress.

Resilience of rice cropping systems to potential global climate change will partly depend on the temperature tolerance of pollen germination (PG) and tube growth (PTG). Pollen germination of high temperature-susceptible Oryza glaberrima Steud. (cv. CG14) and Oryza sativa L. ssp. indica (cv. IR64) and high temperature-tolerant O. sativa ssp. aus (cv. N22), was assessed on a 5.6-45.4 °C temperature gradient system. Mean maximum PG was 85% at 27 °C with 1488 µm PTG at 25 °C. The hypothesis that in each pollen grain, the minimum temperature requirements (Tn ) and maximum temperature limits (Tx ) for germination operate independently was accepted by comparing multiplicative and subtractive probability models. The maximum temperature limit for PG in 50% of grains (Tx(50) ) was the lowest (29.8 °C) in IR64 compared with CG14 (34.3 °C) and N22 (35.6 °C). Standard deviation (sx ) of Tx was also low in IR64 (2.3 °C) suggesting that the mechanism of IR64's susceptibility to high temperatures may relate to PG. Optimum germination temperatures and thermal times for 1 mm PTG were not linked to tolerating high temperatures at anthesis. However, the parameters Tx(50) and sx in the germination model define new pragmatic criteria for successful and resilient PG, preferable to the more traditional cardinal (maximum and minimum) temperatures.

Seed development and maturation in early spring-flowering Galanthus nivalis and Narcissus pseudonarcissus continues post-shedding with little evidence of maturation in planta.

BACKGROUND AND AIMS: Seeds of the moist temperate woodland species Galanthus nivalis and Narcissus pseudonarcissus, dispersed during spring or early summer, germinated poorly in laboratory tests. Seed development and maturation were studied to better understand the progression from developmental to germinable mode in order to improve seed collection and germination practices in these and similar species. METHODS: Phenology, seed mass, moisture content and ability to germinate and tolerate desiccation were monitored during seed development until shedding. Embryo elongation within seeds was investigated during seed development and under several temperature regimes after shedding. KEY RESULTS: Seeds were shed at high moisture content (>59 %) with little evidence that dry mass accumulation or embryo elongation were complete. Ability to germinate developed prior to the ability of some seeds to tolerate enforced desiccation. Germination was sporadic and slow. Embryo elongation occurred post-shedding in moist environments, most rapidly at 20 °C in G. nivalis and 15 °C in N. pseudonarcissus. The greatest germination also occurred in these regimes, 78 and 48 %, respectively, after 700 d. CONCLUSIONS: Seeds of G. nivalis and N. pseudonarcissus were comparatively immature at shedding and substantial embryo elongation occurred post-shedding. Seeds showed limited desiccation tolerance at dispersal.

Advances in seed conservation of wild plant species: a review of recent research.

Seed banking is now widely used for the ex situ conservation of wild plant species. Many seed banks that conserve wild species broadly follow international genebank guidelines for seed collection, processing, storage, and management. However, over the last 10-20 years, problems and knowledge gaps have been identified, which have led to more focused seed conservation research on diverse species. For example, there is now greater ecogeographic understanding of seed storage behaviour and of the relative longevity of orthodox seeds, and we are therefore able to predict which species should be conserved using cryostorage techniques; seed development studies have identified when seeds should be harvested for maximal tolerance of desiccation and longevity in storage, as well as highlighting how seed development can vary between species; and there is now a wealth of literature on the dormancy-breaking and germination requirements of wild species which, as well as enabling better management of accessions, will also mean that their use in restoration, species reintroduction, or for evaluation for other applications is possible. Future research may be focused, for example, on nursery and plant production systems for wild plant species that maximize genetic diversity, so that introduced seeds and plant materials have the resilience to cope with future environmental stresses.

Seeds of alpine plants are short lived: implications for long-term conservation.

BACKGROUND AND AIMS: Alpine plants are considered one of the groups of species most sensitive to the direct and indirect threats to ecosystems caused by land use and climate change. Collecting and banking seeds of plant species is recognized as an effective tool for providing propagating material to re-establish wild plant populations and for habitat repair. However, seeds from cold wet environments have been shown to be relatively short lived in storage, and therefore successful long-term seed conservation for alpine plants may be difficult. Here, the life spans of 69 seed lots representing 63 related species from alpine and lowland locations from northern Italy are compared. METHODS: Seeds were placed into experimental storage at 45 °C and 60 % relative humidity (RH) and regularly sampled for germination. The time taken in storage for viability to fall to 50 % (p(50)) was determined using probit analysis and used as a measure of relative seed longevity between seed lots. KEY RESULTS: Across species, p(50) at 45 °C and 60 % RH varied from 4·7 to 95·5 d. Seed lots from alpine populations/species had significantly lower p(50) values compared with those from lowland populations/species; the lowland seed lots showed a slower rate of loss of germinability, higher initial seed viability, or both. Seeds were progressively longer lived with increased temperature and decreased rainfall at the collecting site. CONCLUSIONS: Seeds of alpine plants are short lived in storage compared with those from lowland populations/related taxa. The lower resistance to ageing in seeds of alpine plants may arise from low selection pressure for seed resistance to ageing and/or damage incurred during seed development due to the cool wet conditions of the alpine climate. Long-term seed conservation of several alpine species using conventional seed banking methods will be problematic.

Ecological correlates of ex situ seed longevity: a comparative study on 195 species.

BACKGROUND AND AIMS: Extended seed longevity in the dry state is the basis for the ex situ conservation of 'orthodox' seeds. However, even under identical storage conditions there is wide variation in seed life-span between species. Here, the effects of seed traits and environmental conditions at the site of collection on seed longevity is explored for195 wild species from 71 families from environments ranging from cold deserts to tropical forests. METHODS: Seeds were rapidly aged at elevated temperature and relative humidity (either 45 degrees C and 60% RH or 60 degrees C and 60% RH) and regularly sampled for germination. The time taken in storage for viability to fall to 50% (p(50)) was determined using Probit analysis and used as a measure of relative seed longevity between species. KEY RESULTS: Across species, p(50) at 45 degrees C and 60% RH varied from 0.1 d to 771 d. Endospermic seeds were, in general, shorter lived than non-endospermic seeds and seeds from hot, dry environments were longer lived than those from cool, wet conditions. These relationships remained significant when controlling for the effects of phylogenetic relatedness using phylogenetically independent contrasts. Seed mass and oil content were not correlated with p(50). CONCLUSIONS: The data suggest that the endospermic seeds of early angiosperms which evolved in forest understorey habitats are short-lived. Extended longevity presumably evolved as a response to climatic change or the invasion of drier areas. The apparent short-lived nature of endospermic seeds from cool wet environments may have implications for re-collection and re-testing strategies in ex situ conservation.

One-step analysis of seed storage data and the longevity of Arabidopsis thaliana seeds.

Seeds of two ecotypes of Arabidopsis thaliana, NW20 and N1601, were aged over a range of saturated salt solutions at temperatures between 6 degrees C and 55 degrees C. For each ecotype, the results from 37 storage experiments were summarized using the Ellis and Roberts viability equations and a modified version of these equations which allows for a proportion of 'non-respondents'. For both models, two approaches were taken in order to model the effect of moisture content (MC) and temperature on seed longevity. The first, a two-step approach, involved fitting individual survival curves and then multiple regression analysis of the fitted parameters on moisture content and temperature. For the second approach, the full viability models were fitted in one step, including the multiple regression for the effects of MC and temperature within the generalized linear model used to describe each survival curve. This one-step approach takes into account the full variability of the data and provides the best predictions of seed longevity based on the original assumptions of the Ellis and Roberts viability equations. As a consequence of taking into account all the variation, this one-step approach is more sensitive and thus more likely to detect changes due to reducing the number of parameters in the model as being significant. Whilst both approaches indicated that seeds from the two Arabidopsis ecotypes have the same response to MC and temperature, parameter values did differ between the approaches, with the one-step approach providing the better fit. The best model for these two ecotypes, from the one-step approach, confirmed a quadratic relationship between temperature and longevity, but the magnitude of the non-linearity is not as large as indicated by the universal value for the quadratic term.