Duration: 10/01/2013 to 09/30/2014

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

I. STATEMENT OF THE PROBLEM: Seeds are the foundation of agriculture. They are critical, strategic resources in commerce and civilization. High quality seeds are needed to produce food, often from plants bred for improved yield and nutritive value. Further, seeds are increasingly important to renew and rejuvenate degraded habitats, provide aesthetic and non-food products, and satisfy fiber and fuel needs. Seeds of weeds and invasive plants cause economic losses and environmental degradation. Seeds are the most economical means to preserve genetic resources for the future and are the delivery system for products generated through genomics and biotechnology. Thus, the ability of seeds to successfully carry out their biological function as propagules is critical in diverse agricultural and environmental contexts.

Seed genetic and physiological traits are critical to their quality and performance as propagules. Seed quality is reduced by adverse environmental conditions during seed development, by innate physiological mechanisms that delay or prevent germination, by the lack of vigor, and by loss of viability in storage. Biochemical, physiological, genetic, and environmental processes that influence seed and seedling quality, as well as performance, are complex and vary by plant species, but likely share common biological underpinnings. Biological processes in seeds need to be understood to reliably achieve field establishment of plants that thrive for their intended end-uses. Seed costs are increasing and represent a substantial recurring investment to agricultural producers. A broad research portfolio is needed to bring sound scientific results to problems that impact the global competitiveness of the U.S. seed industry, food production, commercialization of advanced technologies, protection and restoration of environmentally sensitive habitats, and the deployment of non-traditional plant species for fuel and non-traditional uses. This multistate project is unique in that it focuses on seeds as inputs to both natural and agricultural systems and how seed quality affects the ability of seeds to achieve the desired goals.

II. JUSTIFICATION: Seeds are the primary entities for propagation of food, feed, fiber, bio-fuel, and ornamental plants. America produces copious quantities of seed to grow a vast array of plants whose natural and agro-ecological niches are exceptionally diverse and in environmental conditions that are not always favorable for their germination, emergence, establishment, and persistence. The underlying principles that influence successful plant propagation are biologically based, as are the solutions to overcoming problems that limit establishment of desirable species. Whether at the level of the individual seed, variety, field, habitat, or ecosystem, the needs for seed biology research transcend individual geographic regions. This multistate project has had, and will continue to have, an important role in capitalizing on research opportunities that will successfully address various biological processes that enhance seed performance. Many productive research collaborations have been initiated through this multistate project, and the wide array of seed biology expertise among participants synergistically stimulates new approaches leading to relevant new biological insights. The outcomes from these collaborations foster technical innovations applicable to the wide range of crops and natural species.

The need, as indicated by stakeholders: Our stakeholders are those that produce and use seeds. These stakeholders need high quality seed to produce food, feed, fiber, bio-fuel, and other plant products, as well as validated information useful for the production of high quality seeds. They include academic, commercial and government sector scientists, seed producers, agronomists, horticulturists, land and natural resource managers, educators, extension agents, government agencies, and all industries that deliver seed to growers. Members of this multistate project are educating the current and future generations of scientists that will form the core leadership in the U.S. seed industry to maintain the high standard of seed quality and performance that is a hallmark of U.S. crop production. Maintaining this high standard has made the U.S. crop seed industry the recognized leader in the world with annual sales exceeding $57 and $96 billion from 2007 to 2009 (Anon. 2007; 2012: Table 812, 841). It is also important to encourage adoption of similar standards and enhancements to other species with rising economic potential. Not all species respond equally to the breadth or intensity of available seed technologies, and research is needed to achieve the best balance of treatment cost and seed performance appropriate for the stakeholders' desired uses. Increasingly, crop protection and other traits are delivered via the seed with corresponding increases in cost. Higher seed costs place greater demands and expectations on seed performance. Each farmer is sensitive to the need for rapid and uniform seedling emergence because it is the foundation of successful stand establishment that greatly affects potential yield.

The emphasis of this renewed project is placed on maximizing the quality and performance of seeds to meet the demands of new technologies. For example, the physiological quality of seeds is of increasing importance in conservation tillage and re-vegetation programs, where seeds are often planted under adverse seedbed conditions. In these cases, establishment and survival of the seedlings is the most crucial step. In addition, rapid advances in biotechnology and genomics underscore the importance of maintaining all germplasm resources indefinitely into the future. Genetic resources preserved in seeds will provide diversity upon which future advances in agricultural productivity will depend. Germplasm preservation assumes renewed importance since all genes are now potentially available for utilization in crop improvement. Seeds also deliver plant technology to the field. Farmers are required to invest greater capital in seeds that incorporate seed treatments, such as priming, coating, optical sorting, or value-added traits. The benefits of these sophisticated technologies can best be utilized if seed performance is optimized.

Both domestic and foreign seed companies maintain production and research facilities at various locations throughout the U.S. due to the climatic diversity, the ability to produce high quality seed, skilled farmers who specialize in seed production, and the size of our domestic markets. Seeds are increasingly produced in one location and marketed in another. This interstate and global commerce requires a high quality product capable of withstanding the rigors of shipment and storage, and performing reliably under a diverse range of field conditions. Meeting these demands requires cooperative research efforts in both production and utilization locations.

The importance of the work, and what the consequences are if it is not done: U.S. agriculture is the most competitive and productive agricultural industry in the world and is highly dependent on the quality of seeds utilized. Risk exposure to poor seed quality, even in the background of superior germplasm, is enormous, and would result in disruptive economic and social consequences accompanying yield reductions, fewer exports, higher food prices, and localized commodity shortages. The concept and provision of seed quality is well defined for most familiar agronomic and horticultural crops, but by no means optimized or evenly applied across species. Adoption of seed and seedling quality metrics is important for all utilized plant species. Since such metrics are necessarily species-specific, research is needed at the species level, as well as at the cellular level, where many genetic and environmentally responsive biological processes share common underpinnings but diverse effects. Exploiting traditional or non-traditional species for novel uses, such as those being developed for bio-fuels or high value oils, requires examination (or re-examination) of seed quality metrics to ensure growers and producers have the best chance of providing a high quality product to the consumer. Interruptions or inefficiencies in this supply chain have obvious economic consequences, and can be partially ameliorated by careful scientific attention to seed quality and performance. Loss of genetic resources and diversity through habitat destruction and supplanting traditional varieties and local species could have a long-term impact on the progress of plant improvement.

The unique biology of seeds as life in a suspended state and the specialized nature of the seed industry have given rise to Seed Biology as a distinct scientific and technical discipline. During the last three decades, this discipline has provided the American seed industry with the biological understanding and technical expertise needed to deliver a stable supply of the finest seeds to the U.S. agricultural industry and the world market. Most of the crops contributing to the annual U.S. agricultural productivity are grown from seeds, and the seed industry is a significant agricultural sector in its own right. In addition, exports of agricultural seeds represent a positive balance of trade for the U.S.

The advantages for doing the work as a multistate effort: There is a documented decline in the number of seed scientists graduating from land-grant universities, as well as a decline in the number of seed scientists charged with educating the next generation at these institutions. This is creating a gap of expertise in the seed industry and a declining capacity to meet this need. For example, 44 seed science faculty in 16 land-grant institutions graduated 183 students between 1990 and 2000, but declining support resulted in only 35 students trained in seed science from 2000 to 2005, with a loss of nine faculty and three states with active seed science programs (TeKrony 2006). With declining in-state programs, it is critical to view seed science research in a national context. Rapid progress in basic seed biology research using model species also enables more opportunities for collaborations among seed biologists in multiple states. Preparation and delivery of high quality seed technology as a traditional focus has largely been successfully ceded to the dominant seed production industries; however, these industries also benefit from new approaches and research in the public sector, and still rely upon university research to address complex seed physiology problems that impact product development. The advantage of a multistate project is to integrate individual activities and to leverage information gained from current state programs across the wide range of species and problems faced by seed producers and users nationwide. This multistate project serves as the only mechanism to unify seed science research across the U.S., bringing the national seed science expertise to bear on problems of local and regional significance.

This Regional Research Project was originally initiated in the Western Region over 35 years ago due to extensive seed production of horticultural, forage, and native species concentrated in this region. For example, seeds of cool-season grasses, carrots, beans, alfalfa, sweet corn, and cole crops are produced in the Pacific Northwest. Diverse vegetable and flower seeds, as well as rice, wheat, hybrid sunflower, cotton, alfalfa, and clover seeds, are grown in California and the Southwest. However, the importance of seed production extends beyond this area. Soybeans, corn, and sunflower seeds are grown in the Midwest. Re-vegetation shrub and chaffy grass seeds are produced in the Great Plains states. Peanut, cotton, grass, re-vegetation shrub, and fir tree seeds are produced in the Southeastern states. The diversity of seed production throughout the U.S., and the lack of any other regional projects devoted to seed biology or technology, led the project to expand to encompass land grant researchers across the U.S. Consequently, this project has played a critical role in coordinating public seed biology research at the national level.

Despite the diversity of species and locations involved in this project, fundamental aspects of seed biology are common to all. For example, while some patterns of gene expression and the accumulation of storage products are shared across species, understanding these processes requires an array of examples, due to the diversity of mechanisms and adaptations possible. Measurement and enhancement of seed quality present similar challenges and opportunities regardless of the species or location. It is precisely by examining seed biology from diverse perspectives, from the ecological to the molecular, that the entire biological picture becomes clearer and specific applications can be devised.

Developing solutions to these issues is central to the provision of an abundant supply of high quality seeds for agriculture. These issues are also complex, requiring unique skills, equipment, and methodologies. Utilizing a multistate effort by drawing on the expertise of specialized research scientists is the most efficient approach to addressing these issues on a national level. Despite recent advances in understanding the molecular biology of seeds, relatively little is known about how seeds germinate, why some seeds germinate better than others, why some seeds germinate before harvest, what causes dormancy, and why seeds die in storage. New fundamental knowledge about mechanisms underlying seed development, germination, and storability is required to solve these challenges. Seed performance must be improved: (1) for the U.S. to maintain our global competitiveness as an exporter of seeds as propagules; (2) to increase the efficiency of food production to preserve environmental quality; and (3) to take full advantage of advanced technologies. A clearer understanding of how environmental factors affect seed performance in natural as well as agricultural ecosystems is needed to ensure the continued vitality of native plant populations and the productivity of cropping systems. Successfully completing the stated objectives will provide not only an increased understanding of the factors that influence seed biology, but also practical methods to improve seed performance in the field.

The technical feasibility of the research: We are using the latest technologies, as well as developing new techniques, to investigate the central questions of seed biology and seed delivery systems. In most cases, the technical feasibility of the research procedures is proven as standard practice in the case of field-oriented research, or as an extension of established genetic, biochemical, and physiological principles. Results from genomic and proteomic approaches will likely yield new insights for practical application; however, there will likely be a time lag between discovery and adoption beyond the scope of this proposal.

Members from 12 states are working on projects that relate to Objective 1, two states' activities address issues relating to Objective 2, and two states have technology transfer projects with major focus on Objective 3, with primary attention to more than 25 distinct species. These objectives are not mutually exclusive, but represent the continuum between basic and applied research in meeting seed user needs for the future. We are one of the longest running multistate working groups in the USDA, with origins in the late 1970s. Our members are internationally recognized authorities on seed science, with many demonstrated accomplishments including two major international seed symposia since 2000, dozens of books and book chapters, hundreds of peer reviewed journal articles, and deployment of a series of on-line educational courses. Within the present group, at least 30 collaborations have yielded demonstrated results, and many additional projects are ongoing and planned. The future feasibility of achieving successful results through multistate collaboration is assured, given the prominence and productivity of the groups members in the recent past, as well as over the history of this multistate activity.

What the likely impacts will be from successfully completing the work: The projected impacts from completing this proposed work are as varied as the interests, issues, and species contributed by the members of the multistate project. One major impact of the proposed work results from coordinated research results across species and applications to generalize the innate biology of seeds as a first step to deploying improved technology to the end user. Progress on understanding the intrinsic mechanisms involved in seed development and limiting stand establishment is expected, as are the role(s) of specific genes, the environment, and their interactions. We expect results to increase efficiency and cost effectiveness of crop establishment and habitat restoration. Results will help to understand biological processes involved in seed dormancy and longevity, and germplasm preservation and the maintenance of species diversity. Significantly, transfer and development of seed technologies for the establishment of bio-fuel crops is essential for their widespread adoption, and progress is expected through collaborative efforts among members of this multistate project.

Some examples of prior impacts from this project include the identification of genes and mechanisms specifically associated with stress tolerance during germination, the development and commercialization of new methods to assess seed quality, the identification of mechanisms associated with seed deterioration and methods to delay or prevent this process, methods to alleviate seed dormancy, and models to quantify and predict germination of native and invasive species under natural conditions. The current project will extend these approaches, and by developing greater insight into the underlying genetic and physiological mechanisms, will enable increasingly powerful and effective technologies for improving, assessing and preserving seed quality. Seeds have increasingly become the delivery system for multiple biological and chemical technologies; therefore, expectations for and demands on seeds will require corresponding attention to maintain all aspects of seed quality. New discoveries, such as genes associated with seed dormancy or responses to enhancement treatments, can potentially provide synergistic improvements to seed quality by combining genetic and technological approaches. Similarly, proposed studies on seed coat permeability will enable more effective use of seeds to deliver crop protectants, greatly reducing the amounts of chemical pesticides applied per acre while increasing efficacy. Seeds represent a critical input into agriculture where multiple technologies can be combined for increased efficiency and reduced environmental impact.

Related, Current and Previous Work

Relationship to other projects: A search of the NIMSS database revealed two active multistate projects that peripherally deal with seeds: S1051 (Sustainable Practices, Economic Contributions, Consumer Behavior, and Labor Management in the US Environmental Horticulture Industry) and NC213 (Marketing and Delivery of Quality Grains and Bio-Process Co-products). There is no significant overlap between the objectives of W-3168 and other existing research projects. The NIMSS database review shows that the discipline of Seed Biology is multi-faceted, that a wide range of expertise is required to pursue fundamental scientific advances, and that W-3168 members are contributing the majority of active seed research documented in the database.

Current work of W-3168 member programs:
SEED DEVELOPMENT:
The Bennett group has explored the effects of environmental conditions (temperature, light, water) during seed development on the eventual quality of lettuce seeds. Seeds produced under higher R:FR light ratios had better germinability, poorer storage potential, lower sensitivity to external ABA, and lower ABA concentrations (Contreras et al. 2008; 2009).

The Cohn group investigates seed recalcitrance (desiccation intolerance) in Spartina alterniflora using comparative analysis with the orthodox-seeded S. pectinata. Oxidative stress is not the cause of recalcitrance, based on assays of free radical production, membrane leakage, total antioxidant titer, differential protein oxidation, and DNA fragmentation during drying (Chappell 2008; Chappell and Cohn 2008; 2011; In Press). Ongoing comparative proteomics studies show very different protein profiles (Wang et al. 2011). The results of these studies will provide tools to conserve germplasm of recalcitrant-seeded plants.

The Nonogaki group has been examining the regulatory mechanisms of gene expression in seeds focusing on the regulation of transcription factors by microRNA (miRNA) (Martin et al. 2010a; b; Martin et al. 2010c; Martínez-Andújar et al. 2011a; Nonogaki 2008; 2010; Nonogaki et al. 2008). This focus on an unexplored area of seed science will open the possibility to develop novel gene silencing technology to modify seed development and germination.

Peng and co-workers are focusing on epigenetic regulation of endosperm and seed development in rice by examining how the nutrient metabolic pathway genes and endosperm specific transcription factors are subject to epigenetic regulation, such as histone modifications and DNA methylations. Our research suggests that epigenetic regulation may open a new avenue for seed quality and crop production improvement in addition to advancing our understanding in the regulation of seed development.

Somatic embryogenesis is being used by Perrys lab as an alternative system to study the early stages of embryo development.

Pérez and co-workers have linked the ability of palm embryos to accumulate dry matter throughout development with desiccation sensitivity (Pérez et al. 2012) and are developing a model of allowable cell shrinkage. The model provides a framework to quantify desiccation tolerance via mechanical strain when embryo cells shrink during drying.

SEED DORMANCY:
The Bradford lab has identified a gene involved in regulating thermodormancy (a failure to germinate when imbibed at temperatures above 25-30°C) in lettuce (Argyris et al. 2005; Argyris et al. 2011). Gene/promoter transfer, gene silencing and mutation approaches demonstrated that LsNCED4 encoding a key enzyme in the ABA biosynthetic pathway is necessary for thermodormancy. Future studies will identify additional loci controlling germination and investigate further roles of ABA, GA and ethylene. Identifying genetic loci associated with the sensitivity of seed dormancy to environmental conditions during seed development and germination will enable more consistent production of higher quality seeds (Chiang et al. 2011).

The Geneve group has been studying physical dormancy (PY), which is caused by one or more water-impermeable layers of palisade cells in the seed (or fruit) coat (Baskin and Baskin 2000). The breaking of PY involves disruption or dislodgement of water gap structures that act as environmental signal detectors for germination. Water gap structures have been identified in approximately two-thirds of the families with PY. Less is known about the environmental cues that break PY, and we have a poor understanding of the physical and physiological mechanisms acting to alter the water gap to permit water uptake.

The Gu lab has developed weedy rice as a model system to elucidate genetic, evolutionary and molecular mechanisms that regulate natural variation for seed dormancy in grass species and to identify novel genes to improve resistance to pre-harvest sprouting. A total of 10 quantitative trait loci (QTL) for seed dormancy have been identified from the system and also characterized for phenotypic associations with other adaptive traits (e.g., seed shattering, pigments, and appendages) and for epistasis or genotype-by-environmental interactions (Gu et al. 2003; Gu et al. 2004; 2005a; b; 2006a; b; Gu et al. 2005c; Ye et al. 2010). A marker-assisted genetic approach was developed to determine genes/QTL involved in the regulation of seed dormancy through the embryo or maternal tissue (Gu et al. 2008). The qSD7-1 seed dormancy QTL was map-based cloned from weedy red rice as a transcription factor with pleiotropic effects on ABA and flavonoid biosynthesis in early developing seeds (Gu et al. 2011), and the qSD12 QTL was delimited to a genomic region of several candidate genes (Gu et al. 2010). The lab has also been evaluating the genetic potential for use of wild wheat (Aegilops tauschii)-derived synthetic lines as the donor of seed dormancy genes to improve resistance of common wheat to pre-harvest sprouting (Gu et al. 2010).

The Nonogaki lab has been studying the function of 9-cis-epoxycarotenoid dioxygenase 6 (NCED6), an ABA biosynthesis gene, in seed dormancy and germination using a chemically inducible gene expression system (Martínez-Andújar et al. 2011b). This project has already provided a proof of concept for hormone engineering in seeds and will make an impact on the prevention of precocious germination, such as preharvest spouting in wheat.

The Steber group is using Arabidopsis as model to understand the role of GA signaling genes in controlling seed dormancy by determining whether differences in expression of the GA receptor GID1 control germination both in Arabidopsis and in wheat. The role of ABA in controlling pre-harvest sprouting is being studied in wheat by mapping and cloning genes from an ABA hypersensitive mutant (Zak ERA0).

SEED GERMINATION:
The Bradford lab and others have developed hydrotime and hydrothermal time models to describe seed germination responses to environmental (temperature, water, oxygen, light) and physiological (hormones) factors (Allen et al. 2007). These approaches enable characterization and quantification of the population behavior of seed germination and extend them to both genetic and ecological applications, such as in weed control (Bradford 2005).
The McGrath group has been analyzing sugar-beet germination transcriptomes to identify genes that play a role in increased seedling vigor seen with seeds germinated in hydrogen peroxide. The high germination of beet seeds treated with hydrogen peroxide has been linked to expression of a putative oxalate oxidase and also to the induction of the glyoxylate cycle. These biochemical changes appear to involve MAP kinase cascades and the central modulator mTOR pathway. Understanding this remarkable phenomenon will directly impact creation of highly vigorous varieties that emerge and establish earlier in the season.

The mechanisms of seed germination have been analyzed in the Nonogaki lab using tomato, a crop model, where genes specifically expressed in the micropylar region of endosperm (called endosperm cap) have been identified (Martínez-Andújar et al. 2012) and also using an embryo-arrest mutant that still showed germinability (Kristof et al. 2008). The endosperm cap-specific genes are germination-specific and provide good markers to monitor progression of seed treatment such as seed priming.

The Steber lab has been analyzing the role of GA in germination and in stem elongation; they have identified a GA-stimulated F-box protein that interacts with negative regulators of protein breakdown in the cell and have established that separate regulators exist for germination-related events and stem elongation-related events. This fact suggests that such F-box proteins could be altered to make plants that are shorter and therefore more lodging resistant without causing problems with poor germination and seedling emergence.

The Welbaum group has been evaluating low temperature germination of peanut lines differing in oleic and linoleic acid content to establish whether a relationship exists between fatty acid content and low temperature germination performance.

The Yan group is examining the genetic relationship between dormancy and vernalization in winter wheat, a process that has been little studied. Three vernalization genes have been cloned, VRN1(Yan et al. 2003), VRN2 (Yan et al. 2004), VRN3 (Yan et al. 2006); more recently, they have determined that physiological maturity of seeds might also be regulated by FT-D3 on chromosome 7D in hexaploid wheat (Chen et al. 2010b; Wang et al. 2009). These results suggested pleiotropic roles and various molecular mechanisms of the FT-D3 gene in regulating seed development and maturity in wheat. Mapping studies are also underway in recombinant inbred lines derived from a cross between a cultivar (Jagger) with weak seed dormancy, but earlier physiological maturity, and a cultivar (2174) with strong seed dormancy but later physiological maturity. Preliminary results show that the locus controlling seed dormancy was associated with MFT, the Mother of FT gene, providing the first direct evidence for the linkage between the genes for dormancy and vernalization in winter wheat.

The Downie group identified proteins of the stored proteome in seeds that bind preferentially to enzymes imparting protection or repair implicated proteins of the translational apparatus involved in seed longevity (Chen et al. 2010a; Kushwaha et al. 2012). The 3 end processing and polyadenylation of seed messages have been cataloged for Arabidopsis (Xiaohui et al. 2011). Deficiencies in specific cellulose synthase subunits affect mucilage production on the Arabidopsis testa (Mendu et al. 2011). These investigations permit greater sophistication in the long term storage of germplasm.

The Pérez group examined the interactions between light, nutrients, and carbohydrates on the germination of Bletia purpurea, an orchid of conservation concern, in an asymbiotic germination system. The information translates into development of effective ex situ germination strategies. Studies on the influence of heat stress on the germination of native wildflower germination have also been conducted (Kettner and Pérez 2012). These studies form the basis for continued study on the interaction between seed functional traits and the response to heat stress. Seed desiccation tolerance-cryopreservation studies of sea oats (Uniola paniculata), a keystone dune species, are underway in attempts to determine the feasibility of developing a U. paniculata genebank.

The Goggi group conducted seed germination studies in Miscanthus sinensis, a very promising species for biomass production. Using a thermogradient table, seed germination temperatures were established and a seed germination protocol is being developed.

SEED ECOLOGY:
Brachypodium distachyon is a potentially invasive grass species whose seeds exhibit dormancy (Barrero et al. 2012). In many cases, seed dormancy characteristics are decisive in determining the success of invasive species in new environments (Kimball et al. 2011; Meyer and Allen 2009). In the case of B. distachyon, greater phenotypic plasticity is associated with polyploidy and with greater invasive success (Bakker et al. 2009). The Bradford lab will test whether greater phenotypic plasticity in germination and dormancy characteristics of B. distachyon genotypes is also associated with polyploidy and with increased ability to succeed in stressful environments (Manzaneda et al. 2012). The results will contribute to understanding the nature of invasiveness and developing strategies to prevent it.

The Pérez lab has elucidated the germination ecology and dormancy mechanisms of several native wildflower taxa used in wild land restoration projects throughout the southeastern region. This information has been translated to a management context, thus providing restoration practitioners with guidelines on the establishment of selected taxa for in situ and ex situ propagation.

SEED VIGOR, VIABILITY AND TESTING:
The Bennett and Jourdan group has examined the relationship between internal seed morphology, germination, and vigor of eggplant (Solanum melongena L.) and Portulaca spp. using free space analysis. For P. grandiflora seeds, there is a direct relationship between increased free space and reduced germination, although not for seed vigor (Neumann-Silva et al. 2012 in press).

Both the Bradford group and the Welbaum group have been adopting technological tools for more efficient study of viability and vigor. Bradfords group is using the ASTEC Q2 (an instrument capable of measuring respiration rates from individual seeds during imbibition and germination) to identify respiratory patterns associated with high and low seed quality. In particular, the effects of respiratory inhibitors on initial seed respiration and germination will be assessed in relation to seed quality. Using data analysis approaches developed from population-based threshold models, the Bradford lab will also explore developing the Q2 instrument into a tool for rapid evaluation of controlled deterioration tests to predict potential seed longevity in storage.

The Welbaum team has created a gradient table with a series of parallel aluminum gussets welded lengthwise to connect the ends of the table. The gussets are predicted to maintain a gradient in soil or other growing media so that experiments can be conducted above the table surface with minimal temperature fluctuations. This design addresses constraints of conventional thermogradient tables that generally limit experiments on the effect of temperature on germination to shallow containers.

The Bradford lab is testing whether manipulation of seed isotherms could be used to extend seed longevity in storage. Modified seed drying protocols based on this work could considerably extend seed longevity. Novel zeolite desiccant beads (drying beads) have been developed that can rapidly dry seeds in sealed storage containers. The beads can be regenerated by heating and used repeatedly and indefinitely. The lab is engaged in an international project to make these drying beads available and to provide training in their use to preserve seed quality in both developing and developed countries.

The Pérez lab has developed seed viability screening procedures and optimal germination assays for two ecological keystone species (Aristida stricta, Uniola paniculata) in the southeast region. This information assists in upland and dune habitat restoration and conservation of these important taxa.

The Iowa team studies seed vigor and seed storage of Zea mays and Glycine max. Knapps lab investigates the seed vigor in maize. The Goggi lab investigates soybean seed longevity, seed composition, and seed vigor response to soil fertility, environmental stress, and seed treatment.

STAND ESTABLISHMENT:
Leskovars group emphasizes seed-transplant physiology in response to abiotic stress, particularly drought and high temperature. Mutants exhibiting altered ethylene synthesis or sensitivity have been identified that also exhibit germination tolerance. The team is developing and optimizing application methods in the nursery to mitigate the negative effects of transplant shock that lead to poor stand establishment in the field. The positive effect of ABA on regulating transpiration, while maintaining a favorable water status, and control of root/shoot growth in several species, including pepper (Goreta et al. 2007), tomato, melon, watermelon and artichoke have been documented. Currently the lab is investigating cell division and elongation patterns related to alterations in seedling growth rates, as well as mechanisms involved in transient ABA-driven leaf chlorosis. Understanding morphological and physiological responses of seedlings and transplants to abscisic acid (and other PGRs) can provide a basis to develop transplant conditioning treatments to enhance stand establishment.

Seed treatments are efficient for early season pest management and can reduce pesticide usage by 90%. Taylors lab has developed specialized facilities for seed treatment and coating applications. Many new chemical seed treatments have systemic activity, and the efficacy of systemic seed treatments depends on the ability of applied chemicals to be taken-up and then transported into the developing plant. Seed coat permeability was investigated using fluorescent tracers that provided a range of log Kow values (lipophilicity) and electrical charge (nonionic, cationic or anionic), and avoided the use of labeled pesticides. Seed coat permeability of a particular seed species to solutes was grouped into three categories: 1) permeable, 2) selectively permeable, and 3) non-permeable. Therefore, the ability of a particular compound to diffuse through the seed coat was determined by the chemical nature of the seed covering tissues and the physicochemical properties of the compound applied (log Kow and electrical charge). Systemic tracers that failed to permeate seed coats during seed imbibition were taken up by roots after visible germination (Taylor and Salanenka 2012).

Objectives

1. Identify and characterize biophysical, biochemical, genetic, and environmental factors regulating or influencing seed development, germination, vigor, dormancy, and longevity.
   
2. Determine and model the biotic and abiotic factors affecting seed germination, seedling emergence, and establishment of sustainable populations in natural and agro-ecological systems.
   
3. Develop, evaluate, and transfer technologies to assess and improve seed and seedling quality, health, performance, utilization, and preservation.
   

Methods

Participants in W-3168 represent a wide range of interests and expertise. Among the 14 states participating, 12 have responsibilities examining processes that influence seed development, germination, vigor and dormancy; four have programs targeted towards developing, evaluating and transferring technologies to customers and stakeholders; and two are confirming and extending existing knowledge towards new environments (e.g. habitat restoration, invasive species). Most programs employ biochemistry, physiology, genetics, microscopy, gene expression, transformation, field evaluation, and extension activities. The seed biology models employed are more varied than topic areas, with over 23 plant groups being examined including row crops (maize, sorghum, soybean, sugar beet), field crops (canola and wheat), horticultural crops (carrot, lettuce, pepper, turf grasses, watermelon), and a dozen species with varied uses, habitats, and economic impacts (endangered and invasive species, native forbs and grasses, orchids, weeds and trees, Arabidopsis, and a host of specialty crops for biofuel and other uses). Information gained in one of these topic areas, species groups, or methodological approaches is often directly applicable in other species, at least broadly as is consistent with the goal of W-3168. Specific methods associated with individual projects are described below. Sharing of results from various methods is the essential component during annual meetings, and serves as the primary means by which general principles from novel insights on a wide range of systems are integrated by the group. Methods for Objective 1: Identify and characterize biophysical, biochemical, genetic and environmental factors regulating or influencing seed development, germination, vigor, dormancy, and longevity. OH: Experiments on Asteraceae species (Taraxacum spp., other) will continue the assessment of environmental factors that significantly influence seed development and quality. Greenhouse, growth chamber and field studies will be employed to build on results from lettuce seed research (especially light quality influences). KY: Arabidopsis mutants will be used to elucidate the influence of isoaspartate and light on seed longevity in storage and germination. A better understanding of the stresses imposed upon the seed proteome may suggest techniques to better prepare seeds during development to rapidly and efficiently repair this damage. A thorough investigation of the interaction between CTG10 and PIF1 will elucidate light control over germination. Identification of the gene responsible for the bs1 syndrome will improve understanding of the testa. Using phage display, selected Arabidopsis and soybean late embryogenesis abundant (LEA) protein interactors (client proteins) have been and will continue to be identified for each of the 9 LEA families. WA: The goal is to apply knowledge about ABA and GA hormone signaling to control pre-harvest sprouting and emergence issues in wheat. Too much grain dormancy leads to poor seedling emergence associated with reduced yield, whereas too little grain dormancy leads to problems with pre-harvest sprouting when cool rainy conditions occur before harvest of mature grains. Arabidopsis is used to understand the role of GA signaling genes in controlling seed dormancy. Specifically, Steber will determine whether differences in the expression of the GA receptor GID1 control seed dormancy in Arabidopsis and in wheat. Wheat mutants that are ABA insensitive after-ripen more quickly, whereas ABA hypersensitive mutants show increased seed dormancy (Schramm et al. 2010; Schramm et al. 2012 (In Press)). A specific ABA hypersensitive mutant Zak ERA0 will be mapped, cloned and deployed as a gene to reduce the risk of sprouting in white grained wheat. KY: Research on physical dormancy (PY) will determine the seed water gap in those families where the water gap has not been adequately described. In addition, the mechanism controlling PY in the Geraniaceae will be studied. Treatments (dry or wet heat) will be applied to release seeds from PY. Dye tracking experiments will determine the initial point of water entry (water gap) into the seed. Morphological and anatomical studies will identify the type and putative mechanism of the water gap using EM, light microscopy and paraffin sections on seeds before and after PY break. WV: (a) Fourier Transform Infrared Spectroscopy (FTIR) is used to develop a rapid, nondestructive detection method to identify seeds contaminated with BFB and other pathogens. (b) The mucilage produced by seed coats can be removed using diluted hydrochloric acid. The seeds with and without mucilage can be incubated in different media, to test the function of the mucilage during seed germination. Seeds with and without mucilage will be inoculated with Salmonella, to see if the mucilage promotes or inhibits growth. (c) A one-dimensional thermogradient table is being used to assess germination performance of peanuts with different ratios of oleic / linolenic acid. SD: For seed dormancy in rice, map-based cloning will be used to isolate three additional QTL, and genomics and molecular biology approaches used to characterize regulatory mechanisms of the QTL underlying genes. For seed dormancy in wheat, QTL analysis and marker-assisted selection techniques will be used to transfer dormancy genes into the local cultivars to improve resistance to pre-harvest sprouting. A new project on seed longevity will be initiated to elucidate genetic and molecular mechanisms of seed longevity in the rice model. Segregation populations will be developed from crosses between weedy and cultivated rice; both natural and accelerating aging methods will be used to assay genotypic differences in seed longevity; QTL analysis will be used to map QTL associated with the longevity trait; and the newly mapped QTL compared with seed dormancy loci identified in this and other labs to infer if there is an overlapping genetic basis between these two traits. LA: Putative protective proteins required for desiccation tolerance will be identified. Loss of desiccation tolerance (DT) of orthodox seeds will be compared to recalcitrance (RCT). (a) The cause of recalcitrant seed death in Spartina alterniflora will be determined, which will suggest seed treatments and/or breeding solutions to improve its longevity in storage. S. alterniflora and S. pectinata seeds will be flash dried (0-24h at 23°C) to various moisture contents above and below the critical moisture content (40% dwb) for RCT. Optimization of sample processing and electrophoresis have been completed (Wang et al. 2011). (b) Protein profiles will be compared between the two Spartina species (whole seed total extract; embryo total extract; and respective heat stable extracts), adapting methodology from Boudet et al. (2006). Using these protocols, proteins associated with dormancy will be characterized in both Spartina species by comparing dormant vs. moist-chilled seeds (stratification). MI: Reference transcriptomes will be obtained from the sugarbeet inbred 'C869' at seven specific stages of early season growth, 6 hours post-imbibition, 48 hours post-imbibition (at the time of radicle protrusion), and roots at 1.5-, 3-, 7-, and 10-weeks after emergence, and combined leaf and crown tissue for the later four waypoints. Choice of these time points is dictated by previous work (Naegele 2010; Trebbi and McGrath 2009), and appears to cover the major transitory phases of seedling to adult growth. The approach will be via RNA-Seq using the Illumina HiSeq 2000. One hundred base pair paired-end sequences will be aligned and assembled using the C869 draft genome sequence and the Tuxedo suite of software tools (Bowtie, TopHat, and Cufflinks). Recombinant inbreds have been developed that differ in seedling vigor, and from field phenotypic observations (e.g., germination, field stand counts, stress germination, seedling disease reaction, sucrose content, biomass production), selected RILs will be used to test hypotheses relating gene expression to trait expression. Not all RILs will be tested, but rather those showing extremes of phenotypes present within a RIL population. Molecular phenotypes of selected RILs will be determined /confirmed via qPCR using three biological replicates and two technical replicates per treatment. OR: The GeneSwitch technology, a chemically inducible gene expression system, will be used to engineer hormone levels in developing and mature, imbibed seeds. The chemical ligand methoxifenozide will be applied to siliques containing developing seeds or imbibed seeds to alter hormone balances in seeds. In addition to the GeneSwitch approach, a system to cause spontaneous overexpression of hormone metabolism genes will be created using seed-specific promoters. Database search and a reverse transcription (RT)-PCR approach with degenerate primers will be used to isolate wheat and sorghum genes associated with hormone metabolism and signal transduction. Those genes will be expressed using the chemically inducible gene expression system or spontaneous over-expression using seed-specific promoters. Possible interactions of seed dormancy-associated genes will be tested using an effecter-reporter assay in a transient system, such as Nicotiana bethamiana leaf infiltration assay. KY: To identify DNA sites to which embryonic transcription factors (TFs) bind in vivo, chromatin immunoprecipitation (ChIP) will be utilized to immunoprecipitate these TFs and associated DNA fragments. ChIP is performed by introducing an epitope tag on the TF of interest, validating that the tag does not interfere with function, and then performing the immunoprecipitation using commercially available antibodies. Using ChIP-on-chip, the DNA recovered from immunoprecipitations, as well as control immunoprecipitations, will be converted to hybridization probes to hybridize for the Affymetrix GeneChip Arabidopsis Tiling 1.0R Array. This will allow nearly global mapping of in vivo binding sites for the TFs of interest. Expression microarrays or data publically available in the databases is then used to investigate gene expression changes in response to accumulation of the TF of interest. The results of these experiments will determine the genes that may be directly regulated by the TF or may be farther downstream in the regulatory network. For work in soybean, sequencing approaches to look at in vivo binding will be necessary since a tiling array does not exist for soybean. Protein-protein interactions will be further investigated by yeast 2-hybrid, in planta co-immunoprecipitations, and/or BiFC. Co-regulation of genes by AGL15 and interacting proteins will be examined with ChIP. OK: Seed dormancy of each of 282 RILs and BC populations will be characterized under controlled temperature and light conditions. Dormancy tests will be conducted with the seeds treated with cold, heat, and room temperature (control). The MFT gene will be investigated for its allelic variation in sequence, transcript level and regulation by temperature. Interacting proteins with MFT will be investigated. An intersection between vernalization and seed dormancy in wheat will be established. MS: Methods include mutant screening and characterization, histone modification binding sites identification using ChIP-Seq methods, and DNA methylation site identification with bisulfite sequencing. CA: Lettuce and Arabidopsis mutants exhibiting altered ethylene biosynthesis or sensitivity, as well as high temperature germination capacity, will be characterized phenotypically and genotypically. In lettuce, bulked segregant analysis utilizing DNA sequencing will be used to identify the genes associated with the causal mutations (Laitinen et al. 2010; Schneeberger et al. 2009). Once candidate genes are identified, gene expression analysis, comparison with Arabidopsis mutants and transgenic over-expression and silencing will be used to confirm their roles in regulation of germination. To analyze maternal effects on seed dormancy, recombinant inbred line (RIL) populations will be grown to produce seeds at low (20-25°C) and high (30-35°C) temperatures in greenhouses and also in diverse field environments. Seeds will be phenotypically characterized, and QTL mapping will be performed to identify loci associated with the variation in dormancy phenotypes between the different growth environments. Hydrotime and hydrothermal time analyses will be conducted as previously described (e.g., (Alvarado and Bradford 2005)). FL: Thermal time and hydrothermal time modeling, in conjunction with biophysical analysis, will be used to investigate germination rate limitation of mass-segregated, wild-collected, native wildflower germplasm exposed to heat stress. Survival analysis will be used to develop models of desiccation- and cryo-tolerance of wild germplasm used in restoration and conservation programs. Methods for Objective 2: Determine and model the biotic and abiotic factors affecting seed germination, seedling emergence and establishment of sustainable populations in natural and agro-ecological systems. CA: Both diploid and tetraploid lines of B. distachyon will be obtained (Filiz et al. 2009) and grown in environments varying in salinity. Seeds will be harvested and characterized for germination and dormancy characteristics and for plasticity if those characteristics. The relationship of phenotypic plasticity to genotype, ploidy, and maternal environment will be analyzed. Common garden experiments will be conducted over multiple life cycles to determine relationships between germination phenology and measures of fitness. FL: Germination chamber screenings and in situ germination phenology studies will be used to uncover potential dormancy mechanisms in wild germplasm of Asteraceae and Poaceae, germination timing under lab and field conditions, and the seed interaction with the environment to alleviate dormancy and promote germination. Methods for Objective 3: Develop, evaluate and transfer technologies to assess and improve seed and seedling quality, health, performance, utilization, and preservation. CA: (a) The relationship of seed respiration rates to seed quality will be assessed using the ASTEC Q2 instrument. Individual seeds are imbibed in sealed wells of 96-well plates or individual vials. As the seed imbibes and respires, oxygen is depleted in the well, which is recorded by a moving sensor that shines light on a dye spot on the sealing membrane and records the fluorescence. Indices derived from these data will be compared to other vigor tests. Analysis methods have been developed to utilize population-based threshold models to analyze respiration data, and these will be utilized to extend the applications of the Q2 to seed quality assessment. Metabolomics analyses of respiratory metabolism will be conducted for seeds in specific respiratory states identified in the Q2. For isotherm studies, seeds will be equilibrated at a range of established relative humidities using saturated salts. Seeds will be either pre-hydrated or pre-dried to determine whether they are on their sorption or desorption isotherms. They will then be sealed and aged for various periods at 40 or 50°C and tested for germination and viability. (b) Studies will be conducted on the ratios of drying beads to seeds needed to attain safe storage moisture contents. Modifications to the beads will be tested to improve their performance and reduce costs. NY-Geneva: Seed coating technologies will be adapted and utilized on a wide range of crop and non-crop seeds, including the development of seed agglomeration technology. Coating technology can serve as a carrier of plant protectants and to facilitate sowing. Efficacy of seed treatments for control of insects, nematodes, fungal and bacterial pathogens will be assessed in cooperation with pest management specialists. Controlled release technologies for seed treatment agrochemicals will be developed. Seed treatments to enhance plant growth and development will be studied. Seed enhancement and conditioning technologies will be explored for warm-season grasses and oil crops as feedstocks for bioethanol and biodiesel, respectively. Seed coat permeability will be investigated using nanotechnology and nanoparticles. OH: Combinations of bio-control, PGR and priming treatments will be tested to improve germination and emergence of a range of vegetable and flower species. Imaging of young seedlings will be employed to assess treatment effects. Greenhouse and field studies will evaluate the potential for treatments to mitigate challenges to seedling establishment. TX: Seedling conditioning treatments with ABA and other growth promoters (or inhibitors), such as GA3 and ethylene, will be investigated to regulate plant growth (height), enhance stress tolerance and plant recovery after transplanting. Treatments will include various concentrations and timing of applications under control (well-watered) and drought stress (dehydration). Laboratory and greenhouse studies will be conducted on the following species: pepper (sweet and pungent types), watermelon, melon and artichoke. Shoot growth and its components (stem, leaves) and root growth and its components (taproot, laterals, adventitious and root hairs) will be evaluated. Standard growth measurements will be performed, including chlorophyll content, gas exchange, and scanning techniques with image analysis system for root growth. During the second phase of the project, the involvement of ABA in regulating the antioxidant defense mechanism under drought stress conditions will be investigated.

Measurement of Progress and Results

Outputs

• Research activities will continue to produce peer-reviewed journal articles (e.g. > 150 peer-reviewed articles from 2008-12), books, book chapters, edited books, conference proceedings, patents, educational materials, abstracts, and public outreach materials
• Information regarding gene networks involved in stress responses, dormancy, germination, disease-resistance, and storage longevity
• Practical seed science knowledge for policy and decision making within industry, governmental, and non-governmental agencies
• New seed coating, germination equipment, germination enhancement, and seed storage technologies with patent potential
• New method to identify seed-borne pathogens

Outcomes or Projected Impacts

• Molecular biology research will advance understanding of post-transcriptional regulation of gene expression in seeds
• Understanding of gene expression patterns in developing, dormant and germinating seeds will facilitate development of new hybrids resilient to environmental changes
• Restoration practitioners and seedling producers can better plan and coordinate in terms of seeding activities for restoration of degraded lands
• Conservation practitioners can adapt methods for ex situ activities
• Changes in seed technology and equipment will improve research opportunities, reduce seed losses and associated costs, increase yields, facilitate management of seed inventories, and increase seed industry profits
• Outcome/Impact 6; Identification of seed borne pathogens will protect animal and human health, increase efficiency within supply chain, and limit seed losses

Milestones

(2014): Examine seed permeability to nanoparticles. Determine factors limiting germination of wildflowers. Elucidate protein-protein interactions using phage and yeast assays. Explore functions of proteins of the translational apparatus. Manage PGR applications for vegetable transplants. Test seed treatments for improved establishment of small seeds. Identify genes associated with ethylene in lettuce seed thermodormancy. QTL analyses of phenotypic variation due to maternal environment in lettuce. Apply Q2 respiration assays for seed quality assessments. Develop a mapping population for Zak ERA0 wheat ABA hypersensitive mutant. Determine water gap morphology. Relate FTIR technology to seed quality. Determine role of seed mucilage. Clone gene(s) underlying qSD12 seed dormancy QTL. Identify desiccation tolerance proteins in Spartina. Obtain transcriptome sequence of non-stressed 'C869' sugar beet. Characterize Arabidopsis transgenic lines expressing dormancy-associated genes. Identify NCED6 orthologs. Map binding sites for embryonic TF FUS3. Assess transgenic soybean for effect of 35S:GmAGL15. Fine-map QTL affecting seed dormancy. Develop BC populations for seed dormancy analysis. Identify binding sites of H3K27me3 in rice endosperm

(2015): Develop controlled release seed treatment technologies. Evaluate seed sorting and free space analysis. Describe genes and regulatory networks involved in lettuce thermodormancy. Life cycle and fitness experiments in Brachypodium. Manipulate seed drying via hysteresis. Map the ZakERA0 gene. Prepare a review article summarizing water gap morphologies. Identify relationship between desiccation- and cryo-tolerance in Uniola. Develop a modified thermogradient table. Understand the role of fatty acids in determining peanut seed quality. Publish a new text book on vegetable seed production. Clone the qSD1-2 seed dormancy QTL. Identify proteins associated with the loss of dormancy via stratification in Spartina. Obtain transcriptome sequence of stressed seedlings. Analyze the impacts of altered hormone levels on gene expression profiles in Arabidopsis. Apply Gene Switch system to hormone engineering in crop species. Map binding sites for at least one additional embryonic TF. Investigate proteins that interact with AGL15. Identify allelic variation in MFT-A. Generate rice mutants for selected genes subjected to the regulation of H3K27me3 and DNA methylation.

(2016): Develop seed enhancements to improve plant growth and development under stress. Clarify molecular interactions among regulatory pathways controlling germination and dormancy. Develop improved lettuce germplasm for public release. Identify biophysical methods to address germination rate limitation. Describe relationships between polyploidy, phenotypic variability and fitness for B. distachyon. Determine whether expression of GID1 is regulated by after-ripening or cold stratification. Identify beneficial seed bacteria. Develop a high throughput FTIR system for sorting seeds. Develop cantaloupe cultivar resistant to BFB disease. Clone qSD7-2 seed dormancy QTL. Compare protein profiles associated with the loss of rice seed desiccation tolerance. Examine recombinant inbred selections under stress for changes in gene expression patterns. Identify regulatory mechanisms downstream of ABA biosynthesis and signal transduction. Examine hormone effects on somatic embryogenesis. Characterize MFT genes. Characterize mutants for roles in rice seed and endosperm development.

(2017): Correlate lab and greenhouse studies against field performance. Assess PGRs and bio-controls as seed enhancements. Improved lettuce germplasm will be utilized in breeding industry. Determine whether expression of GID1 is regulated by after-ripening or cold stratification. Identify role of seed mass in germination rate limitation. Develop a natural bacterial treatment to protect plants. Pyramid QTL alleles. Create spontaneous hyperdormancy system in crops species. Examine fun

Projected Participation

View Appendix E: Participation

Outreach Plan

The members of W-3168 comprise a group of highly dedicated seed biologists who excel in the communication of their research findings. All members of the W-3168 project are active participants in seed research at universities and federal facilities throughout the country. They provide leadership in this vital area through undergraduate and graduate instruction, as well as by mentoring graduate and undergraduate research. A number of our members conduct extension workshops to provide the seed industry with a thorough orientation to seed biology fundamentals, as well as the latest cutting edge results. For example, the Seed Biotechnology Center at UC Davis (Bradford) offers courses in seed biology and breeding technologies to the public and seed professionals that incorporate the latest information generated through W-3168 (http://sbc.ucdavis.edu). The Iowa State Seed Center (Knapp, Goggi) also offers regular courses and workshops in topics related to seed biology, conditioning and marketing (http://www.seeds.iastate.edu/).

As documented in the projects annual reports, W-3168 members regularly publish their finding in top-tier, peer-reviewed journals, targeting both the general plant biology and seed biology communities. W-3168 members are also active participants and presenters at various professional society annual national/regional meetings, as well as at the major workshops and symposia sponsored by the International Seed Science Society and the International Society for Horticultural Sciences. W-3168 members serve on journal editorial boards and/or as ad-hoc manuscript reviewers, publish books and book sections on seed biology (Allen et al. 2007; Bewley et al. 2012; Bradford and Nonogaki 2007; Perry and Yuan 2011; Pluskota et al. 2011), and obtain patents for intellectual property (Frey et al. 2009; Madsen et al. 2010; McDonald et al. 2005; Taylor et al. 2011). To date there are 40 labs that have links to their home pages on the American Seed Research Alliance (ASRA) web site (http://dept.ca.uky.edu/asra/).

Organization/Governance

Organization will follow recommendations for the Standard Governance for multistate research activities including the election of a Chair, a Chair-elect, and a Secretary. All officers are to be elected for three year terms, as follows: a Secretary will be elected annually, then become Chair-elect in the second year, and Chair in the third year. Administrative guidance will be provided by an assigned Administrative Advisor and a CSREES Representative. The W-3168 welcomes and encourages participation of expert seed biologists affiliated with State Agricultural Experiment Stations, the Agricultural Research Service, and colleges or universities, as is consistent with the Multistate Research Fund mission of the Agricultural Research, Extension, and Education Reform Act of 1998.

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