Duration: 10/01/2008 to 09/30/2013

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, seed propagules 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. The biochemical, physiological, genetic, and environmental processes influencing seed and seedling quality and 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 W-1168 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, horticulturalists, land and natural resource managers, educators, extension agents, government agencies, and all industries that deliver seed to growers. Members of the W-1168 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 billion (Statistical Abstract of the United States: 2007: Table 812). 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 revegetation programs, where seeds are often planted under adverse seedbed conditions. In these cases, establishment and survival of the seedlings is the most crucial step. Rapid advances in biotechnology and genomics underscore the importance of maintaining all germplasm resources indefinitely into the future. These genetic resources preserved in seeds will provide the 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 reexamination) 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. 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, 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 landgrant 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. To ensure that end users have an abundant supply of high quality seeds and information necessary for their work, this proposal has established three objectives:
1. Identify and characterize biophysical, biochemical, genetic and environmental factors regulating or influencing seed development, germination, vigor, and dormancy.
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.

These objectives are necessarily broad and reflect the diversity of stakeholder needs, the gaps in current knowledge that can be addressed by new technologies, such as genomics, and the promise that proven and new technologies can yield practical solutions to complex seed biology issues related to seed dormancy, germination and seedling vigor. 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, six states' activities address issues relating to Objective 2, and 11 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 in the last five years, 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 members of W-1168 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 W-1168 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. As seeds increasingly become the delivery system for multiple biological and chemical technologies, the 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: There are at least three professional associations (AOSA, SCST, ISTA) dedicated to seed testing and an array of other organizations (AOSCA, AASCO, ASTA) concerned with the genetic, pathological, and physiological quality of seed. While these organizations support seed research, they do not generate fundamental research information. W-1168 is the only research project currently focused solely on seed biology and technology.

A search of the CRIS database revealed two active multistate projects besides W-1168 that peripherally deal with seeds: NC1016 (Economic Assessment of Changes in Trade Arrangements, Bio-terrorism Threats and Renewable Fuels Requirements on the U.S. Grain and Oilseed Sector) and SERA011 (Review and Coordination of Oilseed Rape Research Programs in the Southern Region, IEG-55). In addition, 206 CRIS projects dealing with seed aspects were identified, and of these, 42 deal with seed as propagules, with 21 of these projects associated with W-1168. There is no significant overlap between the objectives of W-1168 and other existing research projects. Most of the projects listed deal with seeds as food and are not targeted to production and quality of seeds for planting. The CRIS 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-1168 members are contributing the majority of active seed research documented in the database.

Current work of W-1168 member programs:

SEED DEVELOPMENT: The Bennett lab has studied the effects of light and temperature during seed development on the subsequent quality of lettuce seeds. Lettuce seeds produced under long days (16 h) were heavier and had a significant reduction in germination (especially at 30°C and in dark), but better longevity in storage than seeds produced under short-days (8 h). Light effects, which may be due to light quality rather than day length, were observed during the last phase of seed development. Lettuce seeds produced at 30/20°C had better germination and longevity in storage than seeds produced at 20/10°C. Studies in the Bennett and the McDonald labs also examined the effects of fruit development and harvest stage on tomato seed quality (Ramirez-Rosales et al. 2004), with special emphasis on high-lycopene genotypes.

Cereal crops have been selected for rapid, uniform germination during domestication, and consequently many cultivars are susceptible to pre-harvest sprouting (PHS) (germination of mature grain on the mother plant when cool moist conditions occur prior to harvest) due to insufficient seed dormancy. It is desirable to regain a certain degree of seed dormancy by manipulating the underlying genes in breeding programs or through mutational/transgenic means. Higher ABA accumulation or sensitivity and lower GA accumulation and sensitivity can result in higher resistance to PHS in white wheat. The Steber lab has identified ABA-insensitive and -hypersensitive mutants in wheat cvs. Brevor and Chinese Spring (Strader et al., 2004), and has characterized a number of genes that may be involved in determining seed dormancy (Itoh et al., 2003; Steber 2007). The Steber lab will determine the effect of these mutations and genes on PHS to support development of white wheats with reduced susceptibility to PHS.

A means of regeneration, either by organogenesis or somatic embryogenesis, is necessary for genetic engineering for most plants. However, somatic embryogenesis is poorly understood, especially early steps in the process. Somatic embryogenesis serves as a model for zygotic processes that are also largely a mystery and difficult to study because early embryo development is relatively inaccessible. Results to date indicate an important role for the MADS-domain transcriptional regulator AGL15 in somatic embryo development. The Perry lab considers how AGL15 promotes embryo development in Arabidopsis. Chromatin immunoprecipitation is used to isolate DNA fragments directly associated with AGL15 in vivo to identify genes directly regulated by AGL15. They have found that a soybean ortholog of AGL15, GmAGL15, can enhance somatic embryo development in soybean when ectopically expressed. A number of genes have been identified in Arabidopsis where either gain- or loss-of-function leads to alteration in the capacity for somatic embryo development (Harding et al., 2003; Tang & Perry, 2003; Wang et al., 2004; Zhu & Perry, 2005; Hill et al., 2007).

SEED DORMANCY: Stand establishment of Spartina alterniflora from seeds is a cost-effective means for coastal stabilization. However, S. alterniflora seeds are recalcitrant. Recalcitrant seeds die when dried, and no solution to this problem exists for any species. Recalcitrant S. alterniflora seeds are dormant and cold tolerant, and can be compared to orthodox (desiccation-tolerant) S. pectinata seeds as a physiological control for nonspecific responses to desiccation (Cohn & Chappell, 2007). The Cohn lab has developed seed harvesting, processing, moist storage, flash drying, germination and viability test protocols for S. alterniflora (Cohn & Gatz, 2002; Cohn et al., 2002). They are using this system to investigate mechanisms of damage and deterioration due to desiccation, including free radical production (TBARS and FOX assays), membrane leakage, and total antioxidant titer, and differential protein oxidation during drying (Chappell & Cohn, 2007). Ongoing research is focusing on DNA fragmentation during dehydration of recalcitrant seeds.

The Gu lab identified seed dormancy genes as quantitative trait loci (QTL) from an accession of weedy rice, and introduced a subset of five dormancy QTL alleles into the genetic background of a non-dormant cultivated line by generations of backcrossing and phenotypic selection. All introgression lines with single QTL alleles from the weedy rice displayed delayed germination or reduced germination rate under controlled conditions. These dormancy QTLs have also been characterized for interlocus interactions (i.e., epistasis) and between genotypes and major environmental factors during seed development (Gu et al., 2003; 2004; 2005a, b, c; 2006a, b).

Lettuce seeds exhibit thermodormancy, or a failure to germinate when imbibed at temperatures above 25-30°C. The Bradford lab has identified an accession of Lactuca serriola, the wild progenitor of cultivated lettuce (L. sativa) that exhibits germination up to 38°C in the light. The majority of this effect is associated with a QTL termed Htg6.1 conferred by the L. serriola accession UC96US23 (Argyris et al., 2005). Near-isogenic lines (NILs) confirmed the QTL effect by introgressing this region into a cultivated (Salinas) background (Argyris et al., 2008). A gene in the ABA biosynthetic pathway, LsNCED4, mapped to the Htg6.1 confidence interval and is highly expressed in Salinas seeds imbibed at thermoinhibitory temperatures, but not in UC96US23 seeds. The Bradford lab is testing whether differential expression of LsNCED4 is responsible for the temperature sensitivity of lettuce seed germination.

SEED GERMINATION: In the Downie lab, mutants (Downie et al. 2003, 2004, Salaita et al., 2005) and biochemical processes (Xu et al., 2004) have been identified bearing on seed longevity in storage and the capacity to complete germination. These are being studied in more detail to understand the factors controlling germination and the mechanisms by which seeds can repair damage suffered during storage.

The Nonogaki lab has characterized seed proteins and genes that are expressed in association with seed germination (Nonogaki et al., 2000; Martin et al., 2005; Liu et al., 2005a, b; Martin et al., 2006; Liu et al., 2007). The factors controlling, or being controlled by, these genes and the mechanistic links among them are being analyzed using molecular techniques. Elucidation of the gene regulatory networks that control or influence seed development, dormancy and germination is being pursued.

STAND ESTABLISHMENT: Environmental stress, particularly in southern regions of the U.S., can greatly reduce stand establishment or impair early growth of vegetable transplants. The Leskovar lab has found that single-dose of ABA can protect seedlings of various vegetable species from dessication injury associated with transplant shock (Goreta et al., 2007; Leskovar et al., 2008). In addition, low-dose ABA exposure impacts the morphology of pepper seedlings and can improve survival of transplants. Application of 1 mM ABA has impacted early seedling root length and tolerance to environmental stress.

Emergence and stand establishment are among the top perennial concerns for sugarbeet growers worldwide, especially in rain-fed agricultural systems. Although growers routinely plant sugarbeet seeds with >90% germination, field establishment averages 60%, and this impacts growers returns. Understanding the genetics underlying sugarbeet seedling vigor is required to enhance this trait in this and other crops, and may assist breeding for superior pathogen resistance, another persistent problem in sugarbeet fields. The McGrath lab has developed an effective seed vigor screening test and identified two indicators of seedling vigor. The first is an oxalate oxidase induced under stress in good emergers, but not poor emergers (de los Reyes & McGrath, 2003). Oxalate oxidase catalyzes the production of H2O2 that acts as a second messenger for a number of stress sensing biological processes (Bienert et al., 2006). The second target is predicted to lie downstream of this trigger and is the activation of the glyoxysomal metabolic pathway (de los Reyes et al., 2003), a marker for lipid catabolism during germination in many seeds. These candidate genes provide specific targets for genetic improvement of sugarbeet seed vigor and stand establishment.

SEED NUTRITION: The digestibility of seed proteins has been investigated in the Buchanan and the Lemaux labs. Sorghum seed proteins and starch are relatively indigestible, reducing the value of sorghum as a food for humans and animals. The B- and y-kafirins, found in the periphery of the protein body, are rich in disulfide bonds, rendering them resistant to protease digestion. The more digestible a-kafirins in the body's center are shielded and hence not efficiently digested. This matrix of B- and y-kafirins also surrounds the starch, thereby explaining its incomplete digestibility (Duodu et al., 2003; Mertz et al., 1984), a problem compounded by boiling the flour as is common practice (Murty et al., 1995). Reducing agents breaking disulfide bonds eliminate this problem (Hamaker et al., 1987; Arbab et al., 1997; Rom et al., 1992). The Buchanan and Lemaux labs are developing genetic approaches to modify the redox potential of the protein bodies and render the kafirins more digestible.

SEED ECOLOGY: Genotype, maternal environment, post-harvest history and germination environment all contribute to the control of population establishment in natural and agro-ecological settings. The environments that a seed experiences during development on the mother plant, in its dormant state, and when it germinates, all directly contribute to viability, vigor, and other factors affecting seed performance. Importance of the environment is illustrated by the observation that establishment for a given population of seeds can range from complete, uniform establishment, to non-uniform establishment for a fraction of the seeds, or to no establishment, each depending on the interactions of temperature, water, light and other physical and chemical influences. Considerable efforts are being made by the group to understand how the environment affects seeds at all stages in both agro-ecological and natural settings.

Temperature and water availability have been identified as important features during seed fill (Dornobos et al. 1989; Vieira et al. 1992; Heatherly 1993; Spears et al., 1997; Egli et al., 2005a, b), and have also been targeted as primary variables related to modeling of seed dormancy (Allen et al., 2007). There is little literature regarding other environmental influences (humidity, light, etc.) on seed fill, nor on conditions that could assist beneficial pathogens that infect seeds. Efforts are underway in the Allen lab to determine whether indigenous seed pathogens can be used as biological control agents for seeds of invasive species (Meyer et al., 2006).

Tissues enclosing the embryo often affect dormancy and germinability. The achenes of pre-varietal Coreopsis basalis, C. floridana, C. leavenworthii, C. pubescens and C. lanceolata have a woody pericarp, a mucilaginous testa and a single cell layer of endosperm around the dicotyledonous embryo. In SEM images, a thick-walled, single-layered endosperm was distinct from embryo and testa tissues. The Norcini lab is studying the roles of the endosperm and/or pericarp in seed dormancy of Coreopsis species.

In natural ecosystems, seeds collected from contrasting populations or in different years often exhibit strong differences with respect to seed behavior (Allen & Meyer, 2002). These differences may have practical implications, including fitness of a given population for use in revegetation of disturbed natural landscapes (Meyer & Allen, 1999). Simulation modeling enables extrapolation from experiments conducted under controlled, usually constant, temperature and water potential conditions in the laboratory to the highly variable field environment. The hypothesis that seeds and seedlings integrate their successive water potential and thermal histories under field conditions in predictable ways has been verified with hydrothermal time and hydrothermal after-ripening time concepts for Bromus tectorum (Allen 2003; Allen et al., 2007; Christensen-Bauer et al., 1998; Bair et al., 2006), but has received limited attention in modeling seedling emergence in the field (Finch-Savage et al., 1998; Roman et al., 1999; Shrestha et al., 1999). The Allen laboratory is developing multifactor models that predict dormancy loss, as well as germination kinetics, in the field driven entirely by measurable environmental variables.

Exotic winter annuals like cheatgrass (Bromus tectorum) represent a major threat to the nation's resource base and environment, infesting over 40 million hectares of western rangelands. Restoration of these lands is extremely difficult without effective cheatgrass control. Meyer and Allen (Utah) initiated research to study the ecology of a native fungal seed pathogen, Pyrenophora semeniperda that has potential for cheatgrass biocontrol. Studies focus on the environmental interactions between cheatgrass and its new enemy, including learning which environmental conditions favor culture of the fungus for application in the field, determining the extent to which the fungus kills seeds of native species, and determining how to use the fungus as a biological control agent. These studies will contribute to knowledge of environmental interactions between seed bank pathogens and their hosts (Beckstead et al., 2007), and will evaluate this pathogen as a biocontrol agent for cheatgrass (Meyer et al., 2006).

SEED VIGOR, VIABILITY AND TESTING: The use of tetrazolium chloride (TZ) has shown promise for estimating viability and germination of pre-variety germplasm of native forbs such as Coreopsis basalis, C. floridana, and C. lanceolata. Apparent differences in membrane permeability and/or seed vigor have been observed among different native Coreopsis species. Studies continue to identify the basis for differential TZ staining of intact achenes of these species.

The Goggi lab found that maize genes are differentially expressed after frost (Hartwigsen & Goggi, 2002) and that these genes could be utilized to identify frost-damaged seed lots. The saturated cold and soak tests were the best predictors of field emergence of frost damaged seed lots, and frost damage could be detected within the first 3 months of storage (DeVries et al., 2007). These tests can be used to identify frost-damaged seed lots prior to distribution and sale.

The McDonald and Bennett labs compared the efficiency of an automated computer imaging system to standard procedures for the assessment of melon (Cucumis melo L.) seed vigor (Marcos-Filho et al., 2006). Indices for vigor and uniformity of seedling growth from the Seed Vigor Imaging System (SVIS) were in accord with results from traditional seed testing methods, resulting in a more rapid, objective and efficient option for melon seed vigor evaluation.

The ASTEC Q2 is an instrument capable of measuring respiration rates from individual seeds during imbibition and germination. Using the Q2, the Bradford lab has identified respiratory patterns associated with high and low seed quality and is assessing the value of the instrument for vigor testing. Studies have also been conducted on the effects of different priming treatments on potential seed longevity (Schwember & Bradford, 2005) and on the genetic basis of seed responses to priming.

Chlorophyll fluorescence (CF) is used to measure stress in leaf tissue and also is an indicator of seed vigor (Jalink et al., 1998). It is unclear whether this is simply a measure of maturity or is indicative of other factors affecting seed quality. The Welbaum lab has measured CF in developing cantaloupe seeds and found a high negative correlation between the development of germinability and CF. Sorting of seed lots using CF could improve seed quality by eliminating immature seeds.

SPECIALTY CROPS (biofuels, forage, ornamental, etc.): Switchgrass is one of the leading candidates for dedicated bioenergy crops. Switchgrass seeds exhibit a high degree of dormancy (Douglas & Grabowski, 1995), often resulting in poor stand establishment. The Chen lab has shown that coating switchgrass seeds with chitosan can enhance germination and seedling vigor. Chitosan has been used as a plant growth regulator (Nge et al., 2006), and its beneficial effects on germination have been reported previously (Bhaskara Reddy et al., 1999). Understanding the molecular basis of dormancy breaking and growth promotion by chitosan may provide important knowledge and tools for utilization of switchgrass as a bioenergy crop.

Eastern gamagrass (Tripsacum dactyloides) seed is encased in a cupule and requires stratification for dormancy release, but subsequent germination rarely exceeds 60%, and results in only 10-15% field emergence. This native, warm-season perennial grass is excellent for forage, biofuel, and conservation plantings, motivating an effort to enhance its performance. Germin (calcium oxalate oxidase) is an enzyme that converts calcium oxalate to Ca2+ and H2O2, both of which may be stimulatory for the alleviation of dormancy in gamagrass. The Geneve lab has isolated a putative TdGermin gene and is now correlating TdGermin expression with dormancy alleviation.

Seeds of some orchid species require a fungal symbiont to complete germination, while some can germinate asymbiotically in vitro on nutrient media. Oxidative enzymes that increase in activity upon infection of the protocorm by the symbiont are also upregulated in response to pathogen attack, suggesting that a defense response is initiated by the orchid. This is supported by the fact that orchinol (a phytoalexin) was found in infected protocorms of Orchis (Beyrle et al., 1995). Using the orchid Cypripedium parviflorum var. pubescens (Cpp) in symbiosis with the mycorrhizal fungus Thanetephorus pennatus (Tp), the Welbaum lab previously identified a trehalose-6-phosphate synthase (CppTPS) that was differentially expressed in roots of Cpp in symbiosis with Tp and is continuing to investigate the putative role of TPS as a key regulator of the symbiotic relationship.

Objectives

1. Identify and characterize biophysical, biochemical, genetic, and environmental factors regulating or influencing seed development, germination, vigor and dormancy.
   
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-1168 represent a wide range of interests and expertise. Among the 15 states participating, 13 have responsibilities examining influencing seed development, germination, vigor and dormancy, 11 have programs targeted towards developing, evaluating and transferring technologies to customers and stakeholders, and six are confirming and extending existing knowledge towards new species (e.g. biofuels, specialty crops) and new environments (e.g. habitat restoration, invasive species). Of 22 topic areas self-identified by the W-1168 group, over 70% of states are involved with issues relating to seed vigor, viability, dormancy, germination, emergence and stand establishment, and seed storage for maintenance of viability and germplasm conservation. Over 40% of states have programs that are targeted towards seed enhancements, seed testing, seed development, stress tolerance, hormonal regulation, and genes. Over 20% of states have programs associated with seed health, structure, and composition, seedling development, oxidative stress, desiccation tolerance, transplant quality, ecology, and biofuels. Two states identify with topics relating to invasiveness, habitat restoration, and endangered 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 (canola, maize, sorghum, soybean, sugar beet, 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-1168. 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 is integrated by the group. Methods for Objective 1: Identify and characterize biophysical, biochemical, genetic and environmental factors regulating or influencing seed development, germination, vigor and dormancy. Bennett & McDonald (OH): Experiments on lettuce will determine the effects of water availability, light (length and quality) and temperature during seed production on seed quality. Greenhouse and growth chamber experiments will be conducted using container-grown lettuce plants cv. Tango. Treatments [no water restriction vs. water deficit; 16 h vs. 8 h day-length; red-rich vs. far-red-rich light; high (30/20°C) vs. low (20/10°C) temperatures] will be applied in the greenhouse (water availability) or growth chambers (light and temperature) from before flowering to seed harvest. Bradford (CA): To test whether lettuce seed thermodormancy is due to temperature-sensitive expression of LsNCED4, a key regulated gene in the ABA biosynthetic pathway, we will develop plants expressing RNAi constructs to suppress expression of LsNCED4 in the seed, which should prevent the induction of thermodormancy. We will also explore mutation (TILLING) as an option to identify mutant alleles in this gene. Crosses will be made to introgress high temperature tolerance into cultivars adapted to warm temperature growing regions. Buchanan & Lemaux (CA): The biochemical and structural basis for indigestibility of starch and protein in sorghum grain will be studied. In Vitro Pepsin Digestion: this assay, using SDS/PAGE to analyze protein remaining after pepsin digestion, was developed and is carried out routinely in our laboratory. In Vitro Starch Digestion: digestibility of starch in sorghum meal is measured using a modified hydrolysis method with bacterial ±-amylase. Microscopy studies: sectioned seeds are prepared for electron microscope analyses that utilize an environmental scanning electron microscope. Chen (TN): A detailed evaluation of chitosan treatment on seed germination and seedling vigor of three switchgrass cultivars (Alamo, Neb28 and Blackwell) will be conducted. Chitosans that differ in molecular weight and solubility will be produced. Different concentrations of chitosans and different periods of seed coating will be tested. Effects of chitosans with different chemical properties on dormancy breaking of switchgrass will be assessed. Microarray technology will be used to identify switchgrass genes critical for germination and seedling growth. A rice whole-genome oligonucleotide array will be used to identify dormancy- and germination-related genes in switchgrass. Taylor (NY-Geneva) and Chen (TN) are cooperating on chitosan seed treatments of switchgrass. Cohn (LA): The severity of DNA/RNA oxidation and its correlation/association with recalcitrant seed death will be assessed in Spartina. Putative protective proteins required for desiccation tolerance will be identified. Loss of desiccation tolerance (DT) of orthodox seeds will be compared to recalcitrance (RCT). 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 flashed dried (0-24 h at 23C) to various moisture contents above and below the critical moisture content (40% dwb) for RCT. DNA will be isolated and purified using ChargeSwitch Technology (InVitrogen); RNA will be isolated and purified using the PureLink Plant RNA Reagent (InVitrogen). DNA and RNA oxidation will be quantitated by GC-MS adapting established protocols (Aruoma et al., 1989; Jenner et al., 1998). Protein profiles will be compared between the two Spartina species (whole seed total extract; embryo total extract; and respective heat stable extracts), as well as profiles of oxidized proteins), adapting methodology from Boudet et al. (2006) and Sheehan (2006). Downie (KY): Tomato and Arabidopsis mutants will be used to elucidate the influence of isoaspartate, light, and a thickened testa 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. TAPa tagged lines of PIF1 and CTG10 will be used to demonstrate physical interaction in vivo between these two proteins. Identification of the gene responsible for the bs1 syndrome will improve understanding of the testa. Molecular markers developed from BAC and EST sequences will be used to fine map bs1. Gu (SD): To investigate genetic, evolutionary, and molecular mechanisms of seed dormancy in rice and wheat crops, the primary objectives are to: 1) develop introgression or isogenic lines for QTL dormancy alleles from weedy rice; 2) map-based clone one to two rice seed dormancy QTLs; and 3) identify dormancy genes imparting resistance to pre-harvest sprouting (PHS) for common wheat. The introgression of QTL alleles with relatively large effects on dormancy will be conducted by recurrent backcrossing using a non-dormant line of cultivated rice as the recipient parent, in combination with the strategies of marker-assisted selection and progeny testing for seed dormancy. The map-based cloning will focus on the rice qSD7-1 and qSD12 QTLs. The wheat dormancy genes will be identified from Aegilops tauschii-derived synthetic hexaploid wheat accessions using QTL analysis and rice-wheat comparative genomics approaches. We will also collaborate with Cohn (LA) to characterize the weedy rice-derived dormancy alleles retained in the introgression lines for physiological functions. Knapp (IA): For seed quality studies, field emergence and laboratory assessments of seed quality will be used to construct the data sets for QTL mapping. If QTLs are identified, potential genes will be assessed relative to their roles in seed quality. For frost damage studies, maize B73 derivatives selected for seed composition will be used to produce single-cross hybrid seed. Seed will be frozen in the ear using a climate controlled growth chamber. A series of biological and molecular tests will be conducted at different times during seed storage to better understand the physiological changes undergone by the frosted seed. McGrath (MI): Linkages between the action of hydrogen peroxide and vigor induction with lipid catabolism in sugarbeet seeds will be investigated by following the signaling cascade predicted from other organisms which proceeds through a mitogen activated protein kinase (MAPK) cascade. Using comparative genomics and bioinformatics, representatives from all MAPK gene families will be sought from beet DNA libraries, and their expression patterns compared among stress and non-stress germination regimes. Transcription factor candidates will be identified from microarray assays. Yan (OK): Seed dormancy of each of 96 winter wheat RILs will be characterized under controlled temperature and light conditions. The physiological and morphological maturity stages of RILs grown in field and greenhouse will be recorded. Dormancy tests will be conducted with the seeds treated with colds, heat, and room temperature (control). Individual markers will be analyzed significant correlation coefficients with seed dormancy. Nonogaki (OR): Loss- and gain-of-function mutants in Arabidopsis will be used for functional genomics of seed germination. For miRNA research, silent mutations will be introduced into the miRNA complementary sequences of miRNA target genes. This will cause de-regulation of transcription factors from miRNA control and will generate transgenic plants expressing miRNA-resistant targets. For tomato seed research, bioinformatic approaches will be used to identify genes related to Arabidopsis seed transcription factors. High throughput approaches such as microarrays and real-time PCR will be used to analyze gene regulatory networks. Obendorf (NY-Cornell): Biochemical and molecular methods will be used to identify and characterize enzyme(s) that convert myo-inositol to D-chiro-inositol in soybean. Perry (KY): To identify DNA sites to which AGL15 binds in vivo, chromatin immuno-precipitation (ChIP) will be utilized to immunoprecipitate AGL15 and associated DNA fragments. Using ChIP-on-chip, the DNA recovered from immunoprecipitation using AGL15-specific antiserum or preimmune serum 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 AGL15. Affymetrix ATH1 arrays will be used to investigate gene expression changes in response to accumulation of AGL15/18. The results of these experiments will determine the genes that may be directly regulated by AGL15 and may be farther downstream in the regulatory network. Loss- and gain-of-function of select downstream targets will aid in determination of function of the products of regulated genes and in identification of proteins that interact with AGL15 to regulate developmental programs. Putative orthologs of AGL15 in Glycine max will be used for testing if they influence somatic embryogenesis. Steber (WA): The effect of ABA-hypersensitive mutants on wheat grain dormancy will be examined using plating assays over a time course of after-ripening and ABA dose-response germination assays. Whether these lines show altered tendency to pre-harvest sprouting (PHS) compared to the wild-type parent will be assessed using the spike-wetting test. In Arabidopsis, we will determine whether DELLA protein activity is altered by ubiquitination or protein-interactions during seed after-ripening using co-immunoprecipitation and immunoblot analysis as in McGinnis et al. (2003), Dill et al. (2004) and Ariizumi & Steber (2007). DELLA activity will be assessed based on germination capacity and on GA-regulated gene expression. Welbaum (VA): Studies will test whether CppTPS plays a key regulatory role in the establishment/maintenance of the symbiotic association between orchids and fungal symbionts. Orchid plants will be transformed using Agrobacterium rhizogenes with TPS:GUS promoter fusion vectors to determine its role in orchid-fungal symbiont interactions. Other constructs will be used to reduce expression of TPS in an attempt to disrupt symbiosis. Tissue culture, tetrazolium testing and other in vitro germination procedures will be used to determine the best procedures for propagating these species listed above. A thermogradient table will be used to determine the base, ceiling and optimum temperatures for germination of each species. 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. Allen (UT) Research focuses on ecology and physiology of seed germination under adverse environmental conditions. Current research methods include regulation of germination timing, simulation models for seed germination in semiarid ecosystems, and soil tests to determine presence and abundance of noxious weed seeds. Baskin (KY): Studies will examine the ecology, biogeography and evolution of seed dormancy and germination. Seed dormancy mechanisms of species present in bio-geographical regions around the world will be identified with a focus on underrepresented species. Bennett (OH): Dormancy characteristics of the non-native invasive species, Phragmites australis, will be studied. P. australis is very successful at vegetative reproduction via rhizomes and runners, but very little is known about seed characteristics of U.S. populations. Recent findings have concluded that P. australis seeds from Lake Erie populations are mostly viable, and they germinate well and at high rates once dormancy is broken, indicating that the weed spreads both sexually and asexually. We will investigate seed production and compare P. australis seed characteristics with those of the non-aggressive, native Phragmites australis subsp. americanus. Duggan (OR): Hybrid carrot seed production will be compared using sprinkler and drip irrigation with the same male/female combination and grown in the same field. Treatments within irrigation methods will be ± fungicide (i.e., azoxystrobin in rotation with chlorothalonil). Soil moisture content will be monitored throughout the growing season. At maturity, the crop will be hand harvested by umbel order and yield composition, and percentage germination from each order will be determined. Seed vigor will also be assessed using the ASTEC Q2 in collaboration with Bradford (CA). Methods for Objective 3: Develop, evaluate and transfer technologies to assess and improve seed and seedling quality, health, performance, utilization, and preservation. Bradford (CA): 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. 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. For priming studies, we are employing genetic approaches to identify QTL associated with priming responses and studying the sensitivity of primed seeds to storage at different moisture contents. Pill (DE): Biological control of various damping off pathogens will be designed for either seed treatment (matric or osmomatric priming) or seed germination. Several species of Trichoderma, either alone or combined and at various rates will be tested for efficacy against selected damping off organisms. Compatibility of biological control measures with crop cultural factors such as growth medium composition, pH, and fertilizers will also be measured. Geneve (KY): A combination of stratification and hydrogen peroxide treatments will be tested to enhance germination of gamagrass via induction of naturally occurring regulating compounds such as nitric oxide and cGMP. The importance of the natural production of hydrogen peroxide, nitric oxide, and cGMP for dormancy release in gamagrass and whether they can be manipulated to enhance germination and stand will be assessed. Germin expression during germination will be measured following dormancy release in gamagrass by stratification or hydrogen peroxide treatment using RT-PCR and Northern analysis. Dormancy alleviation will be attempted using nitric oxide generating compounds, while enzymatic indicators of dormancy release will be assayed. Leskovar (TX): Seedling conditioning treatments aimed at enhancing stress tolerance and increase root mass or length will be evaluated on vegetable species (e.g. pepper, artichoke), known for their sensitivity to high temperature and water deficit stress. We will evaluate phytohormones, such as ABA and ethylene, including their promoters and inhibitors for their role during development of shoot and root morphology, structure and physiology. In roots we will investigate the differential development of primary (taproot), laterals, adventitious and root hairs. Seedlings will be evaluated at various stages of development. McDonald (OH): A system has been developed that captures images of germinated seedlings, which are then evaluated by software that quantifies seedling length and uniformity. This system will be used for vigor assessment and extended to additional species. Norcini (FL): Seeds of pre-variety germplasm of Coreopsis floridana and C. lanceolata will be grown under controlled (growth chamber) and greenhouse conditions. Microscopy and biochemical methods will be used to gain insight into changes in the endosperm that occur during after-ripening. Germination and excised embryo tests (light, GA, 0.2% KNO3) will be conducted during after-ripening. Bahiagrass sod (living, suppressed, and dead) will be evaluated as a native forb/grass sod base that can be used for erosion control. Seeding date, species composition, controlled release fertilizers, and hydrogels will also be evaluated. Emergence, density, percent cover, and aesthetics will be among the parameters recorded periodically. TZ will be evaluated for exposing/extracting embryos to accurately assess viability of C. leavenworthii and Aristida stricta embryos. Time of moist preconditioning, GA, and KNO3 treatments will be assessed for effects on TZ staining of excised embryos. Taylor (NY-Geneva): Studies will be conducted to adapt seed coating technologies as carriers of plant protectants and to facilitate sowing of specialty and other crop seeds. Efficacy of seed treatments for control of insects, nematodes, fungal and bacterial pathogens will be assessed in cooperation with pest management specialists. Controlled release and controlled distribution technologies for seed treatment agrochemicals will be developed. Seed enhancement technologies of warm-season grasses and oil crops as feedstocks for bioethanol and biodiesel, respectively, will be explored. Cooperative projects are in place with Geneve (KY) on development of seed enhancement technologies and stand establishment of warm-season grasses (switchgrass and gamagrass).

Measurement of Progress and Results

Outputs

• Effects of environmental stress during seed development on seed quality
• Influence of seed desiccation rate and time of maturity on seed quality
• Identification and characterization of genes and biochemical processes important in the development and expression of seed quality
• Development of post-harvest methods to enhance seed quality and protect seed and seedlings from pests
• Improvement of seed quality through fundamental knowledge of how light environment at harvest affects seed vigor
• Output 6 The relationship between environmental conditions experienced during seed fill (evapotranspiration, light quality, temperature, etc.) and seed yield, composition and quality
• Output 7 Relationships between seed respiration rates and seed vigor levels
• Output 8 Genetic determinants of seed priming responses and of seed longevity
• Output 9 TZ testing procedures for seed quality in native forbs
• Output 10 The relationship between seed genetics and composition in maize and their influence on seed freezing injury
• Output 11 The relative and interactive roles of ABA and ethylene in shoot and root growth following germination
• Output 12 Improvement in the reliability and uniformity of stand establishment of lettuce crops in warm weather climates
• Output 13 Specific molecular and genetic targets for modification of dormancy behavior of seeds
• Output 14 Nutritionally improved sorghum grain for developing countries
• Output 15 Germination and stand establishment capacity of various species as a biofuel sources
• Output 16 Gene regulatory networks underlying somatic embryogenesis and seed development
• Output 17 Strategies for management of seed bank dynamics in cultivated and natural ecosystems

Outcomes or Projected Impacts

• Seed companies and growers will refine production, harvest and conditioning methods to produce seed lots having high germination percentage and vigor.
• Methods to enhance or alleviate seed dormancy, as needed, to improve seed quality or utilization or to manage weed populations will be developed and tested.
• Management of seed bank dynamics in natural plant populations to enhance establishment of desirable species and reduce invasiveness of undesirable species will be enhanced.
• Convenient, rapid assay systems to assess seed quality and vigor will improve the quality of seeds throughout the seed delivery system.
• Methods to enhance seed germination and seedling development under stressful environments will improve crop establishment and enable utilization of less desirable locations, e.g., for biofuel production or environmental restoration.
• Outcome/Impact 6 Improve germplasm storage and conservation by identifying factors that regulate seed desiccation tolerance and influence longevity in storage
• Outcome/Impact 7 Knowledge of specific genes and alleles conferring desirable or undesirable seed traits will guide molecular-assisted breeding strategies to improve seed quality
• Outcome/Impact 8 Genetic determinants of seed vigor can be incorporated into new varieties to alleviate existing problems with low quality seed

Milestones

(2009): Transgenic plants expressing miRNA-resistant genes will be made that potentially provide novel traits for agriculture. Tomato orthologues of Arabidopsis transcription factors induced by GA during seed germination will be identified to obtain seed germination markers in crop species. LsNCED4 RNAi vectors will be used to transform thermo-sensitive lettuce cultivars/breeding lines to improve high temperature germination. Differences among sorghum varieties for digestibility of uncooked and cooked sorghum grain meal, using corn meal for comparison, will be defined. PIMT target proteins will be evaluated to evaulate succinimide formation as the end point during seed aging. Physiological studies to identify optimum conditions for chitosan treatment will be completed. Isogenic lines for the qSD1 dormancy QTL in rice and fine map the qSD12 QTL region will be developed. Phragmites australis seed characteristics will be compared with those of the non-aggressive, native P. australis subsp. americanus. Effects of increased ABA sensitivity on wheat grain dormancy to solve pre-harvest sprouting problems will be ascertained. Post-harvest seed conditioning that will enable growers to increase percentage of non-dormant Coreopsis seed will be developed. Reactive oxygen species and nitric oxide levels and their effect on germination will be measured. Orchid will be transformed using Agrobacterium rhizogenes with TPS:GUS promoter fusion vectors.

(2010): Results of vigor tests among seedlots of different quality will be correlated against field performance. ABA dosage, timing, and frequency to enhance seedling tolerance to drought and temperature stress will be evaluated. Field experiments investigating the effects of irrigation method upon yield, composition and seed germination of hybrid carrot seed production will be done. The physiological mechanism(s) governing the observed effects for implementing new management practices for high quality lettuce seed production will be clarified. High temperature germination characteristics will be introgressed into cultivars and breeding lines targeted for warm growing regions. RNAi and mutant lines will be evaluated to silence LsNCED4 for effects on seed thermodormancy. Microarray analysis will be used to identify candidate genes critical for dormancy and germination. Brownseed mutant allele bs1 will be mapped at high resolution. Gene networks regulated by the qSD7-1 dormancy gene will be identified in an isogenic background. The qSD12 QTL will be cloned. Stage- and tissue-specific expression of tomato transcription factors will be evaluated. The effect of overexpression of the GID1 GA receptor on DELLA activity and accumulation during germination will be determined.

(2011): RNAi lines of CTG10 will be evaluated for germination and hypocotyl phenotypes to enhance low temperature seed germination. A potential physical interaction between CTG10 and PIF1 will be evaluated. Spartina comparative proteomics will be extended, and differentially expressed proteins will be identified via mass spectrometry. Calcium/calmodulin, guanylate cyclase activity and cGMP levels will be measured during germination. Isogenic lines for the qSD4 QTL will be available, and the gene underlying qSD12 will be characterized. A linkage map for synthetic hexaploid wheat will be completed. Targets of DELLA proteins will be identified by yeast 2-hybrid screening. The necessity of using seed-specific promoters to target silencing to seed will be determined. Seed accumulating different amounts of AGL15 will be tested for altered dormancy.

(2012): Seed dormancy QTLs and rice dormancy gene orthologs will be genetically marked in a wheat mapping population. Candidate genes will be genetically manipulated in switchgrass to ascertain whether germination performance of transgenic seeds is altered. Examining the effect of ABA hypersensitive mutants on pre-harvest sprouting will be conducted in the field. Results of vigor tests among d

Projected Participation

View Appendix E: Participation

Outreach Plan

The members of W-1168 comprise a group of highly dedicated seed biologists who excel in the communication of their research findings. All members of the W-1168 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-1168 (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-1168 members regularly publish their finding in top-tier, peer-reviewed journals, targeting both the general plant biology and seed biology communities. W-1168 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. W-1168 members serve on journal editorial boards and/or as ad-hoc manuscript reviewers, and publish books on seed biology (Bradford & Nonogaki, 2007).

In December 2006, the IV International Symposium on "Seed, transplant and stand establishment of horticultural crops: Translating seed and seedling physiology into technology" was organized, and participants of the W-1168 play a significant role as invited speakers in each of the sessions. A total of 48 papers were printed as a book, with 17 papers as authored by members of the W-1168 (ACTA Horticulturae, 2008, Vol. 782). In September 2007, W-1168 organized a symposium on "Translational Seed Biology: From Model Systems Crop Improvement" at UC Davis (www.plantsciences.ucdavis.edu/seedsymposium2007). This symposium provided high visibility to seed biology and to the activities of W-1168. The group expects to organize another such event in the last year of the renewed project.

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. 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 W1168 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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Land Grant Participating States/Institutions

AR, CA, CO, DE, FL, IA, KY, LA, MI, MS, NY, OH, OK, OR, SD, TN, TX, VA

Non Land Grant Participating States/Institutions

Retired, USDA-ARS/Washington