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W1168: Environmental and Genetic Determinants of Seed Quality and Performance

Duration:
October 01, 2003 to September 30, 2008
Administrative Advisor(s):
Stephen D. Miller (WYO)
NIFA Reps:
Liang-Shiou Lin

Statement of Issue(s) and Justification:

The primary biological purpose of seeds is to propagate the species by successfully completing germination and resuming plant growth. Native species have innate mechanisms that regulate their potential for germination, often delaying or timing germination to coincide with optimal conditions for growth. Domesticated crops have lost some, but not all, of these mechanisms, and there has generally been strong selection for rapid and uniform germination of crop seeds. High quality planting seed is the key to successful crop production, but both biological and environmental factors can reduce seed quality. To ensure that society has an abundant supply of high quality seeds, this proposal has established four objectives:

1. Determine the influence of pre-harvest stress on seed quality. Environmental stress during seed production frequently reduces seed germination and vigor (Spears et al. 1997), thereby increasing seed costs and limiting supplies of high quality seed to producers. Common environmental stresses that occur during seed development and maturation include high or low temperature stress and moisture stress. A better understanding of the impact that environmental stress has on seed quality will result in methods to ameliorate those effects and consistently produce higher quality seeds.

2. Identify the biophysical, biochemical and genetic factors governing seed desiccation tolerance and longevity. The desiccation tolerance of orthodox seeds permits them to survive from one growing season to the next and facilitates long-term storage of plant germplasm and seeds in commercial inventory. It is well known that seed vigor increases until physiological maturity when the seed has acquired its maximum dry weight, although there remains debate about whether vigor continues to increase subsequently during dehydration (TeKrony and Egli 1997). Not all seeds, however, are desiccation-tolerant (termed recalcitrant seeds) and storage of such germplasm that has little or intermediate tolerance of desiccation can be problematic. Successful seed storage is economically important. Approximately 25% of seed inventories are lost annually at a cost of $1 billion. In order to understand the factors that contribute to physiological maturity, desiccation tolerance and storage longevity, it is necessary to understand the physical forces that arise during dehydration and the factors that govern the kinetics of seed ageing. In desiccation-sensitive cells and tissues, these forces cause lethal damage, while tolerant cells and tissues ameliorate these effects to facilitate seed survival (Walters 1998). This objective will improve seed storage and significantly reduce the loss of seeds stored in commercial inventory and in long-term germplasm banks.

3. Identify genes associated with seed development, germination, vigor and dormancy. Genetics and genomics are powerful tools for investigating diverse developmental, physiological and biochemical processes. With respect to seed biology, considerable effort in model systems such as Arabidopsis thaliana and in major agronomic crops has identified a large number of genes involved in embryogenesis, reserve accumulation and seed maturation (Larkins and Vasil 1997). A significant number of genes are known to be associated with seed germination and with its regulation by hormones or dormancy (Finkelstein et al. 2002; Koornneef et al. 2002). However, relatively few of these reveal actual mechanisms by which germination is initiated or inhibited. Very little is known about the genes involved in seed vigor, although there are clearly genetic determinants of vigor (e.g. Eagles and Hardacre 1979). Identifying both regulatory and mechanistic genes involved in seed development, germination, vigor and dormancy would have clear application to the production and utilization of high quality seed for agriculture. Knowledge of specific genes and alleles conferring desirable or undesirable seed traits could guide molecular-assisted breeding strategies to improve seed quality. Greater understanding of the determinants of seed performance as propagules is also required to predict how genetic modifications of seed composition will affect the ability to use such seeds as planting material. The specific projects proposed here will contribute to this overall goal of identifying and characterizing the genes that determine seed quality and performance in the broad sense.

4. Develop technologies to assess seed quality, improve seed performance and enhance seed utilization. Methods are needed that assess the germination potential of a seed lot in a rapid and objective manner. Seeds that do not meet quality expectations can be subjected to enhancement methods to overcome dormancy mechanisms and/or to further improve germination rate (Taylor et al. 1998). These enhancements include physical or physiological treatments, and methods to protect or eradicate seed pathogens and pests. As these technologies are often species- and application-specific, further research is needed to extend and adapt these methods to additional species and markets. Such technologies will better assess and enhance seed quality and result in improved seedling establishment and crop performance.

Developing solutions to these issues is central to the provision of an abundant supply of high quality seeds for successful stand establishment in agriculture. These issues are also complex, requiring unique skills, equipment and methodologies. Utilizing a multi-state effort as described here by drawing on the expertise of specialized research scientists is the most efficient approach to addressing these issues. Successfully completing the four stated objectives will provide not only an increased understanding of the factors that influence seed biology but also improved seed performance in the field.

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 seed certification, laws and trade. These organizations do not generate fundamental research information. W-168 is the only research project currently focused solely on the biology of seeds as propagules. There is no significant overlap between the objectives of W-168 and other existing government-supported cooperative research projects.

The CRIS database revealed seven projects in addition to W-168 that deal with some aspect of seeds as propagules. Cooperative research project NC-202, Biological and Ecological Basis for Weed Management Decision Support Systems to Reduce Herbicide Use, deals with weed management in corn-soybean production systems. One objective of this project is to develop weed emergence and seed bank information needed for corn-soybean weed management. Project S-279, Sustaining and Improving Profitability of Wheat/Stocker Cattle Enterprises in the Southern Great Plains, has one objective dealing with effects of genotype and environmental interactions on wheat seedling emergence at various planting dates. Project NE-124, Genetic Manipulation of Sweet Corn Quality and Stress Resistance, deals primarily with sweet corn breeding and lists the study of physiological mechanisms regulating seed quality as an objective. Projects NC-007, Plant Germplasm and Information Management and Utilization; NE-1010, Breeding and Genetics of Forage Crops to Improve Productivity, Quality and Industrial Uses; S-9, Plant Genetic Resources Conservation and Utilization; S-279, Sustaining and Improving Profitability of Wheat/Stocker Cattle Enterprises in the Southern Great Plains were each listed in the CRIS database as projects dealing with some aspect of seeds, but specific objectives related to seeds were not listed. With the exception of NC-007, these projects deal with seeds only as they relate to problems with the production of specific crops. Other projects do not deal with problems of seed quality, dormancy, or longevity that plague many crop species or are of concern in natural ecosystems. Also, no existing cooperative research project deals specifically with seed quality issues that relate directly to U.S. agricultural industries, the seed industry, or the performance of seeds in nature.

The CRIS database presently lists 119 projects dealing with some aspect of seeds. Roughly 10% of these projects represent the research efforts of participants in the current W-168 Regional Project. There is no significant overlap among these projects. Most of the projects listed deal with seeds as food or livestock feed and are not concerned with the production and quality of seeds for planting or germplasm conservation. Reviewing all projects relating to seeds in the CRIS database makes it apparent that the discipline of Seed Biology is multi-faceted, and a wide range of expertise is required to pursue fundamental scientific advances.

Effects of Preharvest Stress on Seed Quality. Temperature extremes during seed development impact seed fill (Gibson and Mullen 1996) and subsequent seed quality. High temperatures during seed filling reduce germination and vigor of soybeans (Spears et al. 1997). Seeds from high oleic acid soybean lines exhibit low germination and vigor when exposed to moderately high temperatures during seed development. One high oleic line had 100% germination when grown in 22/18C, but germination fell to below 60% when the seeds were grown at 27/22, 33/22, and 38/27C. Since temperatures in this range are common in soybean growing regions, seed quality can be compromised.

Low temperatures also reduce seed quality. Immature maize is partially protected from low temperature (frost) injury by the ear and husk (Fick 1989), but loss of viability and cellular damage can occur, and the level of damage is influenced by seed maturity at the time of exposure and the minimum temperature. Plant exposure to freezing temperatures can also generate cellular water stress. Stress-related dehydrin-like protein and its corresponding transcript were observed in three day-old seedlings from seed exposed to freezing (Hartwigsen and Goggi 2002), suggesting that the effects of stress during seed development can be exhibited in the seedlings.

Studies of wheat and soybean seed development (Rasyad et al. 1990; TeKrony and Egli 1997; Ellis and Pieta Filho 1991; Zanakis et al. 1994) report conflicting results regarding the time of maximum seed vigor expression. TeKrony and Egli (1997) speculated that the differences between these results were due to experimental differences in harvest and drying techniques.

The new USDA National Organic Production (NOP) rules will eventually allow only organic seed or transplants to be used by certified organic crop producers. Current availability of certified organic seed is very limited, while growth forecasts for the U.S. organic food industry are estimated at 25-30% (annual) (Greene 2001). Relatively little research has been conducted specifically on organic seed production, particularly in vegetables.

Seed Desiccation Tolerance and Longevity. Important features of seeds that allow their long-term storage and distribution are their ability to tolerate desiccation to low water contents and to resist deterioration in the dry state. Two physical factors are important to cellular systems in seeds at low water contents: the hydration force at macromolecular surfaces and the viscosity of the solution between the macromolecules. The hydration force, a repulsive force at the surfaces of membranes and hydrophilic macromolecules, gives rise to physical stresses within the cellular ultrastructure. In membranes, these stresses may cause demixing of membrane components, changes in membrane phase behavior, and transitions to non-bilayer structures that compromise membrane integrity (Bryant et al. 2001). Solutes, such as sugars, can modify the hydration force and limit the physical stress that would otherwise damage dry seed membranes (Koster et al. 1994, 2000). Solutes can exert an additional effect if they contribute to the vitrification (formation of a "glassy state") of the solution by mechanically stabilizing the membrane physical state (Koster et al. 2000; Bryant et al. 2001).

Stabilization by the glassy state also improves the long-term preservation of seed embryos (e.g., Horbowicz and Obendorf 1994; Bernal-Lugo and Leopold 1995). Glasses form when the water content and temperature of a solution drop sufficiently to increase its viscosity to ~1014 Pa, forming a non-crystalline, amorphous solid (Franks 1985; Angell 1995). Thus, glass formation is governed by the two physical parameters that have the greatest impact on seed ageing  water content and temperature. However, recent data suggest that the situation is more complex, and that simply ensuring that the tissue has dried and cooled enough to vitrify will not lead to maximal longevity (Leprince and Walters-Vertucci 1995; Buitink et al. 1998; Walters 1998). Instead, there is an optimal water content for storage that changes with temperature in a well-defined manner (Vertucci and Roos 1990; Buitink et al. 1998; Walters 1998). For tissues where these parameters have been characterized, the optimal water content for storage corresponds to the water content at which abrupt increases in the heat capacity of the tissue occur (reviewed by Walters 1998), though the physical basis for this is not understood.

While extensive studies have described the morphological, physiological and biochemical deficiencies at the cellular level associated with seed deterioration, few studies have considered these events at the subcellular level (reviewed by McDonald 1999). Aging has been associated with lipid peroxidation caused by free radicals leading to plasma membrane disruption. Hendry (1993) reviewed the role of oxygen and free radical generation on seed longevity and presented evidence that supported the role of oxygen in favoring the free radical hypothesis. Damage due to free radicals can occur both during dry storage and upon rehydration. Since oxygen is primarily consumed in the mitochondria, McDonald (1999) proposed that free radicals are primarily produced in this organelle, causing disruption of cristae, reduced ATP and slower seedling growth. Free radicals may also assault DNA, leading to its fracture and loss of mitochondrial replication during cell division. Seed antioxidants may protect against free radical attack.

Genes Associated with Seed Quality. Genetics and genomics are powerful tools for investigating diverse biological processes. Two complementary approaches are being used to identify determinants of seed quality: (1) Traditional (or forward) genetics, supplemented with molecular mapping and transcriptome analysis, is used to identify genetic loci associated with seed phenotypes. Once such loci have been identified, various approaches can be used to clone and study the genes responsible for the traits. (2) Reverse genetics or functional genomics alters the expression or activity of specific genes or proteins and determines the phenotypic consequences for seed quality. In this case, the expression of candidate genes or the activity of proteins that are predicted to be important for seed development or quality is manipulated by mutagenesis, transgenic or chemical methods, and the effects on the target process are determined. Together, these approaches provide powerful tools for identifying genes and pathways for improving seed quality.

Genetic analysis: Cold tolerance and seedling vigor are important seed quality attributes in maize. In its native areas in Mexico and Central and South America, land races and cultivated forms of maize have been found growing from 800 m to over 2500 m elevation, and genetic variability exists in maize for both the ability to germinate/emerge and the ability for seedlings to grow autotrophically at low temperatures (Mock and Bakri 1976; Eagles and Hardacre 1979; McConnell and Gardner 1979). Eagles (1982) postulated that the differential seedling growth at low temperatures was due to "a more rapid loss of seed reserves than a more efficient conversion process." Understanding this process could lead to ways to genetically improve maize seed vigor at low temperatures.

For sugar beet, the first eight weeks of growth are most critical for determining yield (Durr and Boiffin 1995). Improving emergence potential genetically is problematic because both the seed germination environment and seed production environment profoundly influence seedling performance (Johnson and Burt 1990). A novel stress test has been developed for assigning relative field emergence potential and identifying higher and lower vigor seedlots. Germination in water showed the same relative ranking of varieties as their emergence in the field, while germination in hydrogen peroxide solution promoted germination and alleviated the effects of excess water (McGrath et al. 2000). Understanding the genetic basis for the dramatic enhancement of sugar beet seed vigor by hydrogen peroxide has potential in breeding for enhanced emergence (de los Reyes and McGrath 2003).

Recombinant inbred lines (RILs) representing segregating populations from crosses between wild and domesticated genotypes in lettuce and sunflower are being analyzed for seed dormancy and vigor (http://compgenomics.ucdavis.edu/). Dormancy, particularly under adverse planting conditions, reduces seed performance in both species (e.g. Valdes and Bradford 1987; Corbineau et al. 1990). Identifying genes and genetic loci associated with dormancy and with high seed vigor provides tools for breeders to select lines having improved seed performance characteristics.

Functional analysis: While many factors contribute to chilling and desiccation tolerance, sugar accumulation is universally associated with both stresses. In plants, sucrose, raffinose and other members of the raffinose family oligosaccharides (RFOs) have been implicated in the acquisition of tolerance to desiccation and low temperature stresses (Anderson and Kohorn 2001; Bailly et al. 2001; Taji et al. 2002). RFOs have also been implicated in extending the longevity of seeds in the dehydrated state and in the vigor with which seeds complete germination (Xiao and Koster 2001; Downie et al. 2003). Downie (KY) is using insertional mutagenesis strategies to produce plants that are deficient in RFOs.

In many seeds, the endosperm enclosing the embryo acts as an obstacle that delays germination and has to be weakened for radicle emergence. Substantial information on the biochemical mechanisms of endosperm weakening has been obtained in the last decade using tomato seeds (Bradford et al. 2000). Several of the cell wall-degrading enzymes, including a germination-specific endo-beta-mannanase gene in tomato (LeMAN2), have been characterized (Nonogaki et al., 2000). Bradford (CA) has constructed similar lines for LeMAN2 and several other germination-associated genes (LeEXP4, LeEXP8, LeXET4, LeSNF4) under the control of an estradiol-responsive inducible promoter (Zuo et al. 2000). Together, the studies proposed here using these transgenic plants should establish whether LeMAN2 and other germination-associated genes are required for endosperm weakening and radicle growth.

Both ethylene production and sensitivity to ethylene are important for germination of lettuce and other species under stressful conditions. Prusinski and Khan (1989) correlated ethylene production of a number of lettuce cultivars with their ability to germinate at both 32C and 35C. Nascimento (1998) also reported that lettuce cultivars, capable of germination at supra-optimal temperature (thermotolerant cultivars), produced more ethylene than cultivars incapable of such germination (thermosensitive ones). Production of endo-beta-mannanase was also higher in thermotolerant lettuce cultivars than in thermosensitive ones (Nascimento et al. 2001). Both priming and maturation of lettuce seeds at high temperatures (30/20C) improved their germination at supra-optimal temperatures and increased both their ethylene production and their endo-beta-mannanase activity (Nascimento et al. 2001). The overall goal is to obtain a better understanding of the hormonal regulation of germination and dormancy for utilization in lettuce breeding and selection programs to improve seed performance under adverse planting conditions.

While genetic evidence implicates the participation of abscisic acid (ABA) in the induction of developmental arrest during seed maturation (Finkelstein et al. 2002), the role of ABA and its signaling components in the maintenance of dormancy after the seed is shed remains controversial. At least two possibilities exist. In dormant seeds (a) ABA may be required continuously or (b) only its signaling components may be needed. Two components of the ABA-signaling pathway (ABI1, ABI2) encode protein phosphatases (PP). Non-specific inhibitors of PP, phenylarsine oxide (PAO) and H2O2, break dormancy of red rice as pulse treatments (Cohn, unpublished). These substances inhibit ABI1 and ABI2 activity in vitro (Meinhard and Grill 2001; Meinhard et al. 2002). PPs are also inhibited by mildly acidic pH (Leube et al. 1998), the nitric oxides (Burke and Zhang 1998), fructose-2,6-bisphosphate (Erickson and Killilea 1992), and low temperatures (Monroy et al. 1997), factors that break dormancy or are early response phenomena of dormancy-breaking. Further research will determine whether ABA-signaling components are involved in the maintenance and release of seed dormancy and identify target genes.

Seed Technology. Seed enhancements are methods that improve germination or seedling growth, or facilitate the delivery of seeds or other materials required at time of sowing (Taylor et al. 1998). Seed enhancements are applied post-harvest, but prior to the completion of germination. Enhancements applied directly to seeds may be physical or physiological treatments. In addition, seeds may be treated for pest management, and seed coatings can serve several purposes. Collectively, many techniques may be employed to enhance seeds. The method(s) employed depends on species and desired outcome with respect to seed performance and utilization.

Physical methods: Eastern gamagrass, a productive and palatable native warm-season perennial grass, is propagated by seeds that are dormant (Ahring and Frank 1968; Anderson 1985). Structures encompassing the embryo have a substantial impact on the expression of dormancy. However, it is unlikely that mechanical or chemical scarification can be successfully applied to this species. Off-colored seeds are routinely present in a seed lot, and subtle differences in seed coat color may be related to seed quality. In addition to visible color changes, seed quality differences may be revealed in the UV and NIR (Lee et al. 1998). Color-sorting technology is available, and further research is needed to explore this technology to physically upgrade seed quality on a commercial basis. In some cases, physical treatments to seeds, such as seed coatings, can improve seed performance. Polymeric coatings can regulate water uptake and thus reduce imbibitional chilling injury in large seeded legumes (Taylor and Kwiatkowski 2001). Coatings may also be able to replace delinting in cotton, which can adversely affect seed quality (Laird et al. 1998).

Physiological methods: Native plant species require effective physiological methods to overcome dormancy. Procedures were developed for the germination of saltgrass, Distichlis spicata (L.) Greene (Bonnart et al 2000). Scarification either by hand or a pressurized air scarifier resulted in higher germination. However, scarification alone was insufficient to obtain greater than 50% germination, so other physiological methods are needed. Crop species can benefit from rapid and uniform seedling emergence, as slow emergence results in smaller plants and seedlings, which are more vulnerable to soil-borne diseases (Ellis 1989). Rapid field emergence is a fundamental prerequisite of increased yield of many annual crops. Various pre-sowing seed treatments are used to reduce the time from sowing and to seedling emergence and to improve seed performance. Seed priming has become a common commercial seed-enhancer process in a number of American and International seed companies (Parera and Cantliffe 1994). Priming, a pre-sowing treatment in which seeds are soaked in an osmoticum, allows them to imbibe water and go through the first stages of germination, but prevents radicle protrusion. The seeds can be dried and distributed for planting, when they exhibit more rapid and uniform germination. The advantage of priming may be lost in seed storage. However, this negative effect can be partially reversed by post-priming treatments including moisture content reduction and heat shock (Bruggink et al. 1999; Gurusinghe and Bradford 2001; Gurusinghe et al. 2002). Physiological treatments may alter growth and development and enhance plant utilization. Bedding plant growth is commercially regulated by synthetic chemicals, but there is strong demand for a natural organic growth regulator that could be applied as a seed treatment to control plant height without causing negative health and environmental impact. Preliminary work has demonstrated that specific sugars can decrease plant growth (Welbaum, unpublished). The germination of triploid watermelon is generally 60 to 80%, compared with to up to 95% for diploid seed. Triploids are very sensitive to soil media moisture content , ranging from 96 to 76% under low moisture, to < 27% under high moisture (Grange et al. 2000, 2003). Rapid imbibition and excess water collected in the seedcoat and air space surrounding the embryo may impair metabolic pathways leading to normal germination and seedling development.

Pest Management: Seed coating technologies provide delivery systems for application of seed treatments to control insects and diseases. Seed treatments result in more than an 85% reduction in pesticide usage compared to in-furrow treatments (Taylor et al. 2001). New chemical compounds with systemic activity have the potential to control both soil and foliar pests in the early stages of the growing season. Foliar pests may be the vectors for viruses (McGee et al. 2000). Seed treatments may also eradicate seed-borne pathogens. Biological seed treatments with beneficial microorganisms can substitute for some chemical protectants, particularly when combined with other treatments (Warren and Bennett 1999).

Seed quality assessment: Current seed testing for germination and vigor utilizes empirical parameters that lead to lack of standardization of results. Computer-aided image analysis is a method to quantify germination percentage, rate and radicle growth. Systems were developed that evaluate images of germinating seeds using computer acquisition of digital images captured using a flat-bed scanner (Geneve and Kester 2001; Sako et al. 2002). Biochemical methods can also provide rapid quantitative tests that are indicators of seed quality. For example, ethanol production from hydrated seeds is a high-resolution index of seed quality (Taylor et al. 1999: Kataki and Taylor 2001). Both the image-analysis and biochemical methods for seed quality assessment require further research and development before widespread commercial adoption is feasible.

Objectives

  1. Determine the influence of pre-harvest stress on seed quality.
  2. Identify the biophysical, biochemical and genetic factors governing seed desiccation tolerance and longevity.
  3. Identify genes associated with seed development, germination, vigor and dormancy.
  4. Develop technologies to assess seed quality, improve seed performance and enhance seed utilization.

Methods:


Effects of Preharvest Stress on Seed Quality. TeKrony (KY) and Egli (KY) will conduct growth chamber experiments to identify the critical growth stage during soybean seed filling when high temperature stress is most deleterious. Plants will be placed in high temperatures (38/27*C) for seven-day periods during seed filling and then returned to the greenhouse. Consecutive seven-day periods to cover the entire seed filling period will be used. Soybean cultivars (DP 4690 and Hutcheson) will also be grown in the field in several states in the southeastern USA under irrigated and non-irrigated conditions to determine the effect of temperature on seed quality in a field production environment.

Spears (NC) will grow soybean and peanut genotypes with enhanced oleic acid concentration in the field under variable production conditions. Seed quality will be measured and related to environmental conditions during seed development. Amino acid synthesis in plants under environmental (drought, high temperature) stress and coincident impact on seed quality will be investigated using soybean and cotton (Meints-MS). Timing and duration of the stress will also be incorporated to determine critical periods impacting synthesis.

Goggi (IA) will use known cold-tolerant and cold-susceptible maize inbreds to produce single-cross hybrid seed. Ears will be harvested at three seed maturity levels and frozen on the ear. A series of biological and molecular tests will be conducted at different times during seed storage to assess susceptibility to freezing damage.

Bennett (OH) will evaluate the cultural, treatment, and testing practices needed to successfully produce organic soybean and food grade open-pollinated corn seed. Soybean and food grade corn seed will be harvested from established field crop transition experiments. Biological control agents (Bacillus spp., Pseudomonas spp., yeast strains, other) will be applied to soybean and corn seed production fields three to four weeks prior to seed harvest. Seed lots produced will be compared for physiological and pathological measures of seed quality (seed weight, vigor testing, pathogen levels, seedling growth and uniformity).

Seed Desiccation and Longevity. Koster (SD) and Walters (ARS-CO) will use biophysical approaches to evaluate the physical factors that affect seed desiccation tolerance and longevity. Techniques such as differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR), and analysis of hydration parameters will be used to measure properties of water and solutions in both embryos and model systems. These measures will be related to the tolerance and longevity of the embryos to determine how hydration forces and viscosity influence the thermodynamics and kinetics of the processes that occur in dehydrated seeds.

McDonald (OH) will study soybean seeds to determine the role of free radicals and antioxidants in seed deterioration. Embryonic axes will be separated from the cotyledons and mitochondria isolated from each (Bakeeva et al. 1999). The rate of lipid peroxidation in the mitochondria will be determined based on the formation of malondialdehyde (Heath and Packer 1968). Mitochondrial DNA integrity will be determined, and glutathione activity will also be assessed (Puntarulo et al. 1988).

Genes Associated with Seed Quality. To determine the genetic basis of physiological differences in maize seed vigor, Knapp (IA) will screen a range of cold-tolerant maize inbreds and populations at 14, 18, and 22C. Emergence will be recorded and apparent photosynthesis by emerged leaves measured via infrared gas analysis. Seedlings will then be harvested for measurement of leaf area, shoot and root fresh and dry weights, and the weight of remnant seed (estimate of endosperm). These studies will characterize lines having superior performance at low temperature and determine whether this is due to their ability to mobilize stored reserves. Based upon the results, analyses of crosses among these lines and within segregating populations can be used to map these traits and identify candidate genes conferring cold tolerance and efficient reserve mobilization (Guse et al. 1988).

McGrath (ARS-MI) will use standard tools of genomics to investigate gene expression during seed development, germination and the first ten weeks of sugar beet growth. Biological materials include over 10,000 accessions of Beta vulgaris including high and low vigor germplasm and populations for genetic analysis. Two high-quality cDNA libraries (at 4 days and 10 weeks), a BAC library with 5X genome coverage, and over 19,000 ESTs are available for expression analyses and molecular mapping. Gene expression will be analyzed during flowering and seed development and during germination and the first 10 weeks of seedling growth. Signaling mechanisms related to perception and response to abiotic stress during the germination and stand establishment phase will be studied, beginning with two-hybrid protein interaction studies using a recently cloned germin-like protein as bait.

Bradford (CA) will identify quantitative trait loci (QTL) associated with seed dormancy and vigor traits in lettuce and sunflower by phenotyping recombinant inbred line (RIL) populations in which a large number of molecular markers are also being mapped. Microarray expression analysis and candidate-gene approaches based upon identification of putative homologs of known dormancy- or germination-associated genes in other species will also be employed (van der Geest 2002). Germination-associated genes identified will also be mapped to determine whether they co-localize with QTL.

Downie (KY) will test maize plants mutated in genes required for RFO synthesis to investigate how mutant seeds or vegetative organs/whole plants are affected by the introduced lesions. Standard germination tests, cold tests, and the capacity and speed with which seeds complete germination under far-red light will be evaluated to determine phenotypic effects of the mutations. The degree to which transcript abundance of various RFO and germination-associated genes is detrimentally affected will be determined using nuclease protection assays, northern hybridization, and PCR analysis. The effects on enzyme activity and on RFO amounts will be assessed by enzyme assays and GC, GC-MS, and HPLC analyses.

Nonogaki (OR) will clone sense and antisense LeMAN2 cDNA into constitutive gene silencing vectors (pHANNIBAL, pART27) (Wesley et al. 2001) and transform them into tomato to create double-stranded RNA transcripts that will silence the endogenous gene. Similar lines are being constructed by Bradford (CA) for LeMAN2 and several other germination-associated genes (LeEXP4, LeEXP8, LeXET4, LeSNF4) under the control of an estradiol-responsive inducible promoter (Zuo et al. 2000). This promoter will allow either overexpression or suppression of specific genes in a controlled inducible manner to test their function during germination. In addition, Nonogaki (OR) will construct enhancer-trap lines (Campici et al. 1999) and reporter gene lines in Arabidopsis to identify additional embryo- and germination-specific genes as candidates for further functional studies of the mechanisms of dormancy and germination.

Cantliffe (FL) will transform a dominant mutant of the ETR 1-1 gene conferring ethylene insensitivity (Bleeker et al. 1988) and overexpression or suppression constructs controlling an ACC synthase gene (e.g, Tarun et al. 1998) into lettuce to manipulate ethylene synthesis during seed germination. Seeds derived from these plants will be evaluated for ethylene production and sensitivity, germination at a range of temperatures, endo-beta-mannanase activity, and responsiveness to other hormones and light.

To test the role of protein phosphatases (PP) in maintenance of dormancy, Cohn (LA) will incubate dormant red rice caryopses and seeds of other selected species with a range of PP inhibitors (okadaic acid, cantharidin, calyculin A, 3,4-dephosphatin, b-naphthyl- phosphate, endothall, and ascorbate analogs) for 24 h, rinse and then transfer them to water for 7 d at 300C. Germination percentages, speed of germination and viability will be assayed.

Seed Technology. Seed enhancement methods will be investigated on selected crop and native seeds including lima beans and parsley (Pill-DE), triploid watermelons (Leskovar-TX), lettuce, and tomato (Cantliffe-FL, Bradford-CA), pepper (Cantliffe-FL), eastern gamagrass (Knapp-IO), saltgrass (Hughes-CO), native grass species including switchgrass, little bluestem, big bluestem, purpletop, and beaked panicum (Panicum virgatum, Schizachyrium scoparium, Andropogon gerardii Vitman, Tridens flavus, Panicum, anceps, respectively) (Meints-MS). Methods examined will vary by species and include controlled hydration by liquid, solid matrix or drum priming. The application of plant growth regulators, hydrogen peroxide, pre-chilling and stratification will be examined singly or in combination. Germination, seedling growth, and field establishment will measure seed enhancement efficacy. Endo-beta-mannanase enzymatic activity (Cantliffe-FL) and morphological studies (Leskovar-TX, Pill-DE, Taylor  NY-G) will be conducted in conjunction with priming treatments. DNA integrity will be examined (Bradford-CA) by pulsed field electrophoresis to separate DNA from seeds and assess its large-scale integrity. Additional techniques, such as the TUNEL reaction and DNase susceptibility assays, may also be employed to quantify DNA integrity. Cell-cycle associated genes and proteins will be assayed for their expression and abundance during and after priming and post-priming treatments and re-imbibition.

Color sorting technology is available for detection in the visible, UV, NIR and by fluorescence. New optical sensor technology is expanding the capability to sort difficult-to-condition seed lots. Misra (IO) and Taylor (NY-G) will study light reflectance characteristics in relation to seed quality with the objective to upgrade seed quality.

Seed treatment and coating technologies will be examined to deliver plant protectants and modify physical parameters of seeds. Laboratory-scale film coating and pan coating equipment is available at Cornell (Taylor-NY-G), and will be used for application of chemical and biological seed treatments. Insecticidal seed treatments will be used to control seed maggot in small and large-seeded vegetable crops (Taylor and Nault-NY-G). Systemic seed treatments will be examined to control the bean leaf beetle, the vector of bean pod mottle virus (McGee-IO). Fungicide seed treatments will be tested to protect lima beans from Alternaria spp. (Pill-DE) and to eradicate seed-borne pathogens in onions (Taylor-NY-G). In conjunction with the seed treatment research, onion seed quality will be examined from coated and treated seeds (Taylor-NY-G), and (McGee-IO) will update the Database on Seedborne Diseases developed in conjunction with CABI International. Other seed treatment research will explore sugars to control bedding plant growth (Welbaum-VT), and polymeric coatings to retard water uptake into lima beans (Pill-DE). The effect of starch-based seed coatings on germination in cotton will be studied by Meints-MS). Potential for incorporation of other seed treatments and amendments will also be investigated for use in cotton as an alternative to acid delinting.

Digital cameras and scanners are used to capture images of seeds and seedlings, which can then be analyzed by software for seed vigor indices, including length, width, height, perimeter, area, color attributes, convex hull area, extent fill and texture. From these determinations, seed/seedling identification tasks can be accomplished as well as evaluations of seedling growth parameters. Digital imaging research will focus on high-value flower seeds (petunia) (Geneve-KY) and field crops (soybeans and field corn) (McDonald-OH).

Biochemical vigor tests will be studied by quantifying volatile compounds from soybeans and snap beans (Taylor-NY-G). Closed systems will be developed to hydrate single seeds to a given seed moisture content and trap the emission of organic volatile compounds. Volatiles will be characterized by GC/MS from aged and nonaged seeds. Volatiles that are common from both lipid- and starch-storing seeds will be further exploited into developing a rapid seed quality test.

Measurement of Progress and Results:

Outputs:
Outcomes or projected Impacts:
Milestones:

(2004): Conduct controlled environment studies during seed development on seed quality during years 1 and 2. Develop methods to quantify free radicals and antioxidants. Identify QTL associated with seed quality traits in selected crop plants. Adapt seed coating equipment for seed treatment application.

(2005): Develop methods to analyze water and solutions in embryos and model systems. Conduct microarray and other analyses of gene expression in genotypes differing in seed quality traits. Refine digital imaging equipment to characterize germination events. Study efficacy of new chemistry plant protectants in years 2  5.

(2006): Conduct field studies on the effect of environmental stress on seed quality in years 3 and 4. Conduct physiological and biochemical analyses of the functional roles of germination-associated genes, including both mechanistic and signaling/regulatory genes, in targeted species. Develop semi-automated systems to assess seed quality based on germination events.

(2007): Correlate free radicals and antioxidants on seed aging and the reduction of seed quality. Examine the physical properties of water on desiccation tolerance. Develop biochemical or genetic screens for seed/seedling vigor based upon physiological criteria. Test specific genetic or chemical manipulations predicted to enhance seed quality.

(2008): Relate biochemical and other markers during seed production under stress on seed quality. Utilize biochemical or genetic screens in cooperation with breeders to improve seed vigor. Develop "proof-of-concept" lines demonstrating that genetic and/or chemical manipulation of specific genes can improve seed performance or reduce dormancy.

Through W-168, a critical mass of researchers in seed biology has been coalesced to address scientific issues critical to agriculture and environmental quality in the U.S. The increasing importance of seeds in the delivery of advanced genetics makes the continued operation of this collaborative project essential to the health and competitiveness of the US Seed Industry through the expertise, information, and services it provid

Projected Participation:

Include a completed Appendix E form

Outreach Plan:

The members of W-168 comprise a group of highly dedicated seed biologists who excel in the communication of their research findings. All members of the W-168 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. All W-168 members regularly publish their finding in top-tier, peer-reviewed journals, targeting both the general plant biology and seed biology communities. W-168 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-168 members serve on journal editorial boards and/or as ad-hoc manuscript reviewers, and they, therefore, help to enhance the quality of published seed research by others.

In August 1997, W-168 organized a symposium to highlight the findings and insights of the group. The proceedings of this meeting were published in the June 1998 issue of Seed Science Research, a peer-reviewed journal published by CABI International. According to the ISI Web of Science, these papers have been cited 126 times since their publication and are still frequently cited at present. The group is considering a second symposium to be held during the next five-year period.

Organization and Governance:

The recommended Standard Governance for multistate research activities include the election of a Chair, a Chair-elect, and a Secretary. All officers are to be elected for at least two-year terms to provide continuity. Administrative guidance will be provided by an assigned Administrative Advisor and a CSREES Representative.

Literature Cited:

Literature Cited

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