W3168: Environmental and Genetic Determinants of Seed Quality and Performance

(Multistate Research Project)

Status: Inactive/Terminating

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Date of Annual Report: 03/07/2017

Report Information

Annual Meeting Dates: 10/10/2014 - 10/11/2014
Period the Report Covers: 10/01/2014 - 09/30/2015

Participants

Bennett, Mark – The Ohio State University, Bradford, Kent – University of California, Davis (joined by video-link), Geneve, Robert – University of Kentucky, Jourdan, Pablo – The Ohio State University, Leskovar, Daniel – Texas A&M University, McGrath, Mitch – USDA-ARS and Michigan State University, Nonogaki, Hiro – Oregon State University, Perez, Hector – University of Florida, Taylor, Alan – Cornell University, Walters, Christina – USDA-ARS and Colorado State University, Welbaum, Greg – Virginia Tech.

Participants Attachment:

W3168-2014.submitted1.pdf

Brief Summary of Minutes

Brief Summary of Minutes of Annual Meeting

Pablo Jourdan welcomed the participants to Ohio State and provided logistical information about the venue. Robert Geneve agreed to chair the meeting. Minutes from the 2013 W2168 meeting were discussed and approved. A general discussion about the new structure for the group and organization of the meeting ensued. Taylor provided some background on the need for the new approach. The overall goal is to foster greater collaboration among the group members. An important element is the identification of funding sources to support the common efforts of the group. Potential new members were identified, and will be contacted by current members explaining the advantages and benefits of participating in the W3168 project. A general consensus was formed regarding the need to keep seed science as an important topic for research and education. There is a perception that there are ‘no problems’ in seed-related areas and that industry takes care of all seed issues, but this is clearly not the case. There is significant industry involvement in the major row crops, but there is much other seed work that is not being addressed. Of particular concern is the training of future seed scientists. These scientists will come from active research and educational programs in seed science at different institutions. Nonogaki suggested that education is a common core activity of the group. Shared educational activity is an area that the group can develop as part of its future collaborative efforts. Discussion of individual objectives followed, stimulated by presentations from individual participants. A discussion was also held about the group’s endorsement of Bradford’s offer to host the 2017 International Society for Seed Science meeting in Monterey, CA. An overview of the proposal to the ISSS was given and there was unanimous enthusiasm for assistance with the scientific program of the meeting. State reports were then presented.

Objective 1 - Identifying key factors involved in the enhancement or loss of seed quality. Seed development through post-harvest losses in storage. Specific topics include seed development, desiccation tolerance, and aging in storage.

CO: Walters. Relayed recent work focusing on seed aging kinetics and seed longevity.

FL: Perez. Related activities supporting Uniola paniculata (Sea-oats) germplasm collection and preservation. Sea oats is a keystone species of coastal dunes throughout the south-eastern US and Caribbean region.

CA: Bradford. Described research on population-based threshold models that describe seed respiration and germination rates.

OH: Jourdan. Described seed research at the Ornamental Plant Germplasm Center on the campus of The Ohio State University.

Objective 2 - Eliminating seed dormancy as a constraint during seed production and germination in agronomic seed production and ecological/biomass seed establishment. Pre-mature sprouting in cereals and other species and the identification dormancy mechanisms to manipulate germination.

KY: Geneve. Discussed work on seeds of the Asteraceae, and showed stunning images of the internal structure of seeds.

CO: Walters. Discussed elimination of seed dormancy in seeds used for restoration projects by seeking to understand the clock mechanism in dry seeds to increase the success of direct- seeded restoration projects such as the germination of Monterrey pine seeds.

CA: Bradford. Presented video ‘A Dual Role for DOG1 in Regulating Seed Thermo-inhibition and Flowering Time in Lettuce’.

OR: Nonogaki. – Recent work on the manipulation of seed dormancy and germination via a new ‘Spontaneous Hyperdormancy’ system was presented. The Spontaneous Hyperdormancy system can be used to prevent pre-harvest sprouting in cereal crops.

SD: Gu. (excused) Submitted report on recent results involving genetic dissection of tissue- based controls on seed dormancy.

Objective 3 - Enhancing seed vigor and germination in agronomic and other species for improved stand establishment. The emphasis of this objective is on post-harvest technologies.

MI: McGrath. Described progress on understanding and genetically improving sugar beet seedling vigor, emergence, and stand establishment.

NY: Taylor. Summarized recent work on chemical and biological seed treatment and seed coating technologies for conventional and organic systems for high value vegetable seed markets.

TX: Leskovar. Relayed progress in vegetable physiology in the 2nd largest winter garden in Texas focuses on adaptation mechanisms to environmental stresses (heat and drought), and development of integrated sustainable vegetable cropping systems particularly for stand establishment of transplants.

VA: Welbaum. Described his work on thermogradient tables using a gusseted soil chamber to study seed germination under realistic conditions, with other modification adding LED lights for illumination, and micro-irrigation research and a greenhouse cover to increase flexibility in the types and age of plants that can be studied.

FL: Perez. Described research on enhancing seed germination of Aristida stricta (wiregrass), a southeastern US native plant in fragmented habitats.

KY: Geneve. Described research on extensive variability of mucilage production and forms from the pericarp of seeds in the Lamiaceae, where it is not clear what critical function such an expensive resource may assume.

Accomplishments

<h1>Accomplishments</h1> <h3>Objective 1 - Identifying key factors involved in the enhancement or loss of seed quality.</h3> CO: Walters. Dynamic mechanical analysis (DMA): measures the tendency of molecules in structures to move, and in peas with 7% water content, there is still motion in seed glasses even at low temperatures. Potential tools to predict longevity and monitor deterioration using small sample sizes have been identified including mechanical analysis, liquid crystallization, volatile assessment, and RNA integrity. In addition, the team is validating predictive tools using  historical experiments, such as the Went/Munz experiment where seeds from 93 California native species were placed in sealed ampoules in 1948 and kept at room temperature, then tested for germination in 2014. Some seeds showed remarkable longevity when stored very dry at room temperature; for example, Clarkia elegans with germinated to 90% after 2 days. Walters also discussed the ambitious genebanking standards approved in 2013. These emphasize longevity in genebanks to mitigate genetic erosion during regeneration. Research is needed to clarify standards on ‘critical’ moisture level, the risk of over-drying, optimum drying protocols, optimum harvest time, and viability monitoring frequency. FL: Perez. Mature seeds of Uniola paniculata (Sea-oats) were collected from north and south Florida populations, and data was collected on the relationship between relative humidity and seed water content (i.e. water sorption isotherms), germination ability of seeds following equilibration to a range of relative humidity (i.e. desiccation tolerance), and the germination ability of seeds following factorial treatments of desiccation and duration of storage in liquid nitrogen. Seeds of both populations had similar water sorption isotherms, and the capacity for germination in both populations was retained to high levels (ca. 63-99%) following equilibration to very low (0.5% RH) moisture conditions. Likewise, germination ability remained high (ca. 59- 94%) following desiccation and storage in liquid nitrogen for up to the maximum duration tested, i.e. 24 hours. CA: Bradford. Respiration associated with seed vigor and vitality: The tetrazolium test indirectly indicates actively respiring tissues. Tomato seed respiration and germination responded to both temperature and water potential in two genotypes (T5 and PI341988). At low temperatures and water potentials, respiration continued at slow rates even when germination would be very slow or completely inhibited. Because of limitations to bulk respiration measurements, it would be better to be able to measure the respiration rates of many individual seeds using the Q2 seed respiration instrument. Germination rates are sensitive indicators of seed quality, and respiration rates reflect germination rates. Some important observations: Seed respiration is initiated immediately upon hydration; Respiration rates are closely correlated with germination rates for many seeds and under diverse conditions; Individual seed respiration rates as determined in the Q2 can be converted into “respiration population time courses” that are similar to a germination time course; Analyses and models that are applied to germination time courses can also be applied to these respiration time courses, describing the effects of temperature, water potential, aging and other factors; Automated measurement of seed respiration on a population basis can reveal diverse components of seed quality. OH: Jourdan. Recent focus has been on developing reliable quantitative germination tests for the very small Begonia seed using an agar substrate and applying this method to compare the germination characteristics of six different species. Other work aims to identify key parameters that affect seed quality in Phlox to generate seeds for germplasm preservation. Also working to enhance Phlox germplasm by interspecific hybridization. Collaborating with Walters (CO) on seed characteristics Begonia and Magnolia virginiana, with the goal to determine potential longevity of these seeds in storage, as well as with Perez (FL) on seed internal structure as related to viability using an X-ray procedure, and with Geneve (KY) examining the pattern of germination of Begonia seeds. <h3>Objective 2 - Eliminating seed dormancy as a constraint during seed production and germination in agronomic seed production and ecological/biomass seed establishment.</h3> KY: Geneve. Discussed work on seeds of the Asteraceae, and presented stunning images of the internal structure of selected seeds of this family. CO: Walters. Discussed elimination of seed dormancy in seeds used for restoration projects by seeking to understand the clock mechanism in dry seeds to increase the success of direct- seeded restoration projects such as the germination of Monterrey pine seeds. CA: Bradford. DELAY OF GERMINATION 1 (DOG1) was identified in the Cvi accession of Arabidopsis as a QTL conditioning a requirement for long after-ripening times for the alleviation of dormancy. DOG1 expression is also sensitive to the temperature of seed maturation. Higher expression of DOG1 is correlated with stronger dormancy.  A DOG1-like gene is expressed during lettuce seed development slightly after expression of NCED4 (encoding an ABA biosynthetic gene) begins.  However, silencing NCED4 expression did not appreciably alter expression of DOG1, similar to results in Arabidopsis.  DOG1 expression in mature lettuce seeds decreased as the temperature during seed maturation increased. Germination at 34°C of freshly harvested Arabidopsis wild type and dog1-1 mutant seeds that were grown at either 10 or 22°C. Regardless of maturation temperature, dog1-1 mutant seeds germinated better at high temperature. Overexpression of the lettuce DOG1 from two thermosensitive genotypes (L. sativa cv. Salinas and L. saligna) and two thermotolerant genotypes (L. serriola and L. sativa PI251246) can rescue the dog1-1 mutant.  Silencing of the lettuce DOG1 in the thermosensitive cv. Salinas improved seed germination at high temperature. In summary, Lettuce DOG1-like genes are functional in Arabidopsis and promote seed dormancy and thermoinhibition; DOG1 action lowers maximum germination temperatures; DOG1 action also delays flowering times, as silencing DOG1 in lettuce results in early bolting and flowering; DOG1 may act on both germination and flowering via potentiating the activity of miR156, which has other effects on the plant life cycle; and DOG1 may be involved more generally in sensing environmental conditions (particularly temperature) and modifying life cycle progression (germination, flowering) through influencing the action of microRNAs. OR: Nonogaki. – For the hormonal regulation of seed germination, the previous studies on the role of abscisic acid (ABA) were expanded. In addition to GeneSwitch technology, a chemically inducible gene expression system which allows induction of seed dormancy by a chemical ligand, a new technology ‘Spontaneous Hyperdormancy’ was developed. In this system, the nine-cis-epoxycarotenoid dioxygenese (NCED) gene driven by an ABA-inducible promoter causes amplification of NCED expression in a spontaneous manner. The Spontaneous Hyperdormancy system can be applied to preventing pre-harvest sprouting in cereal crops. SD: Gu. Completed the anlyses of an endosperm genotype-based genetic approach to determine genes regulating seed dormancy through the embryo, endosperm or maternal tissues. Using this new approach, previously identified seed dormancy loci SD12, SD1-2 and SD7-1 were demonstrated to be involved in the regulation of germinability through the embryo, endosperm and maternal tissues, respectively, in rice. Functional analysis of candidate genes cloned from the SD12 seed dormancy QTL in rice were completed, and map-based cloning of the seed dormancy QTL Sd1-2 and SD7-2 was initiated. <h3>Objective 3 - Enhancing seed vigor and germination in agronomic and other species for improved stand establishment.</h3> MI: McGrath. Germination is crucial to developing healthy, vigorous, and productive field populations of sugar beets. Despite planting high-quality, technically-augmented seed for growers with very high germination (>92%), field emergence and persistence continues to hover at ~60% in Michigan. Previous research suggests this difference is the result of stress during germination in the field environment. The East Lansing sugar beet program has focused on stress responses during germination. One way to identify additional genes involved is to examine expression of all genes during germination in different environments. In this case, we generated four transcriptome datasets of a high vigor variety germinated in stressful germination-in-solution laboratory environments, including water, hydrogen peroxide, sodium chloride, and hydrogen peroxide plus sodium chloride. From over 200 million sequences assessed, and in comparison to water, each treatment showed between 75 and 100 statistically significant, differentially expressed genes, with approximately 10% overlap between any pair of treatments, and only seven shared among treatment comparisons. Approximately 50% of these have no descriptors yet found in nucleotide sequence databases, suggesting germination genes are less well represented in comparative genomics resources. NY: Taylor. Developed rotary pan equipment able to treat as few as 25 grams of seed with  novel coatings, and tested it on onion seeds that were pelleted, film coated, and encrusted. Fillers typically used are diatomaceous earth, clays, and calcium carbonate. Pelleting is often done for precision seeding, especially for seed with irregular shapes or elongated seed, but  adds 6-7 times the weight of raw seed, thus an application and transportation cost to seed producers. Pellets provide a physical barrier, with some pellets splitting to release the seed, whereas others melt away upon watering. Also developing applications using multiple seed pellets (agglomerates) for situations where stand could be improved with numerous propagules per placement. Developed an alternate method for onion testing, an organic peat medium in rolled paper towels, that is accepted as an AOSA rule. A new AOSA rule has expanded from our onion method called the Cornell method to include all crop seeds and there is a new ISTA rule for the same. Systemic movement of insecticides in treated or pelleted seeds are also of concern since their hydrophobicity determines whether they are absorbed through the seed coat and taken up by the embryo or blocked by seed coat and then taken up by root. In onion seeds (also tomato, pepper, switchgrass, field & sweet corn), non-ionic molecules (assessed by the fluorescent tracer dye coumarin) can move through seed coat but ionic (i.e. rhodamine dye) cannot, and is instead taken up by the root as the result of a selectively-permeable testa. Cucumber and lettuce have an impermeable testa, to both rhodamine and coumarin. Systemic insecticides from seed treatments may last 30 days post-emergence, spread apoplastically and move with transpiration stream, and do not accumulate in the fruit. Biostimulants may also be applied as seed coating, such as proteins where gelatin may enhancement growth, soy flour may lead to promotion of growth of Brassica seedlings, but in all cases, side effects of seed treatment may result in increased proportion of abnormal seedling development, perhaps caused by high loading rates on the seed coats. Taylor also highlighted recent collaborations with Ohio State (Bennett, Jourdan) on Taraxacum kok-saghyz seed coating and germination research, W3168 member Brian Nault on entomological treatment of seeds to control leaf hopper (thiamethoxam) and onion maggot, as well as testing organic-approved treatments of seeds (spinosad), and with Sally Miller on bacterial seed treatments (there are few bacteriocidal seed treatments). TX: Leskovar. Development of integrated sustainable vegetable cropping systems for Texas emphasizes seed-transplant production and physiology to increase plant survival and enhance stand establishment, plant hormone applications to modulate seedling growth, determining root/shoot developmental responses to water conservation strategies and irrigation technologies, the impact of cropping systems on antioxidants and sensory attributes of vegetable crops, and genotype selection for drought resistance, high yield, quality, and phytochemical content. A recent emphasis has been examination of root genetic traits using electromagnetic scanning systems to breed for specific root attributes. VA: Welbaum. In addition to the further development of the thermogradient table, research on bacterial fruit blotch in cucurbits was investigated. Nano-chitin seed treatment (during seed priming) was tested to increase disease resistance of plant seedlings with both chitin and nano- chitin seed treatments inducing expression of chitinase in developing and germinating seeds. Welbaum also highlighted recent collaboration with Bradford on the production of a new vegetable production textbook. FL: Perez. Quantified the proportion of filled, unfilled, and infected seeds of Aristida stricta (wiregrass) across seven populations occurring throughout the southeastern US, the germination and presence of contamination and treatment in response to commercial bleach disinfection, and identification of fungal pathogens.  Approximately 55-90% of seeds were non- viable due to lack of seed fill (49-77%) or fungal contamination (2-25%). Populations were similar for seed lot quality when grouped by habitat (e.g. xeric vs. mesic). However, populations were different for seed lot quality when grouped by geographic region. Seeds from all populations displayed infection by Curvularia and most also showed signs of Sorosporium infection, and the use of bleach solutions did not eliminate pathogens on otherwise viable, healthy seeds suggesting that internal tissues may harbor infection. KY: Geneve. Examined numerous species of the Lamiaceae including chia (Salvia hispanica) for mucilage morphology during hydration. Seed mucilage may have a role in adhering to a substrate prior to germination, and may also sequester moisture needed during germination. It was observed that little or no mucilage layer in visible via microscopy in dry seed, but that within a few minutes of hydration, the layer expands to the seed diameter or greater. Further, the unfolding was filmed and each species had it’s own characteristic unfolded structures and morphology. Seed mucilage has potential human health benefits, and production of these species is increasing.

Publications

<h2>Publications</h2> Bewley, J.D., Bradford, K.J., Hilhorst, H.W.M., and Nonogaki, H. (2013) Seeds: Physiology of Development, Germination and Dormancy. Third Edition. (New York: Springer). Boddy, L.G., Bradford, K.J., Fischer, A.J. (2013) Stratification requirements for seed dormancy alleviation in a wetland weed. PLoS ONE 8(9) e71457. Bradford, K.J., Bello, P., Fu, J.C., Barros, M. (2013) Single-seed respiration: a new method to assess seed quality. Seed Science & Technology 41: 420-438. Christian E.J., A.S. Goggi, K.J. Moore (2014) Temperature and Light Requirements for Miscanthus sinensis Laboratory Germination Test.  Crop Science 54: 791-795. Dias, M.A.N., A.G. Taylor and S.M. Cicero (2014) Uptake of systemic treatments by maize seeds evaluated with fluorescent tracers.  Seed Science and Technology: 42, 101-107 Duclos, D.V., Altobello, C. O., Taylor, A. G.    (2014) Investigating seed dormancy in switchgrass (Panicum virgatum L.): Elucidating the effect of temperature regimes and plant hormones on embryo dormancy. Industrial Crops and Products 58: 148–159. Duclos, D.V., Ray, D.T., Johnson D.J., Taylor, A. G (2013) Investigating seed dormancy in switchgrass (Panicum virgatum L.): understanding the physiology and mechanisms of coat- imposed seed dormancy.  Industrial Crops and Products 45 (2013) 377-387. Feng, J., H. Ye, V. Srivastava, X.-Y. Gu. (2014) “Evolutionary and developmental mechanisms  of seed dormancy revealed by map-based cloning of genes underlying a major quantitative trait locus from weedy rice. The 35th Rice Technical Working Group Meeting, New Orleans, LA. Feb. 18-21, 2014. Geneve, R., L.D. Hildebrandt, T. Phillips, N.S. Gama-Arachchige, J. Kirk, M. Al-Ameriy (2014) Seed germination and mucilage production in chia (Salvia hispanica).  Acta Horticulturae. Gu, X-Y. (2014) Challenges and strategies of genetic improvement for pre-harvest sprouting in seed production of hybrid rice. p. 73 in Book of Abstracts for The 11th Conference of the International Society for Seed Science, Changsha, China. Gu, X.-Y., H. Ye, J. Feng, M.S. Mispan. (2014) “Rice green revolution gene: its effects beyond plant height”, The 35th Rice Technical Working Group Meeting, New Orleans, LA., Feb. 18-21, 2014. Gu, X.-Y., J. Zhang, H. Ye, L. Zhang, J. Feng. (2015) Genotyping endosperms to determine genes regulating seed dormancy through the embryo, endosperm or maternal tissues in rice. G3: Genes, Genomes, Genetics 5: 183-193; DOI: https://doi.org/10.1534/g3.114.015362. Huo, H., Dahal, P., Kunusoth, K., McCallum, C.M., Bradford, K.J. (2013) Expression of 9-cis- EPOXYCAROTENOID DIOXYGENASE4 is essential for thermoinhibition of lettuce seed germination but not for seed development or stress tolerance. Plant Cell 28: 884-900. Keaton K., S. Goggi, A.P. Mallarino, R.E. Mullen (2014) Phosphorus and potassium fertilization effects on soybean seed quality and composition. Crop Science 53: 602-610. Leskovar, D.I., Crosby, K.M. Palma, M.A. Edestein, M (2014) Vegetable crops: Linking production, breeding and marketing. In Dixon, G. R. Aldous, D. (eds.) Horticulture: Plants for People and Places, Vol 1: 75-96.  Springer. McGrath, J.M. (2014) Release of EL58 and EL60 sugarbeet germplasm with nematode resistance. USDA-ARS Germplasm Release Notice. Mylor, K., Holton, S., Geneve R.L., Calyskan, S. (2013) Comparison of physical, acid, and hot water scarification on seed germination in Eastern redbud.  ISHS-Acta Horticulturae 1055: 347- 349. Nonogaki H. (2014) Seed dormancy and germination - Emerging mechanisms and new hypotheses. Frontiers in Plant Science 5, 233. doi: 10.3389/fpls.2014.00233. Nonogaki M, Sall K, Nambara E, Nonogaki H. (2014) Amplification of ABA biosynthesis and signaling through a positive feedback mechanism in seeds. The Plant Journal 78: 527-539. Panella, L.W., Kaffka, S.K., Lewellen, R.T., McGrath, J.M., Metzer, M.S., Strausbaugh, C.A. 2014. Sugarbeet. In: Smith, S., Diers, B., Specht, J., Carver, B., editors. Yield Gains in Major U.S. Field Crops. CSSA Special Publication 33. Madison, WI: American Society of Agronomy, Inc., Crop Science Society of America, Inc., and Soil Science Society of America, Inc. p. 357- 396. Pérez, H.E. (2014) Do habitat and geographic distribution influence decreased seed viability in remnant populations of a keystone bunchgrass? Ecological Restoration 32: 295-305. Pérez, H.E., K. Kettner (2013) Characterizing Ipomopsis rubra (Polemoniaceae) germination under various thermal scenarios with non-parametric and semi-parametric statistical methods. Planta 238: 771-784. Puttha R, Goggi, S., Gleason M.L., Jogloy S., Kesmala T., Vorasoot N., Banterng P., Patanothai, A (2014) Pre-chill with gibberellic acid overcomes seed dormancy of Jerusalem artichoke. Agron. Sustain. Dev. (2014) 34:869–878 DOI 10.1007/s13593-014-0213-x. Rosental l, Nonogaki H, Fait A. (2014) Activation and regulation of primary metabolism during seed germination. Seed Science Research 24, 1-15. Rushing, J.B, B.S. Baldwin, A.G. Taylor, V.N. Owens, J.H. Fike, K.J. Moore (2013) Seed safening from herbicidal injury in switchgrass establishment. Crop Science 53: 1650-1657. Schneider Teixeira, A., Ballesteros, D., Molina-Garcia A.D., Walters, C. (2013) Interactions between water and triacylglycerols may explain faster aging rates in stored germplasm at low temperatures. Abstracts #49. / Cryobiology 65 (2012) 339–366. <a href="http://dx.doi.org/10.1016/j.cryobiol.2012.07.050">http://dx.doi.org/10.1016/j.cryobiol.2012.07.050</a>. Sikhao, P., P. Teeraponchaisit, A. G. Taylor and B. Siri (2014) Seed coating with riboflavin, a natural fluorescent compound, for authentification of cucumber seeds. Seed Science and Technology: 42, 171-179. Stevanato, P., Trebbi, D., Biancardi, E., Cacco, G., McGrath, J. M., Saccomani, M. Evaluation of genetic diversity and root traits of sea beet accessions of the Adriatic Sea coast. Euphytica. DOI:10.1007/s10681-012-0775-0. 2012. Von Mark V. Cruz, C.T. Walters, D.A. Dierig (2013) Dormancy and after-ripening response of seeds from natural populations and conserved Physaria (syn. Lesquerella) germplasm and their association with environmental and plant parameters.  Industrial Crops and Products 45 (2013) 191–199.  <a href="http://dx.doi.org/10.1016/j.indcrop.2012.12.018">http://dx.doi.org/10.1016/j.indcrop.2012.12.018.</a> Walters, C (2013) Extreme biology: Probing life at low water contents and temperatures. Abstracts #087. / Cryobiology 67 (2013) 398–442<a href="http://dx.doi.org/10.1016/j.cryobiol.2013.09.093"> http://dx.doi.org/10.1016/j.cryobiol.2013.09.093.</a> Walters, C (2014) Genebanking Seeds from Natural Populations.  Natural Areas Journal 35(1):98-105. Wilson, R. G., S. B. Orloff and A. G. Taylor (2014) Evaluation of insecticides and application methods to protect onions from onion maggot, Delia antiqua and seedcorn maggot, Delia platura, damage. Crop Protection: 67, 102-108. Wood, L.A., S.T. Kester, R.L. Geneve (2013) The physiological basis for ethylene-induced dormancy release and germination in three Echinacea species with special reference to the influence of the integumentary tapetum.  Scientia Horticulturae 156:63-72. Xia, K., L.M. Hill, D.-Z. Li, C. Walters (2014) Factors affecting stress tolerance in recalcitrant embryonic axes from seeds of four Quercus (Fagaceae) species native to the USA or China Annals of Botany   doi:10.1093/aob/mcu193, available online at <a href="http://www.aob.oxfordjournals.org/">www.aob.oxfordjournals.org.</a> Xiang, C., Taylor, A., Frey, M.W. (2013) Controlled release of nonionic compounds from poly(lactic acid)/cellulose nanocrystal nanocomposite fibers. Journal of Applied Polymer Science 27: 1, 79–86. Ye, H., J. Feng, X.-Y. Gu (2014) “Mitigating risk of gene-flow from transgenic cultivars to weedy rice by silencing seed dormancy genes”, The 35th Rice Technical Working Group Meeting, New Orleans, LA. Feb. 18-21, 2014.

Impact Statements

Coating technologies can enhance seed performance, but small seed lots are difficult to treat uniformly. A device was constructed that allows relatively small seed lots to be coated with virtually any potential seed enhancing compound for preliminary evaluations. A wide range of compounds is being tested, allowing seed producers to hone in on the most beneficial seed treatments at minimal expense.

Date of Annual Report: 09/20/2016

Report Information

Annual Meeting Dates: 02/25/2016 - 02/27/2016
Period the Report Covers: 10/01/2014 - 09/30/2016

Participants

Yadegari, Ramin (yadegari@email.arizona.edu) University of Arizona; Bradford, Kent (kjbradford@ucdavis.edu) UC-Davis; Perez, Hector (heperez@ufl.edu) University of Florida; Goggi, Susana (susana@iastate.edu) Iowa State University; Geneve, Robert (rgeneve@uky.edu) University of Kentucky; Cohn, Mark (mcohn@agcenter.lsu.edu) Louisiana State University; Taylor, Alan (agt1@cornell.edu) Cornell University; Jourdan, Pablo (jourdan.1@osu.edu) The Ohio State University; Gu, Xinyou (xingyou.Gu@sdstate.edu) South Dakota State University; Leskovar, Daniel (Daniel.Leskovar@ag.tamu.edu) Texas A&M; Welbaum, Greg (welbaum@vt.edu) Virginia Tech; Johnson, Paul (paul.johnson@usu.edu) Utah State University; Lin, Liang Shiou (llin@nifa.usda.gov) USDA-NIFA; Joshi, Vijay (vijay.Joshi@ag.tamu.edu) Texas A&M; Othman, Yahia (yahia.othman@ag.tamu.edu) Texas A&M

Brief Summary of Minutes

Meeting was presided by Susana Goggi (IA). Minutes of Oct 2014 meeting were reviewed and approved. Discussion of next meeting ensued: will be held in Monterey, CA in conjunction with the ISSS Meeting (coordinated by K. Bradford) between 10-15 September 2017. Participants at the meeting were introduced. Presentation by Dr. Lin, National Program Leader for Plant Science from USDA-NIFA followed, where he described funding opportunities available through NIFA. Dr. Paul Johnson, Chairman, Department of Plant, Soils and Climate at Utah State University, introduced himself as the new Administrative Advisor to the Multistate Committee and shared information about his activities and programs. State reports were presented next, following the project objectives.

Objective 1 - Identifying key factors involved in the enhancement or loss of seed quality. Seed development through post-harvest losses in storage. Specific topics include seed development, desiccation tolerance, and aging in storage.

AZ: Yadegari. A new member of the Multistate Committee, provided his background and description of research on early endosperm proliferation and development in Arabidopsis and maize. Interest in the earliest events in endosperm development; cellularization after double-fertilization; cell differentiation & specification program that leads to endosperm formation. Has been examining gene regulation (OPAQUE 2 regulator) by exploring RNAs, building networks, transcription factors, etc. Also using yeast 1 hybrid analysis; reverse genetic mutants in maize and laser capture micro-dissection (a very powerful tool).

CA: Bradford described his work on seed drying and the ‘Dry Chain’ – emphasizing this concept for maintaining seed quality. Described ‘low-tech’ resources such as RH indicator paper in seed containers to monitor moisture content. This is a critical issue in humid tropics where electricity may not be reliably available. Work includes drying beads and other dessicants. Working with Bioversity International for germplasm storage strategies. Shared a useful motto: Make it DRY - Keep it DRY.

FL: Perez described work on various Florida wildflowers, examining the effect of mass on seed germination (Rudbeckia mollis) by using mass-separated seed lots (heavy, mid, light). Identified potential interaction between aging stress and seed mass, but uncertain about the reason: Is this due to maturity differences? No differences from morphology. Also examining desiccation and aging stress in Gaillardia pulchella, and germination and comparative seed vigor of Eragostis elliottii (promoted by summer temps). Described collaboration with P. Jourdan (OH) on X-ray imaging of Eragrostis seeds.

IA: Goggi shared her studies on genome fluidity in soybean, the capacity of genome to reorganize triggered by environmental stress. The changes are stable and heritable for at least a couple of years. Looked at Aconitase gene changes as a marker.

OH: Jourdan described research on seed production and quality in Phlox, as part of a program to produce seed wild sources that have appropriate quality for long-term storage. Seed production of up to 15 Phlox species in a common garden is challenged by pollination control, harvesting of seeds that are typically forcefully dispersed, and by poor germination. Experiments are underway to explore the factors that will yield high-quality seed for long-term storage.

VA: Welbaum described his teaching of a vegetable seed production course on-line. Also mentioned his collaboration with Bo Wang in soybean seeds with low phytate to address the challenge that low phytate lines are not inherently low in vigor, but in emergence.

Objective 2 - Eliminating seed dormancy as a constraint during seed production and germination in agronomic seed production and ecological/biomass seed establishment. Pre-mature sprouting in cereals and other species and the identification dormancy mechanisms to manipulate germination.

CA: Bradford. Focus of research for many years has been thermodormancy in lettuce. Examining cell wall breakdown very early in germination near the root tip, that does not occur in thermodormant lettuce (at 30c); in a lettuce PI that is not temp-sensitive, the cell wall breakdown occurs also at 30C. Ethylene appears to be involved in the difference in thermosensitivity of the 2 lines. Also reviewed latest on DOG1 (delay of germination) involvement in dormancy; DOG1-like genes promote thermoinhibition and seem involved in dormancy; miR156 levels in lettuce are influenced by this gene. Transition from dormancy to germination is similar to juvenile/mature phase transitions in life cycle of plants (dormancy to germination; embryo to seedling; juvenile to adult; adult to flowering; embryogenesis to seed development; all involve micro RNAs

KY: Geneve. Examining seed dormancy in Sycios, involving anatomical study of seed development. Shared informative movie of a water gap opening and closing. Combinational dormancy (physical and physiological - perisperm-associated). Another study looked at integumental tapetum in Echinacea tennesiensis where the tapetum is ‘sequestering the developing embryo-filial tissue’ from the maternal tissue.

SD: Gu. His lab focuses on the understanding of seed dormancy from natural variation to qtl, genes, and regulatory networks in rice. A system biology approach toward understanding gene regulatory networks of seed dormancy. Seed dormancy trait regulates the timing of germination; have detected 10 qtls for seed dormancy in rice. Noted that seed dormancy is not an independent trait in evolution; it is related to pericarp color, flowering time, plant height, hull color, awn length (co-location of qtl’s for these traits). Also identified association (correlation) between plant height and dormancy (shorter plants, more dormant seeds) - related to GA issues? GA reduction delays seed ABA accumulation and development. The seed dormancy-plant height association is underlain by 2 pleiotropic genes involved in GA biosynthesis. Hybrid rice seed germinates only to 80%; same gene that affects height also increases dormancy.

LA: ‘Cohn’s Last Gasp’ – Soliloquy. Indicated he is retiring in 2016. Shared an historical perspective on seed dormancy. How do non-hormonal chemicals break dormancy of weed seeds? How to rationalize the structural diversity of dormancy-breaking chemicals? Why do recalcitrant seeds die when dried? Are we measuring responses to drying or dying? Spartina alterniflora has recalcitrant seeds; cold tolerant, flooding tolerant, dormant when shed. Related species - S. pectinata is orthodox. Looked at proteins that differ between orthodox and recalcitrant Spartina: dehydrogenase, LEA, chaperonins, ubiquitin & autophagy related proteins, SOD, peroxiredoxins, glutathione peroxidase; 38 proteins different between recalcitrant and orthodox; there are some cis elements in common in 35 of these proteins. Finally, presented a brief history of his lab’s work (since the 1980’s). Left us with the question: When recalcitrant seeds die, are dying because of drying or during rehydration?

Objective 3 - Enhancing seed vigor and germination in agronomic and other species for improved stand establishment. The emphasis of this objective is on post-harvest technologies.

CA: Bradford. Shared work on population-based threshold models that describe seed respiration and germination rates. Using Q2 respiratory instrument to generate population-based oxygen depletion curves - respiratory time courses. There is excellent correlation between respiration and germination. Identified subpopulations within seed lots that behave differently, e.g. in respiration, aging – that could not be detected by germination tests. The system can be used as a very precise vigor test and check for seed lot quality (especially with mixed lots that have populations of different maturity, aging). Can be used to potentially identify the deterioration before it occurs (long lag phase before sudden drop in viability in long-term storage; can be used to predict decline based on models). Respiration rates can substitute for germination rates for all sorts of physiological parameters. A replacement system for germination.

FL: Perez. Research for objective 3 focuses on enhancing seed germination in agronomic and native species for improved stand establishment: Harperocallis flava (narrow endemic in FL). Arare plant with no knowledge of seed biology and desire to increase the limited population; embryos undifferentiated/undeveloped at mature seed release; unique germination pattern described by Geneve involving a haustorial cotyledon and other interesting modified structures; also reported on students and publications (for the group)

IA: Goggi & Manjit Misra. Working on phages that could be used in seed coatings to control bacterial diseases; also an international collaboration in Ghana - USAID for project on technology transfer. This collaboration includes Goggi’s work on biosafety & on the story that biotechnology is safe (e.g. does not cause sterility in males). The project also involves seed testing laboratory design and training, PhD training.

NY: Taylor. His work focuses on seed technology; manages NY seed testing laboratory. Studying the effect of gelatin capsule treatment on cucumber plant growth and stress tolerance. Also looking at plant biostimulants: microbial inoculants; humid and fluvic acid, protein hydrolysate and amino acids; seaweed extracts. In general, most biostimulants applied to foliage; not much with seeds. An observation: multiple seeds inside of a gelatin capsule, resulting plants more vigorous. Trying multiple empty capsules near seeds - are they acting as fertilizers? There seems to be promotion of growth by capsules beyond nitrogen fertilizer. Also testing coating of broccoli seeds with soy flour as a plant-derived coating material. Collaborative work with other W3168 members, Brian Nault (entomologist) and Eric Nelson (plant pathologist) from Cornell who are interested in biostimulants to control insects and pests. Other work involves looking at fluorescent dyes to follow movement of systemic compounds in seed coating/treatments.

VA: Welbaum. Described nanochitin seed treatment of pepper - induces an SAR-like response in seedlings. Organic seed treatments: tea tree oil seems to suppress fungal growth on soybean seeds. Also looking at peanut germination in gusseted thermogradient table to assess temperatures; boron fertilization reputed to be beneficial for peanut seed quality (during production) - no evidence so far that Boron is beneficial.

TX: Leskovar. Enhancing seed germination in agronomic and native species for improved stand establishment. Uvalde region very important for Winter vegetable production; depend on Edwards aquifer - high quality water, but level drops dramatically in drought years. A major area is use of PGRs for enhanced vegetable transplant quality; e.g. ABA or 1-MCP-ethylene inhibitor application enhances drought stress tolerance (physiological conditioning), arrest shoot growth (MCP). Also looking at stand establishment in artichoke - use GA to induce bolting when plants grown in summer; grafting is under consideration. Examined low nitrogen fertilizer to condition the plants for transplant (better root systems); also looking at effect of ethylene on root development. Noted that seed quality and vigor extremely important in leafy vegetable hydroponic production systems.

W3168 Sub-Committee Reports:

New officers : Greg Welbaum, chair (will chair meeting in 2018). Camille Steber nominated for secretary (must confirm with her)

Mitch McGrath, USDA-MSU - will chair W3168’s 2017 meeting in Monterey

2018 meeting - possibility of VA Tech (hosted by Welbaum) or perhaps Chicago area with PanAmerican Seed.

One of W3168’s goals is to host an international seed meeting in 2017. The meeting will be the 12^th International Seed Science Society Workshop - Monterey, CA, Sept 10-15, 2017 coordinated by Dr. Kent Bradford and the Multistate Committee. Discussion followed about the meeting expenses, establishment of scientific committee, strategy and leaders for grants (NSF, Gu; USDA, Perez); potential sponsorship by Seed Companies. Also an initial discussion of the scientific program was held, to be followed-up by e-mail exchanges and phone conferences between W3168 members. Components of the program include pre-conference tours, plenary and concurrent sessions, posters, special presentations. Additional discussion centered on site visits in the Salinas area. The organizing committee will continue discussions throughout 2016.

Accomplishments

Objective 1 CA-Bradford: Methods have been developed to utilize single-seed respiratory data obtained through the ASTEC Q2 instrument in population-based threshold models. This quantitative analysis of the respiratory characteristics of seed populations enables labor-efficient monitoring of seed quality and new insights into seed germination, vigor and aging. Development of a new zeolite-based desiccant for seed drying enables seed drying without heating with major implications for maintaining seed quality, particularly in humid climates. It is scalable from the individual farmer to large companies. When applied to food grains and commodities, desiccant-based drying has the potential to greatly reduce postharvest loss and improve food safety by preventing accumulation of fungal toxins (e.g., aflatoxin) in storage. FL-Perez: We evaluated the germination of mass separated Rudbeckia mollis seeds exposed to favorable temperatures, heat stress and aging stress. We also evaluated the tolerance of Gaillardia pulchella seeds to aging stress and extreme levels of desiccation. We collected data on: 1) the relationship between relative humidity and seed water content (i.e. water sorption isotherms); 2) the germination ability of seeds following equilibration to a range of relative humidity (i.e. desiccation tolerance); and 3) the germination ability of seeds following exposure to conditions of high relative humidity and temperature (aging stress). Germination of R. mollis seeds under simulated seasonal temperatures and constant temperatures was greater or equal to 90% at temperatures up to 29/19C and constant 30C, but decreased similarly across all mass classes at higher temperatures until seeds became thermoinhibited at 37C. Final germination decreased up to 14 and 40% across all mass classes with increasing duration of aging stress. Seeds of G. pulchella displayed initial water potential of about -53 MPa. Germination remained above about 75% following exposure to increasing levels of desiccation and aging stress. We learned that seed mass variation does not reduce germination uniformity in R. mollis following exposure to favorable conditions. However, extended accelerated aging periods are needed to elucidate if higher levels of stress will promote differential germination responses among mass classes. This is important in understanding how mass based germination responses may shift as plants adapt to a warming climate. We also realized that seeds of G. pulchella are exceptionally resilient to desiccation and aging stress. These results suggest that germplasm conservation of G. pulchella seeds under genebank conditions is feasible. Additionally, G. pulchella seeds should be tolerant of variable and stressful establishment conditions. OH-Jourdan: Understanding the seed quality parameters that may influence long-term storage for Phlox has involved studies of phenology, pollination strategy, fruit and seed development, harvesting, conditioning and germination. We have observed that seed quality in Phlox seed is primarily determined by the endosperm itself as the majority of seeds that failed to germinate due to fungal growth during stratification had viable embryos that could germinate when isolated from the seed. We have developed protocols for TZ testing of Phlox that permit us to establish baseline values for seed viability prior to seed lot germination tests, which are challenging due to the high level of fungal development, even after fungicide treatment. We have also developed efficient methods to isolate embryos from mature seed to determine germination potential and response to external signals, such as GA. OR-Nongaki: Abscisic acid (ABA) has negative effects on seed germination and causes slow stand establishment with low vigor. On the other hand, lack of ABA could cause wilty plants and seed maturation problems. It is necessary to control ABA-regulated events in seeds and plants for agricultural production. To this end, it is essential to understand the mechanisms of ABA-regulated biochemical and molecular events in seeds and plants. We took advantage of NCED (nine-cis-epoxycarotenoid dioxygenase; ABA biosynthesis enzyme) increase in seeds in one of our experimental systems, to identify more factors associated with ABA regulation in seeds. We performed RNA sequencing under normal vs. high ABA conditions. We identified DELAY OF GERMINAITON1-LIKE 4 (DOGL4) as one of the ABA up-regulated gene, which we are currently characterizing. In addition to many other coding genes, we also identified ABA-induced long non-coding RNAs, which we named ABAIRs. We are currently characterizing function of ABAIR1.   Objective 2 CA-Bradford: We have demonstrated that seed dormancy and plant flowering times are connected through the action of the DELAY OF GERMINATION 1 (DOG1) gene. This gene is known to be involved in the establishment of seed dormancy and its loss during after-ripening. We have demonstrated in both lettuce (LACTUCA SATIVA) and ARABIDOPSIS THALIANA that DOG1 function is involved in regulating both of these life cycle transitions by influencing the production of microRNAs miR156 and miR172. This work has significant implications for how plants interact with environment to time major life cycle transitions (e.g., germination and flowering) to their reproductive advantage. We have genetically identified a new quantitative trait locus (QTL) in lettuce (LACTUCA SATIVA) that can partially alleviate thermoinhibition, which prevents seed germination following imbibition at warm temperatures. We previously identified a different QTL on chromosome 6 from a wild (LACTUCA SERRIOLA) accession (UC96US23) which was subsequently fine-mapped and identified to be due to differential expression of LsNCED4, a gene in the abscisic acid biosynthetic pathway. Now, using a different germplasm source (PI251246, LACTUCA SATIVA), we identified a QTL on chromosome 9 that confers germination tolerance at high temperature and tentatively identified the causal gene as ETHYLENE RESPONSE FACTOR 1 (ERF1) (Yoong et al., 2016). This provides an additional source of germplasm for this beneficial trait and provides further insight into the role of ethylene in promoting germination under stressful conditions. Dissemination of this germplasm will enable breeders to increase the high temperature limits for lettuce seed germination and improve crop stand establishment in hot seasons and locations. The effects of environmental conditions (particularly temperature) during seed development were characterized through analysis of genetic x environmental interactions influencing seed quality and other reproductive traits in the PI251246 x Salinas mapping population. A distinct QTL was identified as contributing to the influence of temperature during seed development on subsequent seed performance. KY-Geneve & B. Downie: The involvement of enclosing maternal tissue layers in physiological seed dormancy were investigated in three species with different morphologies. The major seed covering tissue layer(s) reported to be involved in physiological seed dormancy maintenance are usually the endosperm and endosperm cap. However, maternal tissue layers also play a role in maintaining physiological dormancy. The current species and tissue types under study include the perisperm envelope in bur cucumber (Sycios), the integumentary endothelium in coneflower (Echinacea), and the pericarp/endothelium in gamagrass (Tripsicum). In bur cucumber removal of the perisperm envelope results in 100% germination. In gamagrass, the germination unit is a caryopsis enclosed in dried floral tissue (cupule). Removal of the caryopsis from the cupule does not change seed germination percentage, but germination in seeds where the other maternal tissue layers are punctured increases germination by greater than 50 percent. In coneflower, there is an integumentary tapetum (endothelium) that lies just below the seed coat and surrounds the embryo. Piercing or removal of the endothelial layer increases germination by 35 to 45% depending on the species. It is plausible that in these three representative species, the described maternally-derived tissue layers present a physical barrier or limit solute movement across the enclosing envelope to participate in control of physiological dormancy. MI-McGrath: Sugar beet growers plant seeds in sub-optimal conditions of moisture and temperature. Previous work focused on moisture stress during germination for which two genetic processes were uncovered that have had good impact in improving the 60% of planted seeds that, on average, survive to produce a harvested sugar beet in Michigan. The current work expands on this to include temperature extremes likely encountered by sugar beet seed at planting in either cool climates such as Michigan or warm climates such as California. In both cases, a marked reduction in overall gene expression was observed, and the suites of genes expressed were not overlapping in their predicted biochemical functions. OR-Nonogaki: We had developed dormancy-inducing technology by using a chemically inducible gene expression (Plant Gene Switch System: PGSS) in collaboration with Prof. Roger Beachy, University of California, Davis. In addition, we developed a new system of spontaneous dormancy, which does not require chemical induction. Both systems successfully induced seed dormancy, which can be applied to preventing precocious germination, such as preharvest sprouting (PHS) in cereal crops. However, these approaches require a counteracting technology to recover germination and rescue viable seeds. For this purpose, another chemically inducible gene expression system was developed using a nitrate-inducible promoter. Briefly, nitrate itself has dormancy releasing effects through upregulation of ABA deactivation genes. If RNA interference against ABA biosynthesis genes are combined by this approach, dual effects of seed germination promotion is expected. This will be a perfect answer to the objective "Eliminating seed dormancy as a constraint during seed production and germination". SD-Gu: Research activities on seed dormancy in rice include: 1) purifying isogenic lines and transgenic lines for cloned seed dormancy (SD) genes SD1-2, SD7-2, SD12a, SD12b and Sd12c and evaluating their dormancy degrees by standard germination testing; 2) sequencing alleles of these genes from a collection of wild, weedy and cultivated rice and genotyping the collected accessions to characterize evolutionary mechanisms of the dormancy genes; and 3) conducing a series of genomic and molecular biology experiments from the RNA to the protein and hormone levels to characterize developmental mechanisms of seed dormancy by the cloned genes. Outputs and short-term outcomes mainly include: 1) completed a multi-year project to clone and characterization of the SD1-2 QTL; 2) completed cloning and validation of candidate genes for the qSD12 QTL, and initiated a transcriptomic analysis for the QTL underlying genes; and 3) advanced projects to fine-map and clone of the qSD7-2 and qSD1o QTL.   Objective 3 CA-Bradford: We have continued to expand on the application of population-based threshold models for the analysis of seed quality and behavior (e.g., dormancy, respiration, viability). These models enable the quantification of the physiological status of seed lots and the variation in quality among seeds in the population. They are being increasingly applied in seed ecology and evolution as well. I contributed to a recent review in Trends in Ecology and Evolution (Donohue et al., 2015) in which applications of this basic model were demonstrated for diverse life cycle applications. We also contributed to a paper published in Ecology (Huang et al., 2015) in which these models were applied to quantify the contribution of seed traits to species diversity and demographics in the Arizona desert. We have also now extended the model to enable it to separate and identify distinct subpopulations within a blended seed lot, for example. These models have become the standard method of quantifying certain aspects of seed performance and we continue to extend the range of seed characteristics and life cycle stages to which they can be applied. FL-Perez: We studied the germination response of two accessions of Elliot's Lovegrass, harvested in different years, to simulated seasonal temperatures and 25C. Seed vigor testing via accelerated aging methods was also conducted. We collected data pertaining to final germination, germination rate and germination uniformity. Final germination was greater than 80% for seeds exposed to spring, summer or fall temperatures. However, seeds exposed to summer temperatures displayed the most rapid (75%in 3 days) and complete germination (89%) in a significantly differnt temporal germination pattern. Germination was reduced by 7% for seeds collected in 2012 and exposed to aging stress. However, germination was reduced in the 2013 lot by 14-27%. We learned that seeds of Elliot's Lovegrass are released from thermal germination constraints in multiple seasons, but actual field emergence may vary based on differences in vigor between seed lots. NY-Taylor: Seed coat permeability was examined using a model that tested the effects of soaking tomato (Solanum lycopersicon) seeds in combination with carbon-based nanomaterials (CBNMs) and ultrasonic irradiation (US). Penetration of seed coats to the embryo by CBNMs, as well as CBNMs effects on seed germination and seedling growth, was examined. Two CBNMs, C60(OH)20 (fullerol) and multiwalled nanotubes (MWNTs), were applied at 50mg/L, and treatment exposure ranged from0 to 60 minutes. Bright field, fluorescence, and electron microscopy and micro-Raman spectroscopy provided corroborating evidence that neither CBNM was able to penetrate the seed coat.The restriction of nanomaterial (NM) uptake was attributed to the semipermeable layer located at the innermost layer of the seed coat adjacent to the endosperm. Seed treatments using US at 30 or 60 minutes in the presence of MWNTs physically disrupted the seed coat; however, the integrity of the semipermeable layer was not impaired. The germination percentage and seedling length and weight were enhanced in the presence of MWNTs but were not altered by C60(OH)20. The combined exposure of seeds to NMs and US provided insight into the nanoparticle-seed interaction and may serve as a delivery system for enhancing seed germination and early seedling growth. Seed agglomeration is a coating technology with the purpose to sow multiple seeds of the same seed lot, or multiple seeds of different seed lots, varieties or species. The objective of this study was to develop agglomeration technology by producing single agglomerates or pellets using lettuce and tomato as model vegetable crop seeds. Physical properties of dry and wet pellets were measured and seedling emergence assessed. Pellets were formed by a molding technique with a mixture of filler, binder, and seeds. Diatomaceous earth (DE) was used as the filler, and two binders were tested: polyvinyl alcohol (PVA) and SOL034, a commercial, organic binder. Each binder solution was mixed with DE, and seeds were added during the agglomeration process. Oval and cylindrical pellets were molded with known compression forces. Pellet strength increased as PVA binder concentration increased from 8% to 16% and pellet strength was greater for pellets produced with 3 kg than 1 kg compression. The percent seedling emergence and speed of emergence were not affected by a compression force of 1 kg and 3 kg for lettuce and tomato, respectively compared to the non-pelleted control. Pellets containing three tomato seeds produced plants with greater leaf area, fresh and dry weight than plants grown from single seeds, 26 days after being sown in the greenhouse. Biostimulants are chemicals that stimulate plant growth and increase plant protection from biotic and abiotic stress. Protein hydrolysates are classified as one type of biostimulant, and have been shown to have a positive effect on plant growth. The effect of gelatin, a kind of animal protein hydrolysate was evaluated on plant growth. Enhanced plant growth was measured in the aboveground portions of plants with the application of gelatin capsules applied at time of sowing adjacent to seeds. Crops tested (cucumber, tomato, broccoli, corn, arugula, pepper) showed increased plant growth as measured by leaf area, fresh and dry weight. The magnitude of plant growth enhancement was crop specific, and cucumber was used as the model crop for all further studies. Plants treated with gelatin capsules exhibited increased nitrogen content, and increased salinity tolerance compared to the nontreated control. Different types of hydrolyzed collagen, including granulated gelatin, gelatin hydrolysate, and amino acid mixtures containing amino acids present in gelatin were compared and revealed that granulated gelatin treatment had the greatest plant growth compared with other treatments. Plants were treated with two gelatin capsules and equivalent amount of nitrogen in the form of urea revealed that increased plant growth from the application of gelatin capsule was not solely due to the nitrogen. RNA-seq results provided some insights on the mechanisms of the growth promotion from the gelatin capsule treatments. Genes were upregulated from the gelatin treatment involved in nitrogen transport including ammonium transporters and amino acid transporters, and genes involved in abiotic stress tolerance such as WRKY transcription factors. Genes involved in detoxification such as Glutathione S-transferase exhibited a high positive correlation with increase leaf area and nitrogen content in plants treated with gelatin capsule.

Publications

Atoum, Y., M. J. Afridi, J. M. McGrath, and L. E. Hanson. (2016) On developing and enhancing plant-level disease rating systems in real fields. J. Pattern Recognition 53: 287-299. Donohue, K., Burghardt, L.T., Runcie, D., Bradford, K.J., and Schmitt, J. (2015) Applying developmental threshold models to evolutionary ecology. Trends in Ecology and Evolution 30: 66-77. Genna, N.G., Kane, M.E., and Perez, H.E. (2015) Simultaneous assessment of germination and infection dose-responses in fungicide-treated seeds with non- and semiparameteric statistical methods. Seed Science and Technology. 43: 168-186. Gu, Lei, · Zhang, Yumin, · Zhang, Mingshuai, · Li, Tao · Dirk, Lynnette M. A., ·Downie, Bruce, · Zhao, Tianyong (2016) ZmGOLS2, a target of transcription factor ZmDREB2A, offers similar protection against abiotic stress as ZmDREB2A. Plant Mol. Biol. 90:157–170. DOI 10.1007/s11103-015-0403-1 Han, Qinghui, Li, Tao, Zhang, Lifeng, Yan, Jun, Dirk, Lynnette M.A., Downie, Bruce, Zhao, Tianyong (2014) Functional Analysis of the 5’-Regulatory Region of the Maize ALKALINE ALPHA-GALACTOSIDASE1 Gene. Plant Mol. Biol. Rep. DOI 10.1007/s11105-014-0840-z Hanson, L. E., Goodwill, T. R., and McGrath, J. M. (2015) Beta PIs from the USDA-ARS NPGS evaluated for resistance to Cercospora beticola, 2014. Plant Disease Management Reports. 9:FC001. Huang Z, Liu S, Bradford KJ, Huxman TE, Venable DL (2015) The contribution of germination functional traits to population dynamics of a desert plant community. Ecology 97: 250-261. Huo, H., Bradford, K.J. (2015) Molecular and hormonal regulation of thermoinhibition of seed germination. In J.V. Anderson (ed.), Advances in Plant Dormancy, Springer, NY, pp. 3-33. McDonald, M.R., Vander Kooi K. and A. G. Taylor (2015) Evaluation of insecticides for control of onion maggot in yellow cooking onions, 2014. Muck Vegetable Cultivar Trial & Research Report 2014. Dept. of Plant Agriculture, Muck Crops Research Station, King, ON. Report No. 64. p. 68-69. McDonald, M.R., Vander Kooi K. and A. G. Taylor (2015) Evaluation of insecticides for control of onion maggot in yellow cooking onions, 2014. Pest Management Research Reports (on-line). <a href="http://phytopath.ca/wp-content/uploads/2015/05/Compiled-PMRR-Final-May-11.pdf">http://phytopath.ca/wp-content/uploads/2015/05/Compiled-PMRR-Final-May-11.pdf</a> McGrath, J. M., Hanson, L. E., and Panella, L. W. (2015) Registration of SR98 sugarbeet germplasm with high levels of resistance to Rhizoctonia seedling damping-off and crown and root rot diseases. J. Plant Registrations 9: 227-231 McGrath, J.M., Townsend, B. J. (2015) Sugar Beet, Energy Beet, and Industrial Beet. Pp. 81-99. in "Handbook of Plant Breeding. Volume 9. Industrial Crops: Breeding for Bioenergy and Bioproducts." Editors: V.M.V. Cruz and D.A. Dierig. Nonogaki M, Sekine T, Nonogaki H. (2015) Chemically inducible gene expression in seeds before testa rupture. Seed Science Research 25, 345-352. Nonogaki M, NonogakiH. (2016) Seed development: Germination. In B. Thomas, B. Murray, D. Murphy eds, Encyclopedia of Applied Plant Sciences, Second Edition Biology, Elsevier, Oxford, in press Pan, L., Zhu, Q., Lu, R., and McGrath, J.M. (2015). Determination of sucrose content in sugar beet by portable visible and near-infrared spectroscopy. Food Chemistry167: 264-271. Pan, L., Lu, R., Zhu, Q., McGrath, J. M., and Tu, K. (2015) Measurement of moisture, soluble solids, and sucrose content and mechanical properties in sugar beet using portable visible and near-infrared spectroscopy. Postharvest Biology and Technology. 102:42-50. Panella, L., Hanson L. E., McGrath, J. M., Fenwick, A. L., Stevanato, P., Frese, L., and Lewellen, R. T. (2015) Registration of ‘FC305’ multigerm sugarbeet germplasm selected from a cross to a crop wild relative. J. Plant Registrations 9: 115-120. 2015. Pérez, H.E. (2014) Do habitat and geographic distribution influence decreased seed viability in remnant populations of a keystone bunchgrass? Ecological Restoration 32: 295-305. Ratnikova, T. R., R. Podila, A. M. Rao, and A. G. Taylor (2015) Tomato seed coat permeability to selected carbon nanomaterials and enhancement of germination and seedling growth, The Scientific World Journal, vol. 2015, Article ID 419215, 9 pages doi:10.1155/2015/419215 Sikhao, P., A. G. Taylor, E. T. Marino, C. M. Catranis and B. Siri (2015) Development of seed agglomeration technology using lettuce and tomato as model vegetable crop seeds. Scientia Horticulturae: 184, 85-92. Wilson, H. T. (2015) Gelatin, a biostimulant seed treatment and its impact on plant growth, abiotic stress, and gene regulation. PhD thesis, Cornell University Wilson, H. T., K. Xu, and A. G. Taylor (2015) Transcriptome analysis of gelatin seed treatment as a biostimulant of cucumber plant growth, The Scientific World Journal, vol. 2015, Article ID 391234, 14 pages. doi:10.1155/2015/391234 Yoong F-Y, O'Brien LK, Truco MJ, Huo H, Sideman R, Hayes R, Michelmore RW, Bradford KJ. (2016) Genetic variation for thermotolerance in lettuce seed germination is associated with temperature-sensitive regulation of ETHYLENE RESPONSE FACTOR1 (ERF1). Plant Physiology 170: 472-488.

Impact Statements

Preparing future seed scientists that can contribute the further development of the seed industry is critical for this sector’s economic health and vitality. Perez (FL) mentored three Master's level graduate students and one undergraduate research associate were mentored during this reporting period. Nonogaki (OR) has trained a PhD student and an international seed industry intern. Committee members as a group are involved in the education and training of a dozen graduate students.

Date of Annual Report: 12/12/2017

Report Information

Annual Meeting Dates: 09/09/2017 - 09/10/2017
Period the Report Covers: 10/01/2016 - 09/30/2017

Participants

Bradford, Kent (kjbradford@ucdavis.edu) - University of California, Davis;
Downie, Allan B. (adownie@uky.edu) - University of Kentucky;
Elias, Sabry (Sabry.Elias@oregonstate.edu) - Oregon State University;
Geneve, Robert (rgeneve@uky.edu) - University of Kentucky;
Gu, Xingyou (Xingyou.Gu@sdstate.edu) South Dakota State University;
Haidet, Megan (Margaret.Haidet@nifa.usda.gov) - USDA-National Institute of Food and Agriculture;
Jourdan, Pablo (jourdan.1@osu.edu) - The Ohio State University;
Perez, Hector (heperez@ufl.edu) - University of Florida;
Steber, Camille (csteber@wsu.edu) - USDA-ARS & Washington State University;
Taylor, Alan (agt1@cornell.edu) - Cornell University;
Welbaum, Greg (welbaum@vt.edu) - Virginia Polytechnic University;
Yadegari, Ramin (yadegari@email.arizona.edu) – University of Arizona;

Brief Summary of Minutes

The 2017 meeting of the W3168 group followed a different format than usual because it was held in conjunction with one of the major committee-sponsored activities: the 12^th Triennial Conference of the International Society for Seed Science. This conference was organized by Prof. Kent Bradford in collaboration with members of W3168 who contributed both logistical and scientific support. As a consequence, the formal meeting of the committee only addressed general business issues since all the scientific activity was incorporated into the conference. The business meeting was held on Sunday, 10 September in the morning.

The meeting was presided by Pablo Jourdan and called to order at 8:00 a.m. Following introduction of those present, the committee heard from Megan Haidet who recently joined NIFA from the BLM-SOS program. Megan has a strong interest in seeds and was very keen to participate in this meeting. Megan detailed various activities and initiatives at NIFA that may bear close relationship to seeds and the expertise of the committee. She indicated the possibility of using seeds of native species as a possible focus for research. Megan informed the group that NIFA is initiating a series stakeholder-wide listening sessions, seeking input on the challenges confronting agricultural systems in the broadest sense. She encouraged the group to participate and share any concerns about support for seed-centric research and development at NIFA

A discussion, led by Kent Bradford, followed on the proposal by Henk Hilhorst, editor of Seed Science and Technology to publish a special edition of the journal focused on short reviews on seed topics – these may or may not be related to the specific topics presented at the conference. W3168 members were encouraged to approach Henk about possible topics of interest.

The structure of the committee was also a topic of extensive discussion, emphasizing the concern about dwindling membership and the need to attract and encourage younger scientists to the group. Suggestions were made to reach out to more seed scientists at the Forest Service as well as at Botanic Gardens and Arboreta; however, since the group depends on Experiment Station support, it is going to be challenging to attract members from non-USDA-supported entities. It was also mentioned that there may be weed scientists with potential interest in the committee and we must make a greater effort to reach out to this group. One possible venue for expanding the membership is to work more closely with the seed-related sections of the Tri-Societies; these include C4 (seed production and technology), C7 (molecular biology) and C8 (plant genetic resources). Chris Walters, a member of W3168, is current chair of the C4 group and she will be approached to seed closer integration between the C4 section and W3168.

A general concerned expressed by the committee is the continued decline in the specialization on seeds. There are few seed science courses at universities; there is a dearth of seed technologists. There is also concern that national review panels for grant proposals often lack knowledge of seeds and thus consider seed-centric research as less relevant. It was also noted that seed companies are really about breeding and not about seeds per-se, even though their principal products is seeds! Many problems related to seeds could be prevented if in breeding new cultivars, more attention is paid to seed attributes and quality early on.

The leadership of the W3168 was established by consensus as follows: Secretary – Ramin Yadegary; Vice Chair (next meeting host) – Sabry Elias; President (chair of the next meeting): Robert Geneve. The next meeting is tentatively scheduled for summer of 2018 in Corvallis, OR. A focus for the 2018 meeting will be addressing the committee objectives in anticipation of the next re-write of the project. It was suggested that we also consider our 2019 meeting in conjunction with the Agronomy meetings to be held in mid-november in San Antonio.

Although the scientific component of the meeting was part of the conference, there was a general discussion about different interests of the group such as seed germination studies specifically focused on the emergent controlled-environment production systems. There was also an interesting discussion about large-scale organic production research for corn, taking advantage of a mutation that provides enhanced seed vigor. A response to the lack of seed scientists could be the development of training grant proposals and it was suggested we make this an important topic for our next meeting.

The meeting was adjourned at 11:30 a.m.

Accomplishments

A major accomplishment of the committee for this reporting period (through 9/30/2017) was organizing and co-hosting the 12th Triennial Conference of the International Society for Seed Science with the Seed Biotechnology Center (SBC) of the University of California, Davis. Organizing this conference was one of the principal proposed activities for the W3168 committee. The conference was held September 10-15 in Monterey, CA. Taking place in the U.S. for the first time since 1989, the conference brought 180 attendees from 24 different countries and enabled top international seed scientists to share research findings and interact with the U.S. and California seed industry. According to conference organizer, W3168 member, and SBC’s Director Dr. Kent Bradford “The International Society for Seed Science is the sole professional and scientific organization dedicated to the scientific study of seed biology, technology, quality, ecology and conservation. Seed scientists play a critical role in enabling high crop yields, in providing high quality seeds for crop propagation and establishment, in controlling weeds and in preserving ecological diversity. In addition, more than 90% of global crop genetic resources are stored as seeds. Thus, seed science is a key component of agriculture’s goal of sustainably feeding the global population.” W3168 members were part of the scientific committee, assisted in raising funds to support undergraduate and graduate student participation, chaired all sessions, and presented numerous talks.   The accomplishments of researchers in individual states is presented below:    Objective 1 - Identifying key factors involved in the enhancement or loss of seed quality. Seed development through post-harvest losses in storage. Specific topics include seed development, desiccation tolerance, and aging in storage     AZ (Yadegari): Our goal is to understand the regulatory mechanisms that underlie accumulation of storage proteins and starch during mid to late stages of endosperm development in maize. During the past year, we have developed a series of datasets related to the expression and function of Opaque-2 (O2), a bZIP transcription-factor protein that is known to regulate zein genes encoding the major seed storage proteins in maize endosperm. Our studies have uncovered the genes that are regulated directly or indirectly regulated by O2. Analysis of these gene sets indicate a broad role for O2 in endosperm metabolism and basic differentiation. A series of experiments to address this hypothesis are currently underway.   CA (Bradford): We developed analysis methods to utilize single-seed respiratory data obtained through the ASTEC Q2 instrument in population-based threshold models. This quantitative analysis of the respiratory characteristics of seed populations enables labor-efficient monitoring of seed quality and new insights into seed germination, vigor, enhancement and aging. We worked with a seed technology company in 2016-2017 to test its applicability to corn (Zea mays) vigor testing and subsequent seed performance in the field. This could be a generally applicable vigor test, and efforts are being made to scale up the analysis system for greater throughput. Elevated seed moisture content due to insufficient drying is the major cause of seed deterioration, particularly in humid environments. When applied to food grains and commodities, drying and packaging (termed the “dry chain”) has the potential to greatly reduce postharvest loss and improve food safety by preventing accumulation of fungal toxins (e.g., aflatoxin) in storage. We have reviewed this field and written a review of strategies to dry and package seeds and dry commodities, particularly in humid regions (in review). We have developed a number of tools and methods to monitor seed/commodity moisture content and use desiccant-based drying followed by hermetic packaging to preserve viability/quality.   FL (Perez): We evaluated the seed viability of the endangered Euphorbia deltoidea ssp. deltoidea. We collected data on the number of seeds from three populations that responded positively to biochemical viability assays. The majority of E. deloidea ssp. deltoidea seeds were filled and apparently fully developed. However, seed viability across populations did not exceed about 5%. Thus, we learned that populations of E. deloidea ssp. deltoidea display low seed viability. However, a seed developmental study would be helpful in determining viability and germination ability.   KY (Downie): Working with colleagues from the Federal University of Sao Paulo, Jaboticaba, Brazil, we have investigated the probable cause of poor seed vigor in reciprocal hybrids of Zea mays (maize; Santos et al. 2017). This was in collaboration with a Brazilian subsidiary of Syngenta whose breeding lines these were. Occasionally, reciprocal crosses produce hybrid seeds with dramatically different seed vigors. This was the case in these two different breeding pairs from Syngenta. In both instances, a possible cause of the poor quality could be linked to greater amounts of reducing sugars in the maize kernels. The debate concerning whether raffinose or raffinose family oligosaccharides (RFO) assist seed viability or vigor has raged since the discovery of these soluble sugars. Using Zea mays (maize) as a model we have demonstrated that raffinose biosynthetic capacity enhances seed vigor (Li et al. 2017, provisional). We continue to use maize as a model using sugar feeding and mutagenesis to try and understand the implications of soluble sugar amount, identity, and balance on seed vigor.   OH (Jourdan): The Ornamental Plant Germplasm Center’s seed research efforts emphasize areas that relate to seed quality, germination, and long-term storage of North American native wild species. We have been working primarily with seeds of perennial species of Phlox that are presenting significant challenges in terms of production, germination and potential for storage. After 3 years of intensive effort we have finally produced enough seed of different species to undertake more systematic studies on parameters that define seed quality, assessment of viability, and efficient germination systems. We have optimized the Tetrazolium test to get some indication of seed viability without relying on germination, as seeds have what appear to be non-deep physiological dormancy and require 2-3 months cold stratification for germination. A major problem is the extensive fungal contamination that is found in these seeds, even after surface-sterilization and/or treatment with various fungicides. The seeds are endospermic and the seed coverings appear to provide excellent substrate for fungal growth. Isolated embryos of most species germinate readily on 1% agar. However, the seeds do not respond to Gibberellic acid treatment by faster germination. We know the GA does get through the seed coverings as embryos isolated from seeds imbibed in 1000 ppm GA show the typical hyper-elongation of internodes. Thus, alternative treatments of whole seeds must be sought to expedite germination tests without the lengthy stratification period. Our progress to date has been possible through the interaction with W3168 colleagues such as Drs. Downie, Geneve, and Taylor who have provided helpful advice and suggested important experiments.   OR (Nonogaki): The genes, which were identified by RNA sequencing (RNA-seq) of NCED6 (nine-cis epoxycarotenoid dioxygenase 6: ABA biosynthesis gene)-inducible lines (described in the previous report), were characterized into details. DELAY OF GERMINATION1 (DOG1)-LIKE 4 (DOGL4), which was identified as an outstanding ABA-regulated gene by RNA-seq, has been particularly focused on during this reporting period. DOGL4-inducible lines have been created using the Plant Gene Switch System (PGSS) and another round of RNA-seq was performed. The sequencing results revealed that DOGL4 is a master regulator of seed storage proteins and has great potential to enhance quality of seeds as the final products in agriculture. An intergenic long non-coding RNA (lincRNA), which was also upregulated by NCED6 induction (termed N6LINCR1), was characterized. For N6LINCR1, a manuscript has been accepted and in press. The DOGL4 manuscript is being prepared in collaboration with the seed biology group at Wageningen University in the Netherlands.   VA (Welbaum): We are studying fundamental aspects of Acidovorax citrulli-cucurbit interactions (bacterial fruit blotch) to elucidate the bacterial determinants that mediate host preferential association towards different cucurbits. The specific objectives of this research are to: (1) to investigate the susceptibility of different cucurbit species to group I and II strains of A. citrulli under field conditions during seed production; (2) to assess the contribution of type III effectors and other virulence factors to A. citrulli virulence and host preference in cucurbit fruit tissues and seeds; and (3) to characterize the mechanism of action of selected type III effectors. The work has provided evidence demonstrating the optimization of a new assay based on surface inoculation of detached melon fruits. We are providing insights into the mechanisms involved in host preferential association in the A. citrulli-cucurbit pathosystem, and developing new strategies for bacterial fruit blotch management through improved screening techniques.     Objective 2 - Eliminating seed dormancy as a constraint during seed production and germination in agronomic seed production and ecological/biomass seed establishment. Pre-mature sprouting in cereals and other species and the identification dormancy mechanisms to manipulate germination.     CA (Bradford): In work conducted in collaboration with the West Coast Metabolomics Center at UC Davis and funded by the American Seed Research Foundation, we characterized the metabolites and storage compounds present in dry and imbibed lettuce (LACTUCA SATIVA) seeds. Several hundred compounds were identified, including small molecules and metabolites, phospholipids and triacylglycerol storage lipids. In addition, we assessed the effects of high temperature and ABA on germination, respiration and metabolites. The work provides a view into how temperature stress can affect respiration and storage reserve mobilization in seeds. This work is described in the PhD dissertation of D. Ardura (2017) and is in preparation for publication.   Seeds of many species are photoblastic, or require light in order to germinate. This can create problems for seed performance, such as for pelleted seeds that require prior hydration and exposure to light in order to germinate. We identified a quantitative trait locus (QTL) associated with dark inhibition of germination in lettuce (LACTUCA SATIVA). Within this QTL is a gene encoding LsGA2ox2. Through gene expression and transgenic experiments, we demonstrated that this gene is a likely candidate gene for this QTL. When it is expressed, GA2ox2 enzyme inactivates gibberellin and prevents germination. Conserved SNPs in its promoter disrupt an ABA-responsive element, transfer of which is sufficient to control germination in the dark. This work is described in the dissertation of M. Niroula (2017) and is in preparation for publication.   OR (Nonogaki): This project had developed a technology, which imposes hyperdormancy through enhanced ABA production and prevents precocious seed germination, such as preharvest sprouting (PHS). As a next step, the germination recovery strategy through expression of ABA counteracting genes, such as GA biosynthesis gene or NCED RNAi was proposed (see previous report). Another germination recovery strategy, through a chemical biology approach, was tested in this reporting period. Application of 3′-hexylsulfanyl-ABA (AS6), an ABA antagonist, was sufficient to recover germination from our inducible hyperdormant seeds. While ABA biosynthesis was still enhanced in the hyperdormant seeds, AS6 caused temporal ABA insensitivity in seeds, which released deep dormancy. These results demonstrate that chemical biology approaches have great potential to eliminate constraints for seed production and germination in agriculture. Combination of hyperdormancy and germination recovery will establish a comprehensive technology for PHS prevention for cereals.   <ul> <li>SD (Gu): Completed cloning and molecular characterization of the seed dormancy loci qSD12. This was a continuous project funded by NSF grants. All candidate genes were confirmed by complementation for function of seed dormancy. Data from a series of research will be released as publications late this to early the next year. </li> <li>Completed an initial experiment to evaluate effects of seed dormancy genes on seedbank longevity. A set of 16 isogenic lines of rice for 4 seed dormancy loci were evaluated for primary dormancy under controlled conditions and also evaluated for seed survivability in soil of a rice field for 7 months from early October to later April the next year. A significant, moderate level of correlation was detected between seed dormancy and seedbank longevity. All these four genes have main and epistatic effects on seed survivability in soil. This would be the first time that relates seed dormancy genes with seedbank longevity.  </li> </ul>   Objective 3 - Enhancing seed vigor and germination in agronomic and other species for improved stand establishment. The emphasis of this objective is on post-harvest technologies.     CA (Bradford): Seed priming (hydration and drying) is used to enhance seed germination and improve stand establishment. We conducted research on seed priming treatments to understand why such treatments can shorten seed longevity and tested some post-priming treatments reported to restore much of the lost storage potential. While we could easily demonstrate a loss of tomato (Solanum lycopersicum) seed storage longevity after priming, none of the post-priming treatments we tested were effective in restoring longevity (Li, 2017).     FL (Perez): Germination ability of Euphorbia deltoidea ssp. deltoidea was also investigated at various simulated seasonal temperatures. Next, we studied the germination response of Linum arenicola. Finally, we investigated the germination ecology of developing Harperocallis flava seeds. All study species are rare and listed as endangered. We collected data pertaining the germination ability of: a) L. arenicola seeds following exposure to a series of constant and simulated seasonal temperatures and b) H. flava seeds following exposure to conditions of low relative humidity and simulated seasonal temperatures. Germination of L. arenicola seeds was delayed for up to 16 days and final percentage varied between 0-4% at simulated winter, spring, and summer temperatures. However, germination increased to about 33% at simulated fall temperatures. In contrast, germination was comparably rapid at constant temperatures. Germination commenced by day 1 under these conditions. Final germination ranged between 28 and 38%, with most germination taking place at constant 29°C. Seeds of H. flava are viable and appear under-developed. Germination is induced at all seasonal temperatures. However, more rapid and complete germination was observed at simulated winter temperatures. Final germination ranged from 5% at simulated summer temperatures to 77% at simulated winter temperatures. Seeds of H. flava germinated to about 24-80% following exposure to 0.5 to 90% relative humidity. 4) Key outcomes or other accomplishments realized – We learned that germination of L. arenicola seeds appears inhibited at alternating temperatures. However, germination progresses rapidly under constant temperatures. Alternatively, seeds of H. flava may possess morphological dormancy and, contrary to anecdotal evidence, have a high capacity for germination. Seeds appear to display some tolerance to high levels of desiccation, which would be an advantage in terms of ex situ conservation. However, testing the desiccation tolerance of mature seeds may provide further insight related to storage under genebank standards.     KY (Downie): In so far as Arabidopsis thaliana is a “native species” somewhere on the planet, we have reported on a gene, previously misannotated, that enhances the completion of seed germination of seeds when overexpressed (Majee et al. 2017). The product produced from the gene of interest somehow plays a role in phytochrome-mediated light signaling in both the completion of germination and hypocotyl elongation.     KY (Geneve): The audience for this project includes private and public seed technologists, seed producers and seed scientists. During the reporting period, invited presentations related to this project were made at the 12th International Society for Seed Science: Seeds as Systems meeting in Monterey, California and the International Plant Propagator’s Society meeting in Tzaneen, South Africa. As a result of these meetings, a review manuscript was submitted to Seed Science Research describing the importance of water gap complexes for controlling physical seed dormancy as well as a proceedings paper related to the impact of seed technology on germination of commercially important horticultural crops.     MT (Stougaard): Falling numbers tests were performed on a set of 18 private spring wheat varieties that are typically marketed in the Pacific Northwest. Entries included materials from Syngenta, Monsanto, Limagrain and Winfield United. Two public varieties from Montana State University were included for comparison. Falling numbers were high and all entries avoided dockage this year. The average falling number for the nursery was 426 seconds. Egan had the highest falling number at 470 while Vida had the lowest at 362.   A separate experiment was conducted to determine the effect of plant population of falling numbers. Six spring wheat varieties were planted at four densities as a complete factorial treatment design. Egan, Expresso, Solano, Soren, Tyra and Vida were planted at densities of 172, 258, 344, and 430 plants/m2. High temperatures and drought typified this season. Falling numbers were high and averaged 396 seconds. The main effect of variety was significant. Egan had the highest at 460 while Tyra had the lowest at 364 seconds. Although falling numbers varied among varieties, the main effect of plant density was nonsignificant and had no effect on the results.     NY (Taylor): A novel method was developed of using plant-derived protein hydrolysates as seed coating materials. The objective of this study was to develop seed coating formulations using soy flour, a sustainable, inexpensive, and green source, as a biostimulant using broccoli as the model system. A 10% suspension of soy flour was used as the seed treatment binder in all coatings. The solid particulate filler was composed of mixtures of soy flour, cellulose, and diatomaceous earth, together termed as SCD. All SCD components were homogenized in water, then dried and ground to a fine particle size <106 mm. The SCD coatings were applied with rotary pan seed coating equipment at 25% of the seed weight. Increasing the proportion of soy flour increased the seed coating strength and also the time for the coating to disintegrate after soaking in water. As a result, the seed coatings reduced the percentage germination and the germination rate compared with the nontreated control. However, the 10-day-old seedling root and shoot growth showed significant improvement for all SCD coating treatments compared with controls. Plant growth and development was also measured after 30 days in the greenhouse. Fresh weight (FW) and dry weight (DW), leaf area, plant height, leaf development, Soil-Plant Analyses Development (SPAD) index (chlorophyll measurement), and nitrogen (N) per plant were all greater from coatings with 30%, 40%, and 50% soy flour than the noncoated control. Nitrogen, from the soy flour applied in the seed coatings, ranged from 0.024 to 0.073 mg per seed, while the enhanced N per plant ranged from 1.7 to 8.5 mg. The coating treatment with 0.063 mg N per seed resulted in the greatest plant leaf area and highest N content. Nitrogen applied in the seed coating only accounted for 1% to 2% of the enhanced N in the plants, indicating the soy flour acted as a biostimulant rather than a fertilizer.   Systemic seed treatment uptake into soybean and corn seeds during imbibition has not been investigated over a broad range of application rates. The objectives of this study were to investigate application dosage rates on uptake, to assess the role of the seed coat on uptake and determine the dosage for greatest uptake efficiency. The model fluorescent tracer compound, coumarin 120, 7-Amino-4-methylcoumarin was applied as a seed treatment in the range of 0.01 to 20.0 mg g-1 seed to study the dose effects on seed uptake of corn lines ‘OH7B’ and ‘B73’, and soybean varieties ‘TMG 312RR’, ‘IAR 1902’, and a blacked-seeded ‘V12-1223’. The uptake by two lines of corn seeds and three varieties of soybean seeds showed a dose dependent process that was described by an exponential model of Y = (r2 ³ 0.93), where y0 is the uptake limit. In general, there was a large increase in seed uptake as dosage increased, followed by a saturated state at higher dosages. The uptake limit (y0) differed between the two corn lines and the three soybean varieties. The maximum uptake efficiency or recovery of applied coumarin 120 was at 0.1 mg g-1. The pericarp/testa of both corn lines and the testa of black-seeded soybean variety ‘V12-1223’, attenuated the uptake in comparison with decoated seeds of each line/variety. Results from this study have implications on the effect of application rate on seed treatment efficacy and phytotoxicity.   Systemic uptake of organic compounds from roots to leaves follows a Gaussian distribution in relation to the lipophilicity, as measured by the log Kow. Quantification of compound uptake with different lipophilicity, and applied as a seed treatment that diffused through the seed coat into the embryo during imbibition has not been reported. The objective of this investigation was to quantify the uptake of nonionic compounds in soybean (Glycine max) and corn (Zea mays). A series of fluorescent piperonyl amides were synthesized and a novel combinatorial pharmacokinetic technique was developed that provided a range of compounds from log Kow 0.02 to 5.7. Seeds were treated with a mixture of amides, imbibed and compounds chemically extracted and quantified by HPLC using a fluorescence detector. The maximum uptake efficiency of the applied amide mixture from whole soybean and corn seeds was 67% at log Kow 2.9, and 43% at log Kow 3.4, respectively. The critical partition coefficient for uptake for both species was <4.2 log Kow. Seeds were dissected and separated as soybean embryo and testa, and corn internal tissues (embryo + endosperm) or seed covering layers (pericarp + testa), and >75% of the amides were found in the soybean embryo or corn internal tissues compared to the covering layers at log Kow <4.2. The distribution of amides showed that corn seed covering layer had similar hydrophilic/lipophilic properties as internal tissues, while soybean tissues had different hydrophilic/lipophilic properties. Collectively, the Gaussian uptake pattern for systemic uptake into plants was not found for either seed species.     TX (Leskovar): Plant growth regulators (PGRs) can positively regulate seed germination and/or root growth and development. Research with PGRs in onion showed ethephon to be beneficial in increasing seedling root surface area but reduced root length. The addition of IAA and/or tZ could potentially control the reductions in root length and diameter in response to ethephon, while combining tZ and ACC can increase root length. Another study assessed the impact of lignite-derived humic substances (HS) on pepper transplant root traits, shoot growth and physiology exposed to water stress. A new study was initiated to evaluate the grafting technology and physiological mechanisms underlying drought tolerance in tomato grafted plants using different Solanum spp. rootstocks.     VA (Welbaum): Standardized germination testing in a laboratory under controlled conditions by placing seeds in rolled paper towels at an optimum temperature. We have developed a new laboratory analysis system called Lab FieldÔ for standardized, realistic seed germination testing in field soils (Welbaum, 2017b). This system provides a more accurate assessment of viability by subjecting seeds to biotic stresses. The Lab FieldÔ was created by welding thin vertical aluminum strips to the face of a thermogradient table while adding micro-irrigation, drainage and LED grow lights, to create a soil environment that can be adjusted to mimic a wide range of field conditions in a laboratory. This allows for realistic seed testing that more accurately predicts how seeds will perform in production fields. Fast germination and vigorous early seedling growth are essential for establishment of pepper (Capsicum annuum), a warm-season crop that germinates poorly under cold conditions. We have developed a novel seed treatment that exposes seeds to “nano” sized chitin molecules created by hydrolysis of chitosan during controlled seed hydration. Effects of hydropriming, nanochitin, and chitosan treatments on ‘California Wonder’ bell pepper were compared for seed germination percentage and mean time to germination (MTG) in soil at 19, 22, 26 and 30°C on a thermogradient table (Welbaum, 2017b). Treatments with nanochitin or hydropriming reduced MTG to 4.9-5.3 days compared with 5.4-6.7 days for other treatments or untreated seeds at 25°C on blotter paper in a standard germination test. Nanochitin, chitosan, acetic acid or hydropriming treatments enhanced low temperature emergence compared with standard Captan fungicide treated or untreated seeds. Treatments with 0.05% nanochitin or hydropriming improved seedling emergence at 19 to 30°C. Hydroprimed seeds germinated faster with improved emergence similar to nanochitin, but nanochitin also visibly reduced fungal growth on the seeds (Samarah et al., 2016).       Impact Statements:       Objective 1 - Identifying key factors involved in the enhancement or loss of seed quality. Seed development through post-harvest losses in storage. Specific topics include seed development, desiccation tolerance, and aging in storage     AZ (Yadegari): A detailed description of endosperm development will provide a roadmap to develop strategies for enhancing seed quality in maize and related crops. Our analysis of the role of Opaque-2 in endosperm development is expected to identify some key processes that can inform research activities in seed quality and germination in grain seeds.     CA (Bradford): A close correlation between single-seed respiration data acquired with the Q2 instrument and germination rates creates an efficient automated method for a seed vigor assay. The Q2 instrument is being modified for scale-up to enable use as a general seed vigor test. Desiccant-based drying systems enable seed drying even in humid environments. Combined with hermetic packaging, this “dry chain” would largely prevent the rapid loss of viability in warm, humid climates. Similarly, it would prevent damage to stored grains due to molds and insects. Desiccant-based drying systems followed by packaging could have a significant impact on food safety and security in tropical regions.     FL (Perez): The impact of our research is that restoration practitioners and seedling producers can better plan and coordinate in terms of seeding activities for restoration and conservation of natural resources. Similarly, conservation practitioners can adapt methods for ex situ activities. We also developed targeted research programs that enhance the wildflower seed industry.     KY (Downie): Based on the response from the industry to the publication (Santos et al. 2017), there exists a fair number of parental lines where a cross in one direction produces seed of acceptable quality while the reciprocal cross produces sub-standard seed quality, sometimes, drastically so. Seed Science; Findings from Sao Paulo State University Broaden Understanding of Seed Science (Reciprocal effect of parental lines on the physiological potential and seed composition of corn hybrid seeds). Agriculture Week, Sep 21, 2017, p.106.   We have established that the response of plant seeds to RFO is amazingly complex depending on whether they accumulate raffinose only (maize) or higher order RFO as well (Arabidopsis). This information may explain why there have been very few publications on the effect of over-expression of RAFFINOSE SYNTHASE or other genes whose transcripts product higher order RFOs while the number of publications over-expressing GALACTINOL SYNTHASE abound. In the first instance the influence on soluble sugar profiles and seed vigor were contrary to our expectations while over expression of GALACTINOL SYNTHASE has universally enhanced the amounts of RFOs and positively influenced seed vigor.     OH (Jourdan): The most efficient way to capture and conserve genetic diversity of native wild species is through seeds. To achieve this in the 25 or so species of Phlox in the germplasm collection at the OPGC, we must be able to produce or obtain seeds of high quality that have the potential for long term storage. Our studies on seed quality parameters of Phlox are providing information that will help us devise strategies to produce the kinds of seeds that can be stored and thus conserve important genetic diversity. In addition, we can make this diversity available to breeders for the development of new cultivars that can be used in diverse constructed landscapes and provide economic benefits to growers and landscapers.     OR (Nonogaki): The discovery of DOGL4 as a master regulator of seed storage proteins and (possibly lipids) provides great potential of crop modification through the manipulation of this gene, which is currently examined in legumes and cereal crops.     VA (Welbaum): Research on the epidemiology of bacterial fruit blotch disease has elucidated the A. citrulli virulence genes as well as resistance genes in Cucurbit species. These discoveries will enable novel genetic approaches to control this economically important disease through continuing collaborative research.     Objective 2 - Eliminating seed dormancy as a constraint during seed production and germination in agronomic seed production and ecological/biomass seed establishment. Pre-mature sprouting in cereals and other species and the identification dormancy mechanisms to manipulate germination.     CA (Bradford): Some seed components, such as raffinose or stachyose, are thought to be involved in seed desiccation tolerance and longevity. How seeds preserve or resynthesize these compounds during hydration and dormancy could reveal mechanisms for seed preservation or elimination of dormancy (e.g., to control weed seed banks). Studies of the metabolism of germinating and dormant seeds are a path to such information.   In genetic and transgenic studies, we identified a gene encoding a gibberellin-inactivation enzyme (GA2ox2) as a key regulator of the light requirement for germination of lettuce seeds. As specific sequences in the promoter of this gene were responsible for this effect, gene editing could be used to transfer the ability to germinate in the dark to different varieties and species.     OR (Nonogaki): The PHS prevention/germination recovery system has direct impacts on food production when translated to crops species. The system is being introduced to cereal crops through international collaboration.       Objective 3 - Enhancing seed vigor and germination in agronomic and other species for improved stand establishment. The emphasis of this objective is on post-harvest technologies.     CA (Bradford): Seed priming enhances germination in the short term, but often reduces the potential seed storage life. Studies were proposed to identify the molecular and biochemical basis of this effect and of the restoration of longevity by post-priming treatments. However, we were unsuccessful in identifying effective longevity-restoring treatments. Thus, the search for the basis of this effect will continue.   The 12th Triennial Conference of the International Society for Seed Science enabled international scientists to network with W3168 members and seed industry representatives in California.     KY (Downie): Demonstrating that the protein encoded by a gene present in Arabidopsis is responsible for allowing faster completion of germination when overexpressed focused our attention on a region of the Arabidopsis genome that was poorly annotated. Deconvolution of the locus responsible has focused our attention on an F-BOX protein as a positive influence on the completion of seed germination through its presumptive targeting of a negative regulator of seed germination that operates through phytochrome.     KY (Geneve): Hemp (Cannabis sativa) is a relatively new crop for U.S. and Kentucky. Cardinal temperatures were established for germination of several potentially important seed cultivars for Kentucky. Optimal germination occurred between 17 and 25C with a lower temperature threshold at 3C and upper limit at 42C. A putative thermal time model was developed that described progress to germination for temperatures between 13 and 30C. These data provide growers with the information required for determining temperature permissive times for optimal spring germination and establishment of hemp.     MT (Stougaard): Screening public and private wheat varieties for falling number will allow for the compilation of a data base which will aid producers in selecting the most appropriate cultivar for their area. This will help in avoiding dockage penalties and at the same time increase economic returns. Similarly, understanding how basic cultural practices, such as seeding rates, influence falling numbers will provide additional opportunities to avoid dockage penalties and help to understand the mechanisms affecting seed dormancy.     NY (Taylor): A novel seed coating method was developed at Cornell by using soy-flour, a sustainable, inexpensive and green source, as a biostimulant using tomato and broccoli. Seedling shoot length, seedling growth uniformity, seed vigor index and seedling biomass were greater from coated seed than the noncoated control for both crops. Nitrogen applied in the seed coating only accounted for 1%to 2% of the enhanced N in the plants, indicating the soy flour acted as a biostimulant rather than a fertilizer. Plant proteins applied in the seed coating can improve early vegetable crop growth and uniformity.     TX (Leskovar): Exogenous PGRs could be useful to enhance germination, root length and surface area of onion seedlings. In addition seed hydro-priming was a simple and viable method to improve onion germination and root traits. These improvements are important since bulb development, uniformity and final size are greatly affected by the time spread of seedling emergence during early establishment. Humic substances applied to soil media rapidly decreased leaf stomatal conductance and transpiration in bell pepper transplants, suggesting that HS transiently ameliorated plants exposed to water stress by reducing moisture losses, which is critical when peppers are established in hot and dry environments. From the grafted project on Solanum spp. we expect to provide growers with recommendations for best scion/rootstock combinations for use in open field and protected culture in Texas. We also expect to identify rootstocks and germplasm lines with improved biotic and abiotic stress tolerance. Results from the studies conducted during the project duration on PGRs and humic substances were presented at annual meetings of the Plant Growth Regulator Society of America (Anchorage, AK), Southern Region of the American Society of Horticultural Sciences (Mobile, AL) and Agronomy (Tampa, FL). Similarly, partial results from the tomato grafted projects were presented in trainings, workshops and field days attracting +200 participants that represented small- and large-scale vegetable growers, as well as retailers interested in the production of home-grown tomato in Texas.     VA (Welbaum): Our new Lab FieldÔ is being adapted by seed companies to provide a more accurate assessment of seed quality. The Lab FieldÔ system will enable more realistic predictions of how seeds will perform in field soils and help in the development of new treatments to enhance seed vigor and improve field performance.   Seed hydropriming enhances germination of pepper seed germination although often reducing the potential seed storage life. Our novel nanochitin treatment appears to naturally increase plant disease resistance by stimulating system natural acquired resistance mechanisms in seeds. Nanochitin treatment will increase seed and seedling disease resistance through preplant seed treatments. Since the treatment uses only natural materials, it may improve plant disease resistance for organic production.

Publications

Adegbola, Y.A. and Pérez, H.E. 2016. Extensive desiccation and aging stress tolerance characterize Gaillardia pulchella (Asteraceae) seeds. HortScience. 51: 159-163.   Amirkhani, M., S. A. G. Avelar, A. R. Quere-Carrillo, and A. G. Taylor. 2016. The effect of film coating on Sideoats grama “Bouteloua curtipendula”. 10th International Rangeland Congress. Saskatchewan, Canada. 10: 727-729.   Amirkhani, M., A. N. Netravali, Huang, W., and A. G. Taylor. 2016. Investigation of soy protein based biostimulant seed coating for broccoli seedling and plant growth enhancement. HortScience: 51(9): 1121–1126. doi: 10.21273/HORTSCI10913-16   Amirkhani, M., A. N. Netravali, and A. G. Taylor. 2016. Using sustainable plant based biostimulant resins and fibers for seed coating. Crops & Chemicals USA, Raleigh, NC, USA.   Ardura, D. (2017) Mass spectrometry-based metabolomics applied to plant chemotaxonomy and to thermoinhibition of germination in lettuce (Lactuca sativa) seeds. PhD disseratation, University of California, Davis.   Avelar, S.A.G., M. Amirkhani, A. G. Taylor. 2016. Upgrading seed quality. Revista Seed News: 20 (3): 8-12. ISSN: 1415-0387. <a href="http://store.seednews.inf.br">http://store.seednews.inf.br</a>  Avelar, S.A.G., D. Yang, M. Amirkhani, and A. G. Taylor. 2016. Soybean seed coat permeability: water imbibition rate and fluorescent tracer uptake. Conference Paper. December 2016. American Seed Trade Association (ASTA), Chicago, IL, Volume: 71st Corn & Sorghum Seed Conference, 46th Soybean Seed Conference, Seed Expo.   Bewley, JD, Nonogaki H. Seed maturation and germination. Reference Module in Life Sciences, Elsevier, Oxford, accepted.   Geneve, R.L., D.F. Hildebrand, T.D. Phillips, M. AL-Amery and S.T. Kester. 2017. Stress influences seed germination in mucilage-producing chia. Crop Science 57:2160-2169.   Geneve, R.L., E.W. Janes and S.T. Kester. 2017. Cardinal temperatures and thermal time for seed germination of industrial hemp. Combined Proceedings International Plant Propagators’ Society 66:325-329.   Genna, N.G. and Pérez, H.E. 2016. Mass-based germination dynamics of Rudbeckia mollis (Asteraceae) seeds following thermal and ageing stress. Seed Science Research. 26: 231-244.   Li, Z. (2017) Seed priming and post-priming treatment effects on germination rates and longevity. MS thesis. University of California, Davis.   Majee Manoj, Wu Shuiqin, Salaita Louai, Gingerich Derek, Dirk Lynnette M.A., Chappell Joseph, Hunt Art G., Vierstra Richard, Downie A. Bruce. 2017. A misannotated locus positively influencing Arabidopsis seed germination is deconvoluted using multiple methods, including surrogate splicing. Plant Gene. 10: 74-85.   Niroula, M. (2017) Environmental sensitivity of quantitative trait loci for seed germination and flowering time in lettuce (Lactuca sativa L.). PhD dissertation, University of California, Davis.   Nonogaki H. 2017. Seed biology updates - highlights and new discoveries in seed dormancy and germination research. Frontiers in Plant Science 8:524. doi: 10.3389/fpls.2017.00524.   Nonogaki M, Nonogaki H. 2017. Prevention of preharvest sprouting through hormone engineering and germination recovery by chemical biology. Frontiers in Plant Science 8:90. doi: 10.3389/fpls.2017.00090.   Nonogaki M, Nonogaki H. 2017. Germination. In B. Thomas, B. G. Murray, D. J. Murphy eds, Encyclopedia of Applied Plant Sciences, Vol. 1, Waltman M. A.,Academic Press, pp. 509-512.   Nonogaki M, Nonogaki H. 2017. Seed dormancy through enhanced ABA biosynthesis and germination recovery by ABA antagonists. Plant Biology 2017 (Honolulu, HI)   Nonogaki M, Nonogaki H. 2016. Germination recovery from the NCED-enhanced hyperdorrmant seeds using nitrate-inducible gene switch and chemical biology with an ABA antagonist. 5th Workshop on the Molecular Aspects of Seed Dormancy and Germination (Vancouver, Canada)   Nonogaki M. 2016. Strategies to recover germination from PHS-resistant hyperdormant seeds. 13th International Symposium on Preharvest Sprouting in Cereals (Perth, Australia)   Nonogaki H. 2016. Prevention of preharvest sprouting through alteration of hormone metabolism, perception and signal transduction. 13th International Symposium on Preharvest Sprouting in Cereals (Perth, Australia)   Nonogaki M. 2016. Prevention of preharvest sprouting through hormonal regulation. Preharvest Sprouting Workshop (Kitami, Japan)   Sall K, Nonogaki H. 2016. Biochemical function of DELAY OF GERMINATION1-LIKE4 in hormone and cell signaling. 5th Workshop on the Molecular Aspects of Seed Dormancy and Germination (Vancouver, Canada)   Sall K, Nonogaki M, Katsuragawa Y, Hendrix D, Dekkers JWD, Bentsink L, Nonogaki H. 2017. DELAY OF GERMINATION1-LIKE4 is a master regulator of reserve accumulation in seeds. Plant Biology 2017 (Honolulu, HI)   Sall K, Hendrix D, Sekine T, Katsuragawa Y, Koyari R, Nonogaki H. 2017. Transcriptomics of nine-cis-epoxycarotenoid dioxygenase 6 induction in imbibed seeds reveals feedback mechanisms and long noncoding RNAs. Seed Science Research, accepted.   Samarah, N.H., Wang, H. and Welbaum, G.E. 2016 Pepper (Capsicum annuum) seed germination and vigour following nanochitin, chitosan or hydropriming treatments. Seed Sci. & Technol.: 44, 3, 1-15. (http://doi.org/10.15258/sst.2016.44.3.18)   Santos, Juliana F., Dirk, Lynnette M.A., Downie, A. Bruce, Sanches, Mauricio F. G. and Vieira, Roberval D. 2017. Soluble sugars, storage proteins and reciprocal effect of parental lines of corn hybrid seeds. Seed Science Research. 27: 206-216.   Tao L, Zhang Y, Wang D, Liu Y, Dirk LMA, Downie B, Wang J, Wang G, Zhao T-Y. 2017. Raffinose but not galactinol in Maize directly improves both drought stress-tolerance and seed longevity. Molecular Plant. Provisionally accepted.   Welbaum, G.E. 2017a. Chapter 2, Seed production. In book: Editors: Brian Thomas, Brian G Murray and Denis J Murphy. Encyclopedia of Applied Plant Sciences, Vol 1, 2nd Edition, Academic Press, pp.546–552.   Welbaum, G.E. 2017b. A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes Training Video <a href="https://www.jove.com/video/54647/a-gusseted-thermogradient-table-to-control-soil-temperatures-for">https://www.jove.com/video/54647/a-gusseted-thermogradient-table-to-control-soil-temperatures-for</a>. JOVE <a href="http://dx.doi.org/10.3791/54647">DOI: 10.3791/54647</a>  Wilson, R., D. Culp, K. Nicholson, S. Peterson, and A. Taylor. 2016. Comparison of insecticides and insecticide application methods to protect spring-seeded processing onions from seedcorn maggot and onion maggot damage. Proceedings 2016 National Allium Research Conference, Savannah, GA, 1-3 December. National Onion Association, Greeley, CO 80631  Zhang L, Lou J, Foley ME, Gu X-Y. 2017. Comparative mapping of quantitative trait loci for seed dormancy between temperate and tropical ecotypes of weedy rice. G3: Genes, Genomics, Genetics 7: 2605-2614 (doi: 10.1534/g3.117.040451)

Impact Statements

Lab Field is being adapted by seed companies to provide a more accurate assessment of seed quality, enhance seed vigor and improve field performance.

Date of Annual Report: 10/24/2018

Report Information

Annual Meeting Dates: 06/28/2018 - 06/30/2018
Period the Report Covers: 10/01/2017 - 09/30/2018

Participants

Bradford, Kent (kjbradford@ucdavis.edu) - University of California, Davis; Downie, Allan B. (adownie@uky.edu) - University of Kentucky; Elias, Sabry (Sabry.Elias@oregonstate.edu) - Oregon State University; Geneve, Robert (rgeneve@uky.edu) - University of Kentucky; Gu, Xingyou (Xingyou.Gu@sdstate.edu) South Dakota State University; Jourdan, Pablo (jourdan.1@osu.edu) - The Ohio State University; Perez, Hector (heperez@ufl.edu) - University of Florida, Taylor, Alan (agt1@cornell.edu) - Cornell University; Welbaum, Greg (welbaum@vt.edu) - Virginia Polytechnic University; Yadegari, Ramin (yadegari@email.arizona.edu) – University of Arizona; Lin, Liang-Shiou (LLIN@nifa.usda.gov) - USDA-NIFA; Chastain, Thomas (Thomas.Chastain@oregonstate.edu) - Oregon State University: Leskovar, Daniel (Daniel.Leskovar@ag.tamu.edu) - Texas A&M University.

Brief Summary of Minutes

Minutes Attachment:

Minutes_W3168_Corvallis Annual Meeting_2018_FINAL.pdf

Accomplishments

Objective 1 - Identifying key factors involved in the enhancement or loss of seed quality. Seed development through post-harvest losses in storage. Specific topics include seed development, desiccation tolerance, and aging in storage. AZ (Yadegari) We continued our efforts in understanding the regulatory mechanisms that control accumulation of storage proteins and starch during mid to late stages of endosperm development in maize (Zea mays). Development of the cereal endosperm involves cell differentiation processes that enable nutrient uptake from the maternal plant, accumulation of storage products and their utilization during germination. We set out to use the previously studies Opaque-2 (O2) bZIP transcription-factor protein, a major regulator of storage protein gene expression in maize, as an opening to dissect further these regulatory programs. We mapped in vivo binding sites of O2 in B73 endosperm, and compared the results with genes differentially expressed in B73 and B73o2. We identified 186 putative direct O2 targets and 1,677 indirect targets, encoding a broad set of gene functionalities. Examination of the temporal expression patterns of O2 targets revealed at least two distinct modes of O2-mediated gene activation. Two O2-activated genes, bZIP17 and NAKED ENDOSPERM2 (NKD2), encode transcription factors, which can in turn co-activate other O2-network genes with O2. NKD2 (with its paralog NKD1) was previously shown to be involved in regulation of aleurone development. Collectively, our results has provided insights into the complexity of the O2-regulated network and its role in regulation of endosperm cell differentiation and function. We plan to further characterize the functions of some of the key downstream target genes of O2 during the next reporting period. CA (Bradford) Elevated seed moisture content due to insufficient drying is the major cause of seed deterioration, particularly in humid environments. When applied to food grains and commodities, drying and packaging (termed the “dry chain”) has the potential to greatly reduce postharvest loss and improve food safety by preventing accumulation of fungal toxins (e.g., aflatoxin) in storage. We reviewed this field and described methods and strategies to dry and package seeds and commodities, particularly in humid regions (Bradford et al., 2018). We have developed a number of tools and methods to monitor seed/commodity moisture content and to use desiccant-based drying followed by hermetic packaging to preserve seed viability and commodity quality. The potential economic impact of implementing these methods for onion seed in Nepal was assessed through surveys of value chain actors (Timsina et al., 2017). We developed methods to analyze single-seed respiratory data obtained through the ASTEC Q2 instrument using population-based threshold models. This quantitative analysis of the respiratory characteristics of seed populations enables labor-efficient monitoring of seed quality and new insights into seed germination, vigor, enhancement and aging. In 2017-2018, we worked with a seed technology company and the Indiana Crop Improvement Association to test the applicability to corn (Zea mays) vigor testing and subsequent seed performance in the field. Individual seed respiration rates could potentially replace the cold test as a vigor assessment, and efforts are being made to scale up the analysis system for greater throughput. FL (Perez) We evaluated the seed viability and initial germination potential of Harperocallis flava, Linum arenicola, Rudbeckia mollis and Uniola paniculata. We collected data on the germination ecology and phenology, response to thermal and desiccation stress, and viability maintenance following storage. Our data indicated that H. flava produces abundant viable seeds with high germination potential (≥ 65%) in the lab. However, germination and seedling establishment in the field is highly reduced (ca. 1%). Seeds germinated readily in sterile culture (≥ 85%). Seeds of L. arenicola also display high levels of viability (≥ 85%) and germination occurs readily at lower constant temperatures (ca. 15°C). Seeds of R. mollis display mass-based separation following aging stress, with germination (i.e. rate and percentage) exceeding observations for larges seeds. Uniola paniculata seeds stored for more than seven years seem to deteriorate in storage as displayed by low (≤ 25%) and delayed germination (> 21 d). Our data indicates that contrary to anecdotes, seeds of H. flava germinate readily in the lab and that recruitment, while limited in our experiments, does occur in the field. Seeds of L. arenicola display a low temperature optimum for germination. We also need to recognize intra-specific seed size as a factor contributing to loss of viability following aging stress. Finally, we need to re-evaluate post-harvest seed handling methods for U. paniculata seeds. KY (Downie) The molecular mechanisms by which seed vigor is lost and which eventually leads to seed death is of considerable importance to those involved in seed production for crop establishment. We have reviewed evidence in support of Job’s Rule (Dirk and Downie, 2018) postulating that, of all components of the proteome, it is those proteins directly involved in translation that are most crucial to maintain in the functional state if the seed is to survive. During seed development, seed quality (or lack of it) is orchestrated. To understand the global purview of a transcription factor important for setting up the capacity to dehydrate ChIP Seq of ABA INSENSITIVE3 (ABI3) was performed and then further investigated with an RNA Seq experiment (Tian et al. 2018). One of the largest categories directly regulated by ABI3 is the LATE EMBRYOGENESIS ABUNDANT proteins. VA (Welbaum) We are studying fundamental aspects of A. citrulli-cucurbit interactions to elucidate the bacterial determinants that mediate host preferential association towards different cucurbits (crops in the family Cucurbitaceae). The specific objectives of this research are to: (1) to investigate the susceptibility of different cucurbit species to Group I and II strains of A. citrulli under field conditions during seed production; (2) to assess the contribution of type III effectors and other virulence factors to A. citrulli virulence and host preference in cucurbit fruit tissues and seeds; and (3) to characterize the mechanism of action of selected type III effectors and find sources of resistance. Objective 2 - Eliminating seed dormancy as a constraint during seed production and germination in agronomic seed production and ecological/biomass seed establishment. Pre-mature sprouting in cereals and other species and the identification dormancy mechanisms to manipulate germination. CA (Brandford) Germination of lettuce (LACTUCA SATIVA) seeds is inhibited at warm temperatures (thermoinhibition). This can reduce crop stand establishment for warm-season plantings. We previously identified a quantitative trait locus (QTL) associated with thermoinhibition of germination in lettuce and subsequently demonstrated that it was due to expression of a gene in the abscisic acid (ABA) biosynthetic pathway (LsNCED4). In cooperation with colleagues at UC Davis, we further demonstrated that mutations in this gene effected using gene editing via CRISPR/Cas9 enabled lettuce seeds to germinate at higher temperatures (Bertier et al., 2018). This should permit incorporation of this high temperature germination trait into existing lettuce cultivars without extensive backcrossing to tolerant lines. Targeting of LsNCED4 can also be used as a selectable marker for the successful action of gene editing in lettuce. KY (Downie) The debate concerning whether raffinose or raffinose family oligosaccharides (RFO) assist seed viability or vigor has raged since the discovery of these soluble sugars. A further contentious issue is the role RFO may play in drought tolerance. Using Zea mays (maize) as a model we have demonstrated that raffinose biosynthetic capacity enhances the seed vigor of both maize and Arabidopsis and positively impacts survival during drought. An unexpected twist in Arabidopsis is the apparent increase in stomatal density in plants devoid of galactinol. A further intriguing result was the heightened sensitivity of the guard cells to drought in plants with more galactinol, potentially linked to greater ABA sensitivity (Li et al. 2018). SD (Gu) Continued work on cloning and molecular characterization of the seed dormancy loci qSD12. New experiments were conducted to characterize qSD12. 1) Molecular characterization of the qSD12 underlying genes SD12a, SD12b and SD12c for cellular localizations. GFP-fusion proteins were detected in both nucleus and cytoplasm for SD12a and only in the nucleus for SD12b, but were not detectable in both nucleus and cytoplasm for SD12c. 2) Molecular characterization for interactions between each pair of SD12a, SD12b and SD12c by bimolecular fluorescence complementation (BiFC) analysis. Preliminary data showed an interaction between SD12a and SD12c in nucleus. 3) Reverse genetic analysis of SD12a by an RNAi-mediated gene silencing approach. This experiment was combined with an on-going project to develop a transgene-mitigation strategy. The gene silencing effect on seed dormancy was evaluated in an F3 population. And 4) started an experiment to determine if qSD12 is involved in an epigenetic control of seed dormancy. Genetic materials prepared for this research were advanced for two generations in a greenhouse. Two experiments were conducted for qSD7-2. One experiment was to collect linkage disequilibrium data to evaluate an association of qSD7-2 with flowering time. Data analysis for this experiment has been completed. The other experiment was to determine the epistasis of qSD7-2 with a candidate gene for qSD10. This experiment was continued to the next year.  Objective 3 - Enhancing seed vigor and germination in agronomic and other species for improved stand establishment. The emphasis of this objective is on post-harvest technologies. FL (Perez) We investigated the germination ability of fresh Linum arenicola seeds at various simulated seasonal temperatures. We collected germination percentage data showing that alternating temperatures delayed germination of L. arenicola seeds by about one month compared to constant temperatures. Similarly, germination did not exceed about 35%. However, germination occurred rapidly (within 14 days) at constant temperatures. Final germination exceeded 50% at constant temperatures. These results indicate that enhanced germination cueing of L. arenicola seeds occurs at constant rather than alternating temperatures. Likewise, temperatures above 25°C seem inhibitory to germination. Seeds of L. arenicola may exhibit physiological dormancy. KY (Downie) In so far as Arabidopsis thaliana is a “native species” somewhere, we have reported on a feedback mechanism whereby the transcription of a gene encoding an F-BOX protein responsible for degradation of the bHLH transcription factor PHYTOCHROME INTERACTING FACTOR1 (PIF1) upon illumination is down-regulated by the transcription factor, assisting in phytochrome-mediated light signaling in both the completion of germination and hypocotyl elongation (Majee et al. 2018). We have elucidated the fate of PIF1 in various light environments illustrating how the transcription of a variety of transcripts encoding enzymes involved in GA production/degradation are indirectly influenced by PIF1’s presence (Dirk et al. 2018). The following stocks have been released to the Arabidopsis Biological Resource Center: CS71627, CS71628, CS71629, CS71630, CS71631, CS71632, CS71633, CS71634, CS71635, CS71636, CS71637, CS71638, CS71639, CS71640, CS71641, CS71642, CS71643, CS71644, CS71645, CS71646, CS71647, CS71648, CS71649, CS71650, CS71651, CS71652, CS71653, CS71654. KY (Geneve) During the reporting period, the revision of the ninth edition of the reference text “Hartmann and Kester’s Plant Propagation: Principles and Practices was completed. Continued research with industrial hemp seed has resulted in submission of a manuscript describing the impact of temperature on seed germination and the recent completion of a solid matrix and osmotic seed priming protocol. NY (Taylor) Systemic seed treatment uptake into soybean and corn seeds during imbibition has not been investigated over a broad range of application rates. The objectives of this study were to investigate application dosage rates on uptake, to assess the role of the seed coat on uptake and determine the dosage for greatest uptake efficiency. The model fluorescent tracer compound, coumarin 120, 7-Amino-4-methylcoumarin was applied as a seed treatment in the range of 0.01 to 20.0 mg g-1 seed to study the dose effects on seed uptake of corn lines ‘OH7B’ and ‘B73’, and soybean varieties ‘TMG 312RR’, ‘IAR 1902’, and a blacked-seeded ‘V12-1223’. The uptake by two lines of corn seeds and three varieties of soybean seeds showed a dose dependent process that was described by an exponential model of Y =  (r2 ³ 0.93), where y0 is the uptake limit. In general, there was a large increase in seed uptake as dosage increased, followed by a saturated state at higher dosages. The uptake limit (y0) differed between the two corn lines and the three soybean varieties. The maximum uptake efficiency or recovery of applied coumarin 120 was at 0.1 mg g-1. The pericarp/testa of both corn lines and the testa of black-seeded soybean variety ‘V12-1223’, attenuated the uptake in comparison with decoated seeds of each line/variety. Results from this study have implications on the effect of application rate on seed treatment efficacy and phytotoxicity. Systemic uptake of organic compounds from roots to leaves follows a Gaussian distribution in relation to the lipophilicity, as measured by the log Kow. Quantification of compound uptake with different lipophilicity, and applied as a seed treatment that diffused through the seed coat into the embryo during imbibition has not been reported. The objective of this investigation was to quantify the uptake of nonionic compounds into seeds of soybean and corn. A series of fluorescent piperonyl amides were synthesized and a novel combinatorial pharmacokinetic technique was developed that provided a range of compounds from log Kow 0.02 to 5.7. Seeds were treated with a mixture of amides, imbibed and compounds chemically extracted and quantified by HPLC using a fluorescence detector. The maximum uptake efficiency of the applied amide mixture from whole soybean and corn seeds was 67% at log Kow 2.9, and 43% at log Kow 3.4, respectively. The critical partition coefficient for uptake for both species was Kow. Seeds were dissected and separated as soybean embryo and testa, and corn internal tissues (embryo + endosperm) or seed covering layers (pericarp + testa), and >75% of the amides were found in the soybean embryo or corn internal tissues compared to the covering layers at log Kow OH (Jourdan) The Ornamental Plant Germplasm Center’s seed research efforts emphasize areas that relate to seed quality, germination, and long-term storage of North American native wild species. We have been working primarily with seeds of perennial species of Phlox that have presented significant challenges in terms of production, germination and potential for storage.  During this reporting period we have examined various parameters that help define the quality of our seed lots.  The first parameter was to describe the viability in seed lots of different species after optimization of the Tetrazolium test.  We estimated viability by TZ in 11 Phlox taxa and found the viability to range from 48% – 85%.  However, germination tests for any seed lot seldom exceeded 50% and most commonly was noted in the 10-20% range.  The most consistent problem was the extensive development of fungi on the seeds; analysis of such fungi indicated they were species of Alternaria, Epicoccum, Fusarium and Phoma.  The potential origin and significance of these different fungi is being assessed to establish more effective control strategies.  Exposure of seed to Gibberellic acid did not result in a significant enhancement in germination;  however, isolated embryos from mature seeds of the different taxa germinate readily when placed on 1% agar, indicating that the dormancy in these seeds is most likely non-deep physiological dormancy imposed by the seed coverings, including the residual endosperm.  These observations indicate that seed storage and longevity in perennial species of Phlox may be problematic and suggest that germplasm preservation of these taxa may be more effective with a clonal strategy using in-vitro maintained material. OR (Chastain) Field trials were conducted on the effects of trinexapac-ethyl plant growth regulator (PGR) and nitrogen on seed development in orchardgrass.  Nitrogen increases the size of the canopy source and the amount of source materials available for seed filling while PGRs increase the preferential partitioning of source materials to seed.  The effects of the PGR and N on the rate of seed growth, the length of the seed filling period, seed set, the rate of progress toward seed maturity, and seed number and seed weight were measured.  Seed weight was greatest in proximal spikelet positions regardless of treatment.  Seed weight was greater with increased N than in the untreated control.  Seed moisture content (SMC) is the most reliable indicator of seed maturity and harvest timing in grass seed crops.  The problem is that the most widely adopted SMC testing methodologies are slow and as a result, it is difficult to make timely harvest decisions.  A portable near-infrared reflectance spectroscopy (NIR) device was field tested as a rapid alternative to the oven test for determination of SMC in perennial ryegrass, tall fescue, and orchardgrass seed crops.  Daily testing of SMC began when the experimental crops were at BBCH 69 and continued through seed development until harvest.  Seed was collected by stripping 30 spikes or panicles into airtight containers from each species until ready for NIR determination of SMC in the field or by laboratory air-oven method.  In perennial ryegrass, NIR determination of SMC over the course of seed development was strongly related to the oven method.  The SMC values provided by NIR also showed a good relationship with the oven method in orchardgrass.  Differences in SMC measurement were observed in tall fescue; the relationship of NIR SMC to oven SMC in turf-type tall fescue was similar to other species but NIR SMC in forage-type tall fescue was more variable in relation to oven SMC.  Spring agronomic practices including PGR, foliar fungicides, and nitrogen had no influence on NIR determination of SMC compared to untreated controls.  TX (Leskovar) Assisted in the organization and convened the VIII International Symposium on Seed, Transplant and Stand Establishment of Horticultural Crops, as part of the XXX International Horticultural Congress, International Society of Horticultural Sciences (ISHS). The conference held in Istanbul, Turkey on August 12-16, brought 80 attendees from 22 different countries and had 39 presentations on topics of seed biology, technology, dormancy, vigor, grafting, seed systems, storage and delivery, mechanization and grafting. A technical 1-day tour depicted onion seed production, onion drying systems, and vegetable transplant nurseries. In terms of research, a study in onion seeds showed that exogenous application of ethylene promoted fine root development and overall root surface area. A follow up onion transplant study evaluated stand establishment strategies (seedling density, planting times, cultivars) to optimize growth, yield and quality. Another tomato transplant study showed the benefits of a soil bio-stimulant (based on humic substances) on root and shoot growth responses. Conversely, the application of 1-MCP to pepper and tomato transplants exposed to heat stress had inconsistent growth responses, which varied greatly with genotypes. The group has initiated studies to phenotype tomato grafted roots using a ground-penetrating radar to facilitate the screening or rootstocks. VA (Welbaum) Standardized seed germination tests used by seed labs is conducted under controlled conditions, usually on moist paper substrates at optimum temperatures.  We have developed a new laboratory analysis system called Lab FieldÔ to provide more realistic seed germination testing using soils controlled at realistic field planting temperatures.  This system provides a more accurate assessment of viability by subjecting seeds to biotic stresses encountered in a field.  The Lab FieldÔ was created by welding thin vertical aluminum strips to the face of a thermogradient table while adding micro-irrigation, drainage and LED grow lights, to create a soil environment that can be adjusted to mimic a wide range of field conditions in a laboratory (https://www.jove.com/video/54647/a-gusseted-thermogradient-table-to-control-soil-temperatures-for).  This allows for realistic seed testing that more accurately predicts how seeds will perform in crop production fields.

Publications

Agehara, S. and D.I. Leskovar. 2017. Growth suppression by exogenous abscisic acid and uniconazole for prolonged marketability of tomato transplants in commercial conditions. HortScience 52(4):605-611. doi: 10.21273/HORTSCI11518-16 Agehara, S. and D.I. Leskovar. 2018. Optimizing spray application rates of abscisic acid for height control of jalapeño transplants. Acta Hortic. 1204, 235-242 DOI: 10.17660/ActaHortic.2018.1204.31 <a href="https://doi.org/10.17660/ActaHortic.2018.1204.31">https://doi.org/10.17660/ActaHortic.2018.1204.31</a> AL-Amery, M., Geneve, R.L., M.F. Sanches, P.R. Armstrong, E.B. Maghirang, C. Lee, R.D. Vieira, and D.F. Hildebrand. 2018. Near-infrared spectroscopy used to predict seed germination and vigour in soybean seed lots. Seed Science Research 1–8   <a href="https://doi.org/10.1017/S0960258518000119">https://doi.org/10.1017/S0960258518000119</a> Anderson, N., Chastain, T.G., Garbacik, C. J.  2017. Irrigation and Plant Growth Regulator Effects on White Clover Seed Crops. Tampa, Florida: 2017 International Annual Meeting Abstracts. ASA, CSSA, SSSA. Anderson, N., Sullivan, C., Chastain, T. G., Garbacik, C. J. 2017. Examining Possible Benefits of Plant Growth Regulator Mixtures in Tall Fescue Seed Crops. 2016 Seed Production Research Report at Oregon State University, USDA-ARS Cooperating (vol. Ext/CrS 153, pp. 1-4). Corvallis, Oregon: OSU Department of Crop and Soil Science. Anderson, N., Chastain, T. G., Garbacik, C. J. 2017. Can Urease Inhibitors Increase Seed Yield in Grass Seed Crops?. Pergamino, Argentina: 9th International Herbage Seed Group Conference. Bertier, L., Ron, M., Huo, H., Bradford, K.J., Britt, A.B., Michelmore, R.W. 2018. High-resolution analysis of the efficiency, heritability, and editing outcomes of CRISPR/Cas9-induced modifications of NCED4 in lettuce (Lactuca sativa). G3: Genes Genomes Genetics 8: 1513-1521. Bradford, K.J., and Chrispeels, M.J. 2018. 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Impact Statements

SD (Gu) Some data from this project has been presented in international conferences for seed biology or weed science. The data and isogenic lines developed were used to develop research proposals submitted to the USDA-NIFA BRAG or Foundational programs.