Brief Summary of Minutes of Annual Meeting

OR (Elias): Oregon State University (OSU) conducts studies in both basic and applied research, the two broad objectives of the current W4168 project. We focused on the application of new technologies to assess and manipulate traits to enhance seed quality.

The seed group at OSU lost two out of its three members, Dr. Hiro Nonogaki for retirement and Dr. Thomas Chastain for becoming the head of the department (100% administration position), only Dr. Sabry Elias  is currently conducting seed research. The following projects have been concluded in 2022/2023, with the publications cited below:

1.     Development of a quantitative DNA test to distinguish between annual and perennial ryegrass.
2.     Evaluation of bioassay methods to screen winter wheat for quizalofop herbicide tolerance.
3.     Diverse Eastern Gamagrass ecotypes: General characteristics, ploidy levels, and biogeography.
4.     Effects of different storage conditions on seed quality of hemp (Cannabis sativa).
5.     Production of Native Seeds (Video): Supplying Restoration (2023). It is a nine-part video series that explores the native seed supply chain in the western United States. (Video 7 Seed Testing and Certification by Oregon State University Seed Lab). Produced by Holden Films, Colorado, USA. brady@holdenfilms.org.
6.     Green synthesis of copper nanoparticles extracted from guar seedling under Cu heavy‑metal stress by Trichoderma harzianum.

KY (Geneve, Downie, Kawashima): (Geneve) Grape seeds showed physiological dormancy and germinated at approximately 60% after 60 days of chilling stratification. Seeds cut at the distal seed end or intact seeds treated with gibberellic acid (GA) germinated at approximately 20% after 30 days. Cut seeds treated with GA at 5,000 mg·L-1  had the highest germination percentages between 63% to 83%. Results from this study provide a system that reduces the need for chilling stratification for grape seed germination by using partial seed coat removal and GA treatment.

(Downie) LATE EMBRYOGENESIS ABUNDANT PROTEINs (LEAPs), intrinsically disordered proteins found in most organisms that can withstand desiccation at some stage of their life-cycle) are hypothesized to provide protection to stressed cells. So too are non-reducing sugars (NRS; trehalose, sucrose, raffinose) thought to provide desiccation tolerance. Together, these molecules are components of the natural protection and repair (NPR) mechanism in orthodox seeds, the focus of our studies.

We found that the means by which plant material was prepared before sugar extraction had a non-trivial influence on the sugar profile retrieved from the tissue. Based on our results we highly recommend avoiding freeze-drying tissue prior to sugar extraction (Dirk, Zhao et al. 2023). Accurate sugar extraction techniques allowed us to demonstrate a role for raffinose in retaining water in stressed maize leaves, helping to avoid wilting during drought (Liu, Li et al. 2023).

Using two orthologous dehydrin LEAPs, one from Arabidopsis (At2G21490; LEA14; (Hundertmark and Hincha 2008)), its soybean (Glycine max) orthologous protein (GmPM12; Glyma.04G009900.1) we have demonstrated their interaction with ABSCISIC ACID INSENSITIVE3 (ABI3; At3g24650). In this endeavor we were ability assisted by Ms. Deepshikha Sai Bassetti, a recent graduate (Summa Cum Laude) of the University of Kentucky Agricultural and Medical Biotechnology (ABT) program who graduated in May, 2023. Ms. Bassetti pursued her ABT research proposal in my lab throughout 2022-2023. Ms. Bassetti expressed and purified both LEAPs (LEA14 and GmPM12) and the transcription factor, ABI3. She and I ran experiments using Temperature Related Intensity Change (TRIC) to assess the binding between the dehydrin proteins, LEA14, GmPM12 and ABI3 mentioned previously. She presented her results on Thursday, April 29, 2021 1:30 PM entitled, “LEA14 and ABI3 effects on the completion of seed germination.” For which she was judged as joint winner of the Glenn B. Collins Research Award for excellence in research.

I was the recipient of a grant from the University of Kentucky Office of the Vice President for Research for $80,000 to upgrade the Dianthus (NanoTemper Inc., Munich, Germany) used for TRIC assays to now also include Isothermal Spectral Shift (ISS) assays also used for molecular interactions. The instrument was sent to Germany for the upgrade and arrived safely back to my lab September 15th, 2023. Once it was reinstalled, hands-on training of 12 individuals from 12 different labs across 5 Departments, 3 Colleges and two universities was conducted on the use and capacities of the Dianthus with both TRIC and ISS (September 21st, 2023). These individuals are now capable and able to use the Dianthus for their various projects. Importantly, the new ISS capacities are a more robust, and independent, measurement of molecular interaction that is directly applicable to our efforts to demonstrate LEAP client protein interactions, with or without NRS and with or without osmotic or ionic perturbations.

We have continued to work to sonoporate protective LEAPs and NRS into Drosophila melanogaster (fruit fly) embryos to enable their successful cryopreservation. Sonoporation is successful in introducing sugars into embryos but only if the latter are dechorionated and dewaxed. The dewaxing step, in particular, is detrimental to fly survival we have found, with approximately 80% mortality during this step. Attempts to sonoporate dechorionated but not dewaxed embryos were not successful. Attempts to identify the causal agent for the high mortality has not been fruitful. However, we noted that those flies expressing a recombinant LEA6 protein from Artemia franciscana (brine shrimp) survive dewaxing better than non-transformed, or flies expressing other brine shrimp, LEAs (unpublished). We are pursuing the cryopreservation of these fruit fly embryos, despite the poor pretreatment survival, as the removal of the chorion and waxy layer are essential to successful sonoporation of naturally occurring, cryoprotective molecules, to quantities sufficient to permit the formation of a glassy state within the embryos. Glass formation is seen as a requirement for survival of both cryopreservation and desiccation. This project supports our efforts to understand LEAP and NRS activities in preserving life in the desiccated cell. If successful, we will have demonstrated that it is possible to create a desiccation tolerant organism by recapitulating within it, aspects of the NPRM.

(Kawashima) The endosperm within a seed is a tissue that supports embryo development and is a product of fertilization. Endosperm cells undergo nuclear divisions without cytokinesis right after fertilization, generating a large multi-nuclear cell. Cellularization subsequently occurs. The duration of this multi-nuclear endosperm phase is correlated with the final seed size. However, the molecular mechanisms and cellular dynamics by which the endosperm contributes to final seed size remains largely unknown. The Kawashima lab has characterized nuclear divisions and movement as well as the role of the cytoskeleton in this unique endosperm development in Arabidopsis thaliana via confocal live-cell imaging technology Both microtubules (MT) and actin filaments (F-actin) generate aster-shaped structure around each nucleus. While MT promotes movement of nuclei at mitosis, F-actin restricts further movement, controlling equidistance of multi nuclei in the endosperm. Furthermore, manipulations of F-actin dynamcis specifically in the endosperm cause changes in seed sizes, providing insights into a new seed size control mechanims.

Using soybean as a crop model, the Kawashima lab investigated what are the differences if any early pahse seed development among cultivars with different seed sizes. Four cultivars (two with small seeds and the other two with large seeds) were grown in a green house and early seed development process were intensively investigated. We found that large-seeded cultivars have longer duration of the early phase of seed development. This results in the increased number of the cotyledon cells, thus pre-determining the max potential of the seed size.

FL (Pérez): We partnered with a group of small-scale farmers interested in developing seed production systems for selected high-value vegetable and wildflower crops. This collaborative research led to assessment of the developmental physiology for selected crops resulting in identification of minimum harvest windows to ensure high-seed quality. For example, in the warm, humid southeastern region of north Florida, seeds of C. moshata ‘Bellevue Butternut’ display appropriate traits for harvest at 50 days after flowering. Brassica rapa ‘Yukina Savoy’ plants planted in the late fall produce high quality seeds by 45 days after flowering. Similarly, V. unguiculata ‘Purple Hull’ seeds should be harvested no earlier than 35 days after flowering. Seed fill of Asclepias tuberosa was high but germination was highly variable (25-80%).

In another study, we investigated the efficacy of scalable, cost-effective desiccant-based drying systems to store seeds of the same crops species. Seeds of all the vegetable crops benefited from drying in the desiccant-based systems and did not display reduced germination capacity or vigor following storage. Dry storage of A. tuberosa seeds under these conditions may be limited to 9 months. However, more studies with fresh seeds are required to verify.

A third project involved assessing the influence of ecotype, prescribed burn timing, and other environmental correlates with seed quality of Aristida stricta (wiregrass). Ecotype and burn month had little comparative effects on germination capacity (range 55-60%). However, exposing seeds to low winter temperatures reduced final germination. The effect of temperature on germination decrease was moderate. Unlike germination, the proportion of filled seeds was strongly influenced by ecotype and burn month. For instance, the odds of plants growing in mesic sites producing filled seeds was three times greater than for plants from xeric sites. Likewise, June appeared optimal for prescribed fire. The odds of a filled seed for plants burned in June was 18% and 8% higher than for plants burned in May or July, respectively. At the same time, for each 10 mm increase in precipitation two months before fires occurred, the odds of seed fill increased by 1%. Moreover, for each 10 mm increase in precipitation during the seed development program the odds of seed fill increased by 6%.

IA (Goggi): Investigation continues into agricultural systems that protect soil and water quality, such as perennial ground covers, often stress seed from cash crops. The emergence, plant development and yield are negatively affected because cash-crop competes with the cover crop.  We seek seed characteristics that allow the cash crop to out-compete the perennial ground cover are essential for the success of these systems. We continue investigating the  effects of seed characteristics on yield at the whole-plot and single-plant level in corn based on emergence date based on plant-to-plant competition. 

TX (Leskovar): This year we summarized two studies conducted previously on the role of humic substances (HS) applied to the growing media on growth and physiology of ‘Micro-Tom’ young tomato plants and ‘Celebrity’.  In the first study we found three significant results:  1) HS increased seedling root development which in turn increased water and nutrient status; 2) HS decreased oxidative stress and cell membrane damage, a response that was related to an increase in ABA and not on antioxidant enzymes SOD and CAT; and 3) HS increase yield, possibly due to improved resource use uptake. Taken together, these results indicate that humic substances helped tomato pants overcome abiotic stresses. In the second study, we found that when HS are applied to the growing media on seedlings exposed to a combination of LED light with a spectral ratio of 10% Blue-90% Red, they had a significant growth improvement that was translated into higher early yield. 

MT (Torrion): In the current year, we conducted a second-year verification study on various irrigation supplementation timings during the seed development of two market classes in winter wheat (soft whites and hard reds genetics). We’ve confirmed that irrigation supplementation during the seed formation leads to late amylase activity. In the same manner, late occurrences of rainfall events (using the center pivot simulation of rain) also lead to more amylase activity. And, irrigation supplementation on top of the rainfall events even more exacerbates amylase activity in the grain. This is more concerning in soft white market class compared with the hard reds market class. 

MI (Fleming): Soil seed banks: MSU’s Baker Woodlot is a protected old growth forest. Periodic floristic surveys in Baker Woodlot have documented the loss of many native species along with increased abundance of invasive species. Soil cores have been collected from nearly 200 sites within Baker Woodlot. They will be examined for the presence and viability of any of the lost native species.

Desiccation tolerance: Seeds of the model plant Arabidopsis thaliana were confirmed to become capable of germinating 10 days after flowering and to acquire desiccation tolerance 14 days after flowering. Seeds at the distal end of a silique mature and acquire desiccation tolerance more quickly than seeds at the basal end, which complicates interpretation of assays done on seeds pooled across several siliques. Desiccation-sensitive seeds are able to survive desiccation if they are dried slowly over several days, but it is unclear whether this is because of a change to their desiccation tolerance mechanisms or because the normal desiccation tolerance pathways have time to be expressed. We are also using wild pea, Pisum elatius, as a complementary system to study desiccation tolerance. Seeds are mature and desiccation sensitive by 21 days after flowering, and become desiccation tolerant 24 days after flowering, when maturation drying starts. Pea seeds have physical dormancy imposed by the seed coat, and the interactions between flower color, seed coat color, dormancy, and desiccation tolerance will be explored further.

We are making protoplasts from spinach and Arabidopsis leaf tissue to do pilot studies using MSU’s Flow Cytometry core facilities, which can sort protoplasts based on size, shape, and other features. We plan to make protoplasts from pea seeds and use flow cytometry to characterize their recovery or death after desiccation and rehydration.

Soybean germination: Michigan farmers are planting soybeans early, at the end of April, because the increased variability in spring weather has made extending the planting season desirable. However, soybeans planted that early are vulnerable to imbibitional chilling injury as well as opportunistic fungal and insect damage. We have shown that prehydrating seeds by moving them to a 100% relative humidity environment at room temperature for 8-48 hours improves germination performance, and prehydrating for 24 hours followed by hydropriming in room temperature water for 18 hours significantly improves germination at 4 C. 

Seed longevity: The RNA Integrity Number (RIN) is a good metric for seed quality. We are working to develop an alternate method as the equipment required for determining RIN is nearing obsolescence. We have shown that digital PCR can quantify integrity of a single mRNA, where degradation of that mRNA was previously linked to lower RNA integrity. The Verbascum blattaria genome is complete and the report is being prepared for publication. 

CA (Khanday): Dr. Khanday has just joined this group and there is no report on accomplishment.

SD (Latvis): In August 2023, Dr. Latvis moved her lab to the University of Arkansas to serve as the director of the UARK herbarium and assistant professor in the Biological Sciences department. She is a co-PI on a USDA-NRCS grant based in South Dakota to investigate the influence of phylogenetic diversity and seed sourcing on pollinator and soil health and continues to make progress on that project. In Fall 2022, we sourced seed for 107 commercially available perennial species native to the Northern Great Plains that had 3 different geographical origins. The seeds were used to create 480 experimental plots at the Oak Lake Field Station near White, SD, varying levels of species richness (8, 16, 32, and 64 species), phylogenetic diversity, and seed source (1 seed origin vs a mixture of 3 different geographic origins). Plots were established in Spring 2023 and maintained throughout the summer. During the process of mapping sources of commercially available restoration seed, we identified important geographic gaps in native seed collection, which influences combination and climate forecasting strategies. We are working on a collaborative manuscript on the gaps in the native seed market.

NY (Taylor): To increase biodiversity and enhance ecosystem services with minimal loss of crop production area, farmers can plant desirable non-crop species near crop fields. Adoption of this practice is limited by inefficiencies in existing establishment methods. We have developed a novel seed-molding method allowing non-crop species to be planted with a conventional corn (Zea mays L.) planter, reducing labor and capital costs associated with native species establishment. Common milkweed (Asclepias syriaca L.) was selected as a model native species, because Asclepias plants are the sole food source for monarch butterfly (Danaus plexippus L.) larvae. Stratified A. syriaca seeds were added to a mixture of binder (maltodextrin) and filler (diatomaceous earth and wood flour) materials in a 3D-printed mold with the dimensions of a corn seed. The resulting Multi-Seed Zea Pellets (MSZP), shaped like corn seeds, were tested against non-pelleted A. syriaca seeds in several indoor and outdoor pot experiments. Molding into MSZP did not affect percent emergence or time to emergence from a 2-cm planting depth. Intraspecific competition among seedlings that emerged from an MSZP did not differ from competition among seedlings that emerged from a cluster of non-pelleted seeds. These findings demonstrate the potential of MSZP technology as a precise and efficient method for increasing agroecosystem biodiversity. 

SD (Gu): Research projects on seed dormancy, germination and soil seedbank longevity in rice were advanced in the past year. Three sets of experiments were continued for genetic and molecular characterizations of interactions between the gibberellin (GA) signaling genes (Slr1, Gid1 and Gid2) and natural genes for seed dormancy. In the first set of three experiments, several thousand of seeds from single plants that are heterozygous only for each of the mutant alleles slr1, gid1 and gid2 were evaluated for the imbibition times (h) to germination under controlled conditions and genotyped for the alleles. Genic (additive and dominance) effects for each of the three loci were evaluated using a quantitative genetic model. In the second set of experiments, three F2 populations, each segregating for five loci (one for each of the GA signaling genes and four seed dormancy genes), were evaluated for the degree of seed dormancy by standard germination testing and genotyped for the five loci. The germination and genotyping data were used for QTL analysis to evaluate for interactions between the GA signaling and seed dormancy genes. The third experiment was a transcriptomic analysis to identify genes regulated by Slr1/slr1 in the embryo tissue of germinating seeds. Transcript abundance was quantified by RNA-sequencing and data analysis is underway. 

Two experiments were conducted to identify the genetic basis of soil seedbank longevity in weedy rice. In the first experiment, a population of >400 recombinant inbred lines were evaluated for the survival parameters of seed samples buried in a rice field at 2 or 20 cm deep for 7 months; and the lines were genotyped with an SNP array. Mapping of QTLs for seedbank longevity was completed and modeling of the QTL G-by-E interaction is ongoing. The second experiment was conducted to evaluate all the nine genotypes at two seed dormancy QTLs for soil seedbank longevity in a total of three years. Field experiments were completed for seed samples buried in a rice field for years one and two, and the experiment for year three is ongoing.  

VA (Welbaum): A new book Vegetable Seeds Production and Technology was completed for the publisher CAB International in hardcover, softcover, and ebook formats.  The book has 17 chapters.  The first six provide general information on the importance of seeds, plant reproductive biology, genetic improvement strategies, seed quality, post-harvest enhancement treatments, and organic seed production.  The remaining 11 chapters describe seed production of major world vegetable crops by family. In collaboration with Dr. Bingyu Zhao also at Virginia Tech, we continue to optimize the ALSV-based virus induced gene silencing (VIGS) system and silence immunity related genes in watermelon and melon as a tool to study host resistance to the seed transmitted disease of cucurbits Acidovarax citrulli. We have found that the needle pin pricking method reproducibly triggers strong PDS gene silencing in different watermelon and melon cultivars. We continue to  generate watermelon and melon DMR6 mutant plants via CRISPR/Cas9-mediated gene editing approach. The mutant plants are being evaluated for improved disease resistance to Acidovarax citrulli.  Virginia Tech Intellectual Properties has applied for an international patent to treat seeds with green tea or its constituents: epigallocatechin-3 gallate, catechin, epicatechin as an organic treatment to prevent bacterial infection of seeds. 

AZ (Yadegari): Research was focused primarily on understanding how mutations that alter basal endosperm transfer layer (BETL) can alter endosperm development and consequently the overall kernel size and quality in maize (Zea mays). We have further analyzed single-mutant and double-mutant combinations in a class of transcription-factor (TF) genes that are responsible for proper differentiation and function of the BETL. One of the single mutants showed a reduction in kernel size and a delay in expression of genes typically associated with proper BETL development. A double-mutant combination of the same gene with a related TF gene showed a more severe phenotype and also a more severe delay/alteration of gene expression programs in BETL and the rest of the endosperm. Current analysis is focused on deciphering the gene networks associated with these mutants.