Plant Posters

P-1009

Metabolomics-based Profiling of Resistant Japanese Plums Infected with Black Knot Fungus. C. SHUM¹, W. El Kayal², D. Ahmad³, I. Sharkawy³, and J. Subramanian¹. ¹Department of Plant Agriculture, Ontario Agricultural College, University of Guelph, ON N1G 2W1, CANADA; ²Faculty of Agricultural and Food Sciences, American University of Beirut, Beirut, 1107, LEBANON; and ³Center for Viticulture & Small Fruit Research, College of Agriculture and Food Sciences, Florida A&M University, FL 32308. Email: shumc@uoguelph.ca

Japanese plums (Prunus salicina) are debilitated by a fungus, Apiosporina morbosa which causes Black Knot (BK) disease. It is a difficult disease to study, due to unpredictable epidemic cycles and long duration before symptoms to appear. Spores germinate on young branches, and over time, the cankers girdle the branches and eventually cause tree death. We hypothesized that there are metabolic differences among plum genotypes that confer resistance to the fungus. Thus, we determined the metabolites involved in BK resistance to provide a marker-based profiling method to accelerate breeding efforts. An untargeted metabolomic study using 2 resistant varieties (Shiro and Vampire) and 2 susceptible varieties (Redcoat and Underwood) was performed. Methanolic extracts of stems of each genotype were analysed using liquid chromatography mass spectrometry (LC-MS). The compounds identified were analysed with Principal Components Analysis (PCA), Partial Least Squares Discriminant Analysis (PLS-DA) and Variable of Importance (VIP) scores to generate associations to the resistant varieties. 471 metabolites in total were discovered, while 6 metabolites were found in greater concentrations solely in the resistant varieties which included polyphenols, benzaldehydes and aromatic compounds. Two unique compounds, Catechin and 3,4 Dihydroxybenzaldehyde were identified in resistant genotypes which are known plant defense metabolites. HPLC analysis of the samples further confirmed the higher presence of these compounds in resistant genotypes. This research paves the way for generating a metabolite marker-based selection of BK resistance in breeding plums and also elucidating mechanisms behind BK resistance.

P-2000

Pyramiding MicroRNAs for Transgene Containment and Broad Plant Abiotic Stress Resistance. Z. CHEN, X. Chen, Q. Hu, and H. Luo. Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634. Email: zhaohuc@clemson.edu

Abiotic stresses such as salinity, heat and drought seriously impair plant growth and development, causing significant losses in crop yield and ornamental value. Plants have evolved different defense mechanisms responding to the varying environmental adversities. Biotechnology approaches manipulating specific genes prove to be an effective strategy to engineer traits that are difficult to obtain with traditional breeding. In perennial grasses, however, the risk of transgene escape and the unforeseen environmental consequences by the use of transgenic technology require development of strategies for engineering male or total sterility for transgene containment. Here, we have developed a novel approach to produce self-contained superior transgenic turfgrass using the dual roles of microRNAs in plant reproductive development and beneficial agronomic trait improvement. Specifically, a microRNA gene miR396 that regulates plant sterility and abiotic stress responses was introduced into turfgrass along with three other miRNA genes miR319, miR528, and miR393, all of which are positive regulators of plant abiotic stress responses. Transgenic lines with stacked miRNA genes are being evaluated for the efficacy of miRNA396-mediated sterility induction and transgene containment, as well as the synergistical effect of all miRNA genes on multiple beneficial agronomic traits. The feasibility of controlling multiple miRNA genes by a single promoter to facilitate biotechnology application will also be evaluated. The results of this study will allow the establishment of a new genetic containment system in grasses and provide new insights into the streamlined strategy for effective miRNA gene stacking for plant genetic engineering.

P-2001

Overexpression of Flowering Locus D (FLD) in Indian Mustard (Brassica juncea) Enhances Tolerance to Alternaria brassicae and Sclerotinia sclerotiorum. ANJANA RUSTAGI¹, Shashi Shekhar¹, Ruby Panwar¹, Deepak Kumar², and Ashis Kumar Nandi³. ¹Department of Botany, Gargi College, University of Delhi, New Delhi-110049, INDIA; ²Department of Botany, Institute of Science, Banaras Hindu University, Varanasi, INDIA; and ³School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, INDIA. Email: anjana.rustagi@gargi.du.ac.in

Productivity of Indian mustard (Brassica juncea) is adversely affected by fungal phytopathogens, Alternaria brassicae and Sclerotinia sclerotiorum. Arabidopsis Flowering Locus D(FLD) positively regulates jasmonic acid signalling and defense against necrotrophic pathogens. In this study, the endogenous FLD (B. juncea FLD; BjFLD) in Indian mustard was overexpressed in B. juncea to determine its role in biotic stress tolerance. We report the isolation, characterization, and functional validation of BjFLD. The transgene expression was confirmed by qRT-PCR. The constitutive overexpression of BjFLD enhanced the tolerance of B. juncea to A. brassicae and S. sclerotiorum, which was manifested as delayed appearance of symptom, impeded disease progression, and enhanced percentage of disease protection. The transgenic lines maintained a higher redox potential under biotic stress and could detoxify reactive oxygen species (ROS) by modulating the antioxidant machinery and physiochemical attributes. The overexpression of BjFLD induced early flowering and higher seed yield in the transgenic lines. These findings indicate that overexpression of BjFLD enhances the tolerance of B. juncea to A. brassicae and S. sclerotiorum by induction of systemic acquired resistance and mitigating the damage caused by stress-induced ROS. We demonstrate the generation of an economically important oilseed crop with higher yield and broad-spectrum resistance. This could potentially reduce the yield loss caused by these pathogens and decrease the dependence on fungicides that are increasingly being wished-off for environmental, health, and economic reasons.

P-2002

Understanding the Regulation of Fruit Abscission in Physalis grisea. ELISE TOMASZEWSKI^1,2 and Joyce Van Eck^2,3. ¹Plant Breeding and Genetics Graduate Program, Cornell University, Ithaca, NY 14853; ²Boyce Thompson Institute, 533 Tower Road, Ithaca, NY 14853; and ³Plant Breeding and Genetics Section, Cornell University, Ithaca, NY 14853. Email: emt236@cornell.edu

Groundcherry (Physalis grisea) is an underutilized species in the Solanaceae family that produces small, sweet, yellow berries enveloped by an inflated calyx (husk). As a semi-domesticated species, groundcherry exhibits undesirable agronomic traits including a sprawling growth habit and extreme fruit abscission. Fruit abscission occurs via detachment of the pedicel along a region of pre-differentiated living cells called the abscission zone (AZ). Compared to other types of plant organ abscission, fruit abscission is not well understood on the molecular level. This project utilizes groundcherry as a model to understand the mechanisms of fruit abscission. It also serves to create improved groundcherry varieties with reduced fruit abscission for large-scale agronomic cultivation. To understand how the abscission zone is regulated, a multifaceted approach is being conducted. Specifically, a reverse genetics approach is being conducted utilizing CRISPR/Cas9 to edit fruit abscission-related genes. This approach will functionally validate abscission development genes for a deeper understanding of the molecular regulation and cellular differentiation behind fruit abscission. We hypothesize that editing specific AZ related genes will disrupt the formation and separation of the AZ, generating mutants with reduced fruit shedding. Lastly, the AZ will be imaged throughout development using paraffin-embedded tissue. This work will characterize and capture the first images of the groundcherry AZ development. It will also aid in illustrating differences in the AZ between abscission mutants and WT. Results from this work will not only lead to an understanding of groundcherry fruit abscission but will also expand the general knowledge of fruit AZ development and detachment.

P-2003

Transformation of American Chestnut Founder Lines with the Wheat Oxalate Oxidase Gene (OxO) for Blight Tolerance. S. REMKO^1,2, A. Tull¹, H. Gladfelter^1,3, and S. Merkle¹. ¹Warnell School of Forestry and Natural Resources, ²Institute of Plant Breeding, Genetics and Genomics, and ³Department of Horticulture, University of Georgia, Athens, GA 30602. Email: skye.remko@uga.edu

Production of American chestnut (Castanea dentata) trees expressing the wheat oxalate oxidase gene (OxO) to provide resistance to the chestnut blight fungus (Cryphonectria parasitica) has been adopted by The American Chestnut Foundation (TACF) as a promising means to restore the tree to forests of the eastern U.S. The primary path chosen by TACF for spreading the transgene to multiple genetic backgrounds for restoration is via pollinating American chestnut trees with pollen produced by Darling 58 transgenic OxO trees. An alternative approach is to directly insert the OxO gene into multiple American chestnut genotypes representing the natural genetic diversity of the tree. The resulting trees would already be adapted for growth in their native regions. We began pursuing this approach by initiating new embryogenic culture lines (“Founder Lines”) from nuts collected by TACF cooperators from large surviving American chestnut trees (LSAs) growing in different parts of the range from Maine to Georgia. In 2020, over 100 new embryogenic cultures representing eight source trees from five regions (New England, Pennsylvania, Maryland, Virginia, Georgia) were captured. Copies of all the new Founder Lines were placed in cryostorage. Then, the cultures were screened for their abilities to produce abundant somatic embryos and high-quality somatic seedlings, to facilitate choosing those to target for transformation with OxO. The selected Founder Lines showed a range of sensitivities to the selection agent geneticin in liquid medium, so selection needed to be customized for each line. Transformation experiments with these lines using the pFHI-OXO and pWIN3.12-OXO vectors have produced over 80 PCR-positive events in nine culture line backgrounds so far. Some of these PCR-positive events are already being grown up in suspension culture for somatic embryo and somatic seedling production. Future plans call for the transgenic lines to be screened for OxO transgene copy number and expression level, as well as for production of the OxO enzyme.

P-2004

Plant Cells-produced Anti-TNFα Biomolecules for Oral Treatment of Inflammatory Bowel Disease. P. PEREZ SANCHEZ¹, J. Trejo Martinez¹, and J. Xu². ¹Department of Biological Sciences, and ²Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR 72401. Email: paula.perezsan@smail.astate.edu

Plant cell culture has emerged as a safe and economical platform for bioproduction of therapeutic proteins. Unlike other bioproduction methods, plant cells can serve as both a bio-factory and a vehicle for oral delivery of recombinant biologics. Recent studies have shown that the cellulose microfibrils of plant cell walls can act as a natural capsule for oral delivery of biologics. This project aims to design and engineer novel anti-TNFα biomolecules in plant cells by leveraging two unique posttranslational modifications: glycosyl-phosphatidylinositol (GPI) anchor and plant-specific hydroxyproline (Hyp)-O-glycosylation. The goal is to develop a new class of oral biologic drugs for the treatment of inflammatory bowel disease (IBD). The designer anti-TNFα biomolecules consist of three functional domains: an N-terminal single-chain fragment variable (scFv) of an anti-TNFα antibody, a proprietary Hyp-O-glycosylation module composed of tandem repeats of the “Ser-Pro” motif, or (SP)n (n= 5 to 30), and a C-terminal GPI anchor. The GPI anchor "displays" the expressed anti-TNFα biomolecules at the plant cell surface, creating a high local concentration of biologics, while the (SP)n glycomodule stabilizes the protein from degradation during bioproduction and oral delivery. The optimal design for the biomolecules is determined to be the (SP)₂₀ module, based on investigations into the accumulation of different sizes of the (SP)_n glycomodule in tobacco BY-2 cells, biological activity, and stability in a simulated gastric fluid. The therapeutic effectiveness of the orally administrated designer anti-TNFα biologic (optimal design) in mitigating the IBD symptom is assessed in a dextran sulfate sodium (DSS)-induced colitis mouse model.

P-2005

Bladderwort Short Sequences Work as Insulators in Nicotiana Plants. EUDALD ILLA-BERENGUER¹, Jubilee Y. Park², Lynsey Kovar², Peter LaFayette^1,3, Jason G. Wallace^1,2,3, and Wayne Parrott^1,2,3. ¹Center for Applied Genetic Technologies, University of Georgia, Athens, GA; ²Institute of Plant Breeding, Genetics and Genomics, University of Georgia, Athens, GA; and ³Department of Crop and Soil Sciences, University of Georgia, Athens, GA; Email: eillaberenguer@uga.edu

Future approaches in plant synthetic biology will rely on the ability to stack multiple genes of interest into compact multigene cassettes. However, enhancers in promoters alter the expression of other genes in the cassette, leading to unpredictable outcomes in gene expression. There needs to be a way to insulate the components of multi-gene cassettes and prevent their interference with each other. Hence a better knowledge of which cis-regulatory elements (CRE) – promoters, enhancers, terminators, transcriptional blockers, and insulators – that can be utilized to restrict expression of transgenes where it is not desired is needed. Bladderwort (Utricularia gibba L.), with one of the smallest known genomes among flowering plants (~82 Mb), is a potential source of compact CREs that could be used for assembling better transgene expression cassettes. Insulators are DNA sequences that could shield expression of genes of interest from spurious transcriptional signals coming from surrounding elements. In animals, many of such sequences have been identified, but only few have been found in plants, and these are too large for routine use in transformation cassettes. To test the potential of the putative bladderwort insulator sequences we used a series of plasmids where CaMV 35S promoter drives the expression of the mCherry fluorescent reporter protein on one direction and a root-specific gene promoter driving the expression of GFP. Bladderwort candidate sequences, as well as other previously described insulators, were placed between the two reporter genes. The constructs were tested in Nicotiana benthamiana leaves by agroinfitration and were evaluated based on their ability to mitigate the ectopic GFP expression caused by the CaMV 35S promoter. As controls, we tested random effects due to fragment size, sequence orientation, or any "stochastic" component. We identified 3 short (< 1kb) bladderwort sequences that are the shortest and most effective insulators described to date, and that can be used as novel insulators in plant transformation.

P-2006

Rapid Propagation Techniques for Indoor Specialty Crop (Strawberry) Production. A. JAMESON and L. Horth. Department of Biological Sciences, Old Dominion University, Norfolk, VA, 23529. Email: ajame018@odu.edu, lhorth@odu.edu

Rational: Strawberries are a 3.4 billion dollar industry annually and the 2^nd most valuable fruit in the USA. Farmers rely on plant plugs from berry plant producers. These take many months to generate. Novel indoor agriculture growers want year-round, rapidly produced plants, and many plants simultaneously. Here, we present three studies comparing indoor propagation methods. Methods: First, 150 plants were grown on a hydroponic (soilless) table to compare berry weight and runner production for five berry varieties. Second, a germination study was conducted to compare seedling emergence in two treatments (soil versus agar petri dishes, 30 seeds/treatment). Third, a germination study was conducted to compare seedling emergence in three treatments (soil, agar and agar plus nutrients, 100 seeds/treatment). Results: In the first study, one plant variety (SAN) produced larger berries (x̄=6.78 g, CI=5.89-.7.66) than all other plant varieties (DAR x̄=4.03g, CI=3.60-4.46; WEN x̄=3.73g, CI=3.30-4.16; HON x̄=3.58g, CI=3.29-3.88; and FLA x̄= 3.52g, CI=3.01-4.04). In total, 776 runners were produced. One variety (HON) produced more runners (x̄=12.50, CI=8.79-16.21) than three of four other plant varieties (SAN x̄=5.79, CI=3.80-7.78; WEN x̄=5.30, CI=3.39-7.22; and DAR x̄=4.48, CI=1.90-6.97). In the second study, no seeds germinated in the soil petri dish treatment but 30% (n=9/30) of seeds germinated in agar (mean time to germination = 29 days). For the third study, there was no germination in soil, but 21% in both agar (n=21/100) and agar with nutrients (n=21/100). Conclusions: Hydroponic growing allows for rapid propagation of high numbers of clonal runners, with runner number varying by plant variety. Agar allows for a higher percentage of seedling germination over soil, providing rapid production of plants from seed.

P-2007

Rapid Shoot Regeneration and High-efficiency Transformation of Leaf Explants in Hybrid Poplar. GARY A. ORR¹, Marina Kalyaeva¹, Kenneth Donsky¹, Michelle Tjahjadi², Alex Crites², Karli Rasmussen², Natalie Dick¹, Jacob W. Hoyle¹, and Matthew J. Heckert². ¹Living Carbon, Charleston, SC 29403 and ²Living Carbon, Hayward, CA 94544. Email: gary@livingcarbon.com

It has been more than thirty years since the first report of a transgenic tree (Fillattii et al. 1987). In the following decades, an abundance of transgenic constructs have been transformed into forest trees, most frequently of poplar (Populus) species. In the world at present, where the pressure for climate change solutions is overwhelming, the demand for large-scale production and planting of forests with improved climate traits -such as enhanced carbon capture and sequestration- is an increasing necessity. Poplar species are the best models for this target and an efficient, rapid regeneration system is highly beneficial for mass production of enhanced seedlings. A novel protocol for de novo shoot organogenesis from the hybrid poplar (Populus tremula x Populus alba) clone 717-1B4 has been developed utilizing meta-Topolin (mT) to stimulate rapid shoot organogenesis in leaf explants. This mT-based regeneration system was able to induce direct shoot organogenesis with 100% efficiency and a shoot formation rate of more than 20 shoots per explant. Obtained shoots rooted with 100% efficiency and had no noticeable morphological changes. This leaf regeneration system was subsequently applied in tandem with Agrobacterium-mediated transformation and transgenic lines expressing the mCherry reporter were recovered under kanamycin selection in less than 3 months. Shoots were rooted on rooting medium with geneticin and young leaf explants of kanamycin-resistant seedlings were also able to regenerate back on mT regeneration medium under geneticin selection. Transgenic nature of obtained lines was further confirmed by qPCR and ELISA testing for the nptII gene. This approach marks the first known application of meta-Topolin in poplar regeneration and transformation.

P-2008

Exploring the Cross-species Applicability of a Tissue Culture Medium Developed for Nuttall's Scrub Oak (Quercus dumosa) for the Ex Situ Conservation of Coastal Chaparral Ecosystem Species. J. F. REE. San Diego Wildlife Alliance, San Diego, CA 92101. Email: jree@sdzwa.org

The coastal sage scrub oak (Quercus dumosa) is an endangered keystone species of the coastal chaparral ecosystem ranging from Southern California, USA, into Baja California, Mexico. Because their acorns cannot be banked conventionally, Q. dumosa must be conserved through other methods, such as tissue culture. Initially, few culture lines grew reliably, with many lines often showing extensive necrosis and considerable phenolic exudation. We tested each component of a 'standard' medium to create a medium that would allow us to cultivate a wide variety of Q. dumosa genotypes. These include novel basal salt formulations, carbohydrate sources, plant growth regulators, exogenous amino acids, antioxidants, and other additives. After extensive experiments, our medium is suitable for over a hundred Q. dumosa culture lines. We then explored if this medium would provide suitable growing conditions for other coastal chaparral species, such as other oaks (Q. tomentella, Q. pacifica, and suspected hybrids) and members of at least 16 different genera of both woody and herbaceous species. Germination of acorns from tested Quercus species ranged between 73-100%. Three manzanita (Arctostaphylos) species and mission manzanita (Xyclococcus bicolor) showed comparatively lower germination rates (11-43%). Shoot tips from field plants showed a range of responses: several genera (Arcostaphylos, Artemisia, Asclepias, Eriodictyon, Eriogonum, others) showed rapid growth and stable establishment of growing cultures, and other genera (Cercocarpus, Ceanothus, Peritoma, Rosa) showed rapid callus development but unstable growth. Though some species might require optimization, this medium provides a robust starting point for conserving other coastal chaparral ecosystem flora.

P-2009

Exploring the Relationship Between Rootstock and Frost Tolerance in Apple Trees Through Soluble Sugars and Reactive Oxygen Species. A. SAINI, J. Liu, Md.-T. Islam, and S.-M. Sherif. Alson H. Smith Extension Research Center, School of Plant and Environmental Sciences, College of Agriculture, Virginia Tech, Winchester, VA 22601. Email: amolpreetkaur@vt.edu

This study reports on the consequences of climate change on apple production, particularly late spring frosts, and the need for growers to adapt to new climatic conditions by selecting rootstocks with better adaptation and tolerance. In the present study, we examined the physiological and molecular mechanisms underlying frost tolerance in apple trees using scion-rootstock combinations, in which 'Fuji' and 'Gala' were used as scions and 'B.9', 'M.26', and 'G.935' were used as rootstocks. Floral buds, leaves were collected from scions and leaves were collected suckers before and after natural frost events, for two consecutive years (2021 and 2022). The accumulation patterns of various forms of carbohydrates (e.g., sucrose, glucose, fructose, sorbitol), and reactive oxygen species (ROS) (e.g., H₂O₂ and O^2-) were studied to understand the role of rootstocks in frost tolerance in apple. Scions on the B.9 rootstocks showed the least flower mortality rates after frost among all rootstocks. Our data showed that scion’s leaves had more levels of glucose and O^2- when ‘B9’ was used. Moreover, leaves from B.9’s suckers and flowers had significantly higher levels of H₂O₂. The variations observed in this study confirm that the differences in cold tolerance among apple rootstocks can be explained, at least partially, by the differential accumulation of soluble sugars and ROS during osmotic stress events. Our future investigations, being done at the transcriptome and metabolome level, would provide better understanding and information regarding the molecular networks linked with spring frost hardiness by uncovering the differentially expressed genes and metabolites in apple.

P-2010

Response of Contrasting Potato Cultivars to Single and Combined Abiotic Stresses Harboring miR156d-3p. ARSLAN ASIM¹, Ufuk Demirel¹, Allah Bakhsh², and Zahide Neslihan Ozturk Gökçe¹. ¹Department of Agricultural Genetic Engineering, Faculty of Agricultural Sciences and Technologies, Niğde Ömer Halisdemir University, Niğde, TÜRKIYE and ²Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, PAKISTAN. Email:arslanasim92@gmail.com

Abiotic stresses are the major constraint to limit crop productivity. Additionally, plants suffer from combined stresses under field conditions. Potato (Solanum tuberosum L.) is sensitive to abiotic stress conditions that result in retarded development, growth, and yield. miRNA’s play key role against abiotic stress, and gene expression regulation studies about stress response of potato are very limited. This study was aimed to use transgenic approach for unveiling the role of miR156d-3p in potato cultivars (sensitive Russet Burbank and tolerant Unica) in response to drought, heat and combined heat and drought stresses. Transgenic plants of both cultivars were generated harboring overexpression of pre-miRNA of miR156d-3p and both transgenic T0 and non-transgenic lines were affected by stress. Comparative studies at molecular levels along with physiological characteristics (gaseous exchange, leaf temperature, chlorophyll content and relative water content) and biochemical traits (proline, malondialdehyde, hydrogen peroxide, antioxidant enzymes) were quantified to compare the performance of wild-type and transgenic plants. The overexpression of miR156d-3p improved physio-biochemical functions in both cultivars under all stress conditions. The escalation in miR156d-3p expression was observed along with a decline in expression of its predicted target Phospholipid-transporting ATPase, that is involved in regulation of antioxidant enzymes in potato. This study showed the development of abiotic stress resilient potato cultivars proving the overexpression of miR156d-3p increased abiotic stress tolerance presumably by repressing the expression of Phospholipid-transporting ATPase. Results of this study can be used in developing tolerant potato cultivars to cope with future climatic changes.

P-2011

Agrobacterium-mediated Transformation and Plant Regeneration of Alfalfa (Medicago sativa). SUMA BASAK¹, Terri Breanley¹, and Seema Dhir². ¹Center for Biotechnology, Department of Plant Sciences, College of Agriculture, Family Sciences and Technology, Fort Valley State University, Fort Valley, GA 31030 and ²Biology Department, Fort Valley State University, Fort Valley, GA 31030. Email: Suma.Basak@fvsu.edu

Alfalfa is cultivated as a fodder crop around the world. The aim of this study was to develop an efficient Agrobacterium-mediated gene transfer protocol for alfalfa (Medicago sativa) that could be used for the large-scale improvement of this plant. We optimized different parameters such as bacterial density (OD_600nm), acetosyringone concentrations, preculture period, infection time, co-cultivation period, and various additives - calcium chloride, silver nitrate, cefotaxime, and hygromycin. Three explants - leaf, stem, and petiole from 3 weeks old in vitro grown alfalfa plants were inoculated with A. tumefaciens GV3101+pCAMBIA1304(1) harboring the β-glucuronidase (GUS), green fluorescent protein (GFP) and hygromycin phosphotransferase II fusion selectable marker gene. Results were tabulated based on the percentage of transient expression of GUS and GFP after 3 days of co-cultivation. Optimal parameters using leaf explants included optimal density (OD_600nm) of 0.4, 2 days of preculture, 25min infection time, 3 days co-cultivation period, 150µM acetosyringone, 75µM silver nitrate, 2mM calcium chloride, 350µM cefotaxime, and 20µM hygromycin. The highest GUS expression on average blue spots with a standard error of 167±6 was observed in leaf explants which were 35±5 and 26±8 spots higher than the stem and petiole, respectively. Subsequently, the explants were transferred onto callus induction and regeneration medium, Murashige and Skoog (MS) basal medium supplemented with 3% sucrose, 350µM cefotaxime, and 20µM hygromycin. Of the three explants, leaf explants had a greater callus induction capability (95%) and regeneration response (85.2%) compared to 40% and 12.5% for the stem, and 21% and 7% for petiole, respectively after 28 days of inoculation. The study results will be helpful in our long-term goal to establish a robust transformation protocol for alfalfa genetic engineering.

P-2012

Developing In Vitro Culture Tools for Mandarin and Sweet Cherry In Vivo Editing. I. FUENTES, J. Rubio, M. J. Montañola, V. Borjas, C. Nuñez, C. Valenzuela, Y. Muñoz, M. Acuña, G. Aguilar, M. Cona, and B. Pollak. Meristem SpA, Fundación Ciencia y Vida, Del Valle 725, Huechuraba, Santiago de CHILE. Email: ifuentes@meristem.bio

Meristem is a start-up company committed to use gene editing to generate new fruit crops for a more sustainable future. Located in Santiago-Chile, our facilities include infrastructure and equipment to implement in vitro culture, molecular biology, protein engineering and bioinformatics. This broad scientific background allows us to develop protocols for fruit trees propagation and transformation, genotyping and molecular diagnostics (phytosanitation). Furthermore, to complete our R&D strategy, we have an experimental station fully equipped with phytotrons and a greenhouse for experimental validation. A major bottleneck for the application of gene editing technologies is the regeneration of plant tissues, which is a particularly challenging process in woody fruit crops. We have been working creatively to implement in vitro culture tools for Mandarin and Sweet Cherry gene editing. For these species, we have already achieved morphogenesis including regular micropropagation, callogenesis, culture tissue from different plant organs (ovules, leaves, roots) and direct embryogenesis. Transient and stable transformation protocols for protoplasts, callus and other plant structures are currently being developed. Additionally, we have managed to complete the full growth cycle for the mandarin W. murcott, obtaining plants from transient (plasmid/protoplast) and stable (Agrobacterium/callus) transformations. In sweet cherry (rootstocks and varieties), we have been able to efficiently obtain transient and stably transformed plant material. These advances in regeneration and transformation protocols, in combination with our customized editing tools, will enable the development of gene edited fruit trees during 2023. We are scaling up our product development platform to bring novel fruits with commercial traits during the next few years.

P-2013

Developmental Sequence of Somatic Embryogenesis Initiated by Differential Expression of Baby Boom and Wuschel2 in Sorghum bicolor L. A. P. KAUSCH and M. Tilelli. Plant Biotechnology Laboratory, Department of Cell and Molecular Biology, University of Rhode Island, West Kingston, RI 02892. Email: apkausch@uri.edu

Genomics, genome editing, and plant transformation technologies comprise a required triad platform for advanced breeding of crop plants. Somatic embryogenesis (SE) is critical and central to most cereal transformation and genome editing protocols, providing the ability to regenerate a fertile plant initiated from a single vegetative cell. Differential expression of morphogenic genes such as Baby boom (Bbm) and Wuschel2 (Wus2) enhance and confer the ability for a vegetative somatic cell to become developmentally reprogramed as an SE initial. Agrobacterium-mediated ‘Altruistic’ transformation was conducted on zygotic immature embryos (IE) using Bbm/Wus:GFP and BAR:DSRED (1:9) and a time course analysis was initiated on co-infection through selection. Three arrays of approximately 150 IEs each were followed using fluorescence microscopy to follow events from initiation to selected colony and on to regenerated to plants. Scanning electron microscopy (SEM) was conducted on cryofractured infected IEs in a separate duplicate series and shows earliest initiation events in the scutellar epithelium followed by a rapid proliferation of early-stage somatic proembryos. A single dominate SE is often observed early followed by formation of many subtending secondary proembryos which often become fused. By day 7 post-infection many asynchronous stages of somatic embryogenesis populate the abaxial side of the IE scutellum. The developmental sequence of somatic embryogenesis from recipient cell initial to late stage embryo development demonstrates scutellar epidermal cell totipotency and confirms previous reports on rapid transformation in Sorghum mediated by Baby boom (Bbm) and Wuschel2 (Wus2).

P-2014

Transient and Stable Transformation of the Model Hornwort Anthoceros agrestis via Biolistics. D. LAFFERTY¹, A. Gunadi², J. Van Eck^1,3, and F-W. Li^1,4. ¹Boyce Thompson Institute, Ithaca, NY; ²J.R. Simplot Company, Boise, ID; ³Plant Breeding and Genetics Section, Cornell University, Ithaca, NY; and ⁴Plant Biology Section, Cornell University, Ithaca, NY. Email: djl34@cornell.edu

Approximately 500 million years ago, land plants diverged into two lineages, vascular plants and bryophytes. The bryophyte clade encompasses mosses, liverworts, and hornworts and although research on mosses and liverworts is rapidly growing, hornworts are under-studied. Hornworts possess unique traits among land plants, such as their carbon-concentrating mechanisms and the ability to form symbiotic relationships with cyanobacteria. Understanding the mechanisms underlying these traits has the potential to translate into approaches for crop improvement by facilitating increased photosynthetic efficiency and providing an additional source of nitrogen. The development of tools such as genetic engineering and gene editing for hornworts research is vital for identifying and characterizing genes that control these traits. The hornwort species, Anthoceros agrestis, is an emerging model species. The aim of this study is to develop a biolistics transformation method for A. agrestis. To develop this method, we used a construct containing the green fluorescent protein (GFP) driven by the CaMV-35S promoter and the hygromycin phosphotransferase gene driven by the native EF1a promoter. For the target material, we used hornwort thallus tissue grown on 2% sucrose AG medium for 7 days. Transient GFP expression was observed within 2 days of transformation. We observed approximately 500 GFP-expressing cells, quantified using CellProfiler. Following bombardment, tissue was cultured on recovery medium without hygromycin. After recovery, the thallus tissue was homogenized and transferred back to AG medium for 2 days before being transferred to AG medium containing 10 mg/L hygromycin. GFP-expressing tissue was observed after 8-10 weeks with 5-15 stable transgenic lines per replicate. Biolistic transformation of A. agrestis provides a valuable tool for functional studies in hornworts.

P-2015

A Novel Templated Editing System for Precise Sequence Replacement via Reverse Transcriptase and Diverse CRISPR-Cas Systems. Q. SHI¹, Y. B. Kim¹, E. B. Pierce¹, M. Brown¹, B. A. Peterson¹, D. Sanford¹, J. Fear¹, D. Nicholl¹, E. S. Pedro¹, G. M. Reynolds², J. E. Hunt¹, D. G. Schwark¹, S. Jali¹, N. Graham¹, Z. Cesarz¹, T. A. Lincoln Chapman¹, J. M. Watts¹, and A. W. Hummel^1,2. ¹Pairwise, Durham, NC and ²Current affiliation: Inceptor Bio, Morrisville, NC. Email: qshi@pairwise.com, ahummel@pairwise.com (corresponding)

Site-specific sequence replacement is a powerful tool for genome engineering. The recently described prime editing tools, which can enable such changes, are based on Cas9, a type II CRISPR-Cas system recognizing G-rich Protospacer Adjacent Motifs (PAMs), coupled with a reverse transcriptase (RT). Here, we describe a novel mechanistic framework utilizing a type V enzyme to achieve RT guided precise sequence replacement, named RNA encoded DNA replacement of alleles with CRISPR (REDRAW). We employed nuclease active Cas12a-RT fusion with a repurposed CRISPR RNA (crRNA) allowing RT-mediated elongation of template-strand DNA following RNA-DNA hybridization. Using a human cell system, REDRAW was shown to tolerate both N- and C-terminal fusions of RT in addition to non-fusion architectures, and the optimal lengths of RT template and DNA template hybridization region were identified. Additional rounds of system iteration allowed REDRAW to have higher crRNA stability, improved RT-mediated polymerization, and permanent edit integration to the genome. All simple base conversions, including all point mutations, precise insertions, and deletions of various lengths, can be achieved by REDRAW. In addition, REDRAW is compatible with EnAsCas12a and RR-Cas12a, the variants with expanded PAM recognition, allowing targeting of sites in the genome inaccessible to prime editing. REDRAW is also compatible with Cas12b and Cas9. Taken together, REDRAW offers broad compatibility with multiple Cas12a enzymes and a novel approach to harness the power of reverse transcriptase towards achieving sequence-specific DNA replacement. Pairwise aims to leverage REDRAW to introduce consumer-oriented plant traits to create healthy food that is more convenient, affordable, and sustainable.

P-2016

Successful Indirect Regeneration of Octoploid Strawberry Varieties through In Vitro Leaf Tissue Culture. CHEOL-MIN YOO and Seonghee Lee. Gulf Coast Research and Education Center, University of Florida/IFAS, 14625 CR 672, Wimauma, FL 33598. Email. cyoo@ufl.edu

Most commercially grown strawberries are of the octoploid type, known for their high level of heterozygosity and genetic diversity between different genotypes and cultivars. The success of in vitro plant transformation and regeneration is largely contingent upon the presence of optimal tissue culture conditions. In this study, we report a successful indirect regeneration of two Florida strawberry varieties, ‘Florida Brilliance’ and ‘Florida Medallion’, using leaf tissue as an explant from in vitro grown plantlets. The explants were cultured on Murashige and Skoog (MS) medium supplemented with different combinations of plant growth regulators. The highest frequency of shoot regeneration was observed on MS medium containing 2.0 mg/L thidiazuron (TDZ) and 0.04 mg/L indole-3-butyric acid (IBA) for ‘Florida Brilliance’, and on the same medium but 0.02 mg/L IBA for ‘Florida Medallion’. It is important to transfer the regenerated shoots to a different MS medium containing 1 mg/L 6-benzylaminopurine (BA) and 0.01 mg/L IBA for the shoot elongation and the development. The root formation was induced by transferring the shoot clumps to MS medium without growth regulators. The regenerated plants were successfully acclimatized and transferred to greenhouse conditions for further growth and development. Further, the 240 regenerated plants from each variety were tested for somaclonal variation by imposing Neopestalotiopsis sp. inoculum in a detached leaf assay. The varying degrees of symptoms in each popluation appeared and the result suggest that the regenerated plants have phenotypic variability that can be exploited to improve the agronomic trait of strawberries. 19 somaclones from 'Medallion' and 10 somaclones from 'Brilliance' were selected for the Neopestalotiopsis tolerance candidates, and they will be field tested in the future. This whole plant regeneration protocol can also be utilized for other biotechnological approaches including CRISPR-based gene-editing to obtain enhanced new varieties.

P-2017

Epigenetic Signatures Defining Gene Editing Efficiency in Bread Wheat (Triticum aestivum L.) ANDRIY BILICHAK, Natalia Bykova, Louie Lopos, Kerry Ward, and Susan Brown. Agriculture and Agri-Food Canada, Morden Research and Development Center, 101 Rte 100 #100, Morden, MB R6M 1Y5, CANADA. Email: andrii.bilichak@agr.gc.ca

Rationale: Improvement of staple crops relies on the natural and inducible genetic diversity of the available germplasm. Frequently, mutations of the selected genes identified through omics approaches (e.g., transcriptomics, proteomics, translational genomics) are unavailable for chosen cultivars. Such edits could be introduced through CRISPR/Cas9. Bread wheat (Triticum aestivum L.) is an allohexaploid crop with six copies of the target gene per diploid genome. The large complex genome and presence of the homeoalleles make it challenging to obtain null mutants in the T₀ generation. Objectives and Methods: Previously, we demonstrated that editing efficiency in cultivar Fielder can be improved selectively through the transgenic lines’ heat shock treatment. This effect could be due to increased Cas9 cleavage activity under elevated temperatures and a change in the expression of the genes involved in DNA repair and chromatin modulation. To further explore the contribution of the epigenetic landscape to the editing efficiency at the target loci, we used Agrobacterium-mediated transformation and generated over 130 T₀ transgenic plants with 17 different gRNAs targeting 51 loci per haploid genome. Results: In this study, we leveraged the wealth of high-resolution epigenomic resources for wheat (DNA methylation, histone marks, ATAC-seq, MNase and RNAPII profiles) to address the impact of chromatin features on the CRISPR/Cas9 mutagenesis using stable transgenic plants. We also examined the effect of the copy number variation of the CRISPR-coding transgene in T₀ plants on gene editing efficiency. We show that Agrobacterium-mediated wheat transformation combined with the delivery of the GRF-GIF chimeric protein results in multiple copy transgene integration events, and editing efficiency correlates with the expression of the CRISPR transgene cassette in wheat cultivar Fielder. Conclusions: Overall, further research is needed to evaluate the effect of chromatin marks on gene editing efficiency in wheat.

P-2018

Establishment of Genetic Transformation in Commercial Cultivars of Theobroma cacao and Stability of Transgene Inheritance in Progeny Plants. M.-J. CHO¹, J. Jones¹, G. Austin¹, E. Zhang¹, D. Tucker¹, D. Rietz¹, M. Gomez¹, D. Dahlbeck¹, C. Garcia², J.-P. Marelli², Donald Livingstone², Ray Schnell², and Brian Staskawicz¹. ¹Innovative Genomics Institute, University of California, Berkeley, CA 94704 and ²Mars, Inc. Email: mjcho1223@berkeley.edu

Genetic engineering tools have the potential to both rapidly and precisely improve the genome of slow-to-breed cacao, which is currently under threat from disease and challenging economic circumstances. A highly efficient transformation protocol is a prerequisite to developing genetically-modified and genome-edited crops. Agrobacterium-mediated transformation of two elite cultivars of cacao has been established using a two-fold strategy of improving transfer-DNA delivery and secondary somatic embryogenesis. Stable transformation with a frequency of 3.7% in INIAPG-038 and 2.5% in Matina 1-6 was achieved using three vectors, each one containing enhanced yellow fluorescent protein (eyfp) and neomycin phosphotransferase II (nptII) genes. Matina 1-6 is the most commonly cultivated variety in West African countries where cacao swollen shoot virus is prevalent and causing major losses to trees and pod yields. INIAPG-038 is a high-yielding, disease-resistant cultivar and breeding line developed by Mars Wrigley, Incorporated, in collaboration with United States Department of Agriculture and Instituto Nacional de Investigaciones Agropecuarias. Analysis of T₀ plants of these two cultivars show that they are phenotypically normal, fertile as both pollen and ovule donor and produce viable seeds with a 98.0% germination rate, indicating our transformation protocol is successful. Further analysis of T₁ progeny demonstrates stable visual eyfp inheritance at the expected Mendelian segregation rates. The presented protocol is being used to generate transgenic and genome-edited plants for cacao improvement. In addition, we are further improving our current transformation protocol using a morphogenic gene approach and testing our tissue culture system to establish a DNA-free genome editing protocol.

P-2019

Optimization of Sorghum Leaf Whorl Transformation and Regeneration. M. KELLY¹ and W. Vermerris^2,3,4. ¹Graduate Program in Plant Molecular and Cellular Biology, ²Department of Microbiology and Cell Science, ³UF Genetics Institute, and ⁴Florida Center for Renewable Chemicals and Fuels, University of Florida, Gainesville, FL 32611. Email: megan.kelly@ufl.edu

Sorghum is a versatile cereal crop grown worldwide and used for food, animal feed and fodder, and renewable chemicals and fuels. Hence, sorghum is a promising candidate for engineering to enhance desirable traits, such as the production of nutraceuticals in the edible seed or greater support of nitrogen-fixing microbes in the aerial roots. The ability to introduce targeted changes using genome editing or novel synthetic pathways using transgenes rests on the ability to produce transformed plants, a process that is often slow and inefficient in sorghum. We recently developed a biolistic transformation protocol using leaf whorl explants, which makes the process faster compared to the use of immature embryos, but transformation and regeneration rates still need to improve further to accommodate the throughput necessary for genome editing. Tissue culture conditions, including the timing of medium changes and the choice and concentrations of phytohormones, were evaluated for improved regeneration rates of leaf whorl explants. The effect of microparticle size and speed (a function of the helium pressure) on biolistic transformation efficiency in leaf whorl and immature embryo explants was evaluated using a GFP reporter gene. Culturing the calli on a hormone-free medium in between callus induction and shoot regeneration produced shoots faster than the original leaf whorl protocol. The combination of 0.8-µm particles and helium pressure of 1350psi produced the most transient fluorescent foci 7-12 days after bombardment in both explant types. Further improvements being evaluated include the use of morphogenic regulators to enhance regeneration.

P-2020

Overexpression of ZmWOX2A Enhances Somatic Embryogenesis and Transformation Efficiency in Maize (Zea mays L.) and Sorghum (Sorghum bicolor L.). JONATHAN MATHEKA^1,2, Frank McFarland³, Nathaniel Schleif¹, Ray Collier², Nathalie Walter², William Petersen², David Animasaun^2,4, Brian Martinell², and Heidi Kaeppler^1,2. ¹Department of Agronomy, University of Wisconsin, Madison, WI; ²Wisconsin Crop Innovation Center, University of Wisconsin-Madison, WI; ³Corteva Agrisciences, Ames, IA; and ⁴Department of Plant Biology, University of Ilorin, Ilorin, NIGERIA. Email: matheka@wisc.edu

Somatic embryogenesis is often a genotype-dependent process in plant tissue culture and transformation, limiting functional genomics research and genetic improvement applications. Tailored expression/excision of genes involved in embryo development (eg. Bbm, Wus2) has recently enabled efficient, genotype-flexible genome engineering of maize and other cereal crops. Through genetic fine-mapping research, we identified a novel embryogenesis-related gene in maize, ZmWOX2A, that, when constitutively expressed, enabled somatic embryogenesis and plant regeneration in recalcitrant maize genotypes. Despite constitutive expression of the developmental gene, T₀ plants and T₁ progeny transformed with the ZmWOX2A overexpression vector were phenotypically normal and fertile. To further optimize the ZmWOX2A-based transformation system, we tested the effects of different promoters and terminator/polyadenylation sequences in the ZmWOX2A expression cassette on transformation frequency and T₀ plant health. Among the sequences tested (RUBQ2, ZmUbi1, NbHSP, NbACT3, EU, EU+NbACT3) the EU terminator gave the highest mean number of somatic embryos (SE) produced per zygotic embryo explant (ZE) at 17.6. We then tested the effects of constitutive (ZmUbi1) versus embryo-specific (PLTP, Aa2m, Dzs18, Expb14, Php20719a) promoter-driven expression of ZmWOX2A on mean SE/ZE. The ZmUbi1 promoter had the highest mean SE/ZE (17.8) followed by PLTP promoter (10.9). Based on these results, we built a “boosted” ZmWOX2A vector with ZmUbi1p and EUt and containing optimized selection/reporter gene cassettes. The boosted ZmWOX2A vector produced significantly higher mean SE/ZE (22.1) than the control vector (3.7) and slightly higher than a comparable BBM+WUS2 vector (20.9). Higher transformation efficiencies were also obtained with the ZmWOX2A-based system in sorghum. Phenotypic and molecular assays of T0 maize and sorghum plants from the promoter, terminator and boosted ZmWOX2A experiments are underway to determine effects on plant health and transformation/editing efficiency.

P-2021

Investigating the Molecular Mechanism Behind In Vitro Fruiting in Solanum lycopersicum cv. Micro-Tom Mutant. C. MAUSS¹, M. Harland-Dunaway¹, A. Narvaez^2,3, M. Orozco-Cardenas^1,2, and R. Jinkerson³. ¹Department of Botany and Plant Sciences, College of Natural and Agricultural Sciences, ²Plant Transformation Research Center, and ³Department of Chemical and Environmental Engineering, Bourns College of Engineering, University of California, Riverside, Riverside, CA 92521. Email: claire.mauss@email.ucr.edu

In vitro regeneration time is a major current bottleneck for plant transformation research as it is time and resource intensive. Fast-cycling plant varieties may allow for accelerated transformation and genetic engineering. We recently engineered fast-cycling cherry tomato plants (Solanum lycopersicum cv. Micro-Tom) that we refer to as Small Plants for Agriculture in Controlled Environments (SPACE) plants. SPACE Micro-Tom (MT) plants were created using CRISPR/Cas9 to knock out a gene encoding an enzyme involved in plant oxidative stress responses that I will refer to as SPACEa in this presentation. When grown both in vitro and in soil conditions, spacea MT plants displayed accelerated development, both flowering and fruiting faster. In soil, this accelerated flowering and fruiting occurred 14 days earlier than wild-type (WT) plants, on average. Members of this enzyme family can catalyze reactions involving nicotinamide adenine dinucleotide (NAD+) upon oxidative stress. Therefore, we hypothesize that the accelerated developmental phenotype we observe in the spacea MT plants is a result of enhanced overall plant energy efficiency due to increased NAD+ pools. To further investigate the spacea phenotype, we utilized RNA-sequencing data to analyze differentially expressed genes between 40-day-old leaf tissues collected from spacea and WT MT plants grown in soil. We found that the most highly enriched DEGs in the leaf tissue are related to pigment binding and photosynthesis. This suggests that spacea plants may display their accelerated developmental phenotype as a result of enhanced photosynthesis, potentially due to increased NAD+ pools. Future work will compare NAD+ levels between spacea and WT MT plants. The results of this study can help to elucidate the mechanism behind engineering accelerated in vitro development in other agroeconomically relevant crop species that may help to advance the field of plant transformation and genetic engineering.

P-2022

Tissue-culture-free Genetic Transformation and Gene-editing in Plants. ARJUN OJHA KSHETRY, Vikas Devkar, Luis Herrera Estrella, and Gunvant B. Patil. Institute of Genomics for Crop Abiotic Stress Tolerance (IGCAST), Department of Plant & Soil Science, Texas Tech University, Lubbock, TX, 79409. Email: arjun.ojha@ttu.edu, gunvant.patil@ttu.edu

Gene editing technologies, especially CRISPR/Cas have revolutionized basic and applied biological research within a decade of its development. It has been profoundly used in crop engineering research thereby transforming the pace of plant breeding and trait discovery. However, the delivery of gene editing reagents and regeneration of the edited progeny are the biggest hurdles in efficient genetic engineering and crop improvement. Current delivery methods and regeneration are largely dependent on tedious, lengthy, and costly in vitro (tissue culture) processes. Moreover, plant regeneration and genetic transformation are highly genotype-dependent and therefore, only very few of all plant species can be genetically modified. Therefore, the development of efficient genotype-independent plant transformation and gene-editing methods in several recalcitrant crops including cotton, soybean, sorghum, common bean, etc. is important in applying this technology for crop improvement. To overcome these challenges, we have created a synthetic cascade to express genes called developmental regulators that are involved in stem cell activity, rapid tissue differentiation, and the regeneration process. This system is successfully tested in vivo, without a need for tissue culture in the model plant Nicotiana benthamiana (tobacco), and applied in tomato with successful results. Our data suggested the successful development of robust technology for gene-editing and regeneration of gene-edited plants as we envisioned.

P-2023

Over-expression of Morphogenic Genes Enhances Plant Regeneration in Cassava (Manihot esculenta). ROSANA SEGATTO^1,2, Gecele M. Paggi², and Nigel J. Taylor¹.¹Donald Danforth Plant Science Center, Saint Louis, MO 63132 and ²Graduate Program in Biotechnology, Federal University of Mato Grosso do Sul, Campo Grande, Mato Grosso do Sul, BRAZIL 79070-900. Email: rsegatto@danforthcenter.org

The Growth Regulating Factor (GRF) and its Interacting Factor (GIF) have been shown to stimulate plant regeneration, and increase efficiency of genetic transformation in multiple species including wheat, beet and citrus. The present work evaluated the effects of overexpression of GRF4-GIF1 and GRF5 on regeneration of transgenic cassava. GRF4-GIF1 and GRF5 derived from Vitis vinifera and Arabidopsis thaliana were cloned into expression cassettes under control of strong constitutive promoters and used to generate plants via Agrobacterium-mediated transformation of friable embryogenic callus in the varieties 60444 and NASE 13. No effect of GRF4-GIF1 and GRF5 was observed on efficiency of transformation or plant regeneration compared to the GFP control through this process. Transgenic plants confirmed to be expressing GFR4 and GRF5 by qRT-PCR were assessed for their morphogenic potential by culturing stem, petiole and leaf-petiole explants on medium supplemented with the cytokinin meta-topolin. Leaf-petioles were the most responsive explant. No shoot regeneration was observed from the GFP controls, or from plants transgenic for AtGRF4-GIF1. In contrast, shoot regeneration took place from greater than 35% and 50% of leaf-petiole explants obtained from 60444 and NASE 13 mother plants transgenic for VviGRF4-GIF1, respectively. Multiple shoots could be regenerated from each explant, with caulogenesis occurring as early as 3-4 weeks after explanting. Shoots were also regenerated from explants expressing AtGRF5, but at lower frequencies. In addition, expression of VviGRF4-GIF1 was seen to accelerate development and maturation of somatic embryos induced from plants expressing GRFs. When established in the greenhouse, VviGRF4-GIF1 expressing plants were shorter in height with an increased total leaf area, while AtGRF5 expressing plants displayed above average results for plant height. The results presented show the potential for GRFs such as GRF4-GIF1 and GRF5 to enhance recovery of modified plants in cassava.

P-2024

Impact of Titanium Dioxide Nanoparticles on Growth, Pigment Accumulation and ATP Synthase Function in a Cyanobacterial Model. MST SAYADUJJHARA, Viji Sitther, Samson Gichuki, D'Avione Jordan, and Yavuz Yalcin. Morgan State University, 1700 E. Cold Spring Lane, Baltimore, MD 21251. Email: mssay1@morgan.edu, viji.sitther@morgan.edu

As photosynthetic prokaryotes, pigments such as chlorophyll, carotenoids and phycobiliproteins in cyanobacteria are exploited for their high commercial value in cosmetics and dyes. In the present study, we evaluated the impact of 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 mg/L nano-titanium dioxide (n-TiO₂) nanoparticles on the growth, chlorophyll a fluorescence, and phycocyanin accumulation in Fremyella diplosiphon strains, B481-WT and B481-SD. In addition, oxidative stress in response to n-TiO₂ was quantified using the 2′,7′dichlorodihydrofluorescein diacetate (DCFH-DA) probe and ATP synthase activity detected using densitometric analysis. We observed significantly higher growth of B481-SD and B481-SD at 2 mg/L n-TiO₂ on day 12. In addition, we observed higher phycocyanin and chlorophyll content at 2 mg/L on day 9 in B481-SD strain while there was no significant enhancement of growth in any of the concentrations and intervals tested in B481-WT. Comparison of oxidative stress using the DCFH-DA probe indicated significantly different ROS in both strains treated with 2 and 16 mg/L n-TiO₂. Densitometric analysis of ATP synthase in B481-SD revealed significantly higher activity at 0.5, 2, and 128 mg/L n-TiO₂ than the untreated control; however significantly higher levels of activity was observed only at 2, and 128 mg/L in B481-WT. Results of the present study indicated 2 mg/L n-TiO₂ to be optimal in enhancing growth, pigment accumulation and ATP synthase activity. Future efforts will be aimed at studying transduction mechanisms that cause metabolic signaling as well as photosynthesis in response to n-TiO₂ in F. diplosiphon. This research was supported by the National Science Foundation’s Nanoscale Interactions Program grant under award number 1900966 and co-supported by the Excellence in Research.

P-2025

Unravelling the Diversity of Nectar Microbiome in Prunus. VIDYA VENUGOPAL, Raizada Manish, Chloe Shum, and Jayasankar Subramanian. Department of Plant Agriculture, University of Guelph, Guelph, CANADA. Email: venugopv@uoguelph.ca

Numerous microbes that play crucial roles in plant growth and health have evolved with plants. At the community level, the plant microbiomes may increase the effectiveness, dependability, and consistency of the growth and health promotion under a wider variety of environmental variables than using a single beneficial species. Therefore, the objective of this study was to extensively characterize the nectar microbiome of six major Prunus species with two varieties in each species (Peach, Japanese plum, European plum, Sweet cherry, Sour cherry and Apricot). The bacterial communities associated with these Prunus spp. were analysed using high-throughput Illumina Miseq sequencing of bacterial 16s rRNA. This is the first report on bacterial communities associated with nectar of Prunus spp. Our results indicated that, different Prunus sps, shared unique bacterial assemblage. Interestingly, Actinobacteria was the most abundant phylum, followed by Proteobacteria and Firmicutes. These results could facilitate to understand the interaction of endophytic bacteriota with the Prunus genome and could support to identify the endophytes among the stone fruit crops and their relationship with disease resistance.

P-2026

Optimization of Nutrient Media, Growth Regulators, Sugar Source and Solidifier for the Rapid and Cost-effective In Vitro Micropropagation of Important Aquatic Plants Hemianthus callitrichoides and Riccia fluitans. E. ÖZCAN and H. H. Atar. Department of Fisheries and Aquaculture, Faculty of Agriculture, Ankara University, Ankara, TÜRKIYE. Email: ozcanesra@ankara.edu.tr

Aquatic plants Hemianthus callitrichoides and Riccia fluitans have an important place in phytoremediation process as well as being used as aquarium plant, food and feed. In this study, 12 different basal nutrient media containing different sugar sources, agar concentrations and various combinations of 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) were tested for the most economical and rapid in vitro micropropagation of these species. These applications had a different effect on each plant species and high rates of in vitro micropropagation could be achieved in a short period of 4 weeks. When shoot formation and rooting rate are considered together, it was observed that nutrient media MS No: 3B for H. callitrichoides and SH+MSvit for R. fluitans provided the highest rate of micropropagation. In different sucrose, fructose and glucose applications, the highest rate of micropropagation results were obtained from 20 g/L sucrose in both species. In addition, 1 g/L agar in the nutrient media produced the highest micropropagation in H. callitrichoides and R. fluitans. Addition of BAP and NAA to the nutrient media adversely affected in vitro micropropagation in the studied species. As a result of this work, high rates of micropropagation were achieved in both species without the need for growth regulators which are the most expensive compounds of nutrient media, and using agar at very low rates. In vitro micropropagated plants were transferred to the aquariums with 100% adaptation rate.

P-2027

Bibliometric Analysis on Extracellular RNA Mechanisms in Pathogen Control. MELEKŞEN AKIN¹, Sadiye Peral Eyduran², Barbara Doyle Prestwitche³, Arne Weiberg⁴, Amy H. Buck⁵, and Guy Smagghe⁶. ¹Department of Horticulture, Iğdır University, Iğdır 76 000, TÜRKİYE; ²Department of Horticulture, Muğla Sıtkı Koçman University, Fethiye/Muğla 48 300, TÜRKİYE; ³School of Biological, Earth and Environmental Sciences, University College Cork, IRELAND; ⁴LMU Munich Biocenter, Großhaderner Straße 4, 82152 Planegg-Martinsried, GERMANY; ⁵Institute of Immunology & Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FL, UK; and ⁶Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Ghent, BELGIUM. Email: akinmeleksen@gmail.com

In addition to its functions inside cells, RNA is also located outside of the cells and can be utilized as a communication tool to deliver information among cells, organisms and species. Pathogens, including viruses, can also use extracellular RNA to enable their infections. Consequently, exRNAs are related to a myriad infectious disease in plants and animals. The knowledge on exRNA mechanisms could be translated into strategies for decreasing parasitism/disease. The objective of this study is to review the literature on exRNA mechanisms in relation to pathogen control within the framework of bibliometrics by displaying the conceptual and intellectual structure of the field as well. The literature published until 2023 was extracted from the Web of Science database and subjected to bibliometric analysis with Bibliometrix package and Biblioshiny interface in R statistical software. A total of 77 documents on the topic were detected, published in diverse journals (64) with various scopes. The first document on the field was released in 2001, and since then there was an 11,6% annual increase of publications over time. The core group consisted of 13 out of 64 sources with PLOS ONE being the most productive in the area (4 documents) followed by Journal of Immunology (3 documents). The highest number of publications was from the Germany with 62 documents, followed by the USA (61 documents), and UK (25 documents). Korea and China showed only single country publications whereas the other most productive countries in this area had also international collaborations. The most frequent author keywords projected after extracellular DNA were inflammation, extracellular vesicle, micro-RNA, biomarker, exosomes, innate immunity, RNA and Covid-19 which represent the current direction of the research area. Other significant keywords were endothelial cells, host-pathogen, Sars-Cov-2, thrombosis, and toll-like receptor. Collaboration network analysis was performed to display collaboration groups on the field between countries, institutions and authors.

P-2028

Wheat Regeneration Efficiency Analysis Approached by the GRF4-GIF1 Chimeric Gene. TAE KYEOM KIM and Jae Yoon Kim. Department of Plant Resources, Kongju National University, Yesan, Chungnam, REPUBLIC OF KOREA. Email: ktk3861@smail.kongju.ac.kr, jaeyoonkim@kongju.ac.kr

Wheat is a highly complex and large-genome allohexaploid crop, which poses significant challenges for genetic breeding research. Furthermore, Improving its low regeneration efficiency remains an important task that still needs to be overcome. Recently, it has been revealed that the fusion protein of wheat, GRF4-GIF1, can improve the efficiency of regeneration, and many researchers are actively interested in and utilizing it. In this study, we developed transgenic wheat with introduced Cas9 targeting the GRF4-GIF1 fusion protein and pre-harvest sprouting (PHS) resistance-related genes. Targeting the TaVDAC, gRNA was designed using an in silico program and inserted into the Cas9 vector containing GRF4-GIF1. Immature embryos of the wheat cultivar 'Bobwhite' grown in a speed breeding growth chamber were collected and transformed via particle bombardment after callus induction. The divided calli were transferred to a selection medium, and a total of 120 individual calli, 30 per plate, were transformed. Hygromycin 30 mg was added to select transformed plants during callus induction, regeneration, and rooting processes. A total of three regenerated plants were obtained, with three of them originating from a single transformed callus (efficiency of approximately 1.7%). The regenerated plants were sampled and screened to confirm the integration of the introduced gene and to verify whether gene correction had occurred. We plan to verify the PHS resistance through generation advancement transfer and are conducting research to shorten the embryogenic callus formation period in wheat. Acknowledgments: This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01652801)” Rural Development Administration, Republic of Korea.

P-2029

A Whole Greater Than the Sum of Its Parts: Host Defense Peptide Synergism Greatly Increases Antimicrobial Activity. NICK SCHIMPF and Dmytro P. Yevtushenko. Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, CANADA. Email: nick.schimpf@uleth.ca

Crop losses due to microbial diseases reduce global food production by one third. Limited efficiency of traditional practices to control plant pathogens hinders food production, especially in developing countries. On the other hand, modes of actions of plant native host defense peptides (HDPs) can allow for durable, long-term host resistance against a broad range of microorganisms without negative impact on the environment. This study seeks to evaluate HDPs for antimicrobial and cytotoxic activities in vitro and to introduce promising candidates in potato plants for enhanced disease resistance. Spores of five economically important fungal species were incubated with various concentrations and combinations of five plant-derived HDPs for 24 h. Spores were considered inhibited if the emerging mycelium was less than twice its length. Antibacterial activity was assessed by measuring the absorbance (OD₆₀₀) of challenged cultures. Cytotoxicity was investigated by challenging mesophyll protoplasts and mammalian kidney cells with the HDPs using colorimetric viability assays. HDP-coding sequences were introduced into plants by Agrobacterium-mediated transformation. Singly, HDPs SM-985, Ib-AMP 1Q, shepherin 1, and P4650 were active against bacterial and/or fungal pathogens. P4650 and BnPRP1 interacted synergistically in combinations with other peptides, decreasing minimum inhibitory concentrations (MICs) by as much as 83%. P4650 was the only single peptide toxic to protoplasts, with more than 96% loss in cell viability at 100 µM. No single HDP nor combinations were toxic to mammalian cells. Although three three-peptide combinations inhibited all fungal spores at 20-50 µM concentrations and bacteria at just 2.5 µM, combination designated as 3D was given precedence for expression in potato plants because of its distinct lack of phytotoxicity. Transgenic plants will be challenged by pathogens in future research to assess host resistance. This study will identify novel HDPs for engineering disease resistance in crops and contribute to improving food security.

P-2030

SIVB Social Engagement Ad Hoc Committee. J. VAN ECK^1,2, A. Belfetmi-Stone³, S. D. Dangol^4,5, H. M. Hashmi⁶, R. Eke Emele⁷, and C. Bagley⁸. ¹Boyce Thompson Institute, Ithaca, NY 14853; ²Plant Breeding and Genetics Section, Cornell University, Ithaca, NY 14853; ³Harvard Medical School, Boston, MA 02115; ⁴Central Department of Biotechnology, Tribhuvan University, Kirtipur, Kathmandu, Bagmati Pradesh, NEPAL; ⁵Nepal Plant Disease and Agro Associates (NPDA), Balaju Chakrapath, Kathmandu, Bagmati Pradesh, NEPAL; ⁶Department of Agricultural Genetic Engineering, Faculty of Agricultural Sciences and Technologies, Nigde 51240, Nigde Province, TURKEY; ⁷Department of Biotechnology, Faculty of Arts and Sciences, Nigde 51240, Nigde Province, TURKEY; and ⁸Inari Agriculture, West Lafayette, IN 47906. Email: jv27@cornell.edu

The recently formed Social Engagement Ad Hoc Committee needs your help promoting SIVB. Leveraging social media will bring broader awareness of the society to help build membership and increase attendance at the annual meetings. In addition, it will allow members to learn more about each other to strengthen connections. The SIVB wants to share this community’s excitement and passion for the exchange of in vitro biology knowledge through the society’s social media platforms. We invite you to share pictures or videos that highlight the people, science, hard work, events and fun that originate from our SIVB community. Let’s show the difference and impact that SIVB members are making in their communities and in the world. We have developed content, however, to sustain the engagement, we need content from SIVB community members to post on YouTube, Facebook, Twitter, LinkedIn, and Instagram accounts. If you are not already following SIVB on these platforms, go to the website (https://sivb.org) and click on the icons in the upper right corner to get started. Instructions for preparing content, including videos for YouTube, are on the SIVB website (https://sivb.org/social-media.html). This content can be about your lab, publications, or anything you think the SIVB community would be interested in knowing. Send it to the Social Engagement Ad Hoc Committee at sivbsocialmedia@sivb.org. In addition, the committee would welcome new members if you would like to help develop social media content on a consistent basis. Feel free to get in touch with any questions regarding development of your content or joining the committee.

P-2031

Engineering “Designer” Proteins and Enzymes in Plants for Biomedical and Industrial Applications. JIANFENG XU^1,3, Paula Perez Sanchez², Berry Dickey², Jonathan Trejo Martinez², and Uddhab Karki^1,3. ¹Arkansas Biosciences Institute, ²Department of Biological Sciences, and ³Molecuar Biosciences Program, Arkansas State University, Jonesboro, AR 72401. Email: jxu@astate.edu

Genetic engineering of plants and plant cells has been used for generating transgenic plants with new traits or for “molecular farming” to produce valuable recombinant proteins. In this study, we explore the potential of two unique post-translational modifications - "glycosylphosphatidylinositol (GPI) anchor" and "hydroxyproline (Hyp)-O-glycosylation" - to design and engineer functional recombinant proteins in plants and plant cells for biomedical and industrial uses. Hyp-O-glycosylation is a plant-specific posttranslational modification that involves the addition of the arabino-oligosaccharides or arabinogalactan polysaccharides to the Hyp residues. It facilitates extracellular secretion of associated proteins and shields proteins from proteolytic degradation. GPI anchoring involves the attachment of glycolipid anchor to a protein's C-terminus, promoting intracellular transport and cell surface display of the protein. GPI anchoring also facilitates N-linked and Hyp-O-linked glycosylation, leading to more homogeneous glycosylation of the anchored proteins. We engineered these two functional modules into an anti-TNFα molecule (single-chain fragment variable of the human anti-TNFα antibody) in tobacco BY-2 cells to create a novel oral biologic for treating inflammatory bowel disease (IBD). For an industrial application, we also introduced GPI anchoring and a Hyp-O-glycosylation module into the thermophilic E1 endoglucanase (cellulase) in tobacco and switchgrass plants to reconstruct the plant cell wall for improved processability of cell wall biomass. Overall, our study demonstrates the versatility and potential of Hyp-O-glycosylation and GPI anchoring as powerful tools for designing and engineering functional recombinant proteins in plants and plant cells for biomedical and industrial applications.

P-2032

Accumulation of Anthocyanin in Brachypodium Seeds by Over-expression of Chalcone Isomerase Gene. M. B. LEE and J. Y. Kim. Department of Plant Resource, College of Industrial Science, Kongju National University, Yesan, 32439, REPUBLIC OF KOREA. Email: jaeyoonkim@kongju.ac.kr

Brachypodium distachyon (B. distachyon), a member of Poaceae, has been used as a model system for important agricultural cereal crops and biofuel feedstocks. Seed color is an interesting agronomic trait associated with seed quality, pre-harvest sprouting, and dormancy. Gamma radiation has been widely adopted to create genetic variations in plants. Seedlings of B. distachyon was subjected to chronic gamma radiation. A mutant line (142-3) showed a significant decrease of anthocyanin content in seeds. Homozygous 14 bp deletion was detected within the exon region of chalcone isomerase, which causes loss of function. Brachypodium immature embryos from WT and the 142-3 mutant line were collected and used for callus induction. Embryogenic callus were used for Agrobacterium-mediated transformation carrying chalcone isomerase over-expression vector. The segregation ratio of T₁ plants was identified by Neomycin phosphotransferase II ELISA assay. Yellow seed color of 142-3 was recovered to brown (WT) in chalcone isomerase over-expressed 142-3 plants. We confirmed that the loss of function of chalcone isomerase was the causal factor of seed color change (to yellow) in 142-3. Single-copy homozygous T₂ plants will be used for further study to discover the gene function of chalcone isomerase. This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01652801)” Rural Development Administration, Republic of Korea.

P-2033

Evaluation of a Tetraploid Somatic Hybrid Produced by Fusing the Salt Tolerant Cleopatra Mandarin with the Salt Susceptible Carrizo Citrange. LAMIAA M. MAHMOUD, Nabil Killiny, Paige Holden, Frederick G. Gmitter Jr., Jude W. Grosser, and Manjul Dutt. Citrus Research and Education Center, University of Florida, Lake Alfred, FL. Email: lamiaa.mahmoud@ufl.edu

Somatic hybridization is an efficient tool for the development of novel genotypes that are used in citrus breeding programs. Somatic hybrids can lead to the direct development of tetraploid rootstocks or be used as breeding parents for the development of tetraploid rootstock and triploid scion cultivars. Cleopatra mandarin is a salt-tolerant rootstock, while the Carrizo citrange rootstock is known to be salt sensitive. Salinity is an issue in the coastal regions of the world, and the development of new citrus rootstock cultivars that can grow in soils with high salinity levels is necessary. In this study, we evaluated a tetraploid somatic hybrid produced from the fusion of Cleopatra mandarin suspension cell derived protoplasts with Carrizo citrange leaf derived protoplasts to understand its performance under in vitro salinity stress. The ploidy of the resultant hybrid was confirmed based on differential leaf morphology, flow cytometry, and Simple Sequence Repeat (SSR) molecular markers. Mature stem pieces were collected from the somatic hybrid and its parents and in vitro shoots produced in tissue culture. The adventitious shoots were transferred into MS medium supplemented with NaCl (0, 50 or 100 mM) for 3 weeks to induce in vitro salinity stress. Physiological and biochemical traits including chlorophyll content, lipid peroxidation, total phenolic compound, proline content, and antioxidant enzymes were estimated in the leaves. Our results indicated that the somatic hybrid is superior to the parents in response to salinity stress as less damage to the cellular membrane, measured by cellular MDA content and electrolyte leakage percentage, was observed in the somatic hybrid under 100 mM NaCl treatment. The total phenolic content (TPC) in the somatic hybrid was greater than in either parent, but Cleopatra mandarin had the highest proline levels. Our results indicate that the somatic hybrid exhibited better salinity stress tolerance than the Cleopatra parent under simulated abiotic stress.

P-2034

High-efficiency Somatic Embryogenesis and Plant Regeneration in Alfalfa (Medicago sativa). SUMA BASAK, Dipika Parajulee, Terri Brearley, and Sarwan Dhir. Center for Biotechnology, Department of Plant Sciences, College of Agriculture, Family Sciences and Technology, Fort Valley State University, GA 31030. Email: Suma.Basak@fvsu.edu

Alfalfa (Medicago sativa) has numerous benefits as a biopharmaceutical product. Recently, this plant has been used in multiple studies to introduce oral plant-based vaccines. A reliable plant regeneration protocol is a prerequisite for large-scale production of alfalfa or any genetic engineering studies. The purpose of this study was to establish an efficient somatic embryogenesis and plant regeneration protocol in alfalfa. Two different media, Murashige and Skoog’s (MS) and Gamborg’s B5 basal (B5H) supplemented with 3% maltose, 0.9µM Kinetin, 4.5µM 2,4-D, were tested for initiating somatic embryogenesis from leaf explants from 3 weeks old in vitro seed-derived plants. Embryogenic callus (EC) from B5H medium had a higher rate of embryo initiation with 3 weeks in dark about 97.9% than that of MS medium at 62.5%. After 3 weeks in light, the EC was transferred onto B5H media supplemented with 3% maltose without growth regulators for EC maturation. The matured EC was placed for 3 weeks in light into the germination medium, MS basal supplemented with 3% sucrose, 1mL/L 1000x Nitsch and Nitsch vitamin, and 0.1 g/L myoinositol in order to increase the mass of EC. The cotyledonary staged embryos were transferred to MS basal medium 3 weeks in light for plantlet formation. The percentage of regenerated plantlets from cotyledonary staged embryos was calculated (94%). Each plantlet was placed into the plantlet acclimatization medium containing half-strength MS medium with 3% sucrose and 0.03% GELRITE^®. After 4 weeks, the developing plantlets were transferred to magenta boxes containing a vermiculite mixture and subsequently the fully developed plant was established in the soil. This somatic embryogenesis and plant regeneration protocol can be used for gene transformation studies in future studies.

P-2035

Accelerating Crop Gene Editing and Metabolic Engineering with Plant Cell-based Design-build-test Loops. M. DILEO and J. Zhou. Elo Life Systems, 3054 East Cornwallis Drive, Durham, NC 27709. Email: mdileo@elolife.com

Gene editing and genetic engineering efforts are throttled both by the uncertainty that accompanies initial target gene selection as well as very long delivery and regeneration timelines. As a consequence, the initial technical demonstration of a gene editing or genetic engineering strategy often occurs on the scale of months or years, delaying commercialization and significantly decreasing potential returns on investments and commercial feasibility. Similar challenges have been successfully addressed in drug development and precision fermentation with rapid, scalable and biologically-relevant assays. Elo’s CULTIVATE® platform enables iterative testing and optimization of genetic elements directly in protoplasts of elite germplasm within a highly-compressed timeline. Here, we demonstrate how rapid design-build-test loops can support 10-100x+ improvements in sequence editing and targeted insertion efficiency as well as regulatory and metabolic network engineering within weeks instead of months. Elo is the next generation ingredient company and is using these approaches as part of our mission to unlock nature's abilities to make consumer's favorite foods more delicious, healthy and planet-friendly.

P-2036

Emerging Factors that Influence Efficiency of T-DNA Gene Transfer into Hemp (Cannabis sativa) via Agrobacterium. TASNIM MOHAMMAD and Sarwan Dhir. Center for Biotechnology, Department of Agricultural Sciences, Fort Valley State University, Fort Valley, GA, 31030. Email: tasnim.mohammad@fvsu.edu

Hemp (Cannabis sativa L.) is an annual typically dioecious crop and has therapeutic potential for human diseases because Phyto- cannabinoids in medical therapy is getting more attention recently. The purpose of this study was to establish a protocol for Agrobacterium-mediated transformation for foreign gene integration in Hemp. Several factors influencing transformation efficiency include the effect of explant type, age of leaf explants, the concentration of silver nitrate and/or calcium chloride, bacteria concentration, infection time, acetosyringone concentration, wounding, and different co-culture periods of bacteria were evaluated to optimize the transformation efficiency. The Agrobacterium-mediated transformation was optimized using the binary vector pCAMBIA1304 (1) encoding the modified GFP and GUS as reporter genes which is driven by the CaMV 35S promoter for early detection of transgene expression. Results obtained were based on the average number of blue spots and percentage of transformed GUS expressing leaf tissue which was observed 3 days post-transformation. The results indicated that 14 and 21 days old fully expanded leaves as explant showed increased transient transformation recorded as the number of GUS blue spots and GFP expression at 7-day-old leaf explant. Enhanced transient GUS expression with an average frequency of 79% was noticed at 30 minutes of the co-cultivation period. The addition of 150 µM acetosyringone, 60 µM of silver nitrate, and 0.5 µM of calcium chloride in the co-cultivation medium increased the transient transformation frequency and the number of blue spots by 77%, 80%, and 76% compared to control treatment in the leaf tissues. The bacterial inoculum concentration of (OD 0.4) at 600 nm showed 90% transient GUS expression. The highest transient gene expression of 74% was obtained from a leaf incubated compared to other explants such as roots, cotyledons, petiole, and/or leaf-derived calli. These optimized conditions have been used to obtain stably transformed explants for subsequent regeneration.

P-2037

Intersection of Undergraduate Research and CUREs on Plant Sciences: A Pilot Study. MARGARET YOUNG¹, Falcon Rankins², and Gloria Payne¹, ¹Department of Natural Sciences, Elizabeth City State University, Elizabeth City, NC and ²PRISSEM Academic Services, LLC, Richmond, VA. Email: mmyoung2@ecsu.edu

High impact teaching techniques include course-based undergraduate research experiences (CUREs). Students’ scientific competence and confidence can be increased with independent research projects such as CUREs. At Elizabeth City State University (HBCU) in North Carolina, we have integrated plant CUREs into two undergraduate biology courses for the past four years (2019 – 2023). These CUREs were developed to intersect with the research interest of the faculty. The PI’s research lab is interested in plant tissue culture and transformation of important crops and educational species. These two techniques can be significant bottle necks in crop improvement. In General Botany (sophomore course), students conducted a CURE in plant tissue culture. In Molecular Biology (junior/senior course), a CURE in plant transformation was completed. Each semester, students are first given a simple peer-reviewed article on the technique for that plant. Students then design their experiments by optimizing one variable (utilizing additional sources). They set up the experiments as groups, take observations, and present the data either in an oral presentation or as a scientific lab report. The information obtained in the CUREs are utilized in formal research by other undergraduates. Sometimes, the undergrad researchers do simple pre-experiments for that plant before deployment to the CUREs. Survey results suggested that students found CUREs to be valuable to their learning and desired more courses that provide opportunities to design and conduct experiments. Survey results and student interviews also suggested self-reported gains in student understanding of the role and importance of plant sciences. Finally, we discuss changes to our CURE approach based both on faculty reflections and accommodations for COVID-19 related uncertainties; and issue recommendations for faculty interested in implementing CUREs in similar institutional environments. Support for this effort was provided by a National Science Foundation grant from the HBCU Undergraduate Program under Award No. 1912192.

P-2038

USAID Feed the Future Global Biotech Potato Partnership. K. ZARKA¹, D. Douches¹, K. Hokanson², M. Ghislain³, P. Wharton⁴, E. Magembe³, J. Marcy¹, D. Zarka¹, J. Fierro¹, and J. Payumo¹. ¹Department of Plant Microbial and Soil Sciences, Michigan State University, East Lansing, MI 48824; ²Department of Horticultural Sciences, University of Minnesota, Minneapolis, MN 55455; ³Center for International Potato, ILRI Campus, Nairobi, KENYA; and ⁴University of Idaho, Department of Entomology, Plant Pathology and Nematology, Aberdeen, ID 83210. Email: zarka@msu.edu

Potato is an essential crop that can play an integral role in achieving global food security. Disease, particularly Late Blight, can be a major issue for farmers often resulting in total crop loss. The United States Agency for International Development (USAID) through the Feed the Future Initiative has awarded funding for a five-year collaborative partnership led by Michigan State University (MSU). The Feed the Future Global Biotech Potato Partnership will bring late blight disease resistant (LBR) potatoes in farmer-preferred varieties to the Southeast (SE) Asian countries of Bangladesh and Indonesia, and the Sub-Saharan Africa (SSA) countries of Kenya and Nigeria. This may expand the Activity into other Feed the Future target countries. The Feed the Future Global Biotech Potato Partnership Activity will continue work of previous USAID investments focused on the research and development of the LBR potato through genetic engineering. The Global Biotech Potato Partnership is a collaboration between MSU, the International Potato Center (CIP), the University of Minnesota, the University of Idaho, African Agricultural Technology Foundation (AATF), and partner country National Agricultural Research Systems (NARS). Building the capacity of the NARS, to conduct this research in the four countries is also a primary goal of the project. The partnership will utilize cutting-edge research to promote a robust potato value chain through the commercialization of the 3 R-gene Late Blight Resistant (LBR) potato as well as developing 2nd generation disease resistant potato products. The goal is to sustainably reduce global poverty, hunger, and malnutrition. This will ultimately help emerging economies in SE Asia and SSA countries accelerate progress towards self-reliance. The Global Biotechnology Potato Partnership expects to complete required research necessary to receive regulatory approvals for general release and commercialization of the LBR potato in each of the four target countries within the next five years.

P-2039

Decoding the Genetic Makeup of Plants: Exploring Ploidy Analysis Methods for Supporting Ornamental Breeding. J. MAHONEY¹, J. Zhao¹, K. Buche², T. Pfeifer¹, C. Konkler¹, and H. Wu¹. ¹Ball Horticultural Company - Ball Helix Central R&D, West Chicago, IL, 60185 and ²Ball Horticultural Company - Ball FloraPlant, Guadalupe, CA, 93434. Email: jonmahoney@ballhort.com

Breeding programs often utilize polyploids or artificially induce polyploids as pre-breeding material or as a new product in ornamental breeding programs. Therefore, ploidy analysis is of critical importance for understanding the genetic constitution of crop species to support breeding. Phenotype characteristics can provide initial insights into ploidy levels, as polyploid plants often exhibit changes in size, shape, and physiology. Cell size measurements are another useful method, with larger cells often indicating a higher ploidy level. Cytology involves examining chromosomes under a microscope and is the most precise method of ploidy analysis but requires expertise and is time-consuming. Flow cytometry is a faster and more efficient method, analyzing thousands of cells in a short period by measuring their DNA content. In this study, we determined baseline ploidy levels of Lantana spp. using traditional chromosome counts for a subset of samples and conducted ploidy level estimates using flow cytometry on 200 samples. Using the baseline cytology chromosome counts, we were able to use flow cytometry to accurately estimate ploidy levels from a diverse and large population of Lantana spp. Both methods had advantages and disadvantages but can be used to complement each other for understanding the baseline ploidy level of a group of related species, subspecies, or varieties. In the second study, we performed genome doubling with Angelonia angustifolia, determined ploidy level changes using flow cytometry along with chromosome counts, cell size and phenotype measurements to obtain a comprehensive understanding of the genome doubling efficiency. Using bead beating method in 96-well format for extracting nuclei has enabled us to successfully screen a large population of plants to determine their ploidy level post-genome doubling in a relatively short period of time. As a result of these two experimental efforts, we found that the combination of ploidy analysis methods can effectively provide a comprehensive understanding of plant ploidy levels to support breeding programs.

P-2040

Adventitious Root Formation in Recalcitrant Woody Specie. X. LU, M. Cuarto, and H. Liang. Department of Genetics and Biochemistry, College of Science, Clemson University, Clemson, SC 29634. Email: Xinyal@g.clemson.edu

The formation of adventitious roots (ARs) is a complicated process with several exogenous and endogenous elements influencing the outcome of the process, which is of great value in successful vegetative propagation. The present study investigated histology and how phytohormone affected AR formation on cuttings of yellow camellia (Camellia nitidissima) (YC), a world-famous ornamental plant with yellow flowers, and American chestnut (Castanea dentata) (AC), an important hardwood species with economic and ecological significance and a native from eastern North America. Our aims are to understand AC’s and YC’s recalcitrance to rooting and uncover plant auxin levels profile. We conducted histological observations to identify AR primordia and location. Using a comparative approach, the phytohormone levels in AC and YC cuttings were compared to easy-to-root poplar cuttings. It was found that AC and YC cuttings showed a relatively lower level of IAA and a higher level of cytokinin, abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), and oxylipin 12-oxo-phytodienoic acid (OPDA). Hormone distribution between leaves and stems also differed. This unfavorite endogenous hormone profile may contribute to AC and YC cuttings’ recalcitrance to rooting. The histological study indicates that ARs can be induced directly from AC and YC cuttings, initiating from cambial derivatives and developing a vascular system connected with that of the stem, rather than forming calli.

P-2041

Roles of Amino Acid Transporters for Metal Transport in Plants and Yeast. A. MAQBOOL¹, S.Thomine², and E. Aksoy³ . ¹Department of Agricultural Genetic Engineering, Faculty of Agricultural Sciences and Technologies, Niğde Ömer Halisdemir University, Nigde, TURKEY; ²Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, 91198 Gif-sur-Yvette, FRANCE; and ³Department of Biological Sciences, Middle East Technical University, Ankara, TURKEY. Email: amirmaqbool767@gmail.com

Plants utilize delicate mechanisms to effectively respond to changes in the availability of different nutrients such as trace metals. Metal transporters play a critical role in ensuring that plants have a sufficient supply of metals for growth and development. Dysfunction of these transporters can lead to metal deficiencies, or toxicities, which may have detrimental effects on plant growth, nutritional value, and yield. Due to redox properties of some metals, living organisms such as plants and yeast always needed to translocate these in the chelated form. Recent studies related to metal homeostasis in plants and yeast defined the roles of amino acids to chelate divalent metals. We are currently characterizing the members of a novel amino acid transporter family in S. cerevisiae and A. thaliana. Mutants and overexpression lines of the family members in Arabidopsis showed distinctive growth phenotypes under different metal availability conditions. S. cerevisiae strains defective in Mn, Fe, Zn and Cu homeostasis showed interesting results when transformed with selected amino acid transporter(s) from A. thaliana. Details of the study will be discussed briefly in the presentation. Determination of the functions of this transporter family in divalent metal chelation and transport may lead to new strategies in plant biofortification and plant nutrient utilization.

P-2042

Embryogenic Stem Cell-specific Genes Profiling via Transcriptome Analysis During Protoplast Culture in Carrot and Ginseng. JONG-EUN HAN¹, Han-Sol Lee¹, Hosakatte Niranjana Murthy^1,2, and So-Young Park¹. ¹Department of Horticultural Science, Division of Animal, Horticultural and Food Sciences, Chungbuk National University, Cheongju 28644, REPUBLIC OF KOREA and ²Department of Botany, Karnatak University, Dharwad 580003, INDIA. Email: soypark7@cbnu.ac.kr

A protoplast culture is a valuable tool in plant biotechnology for various applications. However, protoplast culture has difficulties in cell division and plant regeneration according to various factors such as plant species and explants. Protoplasts were isolated from embryogenic callus (EC) in carrot and ginseng, but cell division was observed only in carrot. In this study, we tried to identify stem cell-specific genes that induce cell division by confirming the pattern of gene expression that changes according to the process of protoplast culture derived from the embryogenic callus of carrot and ginseng. Transcriptome analysis was conducted at three specific points: embryonic callus (EC), protoplasts isolated from EC, and recovery of cell division ability by 7-day cultured. After transcriptome analysis, Differentially Expressed Genes (DEG) analysis and GO analysis was performed. As a result of RNA-seq of carrot, a total number of 13,517 genes was expressed, 10,943 were expressed in EC vs Protoplast, 7,269 in EC vs Recovery, and 8,052 in Recovery vs Protoplast. In ginseng, a total number of 16,046 genes was expressed, 10,182 in EC vs Protoplast, 6,272 in EC vs Recovery, and 10,152 in Recovery vs Protoplast. GO terms related to biological processes showed organonitrogen compound biosynthetic proc., several types of metabolic proc., and cell division in recovery compared to protoplasts in carrot. While in ginseng, GO terms are involved in organ development. Among the three DEGs, SERK2, YUC10, WOX5-like, WOX9-like, and OVA5 were found as stem cell-specific genes that were upregulated in carrot Recovery vs Protoplast. In ginseng, WOX8, BBM, WOX2, and OVA5 were shown at the same time points. Overall, since cell division did not occur in ginseng, YUC10, WOX9, and AGL104 genes, which were highly expressed only in carrots, are expected to stem cell-specific genes that regulate cell division. Acknowledgments: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. NRF-2020R1A2C2102401).

P-2043

Plant Regeneration from Embryogenic Callus-derived Protoplasts and Its Comparative Transcript Profiling by RNA-seq in Angelica gigas Nakai. H. S. LEE¹, J. E. Han¹, D. H. Shim², and S. Y. Park¹. ¹Department of Horticultural Science, Division of Animal, Horticultural and Food Sciences, Chungbuk National University, Cheongju 28644, REPUBLIC OF KOREA and ²Department of Biological Sciences, Chungnam National University, Daejeon 34134, REPUBLIC OF KOREA. Email: soypark7@cbnu.ac.kr

Plant protoplasts can be used as an experimental basis to support the totipotency of plant cells as it can develop into a complete plant through the cultivation of a single cell. We confirmed the difference in gene expression patterns in three phases: embryogenic callus before protoplast isolation (EC), freshly isolated protoplasts, and recovery of cell divison in the embryogenic callus of A. gigas Nakai. The expression pattern of the gene was confirmed through next-generation sequencing to find differentially expressed genes, and the function of the corresponding gene was confirmed through gene ontology (GO). The expression of embryogenesis-related genes, which were highly expressed in ECs, was down-regulated after protoplast isolation, and the expression of the genes was restored at the time of cell division recovery. Similar trends were observed in the cell cycle and hormones as well. In addition, expression patterns of genes related to chromatin accessibility were also different according to each stage. Transfected protoplasts formed cell division and multi-cell formation through culture. As such, we attempted to investigate the differences in gene expression patterns that occur freshly isolated protoplasts and the molecular biological differences in the recovery of cell division. This may be helpful for identifying gene reprogramming events in the protoplasts of various plant species as well as in embryogenic callus protoplasts. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. NRF-2020R1A2C2102401).

P-2044

Genetically Stable Nontoxigenic Aspergillus Strains to Manage Aflatoxin Contamination in Peanuts. ZACHARY JONES^1,2, Ethan Matthews¹, and Sachin Rustgi^1,2,3. ¹Department of Plant & Environmental Sciences, Clemson University Pee Dee Research & Education Center, Florence, SC 29506; ²Center for Human Genetics, Clemson University, Greenwood, SC 29646; and ³School of Health Research, Clemson University, Clemson, SC 29634. Email: srustgi@clemson.edu

Aflatoxin contamination of peanuts is one of the primary challenges facing the peanut industry causing food safety concerns and leading to exportation and domestic use restrictions of peanuts as food or animal feed. Peanut producers in the United States lose an average of $25-58 million annually to aflatoxin contamination. Aflatoxin B1, primarily produced by Aspergillus flavus and Aspergillus parasiticus, is a group one carcinogen that contributes to cancer development and can lead to immune suppression, endocrine disorders, nutrient malabsorption, and infertility. As a result, aflatoxin content is strictly regulated at 20 ug/kg in food in the United States and at 2 ug/kg in the European Union. This research project attempts to control the proliferation of aflatoxin-producing Aspergillus species to mitigate pre- and post-harvest aflatoxin contamination in peanuts. Specifically, this project aims to develop genetically stable nontoxigenic Aspergillus strains with a built-in mechanism to prevent the emergence of toxigenic revertants as a measure to replace the existing toxigenic wild-type strains currently responsible for the field and storage contamination of peanuts. Utilizing a protoplast-mediated delivery method, 22% of tested colonies show successful transformation with a CRISPR-mediated gene drive construct. This biocontrol agent has the potential to benefit peanut producers throughout the southern and southeastern United States as it could reduce input costs and yield loss while establishing a population of nontoxigenic Aspergillus strains in soil. In addition, this nontoxigenic strain has the potential to benefit growers of other crop species, such as corn, tree nuts, and cotton, who also face Aspergillus infestation.

P-2045

Evaluating a Ternary Vector System on the Tropical Maize NAM Line, Tzi8. J. N. UY, T. Amore, and M. Muszynski. Department of Tropical Plant and Soil Sciences, College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, Honolulu, HI, 96822. Email: uyjnr69@hawaii.edu, amore@hawaii.edu

The discovery and utilization of CRISPR technology in genome editing marked a significant turning point in genetic transformation. Genetic transformation of crops is still challenged by low transformation frequencies and poor plant regeneration. Although vectors with the morphogenic genes BBM and WUS2 have been beneficial in increasing transformation and regeneration of recalcitrant maize, these genes can negatively impact plant development and maturity. Therefore, we evaluated the performance of the tropical NAM line Tzi8 and transformed it with a ternary vector system developed for temperate maize line, B104. We transformed immature embryos of Tzi8 and B104 with an auxotrophic Agrobacterium LBA4404Thy- containing a Cas9 binary vector targeting glossy2 and a helper plasmid containing extra copies of virulence genes. The regenerability and transformation frequencies of B104 and Tzi8 were compared. Cas9 positive plants were identified using endpoint PCR and copy number variation of Cas9 positive plants was determined using ddPCR. Plantlet regeneration was significantly higher in B104 with 40% of the embryos regenerating into plants in contrast to Tzi8 with 8% regeneration. Among the regenerated plants, 30% of B104 and 10% of Tzi8 were Cas9 positive. Low copy number variants from the Cas9 positive plants were obtained. We successfully transformed Tzi8 using the ternary vector system developed for B104.

P-2046

Managing Sugarcane Aphid in Sorghum and Armillaria Species in Peach Using Nucleic Acid Molecules. PRASANNA V. SHEKAR, Katherine A. Wakeley, and Sachin Rustgi. Department of Plant and Environmental Sciences, School of Health Research, Clemson University Pee Dee Research and Education Center, Florence, SC. Email: srustgi@clemson.edu

The sugarcane aphid (SCA, Melanaphis sacchari), first discovered in grain and forage sorghum in 2013, has gradually become a major insect pest of sorghum, causing heavy damage to the crops annually, costing millions of dollars in losses throughout the US. Likewise, Armillaria root rot (ARR) caused by Armillaria mellea and A. tabescens, also referred to as oak root rot, has become one of the major causes of precocious peach tree (Prunus persica) death in the southeastern United States. The standard management practices used to control SCA are synthetic pesticides, resistant varieties, and scouting. At the same time, the common management practices for Armillaria are to avoid replanting on the infected grounds and root collar excavations. These practices are somewhat effective in reducing pest pressure and/or disease progression in both cases. No suitable fungicides are available to control ARR pathogens. On the other hand, the pesticides used to manage SCA are Sivanto Prime and Transform; their continued application is expected to develop resistant SCA strains. Additionally, these insecticides cause damage to pollinators. Hence to meet the demand for greener, target-specific, and safer alternatives to manage SCA in sorghum and Armillaria species in peach, this project evaluates the potential of small nucleic acid molecules complementary to the transcript of essential genes in pests and pathogens in preventing their growth in the laboratory, greenhouse, and field studies. The results of the pilot laboratory experiments in both target organisms and future directions will be presented at the symposium.

P-2047

Epitranscriptomic RNA Modification in Plant Development and Environmental Stress Adaptation. RYAN WATTS, Zhaohui Chen, Xiaotong Chen, Morgan Kuess, Emma Jensen, Andrew Fiorentino, Qian Hu, and Hong Luo. Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634. Email: rhwatts@clemson.edu

Epitranscriptomic RNA modification is an essential mechanism to achieve precise regulation of developmental processes or responses to environmental stresses in living organisms. Prior research has shown that the methylation of mRNAs affects many aspects of RNA metabolism, such as stability, splicing, nucleus to cytoplasm export, alternative polyadenylation, and translation. In addition, the methylation or demethylation of specific bases can enhance hydrophobicity and disrupt canonical Watson-Crick base pairing, leading to altered structure of RNAs. The effect of these modifications in plants, especially in perennials, remains to be elucidated. Here, we investigate the role of the RNA modification genes involved in RNA methylation (writer), demethylation (eraser) and methylation marker interpretation (reader) in the development and abiotic stress adaptation of the perennial grass species. Specifically, we have cloned creeping bentgrass homologs of FIP37 (RNA writer), ALKBH (RNA eraser) and ECT9 (RNA reader). Overexpression and knockdown transgenic plants of these genes are being generated for analysis in comparison with the wild type controls. The data obtained would reveal how epitranscriptomic RNA modification impacts plant growth and stress responses, providing information for the development of novel epigenetic tools for crop improvement, enhancing agricultural production.

P-2048

Assessing the Growth and Yield Performances of Tissue-culture-derived Vine and Tuber-raised Gamma-irradiated White Yam (cv. Kpamyo) Under Different Light Regimes. Elizabeth Oboh^1,2, MORUFAT BALOGUN^1,2, Norbert Maroya³, and Michael Abberton¹. ¹International Institute of Tropical Agriculture (IITA) Headquarters, Ibadan, NIGERIA; ²Department of Crop Protection and Environmental Biology, University of Ibadan, NIGERIA; and ³IITA, Cotonou, BENIN. kemtoy2003@yahoo.com, m.balogun@cgiar.org

In white yams (Dioscorea rotundata), staking is crucial for good tuber yield, but at added cost. Genetic improvement of yam for non-staking, photosynthesis-efficient, high yielding types can improve yam productivity. However, conventional hybridization breeding is hindered by irregular flowering. Although mutation breeding has high potentials to create new variants for genetic improvement, low propagation ratios and large sizes of planting materials have limited its application. There has been significant breakthroughs in high ratio propagation technologies (HRPTs) which makes it possible to irradiate large populations of tissue culture-derived small-sized explants, followed by selections from and propagation of desirable types for use as parents in crossing blocks of breeding program. This study investigated effects of interactions among five doses of gamma rays, three light intensities and 2 types of planting material on yield and associated traits in white yam (cv. Kpamyo.) in split-plot design. The M2 generation of small whole minitubers (10g) were irradiated with 0 (control), 10, 20, 30 and 40 Gy. The vine seedlings raised from them (main factor), were grown under no net (2.561×10⁴lux), single net (1.039×10⁴ lux), and double net (0.300×10⁴ lux) shade nets of 75% transmittance (sub-factor). Agronomic and yield data were collected and ANOVA performed. A longer internode (cm) was induced by double net shade (9.66±3.47) which was significantly higher than that of the no net shade (7.52±3.24). The internode of 40 Gy (9.78±2.55) was longer but did not differ significantly from control (8.85±2.36). Mean number and weight of tuber (g) per plant were more (1.83±1.44) for 10 Gy and 30 Gy (191.09±138.62 g) respectively. The vine-raised plants yielded better in number (1.94±1.01) and weight (163.91±142.79 g) of tubers across treatments. Under double net shade, tuber weight at 40 Gy (47.88±35.11 g) was highest and can be said to be relatively light use efficient. It is considered as a putative mutant which can be further evaluated in development of dwarf yam requiring little or no staking.

P-2049

Influence of Media Strength on In Vitro Growth of Lobelia chinensis in Bioreactors and Assessment of Phenolic Content and Antioxidant Activities. XINLEI BAI¹, Ji-Hye Kim¹, Han-Sol Lee¹, Jong-Eun Han¹, Hosakatte Niranjana Murthy^1,2, and So-Young Park¹. ¹Department of Horticultural Science, Division of Animal, Horticultural and Food Sciences, Chungbuk National University, Cheongju 28644, REPUBLIC OF KOREA and ²Department of Botany, Karnatak University, Dharwad 580003, INDIA. Email: soypark7@cbnu.ac.kr

Lobelia chinensis is a perennial herb that belongs to the Campanulaceae family. It contains alkaloids, phenolics, and other active compounds. It has highly valuable medicinal properties and significant beneficial effects on human health. Resource shortages are the result of damage to the growth environment caused by humans. Consequently, it is crucial to apply tissue culture techniques to research L. chinensis in vitro propagation. This study investigated the influence of different Murashige and Skoog media strengths on the growth, production of important secondary metabolites, and antioxidant activity of L. chinensis. Different media strengths including 1/4 MS, 1/2 MS, MS, and 2 MS were tested in airlift bioreactors. The results showed that the plantlets grown under full-strength MS medium accumulated the maximum amount of biomass, compared to other media strengths. The highest shoot length was in MS (6.0 cm), followed by 1/2 MS (4.4 cm) and 1/4 MS (4.2 cm). UV-Vis spectrophotometric analysis showed that the plantlets cultured under 1/4 MS medium accumulated the maximum amount of total phenolics (32.52 mg GAE/g extract) and the highest total flavonoids (7.48 mg GCE/g extract). Meanwhile, in the DDPH, FRAP and ABTS assays, the highest antioxidant activity was observed under 1/4 MS. The plantlets cultured under MS medium accumulated the maximum amount of bioactive compounds and induced rutin and apigenin. In conclusion, full-strength MS medium could promote growth and 1/4 MS medium can promote the secondary metabolite production, and antioxidant activity of L. chinensis cultured in vitro. This work was supported by the Industrial Strategic Technology Development Program (Grant number P0018148) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea).

P-2050

Accelerated Cultivation of Solanum lycopersicum from Callus in Liquid Media. A. SHELTON^1,2,3, A. Narvaez^1,2, C. Mauss³, M. Harland-Dunaway³, M. Orozco-Cardenas^2,3, and R. Jinkerson^1,2,3. ¹Department of Chemical and Environmental Engineering, Bourns College of Engineering, ²Plant Transformation Research Center, and³Department of Botany and Plant Sciences, College of Natural and Agricultural Sciences, University of California, Riverside, Riverside, CA 92521. Email: annie.shelton@email.ucr.edu

Plant calli serve as an essential tool in plant biotechnology for genetic transformation and micropropagation. However, traditional methods rely on a single-use agar gel-based substrate that necessitates frequent media replacement or subculturing every 10-14 days. This process not only consumes a significant amount of time but also generates a considerable amount of waste. To overcome these challenges, modern culturing techniques are exploring more sustainable and efficient alternatives, including the use of liquid media. This study aimed to develop an in vitro protocol for Solanum lycopersicum cv. Micro-Tom callus maintenance, shoot regeneration, and fruiting in liquid medium. Cotyledons and true leaves were transferred onto a liquid MS-based medium supplemented with zeatin, while the same medium solidified with 0.7% agar was used as a control. Zeatin concentration was reduced for both mediums on day 16 and eliminated in vitro liquid day 42; in vitro agar day 52. Callus development occurred in vitro liquid on day 15, while in vitro agar took 30 days. Shoot regeneration initiated, and budding occurred in vitro liquid day 30; in vitro agar day 52. Flowering and fruiting were achieved in vitro liquid day 52; in vitro agar day 74. Fruit(s) fully ripened in vitro liquid day 72; in vitro agar day 110. In both solid and liquid mediums, fruits were mainly parthenocarpic. This study's results reveal that callus regeneration is notably accelerated under liquid conditions compared to agar conditions, and flowering and fruiting can be achieved under liquid conditions. It should be noted that vegetative growth and hyperhydricity were more prevalent under liquid conditions compared to solid. Further work is needed to optimize this protocol by reducing hyperhydricity, vegetative growth, and parthenocarpy, as well as improving fruiting efficiency. Using a liquid medium presents a promising alternative to traditional agar-based culture for plant callus culture, with potential benefits in reducing costs, eliminating waste, and increasing time efficiency.

P-2051

Tragopogon angustifolius as Source of Specialized Metabolites for the Control of Broomrape Weeds. A. O. SIDDIQUI^1,2, J. G. Zorrilla^2,3, A. Cimmino², M. Fernández-Aparicio⁴, and M. Masi². ¹Department of Plant Production and Technologies, Niğde Ömer Halisdemir University, 51200 Niğde, TÜRKIYE; ²Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Naples, ITALY; ³Allelopathy Group, Department of Organic Chemistry, Facultad de Ciencias, Institute of Biomolecules (INBIO), University of Cadiz, C/Avenida República Saharaui, s/n, 11510 Puerto Real, SPAIN; and ⁴Department of Plant Breeding, Institute for Sustainable Agriculture (IAS), CSIC, Avenida Menéndez Pidal s/n, 14004 Córdoba, SPAIN. Email: ahmadomid_siddiqui@mail.ohu.edu.tr

Broomrapes (Orobanche and Phelipanche spp.) are obligate parasitic weeds that drastically reduce the yield and quality of several crop species. They use host-induced germination and form haustorium to infect host plants. To date, no registered weed control methods that can selectively manage broomrape species have been reported. Tragopogon angustifolius is a flowering plant belonging to the family of Asteraceae and known for its antioxidant and anticholinesterase activity. However, its ability to produce specialized compounds with phytotoxic and allelopathic roles has never been investigated. As the essential stage for parasitic weed infection is the induction of seed germination by host roots and seedling growth toward the host, the use of plant metabolites to stimulate suicidal germination in the absence of the host has been proposed as a strategy for the control of these weeds. Therefore, the roots of T. angustifolius, collected in southern Spain, were studied for the first time as allelopathy donors using chromatographic and spectroscopic methods to identify compounds that could be used to develop new bioherbicides. Plant material was extracted by maceration with a hydroalcoholic solution and then three different solvents (namely n-hexane, dichloromethane, and ethyl acetate) were used in sequential order to obtain organic extracts containing metabolites with different polarity. Each extract was studied using specific in vitro bioassays on seeds of different broomrape species (Orobanche crenata, Orobanche cumana, Orobanche minor and Phelipanche ramosa), which resulted in the discovery of a remarked stimulatory activity for all the extracts. Furthermore, the thin-layer chromatography analysis of the active extracts showed a variety of metabolites that could be strongly related to the stimulatory activity. Their NMR analysis revealed the presence of aromatic compounds, Further research will be of interest for discovering new natural products for developing new agrochemicals to control broomrapes through the suicidal germination strategy.

P-2052

In Vitro Regeneration of Passiflora organensis from Leaf and Internodal Segments. CAROLINA R. OLIVEIRA¹, A. P. Ramos¹, M. C. Dornelas² and A. P. Martinelli¹. ¹Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Piracicaba, SP, BRASIL, 13416-903 and ²Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, BRASIL. Email: adriana@cena.usp.br, carolinna.rossi1987@gmail.com

Passiflora species have commercial, ornamental, and phytochemical potential, still unexplored in many species. As the first species of the genus to have its genome completely sequenced, Passiflora organensis, subgenus Decaloba, has good potential to establish itself as a model plant, allowing its use for diverse developmental and phytochemical studies, among others. Well-defined regeneration protocols are essential, mainly aiming at genetic transformation. The objective of this work was to evaluate the regeneration process in P. organensis. Seeds were germinated in vitro and plants were maintained in a double magenta system containing MS medium with half the concentration of salts and vitamins. Leaf and nodal segments were then sectioned and submitted to different combinations of BAP/ANA, respectively (mg/l): T1=control, without plant regulators, T2=0.1/0.1, T3=1.0/0.0, T4=2.0/0.0, T5=1.0/1.0, cultivated under 16 h of photoperiod (light intensity of 100 μe.m^-2.s^-1), or in the absence of light. After 30 days, they were subcultured to MS medium without growth regulators, or to MS medium supplemented with 0.5 mg/L BAP + 0.1 mg/L ANA, and cultivated under 16 h of photoperiod. Shoot buds obtained were transferred to ½ MS medium without growth regulators, in test tubes, for elongation and rooting. Callus and shoot buds were observed in all treatments. A greater formation of callus was observed in T2 and T3 kept in the dark (1 month), both in leaves and in internodes when compared to the other treatments. Explants maintained under a 16 h photoperiod showed less callus formation. In the dark, leaf explants showed greater regeneration of buds, compared to intermodal explants, and when transferred to 16 h of photoperiod, they showed a higher percentage of regeneration when supplemented with plant growth regulators in relation to the control. Shoots were elongated well and produced roots in ½ MS medium. This protocol shows efficiency and good characteristics to be used for genetic transformation of P. organensis.

P-2053

Histological Analysis of the In Vitro Regeneration Process in Setaria viridis Shows Indirect Organogenesis in Callus Derived from Mature Seeds. E. C. R. TAVANO¹, K. R. Silva¹, M. Alves-Ferreira², and A. P. Martinelli¹. ¹Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Piracicaba, SP, BRASIL, 13416-903 and ²Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, BRASIL, 21941-902. Email: adriana@cena.usp.br, tavano@usp.br

Setaria viridis is used as a model plant for C4 metabolism, thus well-defined in vitro regeneration and genetic transformation are important for efficient results. The transformation process is mainly performed via Agrobacterium tumefaciens using callus developed from mature seeds. For improvement of the transformation efficiency, each step, including callus induction, morphogenesis, and plant regeneration, can contribute. In vitro morphogenesis can occur through organogenesis or somatic embryogenesis, either directly or indirectly through callus. Our objective was to investigate the regeneration process from callus of S. viridis accession ME034V, through histological analysis. Callus was obtained from mature seeds cultivated on callus induction medium (CIM), consisting of MS salts, with (mg/l) thiamin (0.1), pyridoxine (0.5), nicotinic acid (0.5), biotin (0.5), 2,4-D (2.0), kinetin (0.5), biotin (1.0), myo-inositol (100), cupric sulfate (0.6), sucrose (30 g/l), phytagel (4 g/l), pH 5.8. After fifty days, two different types of callus were observed, white friable callus and areas of compact and cream-colored callus, the latter considered morphogenic. Morphogenic callus was subcultured to CIM medium and after ten days histological analysis was done. Morphogenic callus showed small cells with dense cytoplasm and prominent nuclei; non-morphogenic regions are formed by large vacuolated cells. After fifty days, we observed the development of several adventitious shoots from callus clumps, composed of leaf primordia and apical meristem, formed from the morphogenic portion of the callus, characterizing the regeneration process as indirect organogenesis from mature seeds. Although a higher auxin:cytokinin ratio may form somatic embryos in different protocols, for this species and culture conditions the structures observed presented only the shoot pole, with roots being developed after subculturing to rooting medium. This finding can contribute to the improvement of the protocols for the regeneration and transformation of this model plant.

P-2054

Production of Polyploid Mandarin Hybrids from Seeds Extracted from Selected Triploids. AHMAD A. OMAR^1,2, Azza H. Mohamed^2,3, and Jude W. Grosser¹. ¹Citrus Research and Education Center, University of Florida, IFAS, Lake Alfred, FL 33850; ²Biochemistry Department, Faculty of Agriculture, Zagazig University, Zagazig 44519, EGYPT; and ³Agricultural Chemistry Department, Faculty of Agriculture, Mansoura University, Mansoura 33516, EGYPT. Email: omar71@ufl.edu

New fresh citrus fruit varieties need to be seedless or nearly seedless to gain traction in the US or international marketplace. Citrus breeders have used multiple strategies for generating new seedless varieties. One of these has been to generate triploids from interploid crosses, as triploids generally produce seedless or nearly seedless fruit (one or a few seeds per fruit). In this study, we used flow cytometry analysis to identify the ploidy level of individuals recovered from triploid-derived seed that were germinated in vitro. We collected fruits from three different triploid mandarin hybrids previously selected in our breeding program for superior quality fruit. Those hybrids are Gator Bite [Clementine x (Nova mandarin + Osceola citrus hybrid), C1-8-29 [FallGlo tangerine x (Murcott mandarin + LB8-8)], and KE9-9 [FallGlo tangerine x (Murcott mandarin + LB8-8)]. After the seeds were extracted from the fruit, they were categorized into large, medium, and small seeds based on their size. For the large and medium seeds, the integument was removed, and the seeds were disinfected before they were placed in the seed germination medium for about two months. In addition to evaluating how many seeds germinated, flow cytometry was also used to determine ploidy. There were 2X, 3X, and 4X plants among the germinated seedlings. For the purpose of confirming the parent's allele source, SSR markers will be used. In a preliminary experiment, 2X, 3X, and 4X seedlings recovered from a superior triploid selection were all of zygotic origin. If all or most of these recovered triploids in the present study are zygotic, this could provide a powerful new breeding method to develop new seedless varieties. In addition, zygotic tetraploids could potentially be used as new breeding parents to produce triploids through further interploid crosses with complementary diploid parents.

P-2055

Chesapeake Watershed Protection from Dairy Runoff: Duckweed Gene Expression Optimization Using Bioluminescent Reporter Genes and Improved Vacuum Infiltration. M. CLIPSHAM, N. Thompson, and W. Curtis. Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802. Email: mjc7108@psu.edu

Duckweed is an aquatic plant that displays rapid uptake of nutrients from farm waste that are problematic for nitrogen management. Our goal is to ectopically express genes to improve amino acid composition in duckweed for livestock feed. To achieve optimal expression, we developed an effective reporter gene that is delivered into Lemna minor duckweed as a transient expression vector and transformed genes into duckweed. In preliminary studies, we observed two issues: (1) damage to duckweed during vacuum infiltration resulting in a lack of transient expression, and (2) autofluorescence that interferes with typical GFP reporters. We switched to using a combination of NanoLuc™ luciferase(NLuc) containing the Arabidopsis AGO intron, a bioluminescent reporter gene that avoids background interference, and the p35s:RUBY construct to be able to make quick high quality visual assessments of the expression in L. minor. These changes to the vacuum infiltration protocol have successfully achieved transient expression in L. minor fronds, something not previously reported in the literature. The working methodology involves agrobacterium-mediated vacuum infiltration of the duckweed with MS media as the infiltration buffer, and co-culture of the agrobacterium and duckweed in MS media with acetosyringone for six days. We have successfully transformed NLuc into L. minor, a gene which has a histidine and methionine level over twice that of L. minor. The current method of infiltration still displays large variability with only a few fronds expressing the gene of interest after an infiltration. Future work includes co-infiltration of anti-gene silencing genes with the gene-of-interest; wounding the duckweed to determine if the cause of decreased expression is low infiltration efficiency or gene silencing in the fronds; and transformation of L. minor with genes with increased histidine and methionine content. Beyond our own efforts in successful initial transient expression in L. minor duckweed, this research represents progress towards a circular nitrogen bioeconomy.

P-2056

Efficient Production of Transgenic Wheat Plants Using Particle-mediated Bombardment and Screening of Gene-edited Lines. S. GAUTAM ^1,2, F. Jiang¹, C. Harvey¹, T. Lam¹, A. Laroche¹, and J. Laurie¹. ¹Lethbridge Research and Development Center, Agriculture and Agri-Food Canada, Lethbridge, AB, CANADA, T1J4B1 and ²Department of Agriculture, Food and Nutritional Science, University of Alberta, Edmonton, AB, CANADA, T6G2R3. Email: sgauta1@ualberta.ca

Wheat (Triticum aestivum) is a complex polyploid crop with a large genome. Genomics research in bread wheat is challenging due to the redundancy present between the three ancestral genomes. Wheat transformation is also difficult, especially when compared to dicots, and is limited to a few germplasm lines. Efficient gene delivery methods and recovery of transformed plants are crucial for genome engineering. This study aimed to develop efficient plant regeneration and recovery of transgenic wheat plants using plant morphogenic genes; Babyboom and Wuschel2 along with our Cas9 constructs. We used these two morphogenic genes to transform immature embryos of two wheat lines; Fielder and AC-Andrew using particle-mediated bombardment. Different levels of hormone were also tested in plant tissue culture and embryos were subjected to heat shock at three weeks post-bombardment. Additionally, the positive transgenic wheat lines were screened for the presence of edits. Results showed that the use of the morphogenic genes facilitated rapid regeneration and recovery of transgenic plants under reduced hormone levels. Heat treatment significantly improved transformation efficiency in both wheat lines. Transformation efficiency in AC-Andrew was approximately 3-fold higher than that of Fielder. This modified tissue culture media and heat treatment can therefore be used to deliver genes of interest into the wheat genome for genome engineering.

P-2057

Inari - We are a SEEDesign^TM Company. J. Beringer, E. Neyrinck, Daelyn Jones, and P. KATHIRIA. Inari Agriculture, Cambridge, MA 02139. Email: pkathiria@inari.com

Inari is designing seeds to help address the need to feed a growing population while reducing the impact of agriculture on the environment. We use our SEEDesign^TM platform to overcome these challenges by understanding the complexity of traits and creating genetic diversity. Our platform integrates Predictive Design and Advanced Multiplex Gene Editing tools to develop resilient seeds that require fewer natural resources and inputs, in a shorter time and lower costs than current approaches. In Predictive Design, we harness the power of data, Artificial Intelligence and cell-based assays to gain an understanding of the genes and pathways that underpin crop performance. Once the target sequences have been identified, we generate new allelic diversity using our Multiplex Editing toolbox to deliver multiple types of changes into elite parental lines. We then provide our improved seeds to our customers through a simple and collaborative go-to-market strategy. We will present illustrations of our technical approaches and product concepts.

P-2058

CRISPR-based Disease Detection Strategy for Candidatus phytoplasma. J. LAGNER¹, Y. Qi¹, Y. Rivera², and E. Newberry². ¹University of Maryland-College Park, College Park, MD 20742 and ²USDA-APHIS, Plant Protections and Quarentine, Laurel, MD 20708. Email: jlagner@umd.edu

CRISPR technology has revolutionized genome editing and transcriptional regulation in living organisms. Beyond these capabilities, CRISPR has also been developed to be a valuable tool in point-of-care pathogen detection. CRISPR-based detection strategies have the capability to produce rapid, robust, and sensitive results on-site and without the usage of expensive technology or trained personnel. Utilizing the trans-cleavage activity of CRISPR-Cas12a nuclease upon cleaving its target sequence, researchers have developed CRISPR-based detection assays to identify the presence of particular sequences of target pathogens. In our lab, we have developed a CRISPR-Cas12a based detection strategy for the emerging and widespread plant pathogen phytoplasma (Candidatus phytoplasma). Many conventional CRISPR-based detection methods require the pre-amplification of a portion of the pathogen genome to increase sensitivity limits, but this often leads to off-target amplification and false positives. Our developed assay is a pragmatic improvement upon these established methods which improved the sensitivity limit without the use of a pre-amplification step. Our system incorporates engineered Cas12a variants, sophisticated redesign of reporter oligonucleotides, and careful genomic consideration from over 7,000 phytoplasma sequences in NCBI database to select the most optimal target sites. We demonstrate that our CRISPR-based detection strategy is as sensitive and specific as current detection methods, like qPCR, with the potential to push the detection limit even further. Based on these developed principles, our system has the potential to be used to detect any DNA sequence from the pathogen of interest, thus preventing spread of plant disease.

P-2059

The Plant Genetic Engineering Network (PlantGENE): Advancing Plant Transformation Through Community Engagement. A. A. MALZAHN¹, J. Van Eck ^1,2, H. Kaeppler^3,4, B. Gordon-Kamm⁵ , K. Lee⁶ , W. Parrott⁷ , N. Taylor⁸, and V. Veena⁸. ¹Boyce Thompson Institute, Ithaca, NY; ²Plant Breeding & Genetics Section, Cornell University, Ithaca, NY; ³Department of Agronomy, University of Wisconsin-Madison, Madison, WI; ⁴Wisconsin Crop Innovation Center, University of Wisconsin-Madison, Madison, WI; ⁵Corteva Agriscience, Johnston, IA; ⁶Department of Agronomy, Iowa State University, Ames, IA; ⁷Department of Crop & Soil Sciences, University of Georgia, Athens, GA; and ⁸Donald Danforth Plant Science Center, St. Louis, MO. Email: aam275@cornell.edu

The NSF-funded Plant Genetic Engineering Network (PlantGENE) is a community-driven, three-year initiative to address the lack of capacity to meet the global demand for plant transformation and gene editing. PlantGENE fosters scientific networks to promote research, training, and knowledge-sharing. It is led by a group of plant genetic engineering experts who represent academia, industry, and non-profit plant research institutes, and is open to anyone with an interest in the topic. Plant transformation is a critical step in research of functional genomics, gene editing, and genomics-based crop improvement. The advent of editing technologies has increased the demand for transformation capacity across an increasing number of species, but current transformation systems are significantly limited by inefficient and complicated methods, along with a lack of training in transformation biology and lack of expertise in technique. Improving capacity will require 1) research advances across transformation technologies; and 2) enhanced knowledge exchange and training in transformation biology and techniques. The PlantGENE community is now over 500 scientists working in academia, federal agencies, government, non-profit, and industry across 53 countries. Several initiatives have been launched as part of PlantGENE’s mission to facilitate research and to ensure sharing of technology, knowledge, and expertise. First, a website (https://plantgene.atlassian.net/l/cp/mybVDHE0) has been established to host resources such as a directory of plant transformation facilities, a member directory, masterclasses, and protocols. Second, PlantGENE hosts open-access workshops organized by members. The attendance of 400+ people at the first workshop attests to the widespread interest in transformation within the plant community. Lastly, PlantGENE facilitates training opportunities and collaborations which includes organizing grant writing groups, or meetings between transformation stakeholders such as transformation facility directors and funding agency representatives.

P-2060

Complementation of Deconstructed REP- Viral Vectors with REP+ Transgenic Plants as a Step Towards Plant Gene Therapy. NATALIE THOMPSON¹, Nathan Vorodi¹, David Samson¹, Rekha Kandaswamy¹, Vijay Sheri¹, Aliya Fathima¹, Michael Ream¹, Maia Clipsham¹, Sairam Rudrabhatla², and Wayne R. Curtis¹. ¹Department of Chemical Engineering, Pennsylvania State University, State College, PA and ²Department of Biology, Pennsylvania State University, Harrisburg, PA. Email: natalie.thompson@curtislab.org

Plant protection through the use of viral vectors allows for manipulation of established crops in the field. This represents an agile strategy to cope with changing environmental conditions with a minimum number of stable gene integrations. This presentation will describe the current status of our plant gene therapy approach using advanced reporter genes such as luciferase. Transgenic tomato plants expressing a viral replication initiation protein (REP) under the control of the tomato dehydrin promoter were generated, selfed, and homozygous single-gene copy plants were selected using digital PCR. A deconstructed tomato mottle virus viral vector was created by mutation of the ATG start codon of the REP gene as the basis of demonstrating complementation with plant-expressed REP function. Plants transgenic for luciferase under the control of a viral trans-activation protein, are being designed for assessment of viral movement. The presentation will also present progress in developing intron containing reporter genes - two fluorescent reporter genes (mNeonGreen & mCherry) and two bioluminescent reporter genes (mushroom luciferase & NanoLuc™ luciferase) - including the unexpected role of the 5’UTR in Agrobacterium expression during transient gene testing during infiltration. Notably, the reduced autofluorescence background for mCherry can provide for fluorometric quantification, while bioluminescence provides 4 orders of magnitude of expression over the control. Choice of the small Arabidopsis AGO intron within NanoLuc provides for a bioluminescent reporter that maintains the viral genome size for encapsidation. This work illustrates the potential and challenge of achieving complementary working components of a viral gene therapy strategy: only those plants experiencing the stress are treated, and only those plants which are transgenic will amplify the viral vector delivering a plant protective gene. The implications of these results towards rapid crop protection for different plant stresses in the face of rapidly changing global climate are discussed.

P-2061

Exploring the Function and Significance of CrRLK1L and RALF Gene Families in Citrus Under Salt Stress Conditions. OMAR ZAYED and Danelle Seymour. Botany and Plant Sciences Department, University of California – Riverside, Riverside, CA. Email: omarz@ucr.edu

We investigated the CrRLK1L (Catharanthus roseus Receptor-Like Kinase 1-Like) family proteins and their evolutionary relationship with their Arabidopsis counterparts in Citrus. The CrRLK1L family plays an important role in the development and growth in plants. Specifically, these proteins are involved in a variety of cellular processes, including cell differentiation, cell wall biosynthesis, pollen tube growth, and root hair development. They also play a role in plant defense against pathogens. Additionally, CrRLK1L proteins have been implicated in salt and drought stress response, indicating their potential importance in plant adaptation to changing environmental conditions. By comparing the sequences of 17 Arabidopsis CrRLK1L proteins with the protein database of Citrus, 255 CrRLK1L-like proteins were identified in 14 different species. Phylogenetic analysis revealed Citrus-specific clades and a total of 11 conserved motifs were predicted among the 255 citrus CrRLK1L members. Moreover, we confirmed that Citrus orthologs of Arabidopsis FERONIA (FER) have similar roles in responding to salt-stress. The tissue-specific expression patterns of CrRLK1L genes in Citrus suggested that they may have distinct biological functions, and further exploration is necessary to determine their roles in specific physiological processes. The small up-regulation of FER, THESEUS1 (THE1), and HERCULES RECEPTOR KINASES 1 (HERK1) in response to salt treatment in Citrus and Arabidopsis suggests that phosphorylation modifications may play a role in salt stress response. This study contributes to our understanding of the roles of CrRLK1L and RALF family genes in Citrus growth and development and their potential involvement in salt response, thus shedding light on the mechanisms underlying the regulation of salt response in this plant species.

P-2062

Nematicidial Activity of Lytic Peptides and Silver Nanoparticles Against the Root-knot Nematode (M. incognita). G. C. BERNARD¹ J. Jaynes¹, D. Bedi², M. Egnin¹, C. Bonsi¹, D. Mortley¹, A. Lockett¹, O. Idehen¹, A. Brown¹, and I. Ritte¹. ¹Department of Agriculture and Environmental Sciences, College of Agriculture, Environment and Nutrition Sciences, Tuskegee University, Tuskegee, AL 36008 and ²Department of Biomedical Sciences, College of Veterinary Medicine, Tuskegee University, Tuskegee, AL 36008. Email: gbernard@tuskegee.edu

Plant-parasitic nematodes (PPNs) are an enormous constraint in agriculture. Each year, billions of dollars in crop losses are attributed to plant diseases caused by PPNs. Conventional control methods may have an environmental impact, necessitating the development of more eco-friendly approaches. This study evaluated a lytic peptide, 4E1, and silver nanoparticles (AgNPs) as potential biopesticides under in-vitro and soil treatment conditions. In vitro study: Juvenile root-knot nematodes (J2s) were exposed to 4E1 at 50 and 100um concentrations for 48 hr under aqueous conditions compared to water-treated controls. The numbers of living and dead nematodes were quantitated to determine the nematicidial activity of the peptide. Soil study: One-month-old tomato (S. lycopersicum) plants were propagated in a 50:50 sterilized field soil/potting soil mix and divided into nanoparticle-treated, nematode-infested, and control groups and replicated five times. AgNPs were synthesized utilizing a naturally occurring biopolymer (chitosan) as a reducing agent through microwave irradiation. Approximately 2,000 J2s were added to the treated, infested groups as plants were watered ad libitum. After two months, the plant roots were collected from all groups, where galling, egg numbers, and nematode levels in the soil were quantitated for statistical analysis. The results from in-vitro studies concluded with a significant (p < 0.05) decrease in living juveniles compared to non-treated controls after 48 hr exposure. Due to nanoparticle treatment, the numbers of eggs, galls, and soil nematode numbers were significantly decreased (p < 0.05) in treated groups. Our investigation highlights two potential alternative nematode control measures to mitigate root-knot nematodes in crop production.

P-2063

Transcriptomic Profiling of Drought Responsive Genes Differentially Expressed Under Water Deficit Stress in Cowpea Cultivars (Vigna unguiculata L. Walp). INOCENT P. RITTE, M. Egnin, O. Idehen, D. Mortley, G. C. Bernard, and C. Bonsi. Plant Biotechnology and Genomics Research Lab, College of Agriculture, Environment and Nutritional Sciences, Tuskegee University, AL 36088. Email: iritte8222@tuskegee.edu, megnin@tuskegee.edu.

Cowpea is a nutritious legume valued for its high protein, vitamins, and minerals content. Drought-stress is one of the major environmental factors limiting production of cowpeas around the world. The molecular mechanisms underlying drought tolerance in cowpeas are not fully understood due to the complexity of drought tolerance trait genes involved. This study investigates the transcriptional changes induced by drought pressure in cowpea leaves to identify drought-responsive genes and pathways involved in tolerance. Four cowpea cultivars (drought tolerant; California blackeye and TVu11987; sensitive Mississippi silver and TVu2428) were subjected to 14-days without watering using a modification of wooden box experiment under greenhouse conditions. Two timepoints gene expression analyses were performed on control and stressed plants at 7- and 14-days following drought treatment initiation. RNAseq analyses of four cultivars resulting in the identification of 6761 and 9897 differentially expressed transcripts at 7- and 14-days drought stress libraries respectively. Enrichment analyses using KEGG and GO indicated that various pathways were affected by drought stress, including MAPK signaling, photosynthesis antenna proteins, ribosome biogenesis, phenylpropanoid biosynthesis, plant hormone signal transduction, nucleotide metabolism, Flavonoid biosynthesis, and Fatty acid biosynthesis. Additionally, five genes possibly involved in regulation of transcription responses to drought stress were discovered. These were Ribulose bisphosphate carboxylase/oxygenase activase, Fructose-bisphosphate aldolase-1, Chlorophyll a-b binding protein-3, Linamarin synthase-2, and NADP-dependent glyceraldehyde-3-phosphate dehydrogenase. The identified DEGs set platforms for uncovering essential genes in cowpea drought-stress responses. Functional analysis of these genes will help decipher their precise roles in enabling cowpea’s adaptation to adverse environmental conditions. Funded by USDA-NIFA Grants to Tuskegee University: #2017-38821-26414-GE and Evans-Allen # ALX-FVC18

P-2064

Investigating the Co-localization of Histone H3.1 and Mitochondria in Arabidopsis thaliana. S. THAO and K. Blee. Biological Sciences Graduate Program, College of Natural Sciences, California State University, Chico, CA 95929. Email: slthao@csuchico.edu, kblee@csuchico.edu

Histone H3 hosts post-translational modifications responsible for nucleosome formation and gene regulation in eukaryotic cells. De novo synthesis of histone H3 occurs in the cytosol before being trafficked to the nucleus. A previous study in 2019 had incubated crude histone fractions with mitochondrial extracts and performed SDS-Page with anti-H3 antibodies. The results indicated a binding affinity between histone H3 and the mitochondria. The experimental protocol involved incubation of histone H3 with purified mitochondria, and therefore may not accurately represent histone H3 activity in living cells. The purpose of this study is to visualize histone H3 and mitochondria to determine any histone H3 trafficking to mitochondria in intact cells. Transgenic Arabidopsis thaliana plants containing mCherry tagged histone H3 proteins have been cross-bred with plants containing YFP tagged mitochondrial transit peptide from yeast cytochrome c oxidase IV. Fluorescent images from our lab suggest the presence of tagged proteins within root hairs and support the success of the fluorescent tags. Anthers of receiving flowers were removed during early development to prevent self-fertilization. Pollen grains of mature donor flowers were then introduced to isolated stigmata in order to perform the cross. Hybrid seeds have been collected for subsequent plant growth and analysis. Images of parent and offspring root cells will be used to determine the location of histone H3 and the mitochondria. We predict that after layering images of the same cell with different filters, we will find that histone H3 co-localizes with the mitochondria in living cells. The co-localization of histone H3 and mitochondria will provide more insight into histone H3’s structure, function, and affiliation with mitochondria.

P-2065

Expanding the Tissue Range for Somatic Embryogenesis Using the LEC2 Transcription Factor in Theobroma cacao, the Chocolate Tree. J. BHATT, S. Maximova, and M. Guiltinan. IGDP Plant Biology, The Pennsylvania State University, University Park PA. Email: Jishnubhatt@psu.edu

The fruits of the tree Theobroma cacao contain cacao beans that are the major raw material of chocolate. It is a commodity that supports a 170-billion-dollar global industry, yet ~38% of potential global production is lost to plant pathogens. Biotechnology is a powerful tool that can be leveraged to mitigate losses. The genetic transformation system of cacao relies on the production of somatic embryos which require tissues from cacao flowers, limiting biotechnology work only to mature flowering trees. This study tests whether the ectopic expression of a developmental regulating transcription factor, Leafy Cotyledon 2 (TcLEC2) can be used to broaden the range of tissues for somatic embryo production. A time course experiment was performed to record somatic embryo production from tissues from a PSU Sca6 (wild-type) tree and a transgenic TcLEC2-GR tree which expresses an inducible LEC2 transcription factor regulated with the hormone dexamethasone (DEX). Petals, A-B leaves, C leaves, E leaves, petioles, and internodes of both genotypes were placed onto our standard tissue culture regimen, with half provided a 72-hour DEX treatment. Tissues were subcultured for 22 weeks, and somatic embryo numbers and quality were recorded and photographed. Our prior work showed that in wild-type trees, only petals were capable of somatic embryo production. Consistent with prior observations, only petals of both genotypes produced embryos without DEX treatment. Remarkably, after DEX treatment, all TcLEC2-GR explants except stage E leaves were subsequently capable of regenerating somatic embryos. This study demonstrates that using developmental regulating transcription factors such as LEC2 can improve the embryogenic potential of previously recalcitrant tissues. Expanding the tissue range for somatic embryogenesis obviates the requirement of mature flowering trees and permits the use of non-sexual somatic tissues from immature seedlings as viable explants for genetic transformation. This has implications for accelerating propagation and genetic improvement of the chocolate tree.

P-2066

Establishment of DNA-Free Genome Editing Protocol in Hexaploid Sweetpotato (Ipomoea batatas L.) Protoplast and Regeneration. ADRIANNE BROWN¹, M. Egnin¹, and F. Bukari². ¹Plant Biotech and Genomics Research Laboratory, Department of Agriculture & Environmental Sciences, Tuskegee University, Tuskegee, AL, 36088 and ²Heliae Development, LLC, Gilbert, AZ. Email: abrown9633@tuskegee.edu

New plant breeding technologies (NPBTs) like CRISPR/Cas systems provide opportunities enable precise alterations to a specific location in the genome of many living organisms which can be used to make improvements. Routine delivery systems of Cas9/gRNA tools are encoded within plasmid vectors and shuttled into plant cells through Agrobacterium-mediated transformation or particle bombardment. However, latest breakthroughs in its delivery involve transfection of Cas9 and gRNAs as pre-assembled ribonucleoproteins (RNPs), which avoids the integration of exogenous DNA. The focus of this investigation was to optimize a protocol for Cas9 RNP transfection using the chemical fusogenic polyethylene glycol (PEG) in hexaploid sweetpotato. Prior to transfection, a total of six out of thirteen sgRNAs were screened to target three classes of the eukaryotic translation initiation factor 4E superfamily (eIF4E, eIF(iso)4E and Cap binding protein (CBP)) using In-vitro assays. Concurrently with protoplast isolation, crRNA and tracrRNAs were hybridized together then complexed with the Cas9-GFP protein using 25% and 40% PEG concentrations. Following transfection, 96 explants were used for nurse culturing putative edited protoplast. Out of the 96 co-cultivated explants, 30% of explants regenerated from 25% PEG transfection samples into plantlets. Regenerants were then screened using T7 mutation detection and Sanger sequencing methodologies. Of the screened regenerants, 15% were detected to display translocation and deletion mutations. Our results demonstrate Cas9 RNPs ability to modify translation initiation factors in sweetpotato. Future studies will investigate disease challenge responses to SPFMV to protect sweetpotato production. Further investigation is required to optimize Cas9 RNP transfection and regeneration in other sweetpotato varieties. Funded by USDA-NIFA Grants: 2017-38821-26414-GE, and Tuskegee University CAENS-GWCAES-NIFA-EVANS-ALLEN.

P-2067

The Impacts of Light Intensity of White LEDs on the Transient Expression of GUS Gene in Soybean (Glycine max) using Agrobacterium-mediated Transformation with Half-seed Method. X. SHI¹, B. Li², and R. Hernández¹. ¹Department of Horticultural Science, College of Agriculture and Life Science, North Carolina State University, Raleigh, NC 27695 and ²2408 Heathlan Farm Drive, Fuquay Varina, NC 37526. Email: rhernan4@ncsu.edu

Soybean (Glycine max) is an important crop with great economic and food security value worldwide. Soybean plants are constantly studied and improved for better agronomic traits through biotechnology techniques. Agrobacterium-mediated transformation is commonly used to deliver desired genes to the meristem of the half seeds. However, the efficiency of the gene delivery is low, hindering the discovery progress of soybean. Light promotes meristem growth. Additionally, light is involved in signaling pathways in plants, especially relating to stem cell activities. The half-seed explants undergo a co-culture phase with Agrobacterium for five days under light prior to transgenic shoot regeneration. A transient expression is a practical approach to studying Agrobacterium infection efficiency in a short period. The present study hypothesizes that increasing light intensity during co-culture increases the transient expression percentage of GUS gene. In this experiment, various light intensities of white LEDs (50, 100, 150, 190 μmol·m^-2·s^-1) and fluorescent light (100 μmol·m^-2·s^-1) were implemented on half-seeds during co-culture for five days. The meristem area of half-seed explants was examined for GUS transient expression by GUS histochemical assay. The meristem area with distinct blue circles was considered a transient event. Results indicated that the percentage of GUS transient expression was low under high light intensity during co-culture. The light intensity of 50 and 100 yielded the highest transient expression percentage, while fluorescent light treatment delivered 10 % less transient expression than white LED at 100 μmol·m^-2·s^-1. Furthermore, this research outcome justifies investigations of the effects of various light intensities during co-culture on stable transformation efficiency. This research project was funded and guided by BASF.

P-2068

Lethal Response of Orally Delivered V-ATPase dsRNA in the Colorado Potato Beetle Leptinotarsa decemlineata. H. TARIQ¹, A. Gökçe¹, M. H. Hashmi², E. Aksoy³, and A. Bakhsh⁴_. ¹Department of Plant Production & Technologies, Ayhan Şahenk Faculty of Agricultural Sciences and Technologies, Niğde Omer Halisdemir University, Niğde, TÜRKIYE; ²School of Science and Technology, Institute of Biology, University of Siegen, GERMANY; ³Department of Biological Sciences, Middle East Technical University, Ankara, TÜRKIYE; and ⁴Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, PAKISTAN. Email: haneeftariq1@gmail.com

RNA interference (RNAi) is a versatile genetic tool that can selectively inhibit the expression of any gene in a targeted organism. The implementation of this technology offers the possibility of preventing crops from being infested by insect pests and diseases. In this study, double-stranded RNA (dsRNA) was synthesized using L4440 vector and Escherichia coli HT115 strain to silence the V-ATPase mRNA in Colorado Potato Beetle (CPB) which is essential for a range of physiological processes. The silencing effect of RNAi was evaluated by feeding potato leaflets treated with dsV-ATPase. The feeding bioassays resulted in significant mortality, reduction in foliage consumption, and weight gain in all the larval instars. The relative transcript expression of the V-ATPase gene was quantified using quantitative reverse transcription PCR in the treated insects. The results indicated a significant reduction in the expression level of the V-ATPase gene in larvae feeding on dsV-ATPase. The findings of this study illustrate that utilizing RNAi-mediated gene silencing to target essential genes represents a potential strategy for managing insect pests.

P-2069

CRISPR-Cas12a Base Editors Confer Efficient Multiplexed Genome Editing in Rice. YANHAO CHENG¹, Yingxiao Zhang^1,2, Gen Li¹, Hong Fang¹, Simon Sretenovic¹, Avery Fan¹, Jiang Li³, Jianping Xu³, Qiudeng Que², and Yiping Qi^1,4. ¹Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742; ²Seeds Research, Syngenta Crop Protection, LLC., 9 Davis Dr, Research Triangle Park, NC 27709; ³Syngenta Biotechnology China, Ltd., 25 Life Science Park Rd., Zhongguancun Life Science Park, Beijing 102206, CHINA; and ⁴Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850. Email: cyh2019@umd.edu

Many Cas9-derived base editors have been established for precise C-to-T and A-to-G base editing in plants. These editors typically rely on a SpCas9 nickase or its engineered variants with altered protospacer adjacent motif (PAM) requisites. CRISPR-Cas12a facilitates highly efficient multiplexed genome editing in plants, with its T-rich PAM preference complementing the G-rich PAM requirement of SpCas9 in genome targeting. However, developing efficient Cas12a base editors has been challenging due to the absence of an effective Cas12a nickase. Despite this challenge, Cas12a cytosine base editors (CBEs) and adenine base editors (ABEs) have been successfully developed in mammalian cells with minimal DNA damage, leveraging deactivated Cas12a (dCas12a). Nevertheless, efficient dCas12a base editors for plants remain to be developed. In this study, we have developed highly efficient Cas12a CBEs and ABEs for multiplexed genome editing in plants. These T-rich PAM-targeting dCas12a base editors, possessing a lower potential to generate DNA breaks compared to Cas9 nickase-based base editors, are ideal for multiplexed promoter editing to fine-tune gene expression without inducing indel mutations. Our success is founded on the integration of an efficient CRISPR-Cas12a expression system, a high-efficiency LbCas12a-D156R variant, high-activity cytidine and adenine deaminases, and optimal linkers. The optimized Cas12a CBEs yielded highly efficient monoallelic editing at high-activity target sites. Notably, the dLbCas12a-D156R ABEs developed in this study appear to exhibit efficiency comparable to Cas9 ABEs, resulting in simultaneous editing of up to six target sites and generating biallelic edits at four target sites in T0 rice lines. These efficient DNA break-free Cas12a CBE and ABE systems hold promise as valuable tools for singular and multiplexed base editing in plants.

P-2070

Trends of Seed Yam Propagation Technologies and Prospects for Enhanced Development and Delivery of Improved Varieties in Nigeria and Ghana. MORUFAT BALOGUN^1,2,6, Norbert Maroya^4,6, James Legg⁵, Lava Kumar¹, Olufisayo Kolade¹, Beatrice Aighewi³, Robert Asiedu¹, and Djana Mignouna⁴,¹International Institute of Tropical Agriculture (IITA), Ibadan, NIGERIA; ²Department of Crop Protection and Environmental Biology, University of Ibadan, Ibadan, NIGERIA; ³International Institute of Tropical Agriculture (IITA), Abuja, NIGERIA; ⁴International Institute of Tropical Agriculture (IITA), Cotonou, BENIN; and ⁵International Institute of Tropical Agriculture (IITA), TANZANIA; ⁶Retired. Email: m.balogun@cgiar.org

The development of sustainable formal seed systems can unlock the potential of yams (Dioscorea spp) for food security and wealth creation, but is hindered by slow rates of vegetative propagation and high disease accumulation. Research efforts have produced significant breakthroughs in high ratio propagation technologies (HRPTs: thermotherapy-TT; conventional tissue culture-CTC; temporary immersion bioreactors-TIBS; aeroponics, hydroponics, leaf bud cuttings; and adapted yam minisett technique). However, irregular breeder seed (BS) availability is a threat. In order to support BS demand planning and catalyze delivery of genetic gains in farmers’ fields, current BS production systems were evaluated. Structured questionnaire was administered to four public institutions and two public-private seed companies. Data were collected on HRPT awareness and functionality, quality control, storage, demand and supply for improved varieties. Data were analyzed using descriptive statistics. Up to 83% of respondents were aware of TT but complained of the slow rate of disease elimination while 17% had facilities for virus testing. IITA-GoSeed, Nigeria and the Crops Research Institute, Ghana has the strongest capacity and were able to access at least 90% of HRPTs. All respondents were willing to pay for clean initial stocks of clean materials of nine identified improved varieties. Capacity development in nutrient optimization, production and business planning were prioritized needs. Only 17% had adequate BS storage facility. High operational costs, inadequate technical know-how, erratic electricity, and irregular availability of nucleus stock plantlets to feed TIBS systems were threats. It will be necessary to incorporate locally available, cheaper sources of supplies and build personnel capacity in TC for sustainable, prompt access to BS. These breakthroughs will enhance phenotyping, conservation, quality control, dissemination and uptake of improved varieties. Partnerships for alternative seed markets and marketing approaches are also crucial.

P-2071

Study of Selected Stevia Plant (Stevia rebaudiana Bertoni) Grown and Selected from the USDA Hardiness Zone 8A, Located at the CAFST, FVSU Research Plots. A. SAHA, A. Howlader, N. Mullen, A. Roberson, and B. Biswas. Specialty Plants by Biotechnology Research Lab., College of Agriculture, Family Sciences & Technology, Fort Valley State University, Fort Valley, GA 31030. Email: biswasb@fvsu.edu

In vitro biotechnology has been used in this study as it is the best tool for rapid plant micropropagation. Stevia plant selection is also used in this study as it is essential for having desired plants with high Reb A, Reb D or Reb M along with cold tolerant potential. Stevia plants are heterozygous as they are self-incompatible, therefore, selection of plants with high steviol glycoside content and cold tolerant plants are desired. After selecting the desired plants, micropropagation is the right method to clonally propagate to have high glycoside and cold tolerant potential in their offspring. Because in vitro propagation ensures to multiply true to type plants not seed propagated stevia. Moreover, in vitro propagation can produce millions of clones (true-two-type) rapidly in small space, in limited time, which is required to supply to the farmers. In this investigation stevia plants were selected based on frost tolerant and high glycoside content grown at the CAFST-FVSU (College of Agriculture, Family Science & Technology, Fort Valley State university) research plots. Plants were collected from the field and were surface sterilized in Clorox solution inside laminar airflow hood. After the last rinse with autoclaved distilled water each explant was taken out from the flask and excised in one-centimeter nodal explant using scalpel blade & forceps. Each nodal explant was inoculated on culture media for multiplication using MS medium with BAP, Kinetin, NAA & Adenine Sulfate in various concentrations (0, 0.1, 0.5, & 1mg/ml) alone or in combination. Cultures were incubated at 25+/- 2 C and 16/8 light/dark photoperiod. Observation was taken every week for responses and some pictures and data were recorded. The data and pictures will be presented during the conference.

P-2072

Leaf Transformation of Different Maize (Zea mays L.) Genotypes and Elite Inbred Lines. RITESH KUMAR¹, Jo Búi¹, Ning Wang², William Gordon-Kamm², and Joyce Van Eck^1,3. ¹Boyce Thompson Institute, Ithaca, NY 14853; ²Corteva Agriscience, Johnston, IA; and ³Section of Plant Breeding and Genetics, School of Integrative Plant Sciences, Cornell University, Ithaca, NY 14853. Email: rk662@cornell.edu

Maize is one of the most important model crops for genetics and biotechnology research. It is used as livestock feed as well as for human consumption. Increasing populations and climate change require maize improvement by the introgression of desirable traits into agriculturally important genotypes. Genetically modified maize development is extremely challenging due to genotype-related recalcitrance to transformation. Transformation of immature embryos is the most widely used method for maize. It is a very labor-intensive method that requires a high level of expertise and efficient greenhouse facilities are needed for growing maize year-round. However, leaf transformation of maize has been recently reported. Unlike maize embryo transformation, leaf transformation uses young seedling leaves, which are more readily available than embryos. The approach utilizes ectopic expression of growth-stimulating morphogenic genes during the early stages of leaf transformation. Our key interest is to study and improve maize leaf transformation of various agriculturally important genotypes and elite inbred lines. For this study, we used a previously published leaf transformation method, which utilizes a construct containing a maize-optimized Baby Boom (Bbm) and Wuschel2 (Wus2) developmental gene combination (Wus2/Bbm). The construct also includes a green fluorescent protein (GFP) gene for assessing transformation progress. A total of nine genotypes are being used in the study: HC69, W22, LH244, PH05F, PH2N0, LH245, LH287, 90DJD28 and 01INL1. To date, we have found that HC69 is most amenable for the recovery of transgenic plants, W22 produces more GFP-expressing calli than other genotypes, and LH244 showed a lower response to the transformation method.

P-2073

Deciphering Hidden Mechanisms in the Biomagnetic Response in Plants: A Study on the Effects of Magnetic Fields on Plant Metabolomic Responses. A. LOCKETT, M. Egnin, E. Bonsi, C. Bonsi, D. Mortley. A. Brown, I. Ritte, O. Idehen, and G. C. Bernard. Plant and Soil Sciences Graduate Program, College of Agriculture, Environment, and Nutrition Sciences, Tuskegee University, Tuskegee, AL 36088. Email: Alockett7364@tuskegee.edu

Plants sense and respond to environmental stimuli (light wavelengths, gravity, touch, electromagnetic stimulation, and geomagnetic fields) with alterations at the molecular level, expressed through physiological changes in growth and development. However, the effects of applied magnetic fields on plants' molecular responses are poorly documented. Thus, a comprehensive understanding of plant magneto-reception dynamism necessitates more in-depth approaches. Our study focused on identifying metabolite expression patterns in tomatoes ( S. lycopersicum) and collard (Brassica oleracea var. viridi) seeds exposed to a uniform magnetic field of 4.7 Gauss for 2hr/day following a six-day duration. Metabolomic extraction was completed immediately after exposures, and analysis was performed by high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS). Using Pathway and Enrichment analysis, 140 compounds for both species and trials were identified as significant (p≤ 0.05) and grouped into metabolite pathways. Over-representation analysis (ORA) identified the biosynthesis of the unsaturated fatty acid pathway as the highest expressed metabolite pathway (p < 0.05), followed by the linoleic acid, starch and sucrose metabolism, and pantothenate and CoA biosynthesis pathways due to treatment. Orthogonal principal least squares discriminant analysis (OPLS-DA) illustrates significant differences in metabolite expression among exposed and control groups. In both collard trials 1,2, C3 H N3 O3 P2, an unidentified compound, was identified as the most significantly expressed metabolite, downregulated due to treatment. In tomatoes, trials 1, 2 Pantothenic acid was identified as the most significantly expressed metabolite, upregulated due to treatment. Overall, this evaluation can contribute to the fundamental understanding of magnetic field effects on plant metabolomics. Developing specific magnetic field exposure protocols may affect the expression of bioactive compounds in plant seeds.

P-2074

Tissue Culture Studies on Strawberries. B. A. DONMEZ¹ and N. E. Kafkas^1,2.¹Biotechnology Department, Institute of Nature and Applied Sciences, Cukurova University, Adana, TURKEY, 01250 and ²Horticulture Department, Cukurova University, Adana, TURKEY, 01250. Email: betulayca96@gmail.com

Strawberry (Fragaria ×ananassa Duch.), a temperate climate fruit, belongs to Rosaceae family emerged as a result of the accidental hybridization of the northern (F. virginiana) and southern (F. chiloensis) hemispheres at the end of the 18th century. Even it was originated in North and South America, cultivated strawberry has been growing throught many temperate regions in the world thanks to its widely adaptation ability. Since its high nutrient composition for human health and the earliest fruit ripened ability in spring among the grape-like fruits, strawberry ranks first compared with other berry fruits in terms of production and consumption in the world. It has been considering that It also has been considering that strawberry is a high source of antioxidants and may be beneficial in the prevention of cancer by showing anti-inflammatory, anti-carcinogenic and anti-mutagenic effects against human diseases. Beside of its high fresh consumption, strawberry has been used as a processed product, frozen fruit and beverages in food industry. Plant tissue culture (PTC), a branch of plant biotechnology, is a technique to obtain large amount of genetical identical and disease free plants through culturing parts of plants on synthetic plant medium under controlled conditions in shorten time. In this review, recent studies on multiplication of strawberry in vitro condition will be summarized. This review could benefit to enhancement of an efficient protocol for in vitro multiplication of strawberry plant, genetic transformation and genome editing studies.

P-2075

Time Series Analysis of the Vegetation Cover Index and Radar in Vineyards of Grapes (Vitis vinifera) for Different Seasons Using Remote Sensing Data. MOSTAFA M. ABDELMEGEED¹ and Shymaa A. Ahmed²_. ¹Alexandria University, 22 El-Gaish Rd, Al Azaritah WA Ash Shatebi, Bab Sharqi, Alexandria Governorate 5424041, EGYPT and ² Faculty of Agriculture, Sohag University, Sohag Al Gadida City, Sohag Governorate 1646130, EGYPT. Email: Kafy202020@gmail.com

Grape vine growth has a physiological variance with seasonal interaction between the environment and the genotype of the plant By studying these changes and monitoring them through satellite images, you can monitor climate changes and their impact on vineyard the model to study the effect of climate changes through phenological changes on grape vines using time series analysis with satellite images of the seasons and the study of the most important vegetative indicators sentinel 2 and radar bands sentinel 1 suitable for studying these changes. The results show the best radar bands combination provided by satellite Sentinel l and 2, So researcher can use the radar bands recommendation from the results to get best results of tracking the plant coverage over the time using remote sensing satellite.