2024 SIVB Student Award Winners

2024 JOHN S. SONG AWARD

Unmasking the Divergent Roles of MYB Transcription Factors of Mungbean Under Waterlogging and Heat Stress

Extreme weather, such as excessive or untimely rain causing waterlogging and high temperatures, poses significant threats to agricultural productivity and food security. Mungbean, a vital crop with nutritional benefits and positive environmental impact, is particularly susceptible to waterlogging-induced hypoxia and heat damage. This study aims to understand the role of R2R3-MYB transcription factors (TFs), known for their involvement in development and plant defense but unexplored in mungbean. Bioinformatics analyses identified 70 R2R3 MYB TFs in mungbean by confirming the presence of the MYB DNA-binding domain, studying evolutionary relationships with other plants, in-silico protein analyses, exon/intron structures, and conserved motifs. Promoter analyses revealed the presence of cis-acting elements related to growth, development, and stress responses. Selected MYB TF genes were then analyzed for gene expressions at different plant developmental stages (seedling, vegetative, and reproductive) and tissues (leaf and roots) through RT-qPCR under hypoxia and high temperature. Potential candidates were further studied using heterologous systems, and genes were silenced via virus-induced gene silencing (VIGS) to identify RNAi-mediated loss of function phenotype. Out of 70 R2R3 MYB TFs, 10 were selected based on in-silico predictions. Three genes displayed enhanced expression in all three developmental stages and both tissues under waterlogging and heat stress. The genes expressed in E. coli exhibited both hypoxia and heat stress alleviation. Transcriptional activity was confirmed by transcriptional activation of GAL4 gene in yeast vector. VIGS plants showed hampered development and reduced resilience against hypoxia and heat stress, supported by biochemical analyses indicating decreased stress tolerance in MYB TF-silenced lines. These findings suggest the potential exploitation of transcription factors to enhance mungbean’s tolerance to waterlogging and heat stress and can contribute to developing stress-tolerant mungbean varieties.

Prashasti Pandey, Banaras Hindu University, Department of Botany, Institute of Science, Banaras Hindu University, Varanasi U.P. 221005, India. Abstract presentation P-2008.

2024 GORDON SATO AND WALLY MCKEEHAN AWARD

Establishment & Scale-up Potential of Embryonic Insect Cells for Cell-cultivated Meat

Cultivated meat is a growing field dedicated to producing meat from cell culture to provide an alternative to large-scale animal agriculture, which is plagued with environmental, animal welfare, and human health concerns. While most research in the field focuses on “traditional” livestock species (bovine, porcine, avian), insect cells are promising cell sources for cultivated meat products. Insect cells are extremely resilient and adaptable in culture, and may be able to reach low-cost and large-scale culture more easily than “traditional” cells. The goal of this project was to establish an isolation procedure to develop a new insect cell line that is scalable, easily adaptable to animal-free media and generates sufficient biomass to assess nutritional properties. Cells were isolated from Manduca sexta embryos and subsequently adapted to single-cell suspension culture in animal-free growth media. Cell growth data is presented in shake flasks and pneumatic bioreactors and spent media analysis was performed using a Cedex metabolite analyzer to better understand cell metabolic processes. Finally, a preliminary proximate nutritional profile is reported using data from the Eurofins nutritional testing platform. The Manduca sexta non-adherent cells (MsNACs) that were developed were spontaneously immortalized and showed robust growth in animal-component free media, reaching densities over 20 million cells/mL in shake flasks. They were also scaled to 2.4 and 10-liter bioreactors and spent media analysis showed that they preferentially consumed glutamate over glutamine and glucose. Finally, nutritional analyses showed that MsNACs had comparable protein, fat, carbohydrate, and ash breakdown to USDA and FDA-approved chicken cells. This study presents a blueprint for developing, scaling, and analyzing novel insect cell lines for food. 

Sophie Letcher, Department of Biomedical Engineering & Tufts University Center for Cellular Agriculture, Tufts University, Medford, MA 02155. Abstract Presentation A-1012

2024 JOSEPH F. MORGAN AWARD AND 2024 STUDENT TRAVEL AWARD

Combined Psilocybin and Eugenol Synergistically Reduce Inflammation via the Gut-Liver Axis

Chronic liver disease is caused by multiple factors including gut dysbiosis-induced endotoxemia. Recently, psilocybin has demonstrated potent, sub-hallucinogenic, and synergistic anti-inflammatory effects on macrophages and within the brain, however, its effects on liver inflammation are unknown. We hypothesized psilocybin could ameliorate liver inflammation and prevent the development of liver diseases. Initially, psilocybin was combined with an array of transient receptor potential channel agonists and was applied to human small intestinal epithelial cells (HSEIC) and 3D EpiIntestinal tissues which were treated with tumor necrosis factor (TNF)-α and interferon (IFN)-γ to induce an inflammatory response. Psilocybin and eugenol synergistically reduced cyclooxygenase (COX)-2 and interleukin (IL)-6 protein levels in HSEIC and was not cytotoxic. Utilizing 3D EpiIntestinal tissue, psilocybin and eugenol significantly reduced TNF-α, (IFN)-γ, IL-6, and IL-8 protein levels. Next, we injected C57BL/6J mice with lipopolysaccharide (LPS) or orally fed dextran sodium sulfate (DSS) to induce liver inflammation, and orally fed psilocybin (0.88 mg/kg), eugenol (17.59 mg/kg), or combinations of psilocybin and eugenol daily for two days as a pre-treatment or once after inflammation induction as a post-treatment. LPS and DSS upregulated mRNA expression of COX-2, TNF-α, IL-1β, and IL-6, while psilocybin and eugenol significantly ameliorated mRNA levels in the post-treatment. In contrast, pre-treatment did not result in significant improvement markers of liver inflammation. Histology in both LPS and DSS-induced models demonstrated an improvement in liver pathology. While both models demonstrated strong anti-inflammatory properties on the liver, our data demonstrates psilocybin and eugenol act directly on liver inflammation and through the gut-liver axis. Post-treatment with psilocybin and eugenol demonstrates strong potential in treating inflammatory liver diseases and should likely be studied further, including in the clinic.

Gregory I Robinson, Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, T1K3M4, CANADA and Gies College of Business, University of Illinois Urbana-Champaign, Champaign, IL 61820. Abstract Presentation: A-1014

2024 PHILIP R. WHITE AWARD

Engineering Novel Anti-TNFα Biomolecules in Tobacco BY-2 Cells for Oral Treatment of Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) represents a group of intestinal disorders that cause prolonged inflammation of the digestive tract. The current therapeutic strategies, including the conventional anti-inflammatory medications and the emerging biologic drugs targeting the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα), have limited therapeutic efficacy and adverse drug reactions resulted from systemic administration. Achieving colon-targeted oral delivery of anti-TNFα agents is crucial for enhancing the treatment of IBD. Plant cell culture has proved to be a cost-effective bioproduction platform for therapeutic proteins, especially for oral biologics, where plant cell walls act as a natural protective capsule for encapsulated proteins. This project aims to leverage two unique posttranslational modifications – glycosylphosphatidylinositol (GPI) anchor and plants-specific hydroxyproline (Hyp)-O-glycosylation – to design and produce GPI-anchored anti-TNFα biomolecule in plant cells for oral IBD treatment. A designer anti-TNFα biomolecule consisting of an N-terminal single-chain fragment variable (scFv) of an anti-TNFα antibody, appended by a Hyp-O-glycosylation module, and C-terminal GPI anchor were stably expressed in tobacco BY-2 cells. While the GPI anchor “displays” the expressed anti-TNFα biomolecule at the plant cell surface to presumably create a high local concentration of the biologic, the Hyp-O-glycosylation module stabilizes the protein from degradation during both bioproduction and oral delivery processes. The bioactivity of expressed anti-TNFα biomolecule was determined in vitro for its functional activity in binding to TNFα and blocking the TNFα-mediated cytotoxicity by using a murine fibroblast cell line. The therapeutic effectiveness of the orally delivered designer anti-TNFα biomolecules in mitigating the IBD symptom was assessed in a dextran sulfate sodium-induced colitis mouse model. This research potentially establishes a novel platform for producing effective oral biologic drugs for the treatment of IBD.

Paula Perez Sanchez, Department of Biological Sciences, Arkansas State University, Jonesboro, AR 72401. Abstract presentation P-2036

2024 MARIETTA WHEATON SAUNDERS AWARD

Investigation of Genetic Determinants Responsible for Citrus HLB Disease Development via Genome Editing

Citrus is a globally important tree crop. However, its production is facing an unprecedented threat caused by Huanglongbing (HLB) in most citrus producing regions. HLB is caused by the phloem colonizing bacterium Candidatus Liberibacter asiaticus (Las) in most places including USA, China, and Brazil. Previous study suggests that HLB is an immune-mediated plant disease and Las causes systemic and chronic immune response in phloem tissues including reactive oxygen species (ROS) production and callose deposition. Wide-spread cell death of phloem tissues is caused by excessive and chronic ROS production (also known as oxidative stress) triggered by Las. Consequently, cell death of phloem tissues causes HLB symptoms and damage to citrus plant. Consistent with our study, multiple reports show that HLB-tolerant citrus varieties contain higher levels of antioxidants and antioxidant enzyme activities that scavenge ROS than susceptible varieties. Here we aim to investigate roles of immune related genes in HLB disease development. For this purpose, we employed CRISPR/Cas9 to edit select genes including BRI1-ASSOCIATED KINASE-1 (BAK1) and SUPPRESSOR OF BIR1-1 (SOBIR1). We will test the homozygous/biallelic mutants in ROS production, phloem cell death, Las titers and HLB disease development compared to wild type controls.  Identification of genetic determinants responsible for HLB development is critical to develop HLB resistant/tolerant citrus varieties.

Javier Dalmendray, University of Florida, 700 Experiment Station Rd, Lake Alfred, FL 33850. Abstract presentation: P-2042.

2024 STUDENT TRAVEL AWARD

Comparative Analysis of Viral Vector Efficiency for Genome Editing in Cas12a-tomato Plant

Virus-induced genome editing (VIGE) uses viral vectors to transiently deliver CRISPR/Cas reaction components into plants. Tomato (Solanum lycopersicum L.) is a mayor horticultural crop grown worldwide, and a model plant for studying the genetics of fleshy fruit. Modern cultivated tomatoes nonetheless have a narrow genetic basis, which hampers breeding of new varieties with improved traits. The main goal of this study was to develop a VIGE strategy in tomato to speed genome editing. For this, we first generated a transgenic Cas9-expressing line of tomato cv. Micro-Tom (MT-Cas9). To deliver single-guide RNA (sgRNA), we used a potato virus X (PVX)-derived RNA viral vector. As a proof of concept, we first edited the reporter gene PHYTOENE DESATURASE (PDS). To achieve fruit color breeding, we targeted STAYGREEN 1 (SGR1), whose knockout produces a green-flesh phenotype characterized by red-brown fruits. First, cotyledons of MT-Cas9 seedlings were agroinoculated with the viral vectors. At 21 dpi, upper uninoculated leaves were collected, and whole plants were regenerated by tissue culture. Regenerated plants were selfed and fluorometric and spectrophotometric analysis of pericarp and locular parenchyma were performed. This VIGE approach was extended to traditional tomato cultivars. In the case of PDS, 70% of regenerated plants harbored mutant alleles, and the progeny of line PDS02.3 presented the homozygous +1 allele, which caused a bleaching  phenotype. In the case of SGR1, 60% of regenerated plants harbored mutant alleles. After progeny analysis, three homozygous lines with a single mutant allele of SGR1 each were identified. The fruits of these plants displayed a green-flesh phenotype. Fluorometric and spectrophotometric quantification revealed a significant increase of chlorophyll a in sgr1 mutant lines. Altogether, these results indicate that the PVX-based VIGE approach can be readily applied for accelerated functional genomics of tomato variation, as well as for precision breeding of tomato traits with horticultural interest.

Arcadio Garcia, IBMCP-CSIC, Avenida de los Naranjos s/n, Valencia Valencia 46022, Spain.  5. Abstract presentation P-2016

2024 STUDENT TRAVEL AWARD

Pyramiding microRNAs Enhances Plant Growth and Broad Abiotic Stress Resistance

Abiotic stress like salinity, drought or heat significantly impacts plant growth and development, and often causes considerable reductions in both crop yield and ornamental value. Plants have evolved various defense mechanisms to cope with these environmental challenges. Small non-coding RNAs such as microRNAs (miRNAs) engage in post-transcriptional target gene regulation impacting plant growth and interaction with environment. Four of the conserved miRNA genes, miR319, miR393, miR396 and miR528 have previously been implicated in plant responses to environmental adversities conferring enhanced tolerance to different abiotic stresses when individually overexpressed in various plant species. Here, we have explored the feasibility of combining multiple favorable traits brought by individual miRNA genes to acquire superior plant performance. To this end, we have simultaneously introduced miR319, miR393, miR396 and miR528 in creeping bentgrass. Transgenic plants overexpressing these four genes exhibited significantly enhanced plant growth and broad abiotic stress tolerance indicating that the four miRNA genes function synergistically to regulate plant development and plant responses to various abiotic stresses. Transcriptomic profiling of the transgenic plants is currently under way and would provide insights into molecular mechanisms underlying stacked miRNA-mediated plant growth and stress responses. Furthermore, our data also revealed that all four miRNA genes exhibited high expression when built into the same expression cassette driven by a single promoter supporting a much-streamlined approach for effective miRNA gene stacking. Overall, our research provides information allowing development of novel biotechnology strategies for crop genetic improvement, enhancing agricultural production.

Zhaohui Chen, Clemson University, Clemson, SC 29634. Abstract presentation P-1010.

2024 STUDENT TRAVEL AWARD

Enhancement of Non-viral Transgene Expression by Photo-biomodulation

Gene therapies are promising therapeutics that seek to deliver exogenous DNA or mRNA to target cells or tissues to express proteins or regulate genes as clinical treatments for various diseases. Non-viral vectors are attractive vehicles for delivering nucleic acids due to their relatively low to non-immunogenicity, with ability to transport larger genes, and flexible modularity, which improves overall cost effectiveness. However, their universal usage in gene therapies is limited by inefficient intracellular trafficking, uncontrolled subnuclear localization, and namely, short-lived, transient transgene expression. Therefore, the purpose of our study was to test the effects of photo-biomodulation (PBM) on transient transgene expression.  PBM employs focused light, usually in the form of red or infrared LEDs or lasers, to non-invasively stimulate cell behaviors such as cell proliferation and wound healing.  For our study, we first transfected mouse C3H10 cells in a 96-well plate with lipofectamine and a plasmid that uses a cytomegalovirus promoter to drive the expression of green fluorescence protein (GFP).  By using fluorescence microscopy and flow cytometry techniques we documented a typical non-viral transgene expression profile that peaked 48 – 72 hrs post transfection and declined by over 50% by day 7 in our system.  We then transfected the cells, but at day 7, exposed the cells to 10 seconds, 20 seconds, or 30 seconds of a class 3B 970 nm laser or a no laser control.  The effects were somewhat variable, but 30 second laser treatments consistently elevated GFP expression by up to 50% on day 8 post transfection compared to non-laser treated controls.  Additionally, we observed that one 30 second PBM treatment on both days 2 and 4 post transfection increased overall transgene expression, suggesting the potential usage of PBM as a therapeutic modality to non-invasively improve gene therapies.

Brandon T. Groff, Midwestern University, 555 31st Street, Downers Grove, IL 60515. Abstract presentation A-1011.

2024 STUDENT TRAVEL AWARD

In vitro Culture of Ceratozamia hildae. A Conservation and Toxicity Study

Plant Tissue Culture has been proposed as a strategy to solve the conservation problem that threats the most vulnerable plant group worldwide, Cycads, considered neurotoxics. In this project, the protocol for Ceratozamia hildae implemented in 1995 is evaluated through physiological, morphological, phytochemical and anatomical aspects. Different stages were selected, the development was observed by taking photographic shots with the Axio Vision Rel 4.7 Program, for 8 months. On the other hand, to determine the presence of azoxiglucosides in the culture medium where somatic embryos of C. hildae were developed, a selection of the extraction and identification techniques of secondary compounds was made in the culture medium and structures from field condition. To corroborate a possible biological activity present in the culture medium, toxicity analysis of the extracts was carried out in 2 biological models (bacteria and crustacean) . It was found that, during the maintenance period, cycles of callus growth, variety of somatic embryo forms, organogenesis have been presented. In some cases reactivation was seen, so the development of mono and dicotyledon embryos was described, as well as the elongation of a circinate leaf in a seedling. Since the identity of black strips of the callus was morphologically unknown, an anatomical study was performed and they were idioblast cells with phenolic content. Thanks to the chromatographic profile made for the methanolic extracts and according to the Rf values, the culture medium presents two types of components that are synthesized in plants in field condition. In the toxicity analysis, there was no response in the model with bacteria to any of the extracts. With artemia, it was possible to observe responses in some concentrations of structures -including the culture medium-, but there were also in the control, so these results are treated as a condition unrelated to the effect of the extracts. In general, these results give rise to new studies to optimice the the protocol; and generation of biomass destined to the production of metabolites of interest.

Isabel Candelaria Velásquez de la Cruz, UNAM, Necaxa 83, Col. Portales. Delg.,Benito Juárez CDMX, 03300 Mexico, DF, Mexico. Abstract presentation P-2001.