2025 SIVB Student Award Winners

2025 WILTON R. EARLE AND STUDENT TRAVEL AWARD

Off-target Effects of the Site-specific Recombinases in Perennial Grasses

Recent advances in biotechnology have enabled efficient plant genetic transformation and expanded the application of genome editing for crop improvement. However, transgenes and other transformation components often require removal due to potential side effects and biosafety concerns, which are central to regulatory approval. Site-specific recombination systems offer a compelling approach for transgene excision, particularly in clonally propagated, self-incompatible, or polyploid crops (e.g., perennial grasses) where sexual segregation is impractical. Although commonly used for DNA excision and transgene containment, these systems can induce unexpected phenotypic changes through off-target genetic and epigenetic modifications. To investigate these effects, we constitutively expressed Cre and PhiC31 recombinases in creeping bentgrass (Agrostis stolonifera). Preliminary data show that PhiC31 significantly enhances plant growth and abiotic stress resistance, whereas Cre modestly improves development but impairs salt stress tolerance. RNA-seq analysis on PhiC31 transgenic plants suggests that an elevated anti-recombinase response may underline increased growth and stress resilience. We also identified numerous putative recombinase recognition sites and are developing a reporter system to confirm their activity. This study will provide insights into the safe and effective use of site-specific recombination systems for producing environmentally responsible transgenic crops.

Xiaotong Chen, Department of Genetics and Biochemistry, Clemson University, Clemson, SC. Abstract Presentation: P-2045

2025 HOPE E HOPPS AWARD

Expanding CRISPR-combo Targeting Scope for A and T Rich PAM Sites

The CRISPR-Combo system enables simultaneous gene editing and activation, offering an orthogonal approach to improve genome engineering in crops. However, its reliance on SpCas9, which recognizes NGG PAM sites, limits its applicability in AT-rich genomic promoter regions. To expand its targeting scope, we integrated iSpyMacCas9, iSpyMacCas9-D10A cytosine base editor (CBE-iSpyMac), and AaCas12b, targeting NAAR-PAM and VTTV-PAM sites. Using these systems, we aimed to facilitate hormone-free regeneration of gene-edited rice lines while improving agriculturally important traits through the activation of BABY BOOM1 (OsBBM1). Our findings revealed that OsBBM1 activation significantly enhanced calli regeneration and editing efficiency, particularly with iSpyMacCas9-Combo, resulting in a higher percentage of heritable mutations while AaCas12b-Combo showed limited improvement. Encouraged by these results, we expanded the approach to develop SpRY Cas9-Combo and CBE-SpRYn-Combo, enabling PAM-less editing and activation. These advancements significantly broaden CRISPR-Combo’s applicability in plant genome engineering, paving the way for highly versatile breeding strategies.

Innocent Byiringiro, Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD. Abstract Presentation: P-2024

2025 GORDON SATO AND WALLY MCKEEHAN AWARD

Development and Utilization of a Rainbow Trout Skeletal Muscle Cell Line to Study the Physiology of Ammonia

In animals, ammonia is a toxic byproduct of protein metabolism and poses challenges to physiological and cellular homeostasis. This study describes a novel cell line, RT1 muscle-UFV1, from rainbow trout skeletal muscle and its use to study ammonia defense mechanisms. RT1muscle-UFV1 was developed over six years ago from a dorsal muscle explant of an adult female rainbow trout. Their species identity has been confirmed as Oncorhynchus mykiss by DNA barcoding and molecular characterization for muscle specific markers are currently ongoing. For defense, the focus is on the ability of glutamine synthetase (GS) also known as glutamate-ammonia ligase (GLUL) to mitigate ammonia induced cellular stress. The endpoint measurement for stress is vacuolization. Vacuolization disrupts lysosomal functions which are key to waste processing and cellular homeostasis. The role of GS in mitigating vacuolization was investigated through GS inhibitors and the addition of substrates and products such as glutamate, glutamine and alpha-ketoglutarate. The cells grow well in L-15 medium supplemented with 10% FBS, and cells up to passage 20 have been successfully cryopreserved and thawed. When exposed to ammonium chloride (NH4Cl) at concentrations of 10 mM to 50 mM at 18-21 °C, nearly all cells exhibited vacuoles, which disturbed the normal organization of cultured muscle cells. From this, RT1 muscle UFV1 is emerging as a useful tool for understanding ammonia toxicity in aquaculture and natural ecosystems.

Caio J. Nicholson De Figueiroa, Faculty of Science, University of the Fraser Valley, Abbotsford, BC, CANADA. Abstract Presentation: A-1001

2025 MARIETTA WHEATON SAUNDERS AWARD

CRISPR-Cas9 Editing of eIF4E for Enhanced Resistance to Wheat Streak Mosaic Virus and Triticum Mosaic Virus in Wheat

Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) significantly threaten wheat production in the Great Plains, where current resistant varieties and cultural practices offer limited protection. Novel approaches like gene editing are necessary to develop long-term solutions. This study seeks to enhance wheat resistance to WSMV and TriMV by editing the eukaryotic initiation factor 4E (eIF4E) gene, which plays a key role in viral replication. Immature embryos of the ‘Bobwhite’ wheat variety were co-transformed with a CRISPR-Cas9 guide RNA vector targeting eIF4E and the pAHC20 vector for glufosinate selection. Regenerated plants were screened for transformation using PCR to detect Cas9 and bar genes. The eIF4E target region was amplified, cloned into a TA vector, and sequenced to identify mutations. Statistical analysis included mutation frequency assessment across sequenced colonies. Gene editing resulted in two successfully edited events. One plant contained a premature stop codon leading to early termination of the eIF4E protein, while the other had a frameshift mutation causing amino acid changes downstream of the edit site. Edits were verified in T₁ plants, and these lines are being advanced for further validation. Preliminary results demonstrate the feasibility of targeting eIF4E to disrupt viral replication pathways in wheat. Future T₂ plants will undergo mechanical inoculation with WSMV and TriMV, and ELISA will confirm viral resistance. Advancing to T₄ generations will enable the evaluation of resistance stability and agronomic performance. This gene editing approach holds promise for developing wheat cultivars with durable resistance to mosaic viruses, offering an effective and sustainable solution to mitigate wheat mosaic complex diseases in the Great Plains.

Giovanna Teixeira Sandoval Moreira, Department of Plant Pathology, Kansas State University, Manhattan, KS 66506. Abstract Presentation: P-2004

2025 STUDENT TRAVEL AWARD

Light Emitting Diodes (LEDs) System-assisted In Vitro Induction of Multiple Shoots in White Strawberry (Fragaria × ananassa) ‘Pinkbell F1’

The white strawberry is one of the most important agricultural and economic premium fruit crops. It is mainly grown in Japan and is not readily available in Malaysia and elsewhere. It has a wealth of nutrients and bioactive compounds with pharmacological properties. They are known for their juicy fruit, sweetness & flavour and are consumed in various forms (ice cream, jam). Strawberries are traditionally propagated by runners, which is difficult (disease-prone and time-consuming). Light plays a decisive role in the in vitro-mediated responses. In vitro propagation is a sustainable approach to increase production efficiency at a lower cost and in less time. To meet the needs of the industry, an efficient micropropagation protocol utilising shoot tips by direct organogenesis using light-emitting diodes (LEDs) has been developed. Of the two cytokinins (6-benzylaminopurine (BAP) & thidiazuron) at concentrations (1-5, 10, 15, 20, 25 μM), 3 μM BAP proved to be optimal for multiple shoot (MS) differentiation in 100% cultures with 4.11 ± 0.39 shoots per explant with 2.44 ± 0.28 cm length after 84 days (d). 2,4-Dichlorophenoxyacetic acid (2,4-D) was tested at concentrations (1, 2, 3, 4, 5 μM). It was confirmed that 1 μM proved to be effective in inducing 13.91 ± 1.53 roots/shoot after 60 d in BAP-treated seedlings. Among the LEDs (White (U0), Red:White::1:32 (U1), Red:White::1:2 (U2), Red:White::5:4 (U3)), U1 was found to be suitable for MS differentiation (2.22 ± 0.33 shoots/explant with 16.44 ± 1.62 cm length after 84 d) on full-strength Murashige and Skoog medium with 3 μM BAP. Biochemical and histological analyses were also performed. The regenerants were acclimatised in hydroponics. The genetic fidelity of regenerants was assessed using start codon targeted and retrotransposon markers (iBPS and ReMAP), which yielded a polymorphic information content of 0.47, 0.29 and 0.47, respectively. This is the first report on shoot tip regeneration in white strawberry ‘Pinkbell F1’. Therefore, the study can be used for programmes to improve cultivation, sustainable agriculture and mass production.

Ayyagari Ramlal, School of Biological Sciences, Universiti Sains Malaysia (USM), Georgetown, Penang MALAYSIA and Division of Genetics, ICAR-Indian Agricultural Research Institute (IARI), Pusa Campus, New Delhi INDIA. Abstract Presentation: P-1007

2024 STUDENT TRAVEL AWARD

Systematic Investigation of Agrobacterium Genes Affecting Transient Expression Efficiency in Arabidopsis Seedlings

Transient gene expression via Agrobacterium-mediated transformation (AMT) offers a simple and fast method for gene functional studies, but many plants remain poor in transient expression. We previously developed a transient expression method, named AGROBEST (Agrobacterium-mediated enhanced seedling transformation), which offers a robust gene function analysis in Arabidopsis seedlings. Although the high- and low- efficiency disarmed Agrobacterium strains derived from commonly used strain C58 were identified but the genetic factors governing the AMT efficiency remain unknown. Here, we aim to systematically identify genetic determinants affecting AGROBEST-mediated transient expression efficiency in Arabidopsis seedlings. Agrobacterium strain C58 has a multipartite genome, including two chromosomes, a tumor-inducing plasmid (pTi), and a pAt cryptic plasmid. Thus, we assessed the effect of pTi, helper plasmid, pAt and chromosomal background on AMT efficiency using AGROBEST. Our results indicate that the same pTi and helper plasmid can exhibit differential AMT efficiencies depending on the pAt and/or chromosomal background. The initial systematic investigation prompted comparative genomic analyses between high- and low-efficiency strains, excluding pTi. These analyses revealed genetic variations in the pAt and chromosomal regions, despite both strains being derived from C58. This study identified candidate genes and loci that may contribute to differences in AMT efficiency. Strikingly, we discovered that deleting an 84-kb region in the pAt of the low-efficiency strain significantly enhanced its AMT efficiency. Furthermore, overexpression of two genes from the low-efficiency strains in the high-efficiency strain reduced AMT efficiency. These findings suggest a shift in focus towards understanding how both the pAt and chromosomal background influence AMT efficiency. Our discoveries may pave the way for developing super-AMT strains for diverse crops and advancing applications in basic research, biotechnology, and agriculture.

Hagos Mohammedseid Juhar, Institute Plant and Microbial Biology, Academia Sinica, 128 Section 2, Academia Road, Nankang, Taipei, TAIWAN. Abstract Presentation: P-2047