2026 Bob V. Conger Plant Biotechnology Student Oral Presentation Competition

First Place

Recombinase Sites for a Targetable Landing Pad in Poplar

Targeted, iterative gene insertion in plants is a major goal for genome engineering, particularly vegetatively propagated species, such as Populus tremula × Populus alba INRA 717 1B4 (717). Sequential, unidirectional integrations would enable controlled trait stacking into genomic safe harbors. Achieving such stacking requires an insertion system that is both efficient and highly specific. Although recombinase systems have been used in plants for decades, the development of a coordinated, multi–recombinase site framework designed specifically for iterative plant transformations would help enable the next generation of feedstocks and other crop modifications. The recombinase protein, Cre, acts at loxP recombination sites, in which spacer sequences and flanking arms determine compatibility. Mutations in these regions alter recombination efficiency and reduce unintended cross-pairing. To expand the toolkit for plant genome engineering, I designed and evaluated mutant loxP variants for use in iterative insertion workflows. Recombination efficiency was assessed by pairing each mutant left/right site with constitutive Cre, using YFP fluorescence to confirm transformation and AmCyan fluorescence to detect recombination. Of the variants tested, Kitm3L/R performed comparably to wild type loxP, while KatL/R showed consistently low activity. KitL/R and Katm7L/R displayed variable efficiencies across experiments, indicating the need for additional optimization. To guard against unintended recombination, each mutated site was tested or ability to recombine with wild type loxP. Several mutated–wild type combinations produced detectable recombination, suggesting that spacer based specificity may also depend on positional context in planta rather than sequence alone. These results highlight design constraints that must be addressed to achieve predictable, iteration-safe gene stacking and support the development of robust recombinase-based engineering platforms.

Katie Toomey, Institute of Plant Breeding Genetics and Genomics, College of Agricultural and Environmental Sciences, University of Georgia, Athens, GA 30602. Abstract Presentation: P2064

Second Place

Anionic Polymers Enhance Ribonucleoprotein-mediated Genome Editing in Plant Protoplasts

CRISPR ribonucleoprotein (RNP)-based gene editing in plant protoplasts is a valuable strategy for plant genome engineering, as edits generated by RNPs do not require integration of a transgene cassette into the plant. However, it remains challenging to achieve consistently high RNP-based genome editing in plant protoplasts across species– with prior work often reporting low or variable efficiencies. In this work, we draw from mammalian literature, where RNP transfection methods have been more extensively optimized, to explore a suite of polymeric and peptide-based amendments to enhancing RNP-based editing workflows in protoplasts. Using in vitro cleavage assays followed by NGS analysis of editing efficiencies in protoplasts, we find that the inclusion of the anionic polymer polyglutamic acid (PGA) significantly improves editing efficiencies in protoplasts relative to standard PEG transfected protoplasts up to 24-fold when tested across several species, N.benthamiana, A. thaliana, and O. sativa. These results were not only replicated across multiple species and cell types, but extended to demonstrate improved editing across several nucleases including Cas9, TnpB, and Cas12a. We observe that PGA-driven improvements in editing often match or outperform improvements derived from from using enhanced nucleases, a more costly amendment to RNP-based workflows. Furthermore, using delivery of luciferase encoding plasmids and mRNA alongside luminescence assays, we demonstrate PGA-driven increases in luminescence, indicating that PGA acts in a cargo independent manner. PGA is a simple, consistent, and affordable strategy for increasing gene editing with potential applications across plant species and macromolecule cargoes.

Sophia Tomatz, Department of Plant and Microbial Biology, University of California, Berkeley, CA 94707. Abstract Presentation: P-1000

Third Place

CRISPR/Cas9-mediated DFR Disruption Reveals Coordinated Changes in Flavonoid Flux and Development in Petunia × hybrida.

Dihydroflavonol 4-reductase (DFR) occupies a critical branch point in flavonoid metabolism, channeling dihydroflavonol substrates toward anthocyanin biosynthesis in competition with flavonol synthase (FLS). While DFR’s role in floral pigmentation is well established, the broader physiological and transcriptional consequences of its disruption remain poorly characterized, particularly in commercially important ornamental species. Here, we report the generation and comprehensive phenotyping of five independent CRISPR/Cas9-mediated dfr knockout alleles in the commercial Petunia hybrida cultivar ‘Carmine Velour’. The allelic series produced different degrees of loss of floral pigmentation that correlated with mutation severity and predicted protein truncation. Beyond pigmentation, dfr mutants exhibited unexpected reductions in floral dimensions (20–40%), leaf biomass (30–50%), and plastidial pigment content, with chlorophyll and carotenoid levels declining 35–60% in petals despite unchanged leaf anthocyanins. Stem anatomy remained unaffected, revealing organ-specific pleiotropic effects. Transcriptional profiling uncovered feedback reprogramming within the flavonoid pathway: upstream genes (CHSA, CHIA) were downregulated while the competing branch enzyme FLS was upregulated almost 2-fold, consistent with metabolic flux redirection toward flavonol biosynthesis. Strikingly, PORA, encoding a key chlorophyll biosynthetic enzyme, was severely suppressed by 60–75%, providing a mechanistic link between flavonoid disruption and tetrapyrrole metabolism. Correlation analyses revealed systemic coordination: floral anthocyanin content predicted leaf chlorophyll and carotenoid levels across genotypes. These findings reposition DFR as a metabolic node whose disruption propagates effects across pigment classes and organ types, with implications for precision trait engineering in floriculture.

Fangchen Liu, Department of Horticultural Sciences, Crop Transformation Center, University of Florida, IFAS, Gainesville, FL32611 and Mid Florida Research and Education Center, IFAS, University of Florida, Apopka, FL 32703. Abstract Presentation: P-1005