The 2026 In Vitro Biology Meeting proudly hosted an oral presentation competition for Plant Biotechnology Postdoctoral Researchers, celebrating the innovative research and outstanding communication skills of our postdoctoral members. Presenters were evaluated on experimental design, data analysis, study originality, and presentation quality by a distinguished panel of judges representing both academia and industry: Dr. Morgan McCaw (Ohalo Genetics), Dr. Ning Zhang (James Madison University), and Dr. Juan Debernardi (University of California, Davis). Six postdoctoral researchers were selected from a highly competitive applicant pool to deliver oral presentations. First place was awarded to Dr. Fiorella Spies (University of Georgia, USA) for her work on genomic safe harbor sites. Second place went to Dr. Kirill Schenstniy (University of California, Berkeley, USA) for advances enabling highly efficient editing in zygotes and egg cells, and Dr. Kelsey Reed (Virginia Tech, USA) earned third place for her use of advanced microscopy techniques to illuminate pluri- and totipotency in protoplasts. In recognition of their outstanding contributions, all winners received certificates and cash awards. We warmly encourage all Plant Biotechnology Postdoctoral Researchers to take part in future competitions, and we look forward to another excellent symposium at next year’s meeting in St. Louis.
Submitted by Zoe Dubrow
First Place
Identification of Genomic Safe Harbor Sites and Development of Site-Directed Genome Editing Tools in Poplar 717
Fiorella P. Spies
Poplar species are increasingly recognized as a leading platform for bioproduct and bioenergy development. Their compact genome, rich genomic resources, and responsiveness to Agrobacterium tumefaciens make them highly suitable for genetic transformation studies. Poplar 717 (Populus tremula × Populus alba) serves as a model due to its sequenced genome and ease of genetic manipulation. Its transformation mainly uses Agrobacterium to introduce foreign DNA, which can integrate randomly into the genome causing disruption of important genes, and variable expression of the transgene. Site-directed genome editing techniques allow DNA to insert at a specific genomic locus, called Genomic Safe Harbors (GSH), minimizing these problems. Our project focuses on designing site-directed genome editing tools that allow precise DNA integration, while also mapping GSH within the poplar 717 genome that can accommodate foreign sequences without disrupting normal cell function. GSH were identified after analyzing T-DNA insertion sites of events that were stably expressed for over 10 years without affecting the poplar phenotype. Sites were analyzed to identify those located away from highly methylated regions, miRNA, or tRNA putative genes, not disrupting any coding genes, and away from known long-terminal-repeats (LTRs). This analysis revealed six candidate SH regions, ranging from 11.1 to 13.2 kb in length, which are undergoing further study to confirm their suitability for precise genome editing. Three alternative site-directed genome editing strategies are currently under evaluation: CAST, which combines the CRISPR/Cas9 system with a codon-optimized transposase for testing in poplar; PrimeRoot, which utilizes the prime editing system alongside CRE recombinase; and nuclease-mediated HDR, which employs Cas9 in conjunction with a viral exonuclease to promote homology-directed repair. A preliminary transformation in poplar was conducted, and PCR followed by sequencing of genomic DNA from transformed calli revealed positive events for the HDR approach.
Fiorella P. Spies, Center for Applied Genetic Technologies, University of Georgia, Athens, GA. Abstract Presentation: P-1010.
Second Place
Highly-efficient Multiplexed Genome Engineering in Crops
Kirill Schenstniy
DNA-free ribonucleoprotein- (RNP-) mediated gene editing in protoplasts has attracted attention because it enables direct delivery of pre-assembled RNPs into protoplasts and avoids transgenesis, but suffers from low genome-editing efficiencies, protoplast fragility, and difficulties in regeneration.
Here, we report that the simple addition of polyglutamic acid (PGA) to standard polyethylene glycol (PEG) transfection protocols reduces protoplast membrane fragility and increases genome editing efficiencies by nearly 25-fold compared to RNPs delivered through standard PEG transfection alone. This modification is particularly advantageous for multiplex genome editing, as increased protoplast survival and editing efficiencies enhance the probability of obtaining cells carrying edits in all targeted genes. Additionally, we report that PGA increases survivability and editing efficiencies in a second transfection protocol: diploid rice zygotes produced via in vitro fertilization (IVF) and haploid non-fertilized rice egg cells. We report successful PGA-assisted PEG-mediated delivery of pre-assembled Cas9 and Cas12a RNPs and gene editing in IVF-produced rice zygotes and non-fertilized rice egg cells. We further show simultaneous multiplexed editing of three rice susceptibility (S) genes in IVF-produced rice zygotes, demonstrating the feasibility of “one-pot” crop bioengineering. We show that edited protoplasts can regenerate into plantlets within 80 days post-IVF. Lastly, we successfully created intergeneric rice (♀) x wheat (♂) zygotes via IVF, delivered pre-assembled RNPs into these intergeneric zygotes, and achieved PGA-enabled high-efficiency gene editing in rice and wheat genomes. We foresee that our technologies: interspecific hybridization via IVF, direct editing of IVF-produced zygotes and non-fertilized egg cells, and PGA-enhanced protoplast transfection, will expedite the process of crop bioengineering by increasing survival, editing efficiencies, and regeneration efficiencies across species and genome editors.
K. Schenstniy, Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA. Abstract Presentation: P-1008
Third Place
Cell By Date: Advanced Microscopy of Plant Pluri- and Totipotency Using Protoplasts
Kelsey Reed
Plant cell culture research continues to lag behind advances in animal systems, leaving key cellular and molecular mechanisms of plant development poorly resolved. However, plant cells’ intrinsic pluri‑ and totipotency offer a powerful but underutilized platform for studying cellular reprogramming and regeneration. To address this gap, we have developed a dynamic protoplast culture system in Arabidopsis thaliana that enables high-resolution, time-resolved imaging of early regenerative events. Our approach integrates matrix-embedded protoplasts with fluorescent subcellular markers to visualize and quantify cell identity transitions, division dynamics, and microcallus formation. Using confocal (Zeiss LSM 880) and lattice light sheet (Zeiss LLS7), we capture cellular and subcellular dynamics, including cell cycle progression, division frequency, organelle distribution, and cell size variability. These approaches reveal substantial heterogeneity in single‑cell responses and illuminate the structural and molecular features associated with successful regeneration. By aligning time‑course imaging across treatments, we rapidly compare division frequencies and track the developmental trajectories of individual cells. This work also addresses a critical bottleneck in plant biotechnology: genotype‑dependent variation in regeneration efficiency. By elucidating the mechanisms underlying protoplast regeneration and identifying quantitative markers of regenerative potential, our system contributes to the development of more robust, genotype-independent transformation strategies. Ultimately, this work advances cellular‑level understanding of plant regeneration and contributes to efforts aimed at improving crop resilience and sustainability.
Kelsey Reed, School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, VA 24061. Abstract Presentation: P-1009
