The 2023 In Vitro Biology Meeting featured an oral presentation competition for Plant Biotechnology Students. Presenters were evaluated on experimental design, data analysis, proper interpretation of the results, originality of the study, technical difficulty, and presentation skills. Our expert panel of judges consisted of Bin Tian (Syngenta, USA), Pamela Vogel (Pairwise, USA), Juliana Almeida (LongPing High-Tech, Brazil), and Lorena Moeller (Bayer, USA). All five students demonstrated knowledge and dedication to their research topics. The judges recognized Xiaotong Chen (Clemson University, USA) with the 1st place award, Vincent J. Pennetti (University of Georgia, USA) with the 2nd place award, and Gregory I. Robinson (University of Lethbridge, Canada) with the 3rd place award. The winners were presented with a certificate and a cash award. We encourage all plant biotechnology students to consider this as an opportunity to develop their presentation skills at future meetings.

Submitted by Carlos Garcia 

First Place

MiR169-NF-Y Module Associates with Creeping Bentgrass Biomass Production and Stress Response

Eleanor Jane Brant
Xiaotong Chen

Abiotic stresses, such as salinity, drought and heat, are important limiting factors for plant growth and development, significantly impacting crop production and agriculture economy. Plants have evolved various protection mechanisms coping with different environmental adversities. Manipulation of genes involved in plant stress regulation to genetically engineer enhanced performance in transgenics plays an increasingly important role in sustainable modern agriculture. MicroRNAs (miRNAs) are endogenous small non-coding RNAs identified in plants that engage in post-transcriptional target gene regulation, crucial for plant development and environmental adaptation. Here, we investigated the role of miR169g, a conserved plant miRNA that targets NUCLEAR FACTOR Y (NF-Y) transcription factors in regulating plant development and stress response and the underlying physiological and molecular mechanisms using transgenic approach to overexpress and knockdown miR169 through target mimicry in an important perennial grass species, creeping bentgrass (Agrostis Stononifera). Our data indicate that miR169 regulates expression of the specific NF-Y transcription factor genes leading to significantly altered plant biomass yield and responses to drought and salt stresses that are associated with modified plant development and physiological and molecular characteristics. The on-going RNA-seq analysis would provide additional information for a better understanding of the miR169-mediated plant development and stress response. The results obtained so far have demonstrated the importance of miR169 as a key coordinator in plant development and stress responses, providing information for the development of novel biotechnology approaches to genetically engineer crops for enhanced agricultural production.

Xiaotong Chen, Masters of Biomedical Science, Department of Genetics and Biochemistry, Clemson University, Clemson, SC 296345. Abstract presentation P-1005.

Second Place

Generation of Auxotrophic Agrobacterium Strains Using a CRISPR-mediated Base-editor

Eleanor Jane Brant
Vincent Joseph Pennetti

Agrobacterium-mediated transformation is an essential tool for plant genetic engineering. Most optimizations surrounding Agrobacterium-mediated transformation have traditionally focused on tissue culture and infection parameter modifications. There has been an increased adoption of auxotrophic Agrobacterium strains in both the public and private sectors as a means of controlling bacterial growth post-infection. Auxotrophic strains help mitigate the use of antibiotics which can have undesirable effects on plant tissue. Historically, auxotrophs have been produced through random mutagenesis, often with unintended consequences, or through homology directed repair, which can be time consuming. Recent developments in Agrobacterium engineering have demonstrated the utility of CRISPR-mediated base-editors for targeted gene knockouts in chromosomes and the Ti(Ri) plasmid. Here, we used the CRISPR base-editing architecture, Target-AID, to induce nonsense mutations in the coding regions of genes relevant for metabolite biosynthesis in laboratory Agrobacterium strains. The Target-AID editor was introduced to Agrobacterium through a binary plasmid via electroporation. Prospective gRNAs were selected either manually or via an in-house developed Geneious Prime wrapper plugin to target codons amenable to nonsensical mutation with minimal predicted off-target binding. In one example, we targeted the thymidylate synthase, thyA/Atu2047, gene in each A. rhizogenes K599-, A. tumefaciens C58-, and transconjugant R1000-derived strains for induced thymidine auxotrophy. Incorporation of a chromoprotein reporter in tandem with sucrose counter-selection enabled rapid identification of putative Agrobacterium mutants that had evicted the editor. Given the shared chromosomal backgrounds of many laboratory Agrobacterium strains, the same gRNA could be reused to produce auxotrophic derivatives in both a time and cost-efficient manner. Further, this strategy could be used to rapidly stack multiple auxotrophies in one strain spanning a variety of metabolites.

Vincent Joseph Pennetti, Institute of Plant Breeding, Genetics and Genomics, University of Georgia, Athens, GA. Abstract presentation P-1007.

Third Place

Copper Sulphate Foliar Applications and Soil Amendments Alters Pathogens Found on Cannabis sativa

Eleanor Jane Brant
Gregory Robinson

Fungal infections of Cannabis sativa are common and can severely affect yields, however, little research has studied the use of fungicides for cannabis production. Copper (II) sulphate is an age-old fungicide that has been used to impede fungal diseases in multiple crops while boosting plant defense systems through increased endogenous ethylene biosynthesis. This study aims to test the efficacy of copper sulphate against cannabis pathogens. Copper sulphate was added as a soil amendment from rooting or applied as a foliar application to 10-week-old C. sativa plants biweekly for four weeks. Leaves were collected, frozen, and genomic DNA was isolated and sequenced. Overall pathogen count was decreased in plants treated with copper sulphate as a soil amendment (1 – 10 mg/L), however, plant growth was unaffected. Divergent results of the abundance of bacteria, fungi, and viruses were seen between soil types and chemotypes of C. sativa. Over 300 pathogens were enriched, while under 200 pathogens were depleted with copper sulphate treatment. In contrast, foliar applications (100 mg/L) inhibited the presence of Penicillium olsonii, which causes penicillium bud rot, an important post-harvest disease in C. sativa, and decreased conidiogenesis on all cultivars tested. To verify the fungicidal activity of copper sulphate, P. olsonii was grown in vitro on potato dextrose agar supplemented with various concentrations of copper sulphate (0 – 300 mg/L). The half maximal effective concentration (EC50) and minimum inhibitory concentration (MIC) was calculated to be 73.1 mg/L and 474.9 mg/L, respectively. This is the first report showing soil amendments and foliar applications of copper sulphate on cannabis can alter pathogen abundance and prevent fungal disease.

Gregory Robinson, Department of Biological Sciences, University of Lethbridge, Lethbridge, AB, T1K3M4, CANADA. Abstract presentation P-1008.

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