The 2021 Vitro Biology meeting featured a virtual prerecorded 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, Pierluigi Barone (Corteva Agriscience, USA), Maria Soto-Aguilar (Syngenta, USA), Veena Veena (Donald Danforth Plant Science Center, USA) and Alex da Silva Conceicao (Calyxt, USA) were very impressed with all the contestants’ knowledge and preparation. They recognized Brett Hale (Arkansas State University, USA) with the 1st place award, Trevor Weiss (University of Minnesota, USA) with the 2nd place award, and Uddhab Karki (Arkansas State University, USA) 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 Alex Da Silva Conceicao

First Place

Induction of Totipotency and Non-gametophytic Morphogenesis within the Soybean Microspore

Brett Hale

Androgenesis-based doubled haploidy is a method used to achieve homozygosity in plant breeding programs. However, its application is limited within the Fabaceae, which has historically demonstrated morphogenic recalcitrance in comparison to other plant families (e.g., Brassicaceae and Poaceae).  In this study, the immature microgametophyte, or microspore, was used to investigate in vitro androgenesis in soybean (Glycine max [L.] Merrill). An induction regimen was constructed to support non-gametophytic growth, and consisted of (i) donor plant temperature stress; (ii) a low-nitrogen, phytohormone-supplemented basal medium; and (iii) step-wise, post-isolation culture conditions. In addition, cytological characteristics in soybean microspores were compared to those reported in model androgenesis systems, revealing that totipotent induction/non-gametophytic morphogenesis within the soybean microspore was marked by (i) symmetrical mitotic division; (ii) vacuolar fragmentation; (iii) production and secretion of intrinsic arabinogalactan proteins; and (iv) stress-induced pollen dimorphism; among other features. Lastly, the transcriptomic landscape of stress-induced microspores was evaluated by mRNA Sequencing, which supported cytological findings and suggested that donor plant temperature stress induced totipotency by suppression of the microgametogenesis (pollen) developmental program. The findings herein provide insight into androgenesis-based doubled haploidy in vitro and may prove useful in legume breeding programs.

Brett Hale, Molecular Biosciences Graduate Program, Arkansas State University, State University, AR 72467. In Vitro Cellular and Developmental Biology, 57:S38 2021

second Place

Differential CRISPR/Cas9 Genome Editing Influenced by Epigenetic Factors

Trevor Weiss

In recent years, newly developed genome editing technologies, such as CRISPR/Cas9, have allowed for advanced functional genomics and accelerated translational research. While previous studies indicated CRISPR/Cas9 editing activities are largely determined by their 20 bp targeted sequences, increasing evidence suggests epigenetic features influence editing efficiency and repair outcomes. In this research, we sought to address this question by examining identical CRISPR/Cas9 target sequences with distinct epigenetic features in Arabidopsis thaliana. By tapping into the high-resolution epigenome information and the wealth of epigenetic resources in Arabidopsis, our results indicate a combinatory effect of DNA methylation and chromatin accessibility could lead to significant variations, i.e. 10-200 fold changes, in editing efficiency. Additionally, comparison of CRISPR/Cas9-induced mutation profiles indicate the same targeted sequences can be differentially repaired at different locations or in different epigenetic contexts. Together, our study provides insight towards better understanding the influence of epigenetic factors on genome editing and DNA repair mechanisms, enabling new strategies to further optimize genome editing at refractory sites.

Trevor Weiss, Department of Plant and Microbial Biology, College of Biological Sciences; Center for Precision Plant Genomics, Microbial and Plant Genomics Institute, and Center for Genome Engineering University of Minnesota, Minneapolis, MN, 55108. In Vitro Cellular and Developmental Biology, 57:S36-S37 2021

third Place

Generating Cell Wall Deficient Plant Cells for Enhanced Recombinant Protein Production

Uddhab Karki

Plant cell-based expression system have been established as a cost-effective alternative production platform for bioactive therapeutic proteins in industrial scale due to their intrinsic safety, low production and downstream cost and the capability of post-translational modification. Due to its central role in bio-production and fundamental research, tobacco BY-2 cell has been referred to as the “CHO-cell in molecular farming” and the “HeLa cell in the biology of higher plants”. However, major challenges exist for the BY-2 cell bioproduction system, such as low protein production and secretion, presence of large vacuoles, and difficulty in cryopreservation, which prevent the commercialization of this bioproduction platform. These problems can largely be attributed to the distinctive plant cell wall structure that is composed of a complex matrix of interconnected polysaccharides forming a thick semi-permeable rigid barrier which protects the cell. Understandably, plant cells devote substantial amounts of energy and resources to the synthesis of cell wall polymers, such as cellulose and pectins. However, complete cell wall structure may not be crucial for in vitro cultured plant cells because optimized culture conditions (medium composition, pH, temperature, etc.) are provided to the cells to support their rapid propagation. The long-term goal of our research is to create cell wall deficient (CWD) plant cells for improving production of recombinant proteins. This could be achieved by cellular engineering of plant cells using modern molecular biology tools, especially genome editing technology. In this research, to provide a quick and easy-access to CWD-like plant cells, pseudo-CWD BY-2 cells were created by providing a cellulose synthesis inhibitor, 6-dichlorobenzonitrile (DCB) to the culture media. The morphology, cell wall structural composition, cell growth, bioproduction properties and transcriptomics of the pseudo-CWD BY-2 were characterized. This study provides a conceptual proof for creating CWD plant cells by cellular engineering in the future.

Uddhab Karki, Arkansas Biosciences Institute and Molecular Biosciences Program, Arkansas State University, Jonesboro, AR 72401. In Vitro Cellular and Developmental Biology, 57:S37 2021

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