There is an urgency to make agriculture more environmentally sustainable and resilient to the changing climate that is exacerbating food insecurity and biodiversity loss. One approach to address this problem is improving crops through biotechnological genome modification, e.g., with CRISPR/Cas9. However, avoiding the regulation associated with genetically modified organism (GMO) labels is necessary for rapid, economical crop development. An alternate approach to transgenic methods of gene editing is the use of protoplasts (cells whose cell wall has been removed) for transient expression and subsequent regeneration of non-GMO, edited plants. However, efficient regeneration of plants from protoplasts is a bottleneck in the implementation of this technique. To create a universal method for protoplast regeneration, there first needs to be a baseline level of regeneration efficiency established in a model organism that is not only easy to work with but can also help us uncover the basic principles governing regeneration. To accomplish this, we are working with Arabidopsis which will allow us to demonstrate enhanced efficiency through culture conditions or ectopic gene expression (e.g., morphogenic transcription factors). Morphogenic transcription factors (MTFs) are a category of genes that coordinate the expression of multiple other genes, guiding the step-by-step formation of organs and embryos. We identified several MTFs that enhance root explant regeneration efficiency through a two-step root-to-shoot regeneration assay, and additionally distinguished the optimal timing of inducing expression of each MTF, either induced early during the callus induction step, late during the shoot induction step, or constantly induced during both steps. Characterizing the optimal induction timing for each MTF that enhances regeneration is crucial for their effective application. For example, when using transient expression in protoplasts for enhanced regeneration together with genetic modification, employing an MTF that boosts regeneration during early induction is likely to be advantageous, given that the MTF is only temporarily present alongside gene editing tools. We additionally investigated the links between these MTFs and their directly and indirectly regulated genetic targets to better understand the mechanistic control each of the MTFs have on regeneration. During these studies, we developed a baseline Arabidopsis protoplast regeneration method. Additionally, we identified five MTFs that enhance root-to-shoot regeneration and analyzed the target genes of the MTF that gave the highest regeneration efficiency. The future aim is to enhance protoplast regeneration using these MTFs. The overall goal of this research is to enhance plant regeneration to make biotechnology for crop trait improvement more broadly applicable. / Doctor of Philosophy / There is an urgency to make agriculture more environmentally sustainable and resilient to the changing climate that is exacerbating food insecurity and biodiversity loss. One approach to address this problem is improving crops through changing the genome in very specific ways. However, avoiding the regulation associated with genetically modified organism (GMO) labels is necessary for rapid, economical crop development. GMO labeling is required for plants that contain genetic characteristics that are not naturally present. To avoid integration of foreign genes while attaining a naturally occurring, beneficial trait, we can use gene editing tools that are temporarily present in a cell, which modify the genome before being degraded. This temporary expression can easily be accomplished using protoplasts, which are individual plant cells that have their cell wall removed. A protoplast with an edited genome must then be regenerated into a non-GMO, edited plant. However, efficient regeneration of plants from protoplasts is a bottleneck in the implementation of this technique.
To create a universal method for protoplast regeneration, there first needs to be a baseline level of regeneration efficiency established in an organism that is not only easy to work with but can also help us uncover the basic principles governing regeneration. We accomplished this using Arabidopsis, which allowed us to demonstrate enhanced regeneration efficiency through optimizing protoplast culture conditions. Another approach for enhancing the regeneration efficiency of protoplasts is to alter which genes are present and govern the role of the cell. Overabundance of a gene responsible for plant growth and development could steer the protoplasts towards division and regeneration, when they are naturally unwilling to do so. Morphogenic transcription factors (MTFs) are a category of genes that orchestrate the levels of many other genes, guiding the step-by-step formation of organs and embryos. We identified several MTFs that enhance regeneration efficiency through a two-step root-to-shoot regeneration method, and additionally distinguished the optimal timing of inducing expression of each MTF, either induced early during the first step, late during the second step, or constantly induced during both steps. The induction timing is significant for the application of these MTFs; for instance, to enhance protoplast regeneration when the gene is only temporarily expressed alongside gene editing tools, using an MTF that enhances regeneration during early induction would be advantageous. We additionally investigated the links between these MTFs and their genetic targets that they directly or indirectly regulate to better understand the mechanistic control each of the MTFs have on regeneration. During these studies, we developed a baseline Arabidopsis protoplast regeneration method. Additionally, we identified five MTFs that enhance root-to-shoot regeneration and analyzed the target genes of the MTF that gave the highest regeneration efficiency. The future aim is to enhance protoplast regeneration using these MTFs. The overall goal of this research is to enhance plant regeneration to make biotechnology for crop trait improvement more broadly applicable.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/120941 |
| Date | 15 August 2024 |
| Creators | Reed, Kelsey Madison |
| Contributors | Horticulture, Bargmann, Bastiaan, Saghai-Maroof, Mohammad A., Wright, Robert Clay, Pilot, Guillaume |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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