Here I am preparing a gel before running DNA through a gel electrophoresis. These are one of my favorite lab procedures because it’s just like following a baking recipe, but on a much smaller scale.

My name is Amy Schexnayder and I am biology and psychology student in the Fulbright college of Arts and Sciences. Over the past year I have continued research to develop tools to test the effect of singlet oxygen accumulation on a model plant, Arabidopsis thaliana. As an aspiring doctor, I found this topic stretched my field of knowledge and furthered my understanding of the research process. I have done this research under my mentor, Dr. Fiona Goggin in the entomology department and with the generous help of PhD candidate Hillary Fischer.

The goal of this research is to eventually determine the effect that reactive oxygen species (ROS) have on plant species and the role that ROS may play in downstream plant mechanisms. We suspect that ROS is a key contributor to plant defense systems, so we are studying how we can manipulate the amount of ROS in a plant and thus develop plant defense systems to defend against common agricultural pests, such as aphids or nematodes. My project is integral to this research in that I am working to develop tools to manipulate the amount of singlet oxygen, a type of ROS, in Arabidopsis. These tools must be developed in order to conduct future research on the effects of singlet oxygen accumulation and its role in signaling for downstream mechanisms that control plant resistance. The three tools that we worked to develop were a) the flu mutant to increase singlet oxygen, b) the SPS1oex transgene to decrease singlet oxygen, and c) the Luciferase reporter gene to detect singlet oxygen accumulation accurately and efficiently. Our final goal is to cross all of the genes and generate a single line that contains all three genes.

This process was much more troublesome than it initially seemed it would be. Each gene had to be individually developed to ensure homozygosity before they could be crossed with one another. In most of the gene lines, this was a time-consuming endeavor due to the simple probabilities of Mendelian genetics. For example, when developing the Luciferase reporter gene, we selected a plant that was able to survive on Hygromycin media. All plants containing Luciferase should be able to survive this media because it is an antibiotic resistant gene. However, there was no way to determine whether the plant we chose was homozygous or heterozygous until we produced offspring of the plant. Similar to rolling dice, we would pick a plant and hope for the best. If proved wrong, we would repeat our experiments and roll again.

After conducting various bioassays to phenotypically assess the zygosity of the plant, we then conducted PCR to ensure that we did indeed have homozygous plants for each gene. PCR can be a tricky process that requires the perfect primer, careful avoidance of cross contamination, and precise attention to detail. I initially struggled immensely with PCR. I would accidently add too little DNA or way too much solution, which lead to many days of throwing away my hard work, only to return the next day to redo it. Overtime I was able to master the process and run successful PCRs, though we also had trouble refining the perfect recipe for PCR and finding the exact primer sequence. After much troubleshooting, frustration, and advice from Hillary and Dr. Goggin, we have almost fully confirmed the presence of desired genes through both bioassay and PCR.

Though we are still working to refine the PCR process for a few of the genes, this project is almost completed. I hope to write the final paper next semester and submit it to an undergraduate journal. This project was only slightly delayed by the pandemic due to the tremendous help of Dr. Goggin and Hillary in making sure that the project was maintained when I had to move home. I am so grateful for their leadership, guidance, and constant patience throughout the project.

The plants certainly weren’t the only things that grew during this research project. I developed my ability to understand scientific literature, perform basic lab techniques, analyze data, and troubleshoot failed experiments. This project was largely dependent on processes such as seed harvesting, seed purification, PCR, and gel electrophoresis. With the exception of seed harvesting, these processes require exceptional attention to detail, which has not always been my strong suit. Though I still make occasional mistakes, I am now able to independently perform all of the procedures and successfully analyze the data for our research.

All my life I have understood research to be this glorious process where epiphanies occur often, and the road is paved with one discovery after another. This past year has shown me that is certainly not the case. Realistically, research involves constant troubleshooting: one day you think you have everything right and then the next day you realize that every process you previously used is incorrect. Research is actually paved by many failures, with small successes. But this is ultimately okay, because you discover more in the failures than you do during the successes.

Without the aid of this grant, I would have been unable to put so much thought, time, and energy into this project. This research has given me the skills to be confident as a scientist and researcher. Research is incredibly overwhelming, but I can confidently say that I am no longer intimidated by it, rather excited by the possibilities that research poses for our world and future development. I know that these skills will carry over into my desired career in academic medicine. I hope to attend medical school in the near future and eventually have a career in which I practice in a hospital, while also engaging in academic and clinical research. I know that the research I have completed at the University of Arkansas has largely prepared me to be successful in this endeavor, and I cannot thank Dr. Goggin, Hillary Fischer, and the Honors College enough for giving me this opportunity.