The Topic: Research Methods for Plant STD: Anther Smut
During the spring semester of my sophomore year, I conducted research on campus in a laboratory at the Biology-Psychology building, spending some time in the Greenhouse as well. I thoroughly enjoyed my time spent at both sites, and I had excellent supervisors, Yanelyn Perez and Dr. Emily Bruns. Yanelyn Perez, whom I worked closely with, was thorough in explaining the lab techniques to me and made sure I understood the meaning behind everything I was doing in the lab, rather than simply providing a step-by-step tutorial for each technique. For future SGC scholars, I would recommend that you look into research that interests you, and it doesn't necessarily have to be within your major. You can explore the department websites of different majors to get in touch with a faculty member conducting research that interests you At my service site, I investigated a plant STD called Anther smut on the Dianthus Pavonius plant. I learned a plethora of techniques that enabled me to grow the pathogen and the host, as well as investigate the system. I completed tasks such as making agar, planting and sterilizing seedlings, growing smut from freezer cultures, completing serial dilutions, counting sporidia under a microscope using a hemocytometer, inoculating adult plants and seedlings, and extracting DNA from smut and using PCR to analyze mating types. When inoculating adult plants, I created a water and smut mixture and sprayed them for a couple of seconds in the greenhouse. For seedlings, it's essentially the same thing, but on a smaller scale. I used tweezers to place seedlings into a water agar plate and then dispensed one drop of a water and smut mixture onto each seed. I completed serial dilutions to make the smut less concentrated and observe conjugation under a microscope. I used an autoclave machine, a device that sterilizes materials and equipment using high-pressure steam, to autoclave potato dextrose agar (PDA) for experiments, specifically fungus growth.
Another method I used for conjugation was a 96 Well Plate. This is a flat plastic plate with 96 individual wells that can be used for various experimental applications. In my research, I used it to mix two mating types together. First, I made a stock solution of my mating types in 1000 microliters of sterile deionized water. Next, I made 10-fold dilutions of each stock solution and counted the sporidia number in the 10-fold dilution under a hemocytometer. I then input all the counts into an excel spreadsheet provided by the lab. The spreadsheet calculated the amount of sterile deionized water and stock solution that I needed to combine. Finally, I mixed the two mating types together and pipetted 75 microliters of the mating type mix into each well of the plate. Once I filled the desired wells, I placed a lid over the plate and parafilmed the edges. After 24 hours, I added blue dye to each well to make it more visible, placed the wells under an inverted microscope, and took pictures.
I learned that Anther-smut is diverging into different species, and researchers do not know what that system looks like. By conducting mating experiments, we can see if there are any barriers in reproduction that help maintain them as different species. Silene Latifolia, one of the plants used in the lab, is a self-mating system, and my research uses a different plant, Dianthus Pavonius, to investigate if it holds true with that species.
I developed an appreciation for critical thinking, attention to detail, communication, and problem-solving skills as a result of my research. Some of the experiments I conducted required a lot of repetition, which forced me to be especially attentive to my work. This experience has inspired me to pursue further research on Anther smut, including more mating experiments that take into account additional variables such as temperature. This fall, I plan to apply the techniques I learned from my supervisor and conduct an independent research project.