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The need for increased crop production is prevalent as population growth continues exponentially. Common methods of increasing production have focused on preventing loss due to insects by creating insecticides. However, insect resistance to those insecticides has become an increasingly common issue in the struggle to combat crop losses. It has also been found that many older insecticides can cause health defects in humans and other important mammals and insect species, building up in the environment and creating unintentional hazards. Newer technologies, such as genetically modified crops, have allowed for greater yields without increased use of multiple insecticides to combat resistance. GM crops have also been shown, over the past few decades that they have been globally used, to have no unintentional health effects on non-target species. One of the most popular GM crops today, Bt crops, have had insect resistance problems cropping up in areas of the United States in the last decade, increasing the need for more environmentally and health conscious GM and insecticide technologies.
A method to screen new potential insecticidal proteins is agroinoculation; a screening method that can determine insecticidal proteins for commercial GM crops by using Agrobacterium as a trojan horse to introduce a virus like DNA sequence that makes the plant produce a large amount of insecticidal proteins. For this study, a tobacco model plant was injected with Agrobacterium containing transfer DNA encoding a modified plant virus that is expressed by the transformed cells but quickly self-replicates to take over plant machinery to produce the desired heterologous protein of interest that is encoded into the virus. This thesis attempts to improve upon injection methods for agroinoculation of tobacco by modifying the ratio of anti-silencing (AS) proteins to protein of interest (POI) injected and the length of incubation for the Agrobacterium and virus-like DNA sequences in the plants before extracting the heterologous protein of interest. Anti-silencing proteins are used to protect the RNA-based virus, which takes over cell machinery to allow for the expression and production of the heterologous protein of interest. A plant can identify the foreign RNA virus and activate the RNA silencing pathway, a common form of antiviral defense. With the injection of Agrobacterium containing virus-like vectors that produce anti-silencing proteins as well as proteins of interest, the plants natural immune system is halted short term while the POI is being produced. The length of incubation is important because after an allotted time the plant cells will become overwhelmed and will begin to die halting the expression of the POI. If the expression of the protein can be increased through altered ratios of AS:POI there can be more protein accumulation, and therefore more protein extracted, prior to plant cell death.
Previous studies conducted tested the ratio of 1:1 for AS:POI versus POI injected alone [7, 8, 9]. This study tested various ratios and determined that there was no significant difference for a AS:POI above 1:1 and anything below 1:1 produced significantly lower levels of protein. Most other studies determined that expression had reached a maximum between 5-7 days, and this study replicated these findings, showing that at injection ratios of 2.5:1 of AS:POI protein expression reached a significant maximum at five days [7, 8, 9]. In addition to determining the maximal expression with these two aspects, agroinoculation was tested on other plants using a fluorescent protein as the POI. Species tested included oats, soybean, Arabidopsis, cotton, rye, spinach, cucumber, and canola. These plant species can be used in bioassays with a greater diversity of insects since they do not contain nicotine or the many other natural defense proteins found in tobacco, compounds that are toxic to most insects. It was found that under this Agrobacterium transfer DNA system to deliver the virus, it did not function in these other species of plants as no heterologous protein fluorescence was observed.
Walters, Stephani, "Optimizing Injection Protocols for a Plant Virus-based Transient Expression System in Tobacco" (2018). Honors Theses. 3029.