Understanding the mechanisms of non-thermal plasma treatments on seeds

The motivation driving plasma-seed treatment research is the renewed importance of sustainable, eco-friendly agriculture. There is a constant interest in finding alternatives to minimize resource use and environmental degradation, while ensuring healthy seed and plant development. Plasma agriculture is being intensively investigated since many laboratories and industries see the potential of plasma-seed treatments. However, the lack of understanding of the mechanisms and lack of standardized protocols has created a situation where interdisciplinary groups work with their own customized setup. Often times, the setup is developed through trial-and-error, and consequently, the process is not shared publicly. This thesis, therefore, addresses both the mechanisms and the design of plasma-seed treatments.For identifying the mechanisms, a short study was first designed to better understand the effect of plasma and its separate components, heat and ozone. This revealed that plasma is unique in its ability to effectively change the seed surface within a short time frame, which was not observed with heat or ozone alone. Next, the plasma conditions were then established for accelerated germination in Arabidopsis thaliana seeds, a plant model organism. These plasma treatment conditions were then analyzed using in situ FTIR to determine the plasma gas chemistry and differences between parameter changes. The results suggest that it is likely short-lived species, such as NO, which are responsible for the effect, although more studies need to be conducted. To understand how the plasma affects the seed and its subsequent development, RNA sequencing was used and it revealed upregulated plant stress and defense responses. Depending on the plasma treatment time and extraction time point, genes were differentially expressed in the phenylpropanoid or glucosinolate pathways. From this information, a tentative hypothesis was proposed; plasma exposure could be interpreted as a wounding and oxidative stress. In terms of plasma-seed treatment design, each variable concerning the biological and physical aspects of this interdisciplinary field is mentioned to raise awareness and explain the relevance. This information was compiled into guidelines for the entire community in hopes that data collection can be done systematically and lead to a meta-analysis in the future. Additionally, specific diagnostics were explored and recommended based on their relative merits. For example, plasma-treated seed surfaces were analyzed and compared using SEM, EDX, XPS, AFM, and ATR-FTIR. A similar comparison was done for the plasma gas chemistry, which revealed that FTIR is mainly useful for long lifetime species and other diagnostics, such as LIF, are more suitable for short lifetime species and are therefore complementary. The importance of analyzing multiple variables through a parametric study was discovered and recommended as an approach for others to follow suit in order to disentangle the complex number of variables in these treatments and minimize the risk of false interpretations and conclusions. Additional studies are needed to understand how to design experiments with reproducible and consistent results from plasma-seed treatments using a systematic approach. My hope is that this work will be the foundation for future studies.