Causes and consequences of experimental variation inNicotiana benthamianatransient expression

Infilitration ofAgrobacterium tumefaciensintoNicotiana benthamianahas become a foundational technique in plant biology, enabling efficient delivery of transgenesin plantawith technical ease, robust signal, and relatively high throughput. Despite transient expression’s prevalence in disciplines such as synthetic biology, little work has been done to describe and address the variability inherent in this system, a concern for experiments that rely on highly quantitative readouts. In a comprehensive analysis ofN. benthamianaagroinfiltration experiments, we model sources of variability that affect transient expression. Our findings emphasize the need to validate normalization methods under the specific conditions of each study, as distinct normalization schemes do not always reduce variation either within or between experiments. Using a dataset of 1,813 plants collected over three years, we develop a comprehensive model of variation inN. benthamianatransient expression, using power analysis to determine the number of individual plants required for a given effect size. Drawing on our longitudinal data, these findings inform practical guidelines for minimizing variability through strategic experimental design and power analysis, providing a foundation for more robust and reproducible use ofN. benthamianain quantitative plant biology and synthetic biology applications.