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<p>Infiltration of <i>Agrobacterium tumefaciens</i> into <i>Nicotiana benthamiana</i> has become a foundational technique in plant biology, enabling efficient delivery of transgenes <i>in planta</i> with 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 of <i>N. benthamiana</i> agroinfiltration 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 1915 plants collected over three years, we develop a model of variation in <i>N. benthamiana</i> transient 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 of <i>N. benthamiana</i> in quantitative plant biology and synthetic biology applications.</p>

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Causes and consequences of experimental variation in Nicotiana benthamiana transient expression

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月14日

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  • Sophia N. Tang,
  • Matthew J. Szarzanowicz,
  • Amy Lanctot,
  • Sasilada Sirirungruang,
  • Liam D. Kirkpatrick,
  • Krista Drako,
  • Simon Alamos,
  • Lyurui Cheng,
  • Lucas M. Waldburger,
  • Shuying Liu,
  • Lena Huang,
  • Sami Kazaz,
  • Emine Akyuz Turumtay,
  • Edward Baidoo,
  • Aymerick Eudes,
  • Mitchell G. Thompson,
  • Patrick M. Shih

摘要

Infiltration of Agrobacterium tumefaciens into Nicotiana benthamiana has become a foundational technique in plant biology, enabling efficient delivery of transgenes in planta with 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 of N. benthamiana agroinfiltration 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 1915 plants collected over three years, we develop a model of variation in N. benthamiana transient 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 of N. benthamiana in quantitative plant biology and synthetic biology applications.