Chapter 6
Transient Transformation of A. thaliana Seedlings
by Vacuum Infiltration
Ce ´ sar Bernat-Silvestre, Vanessa De Sousa Vieira,
Judit Sa ´ nchez-Simarro, Fernando Aniento,
and Marı ´a Jesu ´ s Marcote
Abstract
Transient expression in Arabidopsis thaliana seedlings allows fast expression of fluorescent markers for
different subcellular compartments. This protocol describes a transient transformation assay with five-dayold seedlings using Agrobacterium tumefaciens-mediated vacuum infiltration. Three days after infiltration
of the Agrobacterium containing an expression vector for a fluorescent marker of interest, cotyledon cells
expressing the fluorescent protein can be imaged in a confocal microscope. This assay allows highthroughput screening of new constructs and the study of the localization of a large number of subcellular
markers in Arabidopsis seedlings including wild-type, stable over-expressing and mutant lines.
Key words Seedlings transformation, Arabidopsis thaliana, Agrobacterium tumefaciens, Vacuum
infiltration, Fluorescent markers
1 Introduction
Transient transformation is an easy and powerful method that can
be used in molecular approaches that aim to study gene function,
protein synthesis, and protein subcellular localization. Transient
transformation of plants is a much faster method than stable transformation. The plants can be imaged 3 days after transformation in
contrast to stable transformation, which takes months for the plants
to produce seeds, harvest the seeds, and then select transformants.
Among the transient transformation methods, infiltration with
Agrobacterium containing the DNA to be transferred into the
plant is a simple and widely used technique [1]. Agroinfiltration
of leaves is a method commonly used with many plants though it
has limited application with Arabidopsis leaves and in highthroughput screenings. In contrast, transformation of seedlings
has the advantage of allowing expression analysis “in planta” in
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_6, © Springer Science+Business Media, LLC, part of Springer Nature 2021
147
Transient Transformation of A. thaliana Seedlings
by Vacuum Infiltration
Ce ´ sar Bernat-Silvestre, Vanessa De Sousa Vieira,
Judit Sa ´ nchez-Simarro, Fernando Aniento,
and Marı ´a Jesu ´ s Marcote
Abstract
Transient expression in Arabidopsis thaliana seedlings allows fast expression of fluorescent markers for
different subcellular compartments. This protocol describes a transient transformation assay with five-dayold seedlings using Agrobacterium tumefaciens-mediated vacuum infiltration. Three days after infiltration
of the Agrobacterium containing an expression vector for a fluorescent marker of interest, cotyledon cells
expressing the fluorescent protein can be imaged in a confocal microscope. This assay allows highthroughput screening of new constructs and the study of the localization of a large number of subcellular
markers in Arabidopsis seedlings including wild-type, stable over-expressing and mutant lines.
Key words Seedlings transformation, Arabidopsis thaliana, Agrobacterium tumefaciens, Vacuum
infiltration, Fluorescent markers
1 Introduction
Transient transformation is an easy and powerful method that can
be used in molecular approaches that aim to study gene function,
protein synthesis, and protein subcellular localization. Transient
transformation of plants is a much faster method than stable transformation. The plants can be imaged 3 days after transformation in
contrast to stable transformation, which takes months for the plants
to produce seeds, harvest the seeds, and then select transformants.
Among the transient transformation methods, infiltration with
Agrobacterium containing the DNA to be transferred into the
plant is a simple and widely used technique [1]. Agroinfiltration
of leaves is a method commonly used with many plants though it
has limited application with Arabidopsis leaves and in highthroughput screenings. In contrast, transformation of seedlings
has the advantage of allowing expression analysis “in planta” in
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_6, © Springer Science+Business Media, LLC, part of Springer Nature 2021
147