Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis
Tadeusz Wroblewski,
Anna Tomczak,
Richard Michelmore,
Tadeusz Wroblewski
The Genome Center, University of California, Davis, 1 Shiels Ave., Davis, CA 95616, USA
Search for more papers by this authorAnna Tomczak
The Genome Center, University of California, Davis, 1 Shiels Ave., Davis, CA 95616, USA
Present address: Laboratory of Nematology, Wageningen University, Wageningen, The Netherlands
Search for more papers by this authorCorresponding Author
Richard Michelmore
The Genome Center, University of California, Davis, 1 Shiels Ave., Davis, CA 95616, USA
Correspondence (fax +530-752-9659; e-mail [email protected])Search for more papers by this authorTadeusz Wroblewski,
Anna Tomczak,
Richard Michelmore,
Tadeusz Wroblewski
The Genome Center, University of California, Davis, 1 Shiels Ave., Davis, CA 95616, USA
Search for more papers by this authorAnna Tomczak
The Genome Center, University of California, Davis, 1 Shiels Ave., Davis, CA 95616, USA
Present address: Laboratory of Nematology, Wageningen University, Wageningen, The Netherlands
Search for more papers by this authorCorresponding Author
Richard Michelmore
The Genome Center, University of California, Davis, 1 Shiels Ave., Davis, CA 95616, USA
Correspondence (fax +530-752-9659; e-mail [email protected])Search for more papers by this authorSummary
Agrobacterium-mediated transient assays for gene function are increasingly being used as alternatives to genetic complementation and stable transformation. However, such assays are variable and not equally successful in different plant species. We analysed a range of genetic and physiological factors affecting transient expression following agroinfiltration, and developed a protocol for efficient and routine transient assays in several plant species. Lettuce exhibited high levels of transient expression and was at least as easy to work with as Nicotiana benthamiana. Transient expression occurred in the majority of cells within the infiltrated tissue and approached 100% in some regions. High levels of transient expression were obtained in some ecotypes of Arabidopsis; however, Arabidopsis remains recalcitrant to routine, genotype-independent transient assays. Transient expression levels often exceeded those observed in stably transformed plants. The laboratory Agrobacterium tumefaciens strain C58C1 was the best strain for use in plant species that did not elicit a necrotic response to A. tumefaciens. A wild A. tumefaciens strain, 1D1246, was identified that provided high levels of transient expression in solanaceous plants without background necrosis, enabling routine transient assays in these species.
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