model of Agrobacterium tumefaciens vacuum infiltration into harvested leaf tissue and subsequent in planta transgene transient expression

• Published in: Biotechnology and bioengineering 2009-02, Vol.102 (3), p.965-970
• Main Authors: Simmons, Christopher W, VanderGheynst, Jean S, Upadhyaya, Shrinivasa K
• Format: Article
• Language: English
• Subjects: Agrobacterium tumefaciens; Agrobacterium tumefaciens - genetics; Agrobacterium tumefaciens - metabolism; agroinfiltration; Algorithms; Bacteria; Biological and medical sciences; Biotechnology; Cell Count; Deoxyribonucleic acid; DNA; Fundamental and applied biological sciences. Psychology; Gases - chemistry; Gene expression; Gene Expression Regulation, Plant; Genes, Bacterial; Glucuronidase - genetics; Glucuronidase - metabolism; Lactuca - genetics; Lactuca - metabolism; Lactuca - microbiology; Models, Biological; Plant Leaves - metabolism; Plant Leaves - microbiology; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Stomata - metabolism; Plants, Genetically Modified; Pressure; Tissue engineering; Transformation, Genetic; Transgenes; Transgenic plants; transient expression; Vacuum; vacuum infiltration
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.22118

Agrobacterium-mediated gene transfer, or agroinfiltration, can be a highly efficient method for transforming and inducing transient transgene expression in plant tissue. The technique uses the innate DNA secretion pathway of Agrobacterium tumefaciens to vector a particular plasmid-encoded segment of DNA from the bacteria to plant cells. Vacuum is often applied to plant tissue submerged in a suspension of A. tumefaciens to improve agroinfiltration. However, the effects of vacuum application on agroinfiltration and in planta transient transgene expression have not been well quantified. Here we show that vacuum application and release act to drive A. tumefaciens suspension into the interior of leaf tissue. Moreover, the amount of suspension that enters leaves can be predicted based on the vacuum intensity and duration. Furthermore, we show that transient expression levels of an agroinfiltrated reporter gene vary in response to the amount of A. tumefaciens vacuum infiltrated into leaf tissue, suggesting that vacuum infiltration conditions can be tailored to achieve optimal transient transgene expression levels after agroinfiltration. Biotechnol. Bioeng. 2009; 102: 965-970.