A rapid, highly efficient and economical method of Agrobacterium-mediated in planta transient transformation in living onion epidermis

• Published in: PloS one Vol. 9; no. 1; p. e83556
• Main Authors: Xu, Kedong, Huang, Xiaohui, Wu, Manman, Wang, Yan, Chang, Yunxia, Liu, Kun, Zhang, Ju, Zhang, Yi, Zhang, Fuli, Yi, Liming, Li, Tingting, Wang, Ruiyue, Tan, Guangxuan, Li, Chengwei
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 08-01-2014; Public Library of Science (PLoS)
• Subjects: Agriculture; Agrobacterium - metabolism; Arabidopsis; Arabidopsis - microbiology; Biolistics; Biology; Bombardment; Economic analysis; Efficiency; Epidermis; Equipment costs; Fluorescence; Gene expression; Genetic Techniques - economics; Genetic transformation; Kinases; Laboratories; Life sciences; Localization; Methods; Monomolecular films; Nicotiana - microbiology; Onions - genetics; Onions - microbiology; Oxidative stress; Panicum virgatum; Particle bombardment; Phase transitions; Plant cells; Plant Epidermis - cytology; Plant Epidermis - microbiology; Plants (botany); Plants, Genetically Modified; Populus euphratica; Proteins; Reproducibility of Results; Subcellular Fractions - metabolism; Time Factors; Tobacco; Transfection; Transformation; Transformation, Genetic; Vegetables; China
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0083556

Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.