title: An efficient method for stable protein targeting in grasses (Poaceae): a case study in Puccinellia tenuiflora.
abstract: BACKGROUND
  An efficient transformation method is lacking for most non-model plant species to test gene function. Therefore, subcellular localization of proteins of interest from non-model plants is mainly carried out through transient transformation in homologous cells or in heterologous cells from model species such as Arabidopsis. Although analysis of expression patterns in model organisms like yeast and Arabidopsis can provide important clues about protein localization, these heterologous systems may not always faithfully reflect the native subcellular distribution in other species. On the other hand, transient expression in protoplasts from species of interest has limited ability for detailed sub-cellular localization analysis (e.g., those involving subcellular fractionation or sectioning and immunodetection), as it results in heterogeneous populations comprised of both transformed and untransformed cells.
  
  
  RESULTS
  We have developed a simple and reliable method for stable transformation of plant cell suspensions that are suitable for protein subcellular localization analyses in the non-model monocotyledonous plant Puccinellia tenuiflora. Optimization of protocols for obtaining suspension-cultured cells followed by Agrobacterium-mediated genetic transformation allowed us to establish stably transformed cell lines, which could be maintained indefinitely in axenic culture supplied with the proper antibiotic. As a case study, protoplasts of transgenic cell lines stably transformed with an ammonium transporter-green fluorescent protein (PutAMT1;1-GFP) fusion were successfully used for subcellular localization analyses in P. tenuiflora.
  
  
  CONCLUSIONS
  We present a reliable method for the generation of stably transformed P. tenuiflora cell lines, which, being available in virtually unlimited amounts, can be conveniently used for any type of protein subcellular localization analysis required. Given its simplicity, the method can be used as reference for other non-model plant species lacking efficient regeneration protocols.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Bu
authors.forename: Mengqing
authors.initials: M
authors.lastname: Zhao
authors.forename: Bo
authors.initials: B
authors.lastname: Sun
authors.forename: Xinxin
authors.initials: X
authors.lastname: Zhang
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Takano
authors.affiliation: Key Laboratory of Saline-Alkali Vegetation Ecology Restoration in Oil Field (SAVER), Ministry of Education, Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry University, Hexing Road No, 26, Xiangfang District, Harbin City, Heilongjiang Province 150040, China. shenkuiliu@nefu.edu.cn.
authors.forename: Shenkui
authors.initials: S
authors.lastname: Liu
mesh_terms: D060054:Agrobacterium / Q000235:genetics; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D018929:Cell Culture Techniques; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D059828:Plant Cells / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics; D006109:Poaceae / Q000378:metabolism*; D011523:Protoplasts / Q000166:cytology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D027682:Cation Transport Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C088952:ammonium transporters, plant; D049452:Green Fluorescent Proteins