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Open Access Methodology article

An efficient method for stable protein targeting in grasses (Poaceae): a case study in Puccinellia tenuiflora

Yuanyuan Bu1, Mengqing Zhao1, Bo Sun1, Xinxin Zhang1, Tetsuo Takano2 and Shenkui Liu1*

Author Affiliations

1 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

2 Asian Natural Environmental Science Center (ASNESC), The University of Tokyo, Nishitokyo, Tokyo 188-0002, Japan

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BMC Biotechnology 2014, 14:52  doi:10.1186/1472-6750-14-52

Published: 5 June 2014

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.

Keywords:
Non-model plant; Suspension-cultured cells; Green fluorescent protein (GFP); Agrobacterium; Subcellular localization