Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 119, Issue 1, pp 211–219 | Cite as

A transient gene expression system using barley protoplasts to evaluate microRNAs for post-transcriptional regulation of their target genes

  • Yu Bai
  • Ning Han
  • Jinxia Wu
  • Yinong Yang
  • Junhui Wang
  • Muyuan Zhu
  • Hongwu Bian
Original Paper


Transient gene expression assays using protoplasts have been frequently used in high-throughput screening and functional characterization of plant genes. In barley, however, very few studies have explored the use of protoplasts isolated from green tissues. In this study, a reliable and efficient transient gene expression system has been established using barley green tissue protoplasts. Due to the importance of osmolarity in maintaining protoplast viability and transfection efficiency, different mannitol concentrations were tested to determine the optimal osmolarity suitable for barley protoplast preparation. The method and conditions were also described for efficient isolation of protoplasts from barley leaf and stem tissues and transient expression of exogenous gene constructs. This transient expression system has been successfully demonstrated for protein immunoblot analysis, subcellular protein localization and quantitative analysis of gene expression. Furthermore, a simplified method has been described to quickly evaluate microRNAs for post-transcriptional regulation of their target genes in barley protoplasts.


Barley Protoplast isolation PEG-mediated transfection MicroRNAs MicroRNA target genes 


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Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Yu Bai
    • 1
  • Ning Han
    • 1
  • Jinxia Wu
    • 1
  • Yinong Yang
    • 1
    • 2
  • Junhui Wang
    • 1
  • Muyuan Zhu
    • 1
  • Hongwu Bian
    • 1
  1. 1.Institute of Genetics, College of Life SciencesZhejiang UniversityHangzhouChina
  2. 2.Department of Plant Pathology and Environmental Microbiology, and Huck Institutes of Life SciencesPennsylvania State UniversityUniversity ParkUSA

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