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Plant Cell, Tissue and Organ Culture

, Volume 79, Issue 1, pp 109–114 | Cite as

RT-PCR amplification of mRNAs in nuclei or cytosol of single cells of tomato

  • M. Wada
  • Y. Matsuda
  • K. Fujita
  • A. Nanjo
  • M. Nishimura
  • T. Nonomura
  • K. Kakutani
  • H. Toyoda
Article

Abstract

RT-PCR was used to detect gene expression in situ in single selected cells of tomato callus aggregates. The cytoplasm from one cell was removed with a micropipette viewed under a light microscope and used directly for RT-PCR, followed by nested PCR. This method to remove cytosolic contents prevented the introduction of genomic DNA into the RT-PCR, and only intron-spliced products were amplified when intron-containing genes were used as PCR targets. In addition, transcription of the intron-free gene was possibly detected by simultaneously tracing the intron-containing and intron-free genes using mixed primers for the targeted genes. The present study indicated that some stimuli-activated genes, such as CHI3 and TLC1-LTR, were constitutively transcribed in tomato callus cells.

gene expression Lycopersicon esculentum micromanipulation microsuction 

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Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • M. Wada
    • 1
  • Y. Matsuda
    • 1
  • K. Fujita
    • 1
  • A. Nanjo
    • 1
  • M. Nishimura
    • 1
  • T. Nonomura
    • 1
  • K. Kakutani
    • 2
  • H. Toyoda
    • 1
  1. 1.Laboratory of Plant Pathology and Biotechnology, Faculty of AgricultureKinki University, 3327-204 NakamachiNaraJapan
  2. 2.Pharmaceutical Research and Technology InstituteKinki UniversityHigashi-OsakaJapan

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