A cryohistological protocol for preparation of large plant tissue sections for screening intracellular fluorescent protein expression

Biotechniques. 2012 Jan;52(1):31-7. doi: 10.2144/000113778.

Abstract

In this study, we have developed a robust cryohistological method that allows imaging of virtually any type of plant cell or tissue while preserving fluorescent protein signals and maintaining excellent cellular and subcellular morphology. This method involves modified fixation of plant tissues (i.e., leaves, stems, and petioles), infiltration in a sucrose gradient, freezing, and collection of cryosections directly onto a cryoadhesive tape. Using this method followed by microscopic analysis, we demonstrated a localized accumulation of green fluorescent protein (GFP) in Nicotiana benthamiana plants agroinfiltrated with the movement-incompetent tobacco mosaic virus-based vector and systemic accumulation of GFP in plants infiltrated with the movement-competent vector. Overall, this simple cryohistological procedure reduced sample preparation time and allowed processing of tissue sections for high-resolution imaging of targeted fluorescent proteins in all plant tissues.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cryoultramicrotomy / methods*
  • Green Fluorescent Proteins / analysis
  • Green Fluorescent Proteins / metabolism*
  • Nicotiana / metabolism*
  • Plant Cells / metabolism
  • Plant Leaves / cytology
  • Plant Leaves / metabolism
  • Plant Stems / cytology
  • Plant Stems / metabolism
  • Plants, Genetically Modified / genetics
  • Plants, Genetically Modified / metabolism
  • Solanaceae / cytology*
  • Solanaceae / metabolism
  • Solanaceae / virology
  • Tissue Fixation
  • Tobacco Mosaic Virus / genetics

Substances

  • Green Fluorescent Proteins