A cryohistological protocol for preparation of large plant tissue sections for screening intracellular fluorescent protein expression.
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.
Fraunhofer USA Center for Molecular Biotechnology, Newark, DE 19711, USA.
Elisabeth
E
Knapp
Rosemary
R
Flores
David
D
Scheiblin
David
D
Scheiblin
Shannon
S
Modla
Kirk
K
Czymmek
Kirk
K
Czymmek
Vidadi
V
Yusibov
D016616:Cryoultramicrotomy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism*; D059828:Plant Cells / Q000378:metabolism; D018515:Plant Leaves / Q000166:cytology / Q000378:metabolism; D018547:Plant Stems / Q000166:cytology / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D019657:Solanaceae / Q000166:cytology* / Q000378:metabolism / Q000821:virology; D016707:Tissue Fixation; D014026:Nicotiana / Q000378:metabolism*; D014027:Tobacco Mosaic Virus / Q000235:genetics
D049452:Green Fluorescent Proteins