Whole Mount in situ Localization of miRNAs and mRNAs During Somatic Embryogenesis in Arabidopsis

Frontiers in Plant Science
Anna M WójcikMałgorzata D Gaj

Abstract

Somatic embryogenesis (SE) results from the transition of differentiated plant somatic cells into embryogenic cells that requires the extensive reprogramming of the somatic cell transcriptome. Commonly, the SE-involved genes are identified by analyzing the heterogeneous population of explant cells and thus, it is necessary to validate the expression of the candidate genes in the cells that are competent for embryogenic transition. Here, we optimized and implemented the whole mount in situ hybridization (WISH) method (Bleckmann and Dresselhaus, 2016; Dastidar et al., 2016) in order to analyze the spatiotemporal localization of miRNAs (miR156, miR166, miR390, miR167) and mRNAs such as WOX5 and PHABULOSA-target of miR165/166 during the SE that is induced in Arabidopsis explants. This study presents a detailed step-by-step description of the WISH procedure in which DIG-labeled LNA and RNA probes were used to detect miRNAs and mRNAs, respectively. The usefulness of the WISH in the functional analysis of the SE-involved regulatory pathways is demonstrated and the advantages of this method are highlighted: (i) the ability to analyze intact non-sectioned plant tissue; (ii) the specificity of transcript detection; (iii) the detection of...Continue Reading

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Citations

Apr 1, 2020·International Journal of Molecular Sciences·Barbara WójcikowskaMałgorzata D Gaj
Jul 18, 2020·International Journal of Molecular Sciences·Anna Maria Wójcik
Apr 13, 2021·Frontiers in Plant Science·Ming ZhangLingyun Zhang

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Methods Mentioned

BETA
transgenic
in vitro transcription

Software Mentioned

CellSens
3DHISTECH
Panoramic Viewer

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