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 miRNA; and (iv) a semi-quantitative assessment of the RNA abundance.
Highlights
Somatic embryogenesis (SE) involves the formation of somatic embryos in somatic cells that are cultured in vitro, and it provides a model system to study the developmental plasticity of plant somatic cells
In order to overcome the time-consuming and laborious processes that are required to prepare a specific construct and plant transformation, we present the whole mount in situ hybridization (WISH) protocol for miRNA and mRNA molecules in explants that are undergoing SE induction
Different approaches have been used to determine whether the candidate transcripts that have been selected by expression analysis are present in the cells that are undergoing SE induction within the mass of the explants’ cells
Summary
Somatic embryogenesis (SE) involves the formation of somatic embryos in somatic cells that are cultured in vitro, and it provides a model system to study the developmental plasticity of plant somatic cells. In support of the hypothesis of the involvement of miRNA in SE, mutations in DICERLIKE1 (DCL1), which is required for miRNA biogenesis, have been found to inhibit SE-induction in vitro in cultures of Arabidopsis explants (Wójcik and Gaj, 2016). The hypothesis has been supported by the analysis of more than 190 MIRNA in Arabidopsis, which showed that more than 20 genes were differentially regulated during SE induction (Szyrajew et al, 2017). Similar to Arabidopsis, the differential expression of miRNAs was reported in embryogenic cultures of other plants, including Oryza sativa (Chen et al, 2011), hybrid yellow poplar (Li et al, 2012), Larix laptolerix (Zhang et al, 2012), Dimocarpus longan (Lin and Lai, 2013), Gossypium hirsutum (Yang et al, 2013), and Zea mays (Chávez-Hernández et al, 2015)
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