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Efficient and rapid isolation of early-stage embryos from Arabidopsis thaliana seeds.

Type of publication Peer-reviewed
Publikationsform Original article (peer-reviewed)
Author Raissig Michael T, Gagliardini Valeria, Jaenisch Johan, Grossniklaus Ueli, Baroux Célia,
Project The Genetic and Molecular Basis of Gametogenesis and Maternal Effects in Arabidopsis
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Original article (peer-reviewed)

Journal Journal of visualized experiments : JoVE
Page(s) - - -
Title of proceedings Journal of visualized experiments : JoVE
DOI 10.3791/50371

Open Access

Abstract

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.
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