Efficient and rapid isolation of early-stage embryos from Arabidopsis thaliana seeds
| Autor: | Raissig, M. T., Gagliardini, V., Jaenisch, J., Ueli Grossniklaus, Baroux, C. |
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| Přispěvatelé: | University of Zurich |
| Jazyk: | angličtina |
| Rok vydání: | 2013 |
| Předmět: |
Arabidopsis
food and beverages Plant Biology 2800 General Neuroscience DNA Methylation 580 Plants (Botany) Video article 10126 Department of Plant and Microbial Biology Genes Reporter 1300 General Biochemistry Genetics and Molecular Biology 2400 General Immunology and Microbiology embryonic structures Seeds 10211 Zurich-Basel Plant Science Center 1500 General Chemical Engineering In Situ Hybridization Fluorescence |
| Zdroj: | Journal of visualized experiments : JoVE Scopus-Elsevier |
| DOI: | 10.5167/uzh-89709 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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