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Summary

Abstract

Introduction

Protocol

Representative Results

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Materials

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Biology

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

Published: June 7th, 2013

DOI:

10.3791/50371

1Institute of Plant Biology and Zürich-Basel Plant Science Center, University of Zürich

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

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.

The embryo of flowering plants is surrounded by the endosperm, a nutritive tissue derived from a second fertilization event. Both embryo and endosperm are surrounded by several cell layers of the seed coat. Collectively these tissues form a seed, which develop inside the fruit. Thus, tissue- and cell-specific analyses of Arabidopsis embryos are strongly impaired due their inaccessibility. Nevertheless, embryos at the late-globular or later stages are relatively well amenable to manual dissection by using fine tungsten needles under the stereomicroscope, or by applying slight pressure on the seed using forceps to extract them. Such techniques were successfully....

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The procedure is summarized in the flowchart shown in Figure 1. The microcapillaries and the instrumental setup are shown in Figure 2 and Figure 3, and typical steps of embryo isolation are shown in Figure 4.

1. Material and Buffer Preparation

1.1 Silicon coating of glass microcapillaries

  1. Place the microcapillaries in a 15 ml Falcon tube with ~5 ml of Sigmacote (Sigma) and invert several ti.......

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Our embryo isolation procedure (Figure 1) allows isolation of up to 40 embryos in 4 hr if washes are performed, e.g. for molecular applications, or in less than an hour if washes are omitted, e.g. for cytological applications. Figure 2 displays high and low quality microcapillary tips and Figure 3 shows the setup of the embryo isolation machine. Figure 4 displays the process of embryo isolation on the inverted microscope.

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We developed an embryo isolation protocol that is rapid, effective, and can be easily transferred to other laboratories.

The equipment described here consists of an inverted microscope, a micromanipulator, glass microcapillaries, a vertical filament puller and a microinjector (Figure 3A). The setup is similar to the one described for single animal cell isolation for transcriptomics analyses 17. We also successfully worked with a more basic setup where glass microcap.......

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We would like to thank Tal Nawy and Martin Bayer for their advice on embryo isolation. MTR, VG, UG and CB devised the embryo isolation equipment. MTR, VG and CB developed the embryo isolation protocol. MTR, VG and CB established the protocol, isolated the embryos, and generated embryo cDNA, VG performed the PCR, MTR the GUS staining, JJ the FISH experiments. MTR, VG, CG and UG wrote the manuscript. This work was funded by the University of Zürich, a Fellowship of the Roche Research Foundation (to MTR), and grants from the Swiss National Foundation (to UG and CB).

....

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Name Company Catalog Number Comments
Name of the Reagent Company Catalogue Number Comments (optional)
REAGENTS
Sigmacote SIGMA SL2-100 ml
RNAse OUT Invitrogen (life technologies) 10777-019
First- strand buffer Invitrogen (life technologies) 18064-022 contained in Superscript II package
DTT Invitrogen (life technologies) 18064-022 contained in Superscript II package
Bovine serum albumin (BSA) 100x =10 mg/ml New England Biolabs Inc. Different suppliers will also work
Thin wall Capillaries 1.0 mm World Precision Instruments TW100F-4
DNA LoBind tube 0.5 ml Vaudaux-Eppendorf 0030108.035
CellTricsΔ 30 μm PARTEC 04-0042-2316
5wells 10 mm diameter slides Electron Microscopy Sciences 63421-10
Formaldehyde Solution Sigma-Aldrich F1635
Superfrost Plus slide Thermo Fisher J1800AMNZ Menzel-Gläser
Tris Amaresco 0497
EDTA Applichem A2937
Glycin Fluka 50050
SDS pellets Roth CN30.3
Micro Pestle VWR 431-0094
Microfine insulin syringes BD U-100
DEPC Sigma-Aldrich D5758
Ethanol Schaurlau ET00102500
Forceps N5 Dumont 0108-5
Bioanalyzer Pico Chip Agilent Technologies 5067-1513
EQUIPMENT
Inverted microscope Nikon TMS (Japan),
Micromanipulator Leitz Leica
Micomanipulator Post mount LH1 probe Leica microsystems 39430101 Different brand will also do the work
Vertical filament puller Sutter instrument P-20 model Other model are also suitable
Cell Tram vario Vaudaux-Eppendorf 5176.000.033
Bioanalyzer Agilent Technologies 2100
Qubit Fluorometer Invitrogen (life technologies

  1. Gehring, M., Bubb, K. L., Henikoff, S. Extensive demethylation of repetitive elements during seed development underlies gene imprinting. Science. 324, 1447-1451 (2009).
  2. Muller, B., Sheen, J. Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis. Nature. 453, 1094-1097 (2008).
  3. Gehring, M., Missirian, V., Henikoff, S. Genomic analysis of parent-of-origin allelic expression in Arabidopsis thaliana seeds. PLoS One. 6, 23687-23 (2011).
  4. Xiang, D., et al. Genome-wide analysis reveals gene expression and metabolic network dynamics during embryo development in Arabidopsis. Plant Physiol. 156, 346-356 (2011).
  5. Nawy, T., et al. The GATA factor HANABA TARANU is required to position the proembryo boundary in the early Arabidopsis embryo. Dev Cell. 19, 103-113 (2010).
  6. Baroux, C., Pecinka, A., Fuchs, J., Schubert, I., Grossniklaus, U. The triploid endosperm genome of Arabidopsis adopts a peculiar, parental-dosage-dependent chromatin organization. Plant Cell. 19, 1782-1794 (2007).
  7. Autran, D., et al. Maternal epigenetic pathways control parental contributions to Arabidopsis early embryogenesis. Cell. 145, 707-719 (2011).
  8. Wohrmann, H. J., et al. Identification of a DNA methylation-independent imprinting control region at the Arabidopsis MEDEA locus. Genes Dev. 26, 1837-1850 (2012).
  9. Raissig, M. T., Baroux, C., Grossniklaus, U. Regulation and Flexibility of Genomic Imprinting during Seed Development. Plant Cell. 23, 16-26 (2011).
  10. Rea, M., et al. Determination of DNA methylation of imprinted genes in Arabidopsis endosperm. J. Vis Exp. 47 (47), e2327 (2011).
  11. Breuninger, H., Rikirsch, E., Hermann, M., Ueda, M., Laux, T. Differential expression of WOX genes mediates apical-basal axis formation in the Arabidopsis embryo. Dev Cell. 14, 867-876 (2008).
  12. Kohler, C., Page, D. R., Gagliardini, V., Grossniklaus, U. The Arabidopsis thaliana MEDEA Polycomb group protein controls expression of PHERES1 by parental imprinting. Nature. 37, 28-30 (2005).
  13. Fransz, P., De Jong, J. H., Lysak, M., Castiglione, M. R., Schubert, I. Interphase chromosomes in Arabidopsis are organized as well defined chromocenters from which euchromatin loops emanate. Proc. Natl. Acad. Sci. U.S.A. 99, 14584-14589 (2002).
  14. Morris, J., Singh, J. M., Eberwine, J. H. Transcriptome analysis of single cells. J. Vis. Exp. (50), e2634 (2011).
  15. Nodine, M. D., Bartel, D. P. Maternal and paternal genomes contribute equally to the transcriptome of early plant embryos. Nature. 482 (7383), (2012).
  16. Jefferson, R. A., Kavanagh, T. A., Bevan, M. W. G. U. S. fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901-3907 (1987).
  17. Schmidt, A., Wöhrmann, H., Raissig, M. T., Arand, J., Gheyselinck, J., Gagliardini, V., Heichinger, C., Walter, J., Grossniklaus, U. The Polycomb group protein MEDEA and the DNA methyltransferase MET1 interact in Arabidopsis to repress autonomous endosperm development. Plant J. 73 (5), (1111).

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