Published: Vol 5, Iss 22, Nov 20, 2015 DOI: 10.21769/BioProtoc.1664 Views: 12953
Reviewed by: Samik BhattacharyaYurong XiePengpeng Li
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Abstract
One of the major topics in plant and animal biology is sexual reproduction. It is, therefore, of great interest to isolate and study germ cells and accessory cells. The male gametophyte of the flowering plant Arabidopsis thaliana (A. thaliana), pollen, is the product of two post-meiotic mitotic divisions. Each mature pollen grain consists of two sperm cells contained within the vegetative cell, the non-reproductive companion cell. The tough pollen wall and its special nested structure make it difficult to study pollen cells separately. Here, we describe a simple and efficient method to fractionate A. thaliana sperm and vegetative cell nuclei by fluorescence activated cell sorting (FACS). Our protocol is based on differences in fluorescence intensity of sperm and vegetative cell nuclei stained with SYBR Green I. 100 plants yield about 1 x 106 sperm and 350,000 vegetative cell nuclei. This method can be used for purifying pollen nuclei of various A. thaliana wild-type accessions and mutant lines, and can, in principle, be adapted for pollen of other plant species.
Keywords: SYBR GreenMaterials and Reagents
Equipment
Software
Procedure
Recipes
Acknowledgments
This protocol is based on previously published work (Schoft et al., 2009; Schoft et al., 2011; Ibarra et al., 2012; Schoft et al., 2015). We thank the BioOptics Facility of the Research Institute of Molecular Pathology, Vienna, for setting up and optimizing FACS. We thank Gerald Schmauss for measuring sizes of sorted A. thaliana pollen nuclei. We thank Hisashi Tamaru for his support. This work was supported by Austrian Science Fund (FWF) Grants P21389-B03 and P24918-B21.
References
Article Information
Copyright
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Chumak, N., Mosiolek, M. and Schoft, V. K. (2015). Sample Preparation and Fractionation of Arabidopsis thaliana Sperm and Vegetative Cell Nuclei by FACS. Bio-protocol 5(22): e1664. DOI: 10.21769/BioProtoc.1664.
Category
Plant Science > Plant cell biology > Organelle isolation
Cell Biology > Cell-based analysis > Flow cytometry
Cell Biology > Cell staining > Nucleic acid
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