Sample Preparation and Fractionation of Arabidopsis thaliana Sperm and Vegetative Cell Nuclei by FACS

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 ( ), pollen, is the product of two post-meiotic mitotic divisions. Each mature pollen grain...

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Bibliographic Details
Published in:Bio-protocol 2015-11, Vol.5 (22)
Main Authors: Chumak, Nina, Mosiolek, Magdalena, Schoft, Vera Karolina
Format: Article
Language:English
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Summary: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 ( ), 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 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 10 sperm and 350,000 vegetative cell nuclei. This method can be used for purifying pollen nuclei of various wild-type accessions and mutant lines, and can, in principle, be adapted for pollen of other plant species.
ISSN:2331-8325
2331-8325
DOI:10.21769/BioProtoc.1664