Flow Cytometry

Since the first flow sorter (BD FACStarPLUS) was bought in 1992 the flow cytometry was successfully used for different applications in numerous collaborations with research groups from inside and outside the institute.

Among many other aspects this technique was used for the:

  • determination of absolute and relative DNA content (ploidy, aneuploidy and endopolyploidy) of various plant species;
  • sorting of plant interphase nuclei and metaphase chromosomes based on their differences in the DNA content;
  • differentiation between sexual and apomictic seed formation;
  • analysis and sorting of GFP expressing cells or protoplasts;
  • determination of AT/GC ratios of genomic DNA.

2003 an updated cell sorter, the BD FACSAria, was bought. This instrument allows in the present configuration the simultaneous excitation with three lasers (633 nm, 488 nm und 407 / 375 nm) and therewith a parallel measurement of two scatter parameters and nine fluorescences. The FACSAria enables the sorting of up to four fractions in a bulk of different collection tubes.

 

 

Flow-cytometric analysis and chromosome countings of Arabidopsis thaliana spo11-2 progeny 

 

SPO11-2 is required for proper chromosome segregation and its loss results in aneuploidy in the surviving progeny. 

Left: Flow-cytometric histograms representing the relative DNA content of Col wild-type (top) and two individual spo11-2 offspring plantlets with different amounts of surplus DNA (red peaks) measured in comparison to Raphanus sativus (blue peaks) as internal reference standard.

Right: Corresponding flow-sorted 2C interphase nuclei after FISH with centromeric 180-bp repeat. The number of signals is equivalent  to the chromosome number. 

For further details see Hartung et al. (2007) Plant Cell 19:  3090-3099.