Impedance Flow Cytometry: A Novel Technique in Pollen Analysis.
BACKGROUND
An efficient and reliable method to estimate plant cell viability, especially of pollen, is important for plant breeding research and plant production processes. Pollen quality is determined by classical methods, like staining techniques or in vitro pollen germination, each having disadvantages with respect to reliability, analysis speed, and species dependency. Analysing single cells based on their dielectric properties by impedance flow cytometry (IFC) has developed into a common method for cellular characterisation in microbiology and medicine during the last decade. The aim of this study is to demonstrate the potential of IFC in plant cell analysis with the focus on pollen.


METHODS
Developing and mature pollen grains were analysed during their passage through a microfluidic chip to which radio frequencies of 0.5 to 12 MHz were applied. The acquired data provided information about the developmental stage, viability, and germination capacity. The biological relevance of the acquired IFC data was confirmed by classical staining methods, inactivation controls, as well as pollen germination assays.


RESULTS
Different stages of developing pollen, dead, viable and germinating pollen populations could be detected and quantified by IFC. Pollen viability analysis by classical FDA staining showed a high correlation with IFC data. In parallel, pollen with active germination potential could be discriminated from the dead and the viable but non-germinating population.


CONCLUSIONS
The presented data demonstrate that IFC is an efficient, label-free, reliable and non-destructive technique to analyse pollen quality in a species-independent manner.
Enza Zaden, Research and Development B.V. P.O. Box 7, 1600AA Enkhuizen, The Netherlands.
Iris
http://orcid.org/0000-0002-4389-2120
I
Heidmann
Amphasys AG, Technopark Lucerne, 6039 Root D4, Switzerland.
Grit
G
Schade-Kampmann
Enza Zaden, Research and Development B.V. P.O. Box 7, 1600AA Enkhuizen, The Netherlands.
Joep
J
Lambalk
Amphasys AG, Technopark Lucerne, 6039 Root D4, Switzerland.
Marcel
M
Ottiger
Amphasys AG, Technopark Lucerne, 6039 Root D4, Switzerland.
Marco
M
Di Berardino
D002212:Capsicum / Q000166:cytology / Q000254:growth & development; D002470:Cell Survival; D018553:Cucumis sativus / Q000166:cytology / Q000254:growth & development; D017097:Electric Impedance; D005434:Flow Cytometry / Q000379:methods*; D018525:Germination; D056656:Lab-On-A-Chip Devices; D011058:Pollen / Q000166:cytology*; D011846:Radio Waves; D059010:Single-Cell Analysis / Q000379:methods*; D032322:Solanum / Q000166:cytology / Q000254:growth & development; D014026:Nicotiana / Q000166:cytology / Q000254:growth & development