Abstract
IntroductionAn 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.MethodDeveloping 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.ResultsDifferent 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.ConclusionThe presented data demonstrate that IFC is an efficient, label-free, reliable and non-destructive technique to analyse pollen quality in a species-independent manner.
Highlights
Pollen viability analysis by classical fluorescein diacetate (FDA) staining showed a high correlation with impedance flow cytometry (IFC) data
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
To demonstrate that developmental differences can be detected by IFC we isolated microspores from different bud sizes of tobacco and analysed them by nuclear staining (DAPI), potassium iodine (Lugol) staining for starch detection, and IFC at 0.5 and 12 MHz (Fig 1)
Summary
The aim of this study is to demonstrate the potential of IFC in plant cell analysis with the focus on pollen
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