Dataset Information

Using flow cytometry to estimate pollen DNA content: improved methodology and applications.

ABSTRACT:

Background and aims

Flow cytometry has been used to measure nuclear DNA content in pollen, mostly to understand pollen development and detect unreduced gametes. Published data have not always met the high-quality standards required for some applications, in part due to difficulties inherent in the extraction of nuclei. Here we describe a simple and relatively novel method for extracting pollen nuclei, involving the bursting of pollen through a nylon mesh, compare it with other methods and demonstrate its broad applicability and utility.

Methods

The method was tested across 80 species, 64 genera and 33 families, and the data were evaluated using established criteria for estimating genome size and analysing cell cycle. Filter bursting was directly compared with chopping in five species, yields were compared with published values for sonicated samples, and the method was applied by comparing genome size estimates for leaf and pollen nuclei in six species.

Key results

Data quality met generally applied standards for estimating genome size in 81 % of species and the higher best practice standards for cell cycle analysis in 51 %. In 41 % of species we met the most stringent criterion of screening 10 000 pollen grains per sample. In direct comparison with two chopping techniques, our method produced better quality histograms with consistently higher nuclei yields, and yields were higher than previously published results for sonication. In three binucleate and three trinucleate species we found that pollen-based genome size estimates differed from leaf tissue estimates by 1·5 % or less when 1C pollen nuclei were used, while estimates from 2C generative nuclei differed from leaf estimates by up to 2·5 %.

Conclusions

The high success rate, ease of use and wide applicability of the filter bursting method show that this method can facilitate the use of pollen for estimating genome size and dramatically improve unreduced pollen production estimation with flow cytometry.

SUBMITTER: Kron P

PROVIDER: S-EPMC3448423 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Publications

Using flow cytometry to estimate pollen DNA content: improved methodology and applications.

Kron Paul P Husband Brian C BC

Annals of botany 20120807 5

<h4>Background and aims</h4>Flow cytometry has been used to measure nuclear DNA content in pollen, mostly to understand pollen development and detect unreduced gametes. Published data have not always met the high-quality standards required for some applications, in part due to difficulties inherent in the extraction of nuclei. Here we describe a simple and relatively novel method for extracting pollen nuclei, involving the bursting of pollen through a nylon mesh, compare it with other methods an ...[more]

PMID: 22875815

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Project description:Impedance flow cytometry (IFC) is a versatile lab-on-chip technology which enables fast and label-free analysis of pollen grains in various plant species, promising new research possibilities in agriculture and plant breeding. Hazelnut is a monoecious, anemophilous species, exhibiting sporophytic self-incompatibility. Its pollen is dispersed by wind in midwinter when temperatures are still low and relative humidity is usually high. Previous research found that hazelnut can be characterized by high degrees of pollen sterility following a reciprocal chromosome translocation occurring in some cultivated genotypes. In this study, IFC was used for the first time to characterize hazelnut pollen biology. IFC was validated via dye exclusion in microscopy and employed to (i) follow pollen hydration over time to define the best pre-hydration treatment for pollen viability evaluation; (ii) test hazelnut pollen viability and sterility on 33 cultivars grown in a collection field located in central Italy, and two wild hazelnuts. The accessions were also characterized by their amount and distribution of catkins in the tree canopy. Pollen sterility rate greatly varied among hazelnut accessions, with one main group of highly sterile cultivars and a second group, comprising wild genotypes and the remaining cultivars, producing good quality pollen. The results support the hypothesis of recurring reciprocal translocation events in Corylus avellana cultivars, leading to the observed gametic semi-sterility. The measured hazelnut pollen viability was also strongly influenced by pollen hydration (R adj2 = 0.83, P ≤ 0.0001) and reached its maximum at around 6 h of pre-hydration in humid chambers. Viable and dead pollen were best discriminated at around the same time of pollen pre-hydration, suggesting that high humidity levels are required for hazelnut pollen to maintain its functionality. Altogether, our results detail the value of impedance flow cytometry for high throughput phenotyping of hazelnut pollen. Further research is required to clarify the causes of pollen sterility in hazelnut, to confirm the role of reciprocal chromosome translocations and to investigate its effects on plant productivity.

Dataset Information

Using flow cytometry to estimate pollen DNA content: improved methodology and applications.

Background and aims

Methods

Key results

Conclusions

Publications

Using flow cytometry to estimate pollen DNA content: improved methodology and applications.

Similar Datasets

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