Abstract
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 = 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.
Highlights
Hazelnut (Corylus avellana L.) is a diploid 2n = 2x = 22 (Guo et al, 2009; Falistocco and Marconi, 2013), self-incompatible, monoecious species with peculiar reproductive biology, being wind-pollinated and flowering during late winter or early spring (Germain, 1994)
The analysis of single cells in flow cytometry has enabled a wide range of research opportunities in plant biology (Doležel et al, 2007)
The application of flow cytometry that relies on fluorescent labeling to the analysis of intact pollen has been hampered by some intrinsic characteristics of the pollen grain, such as the highly variable size and wall thickness and composition (Suda et al, 2007)
Summary
Hazelnut (Corylus avellana L.) is a diploid 2n = 2x = 22 (Guo et al, 2009; Falistocco and Marconi, 2013), self-incompatible, monoecious species with peculiar reproductive biology, being wind-pollinated and flowering during late winter or early spring (Germain, 1994). The lack of wild hazelnuts in these areas can hinder fertilization and cropping without the adoption of proper pollination strategies (Ascari et al, 2018). The effect of temperature on hazelnut pollen storage (Ellena et al, 2014; Novara et al, 2017), the influence of spermidine and temperature on pollen performance (Çetinbas-Genç et al, 2019, 2020), and the efficacy of artificial pollination in constrained growth conditions (Ascari et al, 2018) were investigated
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