Hazelnut Pollen Phenotyping Using Label-Free Impedance Flow Cytometry

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 (Radj2 = 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.

Recent Advances in Electrical Impedance Sensing Technology for Single-Cell Analysis

“…Recent IFC devices applied in single-cell analysis are summarized in Table 1 . These applications, discussed in this subsection, are simply classified according to cell species, including blood cells [ 110 , 111 , 112 , 113 , 114 ], tumor cells [ 43 , 52 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 ], stem cells [ 123 , 124 , 125 , 126 , 127 ], plant cells [ 60 , 128 , 129 , 130 , 131 , 132 ] and microbes [ 53 , 62 , 133 , 134 , 135 , 136 , 137 , 138 , 139 , 140 , 141 ]. In terms of blood cells, researchers focused on the identification and counting of normal or diseased blood cells.…”

“…As for stem cells, the main focus is the impedance measurement of their long-term differentiation process. Studies of plant cells include the detection of pollen viability [ 128 , 129 , 130 , 131 , 132 ] and cell screening [ 60 ]. Besides, impedance measurements of microbes are further classified and discussed.…”

“…Furthermore, Heidmann et al predicted the germination rate of tomato pollen population by measuring the amount of viable and dead pollens [ 129 ]. Impe et al [ 130 ] and Ascari et al [ 131 ] assessed pollen viability of hazelnut and wheat, and further identified various factors (sugar, H 3 BO 3 , CaCl 2 ·2H 2 O/Ca(NO 3 ) 2 ·2H 2 O concentration and pH) affecting pollen viability with the same commercial device. Canonge et al utilized the IFC device to track and characterize the developmental process of wheat ( Triticum aestivum L.) genotype Pavon microspores in gametogenesis and anrogenesis [ 132 ].…”

Potential of Impedance Flow Cytometry to Assess the Viability and Quantity of Cannabis sativa L. Pollen

“…IFC characterizes dead and viable pollen cells in a broad range of species such as wheat, corn, hazelnut, tomato, tobacco, etc. [26,27]. In contrast to traditional methods, IFC determines the electrical properties of cells by using microfluidic chips and analyzes a high throughput of pollen cells for viability screening in a standardized method, up to 1000 cells per second [26].…”

Impedance Flow Cytometry for Selection of Pollen Traits Under High Temperature Stress in Pepper

“…Like pollen viability tests, this method is laborious and has low throughput, limiting its usefulness in breeding. There have been reports that IFC is an efficient, label-free, and reliable technique to analyze pollen activity in a species-independent, high-throughput manner (Ascari et al, 2020;Canonge et al, 2020;Heidmann et al, 2016;Heidmann and Di Berardino, 2017;Impe et al, 2020). In IFC, pollen grains in suspension flow through a microchannel, where an alternating electric field is applied between 2 and 12 MHz.…”