Impedance flow cytometry allows the early prediction of embryo yields in wheat (Triticum aestivum L.) microspore cultures

Abstract

Haplomethods are key biotechnological tools that make it possible to rapidly produce perfectly homozygous lines, speeding up plant breeding programs. Under specific stress conditions, microspores are reprogrammed toward sporophytic pathways, leading to embryo formation.Various endogenous and exogenous factors affect embryo yield in androgenesis, so the improvement of androgenesis efficiency requires the development of early, reliable and robust reactivity markers. During the last decade, numerous cytological, cellular and biochemical approaches were carried out to finely characterize microspore development and fate during androgenesis. However, the different available markers are often species-dependent, and their development and application are time-consuming and cumbersome. In this study, we show the suitable use of impedance flow cytometry (IFC) to develop new robust, reliable and strong markers of androgenesis reactivity in wheat, leading to: (i) routine monitoring of the viability of heterogeneous cell cultures; (ii) quick and simple evaluation of stress treatment efficiency; and (iii) early prediction of embryo yields from microspore suspensions. IFC can therefore provide the fine characterization of all of the microspore developmental pathways that occur in a cell suspension, for embryogenic microspores as well as pollen-like microspores. IFC technology has become a very useful tool to track and characterize wheat microspores in androgenesis, but can also be adapted to other species and other in vitro cell culture systems.

Hazelnut Pollen Phenotyping Using Label-Free Impedance Flow Cytometry

“…As a closing remark, IFC turned out to be strikingly useful for the label-free characterization of hazelnut pollen in a high throughput, allowing the fast identification of hazelnut cultivars with high male sterility. Moreover, its versatility holds great promise for numerous research applications in hazelnut pollen biology and pollination ecology and has the potential to simplify haploid plant production for hazelnut breeding purposes ( Gniech Karasawa et al, 2016 ), as demonstrated by recent studies on wheat ( Canonge et al, 2020 ).…”

“…Recently, label-free impedance flow cytometry (IFC) was demonstrated as a useful alternative to the above-mentioned techniques for the analysis of microspore development and characterization of pollen viability in different plant species ( Heidmann et al, 2016 ; Heidmann and Di Berardino, 2017 ; Canonge et al, 2020 ; Impe et al, 2020 ).…”

Microgametophyte Development in Cannabis sativa L. and First Androgenesis Induction Through Microspore Embryogenesis

“…Among the most relevant factors affecting microspore embryogenesis, is the microspore and pollen stage of development. It is widely accepted how vacuolate microspores and young bi-cellular pollen grains are more sensitive to the androgenic induction ( Maheshwari et al, 1980 ; Dunwell, 2010 ; Dwivedi et al, 2015 ; Canonge et al, 2020 ). On the other hand, it has been demonstrated in different species how microspore and pollen stage of development can be correlated with some features of the flower, as is the case of bud length, pedicel length, anther length and petal to anther ratio in Brassica napus L. ( Pechan and Keller, 1988 ), bud length and perianth morphological markers in Solanum lycopersicum L. ( Brukhin et al, 2003 ), pigmentation degree of anthers ( Kim et al, 2004 ) and calyx-corolla ratio ( Bárány et al, 2005 ) in Capsicum annuum L., or more recently, flower bud size in Stevia rebaudiana Bertoni ( Uskutoǧlu et al, 2019 ), and bud length, anther color, and filament length in Opuntia ficus-indica L. Mill ( Bouamama-Gzara et al, 2020 ).…”

Investigating the Use of Impedance Flow Cytometry for Classifying the Viability State of E. coli

“…This technology has shown its potential for single-cell analysis in samples containing a wide range of cell types. It has been used to characterize the electrical properties of Red Blood Cells (RBCs) [6], to analyze plant spores [7], to identify the differentiation state of Mesenchymal Stem Cells (MSCs) [8], to characterize and classify tumor cells [9], to study the effect of chemotherapy treatment on the cell membrane of HeLa cells [10], to monitor the viability of yeast cells [11] and to analyze pollen samples [12]. Due to their small size, it can be difficult to achieve sufficient signal-to-noise when measuring bacteria using IFC.…”