An Evaluation of Nuclei Preparation of the Dormant Axillary Bud of Grapevine for Cell Cycle Analysis by Flow Cytometry.

Michael J Considine,Dina Hermawaty,John A Considine

doi:10.3389/fpls.2022.834977

Michael J Considine, Dina Hermawaty + Show 1 more

Open Access

https://doi.org/10.3389/fpls.2022.834977

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Abstract

Whether the division of cells of a dormant meristem may be arrested, e.g., in the G1 phase, has proven to be an extremely difficult hypothesis to test. This is particularly so for woody perennial buds, where dormant and quiescent states are diffuse, and the organ may remain visibly unchanged for 6–9 months of the year. Flow cytometry (FCM) has been widely applied in plant studies to determine the genome size and endopolyploidy. In this study, we present the application of FCM to measure the cell cycle status in mature dormant buds of grapevine (Vitis vinifera cv. Cabernet Sauvignon), which represent a technically recalcitrant structure. This protocol illustrates the optimisation and validation of FCM data analysis to calculate the cell cycle status, or mitotic index, of dormant grapevine buds. We have shown how contamination with debris can be experimentally managed and give reference to the more malleable tomato leaves. We have also given a clear illustration of the primary pitfalls of data analysis to avoid artefacts or false results. Data acquisition and analysis strategies are detailed and can be readily applied to analyse FCM data from other recalcitrant plant samples.

Highlights

Plant growth and development result from a combination of cell division and cell expansion
Several studies have implicated the regulation of cell division through cell cycle machinery complexes, i.e., the cyclins (CYCs) and CYC-dependent kinase (CDKs), during seed germination and dormancy transition in perennial buds (Hansen et al, 1999; Horvath et al, 2003; Barrôco et al, 2005; Saito et al, 2015; Cembrowska-Lech and Kêpczynski, 2016)
The coefficient of variation (CV) value indicates the integrity of nuclei and the variation of the fluorescence signal, and the debris factor (DF) value assesses the quality of the nuclei suspension preparation by calculating the proportion of high-quality intact nuclei in the nuclei suspension

Summary

Introduction

Plant growth and development result from a combination of cell division and cell expansion. Meristematic activity is discontinuous, in organs that undergo a dormant phase, such as seeds of certain species and the proleptic buds of many temperate perennial plants. In such organs, tight regulation between active growth and arrest is a crucial survival strategy, protecting the meristem from unfavourable seasonal conditions (Anderson et al, 2010). Several studies have implicated the regulation of cell division through cell cycle machinery complexes, i.e., the cyclins (CYCs) and CYC-dependent kinase (CDKs), during seed germination and dormancy transition in perennial buds (Hansen et al, 1999; Horvath et al, 2003; Barrôco et al, 2005; Saito et al, 2015; Cembrowska-Lech and Kêpczynski, 2016).

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