Assessing data analysis techniques in a high-throughput meiosis-like induction detection system

• Published in: Plant methods Vol. 20; no. 1; p. 7
• Main Authors: Cook, Tanner M, Biswas, Eva, Dutta, Somak, Aboobucker, Siddique I, Hazinia, Sara, Lübberstedt, Thomas
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 12.01.2024; BioMed Central; BMC
• Subjects: Analysis; Cell cycle; Channel gating; Cytogenetics; Data analysis; Detectors; Flow cytometry; Gametes; In vitro biology; Information management; Meiosis; Meiosis induction; Methods; Microfluidic devices; Microfluidics; Open source software; Plant breeding; Plant breeding and biotechnology; Polygons; Population changes; Public software; Seeds; Single-cell analysis; Source programs; United States; In vitro biology; Meiosis induction; Plant breeding and biotechnology; Single-cell analysis
• ISSN: 1746-4811; 1746-4811
• DOI: 10.1186/s13007-023-01132-9

Strategies to understand meiotic processes have relied on cytogenetic and mutant analysis. However, thus far in vitro meiosis induction is a bottleneck to laboratory-based plant breeding as factor(s) that switch cells in crops species from mitotic to meiotic divisions are unknown. A high-throughput system that allows researchers to screen multiple candidates for their meiotic induction role using low-cost microfluidic devices has the potential to facilitate the identification of factors with the ability to induce haploid cells that have undergone recombination (artificial gametes) in cell cultures. A data analysis pipeline and a detailed protocol are presented to screen for plant meiosis induction factors in a quantifiable and efficient manner. We assessed three data analysis techniques using spiked-in protoplast samples (simulated gametes mixed into somatic protoplast populations) of flow cytometry data. Polygonal gating, which was considered the "gold standard", was compared to two thresholding methods using open-source analysis software. Both thresholding techniques were able to identify significant differences with low spike-in concentrations while also being comparable to polygonal gating. Our study provides details to test and analyze candidate meiosis induction factors using available biological resources and open-source programs for thresholding. RFP (PE.CF594.A) and GFP (FITC.A) were the only channels required to make informed decisions on meiosis-like induction and resulted in detection of cell population changes as low as 0.3%, thus enabling this system to be scaled using microfluidic devices at low costs.