Large-Scale Analysis of Pollen Viability and Oxidative Level Using H₂DCFDA-Staining Coupled with Flow Cytometry

Area of Science:

• Plant reproductive biology
• Cellular physiology
• Biotechnology

Background:

• Assessing plant reproductive capacity requires accurate pollen viability metrics.
• Flow cytometry is a high-throughput tool, but its application in pollen studies is limited, primarily for DNA content analysis.
• Existing methods for pollen viability assessment are often time-consuming or lack precision.

Purpose of the Study:

• To introduce a novel, rapid, and robust flow cytometry-based protocol for evaluating pollen viability.
• To utilize the fluorescent reporter dye H2DCFDA for assessing reactive oxygen species (ROS) levels in pollen.
• To provide detailed guidelines for implementing this method in plant biology research.

Main Methods:

• Development of a flow cytometry protocol using H2DCFDA staining.
• Analysis of pollen viability and ROS levels in *Arabidopsis thaliana* and *Solanum lycopersicum*.
• Detailed description of staining and analysis procedures for broad applicability.

Main Results:

• The H2DCFDA-based flow cytometry approach enables robust and rapid evaluation of pollen viability.
• Pollen from *Arabidopsis thaliana* and *Solanum lycopersicum* naturally segregates into two subpopulations with differing ROS levels.
• The method is adaptable for various cellular reporters and pollen-related investigations.

Conclusions:

• This flow cytometry method offers a significant advancement for studying pollen physiology and reproductive success.
• The identification of distinct pollen subpopulations based on ROS levels provides new insights into plant reproductive mechanisms.
• The protocol's versatility supports its application in diverse research areas, including stress response studies in plants.