Pollen viability is crucial for plant fertilization and is highly susceptible to temperature changes, particularly to heat stress. A fast and reliable method for screening pollen tolerance to heat stress is critical regarding improving plant’s heat stress tolerance. Previous research disclosing heat stress effects on pollen viabilities were usually less efficiency, given that it required large space, but also predominantly examined only one single heat temperature, thereby inadequately reflecting the dynamic viabilities of pollens under a range of temperatures.

The objectives of this study were to 1) develop an easy and reliable method for investigating wheat pollen viability under different temperatures and 2) identify the best temperature range for screening pollen heat stress tolerance. To accomplish this, mature wheat pollens were harvested and distributed to a 96-well PCR plate filled with pollen culture medium. The PCR plate was then placed in a PCR machine operating under a gradient PCR program to manipulate temperatures.

After four-hour incubation under a series of temperatures (ranging from 21.9°C to 47°C), the pollens were examined under an upright microscopy. A specific image analysis pipeline was employed to quantitatively assess the impact of temperatures on pollen morphological traits, germination rates and tube growth. This method facilitates the screening of a large amount of pollen samples simultaneously, enabling rapid, reliable, and precise determination of pollen viability, germination, and developmental characteristics in response to temperatures.

This system can be employed to screen germplasm and identify quantitative trait loci (QTL)/genes for pollen heat stress tolerance and therefore can be effectively used in breeding programs.