Impedance Flow Cytometry for Selection of Pollen Traits Under High Temperature Stress in Pepper

Abstract

High temperature stress is a major limiting factor for pepper productivity, which will continue to be a problem under climate change scenarios. Developing heat tolerant cultivars is critical for sustained pepper production, especially in tropical and subtropical regions. In fruiting crops, like pepper, reproductive tissues, especially pollen, are the most sensitive to high temperature stress. Typically, pollen viability and germination are assessed through staining and microscopy, which is tedious and potentially inaccurate. To increase efficiency in assessing pollen traits of pepper, the use of impedance flow cytometry (IFC) has been proposed. We conducted three independent experiments to determine the most effective methodology to use IFC for evaluating pollen traits for heat tolerance in pepper. Seven floral developmental stages were evaluated, and stages 3, 4, and 5 were found to best combine high pollen concentration and activity. Flowers in development stages 3, 4, or 5 were then heat treated at 41, 44, 47, 50, and 55 °C or not heat treated (control). The critical temperature to assess heat tolerance using IFC was found to be 50 °C, with a reduction in pollen activity and concentration occurring at temperatures greater than 47 °C. Twenty-one entries of pepper were then accessed for pollen traits using the staining and IFC methods over 2 months, April (cooler) and June (hotter). Growing environment was found to be the greatest contributor to variability for nearly all pollen traits assessed, with performance during June nearly always being lower. PBC 507 and PBC 831 were identified as being new sources of heat tolerance, based on using IFC for assessing pollen. Pollen viability determined by staining and pollen activity determined using IFC were significantly positively correlated, indicating that IFC is an efficient and accurate method to assess pollen traits in pepper. This work provides a basis for further research in this area and supports more efficient breeding of heat-tolerant cultivars.

Exploration of high‐throughput data for heat tolerance selection in Capsicum annuum

“…(2018, 2020) as well as pollen concentration and activity using impedance flow cytometry following the protocol of Lin et al. (2022). In addition, we manually collected leaf temperature, which was recorded between 12:45 and 13:45 p.m. using handheld infrared thermometers held ∼5 cm above the leaf.…”

“…Phenotypic information was gathered through a combination of both manual and automated data collection. The manual data (classical phenotypes) included days to flowering, fruit maturity, yield components of fruit length, width, weight, and yield, as described by Barchenger et al (2018Barchenger et al ( , 2020 as well as pollen concentration and activity using impedance flow cytometry following the protocol of Lin et al (2022). In addition, we manually collected leaf temperature, which was recorded between 12:45 and 13:45 p.m. using handheld infrared thermometers held ∼5 cm above the leaf.…”

Enhancing Cowpea Tolerance to Elevated Temperature: Achievements, Challenges and Future Directions

“…However, advances in high-throughput phenotyping offer automated pollen number and viability analysis. Technologies like Ampha Z30, Z32, and Z40 by Amphasys employ impedance flow cytometry, providing non-destructive and portable options like P20 for use in the field [84,85]. These methods correlate well with classical fluorescein diacetate staining results [86].…”

Chili: Breeding and Genomics

“…The current climate change scenario demands the exploitation of tolerance to high temperatures in cultivated crops for sustainable crop production. High temperature is major abiotic stress affecting crop yields in chili (Lin et al, 2022). A temperature range of 18 to 30°C is ideal for the optimum growth and development of chili and a drastic reduction in vigor and yield of the crop occurs when the mean temperature exceeds 30°C (Berke et al, 2005).…”