Use of digital image analysis, viability stains, and germination assays to estimate conventional and glyphosate-resistant cotton pollen viability

Because the success of labor-intensive hand crosses by breeders is dependent upon pollen viability, quick, simple, and inexpensive methods for viability assessment are of interest. Four such cotton pollen viability assays were compared to determine differences in viability estimates, and relative accuracy by correlation to seed set. The methods compared were Brewbaker & Kwack (B & K) medium, B & K medium plus aniline blue, a fluorochromatic reaction method (FCR), and Alexander's stain. Additionally, digital images of germinated pollen grains were analyzed by means of morphometry software to quantify pollen tube area per pollen grain, as a proposed additional method of assessing viability. Pollen from conventional, nontreated glyphosate-resistant (GR) and glyphosate-treated GR cotton (Gossypium hirsutum L.) plants was tested by each method. Glyphosate treatments to GR cotton reduced pollen viability and corresponding seed set in ail methods tested. Pollen germination measured by the B & K method was most closely related to seed set per boll, while Alexander's stain gave the highest estimates of viability. The FCR method indicated that many pollen grains from glyphosate-treated GR cotton were irregularly shaped and only partially flourescein diacetate (FD) stained. All methods tested showed similar high correlation (0.7-0.8) of pollen viability to seed set. Morphometric analysis of digital images of germinated pollen found the greatest pollen tube area to pollen grain ratio with B & K medium + 30 mM sucrose. Because the B & K method most closely predicted the linear magnitude of seed set reduction to reduced pollen viability, allowed the use of morphometry software analysis, and was one of the simplest and least equipment-demanding methods, it may provide broad utility for those assessing cotton pollen viability.