Establishment of a rapid split-root assay in hydroponic conditions for eight upland cotton varieties

Upland cotton (<i>Gossypium hirsutum</i> L.) is a major crop in the United States. Understanding how cotton roots develop and respond to abiotic and biotic factors is crucial for improving nutrient acquisition, enhancing crop resilience under stress, and optimizing overall crop production. Split-root techniques have been developed for numerous plant species, providing a controlled framework for monitoring root development, and investigating systemic and local plant responses to various environmental factors. However, a standardized cotton-specific protocol optimized for laboratory studies has yet to be established. This protocol facilitates the rapid establishment of split-root systems in eight upland cotton varieties within four weeks after germination. This is accomplished by cutting the primary root and immediately transplanting the seedlings into hydroponic conditions to promote lateral root growth, after which the root system can be divided equally into separate compartments. Once established, each compartment can be subjected to different, independent treatments. This method was validated across all eight varieties by quantifying the difference in root dry weight between the two halves of each plant's root system and analyzing those differences across varieties. Statistical analysis was performed and Kruskal-Wallis and Wilcoxon signed-rant tests confirmed no significant difference between the roots of the two sides for any cultivar, thus confirming this method's reliability.-We developed a standardized split-root protocol tailored for upland cotton using hydroponics.-This protocol was performed on eight varieties within four weeks after germination.-We validated the method by comparing root biomass distribution between compartments to confirm reliability.