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A modified seedling phenotypic assay to identify glyphosate resistance in different alfalfa varieties

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To determine the general applicability of a seedling phenotypic assay, previously developed by Boyle and colleagues to distinguish between conventional and glyphosate-resistant seedlings, we extended the assay to four conventional and four glyphosate-resistant alfalfa varieties. We also tested the assay under more generic laboratory conditions, using petri dishes placed in an incubator. Finally, we tested and confirmed that a 16h light: 8h dark regime, 80 ppm glyphosate concentration and a 14-day germination period, represented the best conditions to separate conventional and glyphosate-resistant seedlings using the phenotypic assay. Contrary to Boyle and colleagues, we did not observe the presence of root hair in all glyphosate-resistant seedlings exposed to 80 ppm glyphosate, although none of the conventional seedlings had root hair. Variation in the percentage of glyphosate-resistant seedlings with root hair was detected among varieties in both the control and the 80 ppm glyphosate treatment. We identified the presence of secondary roots as an additional phenotypic trait useful for this phenotypic assay. Variation among varieties was also detected for percent seedlings with secondary roots. Such variation will affect the sensitivity of the phenotypic seedling assay among varieties. We therefore do not expect the presence/absence of root hair and secondary roots between glyphosate-resistant and conventional seedlings to be 100% accurate when extended to different varieties. We propose a methodology that combines the phenotypic seedling assay with the use of the RUR test strips to detect glyphosate-resistant seedlings. This methodology can be applied to different alfalfa varieties under more generic laboratory conditions and will be useful to detect gene flow events and AP presence in alfalfa seed-production fields.
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