33 SEED TESTING INTERNATIONAL APRIL 2026 • RULES DEVELOPMENT Validation Study for Tetrazolium Viability Test for Glycine max Ignacio Aranciaga1 and Maria Belen Aranguren2 1ISTA Executive Committee Liaison Officer for ISTA Tetrazolium Committee; ignacioaranciaga@gmail.com 2ISTA Tetrazolium Committee member; maria.aranguren@corteva.com Introduction and Objective This study aimed to develop and statistically validate a tetrazolium (TZ) viability test for Glycine max (L.) Merr. seeds, for inclusion in Chapter 6 of the International Rules for Seed Testing (ISTA Rules). Given the economic importance of soybean and the need for rapid decision making, the objective was to establish a reliable, reproducible and standardised TZ procedure for assessing seed viability across laboratories. Materials and Methods Three soybean seed lots with different quality levels were selected. The study was conducted collaboratively by seven laboratories, including six ISTA accredited laboratories and one ISTA member laboratory. Seeds were pretreated by hydration for 18 h at 20 °C, between moist paper in sealed plastic bags. Four replicates of 100 seeds per lot were stained for 6 h at 30 °C in a 1% 2,3,5-triphenyl tetrazolium chloride solution. Seeds were then rinsed, kept submerged during evaluation, manually de-coated, and longitudinally cut through the cotyledons and hypocotyl–radicle axis. Viability was assessed based on staining patterns, tissue turgidity, and location and extent of damaged or necrotic tissues, following the criteria of the ISTA Working Sheets on Tetrazolium Testing. Results Statistical analysis demonstrated clear differentiation among seed lots, with Lot 3 showing lower viability compared to Lots 1 and 2. Data exploration using box plots confirmed consistent trends across laboratories. All laboratory results met ISTA tolerance limits. Repeatability analysis showed an acceptable dispersion factor below 1, while reproducibility analysis demonstrated that interlaboratory variability was below the reference threshold. ANOVA and least squares means analysis confirmed statistically significant differences between seed lots, indicating that observed variability was due to seed lot quality. Conclusions The proposed TZ viability test for Glycine max meets ISTA performance criteria for repeatability and reproducibility. This method proved robust and consistent across participating laboratories, supporting its suitability for routine viability assessment. Based on the results, the proposed method parameters – 18-h hydration, 1% TZ solution, 6-h staining time and evaluation criteria – are recommended for inclusion in the ISTA Rules. Acknowledgements We would like to thank all the laboratories that participated in this validation study, as well as the members of the ISTA Tetrazolium and Statistics Committees for their valuable collaboration. References 1. ISTA Tetrazolium Committee report (1998– 2001). 2. ISTA (2003). ISTA Working Sheets on Tetrazolium Testing, Vol. I. International Seed Testing Association, Bassersdorf, Switzerland. 3. ISTA (2025). International Rules for Seed Testing, Chapter 6. International Seed Testing Association, Wallisellen, Switzerland. 4. Miles, S.R. (1963). Handbook of Tolerances and Measures of Precision for Seed Testing. Proceedings of the International Seed Testing Association, 28(3).
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