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Discussion and Conclusion
The cold test consisting of 7 d at 10 °C and 5 d at  
25 °C clearly identified the same seed lots as high 
or low vigour in six laboratories from four coun-
tries. The repeatability and reproducibility of the 
cold test for maize shown in this comparative test 
indicate that a cold test carried out at 10 °C for 7 d 
followed by 5 d at 25 °C using sand can be applied 
as a vigour test for this species. Germination tol-
erance tables could be used to check outliers.
Cold test results were consistent with field 
emergence results as previously obtained in Italy, 
where more seed lots were used to obtain signif-
icant correlations with the same method of 7 d at 
10 °C in sand (Noli et al., 2008).
Factors influencing cold test results have been 
described in literature. Former comparative tests 
have shown that CT in sand presented fewer 
outliers than CT in soil, and that the method was 
less variable between different regions in the 
world than between different laboratories from 
the same region, especially for in-house methods 
(Nijenstein and Kruse, 2000).
Even though it would have been preferable 
to use more than three seed lots in some field 
trials, the differences between three lots were 
very clear, in both their CT result and their field 
emergence percentage in all three locations 
(Arras, Chappe and Saint Mathurin sur Loire). In 
addition, the same lots emerged well, or poorly 
in each location. The CT also clearly identified 
differences in seed lot performance in the better 
conditions of Haut-Mauco. Thus, the test can 
identify possible differences in emergence in a 
wide range of environments.
Acknowledgements
We gratefully acknowledge Jean-Louis Laffont 
(ISTA Statistics Committee) for his great input 
in statistical analysis and Cassandre Chupeau 
(GEVES) for her helpful analyses on pluriannual 
data using R. We are grateful to Kirk Remund, 
Michael Kruse and Takayuki Okuda for reviewing 
the test plan and validation report.
We also warmly thank all participants to the 
comparative tests in 2023 and 2024, namely: 
Nelly Boinot, Thierry Brunel, Catherine 
Champion, Erik van Egmond, Dorothée Jouany, 
Carey Matthiessen, Enrico Noli, Sandrine Pierre, 
Sandrine Stievenard and Raphaël Suaud. We 
thank Limagrain and Mas Seeds for providing 
seed lots and carrying out field trials. A special 
thanks to the ISTA Vigour Committee who 
improved the validation report with many 
comments and constructive support, especially 
to the former Chair Alison Powell, who did great 
revisions through the review process.
References
1.	ISTA (2025). International Rules for Seed 
Testing. International Seed Testing Association, 
Wallisellen, Switzerland.
2.	Nijenstein, J.H. and Kruse, M. (2000). The 
potential for standardisation in cold testing of 
maize (Zea mays L.). Seed Science and Technology, 
28, 837–851.
3.	Noli, E., Casarini, E., Urso, G. and Conti, S. 
(2008). Suitability of three vigour test procedures 
to predict field performance of maize seed in 
early sowing. Seed Science and Technology, 36, 
168–176.
Method
p_avg
s_Lab
s_Lot × 
Lab
s_r
f_r
s_R
Excluded 
samples %
f_R
f_Miles
7 d @ 10 °C + 5 d @ 25 °C 
in sand moistened at 14%
86
0
1.57
4.32
0.89
2.67
0
1.1
1.66
Table 4. Repeatability/reproducibility estimates using linear mixed model
Figure 4. Mean cold test results per laboratory for six maize seed lots

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