30
SEED TESTING INTERNATIONAL   www.seedtest.org
RULES DEVELOPMENT •
A Reliable Cold Test Method to Identify Differences in 
Vigour of Zea mays
Marie-Hélène Wagner1, Philippe Garreau1, Valérie Blouin1, Loïc Merle2, Pilar Cambet3 and Sylvie Ducournau1
1GEVES, Station Nationale d’Essais de Semences, Beaucouzé, France
2Limagrain, Ennezat, France
3FNPSMS, Germ-Services, Montardon, France
THE COLD TEST IDENTIFIED 
DIFFERENCES BETWEEN SIX SEED LOTS 
FROM TWO CULTIVARS OF MAIZE (Zea 
mays L.), with a standard germination above 90%. 
The seed lots were tested for 7 d at 10 °C followed 
by 5 d at 25 °C by five laboratories comparing 
two highly moistened substrates (paper or 
sand), one laboratory using only paper, and one 
laboratory using only sand, for a total of six cold 
tests per substrate. All laboratories consistently 
identified the same significant differences in 
seed vigour, and the results were repeatable 
within each laboratory and reproducible between 
laboratories. Sand used as a substrate resulted 
in higher correlations with field emergence in 
four locations, providing evidence in support 
of the introduction of a cold test method to the 
International Rules for Seed Testing (ISTA Rules) 
as a vigour test for maize.
Introduction
The cold test is widely used worldwide for maize, 
but many laboratories use in-house methods 
which may differ, leading to differences in 
results between them. Many factors cause 
variability in cold test results, one of which is 
the sowing temperature which depends on the 
world region. Thus, standardisation towards 
one worldwide acceptable cold test appears 
difficult. After reviewing scientific literature to 
analyse the main factors influencing the cold 
test, three laboratories compared their results on 
six different samples, comparing two substrates 
and several combinations of cold duration, 
moistening of the substrate, and growth 
temperature after cold treatment.
In 2024, seven laboratories from four countries 
– Canada, Italy, The Netherlands, France – 
compared a single combination of cold period 
and growth temperature using sand or paper. 
These are the substrates most used worldwide 
for maize germination. Only results on sand are 
reported in this article.
Material and Methods
Seed Material
Six samples of Zea mays were provided, among 
which three seed lots were from one variety and 
three from another variety. All samples were 
above 90% of germination (Table 1).
Conditions for Setting Up Cold Test
Six laboratories compared cold tests in 
sand moistened at 14% of its weight using 
four replicates of 50 seeds. The germination 
temperature was 10 °C for 7 d and 25 °C for 5 d. 
Seedlings were evaluated after 12 d according to 
the ISTA Rules for standard germination.
Field Trials
In three locations, with contrasting soil and 
climatic conditions (Fig. 1; Arras, St Mathurin 
sur Loire, Chappes), three seed lots from the 
same variety were sown at an early sowing in 
experimental plots using four blocks of 100 seeds. 
Due to shipment delay, the three other seed lots 
could not be tested in these suboptimal field 
conditions. The last field in the southwest of 
France (Haut-Mauco) was sown with all seed lots 
on 19 April due to the wet spring (Fig. 1).
Statistical Analyses 
All replicates were computed for their tolerance 
in germination test using 4 × 50 seeds in 
the tolerance tool on the ISTA website (see 
www.seedtest.org/en/services-header/tools/
germination-committee/germination-toolbox.
html). Possible outliers were assessed using 
side-by-side boxplots. The performance of the 
Figure 1. Map of the four locations for field emergence trials in France, with early sowings in  
spring 2024
M1
M2
M3
M4
M5
M6
Mean SG (%)
98
95
95
95
91
96
Standard deviation
0.3
1.2
1.1
1.6
3.9
2.1
Table 1. Details of six lots of Zea mays L.: mean 
of standard germination (SG %)

View this content as a flipbook by clicking here.