Acta agriculturae Slovenica, 118/3, 1–6, Ljubljana 2022
doi:10.14720/aas.2022.118.3.2363 Original research article / izvirni znanstveni članek
Viability of seeds of two varieties of Coffea arabica L. using different pre-
treatments in the tetrazolium test
José Duván BUENDÍA CONTRERAS 1, Laura Yolima MORENO ROZO 2, Seir Antonio SALAZAR MER-
CADO 2, 3
Received October 06, 2021; accepted August 04, 2022.
Delo je prispelo 6. oktobra 2021, sprejeto 4. avgusta 2022
1 Master of Biological Sciences, Universidad Francisco de Paula Santander, San José de Cúcuta, Colombia
2 Department of Biology, Universidad Francisco de Paula Santander, San José de Cúcuta, Colombia
3 Corresponding author, e-mail: seirantoniosm@ufps.edu.co
Viability of seeds of two varieties of Coffea arabica L. using 
different pretreatments in the tetrazolium test
Abstract: This research attempted to determine the effica-
cy of the tetrazolium test in the evaluation of the seed viability 
of two varieties of Coffea arabica L. (‘Castillo’ and ‘Cenicafé’). 
The fruits were obtained from crops located in the municipali-
ties of Salazar de las Palmas and Arboledas (Norte de Santander 
- Colombia). The test was carried out with embryos manually 
extracted from the seeds using tweezers. Three pretreatments 
were established: distilled water, sodium hypochlorite (2.5 %), 
sucrose (10  %), and a control (no pretreatment). Embryos 
were placed in a cysteine solution (0.5 %) to prevent oxidation, 
then immersed in tetrazolium solutions with concentrations of 
0.035 %, 0.075 %, and 0.1 % for a period of 6, 9, and 12 hours 
in darkness. The results of the viability test were validated with 
seed germination, using the wet paper towel method in dark-
ness. The best viability percentages were found with the ap-
plication of sodium hypochlorite (NaClO 2.5 %), with a high 
correlation with the germination percentage. The use of pre-
treatments improved the efficiency of the viability test and al-
lowed the use of low concentrations of the reagent (0.035 %), 
giving the farmer a quick and less expensive alternative to de-
termine germination capacity.
Key words: coffee; germination; optimization; pretreat-
ments
Viabilnost semen dveh sort kavovca (Coffea arabica L.) z upo-
rabo različnih predobravnavanj pred tetrazolijevim testom
Izvleček: V raziskavi smo poskušali določiti učinkovitost 
tetrazolijevega testa za ovrednotenje viabilnosti semen dveh 
sort kavovca, Coffea arabica L. (‘Castillo’ in ‘Cenicafé’). Plodovi 
kavovca so bili pridobljeni z območij Salazar de las Palmas in 
Arboledas (Norte de Santander - Colombia). Test je bil izve-
den na embrijih, ki smo jih s pinceto izolirali iz semen. Upo-
rabljena so bila tri predobravnavanja in sicer: distilirana voda, 
natrijev hipoklorit (2,5 %), saharoza (10 %) in kontrola (brez 
predobravnavanja). Embriji so bili najprej vstavljeni v raztopi-
no cisteina (0,5 %) za preprečitev oksidacije in nato potopljeni 
v različne koncentracije raztopin tetrazolija (0,035 %; 0,075 % 
in 0,1 %) za 6, 9, za 12 ur v temi. Rezulti viabilnosti embrijev so 
bili ovrednoteni s kalitvijo semen v temi na vlažnem filtrirnem 
papirju. Največji odstotek viabilnih semen je bil ugotovljen 
pri uporabi natrijevega hipoklorita (NaClO 2,5 %), z največjo 
korelacijo z odstotkom kalitve. Uporaba predobravnavanj je iz-
boljšala učinkovitost testa viabilnosti in je dopuščala uporabo 
majnih koncentracij reagenta (0,035 %), kar daje kmetom hitro 
in manj drago alternativo določanja kalivosti.
Ključne besede: kavovec; kalitev; optimizacija; predo-
bravnavanja
Acta agriculturae Slovenica, 118/3 – 20222
J. D. BUENDÍA CONTRERAS et al.
1 INTRODUCTION
Coffea arabica represents one of the most important 
crops in the world (RAMÍREZ et al., 2016; Selmar et al., 
2008). Global coffee consumption has been increasing 
due to its pleasant aroma and flavor, as well as its benefi-
cial health effects, leading to a large amount of by-prod-
uct generation (Khochapong et al., 2021). Coffee is one 
of the most traded beverages in the world and in 2016/17 
more than 9 million tons were consumed (da Silva et al., 
2021). The main coffee producers are Brazil, Colombia, 
Vietnam, India, and Indonesia which represents relevant 
economic importance for these countries (da Silva et al., 
2021). 12.5 million households worldwide receive in-
come from coffee cultivation (Montagnon et al., 2021). 
Commercially cultivated coffee species are Coffea ara-
bica L. and C. canephora Pierre ex A.Froehner (Mishra & 
Slater, 2012). Tropical African countries such as Ethiopia, 
Sudan, Kenya, Guinea, or Mozambique are usually point-
ed out as possible centers of origin, although the most 
accepted is Ethiopia (Rojo, 2014).
The coffee plant belongs to the Rubiaceae family and 
there are more than 70 species of the Coffea L. genus (da 
Silva et al., 2021), it is a tropical evergreen tree, grow-
ing at altitudes between 700 and 2000 m.a.s.l. (Tinoco & 
Peña, 2019). The green seeds of Coffea arabica are very 
rich in secondary metabolites. These metabolites have 
antioxidant properties that reduce the incidence of can-
cer and diabetes (Aissaoui et al., 2020). The coffee seed 
is a nut, oblong, flat-convex, of variable size (10-18 mm 
long and 6.5-9.5 mm wide) and mostly constituted by 
a horny endosperm where, at one of its ends and very 
superficially, lies an embryo of 3.5 to 4.5 mm long, with 
a conical radicle and cordate cotyledons (Arcila et al., 
2007). An undesirable feature of coffee seeds is that they 
have slow and asynchronous germination, which pre-
vents obtaining seedlings of desirable quality; this type of 
germination hinders rapid evaluations of viability and/or 
vigor due to the excessive time required to obtain results 
(Da Rosa et al., 2010).
The use of quick methods to know the viability is 
important, to accelerate decision-making regarding the 
management of seed lots, (Medeiros et al., 2015). For this 
reason, the tetrazolium salt test has assumed a prominent 
place for some crops, mainly due to the large amount of 
information that the test provides. Tetrazolium (TZ) can 
be used regardless of the degree of seed dormancy, be-
coming very important for species with this problem. It 
is also a very useful tool for seed producers, graders, and 
traders as it can help in decisions that need to be made 
quickly. (França-Neto and Krzyzanowski, 2019). The 
tetrazolium test is based on the activity of dehydrogenase 
enzymes that reduce the tetrazolium salt in living seed 
tissues by generating triphenyl formazan, a non-diffus-
ible red-colored compound, which indicates respiratory 
activity and viability of cells and tissues; in contrast, dead 
tissues show no coloration (Salazar et al., 2018; Salazar et 
al., 2020a). It is important to indicate that the effective-
ness of the tetrazolium test is mediated by the develop-
ment of preconditioning procedures, which facilitate the 
entry of the tetrazolium solution into the seed (Hosomi 
et al., 2017; Salazar et al., 2019).
Decreasing the concentration of tetrazolium and 
verifying its efficacy allows the evaluator a reasonable 
and optimal use of this reagent, reducing the total cost of 
the test. According to the above, this work aims to deter-
mine the efficacy of the tetrazolium test in the evaluation 
of seed viability of two varieties of Coffea arabica using 
different pretreatments.
2 MATERIAL AND METHODS
2.1 PLANT MATERIAL
The ripe fruits of the Castillo coffee variety (C. ar-
abica) were collected in village Alto del Angulo in the 
municipality of Salazar de las Palmas. The Cenicafé va-
riety drupes were collected in plantations in the village 
of San Onofre in the municipality of Arboledas, both 
belonging to Norte de Santander (Colombia). The mate-
rial was placed in plastic containers with lids, lined with 
newspaper to avoid deterioration, and transported to the 
Biology laboratories of the Faculty of Basic Sciences at 
the Universidad Francisco de Paula Santander where the 
research study was carried out. 
2.2 PRETREATMENT AND VIABILITY OF SEEDS
Solutions of 2,3,5-triphenyl tetrazolium chloride 
were prepared with concentrations of 0.035 %, 0.075 % 
and 0.1  %. To avoid embryo oxidation, a 50 mg l-1 
cysteine solution was prepared (Azofeifa, 2009). The so-
lutions were kept in dark bottles and stored in the refrig-
erator at 5 °C (Salazar et al., 2020b; Salazar et al., 2020c). 
Three pretreatments were established: 6-hour immersion 
in 2.5  % sodium hypochlorite (NaClO), 10  % sucrose 
(C12H22O11), and water (H2O). The fourth group of 
seeds corresponded to the control group. 
The fleshy layers (protective skin, pulp, parchment) 
and the integument were removed from the fruits to ob-
tain the endosperm containing the embryos. The embry-
os were extracted manually, using a dissecting case and 
forceps. One hundred embryos were placed in each Petri 
dish immersed in 5 ml of tetrazolium solution. Each test 
Acta agriculturae Slovenica, 118/3 – 2022 3
Viability of seeds of two varieties of Coffea arabica L. using different pretreatments in the tetrazolium test
had 5 replicates for a total of 500 embryos for each expo-
sure time established (6 h, 9 h, 12 h) in complete dark-
ness. An intense reddish coloration of the embryo was 
determined as positive for the tetrazolium test.
2.3 GERMINATION TEST
The germination test was performed to corroborate 
the data obtained in the viability test. Five replicates of 
100 seeds were used for 500 seeds (Salazar et al., 2020b). 
For the test, the seeds were removed from the endosperm, 
washed with distilled water, and placed to germinate for 
30 days in total darkness on paper towels moistened with 
distilled water in previously disinfected plastic contain-
ers. Germinated seeds were considered to be those that 
presented a root growth at least 4 mm long or that pre-
sented geotropic curvature and the result was expressed 
as germination percentage.
2.4 STATISTICAL ANALYSIS
For the viability test and germination test, data were 
randomly distributed with 5 replicates and 100 seeds per 
replicate. The experimental design consisted of a com-
pletely randomized block analysis. Data were analyzed 
using Infostat statistical software by analysis of variance 
(ANOVA), followed by Tukey’s multiple range HSD 
(Honest Significant Difference) test, to compare aver-
ages and determine significant differences at a level of p 
≤ 0.05. 
3 RESULTS AND DISCUSSION
3.1 PRETREATMENT AND VIABILITY OF SEEDS
The tetrazolium test evaluates the physical and 
physiological conditions of the embryonic structure of 
each seed (França-Neto & Krzyzanowski, 2019). Moreo-
ver, viable seeds are easily identified by their carmine red 
coloration (Figure 1), due to the reductive reaction of the 
tetrazolium solution under the action of dehydrogenase 
enzymes in cellular respiration (Salazar et al., 2020a). 
In the results shown in Table 1, it can be observed 
that the pretreatment with chlorine (2.5 % 6 h) was the 
one that obtained the highest viability in all concentra-
tions and exposure times with tetrazolium, values that 
ranged between 92 % and 100 % viability in C. arabica 
‘Castillo’. However, no significant differences were found 
between the control and sucrose pretreatment at the fol-
lowing concentrations and exposure time with tetrazo-
lium: 0.035 % 6, 9, 12 h; 0.075 % 9, 12 h; 0.1 % 6, 9, 12 h 
(Table 1). The lowest viability values were found in the 
pretreatment with distilled water in most of the concen-
trations and exposure time with tetrazolium, unlike the 
pretreatments with sucrose (0.035 % 12 h, 0.075 % 6 h, 
and 0.1 % 6 h) where the viability using the tetrazolium 
test were lower.
The findings in the Cenicafe variety showed few 
significant differences among pretreatments, concentra-
tions, and exposure times with tetrazolium (Table 2). 
However, the chlorine treatment had a better effect on 
seed viability. The lowest viability value (88 %) was found 
in the sucrose pretreatment, at the tetrazolium concen-
tration and exposure time of 0.035 % 9 h (Table 2). In ad-
dition, the preconditioning with distilled water identified 
statistically homogeneous means with the chlorine pre-
treatment data, unlike the concentration and exposure 
period of 0.075 % 12 h of tetrazolium. 
According to Lamarca and Barbedo (2014), the 
higher the concentration and exposure period to tetra-
zolium, the greater the intensity of embryo coloration, 
which facilitates the analysis and evaluation of viable and 
non-viable seeds. According to Tola et al. (2019), expo-
sure times of 5, 16, and 24 h and concentrations of 0.1 %, 
0.5 %, and 1 % tetrazolium are suitable for viability analy-
sis in coffee seeds. However, in this research, it was pos-
sible to obtain reliable viability levels thanks to the use of 
pre-treatments with chlorine (2.5 % 6 h), in concentra-
tions of 0.035 %, 0.075 %, 0.1 %, and exposure times of 
6, 9, 12 h of tetrazolium. These parameters are decisive 
in the tetrazolium test since they improve the efficiency 
of the viability test. Likewise, Salazar et al. (2020b) in-
creased the efficiency of the tetrazolium test on S. lyco-
persicum L. seeds, using 1  % sodium hypochlorite and 
Figure 1: Viability of Coffea arabica seeds using the tetrazo-
lium test. (A, B, C) non-viable seeds C. arabica Castilla’ (D, E) 
non-viable seeds C. arabica ‘Cenicafe’. (F, G, H): viable seed C. 
arabica ’Castilla’ (I, K) viable seed C. arabica ‘Cenicafe’
Acta agriculturae Slovenica, 118/3 – 20224
J. D. BUENDÍA CONTRERAS et al.
tetrazolium concentrations of 0.25% and 0.15% in 24h. 
However, in a study by Clemente et al. (2011) and Clem-
ente et al. (2012), subjecting sodium hypochlorite (5 %, 
6  %) for 6  h negatively impairs tetrazolium test results 
in C. arabica seeds with a moisture content below 25 %. 
Strobel et al. (2016) state that chlorine doses can generate 
embryo damage, leading to decreased seed viability. In 
addition, Salazar and Maldona, (2020) express, that chlo-
rine generates toxic effects on cells at low doses, because 
it is a strong oxidant compound (Jiang et al., 2017).
In several studies sucrose solution (10 %) has had a 
positive effect on seed viability because sucrose is linked 
to the activation of metabolic processes related to de-
hydrogenase enzymes in cellular respiration, helping to 
maintain an osmotic balance in the seed, which prevents 
damage to the embryo (Mercado and Jaimes, 2022; Sa-
lazar and Botello, 2020; Salazar et al., 2019; Hosomi et 
al., 2017; Hosomi et al., 2012). Moreover, distilled water 
treatments resulted in low viability in the Castillo variety 
at several periods and tetrazolium concentrations. These 
results differ with Mercado et al. (2020), where pretreat-
ment with distilled water on Epidendrum microtum Lindl 
seeds had 100% viability. According to Clemente et al. 
(2012), an imbibition time of 48 hours facilitates embryo 
extraction and does not affect the results of the tetrazo-
lium test. According to Carvalho et al. (2014), hydration 
favors the absorption of tetrazolium and provides the ac-
tivation of enzymatic metabolism.
3.2 COMPARISON OF GERMINATION AND VI-
ABILITY
The viability test should be validated with the ger-
mination test, indicating the consistency of the findings 
concerning the behavior of the physiological quality of 
the seeds (Tola et al., 2019). In the Castillo variety, the 
average germination percentage was 95  %, comparing 
with the viability results, the same trend was maintained, 
where no statistically significant differences were found 
with the hypochlorite treatments. Similarly, in the ceni-
cafe variety, the germination percentage was 96 %, which 
is positively correlated with the viability test. This reflects 
the efficacy of the pretreatments in improving seed vi-
ability. According to Salazar et al. (2020c), the germina-
tion test indicates whether the seeds did not germinate 
because they were dormant or because they showed 
embryo deterioration, being used to corroborate the vi-
Tetrazolium concentration and exposure time
Pretreatments 
0.035 % 
6 h
0.035 % 
9 h
0.035 % 
12 h
0.075 % 
6 h
0.075 % 
9 h
0.075 % 
12 h
0.1 % 
6 h
0.1 % 
9 h
0.1 % 
12 h
Control 90  ± 5.4 a 98 ± 4.4  a 96 ± 4.4 a 94 ± 8.9 a 98 ± 4.4 a 100 a 96 ± 5.4 ab 98 ± 4.4 a 100 a
H2O 6h 72 ± 8.3 b 84 ± 8.9 b 96 ± 5 a 96 ± 5.4 a 88 ± 4.0 b 98 ± 4.4 a 92 ± 8.3 ab 98 ± 3.4 a 92 ± 5.4 b
Sucrose  
10% 6h
92 ± 8.3 a 96 ± 54 a 94 ± 5.4 a 78 ± 7.8 b 98 ± 4.1 a 100 a 82 ± 7.8 a 98 ± 4.2 a 98 ± 1.9 a
Chlorine 
2.5% 6h
92 ± 7.5 a 98 ± 4.4 a 96 ± 8.9 a 98 ± 4.4 a 98 ± 3.9 a 100 a 100 b 100 a 100 a
Table 1: Viability of Coffea arabica ’Castilla’ seeds
Different letters indicate significant differences (p ≤ 0.05)
± Standard deviation
Tetrazolium concentration and exposure time
Pretreatments 
0.035 % 
6 h
0.035 % 
9 h
0.035 % 
12 h
0.075 % 
6 h
0.075 % 
9 h
0.075 % 
12 h
0.1 % 
6 h
0.1 % 
9 h
0.1 % 
12 h
Control 89  ± 4.4 a 98 ± 4.4 a 98 ± 4.4 a 98 ± 4.4 a 96 ± 5.4 a 100 a 98 ± 4.4 a 98 ± 4.4 a 98 ± 3.5 a
H2O 6h 96 ± 5.4 a 98 ± 4.2 a 94 ± 8.9 a 96 ± 5.4 a 98 ± 4.4 a 92 ± 8.3 b 98 ± 4.4 a 98 ± 4.4 a 98 ± 4.1 a
Sucrose  
10% 6h 96 ± 5.4 a 88 ± 4.1 b 98 ± 4.4 a 98 ± 4.4 a 98 ± 4.4 a 100 a 92 ± 8.3 a 92 ± 8.4 a 98 ± 3 a
Chlorine  
2.5% 6h 96 ± 5.2 a 98 ± 4.2 a 98 ± 4.2 a 98 ± 4.4 a 98 ± 4.4 a 100 a 98 ± 5.4 a 98 ± 4.4 a 100 ± 5 a
Table 2: Viability of Coffea arabica ‘Cenicafe’ seeds
Different letters indicate significant differences (p ≤ 0.05)
± Standard deviation
Acta agriculturae Slovenica, 118/3 – 2022 5
Viability of seeds of two varieties of Coffea arabica L. using different pretreatments in the tetrazolium test
ability results generated by the tetrazolium test. However, 
in most cases the viability test generates higher percent-
ages concerning the germination test, agreeing with the 
studies conducted by Tola et al. (2019) on Coffea arabica 
seeds.
According to the results reported by Fantazzini et 
al. (2020), the tetrazolium test is not suitable for seeds 
with germination percentages lower than 60 %. Likewise, 
these authors report that for seeds with a germination 
value greater than 60 %, the results of the tetrazolium test 
were similar to those of the germination test on C. ara-
bica seeds. Likewise, Figueiredo et al. (2017) point out, 
that several investigations have shown the discrepancy 
between the results of the tetrazolium and germination 
test in coffee seeds, specifically in those of lower quality. 
Germination of C. arabica seeds occurs slowly and 
inconsistently. Therefore, the tetrazolium test is present-
ed as a quick alternative to evaluate viability, provided 
that seed preparation and imbibition are established 
(Clemente 2012). In this research, germination percent-
ages were higher than 93 % in the Castillo and Cenicafe 
varieties and indicated a close relationship with viability 
percentages with tetrazolium. According to the above, 
the use of pretreatments can improve the efficacy of the 
tetrazolium test on C. arabica seeds. In this case, the 
seeds exposed to sodium hypochlorite (2.5 % 6 h) gen-
erated high viability percentages, which allows reducing 
the concentrations and exposure periods of tetrazolium 
to 0.035 % and 6 h, respectively, which leads to a reduc-
tion in the cost of the reagent triphenyl tetrazolium chlo-
ride to determine the viability of C. arabica seeds.
4 CONCLUSIONS
Rapid identification of viability in a seed group is 
important because it allows decisions to be made about 
it.  Coffee seed gives germination results in 25 to 30 days, 
but this can be extended up to 40 days. The use of tetra-
zolium determines effectively and with real results the 
viability of the seeds, considering the prolonged germi-
nation time. For the Castillo and Cenicafé varieties, the 
best viability percentages were found with the applica-
tion of sodium hypochlorite (NaClO 2.5 %), with a high 
correlation with the germination percentage. The use of 
pretreatments improves the efficiency of the viability test, 
allowing the use of low concentrations, up to 0.035  %, 
giving the farmer a more economical alternative to the 
expenses generated by the use of the reagent.
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