International Journal of Environment, Agriculture and Biotechnology

Vol-10, Issue-5; Sep-Oct, 2025

Peer-Reviewed International Journal
Journal Home Page Available: https://ijeab.com/
Journal DOI: 10.22161/ijeab

L-Tryptophan Improves Germination and Early Growth

of Glycine max Seedlings Subjected to Cold Test
Samuel Mariano-da-Silva1*, Rafael Dal Bosco Ducatti2, André Luiz Radünz3, Siumar
Pedro Tironi4, Vanderlei Smaniotto5
1,3,4Professor
Course of Agronomy, Federal University of Fronteira Sul, Campus Chapecó, SC, Brazil
2Dugan Agribusiness LTDA, Luís Eduardo Magalhães, BA, Brazil
5PPGCTA, Federal University of Fronteira Sul, Campus Erechim, SC, Brazil

*Corresponding author

Received: 12 Aug 2025; Received in revised form: 10 Sep 2025; Accepted: 15 Sep 2025; Available online: 19 Sep 2025
©2025 The Author(s). Published by Infogain Publication. This is an open-access article under the CC BY license
(https://creativecommons.org/licenses/by/4.0/).

Abstract— The genetic potential of seeds does not inherently ensure uniform establishment and optimal
stand quality in commercial crop fields. This challenge has led to the development of various agricultural
inputs, often applied directly to seeds, including L-tryptophan. The present study was conducted using a
completely randomized factorial experimental design with two factors and four replications. The first
factor comprised two levels: the presence (CT) and absence (ACT) of a cold test. The second factor
included four concentrations of L-tryptophan: 0, 0.02, 0.04, and 0.08 g kg -1 of seeds. Soybean seeds were
sown on Germitest® paper substrate and maintained in a germination chamber. Evaluations included
germination rate (normal and abnormal seedlings, as well as hard or dead seeds), primary root length,
shoot length and dry matter. The application of L-tryptophan exhibited a protective effect on soybean seeds
subjected to cold stress, indicating its potential as an effective seed treatment for crops expected to
encounter low temperatures and high soil moisture during germination.
Keywords— amino acid, cold test, germination test, soybean, seed vigor.

I. INTRODUCTION establishment, as it is the case of the use of the essential

Soybean is highlighted as the sixth most produced staple amino acid L-tryptophan. These inputs have been
crop in the world and occupies the fourth largest area of associated with greater root development and stress
cultivation (FAO, 2025). Brazil stands out as the World’s resistance (Pessoa, 2021).
largest producer of soybeans (USDA-FAS, 2025), with a L-tryptophan is one of the 20 essential amino acids
production in the harvest season of 2024/2025 reaching required for protein biosynthesis. This amino acid contains
approximately 164 million metric tons (IBGE, 2025). an amino group (basic), a carboxyl group (acidic), and a
Nevertheless, the country faces big challenges and risks side chain containing an indole structure, making it an
regarding the production of this crop (Da-Silva et al., aromatic and nonpolar amino acid (Mariano-da-Silva,
2020). 2023). Plants synthesize tryptophan either from shikimic
Seed quality is one of the many factors that considerably acid or anthranilic acid, and when degraded, it is
affect soybean establishment and yield. Although, seeds responsible for producing auxin (Taiz et al., 2021).
bring with themselves high genetic potential, this potential Ahemad & Kibret (2014) demonstrated that auxin levels
does not guarantee uniform establishment and initial increase in plant tissues following an exogenous
soybean stand quality in commercial fields (Mariano-da- application of L-tryptophan. It is well known that L-
Silva et al., 2025; Mógor et al., 2008). Hence, this has led tryptophan is the main precursor in auxin biosynthesis
to the emergence of various agricultural inputs designed to pathways, a phytohormone that plays a fundamental role in
be applied directly to seeds with the aim of improving crop cell elongation (Taiz et al., 2021). In association with

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Mariano-da-Silva et al. L-Tryptophan Improves Germination and Early Growth of Glycine max Seedlings Subjected to
Cold Test

gibberellin, jasmonic acid, salicylic acid, and abscisic acid, bags. L-tryptophan was previously diluted in water in Petri
auxin positively influences the effect of PIN proteins, dishes, generating the four concentrations previously
which regulate radicle protrusion (Mukherjee et al., 2014; mentioned. Once the solution was added to the plastic
Wan et al., 2018). On the other hand, the interaction bags, air was injected, and the bags were vigorously
between auxin and jasmonic acid promotes plant tolerance shaken until the distribution of the treatments was
to abiotic stresses, such as drought, and biotic stresses considered homogeneous. The batches were then placed to
caused by fungi or insects (Hussain, 2024; Wan et al., dry in the shade at an approximate temperature of 25 °C
2018). for 24 h (Marcos-Filho, 2015).
Vigor tests (stress conditions: water deficit, salinity, 2.4 Cold and germination test
flooding, cold, controlled deterioration, and accelerated Each batch was divided into two sub-batches, where one
aging) were developed to identify differences in the sub-batch was allocated to the conventional germination
physiological potential of seed batches (Krzyzanowski et test and the other to the cold test. Seeds designated for the
al., 2020). The cold test is probably the most studied vigor cold test were sown on Germitest® paper substrate
test, assessing the ability of seeds to germinate in cold and previously moistened with a volume of water
moist soils (Cicero et al., 2020). Therefore, this test is corresponding to 3.0 times the dry weight of the paper.
expected to verify the protective effect of L-tryptophan. The rolls were then placed inside plastic bags, sealed with
These cold and humid conditions during sowing are adhesive tape, and stored in a BOD chamber at 10 ± 1 °C
commonly found in Southern states of Brazil, where, for five days (Cicero et al., 2020; Sá et al., 2011). After
according to the Ordinance 886 of the Ministry of this period, the bags were opened, and the germination test
Agriculture and Livestock (MAPA, 2023), soybean was conducted. Seeds designated for the conventional
sowing can begin as early as September 11 (Paraná State), germination test were sown on Germitest® paper
October 01 (Rio Grande do Sul State), and October 02 substrate, previously moistened with a volume of water
(Santa Catarina State). equivalent to 2.5 times the dry weight of the paper. The
The objective of this study was to evaluate the effects of paper rolls were maintained in a germinator at a constant
L-tryptophan on the germination process and seedling temperature of 25 ± 1 °C (Brasil, 2025). Germination
development of soybeans subjected to the cold test, using evaluations were performed on the fifth and eighth days
the parameters of germination, root length, shoot length, after sowing, and the data were converted into percentage
and dry matter. of normal seedlings (Brasil, 2025; Sá et al., 2011).
2.5 Root and shoot length

II. MATERIAL AND METHODS With the aid of a millimeter ruler, the length of the primary
root and shoot length were measured, and the average
2.1 Experimental settings
results were expressed in cm per seedling (Brasil, 2025; Sá
The experiment was conducted at the Seed and Grain et al., 2011).
Laboratory located at the Federal University of Fronteira
2.6 Dry matter
Sul, campus Chapecó. The soybean seeds used during the
experiment were produced during the 2023/2024 harvest The determination of dry matter was performed on normal
season by Limagrain Brasil S/A. These seeds are from the seedlings, quantifying the shoot and root portions. After
cultivar 60163IPRO (category S1) and present a weighing, the seedlings were placed in paper bags and
germination potential of 80% and a purity of 99%. dried in an oven with forced air circulation at a constant
temperature of 80 ± 2 °C for 24 hours (Sá et al., 2011).
2.2 Experimental design
After drying, the samples were weighed again after several
The trial was conducted using a completely randomized hours at room temperature, and the dry matter content was
factorial design with two factors and four replications determined and expressed in grams per seedling.
(Ares & Granato, 2014; Pimentel-Gomes, 2000). The first
2.7 Statistical analysis
factor consisted of two sub-factors: the presence (CT) or
absence of the cold test (ACT). The second factor All ANOVA assumptions were checked (normality,
consisted of four different sub-factors: 0, 0.02, 0.04, and homogeneity and independence) for all data collected
0.08 g of tryptophan per kg-1 of seed. during the trials. When all assumptions were satisfied,
ANOVA was used to analyze the variables (F-test at 95%).
2.3 Treatment with L-tryptophan
A multi comparison test (Tukey’s HSD) or a regression
The soybean seeds used in the trial were divided into eight analysis, both at 95% confidence level, was used to
batches of 200 g each and stored in transparent plastic

ISSN: 2456-1878 (Int. J. Environ. Agric. Biotech.)

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Mariano-da-Silva et al. L-Tryptophan Improves Germination and Early Growth of Glycine max Seedlings Subjected to
Cold Test

compare means that were identified as significantly L-tryptophan doses for all variables (Table 1). A
different (Pimentel-Gomes, 2000; Zimmermann, 2004). significant effect was also observed for the L-tryptophan
dose factor within each CT/ACT condition, specifically at
the doses of 0, 0.02, 0.04, and 0.08 g kg⁻¹ of seed for all
III. RESULTS AND DISCUSSION
analyzed variables. This effect can be observed through
According to the analysis of variance (F-test), a significant the estimates of the second-order polynomial equations
interaction was observed between the CT/ACT factors and presented in Fig. 01 (A, B, C, D, E and F).
L-tryptophan doses for all tested variables, including the
The results obtained demonstrated that increasing the L-
percentage of normal seedlings, percentage of abnormal
tryptophan dose led to a higher percentage of germinated
seedlings, root length, shoot length, and dry matter
seeds, both in those subjected to cold treatment (CT) and
content, indicating a dependency between these factors. By
those without it (ACT), indicating that L-tryptophan
further analyzing the interaction effect through an
positively modulated seed germination. Additionally, L-
additional analysis of variance (F-test), where the levels of
tryptophan treatment reduced the percentage of hard and
the L-tryptophan dose factor were compared within each
dead seeds compared to the control. However, this effect
CT/ACT factor level (and vice versa), a significant effect
was accompanied by an increase in abnormal seedlings in
was observed for the CT and ACT factors within all tested
the cold-treated group.
Table 1. Mean percentage of normal and abnormal seedlings, ungerminated seeds, root length, shoot length, and dry matter
content of soybean seeds and seedlings with and without the application of the cold test and treated with different doses of L-
tryptophan.
L-tryptophan concentration (g kg-1 of seeds)
Variable Treatment
0.0 0.02 0.04 0.08
ACT 77.3 a 81.4 a 85.3 a 85.8 a
Normal seedlings
CT 60.3 b 74.0 b 75.0 b 76.0 b
(%)
CV = 2.741%
ACT 19.8 a 10.0 b 12.5 b 12.5 b
Abnormal seedlings
CT 16.5 b 17.8 a 16.8 a 16.8 a
(%)
CV = 20.425%
Ungerminated seeds ACT 3.0 b 8.6 ns 2.3 b 1.8 b
(dead and hard
CT 23.3 a 8.3 ns 8.4 a 7.3 a
seeds)
(%) CV = 20.233%

ACT 8.05 a 11.07 a 11.18 a 11.88 a

Root length
CT 6.63 b 8.19 b 8.10 b 8.51 b
(cm seedling-1)
CV = 5.532%
ACT 0.93 a 1.33 a 1.47 a 1.68 a
Shoot length
CT 0.40 b 0.57 b 0.54 b 1.03 b
(cm seedling-1)
CV = 12,068%
ACT 0.3138 ns 0.8293 a 0.7793 a 0.7302 a
Dry matter
CT 0.3163 ns 0.2927 b 0.5643 b 0.5918 b
(g seedling-1)
CV = 10.200%
Means followed by the same letter do not differ among themselves within the same column for the same variable, according to Tukey's
HSD test at 95% confidence level.
ns – not significant according to the F-test (5%);
ACT – absence of the cold test;
CT – presence of the cold test;
CV – Coefficient of variance.

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Mariano-da-Silva et al. L-Tryptophan Improves Germination and Early Growth of Glycine max Seedlings Subjected to
Cold Test

Fig. 1. Second-order polynomial equations referring to (A) mean germination rate of normal seedlings, (B) mean
germination rate of abnormal seedlings, (C) ungerminated seeds, (D) mean root length of seedlings, (E) shoot length of
seedlings and (F) dry matter of seedlings, of soybean (Glycine max L), with (CT) and without (ACT) the application of the
cold test and treated with different doses of L-tryptophan.

As the treatments combining CT and L-tryptophan resulted showed a significant improvement in germination rates
in more than 70% normal seedlings, it can be inferred that under salt stress. Hussain et al. (2024), evaluating the
L-tryptophan effectively protected the seeds from the effects of L-tryptophan on sunflower (Helianthus annuus
stress induced by the test (Cicero et al., 2020). L.) seed germination under subtoxic doses of cadmium,
This trend has already been reported by several authors. reported that the amino acid reduced the metal's adverse
Korkmaz et al. (2020) demonstrated that pepper effects on seed germination rates. Queiroz et al. (2023)
(Capsicum annuum L.) seeds treated with L-tryptophan demonstrated that the exogenous application of L-

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Mariano-da-Silva et al. L-Tryptophan Improves Germination and Early Growth of Glycine max Seedlings Subjected to
Cold Test

tryptophan in soybean (Glycine max L.) seeds significantly crops sown under low-temperature and soils with high
increased germination rates, concluding that the amino moisture conditions.
acid can serve as an important inducer of seed germination
and vigor. Hanci et al. (2019) tested the effects of various
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