Published January 22, 2016
Research
A Simple Greenhouse Method for Screening
Salt Tolerance in Soybean
Fernando Ledesma, Cindy Lopez, Diana Ortiz, Pengyin Chen,* Kenneth L. Korth,
Tetsuaki Ishibashi, Ailan Zeng, Moldir Orazaly, and Liliana Florez-Palacios
ABSTRACT
Salinity is an important limiting factor for crop pro-
duction. Over 800 million ha of land globally are
salt-affected. Soybean [Glycine max (L.) Merr.]
is moderately salt-tolerant; however, excessive
salt reduces yield. Developing a quick, reliable,
and inexpensive screening method is critical for
soybean breeding programs. This study aimed
to develop a rapid method for screening salt
tolerance in soybean and to determine the best
growing media and NaCl concentrations for
screening. Four soybean cultivars, known as
Cl – includers (‘Williams’ and ‘Dare’) or excluders
(‘S-100’ and ‘Lee 68’), were screened for salt
tolerance. These four genotypes were grown in
soil, sand, and potting mix and treated with 0,
80, 120, and 160 mM NaCl. Treatment was initi-
ated at the first trifoliate leaf expansion in the
second true node above the unifoliate leaves.
Two weeks later, leaf scorch score on a 1–9 scale
(1  = no chlorosis; 9 = necrosis) was taken.
Additionaly, leaf and root Na+ and Cl- concen-
trations were analyzed. The clearest differences
between tolerant and sensitive cultivars were
obtained using 120-mM NaCl in soil. Once the
best conditions to evaluate salt tolerance were
established, 14 cultivars were screened to iden-
tify those with the most contrasting response.
The most sensitive cultivars were Williams and
‘Clark’; the most tolerant were ‘HBK R5525’ and
‘AG5905’. To validate this method, 97 genotypes
were evaluated under these conditions with
differential responses. The proposed screening
methodology was effective in identifying a range
of sensitive and tolerant genotypes, allowing
confirmation of salt tolerance in some previously
reported genotypes.
crop science, vol. 56, march– april 2016  www.crops.org 1
F. Ledesma, C. Lopez, D. Ortiz, P. Chen, T. Ishibashi, A. Zeng, M.
Orazaly, and L. Florez-Palacios, Dep. of Crop, Soil and Environmental
Sciences, 115 Plant Sciences Building, Univ. of Arkansas, Fayetteville,
Arkansas 72701; K. Korth, Dep. of Plant Pathology, Plant Sciences
Building 217A, Univ. of Arkansas, Fayetteville, Arkansas 72701.
F. Ledesma, C. Lopez, and D. Ortiz contributed equally to this work.
Received 14 July 2015. Accepted 15 Sept. 2015. *Corresponding author
(pchen@uark.edu).
Abbreviations: LSS, leaf scorch score.
S alinity is an important type of abiotic stress that affects plants,
causing significant damage such as a decrease in productivity
and quality and whole-plant death (Chinnusamy et al., 2005).
Saline soil can be caused by the frequent use of irrigation water
with an elevated salt content, improper drainage, rainwater, sea
spray, or air pollution. High salinity levels induce osmotic stress,
nutrient deficiencies, and ion toxicity in plants (Munns et al.,
2002). Saline soils are characterized by the presence and accumula-
tion of Na+ and Cl- ions. Chloride is considered to be an essential
micronutrient for plants that is important during the water-split-
ting reaction of photosynthesis (Marschner, 1995; Clarke and
Eaton-Rye, 2000). However, high concentrations of Cl- or Na+
can cause injury and toxcity in plants (Lessani and Marschner,
1978). In soybean, Cl– accumulation has been associated with the
presence of chlorosis and necrosis in leaves and stems, in addition to
a general loss in productivity (Abel and MacKenzie, 1964; Parker et
al., 1983; Wang and Shannon, 1999; Essa, 2002).
More than 800 million hectares of land across the world are
affected by salt (including both salinity and sodicity), which is
equivalent to more than 6% of the global land area (Arzani, 2008)
and about 20% of the arable land (Sairam and Tyagi, 2004). Accord-
ing to Blumwald and Grover (2006), the percentage of affected land
area could increase drastically in the next years, and it is expected
that nearly 50% of cultivated land will be affected by salt by 2050.
Published in Crop Sci. 56:1–10 (2016).
doi: 10.2135/cropsci2015.07.0429
© Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA
All rights reserved.
Therefore, efforts in plant breeding and genetics will play a
fundamental role in crop improvement facing soil salinity.
Parida and Das (2005) defined salt tolerance as the
ability of plants to grow and complete their life cycle on a
substrate with high concentrations of soluble salts. Soybean
is a moderately salt-tolerant crop. It has been found that not
all soybean genotypes show the same level of salt damage,
which suggests the presence of genetic variability in soy-
bean in response to salt stress (Abel and MacKenzie, 1964;
Parker et al., 1983; Pantalone et al., 1997; Shannon, 1997;
Lee et al., 2004). Therefore, plant breeders could improve
salt tolerance by identifying the genetic variability of this
trait to develop new cultivars that are adapted to salt stress.
Because of the importance of identifying salt-tolerant
soybean cultivars, several screening studies have been
conducted (Parker et al., 1983; Yang and Blanchar, 1993;
Pantalone et al., 1997; Xu et al., 1999; An et al., 2002;
Lee et al., 2004, 2008; Valencia et al., 2008). Field screen-
ing evaluations of soybean genotypes planted in soils with
high salt concentration were unsuccessful because of the
variability of salt levels across the soil and the changing
environmental conditions. Better results were found with
the hydroponics method, in which a NaCl solution was
supplied to the plants under greenhouse conditions. The
advantage of this method is that the nutrient levels and
environmental conditions are controlled (Valencia et al.,
2008). However, during the experiment, nutrient solutions
need to be constantly changed, which is considered to be
expensive and inefficient. Recently, a simpler screening
called the plastic cone-tainer method has been tested with
good results. In this method, sandy soil was used as a growth
medium instead of a nutrient solution (Lee et al., 2008). Even
though the plastic cone-tainer method seems to be easy, less
laborious, and less time consuming than previously reported
methods, it is necessary to test more types of growing media
and evaluate different levels of NaCl to develop an efficient
method of screening soybean cultivars.
Developing a quick, reliable, and inexpensive screen-
ing method is critical for soybean breeding programs and
genetic studies. The objective of this study was to develop
a rapid screening method for salt tolerance in soybean and
to evaluate and identify genotypes with the most contrast-
ing responses to salt stress for future genetic study.
MATERIALS AND METHODS
Plant Materials and Growth Conditions
The experiments were performed in the Rosen Center green-
house at the University of Arkansas, Fayetteville, AR. Plants
were maintained under 14 h light and 26°C/21°C (day and
night respectively) throughout the experiment.
In the first experiment, four soybean cultivars were
screened for salt tolerance: Williams and Dare, known as Cl– –
sensitive genotypes (Ping et al., 2002; Velagaleti and Marsh,
1989), and S-100 and Lee 68, known as Cl– –tolerant genotypes
2 www.crops.org crop science, vol. 56, march– april 2016
Figure 1. View of soybean plants growing in pots filled with sandy
soil, immersed in 120 mM saline solution in a plastic tray 10 d after
treatment with NaCl.
(Essa, 2002; Lee et al., 2004; Pantalone et al., 1997). Six
seeds of each genotype were planted in plastic pots (90 mm
in diameter), which contained three different growing media:
sandy loam soil (sandy loam, Kibler fine-loamy, micaceous,
mesic Typic Dystrudepts), river sand, or potting mix (Redi-
earth, Vermiculita and Canadian Sphagnum peat moss, Sun Gro
Horticulture Distribution Inc., Bellevue, WA). Subsequently,
pots were placed in plastic trays to irrigate soybean plants from
the bottom (Fig. 1). Once the seedlings emerged, four healthy
and uniform plants were selected and maintained in each pot.
At the beginning of V1 stage (expansion of the first trifoliate
leaf ), salt treatment was initiated. Salt treatment consisted of
four NaCl concentration levels: 0, 80, 120, and 160 mM. A
volume of four liters of salt solution from each concentration
was added to the plastic trays containing the pots and main-
tained for 2 h daily for 2 wk. Right after the 2-h treatment each
day, the solution was removed from the trays, and no other type
of irrigation was applied. Nutritional deficiencies were avoided
with the application of water-soluble fertilizer (Miracle-Gro
All Purpose Plant Food, The Scotts Miracle-Gro Company,
Marysville, OH) once a week after the second trifoliate stage.
The same methodology was repeated with 14 soybean
genotypes (Table 1) using the best salt treatment identified in
the first experiment. The genotypes Clark, Williams, S-100
and Lee 68 were included as checks. To validate the results of
this methodology, a third experiment was performed evaluating
a wider range of genotypes (Table 2).
Measurements
Fifteen days after the salt treatment, two criteria were used to
evaluate and classify tolerant and sensitive genotypes. First,
plants were visually rated using a leaf scorch scale (LSS). Leaves
were scored from 1 to 9 where 1 = healthy dark green leaves
with no chlorosis and 9 = necrotic leaves (Fig. 2). After the
evaluation of LSS, leaves and roots of individual plants from
each treatment were harvested separately and oven-dried at
70°C for 5 d. Subsequently, the dried tissue was ground and 0.3
g of tissue was analyzed to determine the Cl- and Na+ content.
Deionized water (Kalra, 1998) was used to extract Cl-. Acid
Table 1. Leaf scorch score and Cl – concentration in 14 soybean cultivars 14 d after 120 mM NaCl treatment in soil.
Cultivar Leaf scorch score† Symptom Response Cl— Classification
mg kg-1
AG4605 8.3 a Necrosis Sensitive 71,370 abcd Includer
Williams 8.2 a Necrosis Sensitive 97,110 a Includer
Dare 8.0 a Necrosis Sensitive 79,710 abc Includer
AG4903 8.0 a Necrosis Sensitive 74,630 abcd Includer
HBK R4924 8.0 a Necrosis Sensitive 74,370 abcd Includer
Clark 7.9 a Necrosis Sensitive 90,590 ab Includer
Hutcheson 6.7 b Chlorosis Segregating 53,640 cde Mixed
Forrest 6.0 bc Chlorosis Segregating 59,880 bcde Mixed
Hartwig 5.7 cd Chlorosis Segregating 55,360 cde Mixed
LEE 68 5.2 cde Light green Tolerant 35,600 ef Excluder
AG5905 5.1 cde Light green Tolerant 31,030 ef Excluder
S-100 4.9 def Light green Tolerant 45,570 def Excluder
AG5605 4.3 ef Light green Tolerant 34,020 ef Excluder
HBK R5525 4.0 f Light green Tolerant 21,120 f Excluder
Mean 6.5 59,778
CV 9.4 16.9
Tukey0.05 1.9‡ 31,028§
† Leaf scorch score (LSS): 1 = green or healthy leaves; 9 = necrosis.
‡ Based on Tukey’s honest significant difference = 1.9, LSS ³6.4 are includers; LSS £5.9 are excluders.
§ Based on Tukey’s HSD = 31,028 Cl- ³66,082 are includers; Cl- £52,148 are excluders. All others are mixed.

Figure 2. Leaf scorch score (LSS) system for evaluating soybean for salt tolerance (1 = no chlorosis; 9 = necrosis). Score 1 shows a
completely healty green leaf from a soybean plant not subjected to salt stress, whereas Score 2 shows a stunted dark-green leaf typically
found in tolerant cultivars under salt stress. Score 3 shows a leaf with slight chlorosis and Score 9 with severe necrosis.

crop science, vol. 56, march– april 2016  www.crops.org 3

Table 2. Leaf scorch score (LSS), Cl – concentration in leaves, Table 2. Continued.
and percentage of dead plants for 98 genotypes after 14 d of
Genotype LSS† Cl- Dead plants Classification
120 mM NaCl treatment in the greenhouse.
R07–5351 5.9 49,440 16.7 ± 40.8 Mixed
Genotype LSS† Cl- Dead plants Classification R04–342 5.8 59,356 27.5 ± 32.5 Mixed
mg kg-1 %‡ R09–209 5.8 65,714 30.0 ± 47 Mixed
R09–1237 8.2 80,723 95.3 ± 48 Includer R09–319 5.8 59,795 37.9 ± 33.9 Mixed
Dare 8.0 81,952 70.4 ± 43.4 Includer AG5606 5.7 61,937 20.0 ± 40 Mixed
UARK-5798 7.7 79,332 58.2 ± 41 Includer UA 5414RR 5.7 52,706 10.0 ± 24.5 Mixed
Desha 7.5 73,363 46.0 ± 36.2 Includer R09–4571 5.7 62,786 27.1 ± 31.8 Mixed
R04–1250RR 7.5 71,948 55.6 ± 39.5 Includer R01–327 5.6 62,552 20.7 ± 27.3 Mixed
R04–572 7.5 66,599 69.8 ± 37.3 Includer R07–1826 5.6 38,893 13.1 ± 21.4 Mixed
R05–1415 7.5 74,933 38.5 ± 41.5 Includer Lee 5.5 56,026 8.3 ± 13.9 Excluder
R05–4969 7.5 73,671 43.7 ± 45.8 Includer R09–1827 5.5 53,024 10.7 ± 20 Excluder
R07–7775 7.5 73,394 66.7 ± 40.8 Includer RM-22590 5.4 66,416 13.9 ± 22.1 Excluder
Walters 7.5 76,726 50.0 ± 54.7 Includer R01–976 5.3 56,316 20.5 ± 32.7 Excluder
R01–2731F 7.4 65,388 62.5 ± 46 Includer R04–1274RR 5.3 56,050 4.2 ± 10.2 Excluder
Clark 7.3 69,945 34.2 ± 39.2 Includer R08–3206 5.3 55,740 8.3 ± 20.4 Excluder
R09–1831 7.3 58,766 40.6 ± 38.3 Includer R07–10322 5.2 63,235 2.4 ± 5.8 Excluder
R98–209 7.3 68,773 50.9 ± 19.6 Includer R08–527 5.2 58,006 26.4 ± 38.9 Excluder
R06–2082RR 7.2 64,105 33.1 ± 38.6 Includer R97–1634 5.1 61,894 21.9 ± 34.1 Excluder
Narow 7.1 68,573 48.6 ± 46.7 Includer RM-1639 5.1 58,469 0±0 Excluder
Ozark 7.1 61,722 47.5 ± 26.7 Includer RM-9508 5.1 45,743 5.6 ± 13.6 Excluder
R08–2776 7.1 68,751 39.6 ± 37.9 Includer AG5905 5.0 55,147 0±0 Excluder
R08–4002 7.1 73,253 69.6 ± 40.9 Includer AG5605 4.9 55,544 3.7 ± 14.9 Excluder
R08–47 7.1 73,072 38.2 ± 35 Includer R06–4433 4.9 61,942 16.1 ± 32 Excluder
R01–416F 7.0 61,088 31.4 ± 37.5 Includer R08–1178 4.9 38,081 27.5 ± 39.2 Excluder
UA 5014C 7.0 67,010 48.4 ± 41.6 Includer UARK-5896 4.9 57,698 0±0 Excluder
R95–1705 7.0 71,158 56.7 ± 37.6 Includer R05–1947 4.8 49,531 9.7 ± 15.3 Excluder
R07–6669 6.9 64,355 35.0 ± 36.3 Includer R07–190 4.8 55,973 13.9 ± 26.7 Excluder
Forrest 6.8 60,416 38.3 ± 26.6 Includer R07–330 4.8 57,996 16.8 ± 22.3 Excluder
Glenn 6.8 69,205 38.4 ± 33.2 Includer R09–2988 4.7 56,369 5.6 ± 13.6 Excluder
Lonoke 6.8 59,154 32.1 ± 31.4 Includer S-100 4.7 58,279 0±0 Excluder
R05–1772 6.8 69,081 43.1 ± 46 Includer 5002T 4.6 58,159 0±0 Excluder
R09–5088 6.8 57,517 31.2 ± 30 Includer R09–2567 4.6 59,230 9.8 ± 15 Excluder
R09–5225 6.8 85,225 52.4 ± 44.9 Includer AG4606 4.5 54,612 5.6 ± 13.6 Excluder
Caviness 6.7 54,671 43.5 ± 31.7 Includer R07–129 4.5 39,910 14.3 ± 35 Excluder
AG4907 6.6 67,625 27.8 ± 27 Includer R07–167 4.5 51,819 7.5 ± 11.7 Excluder
Hutcheson 6.6 70,956 38.5 ± 48.1 Includer R02–3065 4.4 58,189 9.7 ± 15.3 Excluder
R06–4475 6.6 62,582 26.3 ± 30.1 Includer Jake 4.3 54,627 8.3 ± 20.4 Excluder
R08–141 6.6 56,830 33.7 ± 41.5 Includer Osage 4.3 40,105 16.7 ± 40.8 Excluder
R08–3211 6.6 61,302 37.9 ± 42.5 Includer R05–235 4.3 55,106 2.4 ± 5.8 Excluder
R09–1822 6.6 81,275 37.5 ± 44 Includer R08–2797 4.3 56,407 3.3 ± 8.2 Excluder
R09–3742 6.6 74,516 44.4 ± 50.2 Includer R08–107 4.0 45,388 12.5 ± 30.6 Excluder
R09–4010 6.6 77,424 44.4 ± 50.2 Includer R03–1250 3.8 51,787 0±0 Excluder
JTN-5503 6.5 66,028 35.7 ± 42.1 Includer R09–430 3.8 47,521 0±0 Excluder
R01–581F 6.5 65,433 26.7 ± 43.2 Includer UA 5213C 3.7 52,170 0±0 Excluder
R07–1857 6.5 67,159 41.7 ± 49.1 Includer R07–6654 3.5 42,152 0±0 Excluder
UA 4805 6.5 66,347 32.7 ± 40.9 Includer Mean 5.97 61,559.6 27
R01–3474F 6.4 58,912 26.8 ± 35.1 Includer CV 13.18 11.3 75.2
R09–1589 6.4 67,581 55.0 ± 34.3 Includer Tukey0.05 1.96 30,598.4 71.6
R08–3119 6.3 67,560 33.2 ± 44.7 Includer † Leaf scorch score: 1 = Green or healthy leaves; 9 = necrosis; based on Tukey’s
5601T 6.2 63,841 8.3 ± 20.4 Includer honest significant difference = 1.96. LSS ³6.2 are includers and LSS £5.5 are
R04–122 6.2 67,472 30.6 ± 40 Includer excluders. All others are mixed.
‡ Percentage of dead plants ± SD. Genotypes with a percentage close to 50% and
R02–6268F 6.1 64,393 11.8 ± 22.3 Mixed
low SD: probable phenotypic segregation for the salt tolerance trait.
UA 5612 6.1 55,475 32.2 ± 38.7 Mixed
R07–2001 6.0 59,306 9.2 ± 16.6 Mixed
R05–374 5.9 57,211 9.7 ± 15.3 Mixed
Cont’d.

4 www.crops.org crop science, vol. 56, march– april 2016

digestion with nitric acid and hydrogen peroxide was used to
extract Na+ (Plank, 1992). The extracts were analyzed using
a spectrophotometer (Model CIROS ICP, Spectro Analytical
Instruments Inc., Mahwah, NJ).
Experimental Design and Data Analysis
In the first experiment, the experimental design used was a split-
split-plot with four levels of NaCl salt solution as main plot, three
growing media as subplots, and four different genotypes with five
replications as sub-subplots. The second experiment was a split-
plot design with two levels of salt concentration and 14 genotypes
with five replications. In the split-plot design, the salt treatment
was considered as the main plot, and the 14 genotypes were sub-
plots. ANOVA was used to evaluate the LSS and ion (Na+, Cl-)
content. Significant differences among different treatments were
calculated using the Tukey test (P < 0.05). A correlation test
was performed to evaluate the relationship among variables. All
statistical analyses were performed using SAS version 9.2 (SAS
Institute Inc., 2010).
Validation of the Screening Methology
After the evaluation of 14 cultivars in the greenhouse,
additional genotypes were selected to confirm the results
previously obtained with the screening method and to validate
the method with random genotypes ( Table 2). The geno-
types S-100 (excluder), Clark and Dare (includers) were used
as checks. In addition, AG5605 and AG5905 were added as
excluders, representing the lines that had the lowest LSS and
Cl– accumulation values in the previous experiment.
A split-plot design with six replications was performed using
two levels of NaCl- solution (0 and 120 mM) as the main plot
and the genotypes as subplots. As described above, LSS and Cl–
content in leaves were measured. Additionally, the percentage of
dead plants was evaluated to have another parameter to determine
if there was segregation in the salt stress response within each
genotype. The appearance and progression of symptoms were
monitored and recorded throughout the 2 wk at 3-day intervals
after the first day of the salt treatment application.
RESULTS AND DISCUSSION
Leaf Scorch Score
After 2 wk of salt treatment, the four varieties evaluated
presented slower growth compared to the control;
however, foliar symptoms of chlorosis and necrosis were
observed only in sensitive varieties (Dare and Williams).
Statistical differences in LSS were detected across all
growing media and NaCl concentrations (Tables 3 and 4).
The symptoms caused by salt damage were more
severe with increased salt concentration. However, the 160
mM NaCl treatment affected both sensitive and tolerant
cultivars, which did not allow a clear separation between
genotypes in any growing media used in the experiments
(Tables 3 and 4). Similar results have been previously
observed using the hydroponic screening method (Valen-
cia et al., 2008). The damage observed in the sensitive
crop science, vol. 56, march– april 2016  www.crops.org 5
cultivars treated with 80 mM NaCl was not enough to
separate them easily from the tolerant ones. The clearest
visual differences using LSS scale was found at 120 mM
NaCl treatment with either river sand or sandy soil media
(Tables 3 and 4). Previous research also reported compa-
rable symptoms in sensitive cultivars under salt treatments
(Abel and MacKenzie, 1964; Parker et al., 1983; Yang and
Blanchar, 1993; Lee et al., 2004; Kao et al., 2006; Valencia
et al., 2008). Tolerant cultivars (S-100 and Lee 68) dis-
played slight chlorosis and smaller leaf size compared to the
control (0 NaCl level); however, overall they appeared to
be healthier than sensitive cultivars. The stunting observed
in tolerant and sensitive plants has been associated with
shorter height, thinner shoot, smaller leaves, and fewer
nodes (Essa, 2002; Hamayun et al., 2010).
Significant differences in LSS between tolerant and
sensitive cultivars were detected with sand, soil, and
commercial potting mix (Tables 3 and 4). Plants grow-
ing in river sand and sandy soil showed similar visual
damage in the leaves at the end of treatments; sensitive
and tolerant varieties were also clearly differentiated.
Nevertheless, initial chlorosis and necrosis symptoms
appeared 2 d earlier in sensitive cultivars growing in
river sand. The commercial mix was not an appropri-
ate substrate for this screening method because visual
symptoms of salt injury were observed later compared
to other growing media. In addition, in some cases,
sensitive genotypes growing in the commercial mix did
not show foliar damage such as as necrosis or even chlo-
rosis at the end of the treatments compared to the same
genotypes growing in other substrates.
Accumulation of Na+ in Leaves and Roots
Sodium accumulation in leaves was generally greater in
sensitive genotypes than in tolerant ones. The opposite
results were found in roots, where Na+ accumulation
was greater in tolerant genotypes (Tables 3 and 4). These
results agree with other studies, where tolerant soybean
cultivars were able to keep the Na+ in the roots as a strat-
egy to avoid the uptake of this ion by the stem and leaves,
thereby preventing toxic injury in the plant (Phang et al.,
2008). Clearer differences were found in Na+ accumula-
tion in roots and leaves during the experiment performed
in summer season (Table 4). This observation agreed with
the hypothesis that plants exposed to high temperatures
have greater transpiration and take more water in; as a
consequence, a larger ion accumulation was observed
(Yang and Blanchar, 1993; Essa, 2002; Pathan et al., 2007).
Accumulation of Cl- in Leaves and Roots
Significant differences in Cl- concentration between salt-
tolerant and sensitive cultivars were found in leaves and
roots even when no salt solution was applied. The clearest
statistical differences were observed in Cl- concentrations
Table 3. Average leaf scorch score (LSS), Na+ and Cl – content in leaves and roots of four soybean cultivars at different levels of
NaCl treatment in three growing media evaluated in a greenhouse during the spring season.
LSS Leaf Na+ Leaf Cl – Root Na+ Root Cl –
Growing
medium NaCl Tol† Sens‡ Tol Sens Tol Sens Tol Sens Tol Sens
——————————————————————————— mg kg-1 ———————————————————————————
Commercial mix 0 mM 1.2 a§ 1.2 a 25 a 39 a 2,317 b 5,466 a 1,309 a 1,258 a 13,508 a 12,588 a
River sand 1.4 a 1.4 a 20 b 53 a 2,797 b 6,021 a 3,602 a 3,465 a 10,324 a 8,439 b
Sandy soil 1.5 a 1.5 a 22 b 39 a 2,402 b 7,862 a 2,657 a 2,471 a 13,166 a 9,680 b
Commercial mix 80 mM 3.2 b 4.7 a 19,086 a 22,714 a 48,084 b 67,500 a 18,967 a 18,503 a 23,405 a 22,104 a
River sand 2.9 b 7.6 a 33,771 a 30,676 a 76,205 b 82,350 a 27,446 a 22,638 a 29,285 a 35,415 a
Sandy soil 3.0 b 6.6 a 20,085 b 25,869 a 50,870 b 75,985 a 25,887 a 22,070 a 38,742 a 23,190 b
Commercial mix 120 mM 3.7 b 7.6 a 36,688 a 39,826 a 82,370 b 99,180 a 19,119 a 17,264 a 27,805 a 28,968 a
River sand 3.9 b 8.1 a 40,576 a 38,689 a 80,310 b 93,580 a 23,850 a 21,448 a 32,658 a 27,765 b
Sandy soil 3.7 b 8.0 a 29,219 b 38,451 a 70,630 b 95,660 a 28,166 a 28,052 a 37,626 a 42,300 a
Commercial mix 160 mM 5.2 b 7.5 a 32,727 a 36,685 a 72,585 b 99,030 a 25,621 a 20,586 b 39,810 a 47,766 a
River sand 6.6 b 8.8 a 45,743 a 56,804 b 96,600 b 101,125 a 23,909 a 22,957 a 33,150 a 31,253 a
Sandy soil 6.5 b 8.9 a 43,336 b 55,625 a 87,880 b 106,055 a 23,046 a 28,262 a 44,618 a 34,583 a
† Tol, tolerant cultivars: Williams and Dare.
‡ Sens, sensitive cultivars: S-100 and Lee 68.
§ Values followed by different letters are significantly different at the 0.05 probability level.

Table 4. Average leaf scorch score (LSS), Na+ and Cl – content in leaves and roots of four soybean cultivars at different levels of
NaCl treatment in three growing media evaluated in a greenhouse during the summer season.
LSS Leaf Na+ Leaf Cl – Root Na+ Root Cl –
Growing
Medium NaCl Tol† Sens‡ Tol Sens Tol Sens Tol Sens Tol Sens
——————————————————————————— mg kg-1 ———————————————————————————
Commercial mix 0 mM 1.2 a§ 1.2 a 15 a 16 a 267 b 1,759 a 1,049 a 966 a 7,342 a 6,754 a
River sand 1.3 a 1.3 a 20 a 24 a 1,004 b 2,827 a 7,983 a 8,075 a 8,522 a 4,870 b
Sandy soil 1.6 a 1.6 a 34 a 57 a 1,304 b 6,377 a 8,132 a 6,942 a 13,140 a 7,873 b
Commercial mix 80 mM 2.5 b 6.0 a 4,565 b 9,092 a 11,636 b 32,755 a 20,834 a 18,349 b 28,550 a 25,690 a
River sand 2.9 b 8.2 a 27,597 b 36,897 a 51,680 b 78,620 a 32,207 a 27,084 b 36,915 a 33,560 a
Sandy soil 2.6 b 7.8 a 22,280 a 27,093 a 35,705 b 65,160 a 27,878 a 24,098 b 42,150 a 41,730 a
Commercial mix 120 mM 3.7 b 6.9 a 17,237 b 28,232 a 40,655 b 78,460 a 26,066 a 21,154 b 32,140 a 29,905 a
River sand 4.0 b 8.7 a 35,693 b 40,194 a 61,910 a 69,370 a 28,983 a 23,979 b 34,630 a 30,410 a
Sandy soil 3.8 b 8.5 a 35,212 b 40,492 a 68,140 b 96,645 a 28,551 a 24,095 b 48,255 a 37,885 b
Commercial mix 160 mM 5.0 b 7.1 a 28,758 a 29,234 a 49,685 b 66,815 a 25,697 a 25,443 a 33,225 a 30,260 a
River sand 6.9 b 8.9 a 41,613 b 49,927 a 60,770 b 86,520 a 32,306 a 22,948 b 46,055 a 34,350 b
Sandy soil 6.5 b 8.9 a 45,733 b 55,691 a 73,690 b 88,810 a 29,830 a 29,686 a 48,156 a 43,755 a
† Tol, tolteran cultivars: Williams and Dare.
‡ Sens, sensitive cultivars: S-100 and Lee 68.
§ Values followed by different letters are significantly different at the 0.05 probability level.

in leaves. Leaf Cl- content allowed a clearer separation
between salt-tolerant and sensitive cultivars compared to the
Na+ content in leaves and roots and the Cl- concentration
in roots. Moreover, Cl- content in roots was similar in
tolerant and sensitive cultivars; hence, root Cl– is not rec-
ommended as a good indicator for evaluating salt tolerance
response in soybean. The high content of Cl– observed in
sensitive genotypes (Tables 3 and 4) is in agreement with
previous reports in which salt-sensitive genotypes or includ-
ers showed higher translocation of Cl- from soil to leaves
compared to tolerant genotypes or excluders (Wieneke and
Läuchli, 1979; Yang and Blanchar, 1993; Lee et al., 2008;
Lenis, 2008; Valencia et al., 2008).
Correlation between LSS and Ion Contents
The results obtained with LSS evaluation were strongly
and positively correlated with the ion contents in leaves
and roots (Table 5). These results confirmed the accuracy
of LSS in the identification of salt-sensitive and tolerant
genotypes. The lowest correlation values were obtained
between LSS and ion contents (Cl- and Na+) in roots
(Table 5). In addition, the content of ions in roots was
not statistically different in most of the treatments (grow-
ing media and NaCl concentration), indicating that ion
content in roots did not produce consistent results and
therefore is not an accurate parameter for evaluating the
plant response to salinity and to classify cultivars. The
strongest correlation (r = 0.87–0.88, P £ 0.001) was found
between LSS and Cl- content in leaves. However, the

6 www.crops.org crop science, vol. 56, march– april 2016

opposite results were found by Lenis et al. (2011), where
the increase in LSS was correlated with an increase in
Na+ content rather than Cl-. Furthermore, the clearest
statistical differences across all growing media and NaCl
concentrations were obtained with Cl- content analy-
sis in leaves. These data suggest that Cl- concentration
in leaves provided the best indicator of plant response to
salt stress. Similar results have been previously reported
where clearer differences were obtained with Cl- analyses
in leaves compared to those in stems or roots (Wieneke
and Läuchli, 1979; Abel and MacKenzie, 1964; Lee et al.,
2008; Lenis et al., 2011).
Significant differences were found between tolerant
and sensitive genotypes across all growing media and
NaCl concentrations using LSS and Cl- content in leaves.
Nevertheless, the clearest statistical differences in visual
LSS evaluation and leaf Cl- content were obtained with
120 mM NaCl using either sandy soil or river sand growing
media (Table 3). Lee et al. (2008) reported similar results
and stated that leaf Cl- content is a good indicator of soy-
bean response to salt and recommended a 100-mM NaCl
level as the best treatment to find clear differences among
cultivars using LSS or ion analysis. In a similar way, using
the hydroponics method, Valencia et al. (2008) reported
satisfactory results in the separation between salt-tolerant
and sensitive soybean genotypes under a salt treatment of
120 mM and suggested Cl- content as a possible selection
criterion to identify Cl- excluders and includers.
Screening of Soybean Cultivars
Fourteen soybean cultivars were evaluated for salt response
using the methodology previously described. The best
results found in the first experiment were used to separate
salt-sensitive lines from tolerant lines. A good classification
was obtained with both sandy soil and river sand growing
media at a 120-mM NaCl concentration. For the second
validation experiment, a 120-mM NaCl concentration
was used and sandy soil was selected as growing media,
since plants growing in river sand are more susceptible
to nutritional deficiencies, which could be misinterpreted
as salt stress (Fig. 1). In addition, soil is a medium that
closely simulates the real conditions where soybean plants
are grown. As a selection criterion, the LSS scale and the
leaf Cl- content were used.
With this methodology, we were able to classify 14
different genotypes into three groups as tolerant, sensi-
tive, or mixed (Table 1). The inital visual differences
between tolerant and sensitive plants were observed 10 d
after the treatment initiation (Fig. 3). Significant statisti-
cal differences in the LSS score and the leaf Cl- content
were obtained 15 d after initiation of the salt treatment
(Table 1). The most salt-sensitive cultivars were Williams,
Clark, and Dare, since these genotypes had the highest
LSS scores and the greatest Cl- accumulation (Table 1).
crop science, vol. 56, march– april 2016  www.crops.org 7
Table 5. Correlation coefficients (r) among leaf scorch score
(LSS), Na+ and Cl– content across NaCl treatments and media.
LSS‡ Leaf Na+ Root Na+ Leaf Cl – Root Cl –
LSS† – 0.84*** 0.72*** 0.88*** 0.63***
Leaf Na + 0.82*** – 0.82*** 0.93*** 0.78***
Root Na+ 0.75*** 0.83*** – 0.81*** 0.92***
Leaf Cl – 0.87*** 0.96*** 0.88*** – 0.74***
Root Cl – 0.73*** 0.77*** 0.88*** 0.80*** –
*** Significant at the 0.001 probability level.
† Left bottom data from Experiment 1 (Spring).
‡ Right top data from Experiment 2 (Summer).
Similar results were found with AG4903, HBK R4924,
and AG4605. These results agree with the classification
of these cultivars reported previously (Lee et al., 2008;
Valencia et al., 2008). Moreover, HBK R5525, AG5905,
and AG5605 were the cultivars with the lowest Cl- accu-
mulation and LSS scores, followed by Lee 68 and S-100
as excluders.
The results obtained in the third experiment, which
was implemented to validate this methodology, confirmed
the significant differences between salt and non-salt treat-
ment in all the variables measured (P ≤ 0.03). Stunting was
observed in both excluders and includers under salt stress.
The first symptom (light chlorosis) was observed 10 d after
the initiation of the salt treatment; however, symptoms
appeared earlier (7 d after treatment) in some susceptible
genotypes: Clark, Dare, R98–209, Desha, R05–4969,
R04–1250RR, UARK–5798, and Narow. The use of
both LSS and Cl- content demonstrated clear and consis-
tent results for the soybean genotypes classification after
the screening process. The leaf Cl– concentration and the
LSS for the most sensitive lines were about 2.3 times higher
than the values obtained in the most tolerant lines (Table 2,
Fig. 4). Chloride accumulation and mobility are affected by
environmental conditions such as temperature. Some of the
other aspects that largely determine Cl– intake include water
flow, the nutritional status of the plant, and the ionic com-
position of the assay medium (White and Broadley, 2001).
This may cause variability in the Cl– content measured on
the genotypes, not only between tolerant and sensitive cul-
tivars (given the difference in the inherent capacity of the
root to restrict Cl- transport) but also among genotypes
that have similar tolerance levels. However, Cl- concentra-
tion is a very useful parameter of classification and works
very well as it is correlated with visual LSS evaluations to
classify the cultivars.
The percentage of dead plants did not offer a clear dif-
ferentiation between includers and excluders, but showed
potential genetic segregation. Some of the lines where data
with a low SD showed about 50% of dead plants, suggest-
ing a possible phenotypic segregation for salt stress and
therefore classified as mixed (Table 2, Fig. 5). In contrast,
the percentage of dead plants was close to 100% in some

Figure 3. Chlorosis and necrosis symptoms in soybean 10 d after salt
treatment. Plants (a) and (b) correspond to the includers reported in
current and previous works, respectively. Plants (c) and (d) refer to the
excluders reported in the current and previous works, respectively.
includers and 0% in some excluders, as expected. The seg-
regation lines were expected as they were not selected by
breeders for salt tolerance before.
Genotypes Clark and Dare were confirmed to be
includers, with a high LSS and high Cl– content. On the
other hand, AG5605 and AG5905 consistently showed
lower Cl– accumulation compared to S–100 (Fig. 6).
Cultivars such as Osage and Jake showed lower LSS and
Figure 4. Symptoms of stress observed 14 d after salt treatment. Excluders: S–100 (a) and UARK-5896 (c). Mixed: AG5606 (b). Includers:
R05–4969 (d), R04–572 (e), and R09–1237 (f).
8 www.crops.org crop science, vol. 56, march– april 2016
Cl- content than the excluders mentioned above, followed
by the genotypes R07–6654, UA 5213C, R09–430, and
R03–1250, which had the lowest values in the screening
(Table 2). Osage and Jake were previously reported to
be as tolerant cultivars (Huang, 2013). Similarly, Lee is a
well-known salt-tolerant cultivar in which Cl– exclusion
is reported to be controlled by a dominant gene, coming
from its ancestor, S-100 (Abel, 1969; Lee et al., 2004)
These results indicate that this screening methodology
is an effective way to identify salt-tolerant and sensitive
genotypes; furthermore, new sources of tolerant soybean
cultivars could be used for breeding purposes. In addition,
these results suggest that screening the available soybean
germplasm may potentially find other sources of resistance
to salinity. More studies are necessary to confirm the three
classes of genotypes identified and to investigate the genet-
ics of salt tolerance. Identifying the genetic variability of
the salt stress response should be a main goal for breeders.
The current research is a useful source of information and
an important step that contributes to future plant breeding
programs studying the salt response in soybean.
CONCLUSIONS
In this work, we developed a simple, fast, and cost-
effective methodology to screen a large number of soy-
bean genotypes for salt tolerance. Using this greenhouse
method, we identified differences among soybean cultivars
Figure 5. Soybean plants classified as tolerant: Jake (a), moder-
ately tolerant: R09–319 (b), and sensitive: R09–1589 (segregating)
(c) and R04–572 (d).
in response to salt stress. The LSS and Cl- accumulation
results were correlated and represented a good indicator to
evaluate soybean response to salt stress. These parameters
were useful for classifying soybean cultivars as includers
or excluders.
The average LSS observed was 4.7 and 7.0 for exclud-
ers and includers, respectively. The average Cl– content
in leaves for excluders was 54,018 mg kg–1 and 68,473 mg
kg–1 for includers. Most tolerant genotypes showed up to
2.3-fold more Cl– accumulation and LSS than the most
susceptible genotypes. Based on the results of this screen-
ing method, new tolerant lines can be used as checks for
future studies in salt stress or as parents for breeding for
salt tolerance, given the low Cl– accumulation level, and
LSS in leaves observed in some genotypes compared to
the traditional tolerant checks (S-100, Lee, and Lee 68).
These lines include R07–6654, UA 5213C, R09–430,
R03–1250, HBK R5525, and AG5905. The variety UA
5213C is a high-yielding conventional cultivar released by
the University of Arkansas in 2013 (Chen et al., 2014).
Similarly, other susceptible lines such as R09–1237 and
UARK-5798 displayed similar symptoms compared to the
checks (Dare) and can be used as alternative sources to
measure salt inclusion.
The proposed method and the results obtained in
this study constitute a useful guideline to perform future
genetic research and practical breeding for salt toler-
ance. The methodology used was much easier than other
time-consuming and expensive methodologies previously
proposed, including the hydroponics method. The 2-h
daily treatment was enough to observe symptoms in a short
period of time using a LSS and Cl– content as accurate
parameters for response to the stress. By using this green-
house method, it was possible to observe clear differences
among cultivars even 7 d after the initiation of the salt
treatment, which allowed faster and easier classification.
crop science, vol. 56, march– april 2016  www.crops.org 9
Figure 6. Soybean plants used as checks after salt treatment.
Includers: Clark (a) and Dare (b). Excluders: AG5605 (c) and S–100 (d).
Acknowledgments
This work was supported by United Soybean Board and Arkan-
sas Soybean Promotion Board. The authors express their grati-
tude to John Guerber and to the plant breeding soybean team for
their collaboration and support.
References
Abel, G.H. 1969. Inheritance of the capacity for chloride inclusion
and chloride exclusion by soybeans. Crop Sci. 9:697–698.
Abel, G.H., and A.J. MacKenzie. 1964. Salt tolerance of soybean
varieties (Glycine max L. Merr.) during germination and later
growth. Crop Sci. 4:157–161. doi:10.2135/cropsci1964.001118
3X000400020010x
An, P., S. Inanaga, Y. Cohen, U. Kafkafi, and Y. Sugimoto. 2002.
Salt tolerance in two soybean cultivars. J. Plant Nutr. 25:407–
423. doi:10.1081/PLN-120003373
Arzani, A. 2008. Improving salinity tolerance in crop plants: A bio-
technological view. In Vitro Cell. Dev. Biol. Plant 44:373–383.
doi:10.1007/s11627-008-9157-7
Blumwald, E., and A. Grover. 2006. Salt tolerance. In: N.G. Hal-
ford, editor, Plant biotechnology: Current and future uses of
genetically modified crops. Wiley & Sons Ltd, UK.
Chen, P., M. Orazaly, C. Wu, P. Manjarrez-Sandoval, J.C. Rupe,
D.G. Dombek, et al. 2014. Registration of ‘UA 5213C’ soybean.
J. Plant Reg. 8(2):150–154. doi:10.3198/jpr2013.07.0037crc
Chinnusamy, V., A. Jagendorf, and J.-K. Zhu. 2005. Understanding
and improving salt tolerance in plants. Crop Sci. 45:437–448.
doi:10.2135/cropsci2005.0437
Clarke, S.M., and J.J. Eaton-Rye. 2000. Amino acid deletions loop
of the chlorophyll a-binding protein CP47 alter the chloride
requirement and/or prevent the assembly of photosystem II.
Plant Mol. Biol. 44:591–601. doi:10.1023/A:1026528813583
Essa, T.A. 2002. Effects of salinity stress on growth and nutrient
composition of three soybean [Glycine max (L.) Merr.] cul-
tivars. J. Agron. Crop Sci. 188:86–93. doi:10.1046/j.1439-
037X.2002.00537.x
Hamayun, M., S.A. Khan, A.L. Khan, Z.K. Shinwari, J. Hussain,
E.Y. Sohn, S.M. Kang, Y.H. Kim, M.A. Khan, and I.J. Lee.
2010. Effects of salt on growth attributes and endogenous growth
hormones of soybean cultivar Hwangkeumkong. Pak. J. Bot.
42(5):3103–3112.
Huang, L. 2013. Genome-wide association mapping identifies
QTLs and candidate genes for salt tolerance in soybean. Mas-
ter’s thesis. University of Arkansas, Fayetteville.
Kalra, Y.P. 1998. Handbook of reference methods for plant analysis.
CRC Press, Boca Raton, FL.
Kao, W., T.T. Tyng, C.T. Hung, and N.S. Chen. 2006. Response of
three Glycine species to salt stress. Environ. Exp. Bot. 56:120–
125. doi:10.1016/j.envexpbot.2005.01.009
Lee, G.J., R.H. Boerma, R.M. Villagarcia, X. Zhou, T.E. Carter,
Jr., Z. Li, and M.O. Gibbs. 2004. A major QTL conditioning
salt tolerance in S-100 and descendent cultivars. Theor. Appl.
Genet. 109(8):1610–1619. doi:10.1007/s00122-004-1783-9
Lee, J.D., S.L. Smothers, D. Dunn, M. Villagarcia, C.R. Shumway,
T. Carter, Jr., and J.G. Shannon. 2008. Evaluation of a simple
method to screen soybean genotypes for salt tolerance. Crop
Sci. 48:2194–2200. doi:10.2135/cropsci2008.02.0090
Lenis, J.M. 2008. Physiological traits underlying differences in salt
tolerance among Glycine species. Master’s thesis, Missouri State
University, Columbia.
Lenis, J.M., M. Ellersieck, D.G. Blevins, D.A. Sleper, H.T. Nguyen,
D. Dunn, J.D. Lee, and J.G. Shannon. 2011. Differences in
ion accumulation and salt tolerance among Glycine acces-
sions. J. Agron. Crop Sci. 197:302–310. doi:10.1111/j.1439-
037X.2011.00466.x
Lessani, H., and H. Marschner. 1978. Relation between salt tol-
erance and long-distance transport of sodium and chloride in
various crop species. Aust. J. Plant Physiol. 5:7–37. doi:10.1071/
PP9780027
Marschner, H. 1995. Mineral nutrition of higher plants. 2nd ed.
Academic Press, San Diego.
Munns, R., S. Hussain, A.R. Rivelli, R.A. James, A.G. Condon,
M.P. Lindsay, E.S. Lagudah, D.P. Schachtman, and R.A. Hare.
2002. Avenues for increasing salt tolerance of crop, and the
role of physiologically based selection traits. Plant Soil 247(1):
93–105. doi:10.1023/A:1021119414799
Pantalone, V.R., W.J. Kenworthy, L.H. Slauther, and B.R. James.
1997. Chloride tolerance in soybean and perennial Glycine acces-
sions. Euphytica 97:235–239. doi:10.1023/A:1003068800493
Parida, A.K., and A.B. Das. 2005. Salt tolerance and salinity effects
on plants: A review. Ecotoxicol. Environ. Saf. 60:324–349.
doi:10.1016/j.ecoenv.2004.06.010
Parker, M.B., G.J. Gascho, and T.P. Gaines. 1983. Chloride toxicity
of soybean grown on Atlantic coast flatwoods soils. Agron. J.
75:439–443. doi:10.2134/agronj1983.00021962007500030005x
10 www.crops.org crop science, vol. 56, march– april 2016
Pathan, M.S., J.D. Lee, J.G. Shannon, and H.T. Nguyen. 2007. Recent
advances in breeding for drought and salt stress tolerance in soy-
bean. In: M.A. Jenks, P.M. Hasegawa, and S.M. Jain, editors,
Advances in molecular-breeding toward drought and salt toler-
ant crops. Springer, Dordrecht, the Netherlands. p. 739–773.
Phang, T., G. Shao, and H. Lam. 2008. Salt tolerance in soybean.
J. Integr. Plant Biol. 50(10):1196–1212. doi:10.1111/j.1744-
7909.2008.00760.x
Ping, A., S. Inanaga, Y. Cohen, U. Kafkafi, and Y. Sugimoto. 2002.
Salt tolerance in two soybean cultivars. J. Plant Nutr. 25:407–
423. doi:10.1081/PLN-120003373
Plank, C.O. 1992. Plant analysis reference procedures for the south-
ern region of the United States. Southern Cooperative Series
Bull. 368. Univ. of Georgia, Athens.
Sairam, R.K., and A. Tyagi. 2004. Physiology and molecular biol-
ogy of salinity stress tolerance in plants. Curr. Sci. 86:407–421.
SAS Institute Inc. 2010. SAS/STAT 9.22 user’s guide. SAS Institute
Inc; Cary, NC.
Shannon, M.C. 1997. Adaptation of plants to salinity. Adv. Agron.
60:75–120. doi:10.1016/S0065-2113(08)60601-X
Valencia, R., P. Chen, T. Ishibashi, and M. Conaster. 2008. A rapid
and effective method for screening salt tolerance in soybean.
Crop Sci. 48:1773–1779. doi:10.2135/cropsci2007.12.0666
Velagaleti, R.R., and S. Marsh. 1989. Influence of host cultivars and
Bradyrhizobium strains on growth and symbiotic performance of
soybean under salt stress. Plant Soil 119:133–138. doi:10.1007/
BF02370277
Wang, D., and M.C. Shannon. 1999. Emergence and seedling
growth of soybean cultivars and maturity groups under salinity.
Plant Soil 214:117–124. doi:10.1023/A:1004719420806
White, P.J., and M.R. Broadley. 2001. Chloride in soils and its
uptake and movement within the plant: A review. Ann. Bot.
(Lond.) 88:967–988. doi:10.1006/anbo.2001.1540
Wieneke, J., and A. Läuchli. 1979. Short-term studies on the uptake
and transport of Cl by soybean cultivars differing in salt toler-
ance. Z. Pflanzenernaehr. Bodenkd. 142:799–814. doi:10.1002/
jpln.19791420606
Xu, Z., R. Chang, L. Que, J. Sun, and X. Li. 1999. Evaluation of
soybean germplasm in China. In: H.E. Kauffman, editor, Proc.
Invited and Contributed Papers and Posters: World Soybean
Res. Conf. VI, Chicago, IL. 4–7 Aug. 1999. Natl. Soybean
Res. Lab, Champagne, IL. p. 156–165.
Yang, J., and R.W. Blanchar. 1993. Differentiating chloride suscepti-
bility in soybean cultivars. Agron. J. 85:880–885. doi:10.2134/
agronj1993.00021962008500040019x