ARTÍCULOS ORIGINALES

Screening of soybean cultivars for chloride tolerance in Argentina

Tamizado de cultivares de soja por tolerancia a cloruros en Argentina

 

Lúquez J.E.^1,*, Briguglio M.A.¹, Irigoyen F.¹, Eyherabide G.A.¹

¹ Facultad de Ciencias Agrarias, Universidad Nacional de Mar del Plata, Argentina.

* Author for correspondence luquez.julia@inta.gob.ar

Fecha de recepción: 22/02/2015
Fecha de aceptación de versión final: 10/07/2015

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ABSTRACT

Soybean Glycine max [L. (Merrill)] is the most important crop in Argentina, where soil salinization is increasing. Development of salt tolerant cultivars is an effective approach to expand the crop area and minimize yield loss in saline soils. The aim of this work was to screen commercial soybean cultivars for chloride tolerance in three experiments. In the first experiment 37 cultivars were placed in paper rolls wetted with 50 mM NaCl. Germination speed and hypocotyl and radicle length were registered. Twelve cultivars were chloride tolerant as suggested by their hypocotyl and radicle length. In the second experiment, seven cultivars were grown under greenhouse conditions in containers with sandy soil, nutrients and 50 mM NaCl solution. Leaf scorch (LS) and leaves chloride content (LCC) were determined. Variability among cultivars was determined for both characteristics. In the third experiment, 13 cultivars were placed in paper rolls wetted with 100mM NaCl. Germination speed, hypocotyl and radicle length, and fresh plant weight were measured. Five cultivars were chloride tolerant as suggested by their fresh plant weight. Although 100 mM NaCl was the most restrictive treatment for seed germination, common chloride tolerant soybean cultivars were found in the three experiments. Therefore, genetic improvement for soybean chloride tolerance can be promising in Argentina.

Key words: Soybean; Chloride tolerance; Germination; Screening; Variability; Glycine max [L. (Merrill)].

RESUMEN

La soja Glycine max [L. (Merrill)] es el cultivo más importante en Argentina, donde está aumentando la salinización del suelo. El desarrollo de cultivares tolerantes a la salinidad es una forma efectiva de expandir el área de cultivo y minimizar la pérdida de rendimiento en suelos salinos. El objetivo de este trabajo fue probar la tolerancia de distintos cultivares de soja al ion cloruro en tres experimentos. En el primer experimento se utilizaron semillas de 37 cultivares, colocándolas en toallas de papel enrolladas y embebidas en una solución de 50 mM de NaCl. Se registraron velocidad de germinación y largo de radícula e hipocótile. Según estas dos últimas variables, 12 cultivares resultaron tolerantes. En el segundo experimento se utilizaron siete cultivares y se dejaron crecer en invernáculo en macetas con suelo arenoso, regado con solución nutritiva y 50 mM de NaCl. Se determinaron índice de acorchamiento (IA) y el contenido de cloruros en hoja (CCH). Fue posible detectar variabilidad entre cultivares para las dos características. En el tercer experimento se colocaron a germinar semillas de 13 cultivares en toallas de papel enrolladas y embebidas en una solución salina de 100 mM de NaCl. Se determinaron velocidad de germinación, largo de raíz e hipocótile y peso fresco de las plantas, detectándose cinco cultivares tolerantes según su peso fresco. Se encontraron genotipos tolerantes comunes en los tres experimentos, por lo que el mejoramiento genético para tolerancia a los cloruros es promisorio en Argentina.

Palabras clave: Soja; Tolerancia a cloruros; Germinación; Variabilidad; Glycine max [L. (Merrill)].

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INTRODUCCIÓN

To achieve global food security by 2050, primary production must almost be doubled. Climate change continues and good arable land is becoming scarce. There are about 1000 million hectares of saline soils worldwide. Salt stress is reported to inhibit soybean germination and plant growth, nodulation, seed yield and it can also cause severe leaf chlorosis, leaf bleaching and leaf scorching by chloride accumulation in the leaf (Abel and Mackenzie, 1964; Wang and Shannon, 1999; Banzai et al., 2002; Singleton and Bohlool, 1984; Parker et al., 1983; Katerji et al., 2003; Abel, 1969; Yang and Blanchar, 1993). Soybean is salt sensitive (Luo et al., 2005). The threshold salinity for soybean is 5.0 dS m-1 (Chinnusamy et al., 2005). Salt tolerance is thought to be primarily related to the ability of plants to limit accumulation of Na+ and Cl- in leaves by exclusion (Abel, 1969; Lauchli and Wienecke, 1979; Essa, 2002). Genotypic tolerance of the chloride excluders to the acutely toxic effects of chloride per se, is based on visual leafscorching ratings and/or reduced chloride levels in the leaf (Parker et al., 1983; Shao et al., 1995; Yang and Blanchar, 1993). Substantial genetic variation exists for chloride tolerance among cultivars, breeding lines, some accessions of the wild progenitor of soybean (G. soja Sieb abd Zucc) and among the more distantly related perennial accessions (Li et al., 2000; Pantalone et al., 1997; Wang et al., 1997; Zhang et al., 1999).
In Argentina, the third country in the world with soils affected by salinity after Russia and Australia, 19 million ha are cultivated with soybean, which is the major crop in the country. Soybean is cultivated throughout the country, from N to S, and soils affected by salinity include those in arid and semiarid environments with and without irrigation and those soils in humid environments (Lavado, 2007). Development of salt tolerant cultivars may substantially expand the land’s food producing area. Information on plant response to varying salinity levels is important to recommend tolerant cultivars as parents in breeding programs. The objective of this study was to screen Argentinian commercial soybean cultivars for chloride tolerance under varying salinity levels.

MATERIALS AND METHODS

Experiments, cultivars and experimental design
Three experiments were conducted, two in growth chambers and one in a greenhouse at the Unidad Integrada Balcarce, located at 38º S and 58º W, in Argentina. The first experiment was carried out in 2010 in a growth chamber (25º C, 16/8 h light/dark photoperiod, irradiance of 1300 lux). Thirty seven soybean commercial cultivars (cv) (listed in Table 1) were randomly selected for the trial; twenty seeds of each cv were placed in rolls of germination paper wetted with 50 mM commercial NaCl. This concentration was used because the responses of the different cultivars were unknown and we wanted to make sure that germination occurred. A control with distilled water (0mM) was used. A randomized complete block design replicated five times with blocking in time (every 14 days) was utilized. Germination speed, hypocotyl and radicle length were determined.

Table 1. Radicle and hypocotyl length in 37 Argentinian soybean cultivars exposed to 50 mM NaCl salt concentration as compared to the control.

The second experiment was carried out in greenhouse and it was started on August 14th, 2011. Seven commercial soybean cultivars (listed in Table 2) were selected. Seeds from certain cultivars included in the first experiment were unavailable. The new seed included though, are widely distributed in the southeast of Buenos Aires and they belong to the same maturity groups (III and IV) used in this region. Three cultivars which were salt tolerant when exposed to 50 mM NaCl concentration as suggested by hypocotyl length (NA 4413), or by radicle and hipocotyl length (NA 3520) (experiment 1) were included in this experiment. Five seeds from each cv were planted in 8.7 cm tall plastic cones filled with 150 g of a sandy soil with nutrients (according to PC method by Lee et al., 2008) in two replications (one seed per cone). Cones were placed in plastic trays filled with 20 l tap water to keep plant moisture. A 50 mM of NaCl salt solution was added to the plastic trays at the V2 to V3 seedling stage (Fehr and Caviness, 1977), and it was refilled with water and nutrients so that the trays water content of the trays reached one third of the height of the cones. Electrical conductivity (EC) was monitored daily with a Mettler Toledo MC226 Model with temperature probe conductivity meter. EC was 0.6 mS cm-1 and 6.6 mS cm-1 for the 0mM and 50 mM, respectively. Greenhouse temperature was recorded daily every 15 min. In Figure 1, daily average temperatures are presented from the beginning of the salt treatment until day number 11 of salt treatment. The experimental design was split plot, where NaCl level was the whole plot treatment and each cultivar (as a group of five seedlings) was randomized within the whole plot. Chloride content was measured with an Orion chloride ion selective electrode (ISE), 14 days after the initial NaCl treatment (when seedlings were harvested) (Pantalone et al., 1997), and leaf scorching was determined according to Lee et al. (2008).

Table 2. Leaf chloride contents (g/kg-1) (LCC) and leaf scorch score (LSS) in seven Argentinian soybean cultivars.

Figure 1. Average daily greenhouse temperatures (°C) during 11 out of 14 days under salt treatment (50 mM NaCl) (experiment 2).

Once the first trial had been conducted, we were able to see that the different cultivars responded to 50 mM NaCl. So, we decided to increase the salinity level because in Argentina soils can even be more saline. The third experiment was carried out in 2012 in a growth chamber (25º C, 16/8 h light/dark photoperiod, irradiance of 1300 lux). Thirteen soybean commercial cultivars (listed in Table 3) were selected for the trial; twenty seeds of each cv were placed in rolls of germination paper wetted with 100 mM commercial NaCl. A control with distilled water (0mM) was used. Cultivars which were salt tolerant as suggested by hypocotyl length (NA 4413 and NA 3933) and radicle and hipocotyl length (A 3302 RG and NA 3731) in the first experiment in 2010, were included in this trial and some cultivars belonging to the second experiment (DM 4250, NA 4413, NA 4990, DM 4210 and NA 4613), too. There are new seeds because some of the varieties used in the previous experiments were unavailable. Germination speed, hypocotyl and radicle length and plant fresh weight were determined. A randomized complete block design replicated three times with blocking in time (every 14 days) was utilized.

Table 3. Germination speed, radicle and hypocotyl length, and plant fresh weight of 13 Argentinian soybean cultivars exposed to 100 mM salt treatment as compared to the control.

Data collection
First experiment
Germination speed was registered every 24 hours until full germination (Maguire, 1962), and hypocotyl and radicle length were measured at day 14 from the beginning of experiment (Yaver et al., 2009). Each replication lasted 14 days. For more details about germination speed, it was calculated by dividing the number of seedlings per 20 seeds obtained at each counting in the germination test by the number of days seeds have been in the germinator until full germination. The values obtained at each count are the sum at the end of the germination test in this way: (number of seedlings/days to first count +...+ number of seedlings/days to final count).

Second experiment
Salt injury was determined by a leaf scorch score (LSS). As no information was available regarding salt sensitivity of the cultivars used, leaf scorch ratings were made five days after the addition of salt solution, when the first plants (cultivar RA- 424) exhibited salt injury or reached a leaf scorch of three (moderate chlorosis) according to Lee et al. (2008). Fourteen days after the initial NaCl treatment, single trifolioliate leaves, excluding the petiole, were taken and they were dried in a digital oven at 62º C for 24 hours. Dried leaves were ground using a mill and 0.15 g samples were in 30 ml of distilled water using an orbital shaker at 60 cycles min-1 for 1 h. The clear solution resulting after filtering the suspension through a filter paper was analyzed for chloride content using an ion selective electrode (Lee et al., 2008). In ISE determinations, unknown samples are compared to solutions of known chloride concentrations. Known chloride concentration samples were prepared and the electrode potential was determined. A standard curve was established after this potential was graphed in accordance with the logarithm of chloride concentrations. The chloride concentration in the solution extracted from leaves samples was determined using the established standard curve.

Third experiment
Germination speed was registered every 24 hours until full germination (Maguire, 1962), and hypocotyl and radicle length, and plant fresh weight were recorded at day 14 from the beginning of the experiment (Yaver et al., 2009).

Statistical analysis
All experiments were analyzed separately using SAS (SAS Institute, 2004). An analysis of variance (ANOVA) was performed on each of the three experiments and the total variance was partitioned into salinity treatment and genotype effects including their interactions. Means were separated using Duncan´s multiple range tests at 5 % probability level.

RESULTS

First experiment
Germination speed mean values ranged between 5.21 and 7.99 for cultivars RM 039 and AS 4201 for non-salt treatment, and 5.54 and 8.97 for cultivars SPS 4x0 and SRM 4205 for salt treatment. No significant differences among treatments (P>0.05) were found for this characteristic. Means of hypocotyl and radicle length as well as salt detrimental effect (SDE, %) for all soybean cultivars in the experiment 1 and their significance among treatments are presented in Table 1. SDE was calculated as character value at 0 mM NaCl minus character value at 50 mMNaCl/ character value at 0mMNaCl * 100. Hypocotyl length ranged between 1.07 and 5 mm for cultivars DM 4930 and DL 401 RG in non-salt treatment, and 1.55 and 3.65 mm for cultivars DM 4670 and A 3302 RG, respectively in salt treatment. The cv TJ 2049 presented the minor SDE (%) (-51.4) and the cv SPS 4 x 4, the major: 56.4. No significant differences (P>0.05) were found between treatments within 29 cultivars: A 3289, ACA 360, ALM 4930, AS 4201, AS 4810, Champaquí, DM 3700, DM 4200, DM 4930, FN 360, NA 3933, NA 4413 RG, NA 4553 RG, NK 3200, RM 048, TJ 2049, TJ 2145, A 3302 RG, ALM 3530, ALM 4200, ARECO 4330, FN 485, NA 3520 RG, NA 3731, NA 4209 RG, SPS 4 x 0, SRM 3402, TJ 2136 and TJ 2139. Radicle length ranged between 5.50 and 14.50 cm for cultivars DM 4670 and SRM 3402 respectively in 50 mM salt concentration treatment as compared to the control (3.06 and 18.22 cm for cultivars DM4200RG and DM 4930 respectively). The cv DL 401 RG presented the minor value for SDE (24.7 %) and the cv RMO 48, the major value (56.9 %). Fungic contamination in some cultivars in the control probably decreased the plants radicle length. No significant interaction (P>0.05) was found between treatments and cultivars for 15 cultivars: A 3302 RG, ALM 3530, ALM 4200, Areco 4330, DL 401RG, FN 485, NA 3520 RG, NA 3731, NA 4209 RG, NS 4903, RM 039, SPS 4 x 0, SRM 3402, TJ 2136 and TJ 2139. No significant differences were found between treatments for either hypocotyl and radicle length within cvs A 3302 RG, ALM 3530, ALM 4200, ARECO 4330, FN 485, NA 3520 RG, NA 3731, NA 4209 RG, SPS 4 x 0, SRM 3402, TJ 2136 and TJ 2139.

Second experiment
Leaf chloride contents and LSS in the cultivars tested can be observed in Table 2. Leaf chloride contents and LSS ranged between 5.94 and 17.08 g/kg-1 and 1 and 3 for cultivars DM 4250 and NA 4613, respectively, in the salt treatment. Those cultivars exhibiting low LSS also exhibited low chloride contents. Significant variability for chloride sensitivity was observed among cultivars (P<0.05). Non-significant differences in chloride content (P>0.05) were found between cvs RA 424 and NA 4613.

Third experiment
Germination speed mean values were 7.91 and 12.30 for cvs A 3302 RG and NA 3731 in the salt treatment, as compared to the control (13.19 and 17.66 for cvs DM 3070 and NA 3731, respectively). SDE (%) values ranged 21.6 for cv NS 3521 and 45.8 for cv A 3302 RG. Significant interaction (P<0.05) was found between cultivars and treatments for this characteristic. Hypocotyl length mean values ranged between 2.05 and 3.58 mm for cultivars DM 3810 and NA 4613, respectively in salt treatment, and between 3.99 and 5.19 mm for cvs DM 3810 and NA 3933, respectively in nonsalt treatment. The SDE (%) values ranged between 21.3 for cv NA 4613 and 52.7 for cv A 3302 RG. Significant interaction (P<0.05) was found between cultivars and treatments for this characteristic. Radicle length mean values ranged between 10.43 and 15.26 cm for cultivars DM 4210 and NA 3731, respectively in the salt treatment, and between 15.54 and 21.25 cm for DM 3810 and NA 3731, respectively in non-salt treatment. The SDE (%) values ranged between 20 for cv DM 4670 and 43.3 for cv DM 2200. Significant interaction (P<0.05) was found between treatments and cultivars for this characteristic. Plant fresh weight mean values were between 9.26 and 11.44 g for cultivars NA 3933 and DM 3070, respectively, in the salt treatment and between 9.07 and 11.23 g for cultivars NA 3731 and DM 3810 respectively, in nonsalt treatment. The SDE (%) values ranged between 5 for cv DM 3810 and 14 for cv NA 4613. No significant interaction (P>0.05) was found between cultivars NA 4413, NS 3521, DM 2200, A 3302 RG and NA 3933 and the salt treatment.

DISCUSSION

Results found in the different experiments carried out, showed how salinity-induced stress by 50 and 100 mM concentration NaCl, mainly this last concentration, significantly affected speed germination, radicle and hypocotyl lengths and plant fresh weights. This demonstrate that such variables could be very useful for screening salt tolerance at early stages of plant development (Abel and Makenzie, 1964; Alghamdi, 2009; Essa, 2002; Yaver et al., 2009; Neves et al., 2010; Zhang et al., 2011). Similar trends were observed under greenhouse conditions for the variables leaf chloride score and leaf chloride contents, when plants were exposed to 50 mM salt concentration. Cultivars exposed to salt treatment developed more high leaf scorch and higher chloride contents in their leaves than those in the control, which is in agreement with previous findings (Pantalone et al., 1997; Lennis et al., 2011; Lee et al., 2008; Zhang et al., 2011). Results from this study confirm the chloride excluder condition of soybean, because of the significant variability in leaf chloride score and chloride content among cultivars. The ability of a genotype to germinate, to increase radicle and hypocotyl length and to increase its seedling weight under salt stress conditions does not necessarily indicate that the plant could withstand salt stress and complete its life cycle (Pearson and Berstein, 1959; Pearson et al., 1966; Norlyn and Epstein, 1984). However, it is very important to know which the common salt tolerant cultivars are in the different experiments because different variables are measured at different development stages.
In this sense, we can say that 12 out 37 soybean cultivars included in our experiment resulted tolerant to salt stress as expressed by their radicle and hipocotyl lengths when exposed to 50 mM NaCl concentration. Some of the cultivars which were salt tolerant in experiment 1 as shown by hypocotyl length (NA 4413), and by radicle and hypocotyl length (NA 3520), were also included in the greenhouse experiment and showed the lowest LCC and LSS. No significant difference in the plant fresh weight was found in cultivar NA 4413 when exposed to 100 mM salt treatment, as compared to the control. Cultivars A 3302 RG, ALM 3530 and NA 3731 resulted salt tolerant as expressed by its radicle and hypocotyl length when exposed to 50 mM NaCl concentration, and it also resulted salt tolerant as expressed by the plant fresh weight when exposed to 100 mM NaCl concentration, in comparison with the controls. In fact, as it can be observed by the variables germination speed, radicle and hypocotyl length, 100 mM NaCl concentration was more restrictive than 50 mM NaCl concentration for all common cultivars in experiments 1 and 3 (DM 4210, NA 4413, NA 4990, DM 4250 and NA 4613). However, cvs DM 4210, DM 4250 and NA 4413 didn’t present SDE for fresh weight at 100 mM NaCl and had lowers values of LCC and LSS in experiment 2 (50 mM NaCl).

CONCLUSIONS

Two prerequisites for the genetic improvement for salt tolerance include within-species (or cultivars) variability and the access to a reliable method for the screening of large numbers of genotypes. According to our results, it is possible to say that genetic improvement of soybeans for chloride tolerance through traits related with seed germination and other characteristics measured in young plants exposed to 50 mM NaCl concentration is feasible. This is possible since the screening methods used are reliable and that there is genetic variability among cultivars for the traits determined in these experiments in Argentina.

ACKNOWLEDGEMENTS

The authors wish to thank Prof. Viviana Innocentini for the reading and editting of this work.

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