Treatments

The concentration of the salt solutions of NaCl, Na₂SO₄ and NaHCO₃ was calculated based on the theoretical electrical conductivities (CE) and decreasing yield of 0% to 150%, the equation used was proposed by ^{Mass and Hoffman ( 1977)}: Y= 100 - b (CEs - Su): where: Y= is the potential yield (%); CEs= is the electrical conductivity of the solution (dS m^-1); Su= is the threshold salinity (dS m^-1); b = is the decrease in yield per unit increase of salinity.

The treatments investigated were control 0% (T0), 0% threshold (T1), 25% (T2), 50% (T3), 75% (T4), 100% (T5) and 150% (T6) theoretical CE were: T0= 0 dS m^-1; T1= 1 dS m^-1; T2= 2.3 dS m^-1; T3= 3.6 dS m^-1; T4= 5 dS m^-1; T5= 6.3 dS m^-1 and T6= 9 dS m^-1.

Preparation of solutions

The amount of salt in the treatments was calculated by applying the equations proposed by the Salinity Laboratory of the United States of America (^{Richards, 1990}).

Where: mg L^-1= is the salts concentration in the solution; CE= is the electrical conductivity of the solution (dS m^-1 at 25 °C).

For calculation purposes it was considered that 0.64 g L^-1 salt generates a theoretical electrical conductivity of 1 dS m^-1.

Experimental design

A factorial design A×B×C was used, completely random with five replicates. Where A= salt concentration, B = bean variety and C = salt types. The first factor had seven levels (T0= 0 g L^-1; T1= 0.64 g; T2= 1.472 g; T3= 2.304 g; T4= 3.2 g; T5= 4.032 g and T6= 5.76 g), the second had three (Black, Pinto and Azufrado bean) and the third had three (NaCl, Na₂SO₄ and NaHCO₃). As experimental unit it was considered a pot with 10 seeds germinated for the first stage and five seedlings for the second.

Studied variables

In the germination stage it was evaluated with the appearance of the hypocotyl with the cotyledons on the agrolite and in the seedling stage when there was effect of the salts concentration in the first trifoliate leaf. In order to obtain the germination percentage the germinated seeds were quantified compared to the total. In the seedling stage, at the end of the experiment, at 22 days, the length in cm of the seedling and the root were measured. Fresh and dry biomass of the stem and root were evaluated with fresh and dry weight in g, using an analytical balance and stove at 60 °C.

Statistic analysis

Analysis of variance was applied using the SAS program (^{SAS Institute, 1999}). Differences between means were compared by Tukey (p≤ 0.05) and regression analysis were performed.

Germination

In the interaction bean factor by salt concentration it was found that at a concentration of 2.304 g the Black beans decreased in higher value its germination. In the salt type interaction by bean type Azufrado and Pinto beans obtained the greatest germination with Na₂SO₄, but with NaHCO₃ Azufrado and Pinto decreased to near-Black bean values.

Generally the germination percentage decreased as salt concentration increased (Table 1), the theoretical CE of 3.6 dS m^-1 with 2.304 g of the salt decreased 7%, the concentration of 4.032 g 31%and with 5.76 g 49% compared to the threshold, for all concentrations and types of salt. The most resistant bean variety in the germination stage at all concentrations and salt types was Azufrado, followed by Pinto and Negro (Table 2). The type of salt affected particularly each variety, the NaCl affected more the Pinto bean, the Na₂SO₄ affected Black beans and NaHCO₃ the Pinto beans (Figure 1). The type of salt, at all concentrations, that decreased the most the percentage of germination of all beans varieties was NaHCO₃ (33%), followed by NaCl (18%) and Na₂SO₄ (11%) (Table 3).

Promedios en una columna con la misma letra son estadísticamente iguales (Tukey, 0.05). CEt= conductividad eléctrica teórica; PG= porcentaje de germinación; PF= peso fresco; PS= peso seco; DMS= diferencia mínima significativa; CV= coeficiente de variación. Anova general, sin discriminar por tipo de sal ni variedad

Table 1 Effect of salt concentrations on germination, length, fresh and dry biomass of stem and root in beans. 

Promedios en una columna con la misma letra son estadísticamente iguales (Tukey, 0.05). PG= Porcentaje de germinación; PF= peso fresco; PS= peso seco; DMS= diferencia mínima significativa; CV= coeficiente de variación. Los valores corresponden a la respuesta de cada variedad al factor salinidad sin discriminar concentración y tipo de sal.

Table 2 Effect of varieties on germination, length, fresh and dry biomass of stem and root in beans. 

Figure 1 Germination of bean varieties under induced saline conditions with NaCl, Na₂SO₄ and NaHCO₃.  

Promedios en una columna con la misma letra son estadísticamente iguales (Tukey, 0.05). PG= porcentaje de germinación; PF= peso fresco; PS= peso seco; DMS= diferencia mínima significativa; CV= coeficiente de variación. Los valores corresponden a medias generales para todas las variedades y todas las concentraciones usadas para cada tipo de sal.

Table 3 Effect of salt types on germination, length, fresh and dry biomass of stem and root in beans.  

Aerial part

In the interaction bean factor by salt concentration it was found that the length, fresh and dry biomass of the stem of the concentration of 0.64 to 2.304, the Azufrado bean was the most affected with lower value for these variables. In the type of salt by bean type interaction, length, fresh and dry stem biomass, Azufrado showed lower values with NaCl and Na₂SO₄ but with NaHCO₃ the three varieties drastically decreased the values of these variables and were close to each other.

Table 1 shows that as the salt concentration increased, the length and fresh and dry biomass of the stem decreased. At a concentration of 4.032 g L^-1 stem length decreased 3.138 cm on average and 5.760 g 3.942 cm, with respect to the threshold, for all concentrations and types of salt. The variety that reached the highest fresh and dry biomass for all concentrations and salt types was Pinto beans (Table 2). In the seedling stage the salt that most affected the length, fresh and dry biomass of stem and root of bean varieties was NaHCO₃ (Table 3), at a concentration of 2.304 g of NaHCO₃ any variety survived (Figure 2).

Figure 2 Stem length of bean varieties under induced saline conditions with NaCl, Na₂SO₄ and NaHCO₃.  

Underground part

In the interaction bean factor by salt concentration it was found that the length, fresh and dry biomass of root of the concentration 0.64 to 2.304 the Azufrado bean was the most affected with lower value for these variables. In the salt type by bean type interaction, length, fresh and dry root biomass of Azufrado bean showed lower values with NaCl and Na₂SO₄ but with NaHCO₃ the three varieties drastically decreased those values and were close to each other.

The length, fresh and dry biomass of the root decreased as the salinity increased. The root length in relation to the threshold had a difference in average of 8.93 cm at a concentration of 4.032 g L^-1 and 10.532 at 5.76 g (Table 1). Pinto beans obtained higher fresh and dry biomass for all concentrations and salt types, followed by Azufrado and Black (Table 2). Figure 3 shows that NaHCO₃ at a concentration of 2.304 g did not allowed root development. NaCl affects less the length of the root than Na₂SO₄.

Figure 3 Root length of bean varieties under saline conditions induced with NaCl, Na₂SO₄ and NaHCO₃.  

Germination

^{Mass and Hofman (1977)} set, based on the percentage of relative yield, as bean tolerance limit (threshold) in terms of electrical conductivity 1 dS m^-1 (0.64 g L^-1), the data from this research are consistent, however the control (distilled water) had a germination 4% lower than that of the treatment with 0.64 g of salt. Germination in saline conditions where no distilled water is used, greenhouse conditions and inert substrate do not reflect this difference. On this result it is inferred that the concentration of ions in the solutions, up to a certain upper and lower limit, plays an important role in the activation of seed metabolism and function as scarifiers to enhance germination.

Tolerance to salinity in the germination stage is important because it determines rapid implantation of the crop and competitive advantage in the growth and production. The tolerance to salinity between bean varieties was different and in general the increase in salinity decreased the percentage of germination after the tolerance limit. Research in saline germination demonstrate this trend, ^{Yokoishi and Tanimoto (1994)} with Suaeda japónica Makino and Chenopodium quinoa Willd ^{Madueño et al. (2006)} with Rhynchosia minima L., ^{Konak et al. (1999)} and ^{Bagci et al. (2007)} with varieties and genotypes of Triticum aestivum L.

To germinate the seed requires absorbing enough water to activate its metabolism. The reduction in the germination percentage occurs by osmotic stress generated by the salts and the toxicity of specific ions. A lower osmotic potential in the germination medium due to the presence of high concentrations of salts promotes this osmotic stress. The higher the salt concentration the lower the osmotic potential, the water potential will decrease and the water will be less available for imbibition of the seed.

Osmotic stress with certain salts like NaHCO₃ causes toxicity, so that lower germination is even greater. Our germination results with NaCl and Na₂SO₄ match those of ^{Huang and Redmann (1995)} whom in species of barley and canola, found less affection by sulfate salts compared to NaCl.

Aerial part

If the salt concentration of the growth medium exceeds the tolerance of the plant, the height, fresh and dry stem biomass and the water content in the plant would decrease (^{Cachorro et al., 1993}; ^{Hosseini et al., 2002}; ^{Bai et al., 2008}; ^{Keshavarzi, 2012}; ^{Habtamu et al., 2014}). Some plants in order to tolerate high salinity levels in the growth medium perform an osmotic adjustment, which allows them to generate potentials smaller than the medium and to be able to absorb water and nutrients. Osmotic adjustment allows to maintain or increase cell turgor and plant growth (^{Bai et al., 2008}; ^{Bahena-Betancourt et al., 2008}; ^{Pratap and Sharma, 2010}).

The seedlings in order to decrease its osmotic potential absorb the ions in solution and accumulate them in the tissue, thus counteracting the toxic potential generated by the salts. Some absorbed ions have toxic effects when they exceed the specific tolerance limits of each plant. Na⁺, Cl^- and SO₄^2-contribute to osmotic adjustment (^{Ottow et al., 2005}). ^{Bhivare and Nimbalkar (1984)} found that NaCl and Na₂SO₄ salts led to the succulence of leaves, however NaCl reduces the height and dry matter of the plant, and improves cationic balance compared with Na₂SO₄. ^{Cachorro et al. (1993)} concluded that Phaseolus vulgaris is sensitive to NaCl salinity and growth inhibition was due to toxic effects of CI- ion. Bicarbonate at low concentrations had a very toxic effect in Picea glauca where aereal biomass diminished with sodium combined with carbonate at low concentrations (0.5 dS m^-1) (^{Maynard et al., 1997}).

The greatest effect of NaHCO₃ is attributable to the osmotic effect and the generation of an alkaline pH that affects the enzymes activity and metabolic processes in cells (^{Yildirim et al., 2006}; ^{Pratap y Sharma, 2010}).

In this paper, at the end of germination it was observed that the hypocotyl and radicle at 2.304 g of NaHCO₃ were heavily damaged, those that survived concentrations below 2.304 g of NaHCO₃ had a reduction in the stem and root length in the seedling stage. The decrease of fresh and dry biomass of the bean stem is attributed to the saline stress that could cause a reduction in the water absorption.

The plant in order to perform the osmotic adjustment under salinity conditions activates adaptation mechanisms to generate organic compounds. In addition to the accumulation of Na⁺ and Cl^- in the tissues, the plant also produces proline in response to osmotic stress (^{Misra and Gupta, 2005}; ^{Slama et al, 2007}; ^{Bai et al., 2008}). ^{Bahena-Betancourt et al. (2008)} mention that total carbohydrates are also generated in leaves and stems, in order to maintain the turgor in the early stages of the saline stress.

Underground part

Salinity caused by NaCl reduced the length, fresh and dry biomass of the root, which was more noticeable as the salinity of the medium increased and it was different among varieties. It was observed that from 2.3 g is Na₂SO₄ more inhibitory than NaCl regarding the roots length. This trend was observed in forages species (^{Shen et al., 2003}), Glycine species (^{Kao et al., 2006}), in Amaranthus spp. (^{Omamt et al., 2006}), in Phaseolus vulgaris L. (^{Gama et al., 2007}; ^{Bahena-Betancourt et al., 2008}; ^{Radi et al., 2013}; ^{Habtamu et al., 2014}) and Medicago sativa L. (^{Farissi et al., 2013}).

Studies on germination stage of Lactuca sativa L. found that the length of the plumule and radicle decreased when increasing the salinity level (^{Keshavarzi, 2012}). In this research, it was found that from 1.472 g of NaHCO₃ the embryo development stopped and at higher levels they died. ^{Reginato et al. (2014)} in Prosopis strombulifera with NaCl and Na₂SO₄ found that SO₄^2- had a deleterious effect on root growth than Cl^-. In Figure 3 is shown a drastic inhibitory effect of root growth with NaHCO₃ at concentrations of 2.034 g L^-1.

^{Bai et al. (2008)} found that the water deficit of the shoots was greater than that of the roots and had a positive correlation with the salt concentration. Under salt stress water transport was inhibited from the root to the seedling and this kept the Na⁺ in the roots. ^{Hirpara et al. (2005)} with Butea monosperma Taub and NaCl found that root growth was much greater than the stem, and that the water content of all tissues of primary roots and lateral roots was reduced, resulting in lack of internal water and inhibition of succulence. In this research the fresh biomass of the root with respect to the control was smaller and it is attributed to the water stress that could cause a decrease of the water content in the root.