Influences of the NaCl in biochemical indicators evaluated in callus of Stylosanthes guianensis CIAT-184

Leticia Fuentes1, Y. Pérez1, Amalia Domínguez1, A.R. Mesa2 y S. González3

1Centro de Estudios Biotecnológicos-Universidad de Matanzas "Camilo Cienfuegos". CP 44740


2 EEPF "Indio Hatuey", Matanzas, Cuba

3 Facultad de Biología, Universidad de La Habana, Cuba





The accumulation of certain metabolites and the changes in the activity of antioxidant enzymes, such as catalases and peroxidases, can be related to the plant possibilities for counteracting the negative effects of salinity on physiological and morphological processes. In this work, the levels of total proteins and carbohydrates, and the enzymatic activities of catalases and peroxidases were determined in calluses obtained from three explants of Stylosanthes guianensis CIAT-184, cultured in MS medium supplied with 1 mg/L of 2,4-D, 2 mg/L de 6 BAP and different concentrations of NaCl (0, 25, 50, 75 and 100 mM). Although the activity of catalases increased until 50 mM in the three explants and diminished after that, the highest values were detected in true leaves. On the other hand, the hypocotyls showed the highest activity of peroxidases with progressive increments until 100 mM of the evaluated salt, while the rest of explants showed a decrease in the enzymatic activities from 75 mM. No appreciable changes were detected in total proteins, while in the carbohydrates there was considerable increase for the highest values of sodium chloride, which could be related to the accumulation of protecting osmolytes.

Key words: Callus, carbohydrates, enzyme, salinity, Stylosanthes guianensis




The physiological response of plants to the saline stress is multigenic, because several processes linked to the tolerance mechanisms are affected, such as the production of osmotically active compounds, production of species reactive to oxygen, mechanisms of antioxidant defense, ionic transport and compartmentalization of damaging ions in the vacuoles (Sairam and Tyagi, 2004).

The tolerance of different species to environmental stresses is correlated to an efficient antioxidant system, which comprises a group of detoxifying enzymes, such as superoxide dismutases, catalases and peroxidases (Gueta-Dahan, Yaniv, Zilinskas and Ben-Hayyin, 1997; Sreenivasulu, Grimm, Wobus and Weschke, 2000), and non enzymatic antioxidants such as polysaccharides, polyamines and aminoacids, among others.

The common origin of plant species from ancestors that inhabited the sea, suggests the possibility of obtaining more salinity-tolerant plants by means of the repeated cultivation under these conditions, given the possibility of existence of tolerance genes in all plants (Zhu, 2001). The works of genetic improvement by conventional methods, little applicable to the Stylosanthes genus (which encloses a third of the legumes dedicated to livestock production), lead to the combined utilization of biotechnological and selective methods (Consoli, Vieira, Lopes de Souza and García, 1996; Quecini, Oliveira, Alves and Vieira, 2002). This genus is renowned as an in vitro regeneration model, with differences in the response to the cultivation conditions at species level, among cultivars and even among explants of the same cultivar.

The objective of this work was to evaluate the enzymatic activity of catalases and peroxidases, as well as the protein and total carbohydrate contents of different explants of Stylosanthes guianensis CIAT-184, subject to inducing media of organogenic calluses under saline stress conditions.


For the induction of the organogenesis, the one-month old seedlings germinated in inert substratum moisturized with sterile distilled water were divided into 10 mm fragments of their hypocotyls (Hy), cotyledonal leaves (Cl) and true leaves (Tl). They were placed in glass flasks with 30 mL of MS medium (Murashige and Skoog, 1962), supplemented with 7 g/L of agar, 15 g/L of sucrose, 2,4-D (1,0 mg/L), 6 BAP (2 mg/L), MS vitamins and several concentrations of NaCl (0 mM, 25 mM, 50 mM, 75 mM and 100 mM).

Six flasks with six explants (two of each type) were used for each of the five variants of cultivation media, in a completely randomized design, with three replications of each treatment.

Twenty-one days after the explants were sown, the enzymatic activities catalase and peroxidase were determined in the calluses, which were washed with sterile distilled water to eliminate the remnants of agar and they were dried on sterile filter paper. Afterwards they were cold-homogenized, in a porcelain mortar with a buffer solution of sodium phosphate (50 mM, pH 7,0) and then centrifugated at 10 000 rpm. The supernatant was immediately extracted for measuring the enzymatic activity.

The catalase activity was determined through the decomposition rate of H2O2 at 240 nm in a buffer solution of potassium phosphate (20 mm, pH 7,0), which contained 3% (v/v) of H2O2; the final volume of the essay was 3 mL. The peroxidase activity was measured by the guaiacol peroxidase method at 25 ºC (Chance and Machley, 1955). The enzymatic activity was determined in a final volume of 3 mL, which contained: 2,80 mL of buffer solution of sodium phosphate (0,1 M, pH 7,0); 0,05 mL of guaiacol solution (0,018 M) and 0,05 mL of H2O2 solution previously adjusted to an absorbance between 0,40 and 0,41; distilled water was used as blanc. The reading was made at 436 nm in a spectrophotometer (Ultrospect 2000); three replications were taken per treatment.

The protein concentration was estimated through Lowry's method (Lowry, Rosebrough, Farr and Randall, 1951) from the supernatant taken from the homogenized samples.

The total carbohydrates were determined in the supernatant of the homogenized samples by means of the phenol-sulphuric method, using glucose as standard solution (Dubois, Gilles, Hamilton, Rebers and Smith, 1956).

In all the cases samples from ten calluses and three readings of optical density (OD) were taken. The data were processed according to the pack Statgraphic Plus 4 on Windows and the adjustment to a normal distribution was determined by the Kolmogorov-Smirnov adjustment goodness test and the variance homogeneity through Bartlett's tests (Sigarroa, 1985). In the cases in which the data fulfilled the requisites demanded, they were processed by means of simple or multifactorial classification ANOVA, and by Duncan's multiple range test, as needed. The data that did not fulfill these premises were compared through Kruskal-Wallis' test (Kruskal and Wallis, 1952) and the multiple range test of Student-Newman-Keuls (SNK).


A week after being sown the three explants (Hy, Cl and Tl) in MS medium, supplemented with different concentrations of NaCl, the formation of whitish undifferentiated tissue masses was observed in the sites where the cutting was performed. Fifteen days after sowing callogenic masses of friable aspect (Pierik, 1990) were evident in the surface of all the explants (fig. 1), which showed differences regarding the size, color and texture. In the variant of medium supplemented with 100 mM of sodium chloride the calluses were less developed, with a more intense brown color and a more friable texture than in the other cultivation variants.

Thirty days after being sown most of the explants were completely transformed into calluses, although in some cases the hypocotyls in 75 mM maintained little undifferentiated zones. However, the formation of complete seedlings in Hy cultivated at 100 mM was interesting, which indicates direct organogenesis. In the presence of hormones the morphological response, in terms of callus development and formation of growths or roots, depends on the part of the plant from which the callus was obtained (Consoli et al., 1996).

The in vitro callogenic capacity of different species of this genus has been referred by several authors (Meijer, 1982; Consoli et al., 1996; Valarini, Otsuk and Vieira, 1997; Fuentes, Mesa, Ruíz, Peláez de Lucas and Fernández, 2005), as long as a cultivation medium supplemented with auxins and cytokinins is used in a ratio lower than one (Mroginski and Kartha, 1981). Nevertheless, no references have been found regarding the formation of calluses in the presence of sodium chloride.

The formation of calluses from different fragments of plants under saline conditions, has been reported by several authors in other species such as: Cicer arietinum (Pandey and Ganapathy, 1985); Lycopersicum esculentum Mill Peto 863 (El-Enany, 1995); four cultivars of Solanum tuberosum (Rahnama, Ebrahimzadeh and Ghareyazie, 2003), in the species Alhagi graecorum, tolerant to concentrations between 43 and 200 mM (Hassanein, 2004) and in Chrysantemum morifolium (Hossain, Azad, Datta and Biswas, 2007). In the studies carried out in C. arietinum it was possible to obtain cell lines tolerant up to 200 mM, which were capable of accumulating certain metabolites such as proline, unlike the susceptible lines evaluated as control.

Other possible responses to the stress produced by NaCl in the cultivation medium are related to the activation of antioxidant mechanisms. In this sense, in 21-day-old calluses formed under saline conditions, the catalase activity was maintained, in general, without significant variations until 50 mM, with the exception of the calluses obtained from Tl, where a remarkable increase (fig. 2) was observed. Yet, with higher concentrations the enzymatic activity decreased considerably.

These results can be related to the affectation of the enzyme expression or its activity due to changes in the conformation of its structure, when the concentrations are higher than 50 mM of salt. In the studies performed by Rahnama et al. (2003), in four cultivars of S. tuberosum subject to saline stress (between 0 and 150 mM of NaCl) changes were observed in the catalase activity depending on the variety.

In the peroxidase activity statistically significant differences were obtained between the concentration of sodium chloride and the type of explant (fig. 3). An increase of this activity was observed until 50 mM; from this concentration it decreased progressively until 100 mM, with the exception of the calluses from hypocotyls.

Several authors have referred to the correlation between the response of antioxidant enzymes (such as peroxidases) and the tolerance to abiotic stress conditions (Hassanein, Ahmed, Abed-El-Hafez and Soltan, 1999; El-Tayeb and Hassanein, 2000). Hassanein (2004) evaluated the enzymatic activity of several detoxifying enzymes in induced calluses, under hydric and saline stress conditions (176 mM of NaCl), in two plant species with different tolerance levels: A. graecorum (tolerant) and L. esculentum L. (sensitive); this author detected increases in the peroxidase activity of the species under both stressing conditions, motivated by the activation of new isoforms with regards to the control and by the increases in the expression of some isoenzymes, which showed strips with higher intensity in the staining. Nevertheless, the increase percentage in the activity was much higher in the calluses of A. graecorum, species considered tolerant to both conditions. Hossain et al. (2007) obtained similar results in regenerants of Ch. morifolium, obtained from tolerant calluses that were sub-cultivated in progressively higher concentrations of NaCl. In other species, such as S. tuberosum (Rahnama et al., 2003), the peroxidase activity only showed increases under moderate conditions of salinity.

The specific enzymatic activity was related to the protein content of the samples analyzed. The total proteins in the supernatant of the calluses of Stylo 184, after the cold maceration and centrifugation, showed a homogeneous performance for the different variants of salinity and explants (fig. 4).

This result could have been due to the changes in the expression of numerous genes under the influence of sodium chloride. Sairam and Tyagi (2004) found abundant evidence of changes in the gene expression of plants subject to different abiotic factors. In this sense, while certain processes that imply an active synthesis of proteins (such as cellular division) are totally or partially affected by the osmotic stress due to the presence of sodium chloride in the medium (Xiong and Zhu, 2002), another important group of proteins could be over-expressed. Among them are antioxidant enzymes as mechanism for eliminating the free radicals of oxygen, which levels are exacerbated under the effect of saline stress (Hernández, Jiménez, Mullineaux and Sevilla, 2000) as well as ATPase enzymes, protein carriers of ions and aquaporins to maintain the intracellular ionic homeostasis (Xiong and Zhu, 2002).

The increase in carbohydrates as the saline concentration of the medium increased could have been caused by diverse factors. The considerable accumulation of starch in tissues cultivated in media for the induction of organogenic processes, was reported by Mangat, Pelekis and Cassels (1990) and Fortes and Pais (2000). The organogenesis is a high energy consuming process, which is obtained from the degradation of starch, because glycolitic intermediaries are produced that can be catabolized to produce a high quantity of ATP. The studies carried out in different species, such as Nicotiana tabacum (Thorpe, Joy and Leung, 1986), Manihot esculenta (Stamp, 1987) and Humulus lupulus (Fortes and Pais, 2000), have proven this fact.

On the other hand, there have being reference to the possibility that the tissue subject to stress by salinity, synthesizes certain sugars to compensate the difference of osmotic potential between the intra-cellular and extra-cellular medium (Sairam and Tyagi, 2004).

The changes in the carbohydrate concentration and the catalase and peroxidase enzymatic activity, in seedlings as well as in calluses of S. guianensis cv. CIAT-184 obtained under saline conditions, can constitute a part of the physiological mechanism of defense before this stress, for which its research can be considered in future selection studies of somaclons with tolerance to salinity.


The catalase activity was higher in the true leaves than in the rest of the explants, although in the three cases it increased up to 50 mM of NaCl and decreased progressively afterwards. Among the explants, the hypocotyls were the ones with the highest peroxidase activity, which showed sustained increases up to 100 mM of NaCl; while in the cotyledonal and true leaves the activity decreased from 75 mM. No substantial changes were detected in the concentration of total proteins; while in carbohydrates there was a remarkable increase with the highest concentrations of sodium chloride, which could be related to the accumulation of protecting osmolytes.