Development of the experimentand varieties used

The experiments were carried out at the Plant Biotechnology Center of the Center for Genetic Engineering and Biotechnology, Havana, Cuba. Three varieties of common wheat were used. The variety Cuba-C-204 (progenitor), the first variety of wheat obtained in Cuba from the Brazilian wheat variety BH-11 (^{Gutiérrez et al., 2005}) and two radiomutant varieties INIFAT RM 26; INIFAT RM 29 obtained from Cuba-C-204 (^{Argentel et al., 2012}).

Callus formation stage

Callus formation was based on mature seeds previously disinfected (^{Labrada, 1999}). Of each variety studied, 80 seeds were used and grown in MS-based culture medium. Growth regulators 2,4-dichlorophenoxyacetic (2,4-D; 2 mg L^-1) and 6-benzylaminopurine (BAP; 2 mg L^-1) and NaCl concentrations representing electrical conductivities (EC) of 0.002 dS m^-1 (control treatment); 5 dS m^-1, 7 dS m^-1 and 9 dS m^-1. The culture medium was adjusted to pH 5.8, it was solidified with Gelrite agar (2 g L^-1) and autoclaved at 120 °C and 1.5 atmospheres of pressure for 20 min.

Seeding and disinfection of seeds were performed under asepsis conditions in a laminar flow chamber. Two seeds were seeded per test tube (15.5 cm * 2.3 cm), and eight tubes were used per treatment. The seed tubes were placed in a growth chamber under dark conditions and a temperature of 25±2 °C. Three replicates were established for each experimental variant. For each variety in the electrical conductivities, evaluations were performed at 24 h of stress exposure, at 30 days and 70 days in order to evaluate the variability degree of responses as a function of time. The evaluated variables were as follows.

Permeability of cell membranes (PMC)

PMC was quantified in the callus obtained at 24 hours, 30 days and 70 days of stress exposure. 0.05 g of callus was weighed from each treatment in six replicates and washed four times with deionized water. The calluses were then placed in 5 mL of deionized water for 20 h at 18 °C in the dark. The electrolyte output of the samples subjected to different stress treatments was evaluated by the electrical conductivity of the solution after 24 h. A conductivity meter (DD-IIA, China) was used. PMC was estimated with respect to control following the methodology proposed by ^{Blum (1981)}.

Peroxidase activity

0.25 g of plant tissue were homogenized in a mortar with 5 mL of tris-HCl buffer pH 7.4. The homogenate was centrifuged at 20 000 g for 15 min. The supernatant was used to determine the peroxidase activity and was defined as unit of enzyme activity (UAE), a change of 0.01 at absorbance (420 nm) in 1 min per gram of fresh mass. The determination, properly speaking, of the peroxidase activity was performed according to the continuous method described by ^{Martínez (2013)}.

Total soluble protein content

The total soluble protein content was determined by the photometric method. A sample of 1 g of fresh material was deposited in a mortar and macerated. The extraction was performed with 40 mL of a phosphate buffer solution at pH 6.86 prepared from potassium phosphate, a solution which was added slowly while the extraction was being carried out. The extract was decanted in 50 mL centrifuge tubes and centrifuged at 10 000 revolutions per minute for 7 min. Finally the separated supernatant solution was transferred to 50 ml volumetric flasks. Total soluble protein content was quantified by absorption photometry on a Hewlett Packard 8452 spectrophotometer, at a wavelength of 750 nm (^{Galvani and Gaertner, 2006}).

Abscisic acid content

A mass of 5 g of calli of the varieties was taken at each concentration and time of incubation to the stress and these were macerated to make the determination (liquid-liquid extraction) of ABA. The quantification of the phytohormone was performed by an ABA calibration curve and its final concentration was expressed in µm g mf^-1 using a mass spectrometer (^{Bautista and Gallardo, 2008}).

Statistical analysis

The data were processed through analysis of variance of simple classification based on linear models of fixed effects (^{Fischer, 1935}) without interaction between the factors conductivity/variety/time of exposure to saline stress. Independently the response of each variable was evaluated between electrical conductivities, means that were compared by Tukey's multiple comparisons test ^{(Tukey, 1960}) for significance levels of 5% and 1%. The comparisons between exposure times to 30 and 70 days saline stress were performed intravarietally at each level of electrical conductivity using the theoretical distribution of probabilities of t-student (^{Gosset, 1917}) for significance levels of 5% and 1%. The Statistical Professional Statistical Package, version 8.0 for Windows was used for all analyzes.

Permeability of cell membranes

The permeability of cell membranes (PMC) was not significantly affected, although the tendency was to increase when the calli of the three varieties were incubated for 24 h in the saline solutions (Figure 1).

Figure 1 Permeability of the cell membrane (PMC) in calluses of three varieties of common wheat formed in saline medium for 24 h. R2= 0.822; 0.824; 0.831 for Cuba-C-204, INIFAT RM 26, INIFAT RM 29 respectively. ESx= 0.001 ns NS= not significant.  

Several studies try to explain that initially under conditions of saline stress there is a stimulation of the enzymatic activity and the permeability of membranes, such was the result obtained here, an aspect that is known as salinity stimulating effect or euestres (^{Bakry et al., 2012}).

Changes in cell membrane permeability (PMC) generally vary depending on the exposure time of tissues to stress and degree of tolerance of the varieties and has variation between the types of water and saline stress (^{Chávez et al. 2012}). In this paper it was observed that as the electrical conductivity of the culture medium increased, the permeability of cell membranes decreased significantly after 30 days of exposure to stress, perhaps to avoid adverse effects such as ionic toxicity. The largest decrease in PMC was obtained in radiomutants (Table 1). The progenitor variety showed the most marked differences in the response to imposed saline stress and in the times of stress exposure and the highest PMC values at 30 days, which indicates that as the stress exposure period increased, PMC decreased, although to a lesser extent than the mutants. This response has important practical significance because it offers the extent to which membranes become less permeable to ions such as sodium and chlorine that could lead to toxicity.

Table 1 Permeability of cell membranes in callus of three wheat varieties subjected to different electrical conductivities at 30 and 70 days of saline exposure. 

Medias con letras iguales no difieren según la prueba de comparación múltiple de Tukey HSD. ^** y ^*= diferencias para el 5% y 1% respectivamente por t-student; Es(x)=error estándar de la media.

Another important result in the radiomutants was that even in 9 dS m^-1 the PMC values were similar to when the stress was imposed during the 24 h. Perhaps this element is due to the effect of the radioinduction where it shows superdominance of desired characters as the tolerance to the saline stress. In this sense it has been reported that mutation induction has been proposed as an effective biotechnological technique for such purposes in other plant species (Oladosu et al., 2016). The evaluation of PMC in foliar tissues has been an efficient indicator to detect variations in the tolerance levels to saline and water stress in different cereal genotypes (^{Munss et al., 2012}).

Peroxidase activity

The peroxidase activity experienced significant differences with respect to the control at the two evaluation times in the three varieties and the initial tendency was to increase, except for the progenitor variety (Cuba-C-204) in which at 9 dS m^-1 the same value of enzymatic activity as the control. The INIFAT RM-26 variety showed a similar response in the different saline concentrations and in the time of exposure to stress and their values were the highest among the three varieties, although at 9 dS m^-1 the value of the indicator did not show significant differences When compared to that obtained for EC of 5 dS m^-1. In the parent variety the peroxidase activity did not exceed 100 UAE min^-1 at 30 days of exposure while at 70 days it increased to more than twice that at 30 days. In general, the peroxidase activity was higher in the radiomutant varieties than in the progenitor (Table 2). The increase in peroxidase activity in this paper demonstrates the protective ability of the enzyme to prevent oxidative damage caused directly or indirectly by salinity.

Table 2 Activity of the peroxidase enzyme, in calluses of three wheat varieties subjected to different electrical conductivities at 30 and 70 days of exposure to saline medium. 

Medias con letras iguales en una misma columna no difieren según la prueba de comparación múltiple de Tukey HSD. ^** y ^*= representan los tiempos de estrés, diferencias para el 5% y 1% respectivamente por t-student; Es(x)= error estándar de la media; R²= coeficiente de determinación sin ajustar.

When analyzing the values of the determination coefficient for each variety in the two times of exposure to stress evaluated, it was observed that by EC effect explained more than 89% of the variability found.

There is evidence that in maize exposed to water and saline stress the peroxidase activity increases significantly causing the formation of superoxide radicals (O₂ ^-) (Ahmad et al., 2016). The superoxide radical and its reducing agent hydrogen peroxide (H₂O₂) are potentially toxic compounds that when combined form a radical known as hydroxyl radical (OH) and are known as reactive oxygen species (ROS) (Zhang et al., 2016). Stress-tolerant genotypes such as drought and salinity present an efficient active system to prevent oxidative damage as a function of peroxidase activity (^{Young et al., 2012}), perhaps this process was evidenced in this paper.

Concentration of soluble proteins

The concentration of total soluble proteins was similar in the control treatment in the three varieties; however, a significant increase was observed when saline concentration was increased in the two radiomutant varieties but its increase was only up to the EC of 5 dS m^-1 when it was evaluated at 30 days while the parent increased the value of the indicator until 9 dS m^-1. The increase obtained was at least 0.92 mg g mf^-1 of proteins when the incubation period of callus was of 70 days, aspect that denotes the biochemical transformations significant to tolerate the condition of stress. The highest concentrations of soluble proteins were obtained in the INIFAT RM-26 mutant, although the value of the evaluated indicator decreased (at 30 days of stress exposure) from CE= 7 dS m^-1 (Table 3).

One way to maintain the stability and integrity of cell membranes is through the increase in structural protein synthesis. This increase could be associated with the synthesis of new proteins under stress conditions (^{Valifard et al., 2012}). Such an assertion demonstrates that the varieties studied synthesize new proteins in response to the imposed stress.

Table 3 Activity of the peroxidase enzyme, in calluses of three wheat varieties subjected to different electrical conductivities at 30 and 70 days of exposure to saline medium. 

Medias con letras iguales no difieren según la prueba de comparación múltiple de Tukey HSD. ^** y ^*= representan diferencias para el 5% y 1% respectivamente por t-student; Es(x)= error estándar de la media.

In salinity conditions, in many species of cereals, there is an increase in the content of total soluble proteins, but these values are decreasing due to the activity of the proteases to use the amino acids for protein exchange, as energy source, as a carbon or nitrogen source (^{Mollasadeghi et al., 2011}), as well as using amino acids in the first step to increase the concentration of osmotically active compounds, among others, proline and glycine, glycine-betaine, and thus to ensure a decrease in the osmotic potential and consequently the cellular water potential (^{Argentel et al., 2013}).

Abscisic acid content (ABA)

The concentration of ABA was significantly increased in calli of all varieties under salinity conditions of the incubation medium. In addition, differences between their concentrations were observed in the two incubation times evaluated, being the highest concentration at 70 days of stress. Increases in ABA concentration were found in more than 25 units in established electrical conductivities. The ABA concentration was similar for each of the three varieties studied when the exposure time to the salinity was 70 days to the electrical conductivities of 7 dS m^-1 and 9 dS m^-1 (Figure 2).

Figure 2 Concentration of ABA in callus of three wheat varieties subjected to different electrical conductivities at 30 and 70 days of saline exposure. 

In most of the higher plants, the ABA biosynthesis is indirectly carried out by degradation of some carotenoids (C40) present in chloroplasts (Wang et al., 2016); however, the results shown here demonstrate that its synthesis occurred fully in callus, not in vascular cells or from carotenoids, perhaps from non-pigmented precursors, showing the ability to synthesize ABA by any plant cell under saline stress conditions.

Among the effects of ABA on plants in addition to stomatal regulation and opening (^{Fujita et al., 2011}), bud dormancy (^{Nakashima and Yamaguchi-Shinozaki, 2013}) and seeds (^{Kadri et al., 2014}) the inhibition of RNA synthesis and some proteins is shown (Alhasnawi et al., 2016). Therefore, it would be interesting to test if from the callus with high concentrations of endogenous ABA, as in this paper, the capacity of regeneration of plants is affected.

The results of the evaluated biochemical variables in this paper show the effect of the induction of mutations to create response variability and superdominance from varieties considered as tolerant in agricultural production under conditions of stress such as saline (Jimenez et al. 2016). Genetic improvement programs are currently involved in the creation, identification and characterization of such variability by rapid and precise techniques such as mutation induction, since breeding for abiotic stress conditions in general is a long process (Oyiga et al., 2016), and sometimes when a promising variety is obtained the edaphic conditions have changed and the new variety does not express its productive genetic potential (Al-Mashhadani et al., 2016).

The research was a feasible and efficient way to determine the effect of saline stress on these tissues and at the same time to define the variability of the response of mutants regarding to their parent, which showed greater tolerance. The characterization of these radiomutants will allow their recommendation as future parents for the genetic improvement since their permeability decreased even when the saline concentrations were high, at the same time they demonstrated to develop mechanisms to mitigate the saline stress through the synthesis of osmotically active compounds to ensure a good water status and prevent the physiological drought that occurs under conditions of saline stress.