Introduction

During germination and metabolic signal transduction leading to the expansion, cell division and differentiation pathways are activated (^{Tnani et al., 2012}). The levels of sugars and starch in the seed should be regulated to ensure a good contribution of biomolecules and energy to the embryo and control the distribution of water in the expanding tissues. The micronutrients escaping from the seed during the imbibition are reabsorbed and mobilization depends on the growth rate of the seedling, solubility thereof and concentration of these in the solution surrounding the seed (^{Melo et al., 2009}). The presence of compounds in the seed coat, such as flavonoids, decrease the loss of solutes, are a barrier against fungi and have antimicrobial effect (^{Angelovici et al., 2010}).

The seed size may be one of the factors controlling the efficiency of germination. According to ^{Limami et al. (2002)}, maize lines with smaller seed size have higher germination efficiency;larger seeds produce larger seedlings capable of emerging at greater planting depth and have a higher rate of root growth. The de novo synthesis of proteases and cell wall hydrolases is required for the radicle protrusion (^{Dogra et al., 2013});other events, such as oxidation of disulfide bridges of enzymes, already present in the seed, may be important for metabolic regulation during germination.

They were detected post-translational modifications in proteins during germination, such as oxidation, deamination, acetylation and truncated isoforms (^{Angelovici et al., 2010}; ^{Tnani et al., 2012}). An increase in gibberellins and isopentenyl adenine has been correlated with the onset of water absorption. After imbibition and perception of gibberellins, many enzymes are activated and degrade starchy endosperm, whose products are absorbed by the scutellum (^{Preston et al., 2009}).

The maize seeds that take longer to germinate tend to produce a greater number of abnormal seedlings.The deterioration causes a delay in seed germination in different processes, from the first signs of imbibition and germination until the fourth leaf stage (^{Khajeh-Hosseini et al., 2009}). In the phase of seed desiccation sugars they accumulate such as sucrose, raffinose, galactinol, trehalose, cycle intermediates tricarboxylic acids, some amino acids and free fatty acids (^{Angelovici et al., 2010}). In cells there are molecules such as sugars, amino acids, organic acids, which include malic, citric and succinic acid.

The concentrations in which they are found do not allow them to be considered only as intermediates of the metabolic pathways. The proposal is to form a medium other than aqueous and lipid, which are considered the two media in the cell (^{Choi et al., 2011}). The organic acids are metabolized by the Krebs cycle (breathing), gluconeogenesis, ethanol fermentation, synthesis/interconversion of aminoacids and as a substrate for the production of secondary metabolites, such as pigments (^{Famiani et al., 2015}). It has been reported that the microwave treatment enhances germination imbibition rate increases and water redistribution of seed cells is promoted (^{Anand et al., 2008}).

In this research was evaluated the effect of osmoconditioning on germination, vigor, reserve proteins, electrophoretic patterns and titratable acidity of the seed of a popcorn maize, since this treatment has been recommended for seeds that will be planted under stress conditions low temperatures, shortage or excess water (^{Finch-Savage et al., 2004}).

Materials and methods

The genetic material that was used in this research was a popcorn type maize, which was acquired in the local market. The four treatments were tested, polyethylene glycol 8 000 at 30% (P/V) (^{Tiryaki and Buyukcingil, 2009}) and potassium nitrate at 3% (P/V) over gibberellic acid (RaliGeb^MR) at 0.01% (P/V); both solutions were used during imbibition of the seed at 6 and 12 hours. The initial weight of each sample was recorded and 25 ml of the corresponding solution was added. Four replicates of 25 seeds each were made. After the treatment, the seeds were placed in paper towels to drain them and the final weight was recorded, this to calculate imbibition rate. The seeds were placed in Petri dishes and dried for seven days at room temperature.

The weight was recorded at the end of the drying period. For the germination test, Captan at 1% was used and the seeds were immersed for 5 min (^{ISTA, 2005}). As a control, non-osmoconditioned seed was used. Soak test: 25 seeds were placed in a 250 mL plastic vessel with water at 25 °C for 24 h. These had already been subjected to osmoconditioning, except in the case of control seeds. They were immersed in Captan at 1% for 5 min and four replicates of 25 seeds were made for the germination test. Four seedlings were taken per repetition and treatment and the length of plumule and radicle was measured, as well as the weight of each one to the fourth and seventh day. The vigor test (average length of plumule and radicle) was carried out according to the method described by ^{Moreno (1984)}.

It was carried out in the germination chamber at 25 °C with 4 replicates with 25 seeds per treatment. Ten seedlings were taken per repetition for fresh weight; were oven dried at 80 °C for 48 h to determine dry weight. Extraction of protein. Ten seeds were finely milled per treatment and 0.25 g flour was taken to which 1.5 ml of extraction buffer pH 7.5 containing 250 mM sodium chloride, 50 mM potassium phosphate and 0.03% dithiothreitol were added. They were mixed and incubated in a water bath at 80 °C for one min, cooled and refrigerated at 5 °C for 24 h. It was centrifuged at 13 000 x g at microfuge (Eppendorf^MR) for 15 min, the supernatant was taken and glycerol was added to obtain a concentration of 10% and frozen at -20 °C until use. The protein quantification was carried out with a Nanodrop Model 2000 C equipment by the method of ^{Bradford (1976)}. For titratable acidity, 2 g of flour sample was taken and a ratio of 1: 4 flour/distilled water was added; was titrated with sodium hydroxide solution 0.1 N (Karal^®).

The acidity in the sample, expressed as malic acid, is calculated with the following formula: acidity g/L (malic acid) = (V*N*67)/M, where: V= volume of sodium hydroxide solution 0.1 N spent on titration of the sample, in mL. N= normality of the sodium hydroxide solution. M= volume of the sample, in mL. 67= malic acid equivalent. The electrophoretic patterns were obtained in triplicate using the method of ^{Schagger and von Jagow (1987)}. The evaluation of the standard germination, vigor (plulet length), soak test and titratable acidity tests were done by a completely randomized design with four replicates; the Anovas and the comparison of means were performed using the SAS 9.0 program.

Results and discussion

Germination test. In the Table 1 shows the analysis of variance for the variable germination, where it was observed that there were highly significant differences between treatments for normal seedlings of the first count, which defines the emergency rate. In the second count, the percentage of abnormal seedlings, hard seeds and imbibition rate also showed highly significant differences between treatments; at least one treatment produces different effects. The coefficients of variation are acceptable because of the variation expressed by these characters between the first and second counts.

Table 1 Mean squares, degrees of freedom and coefficient of variation of ANOVA for the germination test in popcorn maize. Roque, Guanajuato.  

^*, ^** = indica significancia estadística al nivel 0.05 y 0.01 de probabilidad, respectivamente; ns= indica no significativo; Trat= tratamiento; NOR= semillas normales; MUER=semillas muertas; ANOR= semillas anormales; DRS= semillas duras y TI= tasa de imbibición.

In the comparison of means (Table 2), the control and treatment with nitrate 12 h were statistically the same in the percentage of germination, but in the other treatments the percentage of germination fell and the difference was statistically significant. The water uptake was higher in nitrate treatment at 6 h and was statistically different from the other treatments. The PEG treatments 6 and 12 h treatment with KNO₃ + AG_3-6 h of imbibition were statistically equal and are below 84% germination of the control sample. The value of KNO₃+AG₃ at 12 h of imbibition was statistically equal to the control; among pretreatments, irradiation with microwaves with a low energy dose improves germination; high doses damage the seed (^{Anand et al., 2008}).

Table 2 Comparison of means by DMS test (p< 0.05) for germination variable in maize type popcorn. Roque, Guanajuato.  

Medias con la misma letra dentro de cada variable son estadísticamente iguales, con base en la comparación de medias con DMS (p< 0.05). NOR= semillas normales; MUER= semillas muertas; ANOR= semillas anormales; DRS= semillas duras y TI= tasa de imbibición.

Most results show a positive effect of osmoconditioning, promoting faster and synchronized germination (^{Moosavi et al., 2009}), which is also observed in other results with other genetic material of native maize (results not shown). ^{Méndez et al. (2008)} also obtained very low percentages of germination in maize seed pretreated with PEG 4000.

The treatment with PEG at 6 h of imbibition, at the first count, generated a higher percentage of abnormal seedlings (15%), which is very similar to PEG at 12 h (12%); these treatments are the ones that gave the highest percentage of abnormal seedlings. Treatment with KNO₃+AG₃ to 12 h (1%) was similar to the control (4%). No treatment was superior to the control for the variables dead seeds and hard seeds. The osmoconditioning treatments had a higher rate of imbibition relative to the control, which was not reflected in the percentage of germination these results are consistent with ^{Méndez et al. (2008)} who did not find a relation between the rate of imbibition and the percentages of germination. The popcorn is considered an ancient race, so you may need a deeper or prolonged treatment to germinate, like seeds in nature (^{Preston et al., 2009}); however, it can recommend treatment with KNO₃+AG₃12 h before planting, to improve their behavior.

Soak test

The analysis of variance for this variable is shown in Table 3, there were significant differences in the percentage of dead seeds. Highly significant differences were observed in the imbibition rate (TI1) in the second count; at least one treatment was different. The coefficients of variation were good, which indicates that the experiment was well conducted.

Table 3 Average squares, degrees of freedom and coefficient of variation of Anava for soak test in maize type popcorn. Roque, Guanajuato.  

^{* **} = indica significancia estadística al nivel 0.05 y 0.01 de probabilidad, respectivamente; ns= indica no significativo; Trat= tratamiento; NOR= semillas normales; MUER=semillas muertas; ANOR= semillas anormales; DRS= semillas duras y TI= tasa de imbibición.

In the comparison of means (Table 4), all treatments except PEG 6 h, gave a lower percentage of dead seeds, compared to the control, which is positive.Imbibition rate, all treatments had higher osmoconditioning imbibition rate compared to the control without it would be reflected in the percentage of germination, a result consistent with ^{Méndez et al. (2008)}. It is possible that under field conditions the highest rate of imbibition could mean an advantage for seeds as the endosperm takes longer to rehydrate the embryo (^{Finch-Savage et al., 2004}).

Table 4 Comparison of means of the soak test in maize type popcorn. Roque, Guanajuato 

Valores con la misma letra dentro de columnas, son estadísticamente iguales con base en la comparación de medias con DMS= 0.05; MUER= semillas muertas (primer conteo); TI= tasa de imbibición (segundo conteo).

The analysis of variance for the variables length of radicle and plumule (Table 5) showed significant differences in all its components;there were highly significant differences for length of plumule in first and second counting and root length in the second count;was significant for root length at the first count and for total seedling weight at the first and second counts.The effect of osmoconditioning for these variables was manifested from the first count. This is consistent with the findings of ^{Zhang et al. (2015)} regarding the osmoconditioning increases the rate of germination and vigor.

Table 5 Average squares, degrees of freedom and coefficient of variation of ANOVA for radicle length and plumule of the first and second soak test counts in maize type popcorn. Roque, Guanajuato.  

^*, ^**= indica significancia estadística al nivel 0.05 y 0.01 de probabilidad, respectivamente; LP= longitud de plúmula; LR= longitud de radícula y PT= peso total de la plántula.

In general, there was no effect of the treatments on the total weight of the seedling, compared to the control (Table 6), in relation to the seedling length variable, all treatments were superior to the control; for radicular length, treatments with PEG at 6 and 12 h in both counts and were superior to the control. The treatments with PEG are those that gave the best result for length of plumule and radicle (Table 6). In Mexico it is customary to sow maize at great depth, to ensure the emergence in well-moist soil, this practice could be favored by the osmoconditioning.

Table 6 Comparison of means by DMS test (p< 0.05) for variable length plumule soaking treatment first and second count in maize type popcorn. Roque, Guanajuato. 

Medias de tratamiento con la misma letra dentro de cada variable son estadísticamente iguales, con base en la comparación de medias con DMS (p< 0.05); TRAT= tratamientos; LP= longitud de plúmula; LR= longitud de radícula y PT= peso total de la plántula.

In the Table 7 shows the anava for protein content in the seed and its components. There was statistical significance between treatments for the three characteristics. The coefficients of variation were good. The treatments with KNO₃ were higher than the control and other treatments this could be due to the increased availability of soluble protein to the embryo when the germination process starts; since the PEG has an effect on the availability of water, there are fewer available to the seed by the osmotic effect (^{Moosavi et al., 2009}), explaining that these treatments the extracted protein does not increase (Table 8), in the endosperm the treatments in general were superior to the control, since the protein of this structure is the first to be mobilized, it would explain in part, the positive effect of the osmoconditioning. In embryo, a decrease in the protein content is observed in the treatments at 12 h, which could be due to the use of these for the generation of amino acids and synthesis of new proteins. ^{Zhang et al. (2015)} found an increase in soluble proteins and free amino acids in osmoconditioned seeds; also found that genes related to the degradation of peptides and proteins are activated. Determinations performed by ^{Narváez-González et al. (2006)} show that the total protein content among native varieties is very similar (8.8-9.5% in the case of the popcorn).

Table 7 Average squares, degrees of freedom and coefficient of variation of ANOVA or protein content by the Bradford method in maize type popcorn. Roque, Guanajuato.  

^*, ^** = significativo al 0.05 y 0.01 de probabilidad; ns= no significativo.

Table 8 Comparison of means by DMS test (p< 0.05) for protein content by the method of Bradford in maize type popcorn. Roque, Guanajuato.  

Medias de tratamiento con la misma letra dentro de cada variable son estadísticamente iguales con base en la comparación de medias con DMS (p< 0.05).

There were differences between treatments for titratable acidity in the embryo and the endosperm only (Table 9), the osmoconditioning treatments reduced the titratable acidity in the embryo, compared to the control, indicating a change in the set of low molecular weight molecules. Since the embryo is the living part of the seed and the one that directs the changes that occur throughout it at the beginning of germination, these changes would first be performed in the embryo itself (Table 10). They have been documented differences in the distribution of water within the seed, which would affect the metabolic activities (^{Anand et al., 2008}).

Table 9 Average squares, degrees of freedom and coefficient of variation of ANOVA for titratable acidity in maize type popcorn. Roque, Guanajuato.  

^*, ^**= significativo al 0.05 y 0.01 de probabilidad; ns = no significativo.

Table 10 Comparison of means by DMS test (p< 0.05) for titratable acidity in maize type popcorn. Roque, Guanajuato.  

Medias de tratamiento con la misma letra dentro de cada variable son estadísticamente iguales, con base en la comparación de medias con DMS (p<0.05)

Above the molecular mass marker of 66.2 kDa, a band (arrow rail 4 Figure 1) appears in the treatments but not in the control. Below this marker (66.2) is a band (arrow rail 4) and another in lane 5 that are not seen in the control. In lane 3 above the 31 kDa marker a band, indicated with arrow in the lane 5 is not visible. In lane 4 a band is noted at the bottom of the gel, which does not appear on the other lanes (Figure 1). The treatments caused not very noticeable alterations in the electrophoretic patterns of the seed.

Figure 1 Electrophoretic pattern of protein of maize type popcorn in whole-seeded. Lane 1, molecular weight markers (MPM); lane 2 indicator; lane 3 PEG 6 h; lane 4 PEG 12 h; lane 5 KNO₃ 6 h; lane 6 KNO₃ 12 h.  

In Figure 2 between the marker of 97.4 and 66.2 kDa lane 4 an arrow is indicated with a band present in the treatments but not in the control. Below this marker, 66.2 kDa, a band is indicated that is not in the control but in the osmoconditioning treatments. In Brassica napus seeds in the drying stage, after treatment of osmoconditioning, only a decrease was observed in abundance cruciferin (^{Kubala et al., 2015}).

Figure 2 Electrophoretic pattern of maize type popcorn in embryo protein. Lane 1, molecular weight markers (MPM); lane 2 empty; lane 3 control; lane 4 PEG 6 h; lane 5 PEG 12 h; lane 6 KNO₃ 6 h; lane 7 KNO₃ 12 h.  

In Figure 3, lane 6 indicates a band almost at the height of the marker of 66.2 kDa, which is observed in this treatment, but not in the others, including the control. In this same figure it is observed that the front of the lane of the control goes lower with respect to the treatments, this could be due to that the proteins underwent modifications or even cuts that result in peptides or proteins of smaller molecular mass.

Figure 3 Electrophoretic pattern of maize type popcorn in endosperm protein. Lane 1, molecular weight markers (MPM); lane 2 empty; lane 3 control; lane 4 PEG 6 h; lane 5 PEG 12 h; lane 6 KNO₃ 6 h; lane 7 KNO₃ 12 h.  

The proteases found in wheat is proposed that would be involved in the growth of the radicle and the seedling during germination and proteases and hydrolases cell wall are required for the protrusion of the radicle (^{Tamura et al., 2007}; ^{Dogra et al., 2013}).

Conclusions

In the KNO₃ 12 h treatment only had no negative effect on germination compared to the control in normal and abnormal seedlings in the first count. For the second count, these differences were no longer observed. As mentioned above, it is probable that in the field the effects of osmoconditioning may be better appreciated, because although it is of value, the laboratory germination test is limited.

In the soak test, no differences were observed between the control and the osmoconditioning treatments; this indicates that the popcorn maize is resistant to water immersion (soak test), under the tested conditions. When irrigation is used for sowing it is common to flood the field and the seed is subjected to hypoxia, this allows us to indicate that even under these conditions the popcorn would have a good emergency.

There was no effect of the osmoconditioning treatments on the total weight of the seedling, compared to the control; in the variable length of seedling, all treatments were superior to the control; for radicular length, treatments with PEG at 6 and 12 h in both counts and were superior to the control. The greater elongation of the seedling can enable it to reach light and, therefore, self-infliction; the longer root length would allow it to reach the moist soil.

The KNO₃ treatments increased the amount of protein extracted from whole seed. In the endosperm in general, an increase in the extracted protein was observed with respect to the control.

There were differences between treatments for titratable acidity in the embryo and the endosperm alone. Changes in seed status have been studied; the redistribution of water and the phase change in the membrane allow the seed to germinate and establish itself; the osmoconditioning seems to give advantages in this sense.

The electrophoretic patterns were obtained and changes were observed in the osmoconditioned seeds with respect to the control. Other authors have reported changes in proteins; it is desirable to delve into this to try to identify them and know their function, as well as whether there is a correspondence between the amount of protein, its activity and the amount of transcript.