Introduction

The osmo-conditioning consists of the controlled imbibition of the seed in osmotic solutions or water, interrupting this process before the protrusion of the radicle. The treated seeds increase their germination percentage, as well as the seedlings growth, improving the nutritional quality of the crops like maize. With seed imbibition and enzymes activation due to the effect of gibberellins, glucanases are activated which degrade starchy endosperm, whose products are absorbed by the scutellum (^{Preston et al., 2009}). During germination there is a passive absorption of wáter that is divided into three phases, the third phase does not occur in dormant seeds (^{Méndez-Natera et al., 2008}). Shortly after imbibition there is a decrease in abscisic acid content (ABA).

An increase in gibberellins seems to correlate temporally with the onset of water absorption. After three hours of imbibition, more than 2 000 genes in Arabidopsis show changes in their activity, some positive, some negatively. Among the first there are genes for the metabolism of carbohydrates and ribosome proteins. In Brassica napus, the osmoconditioning affected 952 genes and 75proteins; further more, dipping, drying and germination turn on specific paths, with a limited number of genes and proteins involved in all phases, the vast majority has a participation in only one of the pathways (^{Kubala et al.,2015}). An important part in the germination process is played by the H⁺ATPase that provides the biasing force for entrance and exit of ions and metabolites across the plasma membrane and is activated at two hours after imbibition (^{Mei and Song, 2008}; ^{Sveinsdottir et al., 2009}).

The previous immersion of the seed in osmotic solutions, even in water, can improve seed behavior during germination. Several results show that osmo-conditioned seeds improve its germination rate, as well as the behavior of the seedling, even under stress conditions, it is recommended for water scarcity, to accelerate growth in transplanting seedlings and even to improve nutrimental quality of crops such as maize (^{Finch-Savage et al., 2004}; ^{Harris et al., 2007}; ^{Zhang et al., 2015}).

Substances such as gibberellic acid, potassium nitrate or calcium, polyethylene glycol (PEG) and polyamines, zinc sulfate, hydrogen peroxide (^{Ahmmad, 2014}) have been used for osmo-conditioning. Osmo-conditioning has been associated with the stimulation of antioxidant enzymes; it is proposed that it favors imbibing and increases the extensibility of the cell walls of the radicle and weakens the endosperm, facilitating mobilization, which starts at six hours of imbibition (^{Mei and Song et al., 2008}), although the benefits of osmoconditioning are not clearly related to efficiency in reserves mobilization (^{Zhang et al., 2015}), it shortens the protrusion of the radicle (^{Moosavi et al., 2009}). The objective of this research was to evaluate the effect of osmotic conditioning on the germination and physiological quality of two pigmented maize populations, given the great diversity of maize in México and the conditions under which it is grown. The hypothesis was that there are differences between maize races in the response to osmoconditioning.

Materials and methods

Two races of maize were evaluated: Elotes Occidentales of red color and Bofo of purple color, the seeds were obtained from the maize breeding program of the Technological Institute of Roque, stored under normal conditions of temperature and humidity, semi-dry, semi-cold, average temperature of 18 °C. The seed came from the previous cycle, a homogeneous sample was taken and from this, samples were taken for the experiments. Both osmotic solutions used were prepared with 30% (p/v) polyethylene glycol 6000 (SIGMA^®) and 3% of potassium nitrate (Golden Bell^®) plus 0.01% gibberellic acid (Rali Agropecuaria^®) (^{Tiryaki and Buyukcingil, 2009}). The initial weight of each sample was recorded. A completely randomized design was used with 4 replicates of 25 seeds each. As a control, seeds without osmo-conditioning. The immersion was of 6 and 12 h in an open vessel; at the end of the imbibition time, the seeds were placed in paper towels to remove water excess and then weighed.

They were dried in Petri dishes for 7 days in the environment. Weighed at the end of the drying period. The germination test was carried out according to the method of ^{ISTA (2005)} in a Control System Labs^® germination chamber at 25 °C with 80% of relative humidity. A 1% Captan solution was prepared and the seeds were immersed in it for 5 min. Soak test: 25 seeds were placed in a 250 mL plastic vessel, purified water was added at 25 °C for 24 h at controlled temperature; the seeds were covered with a water layer of 2 cm. Subsequently, they were immersed in 1% Captan for 5 min. Four replicates of 25 seeds were seeded for the germination test. Four seedlings per replicate were taken, as it was considered adequate to represent the populations, this was corroborated in the analyzes of variance since they had good variation coefficients; the plumule and radicle length as well as the total weight at the fourth and seventh days were measured to determine growth.

Strength test (average length of plumule and radicle): it was carried out according to the method described by ^{Moreno (1984)}, seeds were placed in polyethylene bags and then placed inside the germination chamber at 25 °C. Ten seedlings were taken per replicate and the radicles were separated and weighed separately to obtain its fresh weight, each was then placed in paper sachets and then dried in the oven (laboratory apparatus model H 62) at 80 °C for 48 h to determine the dry weight. Titratable acidity. 2 g of flour sample was taken and a 1:4 flour/distilled water ratio was added; it was titrated with a solution of sodium hydroxide at 0.1 N (Karal^®). The acidity in the sample, expressed as malic acid, was 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 the titration of the sample, in mL, N= normality of the sodium hydroxide solution; m= volume of the sample, in mL; 67= chemical equivalent of malic acid.

For protein extraction, 10 seeds were milled with a KRUPS GX4100 mill; 0.25 g of flour from each sample was weighed and 1.5 ml of extraction buffer (pH 7.5 with 50 mM sodium phosphate, 250 mM sodium chloride 2% DTT (dithiothreitol) were added to each tube. The samples were mixed by agitation in the Vortex® for 5 min. Samples were incubated in a water bath at 80 °C for one minute; then cooled and refrigerated at 5 °C for 24 h the supernatant was decanted and centrifuged at 13 000xg in the microfuge for 15 min, the supernatant was removed again and glycerol was added to each sample for a concentration of 10% and finally frozen at -20 °C. Protein quantification was carried out by the method of ^{Bradford (1976)}. The electrophoretic patterns were obtained in triplicate using the method of ^{Schagger and von Jagow (1987)} .The evaluation of the standard germination, vigor (plumule length), soak test and titratable acidity tests were done under a completely random design, and the statistical analysis was performed with Andevas using the SAS 9.0 program.

Results and discussion

In the Bofo breed (Table 1), the imbibition rate of all treatments was higher than the control. In this regard, ^{Mendez-Natera et al. (2008)} found no relationship between the imbibition rate and seed germination rates of common vean seeds (Phaseolus vulgaris), pigeon pea (Cajanus cajan) and maize (Zea mays). It has been attempted to control the amount of water imbibed by the seed, but is difficult to achieve in addition, the behavior would depend on the species, the lot and the conditions under which the treatment is carried out (^{Sánchez et al., 2001}). The maize embryo is hydrated at 15 h, the endosperm at 48 h, most imbibition may improve field establishment according to ^{Finch-Savage et al. (2004)}.

Table 1 Comparison of means by the DMS test for soaking treatments of Bofo maize. Roque, Guanajuato, 2015. 

Medias de tratamiento con la misma letra dentro de cada variable son stadísticamente iguales con DMS (p< 0.05). TRAT= tratamientos; NOR= semillas normales; MUER= semillas muertas y TI= tasa de imbibición.

Table 2 shows that all the treatments were superior to the control in the variables length of plumule and radicle, there were no differences between treatments of osmo-conditioning. The treatments had no negative effects on the evaluated variables; some researchers (^{Campos-Álvarez et al., 2002}; Moosavi et al., 2007) indicate that the percentage and germination rate decrease when the osmotic potential of the solution is increased, indicating that in this case the amount of salts used did not have stress effects on the seed. In the second count a similar behavior was observed, the positive effect of the osmo-conditioning was shown again in these variables; in the length of secondary root only some treatments were superior to the control (PEG 12 h y KNO₃+AG₃ 6 h).

Table 2 Comparison of means by the DMS test for the variables length of radicle and plumule of the first and second counts in the soaking test of Bofo maize. Roque, Guanajuato, 2015. 

Medias de tratamiento con la misma letra dentro de cada variable son estadísticamente iguales con DMS (p< 0.05). TRAT=tratamientos, LP= longitud de plúmula, LR=longitud de radícula, LRS1 y 2= longitud de radículas secundarias 1 y 2.

These results could be related to the activation of genes that modify and make the cell wall more lax and the organization of the cytoskeleton, which facilitates or helps the elongation of the seedling structures. By increasing the extensibility of the cell walls of the radicle and weaken the endosperm they facilitate mobilization, as indicated by ^{Mei and Song et al. (2008)}. Genes involved in cell division are regulated positively during osmoconditioning, although it has been reported that DNA synthesis occurs at this stage to repair itself (^{Zhang et al., 2015}). The observed positive effects suggest that these pre-treatments are proposed to improve germination and increase stress tolerance (^{Moosavi et al., 2009}), as conditions of water scarcity or to accelerate seedlings transplanting (^{Finch-Savage et al., 2004}; ^{Harris et al., 2007}; Zhang et al., 2015).

It has been observed that rapid growth after osmocoditioning coincides with an increase in the pool of UDP-glucose synthesis precursor of cell wall cellulose (^{Bray et al., 1989}). The variables evaluated in the soaking test for Elotes Occidentales are shown in Table 3. The percentage of germination of normal seeds at the first count was statistically higher than the control in seeds treated with PEG at 6 and 12 h. PEG osmotic solutions have been used to impose water stress on plants because it prevents excessive absorption of water by the seed. Abnormal seedlings showed differences in the control sample and in the treatment with PEG at 12 h. In dead seeds the treatment with KNO₃+ AG₃ at 12 h showed the highest value; KNO₃+ AG₃ at 6 h and the control did not show differences. For TI2 the highest treatment was PEG at 12 h and the lowest was KNO₃+ AG₃ at 6 h. Slowing imbibition using PEG prevented the occurrence of dead areas in cotyledons of peas (^{Méndez-Nátera et al., 2008}). In this case the treatments with PEG showed greater germination.

Table 3 Comparison of means of soaking test in Elotes Occidentales maize. 

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, NOR= semillas normales; MUER= semillas muertas; ANOR= semillas anormales; DRS= semillas duras y TI= tasa de imbibición.

Table 4 shows the measurements on the fourth and seventh days in Elotes Occidentales. Plumule length of treatments was superior to the control, ^{Mora-Ojendiz et al. (2012)} reported a range of 9.24 to 12.54 cm. In taproot length of the PEG treatments at 12 h and KNO₃+ AG₃ to 6 and 12 h were statistically equal. All treatments were lower than the control seed. There were no differences for total seedling weight. In the second count there were no differences between treatments and all were above the value of the control sample in plumule length. The total weight of the seedling in the treatments was lower than the control treatment. In maize seed the treatment with hydrogen peroxide increases the length and seedling dry weight (^{Whaid et al., 2008}).

Table 4 Comparison of means of root and plumule lengths from the first to the second count of Elotes Occidentales maize seedlings in soak test. 

^a Medias de tratamiento con la misma letra dentro de cada variable son estadísticamente iguales con DMS (p< 0.05). TRAT= tratamientos; LP= longitud de plúmula; LR=longitud de radícula; LRS2= longitud de radícula secundaria y PT= peso total de la plántula.

Table 5 shows the variables evaluated in Bofo maize. KNO₃+ AG₃ at 12 h was statistically equal to PEG at 12 h and showed the values with respect to the other treatments and the control. The plumule length with PEG treatment at 6 h was 11.7 cm, which is below the value of the control seeds but which lies between the values 11.05 to 11.86 cm reported by ^{Ruíz-Torres et al. (2012)}. In the radicle length, PEG value at 12 h proved to be the highest with 18.05 cm and similar to the 21.2 cm reported by ^{Martínez et al. (2010)}. The PEG value at 6 h for fresh weight of the plumule is below the control sample. The highest value was 0.884 g which corresponded to the seed treated with KNO₃+ AG₃ at 12 h. The control showed the highest radicle fresh weight and was different to the lowest, KNO₃+ AG₃ at 6 hours; the other treatments were statistically the same.

Table 5 Comparison of means of plumule and radicule lenghts of Bofo maize. 

Medias de tratamiento con la misma letra dentro de cada variable son estadísticamente iguales con DMS (p< 0.05). TRAT= tratamientos; LMP= longitud media de plúmula; LMR= longitud media de radícula y PFP= peso fresco de la plúmula; PSP= peso seco de la plúmula; PFR= peso fresco de la radícula; PSR= peso seco de la radícula y TI= tasa de imbibición.

The greatest length of plumule and radicle in Elotes Occidentales was observed in the treatment with PEG at 12 h (Table 6); measures are below the 25.6 cm reported by ^{Espinosa-Calderón et al. (2009)}. The lowest value corresponded to PEG treatment at 6 h. In the imbibition rate (TI), KNO₃+ AG₃ at 12 hours with 2.62 g was superior to the other treatments. For the second count of normal seeds, it was found that 11% germination of the control was superior to treatment, showing statistically significant difference, the lowest value was observed in the treatment with KNO₃+ AG₃ at 12 h imbibition, these low values in the second count indicate that the highest percentage of normal seeds was shown at the first count; therefore, for the second count the germination percentage was very low.

Table 6 Comparison of means of plumule and radicle lengths of Elotes Occidentales maize. Roque, Guanajuato, 2015. 

Medias de tratamiento con la misma letra dentro de cada variable son estadísticamente iguales con DMS (p< 0.05). TRAT= tratamientos; LMP= longitud media de plúmula; LMR= longitud media de radícula TI= tasa de imbibición; NORM= plántulas normales.

The imbibition rate was similar in the PEG treatments at 12 h and KNO₃+ AG₃ at 6 hours; KNO₃+ AG₃ at 12 h was higher than all treatments, including the control (Table 6). Genes, such as transcription factors MYB, ERF/AP2, encoding proteins involved in modifying the cell wall, cytoskeleton organization and cell division were regulated positively during the osmoconditioning and germination (^{Zhang et al., 2015}) and the membrane H⁺ATPase is active in maize seeds imbibed for two hours (^{Yu-Qin and Song-Quan, 2008}). This would explain the greater germination at four days. The Elotes Occidentales breed showed 98% germination with nitrate treatment at 12 h and was statistically different from the control, which showed 84%. The highest water uptake by seeds was in the treatment with nitrate at 12 h and the lowest with the treatment of PEG at 6 h, being the difference statistically significant (Table 6). Most germinating osmoconditioned seeds is consistent with that reported by ^{Sánchez et al. (2001)}.

Table 7 shows that the extractable protein of the whole seed is greater in the treatment with KNO₃+ AG₃ at 12 h and lower in PEG at 6 h; something similar happened with the protein that was extracted from the embryo and the endosperm in the Elotes Occidentales breed. The germination process has broad effects on the seed and to increase protein is one of them (^{Afify et al., 2012}). Although the time of exposure to the liquid was 12 hours in both PEG and nitrate, a greater effect was observed with the latter, possibly due to its chemical nature, since the PEG decreases the available water for the seed due to its osmotic effect.

Table 7 Comparison of means of protein content in Elotes Occidentales maize, Roque, Guanajuato, 2015. 

Valores con la misma letra dentro de columnas, son estadísticamente iguales con DMS= 0.05.

Table 8 shows that from the treatments, whole seeds, almost twice the protein was extracted than the control. This could be explained by the partial solubilization of the protein due to the osmo-conditioning. As noted earlier germination increases the content of soluble protein in sorghum (^{Afify et al., 2012}).

Table 8 Comparison of means of protein content in Bofo maize, Roque, Guanajuato, 2015. 

Valores con la misma letra dentro de columnas, son estadísticamente iguales con (DMS= 0.05).

Table 9 analyzed a decrease in titratable acidity in treatments in Elotes Occidentales, which could indicate the removal of compounds towards metabolic pathways or a recompartamentalization in such a way that they remained less accessible. In the embryo, however, titratable acidity increased. Since the embryo is hydrated before the endosperm, this could be indicative of the resumption of metabolic activity in the embryo. According to Afify et al. (2016) in sorghum seed, after germination, there was an increase in free amino acids, which participate in pH regulation.

Table 9 Comparison of means of titratable acidity in Elotes Occidentales maize, Roque, Guanajuato, 2015. 

Valores con la misma letra dentro de columnas, son estadísticamente iguales con (DMS= 0.05).

In Table 10 it was verified that the complete seeds showed a tendency to contain greater acidity than the treatments. This is likely to reflect the mobilization of some seminal components; the increase in molecular mobilization in the cytosol of seeds has been correlated with a decrease in viability during accelerated aging (^{Gurusinghe et al., 2001}), but could also be related to germinal processes. In the embryo is where the major changes were observed, perhaps because this is the most active part of the seed when rehydrated.

Table 10 Comparison of means of titratable acidity content in Bofo maize, Roque, Guanajuato, 2015. 

Valores con la misma letra dentro de columnas, son estadísticamente iguales con (DMS= 0.05).

The treatments did not cause noticeable alterations in the electrophoretic patterns of the seed (Figure 1 and 2). According to ^{Bray et al. (1989)}, during the osmoconditioning occurs protein synthesis in Allium porrum and the identity of these proteins may be important for the germination but in osmoconditioned Brassica napus seeds only a decrease in the abundance of cruciferin was observed (^{Kubala et al., 2015}). In a previous work (^{Valle Moysén et al., 2017}) electrophoretic patterns were obtained in popcorn maize, allowing to compare and note that they are very similar, showing similarities at biochemical level among the studied breeds.

Figure 1 Electrophoretic patterns of Bofo maize with osmoconditioning (embryo), on the left are markers of molecular weight. Lane 1, MPM; lane 2, ampty; lane 3, control; Lane 4, PEG 6 h; lane 5, PEG 12 h; lane 6, KNO₃+AG₃ 6 h; lane 7, KNO₃+AG₃ 12h. Arrows indicate differential bands and indicating the calculated weights. 

Figure 2 Electrophoretic patterns of Elotes Occidentales maize with osmotic conditioning (full seed) on the left the molecular weight markers are shown. Lane 1, MPM; 2 empty; 3 control; 4 PEG 6 hr; 5 PEG 12 hr; 6 KNO₃+AG₃ 6 h; 7 KNO₃+AG₃ 12 h. 

^{Liu et al. (2011)} reported that the RNAs and proteins required for germination are already found in the endosperm and embryo. It is expected that there will be no conspicuous changes in protein patterns, quantitatively or qualitatively, although maize oleosins gradual degradation during imbibition has been described ^{Tnani et al. (2012)}. Finer fractionation of proteins is required when extracting them and implementing more sensitive techniques for its detection (^{Wang et al., 2016}). ^{Wu et al. (2012)} extracted prolamins and non-prolamins from maize grains; the latter give a large number of bands of about 100 to 10 kDa, emphasizing the relative abundance of some bands in certain maize lines.

Conclusions

Seeds of Bofo breed subjected to hypoxia by water excess (soak test) showed a positive effect on the germination in PEG 12 h and KNO₃+ AG₃ 6 h treatments. The treatments had a positive effect on the plumule and radicle length on the fourth day and remained in the latter up to the seventh day.

In the Elotes Occidentales breed, treatment with PEG at 6 and 12 h had a positive effect on germination when the seeds were subjected to hypoxia. PEG at 6 h and KNO₃+ AG₃ in both incubation times had a positive effect on seedling length at the fourth day.

All treatments showed a positive effect on seedling length at the seventh day. In the vigor test, KNO₃+ AG₃ at 12 h and PEG 12 h had a positive effect on plumule and radicle length, in both breeds. Protein extracted from whole seed of Bofo breed was higher than the control in PEG 6 and 12 h and KNO₃+ AG₃ 6 and 12 h treatments. In Elotes Occidentales breed more protein was extracted from the embryo, endosperm and complete seed in the treatment with KNO₃ + AG₃ 12 h. Titration of acidity in Bofo breed, in whole seed, more acidic species in the control were found, which overlapped with KNO₃+ AG₃ 12 h, in the embryo, treatment with PEG12 h decreased the amount of titratable acidity. In the endosperm the amount of titratable acidity decreased with PEG 6 h. Elotes Occidentales breed showed a decrease in full seed with PEG 6 h and both treatments with KNO₃+ AG₃. In the embryo the titratable acidity was increased in all treatments.