In this study, two varieties of maize grains from the Conical Group were used, purchased from two different locations and harvest years as follows: a) grains of plants grown in the Chalco Valley (CH), State of Mexico (19˚15'00''N 98˚50'00''O/19.25, −98.83333) during the 2016 planting cycle, and b) plants grown in the Municipality of Milpa Alta (MA) in Mexico City (19˚11'39''N 99˚01'17''W/19.19405, −99.02149) and obtained during the 2018 sowing period. The mature grains were acquired from producers in the local market. The grains were transported to the laboratory, cleaned to remove residues, placed in airtight plastic containers and stored in darkness at 7˚C and 40% relative humidity until use [23]. For the study, it was ensured that the grains of each race used had similar weights (from 0.5 to 0.6 g).

The chemical reagents, substrates, inhibitors, and solvents used in this study were obtained from Sigma-Aldrich, Merck, Baker (USA).

The embryos were manually dissected from the grains with similar weight of each race with no damage and then were superficially disinfected with a 3% NaClO solution for 5 min and washed three times with sterile water. Five embryos with similar dry weight were placed per germinator, ensuring that the scutella have no damage; the embryo were placed into direct contact with the base of the germinator. Each germinator consisted of a Petri dish with two layers of filter paper on both the base and the cover. To each germinator, 10 ml of the corresponding imbibition medium was added at the base to moisten it while ensuring that there was no excess. The imbibition medium used was H₂O (pH 6.8) and was contrasted with four sodium phosphate (Pi) media at a concentration of 1 mM or 10 mM at pH 6.8 or pH 4.5. Sodium phosphate media were calculated using the Henderson-Hasselbalch equation and adjusted on a potentiometer with concentrated NaOH or HCl in case of needing.

The embryos were incubated for 24 h in darkness at 25˚C - 26˚C in a Riossa bacteriological incubator (NY, USA). Once the imbibition time was completed, the fresh weight of the embryo was determined, and the length of the radicle from the base of the scutellum to its apex was measured.

At the end of the 24 h of imbibition on H₂O (pH 6.8) or sodium phosphate media (1 mM or 10 mM at pH 6.8 or pH 4.5), from each germinator four of the most vigorous embryos were selected, and their fresh weight and radicle length were determined; from these embryos, the scutella were dissected and the fresh weight is obtained. The scutella were placed on ice bed until use; the embryonic axis was removed to rule out the high activity of class III peroxidases in the expanding radicle [73]. To obtain the enzymatic extract, the procedure of Corona-Carrillo et al. [19] was followed. Briefly, each enzymatic extract consisted of 4 scutella that were homogenized in 3 ml of a 100 mM sodium phosphate buffer (pH 6.8). The homogenate was centrifuged at 10,000 × g for 30 min at 4˚C in a Sorvall RC-5 refrigerated centrifuge (Waltham, MA, USA). The resulting supernatant was the enzymatic extract and was stored for short time at 4˚C until use [19][23][74].

The activity of class III peroxidase (POX) was quantified by the method described by Corona-Carrillo et al. [19][23]. Briefly, two reaction mixtures were used: a) guaiacol peroxidase (POXgua) in a reaction medium consisting of 870 μl of a 50 mM sodium phosphate buffer (pH 6.8), 10 μl of enzymatic extract and 10 μl of 1 M guaiacol; to start the reaction, 9 μl of 3% H₂O₂ was added, and the absorbance was determined at 475 nm every 20 s for 2 min; and b) catechin peroxidase (POXcat) in a reaction mixture consisting of 440 μl of a 50 mM sodium phosphate buffer (pH 6.8), 20 μl of enzymatic extract, and 440 μl of 20 mM (+) catechin; the reaction was initiated by adding 4 μl of H₂O₂ at 3%, and the absorbance at 475 nm was recorded every minute for 5 min.

The effect of KCN as an inhibitor of POX activity was determined [19][73], and 10 μl of 0.1 M KCN was added to each reaction mixture removing the same volume of the 50 mM sodium phosphate buffer (pH 6.8). The absorbances were recorded using a Mecasys Optizen Pop UV/Vis spectrophotometer (Korea). The enzymatic activity results are expressed as the change optical density (OD) for minute in each scutellum (OD/min scutellum).

The quantitative results are expressed as the mean ± standard error of 6 to 11 independent test, each test consists of four scutella. Results were analyzed as a factorial experiment, using maize race, phosphate concentration and pH as factors. A one-way analysis of variance (ANOVA) was conducted, followed by a Tukey-Kramer multiple comparison test, with a significance level of p < 0.05 for parametric data, and a Kruskal‒Wallis one-way ANOVA on ranks followed by a Bonferroni multiple comparison test with a significance value of p < 0.05 was conducted for nonparametric data. The statistical analyses were performed using the NCSS 2020 software.

To compare the grains from the two locations, grains weighing between 0.5 and 0.6 g that exhibited high vigor (data not shown) were selected. The embryos of both grains were imbibed in five different media for 24 h. At the end of this time, the embryos were found to be in the germination phase and in transition to seedling growth, associated with the beginning of radicle elongation. It is important to note that the imbibition was performed using isolated embryos, which favored homogeneous imbibition through direct diffusion of the imbibition medium without any restrictions due to the removal of impermeable pericarp and the endosperm. Thus, the real-time of the germination was synchronized under the different imbibition conditions and allow association with the of scutellum/embryonic axis characteristics of the race from which they originated.

The fresh weight of the scutellum isolated from the embryos following imbibition for 24 h in the different imbibition media varied (Figure 1(A)). The average fresh weight of scutellum (FWS) of the grains of plants grown in the Chalco Valley (CH) imbibed in the different media was 0.183 ± 0.003 g, while the fresh weight of the scutellum of the grains from the Municipality of Milpa Alta (MA) was 0.170 ± 0.003 g, resulting in an average difference of 7.1%. With respect to imbibition in H₂O (pH 6.8; H₂O/6.8) resulted in a slight increase in the FWS of the CH compared with that of the MA (0.177 ± 0.007 g and 0.164 ± 0.007 g, respectively) resulting in a difference of 7.92%. The imbibition of CH maize embryo in various phosphate imbibition media produced a differential increase in scutellum fresh weight from 1.81% in 10 mM phosphate medium (pH 4.5; Pi 10/4.5) to 8.66% with 1 mM phosphate medium (pH 4.5; Pi 1/4.5) compared to the H₂O/6.8 imbibition medium (Figure 1(A)). In comparison, the fresh weight of the MA scutellum was slightly lower than that of CH scutellum, although the changes were not significant. In comparison, the scutellum of the MA maize embryo imbibed in various phosphate media produced an increase from 10.7% to a slight decrease of 0.3% in fresh weight (Pi1/6.8 and Pi10/4.5 respectively) compared to imbibition in H₂O/6.8 (Figure 1(A)). This variation in the results shows very small changes and does not differ significantly between the different imbibition media or between the two types of maize from different locations (Figure 1(A)). This implies that the scutellum of the grains from the two locations exhibits similar characteristics with respect to imbibition in different media. Furthermore, the scutellum represents approximately 80% of the embryo mass in both grains.

The maize grains used in the present study have high viability, vigor and germination capacity. In most embryos, radicle elongation had begun, while in others, the expansion was very small or not yet evident (Figure 1(B)). The radicle length of embryos from grains from the Chalco Valley (CH) imbibed in H₂O/6.8 for 24 h was 1.53 ± 0.27 mm which was intermediate compared with the values obtained under other conditions; however, radicle elongation had not yet been initiated in a proportion of the embryos, while the radicles of very few embryos reached a maximum length of 5 mm. When the embryos were imbibed in Pi 1/6.8, the average length of the radicle was 0.7 ± 0.15 mm, with a greater percentage of embryos that had not yet initiated radicle elongation or that were very small for this race. An increase in radicle length was observed when the embryos were imbibed in Pi 10/6.8 with the average length being 1.86 ± 0.30 mm. The difference in radicle length of embryos imbibed in Pi 10/6.8 was only 1.21 times greater with respect to H₂O/6.8 or 2.6 with respect to Pi 1/6.8. Whereas the size difference implies only 0.45 times when comparing the radicle length in imbibition with Pi 1/6.8 with respect to that of H₂O/6.8. Therefore, at a pH of 6.8, the radicle length was statistically different between embryos imbibed in 1 and 10 mM phosphate media. In Pi 10/6.8 medium a higher number of embryos having elongated radicles and one embryo that achieved the maximum length for this race (Figure 1(B)-(D)). The radicle of the embryos imbibed in two different concentrations of phosphates (pH 4.5) was similar, with a length of 1.0 ± 0.19 or 1.06 ± 0.25 mm when the embryos were imbibed in 1 mM phosphate or 10 mM phosphate medium respectively (Figure 1(B)). Imbibition in phosphate media at a pH of 4.5 resulted in inferior radicle growth compared with radicle length of embryos imbibed in H₂O (pH 6.8). However, it is important to note the variation in the radicle length since very few embryos achieved a radicle length of 5 to 6 mm, and several embryos had not yet initiated radicle elongation (Figure 1(C), Figure 1(D)). Thus, for the CH embryos, imbibition in Pi 10/6.8 favored the asynchronous transition from germination to seedling growth. The smallest radicle length was observed for embryos imbibed in Pi 1/6.8 indicating that these embryos were at the end of the germination stage.

Figure 1. Morphometric parameters of the embryo in different imbibition media. Embryos from grains from the Chalco Valley or the municipality of Milpa Alta were incubated in different imbibition medium: H₂O (pH 6.8; H₂O/6.8), 1 mM phosphate medium (pH 6.8; Pi 1/6.8), 10 mM phosphate medium (pH 6.8; Pi 10/6.8), 1 mM phosphate medium (pH 4.5, Pi 1/4.5), or 10 mM phosphate medium (pH 4.5; Pi 10/4.5), at 25˚C - 26˚C for 24 h. (A) Fresh weights of the scutellum. (B) Radicle length of the embryos. (C) Radicle length and its dispersion in Chalqueño embryos. (D) Radicle length and its dispersion in Cónico embryos. In (A), the bars represent the mean ± standard error of 6 to 11 independent tests; 4 scutella were used for each test to determine the fresh weight of the scutellum; (B)-(D), the bars represent the mean ± standard error of 30 independent measurements to determine radicle length. The values represent the mean ± standard error of 150 total embryos. A one-way ANOVA was performed, followed by a Tukey-Kramer test, with a value of p < 0.05 considered to indicate statistical significance, the lowercase letters denote statistically significant changes.

When the MA embryos (Figure 1(B)) were imbibed for 24 h in H₂O (pH 6.8) the radicle length was 1.66 ± 0.43 mm, which indicates that the length is slightly greater than the radicle length under the same imbibition condition of the CH embryo. The shortest radicle length is induced in the Pi 1/6.8 medium (1.16 ± 0.24 mm), which is 1.65-fold greater in relation to the same imbibition medium in the CH embryo.

Radicle length was similar between CH embryos imbibed in a 10 mM phosphate medium (pH 6.8) compared to MA embryos imbibed in H₂O/6.8 (1.66 ± 0.39 mm and 1.66 ± 0.43 mm MA vs CH). A similar length in the MA radicle when imbibed in Pi10/6.8, H₂O/6.8 and Pi1/4.5 (1.66 + 0.39 mm, 1.6 + 0.35 mm respectively). However, imbibition of embryos in Pi10/4.5 induced the greatest radicle length, representing the maximum length values of the entire study with an average value of 2.03 ± 0.39 mm, with a marked decrease in the number of embryos that have not yet begun radicle elongation, while other embryos have radicles that reached lengths of 7 to 9 mm (Figure 1(C), Figure 1(D)). The increase in radicle length induced by this imbibition medium Pi 10/4.5 between the two races was 1.96 higher in the MA than CH. However, the differences in the values were not significant, but the results revealed that some of the MA embryos exhibited accelerated germination at 24 h and were already in the seedling growth phase (Figure 1(C), Figure 1(D)). Accordingly, the imbibition medium that resulted in the greatest radicle elongation in the embryos of MA maize race was Pi 10/4.5, while the smallest radicle elongation was observed for embryos imbibed in Pi 1/6.8 (Figure 1(B)).

The larger length of the radicle of the MA embryos imbibed in Pi 10/4.5 or of the Chalqueño embryo imbibed in Pi 10/6.8 compared with that of embryos imbibed in H₂O/6.8 revealed small differences between 1.22 to 1.21-fold respectively, although this increase was not significant. The imbibition of the embryos in 10 mM phosphate medium tended to accelerate the germination process and with it, radicle elongation, but this effect was associated with the pH of the imbibition medium and the maize race; thus, a pH of 6.8 better induced radicle elongation in the CH embryos, whereas a pH of 4.5 better induced radicle elongation in the MA embryos. In contrast, imbibition of the embryos in Pi 1/6.8 tended to produce the smallest elongation of the radicle for both races.

The degree of radicle expansion for both races indicated that after 24 h of imbibition, the embryos were at the end of the germination stage toward the transition to the beginning of seedling growth. Although in some, the size of the radicle indicates that they were properly in the seedling growth phase (Figure 1(C), Figure 1(D)).

The use of catechin and H₂O₂ as substrates allowed the determination of POXcat activity in the scutellum of the two maize races. POXcat activity was higher in the CH scutellum than in that of the MA scutellum when the embryos were imbibed in the different imbibition media (Figure 2(A)). The POXcat activity in the CH scutellum induced by the different imbibition media exhibited the following trend: Pi 1/6.8 > Pi 1/4.5 > H₂O/6.8 > Pi 10/6.8 > Pi 10/4.5. The difference in activity was significant between the first and the last imbibition media. For the same concentration of phosphates, there is a tendency to induce greater activity at pH 6.8 compared to pH 4.5 and 1 mM phosphates are approximately 1.4-fold more potent than 10 mM phosphate at the same pH. When the embryos of the MA maize race were imbibed for 24 h in the different media, the POXcat activity in the scutellum fluctuated slightly, with small differences that were not significant, as follows: Pi 1/4.5 > Pi 1/6.8 > Pi 10/6.8 > H₂O/pH 6.8 > Pi 10/4.5. The POXcat activity when the embryos were imbibed in 10 mM phosphate medium tended to be lower than that when the embryos were imbibed in 1 mM phosphate medium, but no association was found between POXcat activity and the pH of the imbibition media, showing that the small differences are not statistically significant (Figure 2(A)). There are significant differences when comparing the activity of POXcat from imbibition in two media; in Pi1/6.8, and in H₂O, in which the activity in the MA scutellum represents 56.6% or 55.85% of the activity of the CH scutellum respectively. The POXcat activity in the CH scutellum was higher than in the MA scutellum in the other imbibition media, where the proportional difference (CH vs MA) was less than 1.3 to 1.5 times, indicating that the changes were not significant.

The addition of KCN to the reaction mixture caused a significant decrease in POXcat activity for each imbibition condition in both maize (Figure 2(B)). The residual POXcat activity with respect to the total POXcat activity without KCN in the of the CH scutellum represented between 2.12 ± 0.19 to 3.56 ± 0.78% when the embryos were imbibed in the different media. Similarly, the residual POXcat activity in the MA scutella fluctuated between 2.77 ± 0.58 to 3.69 ± 0.43% in the different media used. These results show that KCN generated an inhibitory effect greater than 96%; this effect was replicated under all imbibition conditions and in both races. This decrease was significant compared with the total POXcat activity under the same imbibition conditions (Figure 2(B)); this indicates that the observed activity is the result of catechin peroxidation.

A tendency towards an inversely proportional relationship was observed between POXcat activity induced by the type of imbibition medium and incipient radicle elongation. Under the conditions of imbibition at the extremes of this trend for the CH scutellum (Figure 2(C)), imbibition at Pi 1/6.8 resulted in the highest POXcat activity with the shortest radicle length, while the lowest POXcat activity was observed when embryos were imbibed at Pi 10/6.8 with a tendency to develop longer radicles. Although the differences in POXcat activity and radicle length in MA embryos do not show a statistically difference among them, there is a similar propensity (Figure 2(D)) for greater POXcat activity was observed in embryos imbibed at Pi 1/4.5 and that had shorter radicles, conversely, the lowest POXcat activity was observed in scutellum of embryos imbibed in Pi 10/4.5 having larger radicles (Figure 2(D)). For both embryos, an inverse correlation is maintained between the POXcat in the scutellum and the radicle length.

The POX activity in the CH scutellum in the presence of guaiacol and H₂O₂ (POXgua) tended to be slightly higher under 4 of the 5 imbibition conditions compared with the activity of the MA scutellum (Figure 3(A)). Imbibition of the CH maize embryos in H₂O/6.8 for 24 h resulted in a POXgua activity of 50.25 ± 8.08 OD/min scutellum, representing the highest value across all the imbibition conditions and between the two scutella from different locations. Under the same imbibition conditions, the POXgua activity in the MA scutella was 31.86 ± 3.23 OD/min scutellum, indicating that, on average, POXgua activity was 1.57-fold greater in the CH scutellum than in that of MA scutellum under the same imbibition conditions (Figure 3(A)).

Figure 2. Effect of the imbibition medium on class III peroxidase activity in the scutellum when catechin + H₂O₂ was used as substrate. (A) POXcat activity in CH scutellum and MA scutellum extracted from embryos imbibed for 24 h in different media: H₂O (pH 6.8; H₂O/6.8), 1 mM phosphate medium (pH 6.8; Pi 1/6.8), 10 mM phosphate medium (pH 6.8; Pi 10/6.8), 1 mM phosphate medium (pH 4.5; Pi1/4.5), or 10 mM phosphate medium (pH 4.5; Pi 10/4.5). (B) Residual activity of POXcat upon addition of KCN to the reaction mixture. The results represent the percentage of residual activity compared to the total POXcat activity from the same enzyme extract without the addition of KCN. (C) Relationship between POXcat activity in the scutellum and radicle length of CH embryos imbibed for 24 h in different media. (D) Relationship between POXcat activity in the scutellum and radicle length of MA embryos imbibed for 24 h in different media. The data represents the mean ± standard error of 6 to 11 independent tests; for each test, 4 scutella were used. An ANOVA was performed, followed by a Tukey-Kramer multiple comparison test, with a value of p < 0.05 considered to indicate statistical significance. The results reveal significant changes in POXcat activity in the scutellum induced by the different imbibition media at 24 h between the two maize races, statistically significant difference between the two imbibition conditions in the same race is represented by Φ; the lowercase letters denote statistically significant changes under the same imbibition condition between the two grains.

POXgua activity in the CH scutellum imbibed in different phosphate media varied from a maximum of 42.86 ± 6.18 OD/min scutellum for Pi 10/6.8 to a minimum of 32.49 ± 4.01 OD/min scutellum for Pi 1/4.5. The activity trend between the different imbibition conditions was as follows: H₂O/6.8 > Pi 10/6.8 > Pi 10/4.5 > Pi 1/6.8 > Pi 1/4.5. POXgua activity in the MA embryo fluctuated from a maximum of 40.23 ± 6.35 OD/min scutellum when imbibed in Pi 10/6.8 to a minimum of 30.96 ± 3.35 OD/min scutellum when imbibed in Pi 1/6.8, with a decreasing trend in the following order: Pi 10/6.8 > Pi 1/p4.5 > Pi 10/4.5 > H₂O/6.8 ≥ Pi 1/6.8. The differences in the POXgua activities under the different imbibition conditions and between the two embryos were not significant (Figure 3(A)). However, in the presence of phosphates, there was a tendency in both grains to exhibit the maximum POXgua activity in the scutellum when the embryos were imbibed in Pi 10/6.8, in contrast, the scutellum of the embryos imbibed in Pi 1/6.8 tended to exhibit the lowest POXgua activity. The proportional difference in the POXgua activity for the same imbibition condition between the scutella of the two embryos, considering the activity for each imbibition based on the MA scutellum, ranged from a maximum of 1.57-fold in the imbibition medium in H₂O/pH 6.8 to minimum of 0.85-fold that in Pi 1/4.5 medium. Moreover, the trend in the proportional change in the POXgua activity according to the imbibition condition between the races was as follows: H₂O/6.8 > Pi 1/6.8 > Pi 10/6.8 > Pi 10/4.5 > Pi 1/4.5.

Figure 3. Effects of the imbibition medium on class III peroxidase activity in the scutellum when guaiacol + H₂O₂ was used as substrate (POXgua). (A) POXgua activity in scutellum extracts of the CH and MA embryos imbibed for 24 h in different media: H₂O (pH 6.8; H₂O/6.8), 1 mM phosphate medium (pH 6.8; Pi 1/6.8), 10 mM phosphate medium (pH 6.8, Pi 10/6.8), 1 mM phosphate medium (pH 4.5, Pi1/4.5) or 10 mM phosphate medium (pH 4.5, Pi 10/4.5). The differences in POXgua activity under the various imbibition conditions in both races were not significant. (B) Residual POXgua activity upon addition of KCN to the reaction mixture. The results represent the percentage of residual activity compared to the activity of POXgua from the same enzyme extract without the addition of KCN. (C) Correlations between the POXgua activity in the scutellum and the radicle length of CH embryos imbibed for 24 h in different media. (D) Correlations between the POXgua activity in the scutellum and the radicle length of MA embryos imbibed for 24 h in different media. The data represents the mean ± standard error of 6 to 11 independent tests; for each test, 4 scutella were used. A Kruskal-Wallis one-way ANOVA on ranks was conducted followed by a Bonferroni multiple comparison test with a value of p < 0.05 considered to indicate statistical significance. No significant differences were found in the quantification of this biochemical parameter.

The residual POXgua activity in the presence of KCN in the CH scutellum fluctuated on average from 2.73 ± 1.14% when imbibed in H₂O/6.8 to 1.02 ± 0.28% when imbibed in Pi 1/4.5. In contrast, the residual POXgua activity in the MA scutellum varied from 1.55 ± 0.37% when imbibed in H₂O/6.8 to 0.79 ± 0.26% when imbibed in Pi 1/6.8. This indicated an inhibition of greater than 96%. These results confirm the presence of a class III peroxidase in the scutellum of both maize races that is strongly inhibited in the presence of KCN (Figure 3(B)).

When the radicle length and the POXgua activity for each imbibition condition for the two races were correlated (Figure 3(C) and Figure 3(D)), it was found that a difference in radicle length did not lead to a significant increase in POXgua activity. Thus, CH embryos imbibed in H₂O/6.8 showed the highest POXgua activity in the scutellum but had intermediate radicle lengths, similarly in Cónico race, the embryo shows an intermediate radicle length with the maximum POXgua activity in the scutellum imbibed in Pi 10/6.8.

The POX activity index (AI) between the two substrate mixtures was determined by obtaining the ratio between the POXgua activity and the POXcat activity in the scutellum (POXgua/POXcat) after 24 h of imbibition in different media. Imbibition in H₂O/6.8 resulted in an AI value of 5.45 ± 1.18 for CH scutellum and 7.28 ± 0.91 for MA scutellum indicating a 1.33-fold increase between the two races (Figure 4(A)).

The AI value when the CH scutellum was imbibed in phosphate media ranged from a minimum of 3.84 ± 0.38 for Pi 1/4.5 to a maximum of 6.33 ± 0.38 for Pi 10/4.5, with the following trend: Pi 10/4.5 > Pi 10/6.8 ≥ H₂O/6.8 ≥ Pi 1/6.8 > Pi 1/4.5 (Figure 4(A)). In contrast, for MA scutellum, the observed AI tended to be higher than for the CH scutellum (Figure 4(A)), and this difference was not significant for the same imbibition condition between races. Therefore, the AI value varied between 6.42 ± 0.49 to 9.15 ± 0.97, for Pi 1/6.8 and Pi 10/4.5 respectively; however, the difference was not statically significant. The trend observed for the different media was as follows: Pi 10/4.5 > Pi 10/6.8 ≥ H₂O/6.8 > Pi 1/4.5 ≥ Pi 1/6.8 (Figure 4(A)). In the scutella of both grains, the differences in AI were due mainly to the discrepancy in the POXcat activity under different imbibition conditions; and only the difference in AI values was statically significant between the MA scutellum imbibed in Pi 10/4.5 compared with CH scutellum imbibed in Pi 1/4.5 or with Pi 1/6.8.

The residual activity index (RAI) when the CH embryo was imbibed in H₂O/6.8 was 4.66 ± 1.34 and the lowest RAI of 2.1 ± 0.52 was achieved when the embryos were imbibed in Pi 10/4.5. Thus, the trend of the RAI for this maize was as follows: H₂O/6.8 > Pi 10/6.8 ≥ Pi 1/6.8 > Pi 1/4.5 ≥ Pi 10/4.5. The RAI in the MA scutellum was maximum when the embryos were imbibed in Pi 10/6.8, with a proportion achieving a value of 3.86 ± 1.6, and the minimum value of 2.20 ± 0.35 when the embryos were imbibed in Pi 1/4.5. The trend of the RAI for this scutellum was as follows: Pi 10/6.8 ≥ H₂O/6.8 > Pi 10/4.5 > Pi 1/6.8 ≥ Pi 1/4.5. The differences in RAI between the imbibition conditions for the same race or between races were not significant (Figure 4(B)).

Figure 4. POX activity index (AI) of the scutella. The data represents the AI values calculated from the ratio between the activity of POXgua/POXcat for the scutellum of embryos imbibed for 24 h in different media. (A) The activity index for the scutellum of grains of different locations at 24 h after imbibition in different media. (B) The residual activity index for the scutellum at 24 h, involving the addition of KCN to the activity assay. The scutellum used were obtained from embryos of each race after being imbibed for 24 h in different media: H₂O (pH 6.8, H₂O/6.8), 1 mM phosphate medium (pH 6.8; Pi 1/6.8), 10 mM phosphate medium (pH 6.8, Pi 10/6.8), 1 mM phosphate medium (pH 4.5, Pi1/4.5), or 10 mM phosphate medium (pH 4.5, Pi 10/4.5). (C) Correlations between the POX activity index of the scutellum and the average radicle length of the CH embryos imbibed for 24 h in different media. (D) Correlations between the POX activity index of the scutellum and the radicle length of the MA embryo imbibed for 24 h in different media. The data represents the mean ± standard error of 6 to 11 independents tests, with 4 scutella used for each test. A Kruskal-Wallis one-way ANOVA on ranks was conducted followed by a Bonferroni multiple comparison test with a value of p < 0.05 considered to indicate statistical significance. Bars with the same letter indicate a statistically significant difference.

In terms of the activity index, the scutella of CH maize presented a fluctuating baseline value at 24 h after imbibition across the different imbibition conditions independent of the radicle length (Figure 4(C)). For the MA scutella, the observed values of the AI in relation to the radicle length fluctuated around a basal value for 4 of the 5 conditions, whereas for Pi 10/4.5, the highest AI corresponding to a greater radicle length was obtained (Figure 4(D)).