Physiological behavior of bean ’ s seeds and grains during storage

Beans are one of the most used foods to meet the energy needs of the Brazilian diet, requiring farmers to use high seed physiological potential. The aim was to evaluate the physiological quality of beans stored for 360 days. Analyses were performed at 0, 30, 90, 180, 270, and 360 days after receiving the seeds (S1 and S2) and grains (G1 and G2) of BRS Splendor. Tests of germination, accelerated aging, cold, speed of germination, average length of shoots, and root were performed. The experimental design was completely randomized split-plot in time and the means were compared through Tukey test at 5% probability. Seed germination was not affected in S2, while the drop in S1 and G1 was significant. The vigor of grains from field 1 declined from 91 to 50% and from 93% to 76% by accelerated aging and cold, respectively, after 360 days. The germination speed tests performed showed a decreased during the experiment. The grains from field 1 had lower physiological quality. The accelerated aging and cold tests, through the speed of germination parameter, showed decrease in the vigor of the Splendor BRS. The storage period influenced the physiological quality of the beans tested.


INTRODUCTION
Beans (Phaseolus vulgaris L.) are one of the most important foods used to supply Brazilian's energetic needs (Toledo et al. 2009).Brazil is the biggest producer of this legume, producing approximately 3500.4 thousand tons with productivity of 907 kg ha -1 for harvest 2011/2012(CONAB 2012)).
Bean low productivity, when compared to other producing countries, may be related to the low rate of certifi ed seeds used on planting (Marcos Filho 2005).According to Carvalho and Nakagawa (2000), seeds are a living entity, and when carried in the fi eld, aim to ensure specie's reproduction and endurance, while the grains are produced intended to consumption as food.
Seeking more income, bean producers are searching cultivars with faster germination and high vigor (Maia et al. 2011).Thus, the physiological potential evaluation, is a very important factor and aims to guarantee that the seeds have necessary vigor that allows a direct relationship with the culti-FLÁVIA D.R. CASSOL et al. vation (Coimbra et al. 2009), under a wide range of environmental conditions, through reliable, quick and reproducible tests (Binotti et al. 2008).
Germination test is a standard method for determining physiological potential of seeds, being conducted in optimum conditions (Marcos Filho 2005).Along with this test, said Nakagawa (1999), seedling evaluation can be performed, such as germination velocity and seedling growth.The result of these evaluations expresses the assessed seeds vigor.
For Soares et al. ( 2010) accelerated aging and cold are considered important vigor tests.They evaluate seeds behavior under stress conditions and are performed in high humidity conditions.The fi rst one is conducted in high temperature, which is related to their deterioration rate increase (Marcos Filho 2005, Binotti et al. 2008).While the cold test is performed in low temperature, simulating factors that can occur on the planting season (Marcos Filho 2005).
Therefore, quick evaluations allows greater promptness on the results, thus, the decisions regarding production, storage and marketing can be made more effi ciently (Fessel et al. 2010, De Bittencourt et al. 2012).Because the seed aging results from physiological potential decay, occurs germination ability decrease and an environmental stress sensibility increase.(Marcos Filho 2005).
After reaching the maximum physiological quality, on beans and other species, naturally an irreversible and continuous process of deterioration begins.This can be delayed trough appropriate process of storage (Carvalho and Nakagawa 2000, Palabiyik and Peksen 2008, Cardoso et al. 2012).According to Resende et al. (2008), usually bean storage happen in inappropriate conditions, resulting qualitative and quantitative losses.
Thus, the aim of this work was to evaluate the physiological behavior of bean's seeds and grains of cultivar BRS Splendor, stored under not controlled environmental conditions for 360 days.
The fields were being used for seeds reproduction and, as established by Agriculture Ministry (Brasil 2011), the three meters around the area, can only be marketed for consumption (grain).Thereby, the beans were classifi ed as: fi eld seeds 1 (S1), fi eld seeds 2 (S2), fi eld grains 1 (G1) and fi eld grains 2 (G2).
At the processing unit, the beans were submitted to a pre-cleaning, drying and classification.Next, they were separated and stored in the lab, into paper Kraft bags, under not controlled environmental conditions for 360 days.When arrived and after 30, 90, 180, 270 and 360 days the samples were submitted to the following evaluations.
In accordance with the indicated method of Rules for Seeds Analyze -RAS (Brasil 2009), these tests were conducted: Mass of 100 seeds; Water content: through standard oven method, also verifi ed after the accelerated aging test, as recommended by Marcos Filho (2005).
For the germination test, "germitest" paper was used as substrate, dampened with distilled water (2.5 times weight), 50 seeds were laid on it; the resulting rolls were placed in a sprouting camera at 25 ±1 ºC, for seven days (Brasil 2009).Germinated beans counting was daily, until seedlings number stabilization (Nakagawa 1999) and the germination velocity index (GVI) was calculated according to Maguire (1962).On the last day of the germination test evaluation, fi ve normal seedlings were taken from every replicated; then average root length (ARL) and average length of aerial part (ALAP), were measured.
On the accelerated aging test, beans were distributed on single-layer, atop a stainless steel surface, and placed in plastic boxes (gerbox) containing 40 mL of distilled water on bottom and kept at 42 ºC of temperature, for 24 hours, in sprouting camera.After that, they were evaluated according to the germination test (Marcos Filho 1999).
The cold test was performed trough Barros et al. (1999) method, in accordance with the germi-nation test and the rolls were kept under a 10 o C temperature for three days, as indicated by Miguel and Cícero (1999).After this time, the camera temperature was increased to 25 o C and the evaluations were done according to the germination test.
Meteorological data of precipitation, relative humidity and maximum and minimum temperature are presented on Figure 1.The results were submitted to analysis of variance (ANOVA) and the average compared trough Tukey test, 5% probability level, software R, version 2.15.1 (R Development Core Team 2011) and the regression analysis was tested trough Sisvar® software, version 5.3 (Ferreira 2008).

RESULTS AND DISCUSSION
The average values found to variable mass of 100 seeds did not show interaction with storage period.
The means were statistically equals and varied between 18.8 and 20.6 g to S1; 20.4 and 22.3 g to S2; 20.9 and 22.9 g to G1; and 20.4 and 21.9 g to G2.Similar results were found by Avaci et al. (2010), whose variable showed no statistically difference after 30 days and by Cassol et al. ( 2012), whose 90 days of storage period didn't affect the beans batches.
There was significant interaction between the factors to water content, represented in the regression model, Figure 2. It should be noted decrease of this variable until the 90 days of evaluation, also observed on storage beans of "red" cultivar by Resende et al. (2008)  The initial analysis of germination test indicated lower quality of G1 (Table I), which persisted until the end of the experiment with 74% germination.From the second evaluation period (90 days) differences on the germination potential were showed and by 360 days of storage the classifi cation of physiological quality between beans was possible, S2 and G2 kept high germination percentage.
As presented on Table I, GVI showed similarity until 180 days of storage, followed by a signifi cant decrease after this period; S1 and G1 were lower during all the experiment, the same tendency was observed on the germination test, which confi rm the loss of quality of storage beans.
The difference observed between the two areas could be because of environmental conditions of the time of the harvest.On fi eld 2 (low precipitation and ±70% UR) and on fi eld 1 (after rain period and ±90% UR), factors that affected initial humidity and beans quality.On field, the seeds are infl uenced by environment, being susceptible to adverse conditions (Coimbra et al. 2009).The similarity between grains and seeds from the same location can be explained by the fact that the seeds production area border have been intended to consumption (grain), as established by Agriculture Ministry (Brasil 2011).
Germination bean reduction, during storage, have already been reported by several researchers for the varieties: PS Beautiful, TPS Noble, For the signifi cant regression model (Figure 3) of seeds and grains vigor after exposition to accelerated aging test, it can be verifi ed that although the decrease on germination potential have been observed on all beans, those from fi eld 2 showed more viability.G1 suffered bigger infl uence under heat stress, having decreased germination from 91% to 50%, indicating its lower quality.Until 90 days of storage, after being stressed, the index was kept; however, after 270 days this variable dropped signifi cantly, indicating decrease of vigor.
The GVI of beans submitted to heat stress (Figure 3) suffered interaction during the experi-ment, being G1 the sample that was most affected by storage.The values of this variable were stable until 90 days of storage.After this period, S2 and G2 had the biggest index, followed by S1 and G1, indicating a direct relation with beans vigor.
Silva et al. ( 2012), using the same beans test, found signifi cant decrease in vigor after one year of experiment, maybe due the bigger time of exposition to the test (42 °C for 72 horas).But Cassol et al. ( 2012), after 6 and 12 hours of stress by the accelerated aging test, at 42 °C, were able to distinguish the lots without interference of storage time.
Binotti et al. ( 2008) showed a more intense decrease in beans vigor after 72 hours of heat exposition.Also Avaci et al. (2010) saw this decrease and found that, with 15 and 30 days of accelerated aging at 40 °C and 76% de UR, the vigor was null.As well as Bertolin et al. (2011) who verifi ed decrease and increase of the deterioration rate due bigger exposition to the test.
The water content of the beans after exposition to accelerated aging test remain about 24% for time 0 and 30, 22% for 90, 180 and 270 and 23% for 360 Means followed by case letter: comparison between columns and rows differ between them by Tukey test (5% of signifi cance).S1 and G1 seeds and grains from fi eld 1, S2 and G2 seed s and grains from fi eld 2, respectively.As showed in Figure 4, when submitted to cold stress, S1 and G1 were more sensitive.In grains (G1), the initial 93% dropped to 76% on fi nal vigor.There was no statistically difference on vigor until 180 days of storage.After this period, this variable decreased for the beans from the two fi elds.
Results that support with Fessel et al. ( 2006), which observed decrease on maize seeds vigor submitted to the same stress after 4 months of storage.Also Fessel et al. ( 2010) detected an initial difference between soy seeds studied and after three months the less vigorous lots showed signifi cantly reduction of this variable, while on other the decrease was more gradually.So Guiscem et al. (2010), evaluating cowpea seeds, by cold test without soil, were able to identify different vigor levels between studied materials.
It can be observed on Figure 4 that G1 shows lower values, being statistically different than the others.In evaluation period oscillations were noticed until 270 days, with a drop on GVI of all beans, at the last analyze.The cold test didn't show the same tendency of GVI, however, the vigor dropped substantially.The GVI of the tests performed showed decrease during experiment, indicating a reduction on beans vigor because of the storage, agreeing with the study performed by Cardoso et al. (2012) on crambe seeds, whose GVI (from the germination test) decreased with the storage in a conventional package.Marcos Filho (2005) commented that the seed aging results in a decrease of the physiological potential: a reduction in the germination capacity and an increase of environmental stress sensibility occurs.
The average length of aerial part (Table II), of beans seedlings studied suffered interaction throughout the study.The G1 ALAP decreased while the others had oscillations along the experiment.G2 suffered bigger variation during storage, while S1 did not show variation at all.
Seedlings with bigger aerial part and root length derived from vigorous seeds were observed in soy seeds by Vanzolini and Carvalho (2002), when evaluating the effect of lots with different vigor on the emergence in fi eld and by Henning et al. (2010), by evaluating the chemical composition and the reserve mobilization in low vigor seeds.Such affi rmation are confi rm by the GVI results showed in this work.
The ARL of G1 decreased (Table II), while the others showed variations along the experiment; however, can be observed the statistically similarity between seeds and grains.All of them were infl uenced by storage, which caused varied lengths between the periods.Santos et al. ( 2005) observed reduction in this parameter, an average of 40-50%, in their study.While, in Cassol et al. (2012) study, the average length of root showed difference between the lots and by 90 days of storage an increase was noticed.
The storage period of 360 days under natural conditions, affected the physiological behavior of seeds and grains tested.
The accelerated aging tests, cold and GVI were the most sensitive in the detection of vigor

Figure 1 -
Figure 1 -Daily precipitation data (mm), relative humidity (%) and maximum and minimum temperature (ºC) during experimental time l (SIMEPAR 2012).Cascavel, PR-2013 after 112 and by Rigueira et al. (2009) after 120 days.After 180 days of storage the hydration of seeds and grains occurred, reaching the same level FLÁVIA D.R. CASSOL et al. statistically until the end of the study, when showed, on average, 16% of water content.According to Marcos Filho (2005), the seed physiologically behavior is affected by water vapor of atmospheric air, and so, the water content increase with relative humidity and vice-versa.In this study, oscillation of water content in relation to relative humidity modifications of storage period was observed (Figure 1), showing this water vapor exchange capacity to reach hygroscopic balance.This water content variation was found by Santos et al. (2005) for bean varieties, TPS Beautiful; TPS Noble; Macotaço, IAPAR 44 and TS Bionoble stored in natural conditions, by Cassol et al. (2012) for cultivar IPR Tangará stored for 90 days, on the same environmental conditions and fi nally by Avaci et al. (2010) on Uirapuru and Red river varieties, submitted by accelerated aging for 30 days.

Figure 2 -
Figure 2 -Signifi cant regression model to water content data of beans seeds (S1 and S2) and grains (G1 e G2), of variety BRS Splendor, during storage periods.Cascavel, PR-2013 FLÁVIA D.R. CASSOL et al. days of storage.The similarity between the beans is desirable, since it proves the uniform conditions of the test execution (Marcos Filho 1999).Similar results were also found by Toledo et al. (2009) in beans of Pearl cultivar, by Bertolin et al. (2011) who observed differences lowers than 4% between tested samples of beans and also by De Bittencourt et al. (2012) between the maize lots studied.

Figure 3 -Figure 4 -
Figure 3 -Signifi cant regression model to vigor by accelerated aging test of 24 hours (a), and germination velocity index (b) of beans seeds and grains, of BRS Splendor variety, during storage periods.S1 and G1 seeds and grains from fi eld 1, S2 and G2 seeds and grains from fi eld 2, respectively Cascavel, PR-2013