Electrical conductivity of different common bean seeds genotypes 1

The electrical conductivity test is a fast and non-subjective method used to assess seed physiological potential. Thus, the objective of this study was adapting the methodology for conducting the electrical conductivity test in common bean seeds and investigate effects of genotype in results of the test, comparing cultivars of the two trade groups: Carioca and Black. The study was conducted in two phases: 1tests with combinations of different temperatures (20, 25, 30, and 35 °C) and five different inhibition periods (4, 8, 16, 20 and 24 h); and 2assessment of influence of genotype by comparing results obtained on the seeds of both groups. Tests of accelerated aging and seedling emergence in field were also performed. It was concluded that, for seeds of common beans, electrical conductivity test may be performed with samples of 50 seeds per replication, soaked into 75 mL deionized water, incubated at 25 °C, and performing reading of results after 20 or 24 h. It was also concluded that test efficiency is variable according to genotype, and that for the trade group Carioca, there is not genotype effect; although the same results have not occurred for results obtained for the trade group Black.

Nevertheless, some factors inherent to methodology of this test are able to affect its results, such as: amount of moisture (Dutra and Vieira, 2006;Soares et al., 2010); temperature, as well as imbibition periods (Dutra and Vieira, 2006;Carvalho et al., 2009a); number of seeds (Vidigal et al., 2008;Lopes et al., 2012); and the initial moisture content of seeds (Vieira et al., 2002;Barbosa et al., 2012).
In the literature, there are several studies indicating use of the test of electrical conductivity to assess vigor of common bean seeds (Phaseolus vulgaris L.) (Santos et al., 2003;2004 and2005;Botelho et al., 2010;Coelho et al., 2010), but using the same methodology recommended to assess soybean seeds (Hampton and TeKrony, 1995;Vieira and Krzyzanowski, 1999).Therefore, further studies are needed aiming at determining a specific methodology for this species.
In addition, there are indications that genotype might affect electrical conductivity test results on common bean seeds.In a study with several cultivars and lines of common beans, each represented by one single seed lot, Vieira et al. (1996) have found that some genotypes with high performance, when assessed by the tests of germination and accelerated aging, were sorted as low quality by the electrical conductivity test; although these authors have also found opposite results.Therefore, more detailed and careful studies on behavior of different commercial groups of common bean cultivars are needed; once that the genotypes of this species are sorted in several categories for commercialization.
In this study, the main objective was to adapt the methodology aiming at the suitable conduction of electrical conductivity test on common bean seeds and, thereafter, investigating influence of genotypes of this species in results of this test, by comparing two distinct commercial cultivars belonging to the trade groups: Carioca and Black.
This study has been carried out in two distinct phases according to the methodologies following described:

Phase I
During this phase, the experiments have been conducted in the laboratory of seed analyses of Department of Plant Sciences and Plant Health of the Federal University of Paraná, headquartered in the municipality of Curitiba, State of Paraná, Southern Brazil.The studies for determining the suitable methodology to the electrical conductivity test with common bean seeds were conducted with four different lots of seeds of the cultivar IPR Tuiuiú, which were produced in the harvest season of 2010/2011.To this, the seeds were submitted to different determinations, as following described: Moisture content: this variable was assessed by the oven method, at 105 ± 3 ºC, during 24 h (Brasil, 2009) by using two subsamples of 5.0 g seeds each, per each seed lot.Results were expressed in percentage of moisture content (wet basis); Germination test: for this test, a sample of 200 seeds was split in eight subsamples of 25 seeds each.The seeds of each subsample were evenly distributed on top of two sheets of paper towel previously moistened with distilled water, in a volume equivalent to 2.5 times the mass of dry substrate, covered with another sheet of same paper likewise moistened, which were then made into rolls.Immediately after, these rolls were placed into a seed germinator (Brand Mangelsdorf ® ) and kept at 25 ºC.The counting of emerged seedling was performed at the fifth day after sowing, by computing the percentage of normal seedlings emerged in each subsample (Brasil, 2009); Accelerated aging test: for this test, a sample about 220 seeds.These seeds were then evenly distributed in a single layer upon a stainless screen, firmly fixed to the upper edge of plastic germination transparent boxes (Gerbox) measuring 11 cm x 11 cm x 3.5 cm, containing 40 mL of water at the bottom, thus forming a moist chamber.Immediately after, the Gerboxes were lidded and then kept into a germination chamber, at constant temperature of 41 ºC, during 72 h (Hampton and TeKrony, 1995).After this aging period, the seeds were subjected to germination test, according to procedures already described in previous item.The moisture content was determined before and after the accelerated aging test; Seedling emergence in field: this test has been performed in seedbeds installed in the experimental area of the Department of Plant Sciences and Plant Health of the Agrarian Sciences Sector of the Federal University of Paraná, in November, 2011.To this, were used four subsamples of 100 seeds each, per lot.The seeds were manually sown in rows of 1.0 m long and approximately 3 cm deep.Irrigations were performed whenever needed, according to rainfall intensity during test conduction; this way assuring ideal water supply for germination of the seeds and emergence of seedlings.The assessment was carried out at the day fourteenth after sowing and results were expressed as the percentage; Electrical conductivity test: through this test, it was studied the interaction between four different temperatures (20, 25, 30 and 35° C) and five different periods of seed imbibition (4, 8, 16, 20, and 24 h) aiming at determining the best combination of temperature x imbibition period to be used specifically for common bean seeds.This way, four subsamples physically pure of 50 seeds each were used for each seed lot.The seeds were weighed in an analytical balance with 0.01 g precision and placed to soaking in plastic cups (180 mL capacity) containing 75 mL deionized water, which then were kept into a germination chamber under the previously determined temperatures.After each imbibition period (conditioning), the electrical conductivity of imbibition solution was determined by reading the values obtained, with aid of a digital conductivimeter.Results were expressed as µS.cm -1 .g - seed.

Phase II
After determining the best interaction between suitable temperature and the ideal imbibition period for conducting the electrical conductivity test with seeds of common beans, the effect of genotype on this test results also has been assessed.
To this phase of research, were used two common beans cultivars from the trade group Carioca (cv.IPR 139 and cv.IPR Juriti) and two cultivars from the trade group Black (cv.IPR Gralha and cv.BRS Campeiro); each group represented by four different seed lots.All seeds used during this phase were also subjected to determination of moisture content and to tests of: germination; accelerated aging; and seedling emergence in field, using the same methodology previously described.Nevertheless, for the test of electrical conductivity, was utilized only the combination 25 ºC x 24 h of imbibition; and this combination was assessed with four replications of 50 seeds each, to each cultivar.
To this phase of research, a completely randomized experimental design was used with four replications per treatment and ANOVA has been separately computed for each genotype; except for data on moisture content.Means were compared by the Tukey test, at 1% probability.

Phase I -definition of specific methodology for common bean seeds
For all four seed lots assessed, data on initial moisture content of seeds for cv.IPR Tuiuiú were similar, with a maximum variation of 0.8 percentage points (Table 1).As emphasized by Marcos-Filho (2005), the knowledge of the initial moisture content of seeds is a very important aspect for obtaining reliable results, when the procedures are aimed at standardizing methodology for the electrical conductivity test.
Through test of seedling emergence in field, it was possible to show better performance of the seeds from lot 1 in relation to seeds from the remaining lots (Table 1).It was also possible to observe that, for lots 2 and 3, the seeds presented intermediate vigor levels, and that the seeds of lot 4 have been the seeds presenting the lowest vigor; being this classification similar to that obtained by test of germination.
By results obtained in the electrical conductivity test, which was conducted with 4, 8, 16, 20, and 24 h of imbibition periods and under temperatures of 20, 25, 30, and 35 °C (Table 2) it was found that, under the temperature of 20 °C, regardless the imbibition period assessed, the seeds have been sorted in three vigor levels: high (lot 1); intermediate (lot 2); and low (lots 3 and 4).
As previously discussed, it was observed that when the electrical conductivity test was conducted at 25 ºC and starting from the imbibition period of 20 h, were found results similar to results obtained for emergence of seedlings in field (Table 1), and so, the seeds of lot 1 were sorted as the most vigorous, the seeds of lots 2 and 3 with an intermediary vigor, and the seeds of lot 4 as the less vigorous ones (Table 2).An identical ranking was maintained after 24 h imbibition period, thus demonstrating methodology efficiency.Such fact is relevant because the electrical conductivity test simulates the behavior results that can affect the vigor of the seeds in its establishment of seedling in field (Comiotto et al., 2011).
It is worth mentioning that the combination of 25 ºC temperature with an imbibition period of 24 h, already was successfully used to other species of Fabaceae such as: soybean (Hampton and TeKrony, 1995;Vieira and Krzyzanowski, 1999;Santos et al., 2011) and peanuts (Arachis hypogaea L.) (Barbosa et al., 2012).
With the increase on the conditioning temperature to 30 ºC (Table 2) it has been found that there has been higher release of exudates, in relation to temperature of 25 ºC, and that the test of electrical conductivity has established a ranking for the lots well different from ranking observed for emergence of seedlings in field as previously discussed; and so has not detected statistically significant differences for quality of the seeds originating from seed lots 3 and 4. Similar results were obtained by Albuquerque et al. (2001) in a study with sunflower seeds (Helianthus annuus L.) in which it was detected that the high incubation temperature of 30 ºC caused a significantly higher release of electrolytes into imbibition solution in relation to temperature of 25 ºC.
Within this study, results of this test were likewise unsatisfactory when incubation was performed at temperature of 35 ºC (Table 2), once the test was not sufficiently sensitive to detect statistically significant differences between seed lots 3 and 4 and the seeds were then sorted as bearing the lowest physiological potential; what does not coincide with the results obtained for emergence of seedlings in field, as previously discussed.This response has probably occurred in function of release of higher amounts of leachates into the imbibition solution, due to smaller structuring and selectivity of cellular membranes, as previously reported by Vieira and Krzyzanowski (1999).
In a general way, in observing data achieved by test of electrical conductivity (Table 2) it is possible observing that, at the temperatures 30 °C and 35 °C, and as the imbibition periods were gradually increasing from 4 until 24 h, the values obtained were also increasing; clearly showing there was a higher release of exudates into the seed imbibition solution.This event may be related to the temperature increase at the moment of seed incubation, once temperatures above 30 ºC can influence water absorption speed (Taiz and Zeiger, 2004) and consequently increase the amount of solutes leached by the seeds into the imbibition solution.
For soybean seeds, Carvalho et al. (2009a) have found that the increase on temperature during the seed imbibition process for test of electrical conductivity, decreases seed conditioning period for the identification of vigor levels.However, the results herein achieved have not demonstrated the same efficiency of this test in assessing common beans seeds when the temperature is increased beyond 30 ºC (Table 2).
In this context, the combinations of 25 ºC x 20 h of imbibition as well as of 25 ºC x 24 h of imbibition, have shown to be adequate for conducting the electrical conductivity test, once results have demonstrated a behavior similar to the results found for emergence of seedlings in field.It is worth mentioning that by the easiness in the routine analyses in seed testing laboratories, the soaking of seeds during 24 h has have been the most recommended (Vieira and Krzyzanowski, 1999).In addition, the temperature of 25 ºC is fairly promising for conduction of this test, since it is closest to the environmental conditions found in majority of the seed testing laboratories in Brazil.

Phase II -Genotype effects A -Results for the trade group Carioca
Results obtained for seed quality of the cv.IPR 139 and cv.IPR Juriti are presented on Table 3.Through these data, it is possible to observe that values for the initial moisture content of seeds were similar between the different seed lots, which have presented maximum variation of 0.4 (cv.IPR 139) and 0.7 (cv.IPR Juriti) percentage points.Another important aspect is that a moisture contents very low (≥10), as well as a moisture contents very high (≤17) have influence on electrical conductivity test results (Vieira et al., 2002).Nevertheless, by results herein obtained for moisture content, the common bean seeds were within the recommended range, i.e., around 13% (Table 3).
As for the seeds of cultivar IPR 139, the germination percentage has allowed sorting seed lots as high quality (lots 5 and 6) and low quality (lots 7 and 8).By the accelerated aging test and emergence of seedling in field, which provide data on the seed vigor, allowed the seed lots were considered of high vigor (lot 5), intermediate vigor (lot 6), and of low vigor (lots 7 and 8) (Table 3).Concerning seed quality, on analyzing results of electrical conductivity test, it was found similarities with results obtained in the tests of germination, accelerated aging, and emergence of seedling in field in relation to the sorting of the lots, thus allowing the differentiation between the lots with lower vigor (lots 7 and 8) from the other two lots (lots 5 and 6), indicating that test was efficient in estimating physiological potential of the seeds of this common bean cultivar.
Thus, it was possible to verify efficiency of electrical conductivity test, for seeds of the two common bean cultivars belonging to the trade group Carioca, only when the test was carried out at 25 ºC temperature and a soaking period of 24 h for conditioning; but, without allowing the detection of the influence of genotype on test results.Nonetheless, these results differ from those results achieved by Vieira et al. (1996), who have reported that the results for vigor of common bean seeds are very expressively influenced by the genotype factor, when these seeds are assessed by the test of electrical conductivity.

B -Results for the trade group Black
Data referring to the moisture content of seeds of the cv.IPR Gralha and cv.BRS Campeiro, assessed before and after the accelerated aging test, as well as data of the tests of: germination; accelerated aging; seedling emergence in field; and electrical conductivity are presented on Table 4. Through these data it is possible to verify that the values for initial moisture content of seeds were similar, with maximum variation of 0.5 percentage points for cv.IPR Gralha and 1.0 percentage points for cv.BRS Campeiro.However, when the moisture content of the seeds was assessed after the accelerated aging test, these seeds presented a maximum variation of 1.5 percentage points for the moisture content (Table 4).
It is worth mentioning that the knowledge of moisture content is important for performing seed assessment tests, once the use of a pattern of moisture is indispensable for the reliability in the results of the assessments (Marcos-Filho, 2005); and that data obtained for the moisture content (Table 4) also have agreed with what has been proposed by Vieira et al. (2002) for conducting the electrical conductivity test.The influence of moisture content of seeds, before conducting the electrical conductivity test, already was the target of a study with seeds of peanuts (Arachis hypogaea L.), where there would be were sorted as having the best performance in field and the lots 11 and 12 as bearing intermediate and low quality, respectively.Table 3. Means values for the initial moisture content of seeds (IMC) and moisture content of seeds after the accelerated aging test (AAA); germination, accelerated aging, seedling emergence in field, electrical conductivity obtained for four seed lots of common beans, cv.IPR 139 and cv.IPR Juriti, belonging to trade group Carioca.
In contrast, it was not possible separating the seed lots in different vigor levels by the test electrical conductivity (Table 4).Therefore, the means obtained for the seeds of the lots classified as of low performance by the tests of germination, accelerated aging, and seedling emergence in field (lots 15 and 16) were not statistically different from the means obtained for lots 13 and 14.As highlighted by Schuab et al. (2006), it is necessary that results achieved through this test have similarity with results obtained by the test of seedling emergence in field, once climatic conditions prevailing in the field are quite variable.
By the means obtained for germination of the seeds of the cv.BRS Campeiro, it can be seen that the seeds of this cultivar have presented differences in physiological quality; once the seeds of lot 17 have presented the highest values for germination (97%), lots 18 and 19 have presented intermediate germination (94% and 90%, respectively), and the lot 20 has presented the lowest values for physiological quality (81%) (Table 4).
Through the means computed from the data obtained after accelerated aging test (Table 4), the seeds of lot 17 have shown the highest vigor level (88%), the seeds of lots 18 and 20 have shown an intermediary vigor level (80% and 81% respectively) and the seeds of lot 19 have shown the lowest vigor level (77%).Nevertheless, through the means obtained by the seedling emergence in field, the seeds from lots 17 and 18 were sorted as the most vigorous ones (88% and 89% emergence, respectively), the seeds from lot 19 were classified as having intermediate level of vigor (80%), and the seeds from lot 20 as having the worst vigor level (73%) (Table 4).
When the means obtained by the electrical conductivity test are examined, it is possible to verify that the seeds from the lots that have revealed lower performance, when assessed by the tests of germination, accelerated aging, and seedling emergence in field, i.e. lots 19 and 20, were the ones that have presented with the smallest amount of electrolytes released into the imbibition solution, allowing to classify them as the seeds with the lowest vigor (Table 4).However, the seeds of lot 17, which were considered as of the highest quality when

Table 2 .
Means values of electrical conductivity of four seed lots of common beans, cv.IPR Tuiuiú, at different temperatures and periods of imbibition.

Table 4 .
Means values for the initial moisture content of seeds (IMC) and moisture content of seeds after the accelerated aging test (AAA); germination, accelerated aging, seedling emergence, electrical conductivity obtained for four seed lots of common beans, cv.IPR Gralha and cv.BRS Campeiro, belonging to trade group Preto.