Electrical conductivity test in Melanoxylon brauna Schott . seeds ( Fabaceae-Caesalpinioideae )

The electrical conductivity test is a simple and rapid method for evaluating seed vigor. The aim of the present study was to optimize the electrical conductivity test for assessing the vigor of Melanoxylon brauna seeds by investigating the effects of seed number (25, 50, or 75), water volume (25, 50, or 75 mL), and soaking duration (24, 48, or 72 h). The seeds belonged to two lots (I and II) that were collected in 2010 and 2012, respectively, and were incubated in a germination chamber at 25 oC. Electrical conductivity was determined using a conductivity meter Micronal model B220. The Lot II seeds performed better, in terms of vigor and germination, than the Lot I seeds, regardless of other parameters. In addition, electrical conductivity decreased with increasing water volume and soaking duration, regardless of seed lot. In order to most effectively evaluate the physiological quality of M. brauna seed lots, the electrical conductivity test should be performed using 50 seeds, 50 mL of water, and a 48-h soaking period.


Introduction
The restoration of damaged forestry ecosystems and maintenance of biodiversity has recently become a major focus of government policies and social interest, and in the Atlantic Forest, Melanoxylon brauna, a highvalue arborous species that is used in construction and landscaping, has received particular interest (Lorenzi, 2009).Currently, M. brauna is categorized as "vulnerable" on the "list of Brazilian flora species threatened of extinction" (MMA, 2008).
The propagation of M. brauna is by seeds, and the analysis of seed quality is important for seedling production.Physiological quality, which can be measured as germination capacity or seed vigor, is essential for the use and preservation of seed lots.According to Rajjou et al. (2012), seed vigor can be defined as the intrinsic characteristics of specific seed lots that determine the capacity of seed lots in the field, under a large set of environmental conditions.
Electrical conductivity is a measure of exudates leached by seeds during a soaking period and is based on the principle that cell membranes are restored slower in damaged seeds and that more solutes (e.g., sugars, amino acids, fatty acids, proteins, enzymes, and organic ions) are lost into aqueous solutions by damaged seeds than by vigorous seeds (Marcos Filho, 2005).
However, the methodology of electrical conductivity tests often differs, depending on the plant species being examined.The assessment of seed vigor can be affected by seed lot and seed number, as well as by soaking duration and temperature (Vieira and Kryzanowski, 1999.Therefore, it is important to investigate optimal methods, when information is lacking, especially for forestry important species. According to Vieira and Kryzanowski (1999), the electrical conductivity test is a simple and practical method for assessing seed vigor and for rapidly providing insight into the proper use and management of seed lots.
The aim of the present study was to optimize the electrical conductivity test for assessing the vigor of M. brauna seeds by investigating the effects of seed number, water volume, and soaking duration.

Material and Methods
The present study was conducted in the Forestry Seeds Analysis Laboratory of the Federal University of Viçosa, Brazil, from October 2012 to January 2013, using two groups of M. brauna seeds (lots I and II) from fruits that were collected from trees in Leopoldina, MG, Brazil, in September 2010 and 2012, respectively.After collection, the fruits were dried in the sun, and the seeds were manually removed.During this procedure, immature, rotten, and damaged seeds were removed, and the selected seeds were stored in fiber drums in a cold chamber (5 °C and 60% relative moisture) until the initiation of the experiments.
Seeds from both lots were germinated over 10 days in Petri dishes with germitest paper, in a germination chamber at 25ºC and constant light.Germination was calculated daily as the number of seeds with newly emerged radicals, and the germination speed index (GSI) was calculated using the formula proposed by Maguire (1962).
Electrical conductivity tests were performed using different combinations of seed number (25, 50, or 75), water volume (25, 50, or 75 mL), and soaking duration (24, 48, or 72 h).For each combination, the seeds were weighed using a 0.01 g precision balance, transferred to disposable cups that contained the appropriate volume of distilled water, and incubated in a germination chamber that was maintained at 25 ºC.
After soaking, the electrical conductivity of each replicate was measured using a B 330 MICRONAL conductivity meter, with K = 1.0 and results were expressed in μS cm -1 g -1 .Each treatment was represented by five replicates.
The experimental design was randomized, with a 3×3×3 factorial scheme (seed quantity, distilled water volume, and soaking duration).After verifying data normality, using the Kolmogorov-Smirnov and Lilliefors tests, and variance homogeneity, using the Cochran and Bartley tests, the data were subject to analysis of variance (ANOVA).The averages of germination, GSI, and electrical conductivity, between both lots was provided by the Tukey test at 5% of probability.
Correlation between electrical conductivity values and germination and GSI were investigated using the Ftest, at 5%, in Statistica 8.0 (Statsoft, 2009).

Results and Discussion
The average percent germination of the Lot I seeds (54%) was significantly less than that of the Lot II seeds (92%; Figure 1a), and a similar pattern was observed for GSI, with the Lot II seeds being more vigorous (Figure 1b).These differences indicated that the Lot I seeds, collected in 2010, were less viability and vigorous that the Lot II seeds, collected in 2012.
According to Bewley et al. (2013), seed deterioration is affected by environmental conditions (temperature and relative moisture), genetics (species, seed lot, or initial seed quality), and the presence of fungi or bacteria, and the physiological and biochemical alterations that occur in seeds during deterioration are associated with tissue damage and reduced germination capacity.
Revista de Agricultura Neotropical, Cassilândia-MS, v. 5, n. 4, p. 7-12, out./dez.2018.The disruption of membrane system was observed in Phaseolus vulgaris seeds (Lee et al., 2012), and the increase of lipid peroxidation and decreasing of enzyme activities from the antioxidant complex was observed in M. brauna (Corte et al., 2010), resulting on seed quality loss.
The electrical conductivity of the Lot I seeds was greater than that of the Lot II seeds, regardless of seed number, water volume, and soaking duration (Table 1).
Increasing water volume significantly and consistently reduced the electrical conductivity of Lot I.However, for Lot II, such behavior was not observed when soaking 50 seeds for 48 h or 25 seeds for 72 h, since the average electrical conductivity values for the 50-and 75-mL combinations were statistically similar.In general, increasing seed number from 25 to 50 or 75 did not increase the exudates leaching, except when soaking Lot I seeds for 48 h in 50 or 75 mL distilled water.For example, in 50 mL water, increasing seed number from 25 to 50 or 75 significantly increased electrical conductivity from 13.26 to 16.77 and 16.28 µs cm -1 g -1 , respectively.In 75 mL water, increasing seed number from 50 to 75 increased electrical conductivity from 7.87 to 10.62 µs cm -1 g -1 .
The difference observed in the exudate leaching of the lots can be explained by the deterioration of cell membranes in the seeds.The membrane permeability of damaged seeds is greater than that of intact seeds, due to disorganization and low reorganization capacity, which favors solute output during electrical conductivity tests.
According to Menezes et al. (2007), both vigorous, intact seeds and damaged seeds initially exhibit solute leaching, thereby hindering the comparison of lot quality during the initial hours of soaking.According to these authors, the leached solutes liberated by the vigorous seeds is stabilized during the process, due to the membrane reorganization.
The membranes of vigorous seeds may require 48 h to return to their liquid-crystal state, acquiring the semipermeability, differing from the seeds with low vigor.Afterward, the solute output of the cells to the aqueous solution will be less intense than that observed during the initial hours.However, the heavy exudate leaching by Lot II seeds, alone, might compromise seed quality, due to the loss of fundamental components of metabolism.
Soaking periods of 36 h or more allowed to divide the Dalbergia nigra seeds lots (Marques et al., 2002), whereas soaking for 24 h enabled increased the electrical conductivity curves distancing of Helianthus annuus lots (Oliveira et al., 2012).
On other hand, studies of Senna siamea, Ricinus communis, and Jatropha curcas were able to distinguish between seed lots during the initial hours of soaking and found that the optimum soaking period was 6 h (Dutra et al., 2007;Souza et al., 2009;Araujo et al., 2011).Similarly, studies of Solanum sessiliflorum and Brassica napus identified optimum soaking periods of 2 and 8 h, respectively (Pereira and Martins Filho, 2012;Milani et al., 2012), which indicated that the immersion period was more appropriate for assessing seed characteristics than variation in lot vigor.
Increasing soaking duration increased the electrical conductivity of both Lot I and II seeds (Figure 2a).Lot I seeds, which were less vigorous than Lot II seeds, yielded greater mean electrical conductivity values, regardless of soaking duration, with a mean of 19.82 µs cm -1 g -1 , after 72 h, whereas the mean observed for Lot II seeds after 72 h was 9.35 µs cm -1 g -1 .
Increasing seed number was also observed to increase electrical conductivity of Lot I seeds, when 50 seeds were used, but did not affect the electrical conductivity of Lot II seeds (Figure 2b).Higher seed mass implies in a higher amount of leached solutes liberated in the soaking solution, accounting only the compounds liberated in the conductivity.However, the similar electrical conductivity values observed for combinations with different numbers of seeds might be explained by dividing the mass of the seeds (µs cm -1 g -1 ), indicating an equivalence on the solute's liberation per gram of fresh mass.In both Lot I and II, increasing water volume gradually reduced electrical conductivity, with means approaching 8 and 5 µs cm -1 g -1 in the 75-mL combinations, respectively (Figure 2c).For forestry species with significant genetic variation in maturation and seed size, authors have reported that increasing seed number improves discrimination between lots, with 75 seeds providing consistent results in Sebastiania commersoniana (Santos and Paula, 2005) and Guazuma ulmifolia (Gonçalves et al., 2008) and 50 seeds providing consistent results in Pterogyne nitens (Ataíde et al., 2012).
When seed mass is held constant, the liquid-crystal structure of hydrated cell membranes allows the passage of a definite quantity of exudates into the water, due to its semi-permeability characteristics.Therefore, greater water volumes yield lower concentrations of liberated solutes, through dilution, and, consequently, yield lower electrical conductivity values.This hypothesis was verified in Dictyloma vandellianum seeds, in which 100-mL soaking volumes yielded lower mean electrical conductivity values than 50-and 75-mL volumes (Flavio and Paula, 2010), and in Solanum melongena seeds, in which 75-mL soaking volumes yielded lower mean electrical conductivity values than 50-mL volumes (Alves et al., 2012).Accordingly (i.e., due to solute dilution), the 75-mL soaking volume was less efficient in distinguishing between the vigor of the two seed lots.Pereira and Martins Filho (2012) observed a similar pattern, with lower soaking volumes facilitating the discrimination of Solanum sessiliflorum seed lots with different qualities, and both Lopes and Franke (2010) and Milani et al. (2012) reported that lower water volumes were more appropriate for measuring the electrical conductivity of Lolium multiflorum and Brassica napus seeds, respectively.Therefore, there appears to be a direct relationship between the seed vigor differentiation and soaking volume.
Negative correlation coefficients were observed between electrical conductivity test and both germination and GVI, with values ranging from -0.59 to -0.88 (Table 2), and the most significant correlations were observed when soaking 50 seeds in 50 mL for 48 h (r = -0.86 and -0.84, respectively; Table 2).
Because the 25-mL soaking volume may hamper the measurement of electrical conductivity, depending on seed size and quantity, 50 mL is recommended as the optimum volume for measuring and comparing the electrical conductivity of M. brauna seed lots.

Figure 1
Figure 1 -Germination (a) and germination speed index (GSI) (b) of Melanoxylon brauna seeds of lots I and II, collected in the years 2010 and 2012, respectively.

Figure 2 -
Figure 2 -Electrical Conductivity (µs.cm -1 .g -1 ) of Melanoxylon brauna seeds of lots I and II, collected in the years 2010 and 2012, respectively, as a function of the soaking time (A), the number of seeds (B) and volume of water (C).

Table 2 -
Simple correlation coefficients (r)  between the mean values of germination (G) and germination speed index (GSI) and electrical conductivity varying the soaking time, the amount of water and seeds.at 5%, by the t-test.

Table 1 -
Values of electrical conductivity (µs.cm -1 .g -1 ) of Melanoxylon brauna seeds of lots 2010 and 2012 with changes in the quantities of seeds, amount of water and time of soaking.Means followed by the same uppercase letter un the column, for each imbibition time and seeds quantity, and by the same lowercase letter in the row, for each lot and distilled water volume, do not differ statistically by the Tukey´s test at 5%. *