ELECTRICAL CONDUCTIVITY AS AN INDICATOR OF PEA SEED AGING OF STORED AT DIFFERENT TEMPERATURES

Research on soybean seed has revealed that conductivity test results may be influenced by storage temperature and that an apparent direct relationship between deterioration at low temperatures and loss of cellular membrane integrity is not evident. The objective of this study was to verify the influence of low storage temperatures on electrical conductivity results in other species, such as seeds of two pea cultivars studied temperatures were: 10, 20, 25, 20/10 and 25/10oC. The physiological potential of the seeds was evaluated every three months during an 18 month storage period using germination, accelerated aging and electrical conductivity tests. The electrical conductivity test was not a good indicator for the degree of seed deterioration stored at low temperatures. Seed deterioration at 10oC did not seem to be directly related to the loss of membrane integrity, possibly due to membrane repair or reorganization during storage at this temperature.


INTRODUCTION
Organization of cellular membranes is at its peak by the time a seed reaches physiological maturity (Abdul-Baki, 1980).Seeds undergo a structural disorganization process during the drying period before harvest; the lower the water content, the greater the disorganization.As a result seeds temporarily lose their integrity (Bewley & Black, 1994).During the initial phase of the soaking process, the capacity of a seed to reorga-nize the cellular membrane system and repair physical and/or biological damage that may have occurred, will influence the quantity and nature of the lixiviates released into the environment (Simon & Raja Harun, 1972;Bewley & Black, 1994;Vieira & Krzyzanowski, 1999).Thus, seeds with less physiological potential as a result of the deteriorative process have a reduced capacity for membrane reorganization and a greater loss of solutes to the environment, resulting in decreased seed reserves and reduced germination uniformity and speed.Soaking liberates sugars, amino acids, fatty acids, enzymes and ions in quantities varying according to the organizational state of the cellular membrane systems (Simon & Raja Harun, 1972;Short & Lacy, 1976;Givelberg et al., 1984;Woodstock, 1988;Bewley & Black, 1994;Taylor et al., 1995).Release of solutes and water absorption are very high at the beginning of the soaking process; however, the rate at which the membranes reorganize decreases with seed age (Abdel Samad & Pearce, 1978;Simon & Mathavan, 1986).
Electrical conductivity was proposed as a test to determine the physiological potential of seeds.Its objective was to indirectly evaluate the degree of cellular membrane damage resulting from seed deterioration, by means of determining the quantity of lixiviated ions in a solution with a fixed volume of deionized water, in which a known number of seeds are soaked at a controlled temperature for a pre-established period of time.Seeds with lower physiological potential liberate a greater amount of electrolytes as a consequence of lower cellular membrane selectivity.Decreased physiological potential and seed germination are directly related to increased quantities of lixiviated solutes resulting from a loss of membrane integrity (Loeffler et al., 1988;Marcos-Filho et al., 1990 andVieira et al., 1999).
The electrical conductivity test has been recommended to evaluate seed vigor for peas.It has also been suggested for soybean seeds (Hampton & TeKrony, 1995).Despite specific recommendations for these two species, the test has been studied for use with other crops due to its simplicity in execution and rapid response (Vieira & Krzyzanowski, 1999).Although the electrical conductivity test is often done on a routine basis in seed laboratories, the technique has been applied in only a few studies to estimate the effect of storage on seed vigor.Some studies have indicated that electrical conductivity results may be influenced by storage temperature especially the lower ones.Ferguson (1988) observed that soybean seeds stored at 10ºC (temperature normally utilized in a cold chamber) had a reduced physiological potential when evaluated by germination and accelerated aging tests; however, this reduction was not detected by the electrical conductivity test.
With the objective of investigating the effect of storage temperature on seed deterioration in soybean, Vieira et al. (2001) evaluated six seed lots with distinct physiological potentials.They observed that at 10ºC, germination obtained after accelerated aging was reduced for all stored lots, while electrical conductivity data did not vary during the same period.
Seed samples stored at 20ºC presented rapidly decreasing values for standard germination and accelerated aging tests, accompanied by increased electrical conductivity values.
When seed samples were transferred from 20ºC to 10ºC after six months storage, accelerated aging test results showed that there was a continuing decline in vigor but that the electrical conductivity values remained stable.This fact led the authors to question the use of electrical conductivity as an indicator of deterioration and seed vigor in soybean after storage at low temperatures.In a more recent study, Fessel (2001) sought to confirm the influence of storage at 10ºC on electrical conductivity results and verified that this test was also not indicated for evaluating seed vigor in soybean under cold storage.On the other hand, seed deterioration at 10ºC does not seem to be directly related to loss of membrane integrity, which may be due to its repair or reorganization during storage at lower temperatures.
Although there are several studies on the effect of storage temperature (10ºC in particular) on electrical conductivity test results in soybean, more detailed studies are needed for other species, especially to verify the influence of low storage temperatures.Consequently, the present research study was carried out on pea seeds with the objective of studying the effect of different storage temperatures on electrical conductivity results.The choice of this species is justified by the fact that this test is already considered to be sufficiently standardized for pea (Hampton & TeKrony, 1995).

MATERIAL AND METHODS
The study was made between November 2003 and July 2005 in Jaboticabal, SP, Brazil.Lots with distinct physiological potentials (one superior and one inferior) were selected from the pea cultivars DILETA and MARINA (Table 1).
Seed water content was initially adjusted around 12% using the following procedures: a) Drying (when initial water content was greater than 12%): each sample was spread on a polystyrene tray and placed in a dryer with forced air circulation at a temperature varying between 30 and 33 o C; b) Moistening (when initial water content was less than 12%): a single layer of seeds was laid on an internal screen inside adapted plastic boxes (26.0 × 16.0 × 8.5 cm), filled with 500 mL water, covered and maintained in an incubator at 20 o C. Seed water content was monitored during artificial drying and moistening by successive weighing until the desired level was obtained.As soon as the desired water content was reached, each sample was transferred to an aluminum container, hermetically closed and duly identified.
Samples were stored at the following temperatures: a) 10ºC (constant); b) 20ºC (constant); c) 25ºC (constant); d) 20ºC for six months then 10ºC until the end of storage; e) 25ºC for six months then 10ºC until the end of storage.The use of the latter seeks to verify if seeds stored at a higher temperature maintain their physiological quality or delay their deterioration process when transferred to an environment at lower temperatures.
The following tests were used to evaluate the pea seeds every three months during the 18 month storage period: Germination -four replications of 50 seeds of each lot were sown in rolled paper towels (Germitest, specifically made for germination) moistened with a quantity of water equal to 2.5 times the weight of the substrate, and incubated at 20ºC.Evaluations took place five and eight days after sowing, following the criteria established by the Rules for Seed Analysis (Brasil, 1992); Accelerated Agingconducted inside plastic boxes (11.0 × 11.0 × 3.5 cm) as individual compartments (mini-chambers) with an aluminum mesh screen on which seeds were distributed in a single layer, and 40 mL deionized water was added to each individual compartment.The boxes were then covered and maintained in an aging chamber ("water jacketed" model) at 42ºC for 48 h (Caliari & Marcos-Filho, 1990).After aging, the seeds were submitted to a germination test.Evaluation took place five days after sowing by calculating the percentage of normal seedlings; Bulk electrical conductivityfifty seeds of each replication were weighed (0.01 g precision) and soaked in 500 mL plastic cups contain-ing 250 mL deionized water, for 24 h at 20ºC (Hampton & TeKrony, 1995).Afterwards, the electrical conductivity of the solution was determined through reading in a conductivimeter (DIGIMED DM-31) and the average values obtained for each lot were expressed as µS cm -1 g -1 .
Trials were set up in a randomized complete design with four replications per treatment.Results were submitted to an analysis of variance and means were compared by a Tukey test at a 5%.Correlation analyses were established between storage periods (months) and the results of each test.

RESULTS AND DISCUSSION
Germination of DILETA (Figure 1) and MA-RINA (Figure 2) pea cultivars decreased over the storage periods (3, 6, 9, 12, 15 and 18 months) and storage temperatures (10, 20, 25, 20/10 and 25/10ºC).The higher the storage temperature, the lower was the physiological quality of the seeds.Transfer to a lower temperature after six months storage at 20/10ºC and 25/10ºC decreased germination further.This behavior was observed in seeds with superior or inferior physiological potential of both cultivars.
Accelerated aging reduced seed germination in all treatments (Figures 1 and 2).Decrease in germination was accentuated by the higher temperature treatments.In the case of storage at 20/10ºC and 25/ 10ºC, transference to the lower temperature (10ºC) after six months did not delay the seed deterioration process.
Storage at 10ºC did not have any effect on electrical conductivity test results through the storage periods (3, 6, 9, 12, 15 and 18 months).Contrary to results of the germination and accelerated aging tests, electrical conductivity apparently indicated that seeds maintained their physiological quality when stored at the lowers temperature (10ºC).The higher temperature treatments (20 and 25ºC) resulted in changes in electrical conductivity for all storage periods, revealing increased loss of lixiviates over time.In the above test, liberation of a greater quantity of exudates to the soaking solution was related to seeds with lower physiological potential, revealing greater intensity of disorganization of the cellular membrane systems.
Treatments in which seeds were transferred to 10ºC after six months at either 20ºC or 25 resulted in lower electrical conductivity readings after nine or more months storage as compared to treatments in which seeds were stored at a constant temperature, i.e., 20ºC and 25ºC.However, this transference not result in delayed seed deterioration; electrical conductivity values increased through the evaluated periods.The data obtained with pea seeds stored at 10ºC agree with those found in the literature for soybean (Ferguson, 1988;Vieira et al., 2001).This indicates that the release of ions by seeds may be influenced by the storage temperature; consequently, this test may not be the best indicator for the degree of deterioration for seeds stored at low temperatures.
Seed hydration allows the cellular membrane structure to be reorganized, thereby reducing permeability and consequently the liberation of lixiviates (Bewley & Black, 1994).On the other hand, results of the present study suggest that, during imbibition, membranes are also reestablished in pea seeds when stored at low temperatures, since no increase was observed in electrical conductivity values over time.

CONCLUSIONS
The electrical conductivity test was not a good indicator for the intensity of deterioration of pea seeds stored at low temperatures.The accelerated aging test was more sensitive in detecting seed deterioration at lower storage temperatures than electrical conductivity.Seed deterioration at 10ºC appeared not to be directly related to loss of membrane integrity.

Table 1 -
Initial water content (WC) of the seeds, standard germination (SG), accelerated aging (AA) and electrical conductivity (EC) of pea seeds of the cultivars DILETA and MARINA.