Effect of storage conditions on seed quality of soybean (Glycine max L.) germplasm

1.   Introduction

Soybean is the world's leading legume ^[1] among oil seed crops and its cultivation is widespread in the tropical, sub-tropical and temperate region ^[2,3]. Soybean seeds are structurally weak, inherently short-lived and easily subjected to damage ^[4]. Seed storage life is influenced by the genotype as well as the storage conditions, mainly referring to temperature and duration ^[5]. Seed deterioration during storage is largely attributed to lipid peroxidation ^[6], subsequently affecting seed viability and storage longevity. Among factors influencing seed deterioration, temperature and seed moisture content are most important in terms of loss of viability during storage, ultimately affecting seed vigour and germination potential ^[7]. Most importantly, deterioration is an irreversible catabolic process that, once occurred, cannot be reversed. However, the seed potential storage life as well as the deterioration rate are greatly subjected to both species and genotype dependency. Soybean is generally viewed as a species of weak seed structure, short-lived and prone to mechanical damage, thus their quality being greatly depended on maturity stage which is related to their longevity during storage ^[8].

Overall seed longevity during storage is influenced by four major factors i) genotype, ii) seed quality at storage time, iii) seed moisture content and iv) temperature during storage ^[9]. Several studies related to the physiological quality of soybean seeds during storage for a period of six months under different temperature conditions point to the conclusion that air-conditioned environments positively affect the conservation of seed quality ^[10,11]. To determine the long-standing storage effects on seed quality, three individual tests were employed: i) seed germination test, ii) electric conductivity test and iii) measurement of the free fatty acids percentage. Such tests collectively provide an excellent means of assessing seed vigour during storage ^[12], especially in species whose seed longevity is subjected to strong genotypic dependency ^[13].

Germination is defined as the appearance of the first visible signs of growth or emergence of embryonic root, termed radicle. Germination is affected by several factors, including environmental conditions, mainly temperature and relative humidity ^[14]. The germination percentage has been widely employed as an indicator of deterioration in various grains during storage. According to Abba and Lovato ^[15], seed germination is adversely affected by high storage temperature, with the germinated fraction proportionally decreasing as temperature increases. Relative are the findings that popcorn seeds showed a trend of a reducing seedling emergence as exposure period to accelerated ageing increases ^[16], thus further reinforcing previous conclusions that the prolonged exposition period to accelerated ageing proportionally enhances deterioration rate ^[17].

Deterioration of grain refers to any degenerative changes occurring after the grain has reached its maximum quality, involving genetic damage, lipid peroxidation, loss of membrane integrity and cellular compartmentalization, selective reduction of capacity and solute leaching ^[18]. The sequel of cellular, chemical and metabolic alterations is primarily initiated by loss in cellular membrane integrity, thus the deterioration process of grains and seeds is usually associated with poorly structured membranes and damaged cells. Given that the value most affected by genetic factors is the seed's electric conductivity ^[19], referring to the amount of ions leached into the soaking solution of seeds, it has been proposed that its evaluation provides an accurate means of estimating germination ^[20]. As such, an increased value of electric conductivity is associated with a proportional decreased seed vigour as a result of loss of cell membrane integrity ^[17].

Given that free fatty acid content is a factor directly linked to seed vigor and viability, its estimation provides a significant qualitative indicator of seed deterioration during storage ^[21]. The free fatty acids percentage increases as the storage temperature is rising ^[22] and vegetable oils are often characterized by high free fatty acid content due to mechanical injury and/or sub-optimum storage conditions of grains or seeds ^[23]. High free fatty acid content has been associated with excessive losses in refining, while it has been further suggested that its increase contributes to seed deterioration via cell membrane disruption and/or peroxidation-mediated toxicity ^[24].

Seed ageing is directly associated with the seed moisture content and temperature, thus their manipulation provides a means of technically inducing the seed deterioration process. Under storage conditions, seeds typically lose their viability within a few days or weeks ^[25]. In this line, it has been evidenced that accelerated ageing causes a remarkable decrease in germination percentage ^{[26,27,28,29]} while, at the same time, ageing seed is characterized by loss of germination, reduced germination rate and poor seedling development ^[30,31]. In soybean, it has been confirmed that the accelerated ageing test provides the possibility of predicting the actual seed germination rate during natural ageing ^[32].

There are many circumstances under which it is important to predict the environmental effects on seeds' longevity, ranging from the rapidly occurring loss of viability due to the hot air-drying of wet seeds to slowly occurring deterioration as a result of long-term storage for genetic conservation purposes. Such equations allow for an accurate prediction of the expected percentage of seed viability formed at any given period of medium-term storage and any combination of temperature and moisture content ^[33,34].

Given that soybean seeds are unusually sensitive to storage conditions ^[35], this study aimed at investigating the effect of storage conditions and duration on seed quality and longevity of ten commercial soybean varieties, using the accelerated ageing technique.

2.   Materials and methods

Soybean germplasm consisted of ten soybean varieties, namely Adonai, Celina, Neoplanta, P21T45, PR91M10, PR92B63, PR92M22, PR92M35, Sphera and Zora. The genotypes were provided by the Institute of Industrial & Forage Crops, Larissa, Greece, while the experiment was conducted in the Laboratory of Plant Breeding, Department of Agriculture Crop Production and Rural Environment, University of Thessaly. A sample of 2 kg per variety was stored either at room temperature (18–22 ℃) or at cooling conditions (2–6 ℃) for a period of 12 months. In order to determine the seed storage behavior, based on the seed viability equation, seed samples from both storage conditions were imported in the accelerated ageing chamber (40 ℃) for a period of 48 days. During accelerated ageing, samples were removed at 3-days-intervals and the long-standing storage effects were determined on the basis of germination, electric conductivity, free fatty acid content and seed viability equation.

2.1.   Germination test

A sample of 200 seeds per variety was initially surface-sterilized in a 10% sodium hypochlorite/dH₂O solution, while gently mixing for 5 min, and washed (4x) with sterile dH₂O. Sterilized seeds were subsequently placed in plastic trays and incubated in a germination chamber (17 ℃). Germination scoring was performed every 2 days and, after counting, germinated seeds were removed. Scoring was continued until there were no seeds left or the remaining seeds were incapable of germination.

2.2.   Electric conductivity test

A sample of 50 seeds per variety were placed into a plastic glass filled with 75 mL of dH₂O ^[38] and covered with plastic membrane to avoid the entrance of dust. Following 24-hours incubation under shady conditions, the electric conductivity value was determined in μScm⁻¹g⁻¹, using a conductivity meter. The value of electric conductivity is gradually increasing as the germination percentage decreases ^[40].

2.3.   Free fatty acids

The free fatty acid content was estimated based on the AOCS Official Method Ca 5a-40 ^[36]. Such measurements were performed in collaboration with Bios Agrosystems.

2.4.   Viability equation (Ki)

The Ki value of each genotype was calculated using the formula created by Warren H.J. and Y.W. Wang, National Taiwan University. Based on Ellis and Roberts equation ^[41], seed survival curve was described as:

where v represents probit percentage viability, 1/σ represents seed survival curve, p represents storage period (days) and Ki represents probit percentage viability at the beginning of the storage.

The slope $\left( {}^{1}\!\!\diagup\!\!{}_{\sigma }\; \right)$ of the survival curves is not affected by the genotype or the seed quality, instead it is the intercept Ki of the survival curve that is affected by such factors ^[37]. A higher Ki value is generally related to an enhanced absolute longevity ^[36].

2.5.   Statistical analysis

The experimental layout was completely randomised, with four replications, each consisting of 50 seeds. All analyses were measured in four replicates and expressed as a mean. The differences between mean values were evaluated using Duncan's test at the level of significance p < 0.05. Hierarchical Cluster analysis was performed using Ward's method in order to explore the trends and relationships between genotypes. Principal component analysis (PCA) with varimax rotation was performed to explore relationships between traits. All statistical analyses were performed using SPSS statistical software v.20.

3.   Results

Traits most affected by storage duration were germination percentage and electric conductivity (Table 1). The storage conditions prior to the accelerated ageing, referring to either room temperature or cooling conditions, was the second most influencing factor in terms of effect on both germination percentage and electric conductivity. On the other hand, the percentage of free fatty acids was mostly affected by the genotype and the interaction between storage conditions and genotype.

3.1.   Germination test

Storage duration in the accelerated aging chamber, as expected, drastically affected germination percentage of seeds that were previously stored in both storage conditions. Seeds that were previously stored under room temperature conditions, showed no significant difference at 6, 9 and 12 aging days (Table 2). Accordingly, no significant differences were noted in seeds that were treated for 18 and 21 aging days, 21 and 24 aging days, 36 and 42 aging days. However, all aging treatments differed significantly from the control (0 aging days). Among varieties, Adonai was least affected compared to all other varieties, as evidenced both by the highest final germination percentage (48 aging days: 10, 5 %) and the highest mean germination percentage (57.1%). Seeds that were previously stored under cooling conditions, showed no significant difference at 0 (control), 6 and 9 aging days as well as at 21 and 24 aging days. At the variety level, Celina, Neoplanta and Adonai presented the highest final germination percentage (48 aging days), while Adonai showed also the highest mean germination percentage (76.7%), followed by Celina (74.7%) and Neoplanta (73%). In relation to the previous storage conditions, it was evidenced that a significant decrease in germination percentage was noted at 6 days and 12 days of aging for the seeds stored under room temperature and cooling conditions, respectively. Further, it was shown that the previous storage conditions affected differently the germination potential of all varieties under study. As such, the cooling storage conditions yielded a significantly higher germination percentage in seeds that were aged from 0 up to 24 aging days, and thereafter a drastic decrease in germination percentage was observed. To the contrary, seeds that were stored at room temperature showed a gradual decreasing trend as the aging treatment increased.

The mean germination percentage of seeds previously stored under room temperature and cooling conditions, within each variety tested, are presented in Table 3. Significant differences were noted in varieties Celina, Neoplanta and PR92M22, exhibiting a higher mean germination percentage upon storage at cooling conditions, whereas all other varieties did not differ significantly. Despite the absence of significant differences, overall data point to a general trend of increased mean germination percentage of seeds previously stored under cooling conditions as compared to the respective values of seeds stored at room temperature.

3.2.   Electric conductivity test

Our data are in accordance with the well-known negative association between germination percentage and electric conductivity, as reflected in the lowest electric conductivity value of the varieties Adonai and Celina after treatment of 48 aging days, which were previously characterized by the highest final germination percentage (48 aging days) in seeds that were stored at room temperature (Table 4). Such findings were further supported by the lowest mean electric conductivity value of Adonai and Celina. The negative association among the aforementioned values was also confirmed in seeds that were stored under cooling conditions. In this case, the varieties Adonai, Celina and Neoplanta, which presented the highest final germination percentages, showed the lowest electric conductivity values after treatment of 48 aging days, while the former two had also the lowest mean electric conductivity values (Table 4). Further, the findings point to a trend of increasing electric conductivity during storage. Seeds that were previously stored under cooling conditions had an electric conductivity value of at least 20 μScm⁻¹g⁻¹ lower than those stored at room temperature. Although this difference was preserved until 24 aging days, thereafter declined to the point of neutralization. The observed decreasing difference at 36, 42 and 48 aging days is in agreement with the decreasing difference in the respective values for germination percentage.

As expected, the percentage of free fatty acids (%) was lower in seeds that were stored at cooling conditions as compared to those stored at room temperature throughout the aging period. These findings are indicative of the fact that storage under cooling conditions enhances the longevity of seeds. At the variety level, Adonai variety showed the lowest free fatty acids percentage (Table 5) and, by extension, the lowest deterioration of seeds previously stored at both conditions under study.

3.3.   Viability equation (Ki values)

Viability equation (Ki values) indicates the storage potential of each variety for the two storage conditions under study. Storage under cooling conditions had a positive impact on the Ki value, thus enhancing the storage longevity, of all genotypes. At the variety level, Adonai had the highest Ki value in both storage conditions (Figure 1).

Overall data from germination percentage, electric conductivity and free fatty acids were subjected to cluster analysis, which revealed the classification of genotypes into 7 different clusters. As evidenced, genotypes Adonai, Celina and Neoplanta, which showed the highest mean germination percentage and the lowest mean electric conductivity and free fatty acids percentage after storage at cooling conditions, formed Cluster 2 (Figure 2). To the other end, genotypes PR91M10 and PR92B63, which were previously stored at room temperature and presented the lowest mean germination percentage and the highest mean electric conductivity and free fatty acids percentage, were classified into Cluster 3. Such findings indicate that genotypes belonging to Cluster 2 were the most tolerant to accelerated ageing, whereas those grouped into Cluster 3 were the most sensitive to accelerated ageing and, by extension, to storage. Moreover, overall data underline the significant positive correlation between electric conductivity and free fatty acids percentage as well as their significant negative correlation with germination percentage.

4.   Discussion

Soybean belongs to the species whose seed germination and vigour is lower compared to other grain crops, thus often being reduced prior to planting as a result of harsh environmental conditions. The loss in seed vigour is evident by delayed emergence, slow growth and ultimately decline in seed germinability ^[42]. Under these circumstances, the possibility to robustly predict the relative storability of different seed lots becomes of paramount importance from a scientific, industrial and practical viewpoint. To this direction, the accelerated ageing technique provides an accurate estimation of seed storage longevity, thus enabling rational decision making related to the seed lots which may be retained or should be directly disposed to the market. In this framework, the present study focused on investigating the effect of storage conditions on seed quality and storage longevity in soybean germplasm, consisting of ten commercial varieties.

It is well known that the extent of seed deterioration, caused by seed ageing during storage, strongly depends on storage conditions ^[43,44] as well as on seed genetic traits ^[45]. In our study, accelerated ageing adversely affected germination percentage and electric conductivity, with its effects being proportional to the duration of the accelerated ageing, while the free fatty acids percentage was mostly affected by the genotype. Such findings are in accordance with previous reports on loss of viability during accelerated ageing ^[30,31]. Worth mentioning is the fact that according to de Alencar et al. ^[49] at 40 ℃ the soybean seeds were classified as out of market after 45 days of accelerated ageing.

In relation to the effect of storage conditions, prior the seed entrance into accelerated ageing chamber, it was revealed that previous storage under cooling conditions was positively associated with a higher germination percentage in all genotypes. The superiority of storage under cooling conditions was further evidenced in seeds exposed to accelerated aging, as they retained a high germination percentage, compared to seeds previously stored at room temperature, thus prolonging storage longevity and delaying seed deterioration. Such findings further reinforce previous reports related to the fact that seed storage at room temperature enhances seed deterioration in soybean, whereas storage under cooling conditions favours seed viability ^[8,46,47,48]. Despite the observed differences relating to the previous storage conditions, differences were also noted in the response of genotypes to accelerated aging. As such, Adonai showed a relative superiority as evidenced by a higher germination percentage throughout the observation period as well as a higher mean germination percentage in seeds that were previously stored under both storage conditions. On the other hand, PR91M10 and PR92B63 exhibited the lowest germination percentage in seeds that were previously stored at room temperature and under cooling conditions, respectively.

In accordance with previous studies, our data point to a negative correlation between electric conductivity and germination percentage, thus indicating that electrolytic leaching is directly linked with germination loss ^[49]. As expected, electric conductivity increased over time ^[50], its value being further subjected to dependency on the temperature storage conditions though. Specifically, electric conductivity was consistently maximized in seeds that were previously stored under room temperature conditions, thus confirming its previously reported association with storage temperature ^[51]. Differences were further observed among genotypes, with Adonai and Celina showing the lowest mean electric conductivity in seeds that were stored at room temperature, while Adonai and Neoplanta presented the lowest values upon seed storage under cooling conditions. In contrast, PR92B63 presented the highest electric conductivity values in seeds that were previously stored under both storage conditions.

Another factor adversely affected by storage duration was free fatty acids percentage, which showed a gradual increase over time. In relation to free fatty acids, previous studies provide evidence that damage of grains or seeds, due to inappropriate storage practices, lead to their increased content in vegetable oils ^[23], the increase being positively related to storage temperature ^[46]. Relative in this manner are our findings that the percentage of free fatty acids was higher in seeds stored at room temperature as compared to those stored under cooling conditions. Among genotypes, Adonai and PR92M22 exhibited the lowest free fatty acid content in seeds that were stored at room temperature, whereas in the other group of seeds Adonai showed the lowest respective values. In contrast, PR92B63, as expected based on abovementioned findings, showed the highest free fatty acid content in seeds that were previously stored under both storage conditions.

To further examine the effects of storage conditions on seeds' longstanding viability, the viability equation was calculated. Overall data support the conclusion that storage longevity is significantly affected by storage conditions in all genotypes, with storage under cooling conditions giving rise to higher Ki values, thus indicating its positive effect on storage longevity via delaying seed deterioration. Such negative correlation between seed viability and storage temperature in soybean has been also previously reported ^[51]. Among genotypes, Adonai consistently presented a higher Ki value, under both storage conditions, providing strong evidence for its suitability for long-duration storage. Further supportive of this conclusion are the clusters defined on the datasets of germination percentage, electric conductivity and free fatty acid content for all genotypes, which place Adonai, along with Celina and Neoplanta, as the best performing cultivars in terms of retaining a relatively high germination potential under cooling seed storage conditions.

5.   Conclusions

Collectively, our findings underline that storage under cooling conditions set a ground for retaining a high germination percentage combined with low values of electric conductivity and free fatty acid content. More importantly, the superiority of such storage conditions was evidenced in artificially aged seeds which maintained their seed quality for longer storage period, due to a delay in seed deterioration processes. In relation to the comparative variety performance, Adonai, Celina and Neolplanta were classified as most superior cultivars, thus providing prospects for maintaining a high seed quality even under long-term storage conditions.

Conflict of interest

All authors declare no conflict of interest in this paper.