Effects of Pre-Stratification Storage Conditions on Black Walnut Seed Post-Stratification Germination Capacity

ABSTRACT

The objective of this work was to quantify changes in the germination capacity of Juglans nigra seeds following storage and then artificial stratification. Seeds were stored at 5 and –20 °C under 5% and 14% internal moisture content (IMC) for 0, 8, 10, and 12 months under conditions with oxygen or under vacuum. Germination percentage (GP) was affected by storage temperature, IMC, and storage period. Seeds stored with 5% IMC succeeded in germinating regardless of the temperature. Seeds stored with 14% IMC did not germinate (–20 °C) or germinated during storage (5 °C). The GP was reduced with the time of storage in seeds at –20 °C and 5% IMC and this effect was avoided in storage under vacuum. In conclusion, the germination capacity of black walnut seeds did not change for up to 12 months at low above-zero temperature and low IMC (5 °C, 5% IMC), being the most cost-effective alternative for long-term storage of black walnut seeds in commercial nurseries. Vacuum storage avoided the decrease of around 50% in average germination capacity of seeds stored for a year at –20 °C.

KEYWORDS:

• Internal moisture content
• Juglans nigra
• seed deterioration
• vacuum conditions
• fruit storage
• seed storage

Introduction

Black walnut (Juglans nigra L.), also called eastern black walnut and American walnut, is one of the most coveted hardwood tree species. Native to the deciduous forests of the eastern United States, its best known use is for its lumber. The wood is used for furniture of all kinds, cabinetwork, musical instruments, and gunstocks (Voulgaridis and Vassiliou, 2005; Williams, 1990). In Argentina it is mainly used as a rootstock for European walnut (Juglans regia), since it induces precocity to the grafted variety (Lemus, 1997) and confers tolerance to Phytophthora cinnamonii, root nematodes, Armillaria, and crown gall (Iannamico et al., 2006).

Black walnut seed production can be unsteady, with large seed crops occurring at irregular intervals followed by years of poor yield. Thus, storage of the seeds is of paramount importance to cover the annual needs of nurseries, since it ensures availability of seeds in years with insufficient seed production (MINAGRI, 2014).

Storage may be defined as the preservation of viable seeds from the time of collection until they are required for sowing (Holmes and Buszewicz, 1958). Inadequate storage causes seed deterioration, which results in lower seed quality, viability, and vigor (Kapoor et al., 2010). Changes during storage involve important seed physiological modifications in the cell tissues, such as the loss of food reserves, build-up of toxic metabolic by-products, aging of cell membranes, and nuclear DNA alterations (Copeland and McDonald, 2001; Palma et al., 2000; Rivera et al., 2007; Zhou et al., 2009). Most oil-bearing seeds like walnut and cashew are very susceptible to microbial attack as a result of improper long-term storage. Microbial attack leads to hydrolysis of the oil in the seed and hence to the formation of free fatty acids, which are responsible for the rancid flavor/odor and seed deterioration (Onilude et al., 2010). These authors identified eight fungal species and three bacteria with lipase activity, which was enhanced when cashew nuts were stored in an atmosphere with >30% relative humidity.

According to their behavior during storage, seeds are classified into orthodox and recalcitrant (Roberts, 1973). Orthodox seeds are those that can be dried down to a low internal moisture content (IMC) of around 5% (wet basis) and successfully stored at low or sub-freezing temperatures for long periods without seed injury (FAO, 1991). Recalcitrant seeds, on the other hand, are those that cannot survive drying below relatively high moisture content (often in the range of 15–50%, wet basis). Intolerance to desiccation is an important agronomic disadvantage because it makes long-term seed conservation difficult (FAO, 1991; Farrant et al., 1993; Gentil, 2001).

The last stage in the ripening of orthodox seeds involves an extensive cell desiccation, which starts with the disruption of the vascular connection between the mother plant and the seed (Bewley and Black, 1994; Kermode and Finch-Savage, 2002). During this period seeds acquire the desiccation tolerance, which improves their viability and storage potential (Hoekstra et al., 1994; Nkang, 2002). The decrease in the seed’s IMC reduces the respiration rate and triggers several other biochemical processes, which protect the organelles and the cell walls (Walters et al., 2005), delaying seed aging and extending viability (Pérez Camacho et al., 2012). Recalcitrant seeds, on the other hand, do not undergo dehydration while on the mother plant, and must be sown immediately after harvest, or artificially stratified over winter before sowing in the spring, to maintain their viability (Farrant et al., 1993).

Regardless of the cause of deterioration, viability of orthodox seeds can be prolonged by reducing their respiration rate during storage (Pérez Camacho et al., 2012), either by lowering the temperature, the IMC, or storing in sealed containers (FAO, 2013). In the latter case, respiration decreases oxygen concentration around the seed. However, a minimum threshold of respiratory rate is necessary to support the multiple cell changes that occur during stratification and that are correlated with the breaking of seed dormancy, and consequently with seed germination and seedling growth (Hopper et al., 1985). These changes involved a reduction in stored protein and lipids and an increased number of mitochondria and endoplasmic reticulum in the hypocotyl cells of black walnut embryos (Somers and Van Sambeek, 2003). If these changes do not occur during seed storage, e.g., by the absence of oxygen, seed can remain viable for a long time but will require additional stratification (Williams, 1971).

The objective of this work was to quantify the changes in the germination capacity of black walnut seeds under different pre-stratification storage conditions in order to improve the time of conservation while maintaining their post-stratification germination capacity.

Materials and methods

Fruits were obtained from a 50-year-old J. nigra stand at the J.F. Villarino Experimental Field of the Facultad de Ciencias Agrarias, Universidad Nacional de Rosario, located in Zavalla (33° 01′ S, 60° 53′ W), Santa Fe province, Argentina. Fruits were collected following the FAO recommendations (FAO, 1991) from at least 15 trees after natural fruit drop, and then mixed to form a single sample. Fruits were husked and washed over sieves, then immediately soaked in water to remove floaters, usually non-viable seeds (Flores et al., 2013).

The first experiment was performed following a randomized complete block design with a factorial arrangement and four 50-seed replications per treatment. The three factors assessed were storage temperature (with two levels, 5 and –20 °C); internal moisture content (IMC) (two levels, 5% and 14%); and storage period (three levels, 8, 10, and 12 months). To lower the IMC of freshly collected seeds (~20%) down to the IMC of each treatment, seeds were exposed to the air and frequently shaken to promote uniform drying (Turnbull, 1975). IMC was determined by change in weight. Seeds that were to be stored at 5 °C were placed in 3-L glass jars filled to the brim and sealed (FAO, 1991). The jars were placed on shelves in the storage chamber at 5 ± 1 °C. Seeds for storage at –20 °C were placed in paper bags, then in coextruded heat shrinkable polyethylene bags (50 × 25 cm) to avoid moisture loss.

The second experiment was performed following a completely randomized block design with a factorial arrangement, with four 50-seed replications for each treatment. Seed were stored at –20 °C and 5% IMC and the two factors assessed were the effect of the storage atmosphere (two levels, vacuum and no vacuum), and storage period (three levels, 8, 10, and 12 months). For vacuum treatments, bags were sealed with an ST 120 sealer equipped with a Busch vacuum pump, whereas in treatments with no vacuum, polyethylene bags were sealed with adhesive tape. In both experiments, seeds were stratified in moist sand at a constant temperature (5 °C) for 4 months after the storage treatments to overcome physiological dormancy (Baskin and Baskin, 1998). Control seeds were stratified immediately after collection. After stratification, seeds were tested with the standard germination test for 28 days (ISTA, 2003). The results were expressed as percentage of normal seedlings, and those with stubby primary root, shortened epicotyl, primary root missing, and damaged epicotyl were considered as abnormal. The variables analyzed in both experiments were germination percentage (GP), germination velocity index (GVI), mean time to maximum germination (MTMG), percentage of normal seedlings (%NS), and mean dry weight of normal seedlings (DWNS). GVI represents the percentage of the hypothetical maximum germination velocity that would be reached if all seeds germinated within the first days of sowing (Silva and Nakagawa, 1995). MTMG expresses the average time needed to reach maximum germination (Edmond and Drapala, 1958). To determine mean dry weight, seedlings with their cotyledons removed were oven dried at 80 °C for 2 h, and weighed on a precision scale (AOSA, 2002; Nakagawa, 1994).

Statistical analysis was performed with Infostat software. Data were analyzed with analysis of variance (ANOVA) and means were compared using the Tukey test at the 5% probability level. Normality and homoscedasticity were tested graphically (Q-Q plot, and residuals versus predictors plot, respectively).

Results

In the first experiment, GP was affected by storage temperature (p < 0.0001), IMC (p < 0.0001), and storage period (p = 0.001). Interactions between temperature and storage period (p = 0.0002), temperature and IMC (p < 0.0001), and storage time and IMC (p = 0.001) were also observed.

Control seeds, which were stratified immediately after collection, had an average 87% GP. IMC was the variable with the greatest influence on the GP during storage. Seeds stored with low IMC reached 74% GP on average. On the other hand, seeds kept at 5 °C and high IMC started to germinate inside the jars after 3 months, and were deteriorated and covered with fungi at the end of the 8–12 months storage periods evaluated in this experiment. Thus, the GP of seeds at 5 °C and high IMC could not be determined (seeds germinated during storage) (Figure 1a).

Figure 1. Influence of pre-stratification storage at different internal moisture content (IMC) (a), storage temperature (b), and storage time (c), on the post-stratification germination capacity of black walnut seeds. Seeds were stratified for 4 months after each storage treatment. Control seeds reached 87.8% of germination. Data of high IMC treatments was calculated only from seeds stored at –20 °C since values from those stored at 5 °C could not be determined because germination occurred during storage. Columns with different letters indicate that the mean value significantly differs according to the Tukey’s test (p ≤ 0.05).

Storage temperature also affected GP, which dropped by 65% when storage temperature was lowered from 5 °C (87%) to –20 °C (30%) on average (Figure 1b). In relation with the variable storage period, GP decreased by 8% on average when the storage time was increased from 10 to 12 months (Figure 1c).

Storage conditions at 5 °C and low IMC, followed by 4 months of stratification, proved best, reaching a germination percentage near 90% for all of the storage periods evaluated (8 to 12 months) (Table 1). This value did not show significant differences in comparison with control seeds. On the other hand, seeds stored at –20 °C and low IMC reached 60.5% of GP on average, which was 30.8% lower in comparison with the GP of seeds stored at 5 °C and low IMC. Also, at –20 °C storage temperature, germination decreased sharply (–31.7%) when the storage period was increased from 10 to 12 months. This effect was not observed in seeds kept at 5 °C and low IMC, which explains the interaction between temperature and storage period (Table 1).

Table 1. Effects of the time of storage on the germination (%) of black walnut seeds kept under different temperature, internal moisture content (IMC), and vacuum during storage.^z

	Storage time (months)
Treatments	8	10	12
First experiment	Germination (%)
5 °C, Low IMC^y	88.38a^v	90.63a	83.5a
5 °C, High IMC^x,w	—	—	—
–20 °C, Low IMC	68.13b	66.88b	46.50c
–20 °C, High IMC	0.00d	0.00d	0.00d
Second experiment
–20 °C, Low IMC, vacuum	90.00a	88.50a	87.50a
–20 °C, Low IMC, no-vacuum	71.50b	68.50b	41.50c
Control (non-stored seeds)		87.8a

^zSeeds were stratified for 4 months after each treatment and control seeds were stratified immediately after harvest.

^yLow IMC = 5%.

^xHigh IMC = 14%.

^wData of 5 °C and high IMC treatment could not be determined because seed germinated during storage.

^vMeans followed by different letters in the same column and line of each experiment significantly differ according to Tukey’s test (p ≤ 0.05).

Similarly, the lower GP observed in seeds kept at –20 °C and low IMC as compared to seeds stored at 5 °C and low IMC explains the interaction between temperature and IMC (Table 1). Seeds kept at –20 °C and high IMC failed to germinate, regardless of storage period. However, seeds in these treatments, unlike those kept at 5 °C and high IMC, were not deteriorated at the end of the storage time, although they were covered by an ice layer. Therefore, only seeds stored with a low IMC succeeded in germinating after the storage treatments (8, 10, and 12 months), regardless of the storage temperature (Table 1).

The other variables analyzed, namely GVI, MTMG, %NS, and DWNS, were also affected by storage temperature, storage period, and IMC. The effects and interactions were similar to those found for the GP (Table 2). The best result for these variables was obtained in seeds kept at 5 °C and low IMC. Under these storage conditions, GVI was as high as 21% and MTMG was 5 days on average. Seeds stored at –20 °C and low IMC decreased their germination velocity by 48%, reaching a GVI of 11% and a MTMG of 10 days on average. These variables were not assessed in treatments with high IMC, since seeds either germinated in the jars during storage (5 °C) or failed to germinate (–20 °C).

Table 2. Influence of storage temperature, internal moisture content (IMC), and vacuum for different periods of storage (months) on germination velocity index (GVI), mean time of maximum germination (MTMG), percentage of normal seedlings (NS), and mean dry weight of normal seedlings (DWNS) of black walnut seeds.^z

	GVI (%)			MTMG (days)			NS (%)			DWNS (mg)
	Storage time (months)
Variable	8	10	12	8	10	12	8	10	12	8	10	12
First experiment
5 °C, Low IMC	22.1a^x	21.8a	19.6a	3.4a	4.1a	5.9ab	75.6ab	78.7a	71.2ab	618a	608a	584a
5 °C, High IMC^y	—	—	—	—	—	—	—	—	—	—	—	—
–20 °C, Low IMC	13.0b	12.5b	7.7c	8.22b	8.6b	13.5c	59.9c	57.3c	39.8d	526b	495b	433c
–20 °C, High IMC	0.0d	0.0d	0.0d	0.0d	0.0d	0.0d	0.0d	0.0d	0.0d	0d	0d	0d
Second experiment
–20 °C, Low IMC, vacuum	19.7a	22.1a	20.9a	5.2a	4.5a	4.7a	77.0a	76.1a	74.1ab	600a	607a	600a
–20 °C, Low IMC, no-vacuum	13.5b	12.9b	6.9b	7.6b	8.2b	14.6c	63.0b	58.01bc	35.0c	539b	498b	417c
Control (non-stored seeds)	19.6a			5.0a			81.2a			599ª

^zSeeds were stratified for 4 months after each storage time treatments. In the control, seeds were stratified immediately after harvest.

^yData of 5 °C and high IMC treatment could not be determined because seed germinated during storage.

^xMeans followed by different letters in the same column and file for each variable significantly differ according to Tukey’s test (p ≤ 0.05).

The %NS of seeds stored at 5 °C and low IMC was near 75% regardless of storage period. This value decreased by 30% in seeds stored at –20 °C and low IMC (52% on average). In seeds kept at –20 °C, a marked decrease (34%) in %NS occurred when the storage period was increased from 10 to 12 months, showing an interaction between temperature and storage time (Table 2).

DWNS from seeds stored at 5 °C and low IMC was 603 mg on average, a value that did not differ from that of seedlings obtained from control seeds (599 mg). This value decreased by approximately 20% when the storage temperature was lowered from 5 °C to –20 °C, and a further 17% when the storage period at –20 °C was increased from 8 to 12 months (Table 2).

In the second experiment, GP was affected by storage conditions and by storage period. Also, there was an interaction between both variables (p < 0.0001) (Table 1). The GP of seeds stored at –20 °C under vacuum conditions was 32% higher on average than the GP of seeds stored without vacuum. Thus, vacuum treatments allowed seeds stored at –20 °C to reach germination percentages (>87.5%) similar to those of non-stored (control) seeds (87.8%), preventing their deterioration even under the longest storage period evaluated (12 months). Vacuum also prevented the decline in the GP observed in seed kept without vacuum, mainly when the storage time increased from 10 to 12 months. This explains the interaction observed between the storage atmosphere and storage time variables (Table 1).

GVI, MTMG, %NS, and DWNS, were also affected by vacuum conditions. The best results for these variables were obtained with –20 °C and vacuum, regardless of storage time. The GVI increased 89% by vacuum, whereas the TMG decreased by 50%. Vacuum also increased the %NS (+44%) and the DWNS (+24%) (Table 2).

Discussion

Loss of seed viability during storage is largely governed by their respiration rate. Thus, any method that reduces the rate of respiration without otherwise damaging the seed generally results in longer seed viability (Pérez Camacho et al., 2012). The usual methods for decreasing the seeds’ respiration rate include lowering the internal moisture content as well as the temperature and oxygen concentration in the storage atmosphere (Copeland and McDonald, 2001; FAO, 1991; Melgoza et al., 2003; Ruiz et al., 2008). However, the most widely used technique is the reduction of the internal moisture content because it is the most cost effective (Pérez Camacho et al., 2012; Walters et al., 2005). Williams (1971) was able to store black walnut seed in outdoor stratification pits (90–120 cm deep) for as long as 4 years (40% germination) compared with 2 years in cold chamber both under dry and moist conditions (15% germination). In the mentioned experiment there was no subsequent seed stratification, so the germination capacity of those treatments that cause a sharp decrease in the seed metabolic activity during storage could be underestimated. The progressive morphological, anatomical, physiological, and hormonal changes that occurred during stratification and that were necessary for normal seedling germination could be limited in treatments with a minimal metabolic activity during storage (Somers and Van Sambeek, 2003). Instead, in our work all treatments included pre-stratification with the objective to retain seed viability followed by stratification to determine their germination capacity.

Our results confirm that IMC was the most important storage factor in extending seed viability. Seeds stored with low IMC (5%) presented high germination percentage, faster germination, and greater proportion of normal seedlings regardless of the storage temperature. However, it is important to point out that no high-temperature storage treatments were included in this study since the two temperatures evaluated (5 and –20 °C) are able to restrict or virtually stop seed metabolism (Harrington, 1972).

Since seeds kept at high IMC (14%) and low temperatures overcame dormancy and germinated inside the jars, storage under these conditions is suitable only for short storage periods and for tree and shrub species with dormant seeds (FAO, 1991; Hong and Ellis, 1992). Under these conditions, storage time should be similar to the period required by seeds to overcome their physiological dormancy (Baskin and Baskin, 1998), which in black walnut should not exceed 4 months (Flores, 2013; Van Sambeek et al., 1989).

Another result that emerged from our data was that black walnut seeds were able to overcome seed dormancy without the requirement of placing them on a moist substrate, and that their own internal moisture content was sufficient when its value was 14%. Consequently, storage under low temperature (5 °C) and high IMC (14%) conditions replaced the requirement of seed stratification because the storage conditions were similar in both cases (Hong and Ellis, 1992).

Seeds stored at freezing temperatures (–20 °C) and high IMC were also non-viable at the end of the 8–12 month periods of storage, but in this case the more likely cause of seed death was the formation of ice crystals inside the cells, which causes a direct damage on membrane and nucleic acid structures (Levitt, 1980). In contrast, ice formation does not occur at low IMC conditions because dehydration is a strategy of seeds to avoid ice formation and chilling injury (DeVries et al., 2007; Woltz et al., 2005). This explains the large differences in chilling tolerance found in seeds with different IMC levels when they were stored at –20 °C.

In the second experiment, it was demonstrated that the exclusion of oxygen, when seeds were stored at low IMC and freezing temperatures, allowed obtaining a GP similar to that of non-stored seeds (control), avoiding a declination of around 50% in the main seed’s quality attributes after a year of storage. Seed deterioration involves the oxidation of lipids, cell and mitochondrial membranes, DNA, and RNA (Bailly, 2004; Osborne, 1994; Rajjou and Debeaujon, 2008; Rajjou et al., 2008), which points to the key role of oxygen in seed deterioration during storage (Groot et al., 2012). In addition, it has been reported that the increase in oxygen levels during storage triggers chromosome aberrations during cell division (Abdalla and Roberts, 1968). Even though the detrimental effect of the presence of oxygen during storage is well established, its role has hardly been considered in the practice of commercial or gene-bank seed storage (Groot et al., 2012), and only low moisture content and low temperatures during storage have long been considered as the key factors in prolonging seed viability (Cromarty et al., 1982). Despite the adequate behavior of black walnut seeds at –20 °C, desiccated orthodox seeds should be stored in the range of –5 °C and +5 °C in order to save a considerable amount of energy (Pérez-García et al., 2007).

In conclusion, our research showed that the germination capacity, GP, GVI, MTMG, %NS, and the DWNS, of black walnut seeds did not change for up to 12 months of storage at low above-zero temperature (5 °C) and low IMC (5%). Furthermore, vacuum avoided the decrease of around 50% in average of the germination capacity of seeds stored for a year at –20 °C and low IMC.

Acknowledgments

The authors thank Gabriela Venturi for her assistance in writing the manuscript in English, and Jerry Van Sambeek for his assistance in the discussion of results.

Funding

This work was principally supported by the Universidad Nacional de Rosario and the Universidad Nacional del Litoral (CAI+D 50120110100002 LI).

Additional information

Funding

This work was principally supported by the Universidad Nacional de Rosario and the Universidad Nacional del Litoral (CAI+D 50120110100002 LI).