Physical, biochemical, and Physiological changes on oil palm (Elaeis guineensis L. Jacq) seeds throughout short-term storage

Seed deterioration is an inexorable process, including on oil palm (Elaeis guineensis L. Jacq) seeds which are categorized as intermediate seed type. Seed deterioration rate can be predicted through physical, biochemical, and physiological parameters. This experiment aimed to study physical, biochemical, and physiological changes in oil palm seeds in short-term storage. The experiment was conducted in a 2-factors of nested design. The first factor was storage temperature: open storage (24.2 − 30.5°C; RH 48.6 − 82%) and controlled storage (22.0 − 27.7°C; RH 43.9 − 82.1%). The second factor was storage packaging: single polyethylene (PE) plastic and double PE plastic, which was nested to the first factor. Oil palm seeds of Dura (D) x Pisifera (P) variety DxP PPKS 540 were obtained from the Marihat Business Unit of IOPRI. Results showed that during 6 months of storage, the seed moisture content (SMC) decreased in all treatments, even though the final SMC (11.2-11.4%) was still at a safe level for oil palm seed storage. Significant changes on fatty acid compositions of the oil palm endosperm were not found. Seeds could be stored for 3 months under controlled storage without a significant reduction in the germination percentage compared to those of fresh seeds, while open storage significantly reduced germination 2 months after storage. Germination percentage was not correlated with fatty acid content in the endosperm. The highest germination speed index was detected 2 months after storage in both storage conditions.


Introduction
Based on their storage type, seeds are categorized into three groups, i.e. recalcitrant, intermediate, and orthodox types [1].Seed deterioration rate, especially during storage, is determined by the type of seed.
Ellis et al.
[2] classified oil palm seed as an intermediate seed type.In more detail, Norziha et al.
[3] stated that water content of oil palm seeds can be reduced to 10% and stored at low temperatures.However, storing oil palm seeds for a long period can reduce seed moisture content (SMC) lower than the threshold mentioned and thereby affecting the viability of the seeds.Research by Martine et al. [4] showed that after 6 months of storage in an air-controlled room, the SMC of oil palm seed decreased from 15.33%, which was the SMC of fresh seeds, to 9.31%.To maintain the SMC of oil palm seed during storage, Arif and Sihombing [5] utilized polyethylene (PE) plastic bags which utilization slowed down the declining rate of oil palm SMC.However, the SMC movement during seed storage was not associated with seed viability changes.
A decrease in seed viability during storage is also caused by high lipid content in the seed endosperm, which functions as a seed food reserve.Sehgal and Sharma [6] stated that oil palm kernel contains 32.6-49.4% lipids, 7.5-10.9%protein, and 33.4-35.1% carbohydrates, showing that lipids as major component in the endosperm.Faster seed deterioration can occur due to oxidation of the contained lipids.This research aimed to study physical, biochemical and physiological changes that occur in oil palm seeds during short-term storage, both under controlled and open storage conditions.

Time and place
The experiment was carried out from July 2020 to June 2021 in the oil palm seed storage room of the Plant Breeding and Biotechnology Research Group and the seed processing unit (SPU) of the Seed Production Division.The two facilities are units of the Indonesian Oil Palm Research Institute (IOPRI), North Sumatra, Indonesia.

Plant material
Plant materials utilised were oil palm Dura (D) x Pisifera (P) seeds, variety DxP PPKS 540, obtained from the Marihat Business Unit of IOPRI.Seeds for the experiment were derived from bunches that were harvested between 5.0 and 5.5 months after pollination.

Experimental design
The experiment was conducted utilizing randomized complete block design (RCBD) with nested design.The major factor was storage condition, which determined the microclimate condition of the storage room, with two levels i.e. controlled and open storage.In the controlled storage, the experimental units were placed in a room with air conditioners (AC), with a temperature range between 22.0 and 27.7 o C (22.8 o C average) and relative humidity (RH) ranging from 43.9 to 82.1% (64.6% average).Open storage was carried out in an ambient room condition with a temperature range between 24.2 and 30.5 o C (27.4 o C average) and RH ranging from 48.6 to 82% (73.2% average).The temperature and RH were measured by Elitech digital temperature and humidity data logger type GSP-6.The nested factor was seed packaging with two levels i.e. single polyethylene (PE) plastic bags and double PE plastic bags, which was nested into the storage condition.The PE plastic bags used were 60 x 30 cm with a thickness of 0.2 mm.Analysis of the variance of obtained data was carried out using SPSS ver.18.0 (IBM) to determine the effect of treatment on the observed parameters.If the analysis of variance shows that there is a significant effect on the treatment, data analysis was continued with Duncan's Multiple Range Test (DMRT) at 5% level to compare the treatment mean values and to find statistically different treatment levels.

Dormancy breaking and germination
Every month for 5 months of storage, 100 seeds from every experimental unit were randomly taken for germination process, while the remaining seeds were stored in the seed storage room according to the storage condition and seed packaging as described above.Seed dormancy breaking was carried out using the dry heat method [7], which process was started by soaking the oil palm seeds for 5-7 days, and drying them after.Then the seeds were placed in a hot room which air temperatures ranging between 38 and 40 o C for 60 days.After that, the seeds were soaked for a second time for 3 days to increase the SMC to 21-23%.Finally, the seeds were placed in a germination room with ambient temperatures between 26-28 o C. Observation on seed germination began on the 14 th day after the seeds were placed in the germination room up until day 95.
IOP Publishing doi:10.1088/1755-1315/1308/1/0120123 2.5.Observations For physical changes, observations were conducted on variables (1) SMC which was performed weekly, and (2) electrical conductivity (EC) which was done fortnightly.Biochemical changes were observed through changes in fatty acid content in the seed endosperm which were performed monthly.While for physiological changes, observations on total germination percentage (GP), time required for 50% of processed seeds to germinate (GP50), and germination speed index (GSI) were completed monthly.SMC was determined by utilizing 10 randomly selected seeds from each experimental unit.The seed samples were placed inside each experimental unit and weighed (using Sartorius analytical balance type BL-2105) weekly during the storage process.At the end of the observation, every seed sample was ovendried by the constant low-temperature oven method of 103 ± 2 o C for 48 ± 3 hours [5] using Memmert oven type UNE-800, with the formula: EC was measured using 10 seeds from each experimental unit.The seed sample was weighed, put in a glass beaker, and soaked in 100 ml of distilled water for 24 hours at room temperature while the container was covered with aluminium foil.In parallel, controls were also made with each 100 ml of seedless distilled water.After 24 hours of immersion, the EC of each experimental was measured using the formula: Fatty acid content was carried out by gas chromatography (GC) utilizing services of the Postharvest Laboratory of IOPRI.GP and GSI were calculated using the formula:

Seed physical changes
Seed water content.During six months of storage, every experimental level was showing a decrease in SMC, which reduction varies between levels arranging from 0.11 to 0.32% (Figure 1).Based on its storage characteristics, oil palm seed is categorized as an intermediate seed [2] which is tolerant to desiccation of up to 7-10% [8].Final SMCs of oil palm seeds which were stored within 6 months period in controlled and open storage conditions ranged from 11.2-11.4%,which are classified as safe moisture contents for oil palm seeds to be stored.Seeds stored in controlled storage appeared to experience a faster decrease in SMC than those in the open storage, both for single PE and double PE treatments.Based on seed packaging, the usage of double PE bags was more able to maintain SMC than those with single PE bags, both in controlled and open storage.
Differences in the reduction rate of SMC between levels of storage rooms were due to differences in RH inside the rooms as open storage had higher RH than controlled storage.The average RH value in open storage conditions was 73.2% (48.6% -82.0%) while the average RH in controlled storage was 64.6% (43.9% -82.1%).SMC decreases gradually to adjust the moisture inside the seeds with the humidity in the air due to the hygroscopic nature of the seeds [9].Water content in the seeds which was relatively higher than water content in the air inside the storage rooms causes water mass movement from the seeds into the air until an equilibrium occurs, which is known as moisture equilibrium [9].Data shown by this study revealed that the average RH in the controlled storage was lower than that in the open storage, leading to a faster reduction in SMC for seeds that were stored in the controlled storage than those stored in the open storage.Even though SMC decreased at all treatment levels, double PE bag usage was able to retain SMC better than the use of a single PE plastic bag.This can be seen from the trend lines shown in Figure 1.The result of this experiment is in line with the results found by Ali et al. [10] who showed that PE plastic can be used for seed storage.Agha et al. [11] stated that effective storage packaging is a packaging which able to suppress the effects of surrounding air on the stored seeds, hence double PE plastic bags is a better packaging method than the utilization of single PE plastic bag to store oil palm seeds.
In this experiment, the random movement of EC value was due to exudates which are released by the endocarp (shell) of the seeds, which is in line with the statement of Szemruch et al. [12] who affirmed that the pericarp layer could be a source of contamination in EC observation.Therefore, by using sunflower seeds, Szemruch et al. [12] suggested removing the seed coat before carrying out the EC test.In oil palm seeds, removing the seed endocarp before the EC test should be done with caution since the endosperm of the seed can be damaged, causing further bias for EC measurement.
Regarding the increasing trend of EC value during storage, Wahyuni [13] noted that several factors influence EC value, i.e.SMC, size of the tested seeds, the temperature of the distilled water utilized, duration of seed immersion, and water temperature when EC is measured.Furthermore, Nuraini et al. [14] noted that the EC value was also influenced by the chemical composition of the tested seeds, while Corley and Tinker [7] stated that the endosperm of oil palm seed has a high content of lipids.Hudiyanti [15] acknowledged that fat decomposition during seed storage into fatty acids may cause the loss of phospholipids, which are components of cell membranes.Damage on cell membranes may increase EC value since cell metabolites such as sugars, lipids, and amino acids are translocated from the cell.Electrical conductivity.The EC value changed randomly during storage (Figure 2).Slow increments of EC values were presented by all experimental units.

Seed biochemical changes
Changes in the proportion of fatty acids through the oxidation process during storage can be an indication of a decrease in seed viability.López-Fernández et al. [16] explained that oxidation affects all types of fatty acids, but oxidation effects are greater on unsaturated fatty acids (UFAs), which have double bonds in their structure.The proportion of fatty acid content in the endosperm of oil palm seeds changed during storage (Table 1).The result shows that the main fatty acid in the endosperm was saturated fatty acids (SFAs) with a composition of around 78.5 -82.6%.Monounsaturated fatty acids (MUFAs) were in the proportion of 15.4 -18.8%, while polyunsaturated fatty acids (PUFAs) were in the range of 1.3 -5.3%.
Cui et al. [17] explained that the endosperm of oil palm seeds contains medium-chain fatty acids (C6-C12) and contains more SFAs than those contained in the mesocarp.Uddin et al. [18] observed that fatty acid composition in crude palm oil (CPO) as follows 43.14% of C18:1, 31.33% of C16:0, 11.5% of C18:0, 10.18% of C18:2, and 3.12% of C14:0, showing balance proportion between SFAs and UFAs, in contrast to the content of SFAs and UFAs in the endosperm shown in this experiment.In general, the content of SFAs and UFAs endosperm of the DxP PPKS 540 variety were 80.22% and 19.78%, respectively.
Based on the trend line of the five main fatty acids in the endosperm, the proportion of SFAs, i.e. lauric (C12:0), myristic (C14:0), and palmitic acid (C16:0) tended to increase corresponding to the storage duration, in contrast to the proportion of UFAs, namely oleic (C18:1) and linoleic acid (C18:2) which slowly declined.
Based on changes in the proportion of fatty acid content during storage, the results shown in Table 1 are in line with those obtained by Zarifikhosroshahi and Ergun [19] using watermelon seed oil extract (Citrullus lanatus var.lanatus) where there was a decrease in linoleic acid (PUFA) content during storage, especially those stored in open storage.In addition, the authors also stated that the oil extracted from fresh watermelon seeds had low palmitate and stearate (SFA) content which increased during storage.Therefore, Zarifikhosroshahi and Ergun [19] concluded that during watermelon seeds storage, there was a conversion from PUFA to SFA to maintain the homeostatic condition of the seeds.

A B
Five main fatty acids in the endosperm (lauric, myristic, palmitic, oleic, and linoleic) showed no significant difference based on storage condition treatment (Figure 3).Cui et al. [17] stated that lipase enzyme activity and fat remobilization increased significantly at stages 5 and 6 of the oil palm germination process, an advanced stage when secondary root formation occurs and plumule extends.In addition, the fatty acid content was not significantly different in seeds stored in the open and controlled storages, presumably because the average temperature in the open storage (27.4 o C) was not high enough to trigger fatty acid degradation [20].

Seed physiological changes
The longer the storage duration, the lower the GP.The occurrence appeared both for seeds stored in controlled and open storage (Figure 4).In controlled storage, GP significantly decreased in 4 th month of storage.While in the open storage, the happening occurred faster, in the 2 nd month of seed storage.Figure 4 also shows that storage periods of 4 and 5 months provide lower GSI values than other storage period levels.Oil palm seeds are categorized as intermediate-type seeds [2] whose moisture content can be reduced to a certain level and stored at low temperatures.Yulianti et al. [21] explained that although intermediate seeds can be stored for a relatively longer time than recalcitrant seeds, their storage duration is shorter than orthodox seeds.The authors further stated that intermediate seeds can be stored for less than 1 year.This is in line with the result which is shown in this experiment where the GP of fresh seed (83.8%) decreased since the first month of storage and reached 25.9% and 45.0% of GP after being kept for 5 months for controlled storage and open storage, respectively.GSI analysis (Figure 4) showed that oil palm seeds stored for 2 months generated high GSI values (2.49%/etmal in the controlled storage and 2.36% in the open storage), significantly higher than the GSI of fresh seeds (1.42%/etmal).However, the GP value was declining since the first month of storage, indicating that oil palm seeds should be germinated as soon as possible after physiological maturity is reached.Correlation analysis (Table 2) displays no significant correlation between GP and fatty acid content in the endosperm, neither SFAs nor UFAs.GP correlation value with fatty acid content only ranged from -0.16 to 0.18.C6:0 C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 C20:0 C20:1 GP -0.16 0.05 0.11 -0.16 0.02 0.09 0.16 0.18 -0.13 -0.11 0.11 Purwanti [22] stated that hydrolysis during storage led to an increase in SFAs, and in parallel also produced toxin products during the process, causing a decrease in seed viability.The absence of correlations between GP and fatty acid content indicates that fatty acid composition cannot be an indicator to determine the physiological state of the stored seeds.

Conclusion
Based on physical parameters, storing oil palm seeds (DxP PPKS 540 variety) with single or double PE plastic bags (60 x 30 cm x 0.2 mm) was able to maintain SMC at a safe-storage level for 5 months both in open storage (temperature 24.2 -30.5 o C and RH 48.6 -82%) and controlled storage (temperature 22.0 -27.7 o C and RH 43.9 -82.1%), while electrical conductivity of the seeds fluctuated during storage.During seed storage, there were no significant changes in the fatty acid composition of the oil palm endosperm.Seeds could be stored for 3 months under controlled storage without significant reduction in GP, while open storage significantly reduced germination 2 months after storage.Germination percentage was not correlated with the fatty acid content in the endosperm.

Acknowledgments
The authors acknowledge the assistance provided by the Indonesian Oil Palm Research Institute (IOPRI) and The Southeast Asian Regional Center for Graduate Study and Research in Agriculture (SEARCA)

Figure 1 .
Figure 1.The declining of seed moisture content: in the controlled storage (A); in the open storage (B).

Figure 2 .
Figure 2. Electrical conductivity of oil palm seeds during storage: in the controlled storage (A); in the open storage (B).

Figure 3 .
Figure 3. Fatty acid composition in the oil palm endosperm based on storage conditions.

Figure 4 .
Figure 4. Physiological parameters of stored oil palm seeds: in controlled storage (A); in open storage (B).
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Table 1 .
Changes in fatty acid content in the endosperm of oil palm seeds during storage.

Table 2 .
The correlation value of GP to fatty acid content in the endosperm of stored seeds.