Introduction
Seeds are the basis for a new generation of life for most plants. Storing seeds from the same and different species for long periods serves to preserve genetic diversity, akin to keeping treasures for the benefit of future generations. To preserve the seeds for short- or long-term storage, plant germplasm scientists need to maintain seed viability. Maintaining the viability of seeds or clonal materials over prolonged periods is undoubtedly one of the main responsibilities for germplasm scientists (Sallon et al., Reference Sallon, Solowey, Cohen, Korchinsky, Egli, Woodhatch, Simchoni and Kislev2008). The longevity (the half-viability period, P50) of seeds from different species stored in gene banks has been well documented (Priestley et al., Reference Priestley, Cullinan and Wolfe1985; Walters et al., Reference Walters, Wheeler and Grotenhuis2005). A wide range of P50 from as low as 13 years to more than 450 years (extrapolated) among 276 species was observed with an average of 54 years. Within a single species, genetics and seed chemical composition may affect seed viability, longevity and seedling performance. In rapeseed (Brassica napus L.), variability in fatty acid and glucosinolate composition significantly contributed to P50 variations from 15.2 to 50.7 years and also affected the rate of normal seedling growth (Nagel et al., Reference Nagel, Holstein, Willner and Börner2018). The correlation between fatty acid composition and seedling growth rate was also observed in soybean. Higher palmitic (C16:0) and stearic (C18:0) acid content significantly decreased the seedling growth rate (Wang et al., Reference Wang, Harp, Hammond, Burris and Fehr2001). Additionally, different cultivars within the same species may be impacted differently by storage conditions leading to different longevities, germination rates, and seedling emergence and normality rates. Significant differences in field germination and emergence rates among four peanut cultivars were observed when the cultivars were stored at different conditions before planting (Morton et al., Reference Morton, Tillman, Gorbet and Boote2008). To maximize yield, farmers need to get good plant stands (plants/acre). At planting time, they determine seedling rates (seeds/acre) based on the results of seed germination and seedling emergence tests (Carlson, Reference Carlson2016). In chickpea, early seedling emergence produced more pods and led to higher seed yield (Auld et al., Reference Auld, Bettis, Crock and Kephart1988; Gan et al., Reference Gan, Miller, Liu, Stevenson and McDonald2002). Therefore, seed germination and seedling emergence rates and seedling vigour can significantly affect the crop yield.
The USDA cultivated peanut germplasm collection is maintained at the Plant Genetic Resources Conservation Unit (PGRCU) in Griffin, Georgia. A screen of nearly the entire collection (about 8,466 accessions) for seed fatty acid composition revealed that the -01 inventory of PI 268941 deposited into the gene bank in 1989 was relatively high for oleic acid (71.7%). Additional chemical analysis of individual seeds indicated that this inventory contained two types of seeds (high oleic seeds averaging ∼80% and normal oleic seeds averaging <50%). Based on field observations, the seeds from this inventory produced plants with a uniform morphology, and the harvested seeds showed uniform seed coat colour and size. Since the two seed morphotypes had very different levels of oleic acid, sequence analysis of the FAD2A and FAD2B genes was performed to determine the genotype of these two groups of seeds. A novel functional deletion was found in the coding sequence of the FAD2B gene (435DelT) resulting in a frameshift mutation and premature translation termination downstream in the high oleate seeds. Normal oleate seeds exhibited the FAD2B wildtype genotype without the 435DelT mutation (unpublished results). A spontaneous mutation may have happened resulting in the mixed genotypes. Since seeds of this inventory with different levels of oleic acid and different FAD2B genotypes were stored for 30 years (1989–2019) within the same aluminium bag, it provides good genetic materials to conduct individual seed analysis and determine the relationships between the seed fatty acid composition and the seed germination rate and seedling performance after long-term storage and current greenhouse planting. Therefore, the objectives of this study were to determine (1) the FAD2B genotype of individual seeds by real-time PCR; (2) the fatty acid composition of individual seeds by gas chromatography (GC) analysis; (3) seed germination and seedling normality rates by planting in greenhouse and observation; and (4) the correlation between fatty acid composition and seedling performance after long-term storage and current greenhouse planting.
Experimental
Germplasm accessions are assigned different inventory numbers when harvested at different locations or in different years and stored at different times. To test seed germination and seedling performance, seeds from the same -01 inventory of PI 268941 which had been stored at −18°C for 30 years were requested from the PGRCU gene bank. A total of 200 seeds were acquired and numbered individually. Seeds from the sample were all the same colour. Before the germination test, a small portion (∼150 mg) of seed tissue was chipped off from the distal end (tip of the cotyledon) using a scalpel. The chipped seed tissue was used for fatty acid analysis and real-time PCR genotyping. The large portion of seed containing the embryotic shoot and root was used for the seed germination test and seedling performance observation. When the tip of the radicle has grown free of the seed coat, a seed is considered germinated. Seed germination was performed following the rolled-towel germination method. Before germination, seeds were treated with the fungicide Dynasty-PD and spread individually on a wet standard-weight paper towel (35.5 × 63 cm). The seeds were then covered with a second wet towel, and the two sheets were loosely rolled with the seed inside. They were placed upright in a plastic container and transferred into a growth chamber. The growth chamber was set to diurnal conditions of 29°C/12 h and nocturnal conditions of 25°C/12 h with 60% relative humidity. The germination paper was checked daily and moistened with filtered water as needed. After 7 days, seed germination was examined and recorded.
Seed emergence is defined as the coleoptile of a planted seed emerging from the soil following germination. Seedlings with a hypocotyl-radicle axis >3 cm are defined as emerged. The germinated seeds were planted in small trays filled with soil (Metromix-360, Griffin House and Nursery Inc., Griffin, GA) in the greenhouse (30°C/diurnal for 12 h and 20°C/nocturnal for 12 h) to observe seedling emergence and normality. Seven days after germinated seeds were planted in the greenhouse, seedling emergence was observed and recorded. Among 200 seeds tested, some germinated seeds did not emerge at all. Some seeds emerged normally, while some emerged abnormally. All germinated, planted seeds were classified as either normal or abnormal emerged seedlings. The seedlings were established approximately 3 weeks after planting, and the emerged, growing plants were classified into one of two categories: normal growth or abnormal growth. Abnormally growing seedlings were defined as having stunted or slow growth, irregular leaf shape or other unusual growth observations. Plants that grew normally from normal and mutant genotypes had the same morphology.
Fatty acid composition analysis followed a previously published method with minor modifications (Wang et al., Reference Wang, Chen, Tonnis, Barkley, Pinnow, Pittman, Davis, Holbrook, Stalker and Pederson2013). Fatty acid composition was measured on an Agilent 7890A GC. Fatty acid percentages were determined by calculation of the corresponding peak area within the total peak area. Two measurements were taken for the chipped seed tissue, and the results were averaged. Based on preliminary sequence data from individual plants of PI 268941, a FAD2B functional mutation (one base pair deletion at position 435, or 435T) on the B sub-genome was identified in high oleic seeds. No deletion was found in normal oleic seeds of the same accession. A real-time PCR assay was designed using PCR primers and TaqMan® probes (Applied Biosystems) to amplify a 58 or 59 base pair fragment of the FAD2B gene depending on the presence or absence of the deletion and then determine the genotypes (BB or bb) of each individual seed following the adapted method (Barkley et al., Reference Barkley, Chenault-Chamberlain, Wang and Pittman2009). A Pearson’s coefficient analysis was performed to determine significant correlations among different seed chemical composition, seed germination and seedling performance traits. An analysis of variance was performed on the data, and means were separated using Tukey’s multiple comparison procedure (SAS, 2008, Online Doc® 9.2. Cary, NC: SAS Institute Inc).
Discussion
Significant fatty acid composition variation was identified among 200 individual seeds, and the results are listed in Table S1. The fatty acid profiles of the individual seeds were classified into two distinct groups, which were designated as high oleate and normal oleate. An example of each is shown in Fig. 1a where the high oleate seed had 79.6% oleic acid, 3.7% linoleic acid and 5.0% palmitic acid, whereas the normal oleate seed had 43.2% oleic acid, 32.5% linoleic acid and 10.2% palmitic acid. Furthermore, only the normal oleate profile had additional, unidentified peaks (circled in Fig. 1b and enlarged from Fig. 1a). From the real-time PCR genotyping of FAD2B, 100 seeds had the ‘T’ deletion at position 435, and 100 seeds had no ‘T’ deletion (wild type). When the genotypes were compared with their corresponding fatty acid profiles, 98 of 100 seeds with ‘T’ deletion had the high oleate profile (>74% oleate), and all 100 wildtype seeds had the normal oleate profile (<58% oleate). Two seeds with the ‘T’ deletion had mid-oleate profiles (67.7% and 72.5%), higher than normal but slightly below what is considered high. Therefore, this ‘T’ deletion which causes a frameshift and functional mutation in the FAD2 enzyme is very likely responsible for the oleate differences. The seeds with the ‘T’ deletion were designated as high oleate genotype (bb), and the seeds without the ‘T’ deletion were designated as normal oleate genotype (BB). The ratio of high and normal oleate genotypes was 1:1 (100:100). This segregation ratio may indicate that the point deletion happened spontaneously, and both genotypes were subsequently included in the inventory for storage.
Comparison of major fatty acids, extra peaks, germination differences between high and normal oleic acid genotypes. (a) Comparison of normal-size chromatograms. X-axis is retention time, and Y-axis is FID response value. PA, picoamperes; high oleic, ∼80% oleic acid; normal oleic, ∼45%. (b) Comparison of enlarged-size chromatograms from the left panel. The extra peaks are circled and the percentage of 4.2% is the total area of extra peaks. (c) Comparison of germination rate, seedling emergence normality rate and seedling growth normality. X-axis is two different genotypes, and Y-axis is the percentages of each trait rate. Blue bar represents genotype BB and orange bar represents genotype bb. If the letters above two bars are different, there are significantly statistical differences in rates (%) between two genotypes.
There were significant differences in each fatty acid content between high and normal oleate genotypes, and the results are shown in Figures S1 and S2. The high oleate genotype had significantly higher levels of oleic (78.90% vs 48.19%), gadoleic (2.94% vs 1.87%) and lignoceric acids (2.25% vs 2.11%) than the normal oleate genotype. But the normal oleate genotype had significantly higher levels of palmitic (9.52% vs 5.08%), stearic (2.05% vs 1.88%), linoleic (28.53% vs 4.75%), behenic (3.48% vs 3.09%) and arachidic acids (1.25% vs 1.11%). The additional unidentified compounds (extra peaks 1 and 2) averaged 1.31% and 1.69% of the total in normal oleate genotype, while they were not present in any of the seeds with the high oleate genotype except for a small amount of EPk1 in one seed with mid-level oleate (67.74%).
Significant morphological variation during seed germination, seedling emergence and seedling growth was observed among seeds, and examples of each are shown in Fig. 2. For seed germination, normal seeds had extended primary roots with root hairs, whereas abnormal seeds had very short primary roots with no root hairs. For seedling emergence, normal seeds had true leaves, whereas abnormal seeds had very tiny or no true leaves. For seedling growth, normal seeds had a good plant and stem growth with many expanded true leaves, whereas abnormal seeds had a very slow plant growth with a short stem and small leaves. Based on the seed FAD2B genotypes, the seed germination, seedling emergence normality and seedling growth normality rates were calculated, and the results are listed in Table S2 and shown in Fig. 1c. Seed germination and seedling emergence normality rates were significantly higher in the high oleate seeds (89.11% and 89.11%) than the normal oleate seeds (64.65% and 60.61%). Seedling growth normality rate was significantly higher in the high oleate seeds (76.24%) than the normal oleate seeds (28.29%). This suggests that the ‘T’ deletion in the FAD2B gene may be an important factor affecting seed germination, seedling emergence normality and seedling growth normality.
Comparison between normal and abnormal germination, seedling emergence and seedling normality. (a) Seeds germination. Seeds germinated in growth chamber for 1 week following our laboratory Standard peanut germination procedure. (b) Seedling emergence. After germination in growth chamber, the germinated seeds were transferred to pit soil and grown for 1 week in greenhouse. (c) Seedling normality. Seedlings were grown in the greenhouse for 3 weeks. Red bars in each picture are equal to 1 cm.
To see how fatty acid composition affected seed germination rate, seedling emergence normality rate and seedling growth normality rate, the correlation coefficients among the investigated traits were calculated, and the results are listed in Table S3. Seed germination rate was highly and significantly correlated with seedling emergence normality rate (r = 0.95, p < 0.0001), and seedling emergence normality rate was significantly correlated with seedling growth normality rate (r = 0.61, p < 0.0001). This makes intuitive sense because seedling emergence and growth do not happen if the seeds do not germinate initially. But the correlation of fatty acids with these observations was more nuanced. Three fatty acids (stearic, arachidic and lignoceric) did not have significant correlations with seed germination and seedling performance traits. Gadoleic acid had a positive significant correlation with seedling growth normality rate (r = 0.30, p < 0.0001) but not with seed germination rate or seedling emergence normality rate. Oleic acid had significant positive correlations with seed germination rate, seedling emergence normality rate and seedling growth normality rate, respectively. Three other fatty acids (palmitic, linoleic and behenic acids) had significant negative correlations with seed germination rate, seedling emergence normality rate and seedling growth normality rate, respectively (see Table S3). In soybean, a negative correlation between seedling growth rate and enhanced palmitic acid (C16:0) was also observed (Wang et al., Reference Wang, Harp, Hammond, Burris and Fehr2001). This correlation was consistent with our results in peanut. Soybean also exhibited a significant negative correlation between seedling growth rate and enhanced stearic acid (C18:0), but this negative correlation in peanut was not significant (r = − 0.06, p = 0.4063). There is a significant difference in the range of fatty acid composition between peanuts and soybeans. Differences in fatty acid composition can significantly affect seed germination and seedling performance, and this may explain the correlation differences between the two species.
The unidentified compounds (EPk1 and EPk2) also had significantly negative correlations with seed germination rate, seedling emergence normality rate and seedling growth normality rate (EPk1: r = −0.24, p = 0.0007; r = −0.28, p < 0.0001; r = −0.49, p < 0.0001; respectively) (EPk2: r = −0.27, p < 0.0001; r = −0.32, p < 0.0001; r = −0.53, p < 0.0001; respectively). Interestingly for all the significant positive and negative correlations, the absolute r-value for each became either more positive or more negative progressing from seed germination rate to seedling emergence normality rate to seedling growth normality rate. For example, the r-value correlations with oleic acid increased from 0.35 to 0.39 to 0.56. This indicates that as the seeds germinated and grew into seedlings, the apparent effects of the fatty acid on plant health and normality increased. This may explain why seedling growth normality rates were significantly lower than the seed germination rates (Table S2, for BB genotype, 28.29% and 64.65%; for bb genotype, 76.24% and 89.11%). In other words, seedling growth normality rate rather than germination rate may be a good parameter to reflect the effect of seed fatty acid composition on plant performance.
The unidentified compounds were also significantly correlated with several fatty acids (palmitic, oleic acid, linoleic, gadoleic and behenic acids). Furthermore, these compounds were also significantly correlated (r = 0.97, p < 0.0001) to each other (Table S3). They have not yet been conclusively identified in our laboratory, but analyses of freshly harvested seeds do not show the presence of these compounds, so they must have been produced over time during seed storage either by oil oxidation or some other bioprocess. Both compounds are negatively correlated with oleic and gadoleic acids, the two monounsaturated fatty acids identified in peanut seeds. Our results show that EPk1 and EPk2 are only present in stored seeds with lower percentages of oleic and gadoleic acids (i.e. non-high oleate) which would explain the observed negative correlation. Oleic and linoleic acids make up a large proportion (∼80%) of the total oil in peanut seeds, and the amount of each is largely controlled by the genotype of the FAD2A and FAD2B genes. Functional mutations in these genes can affect the activity of the desaturase enzyme they encode that converts the monounsaturated oleic acid to polyunsaturated linoleic acid. Therefore, the ratio of oleic to linoleic acid will have a large effect on the overall amounts of monounsaturated and polyunsaturated fatty acids. Monounsaturated fats have higher oxidative stabilities than polyunsaturated fats. Peanut seeds with lower amounts of oleic acid and higher amounts of linoleic acid would be more prone to oil oxidation resulting in the generation and build-up of oil breakdown products which could explain the presence of EPk1 and EPk2 in these seeds. Since oleic acid is positively correlated with seed germination, emergence and normal plant growth of long-stored seeds, it may be the result of the protective effects of this fatty acid on overall seed stability against oxidative damage and breakdown.
In summary, one inventory of peanut accession PI 268941 having been stored for 30 years provided a unique experimental material to estimate the effects of long-term storage and varying seed fatty acid composition on subsequent seed germination, plant emergence and plant growth. The accession contained a natural genetic mixture of seeds with and without a T deletion at the nucleotide position 435 of the FAD2B gene resulting in half the seeds being high oleate and half being normal oleate. Chemical analysis revealed that only normal oleate seeds had additional, unidentified compounds in their fatty acid profile. These compounds may be oil breakdown products resulting from lower oxidative stability in these seeds versus high oleate seeds (O’Keefe et al., Reference O’Keefe, Wiley and Knauft1993). The biosynthesis and genetic control of fatty acids, especially for oleic acid in peanut are well documented (He et al., Reference He, Qin, Wang and Ding2020; Jung et al., Reference Jung, Powell, Moore and Abbott2000a, Reference Jung, Swift, Sengoku, Patel, Teule, Powell, Moore and Abbott2000b). The significant difference between the levels of high oleate (78.9%) and normal oleate (48.19%) was therefore most likely caused from the mutation of FAD2B gene. The results were confirmed by both single nucleotide polymorphism genotyping and GC analysis. More importantly, the high oleate seed class had higher germination, emergence and normal plant growth rates than the normal oleate seeds meaning they remain viable longer and incur less oxidation damage during long-term storage. These results indicate that seed storage duration, genotype and fatty acid composition (an example from canola; Seyyedi et al., Reference Seyyedi, Afshari and Daneshmandi2018) should be considered by curators and farmers in determining when seed lots should be planted as well as what potential seedling rates (seed/acre) and yield can be expected.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S1479262125000097.
Acknowledgements
The authors thank Dr. Zhenbang Chen and Dr. Brad Morris for reviewing the manuscript and Ms. Phiffie Vankus and Sarah Moon for assisting seed germination.
