High oxygen seed test

The ‘controlled deterioration seed ageing tests’ currently used accelerate the deterioration by storing seeds at both higher temperatures and seed moisture levels. Seed companies find that the tests do not correlate well with the actual shelf life in practice when stored at low seed moisture levels. The likely reason for this poor correlation is the difference in physiology between dry and moist seeds. Noticing that the damage induced during seed ageing is due to oxidation, we looked to accelerate the ageing by increasing the oxygen concentration instead of the moisture level. As a result Wageningen University & Research succeeded in developing a more realistic ageing test, storing the seeds under an elevated partial oxygen pressure created through compressed air in a steel tank.

Improving seed storage

The prevention of quality loss during storage is of major economic importance for seeds, which are used by farmers to produce new crops and by the malting industry as input for beer production. It is important for seed suppliers and maltsters that they can estimate the shelf life of seed batches and determine the effect of seed treatments. It is known that seeds from different crops can vary largely in their shelf life. The viability of seeds from lettuce, for example, declines within a couple of years, while barley seeds can survive storage for several decades. Even seed lots from the same crop but different production locations may vary in their shelf life. Seed companies want to know which seed lots to sell first and which can be stored longer.

Ageing of seeds

Less well known is the fact that seed quality gradually declines during storage. Although accumulation of damage will ultimately result in a lack of germination and emergence, long before that the damage will lead to slower germination and the seeds and seedlings having a reduced tolerance to biotic and abiotic stress. This early damage will also result in a less productive start of a crop and a lower efficiency in malt production.

To determine the shelf life of a seed lot or study what occurs during ageing, one can take samples at regular intervals during storage and measure the quality. However, by the time the quality drops below the threshold it is too late to take precautions. This is why seed researchers have developed tests for estimating the shelf life of seed lots. An elevation of storage temperature and seed moisture level increases the rate of seed ageing. Researchers applied that experience to create survival tests that estimate the shelf life of a seed lot under commercial storage conditions in a shorter time frame.

These so-called ‘controlled deterioration’ tests work reasonably well when comparing seed lots with a major difference in shelf life under normal storage conditions. However, there is a poor correlation when it concerns samples with more subtle differences in shelf life. One of the main reasons is that there is no or hardly enzyme activity under dry conditions (below a relative humidity of 40%), whereas enzymes will be active at the higher seed moisture levels as used in the ‘controlled deterioration’ tests. Moreover, food science teaches us that the kinetic model for lipid oxidation at 60°C deviates from that at temperatures of 40°C or less. This created the need for a more realistic seed shelf life test that can be performed at ambient temperatures and at seed moisture levels which are not higher than used in practice. Of course the test should still give a faster result compared to waiting for the actual decline during commercial storage.

Shelf life test

We have shown that dry seed storage under anoxia can result in a very significant increase in shelf life under the same seed moisture level and storage temperature (Groot et al., 2015). We thought about using this the other way around and hypothesised that if anoxia increases shelf life, than elevated oxygen levels might accelerate ageing and enable a fast shelf life test. Indeed, seed storage under 100% oxygen accelerated the ageing, but it was still too slow to be used as a test.

As an exploratory experiment we stored lettuce seeds in a SCUBA tank, normally used for exploring the underwater world, with compressed air under a pressure of 200 bar. This means that the oxygen concentration in the tank was 200 times that of ambient storage conditions, while seed moisture level and storage temperature were the same as with commercial storage. Most of the seeds failed to germinate properly after only three weeks of storage under these conditions. Indeed, the seeds had aged very fast due to the high oxygen pressure and the symptoms observed with the poor quality seedlings were comparable to those observed after a few years storage in a seed company warehouse.

Using this result we developed an experimental setup to store seeds under an Elevated Partial Pressure of Oxygen (EPPO) and study ageing processes under dry conditions (Groot et al., 2012). Experiments with seeds from barley, soybean and cabbage showed that all responded to the higher oxygen concentration with faster ageing of the seeds. This test has since been employed with several crops, including the study of genetic variation in shelf life with barley grains (Nagel et al., 2016). Metabolomics studies showed that the levels of vitamin E, an essential anti-oxidant for the protection of cell and mitochondrial membranes, declines during EPPO storage in correlation with the reduction in seed germination. In addition, ethanol synthesis by the seeds upon wetting – an indication of induced damage to the mitochondria – correlated well with the decline in seed quality. Both these correlations were lacking with seeds stored under moist and warm ‘controlled deterioration’ conditions.

The research was funded by the Dutch Ministry of Economic Affairs.