Cold plasma-treatment has proven to be an effective technique to improve the germination and growth of different crops. Various studies showed a significant improvement in germination and growth of agricultural seeds after cold plasma-treatment. Hitherto, germination and growth tests of the seeds were conducted immediately after the plasma-treatment. The longevity of these plasma-treatments is essential to be ascertained because storage and distribution requirements delay immediate seeds plantation.
The current study used different characterisation techniques to represent seed germination and growth. The study further elaborated on the factors responsible for crop improvements and their deterioration in these proxy tests, in a process known as ageing, similar to those observed in synthetic polymers or plasma polymerised biomedical surfaces 11,13. Plasma diagnostic with OES was included in this study. Figure 6 shows the species generated during plasma-treatment used in the surface modification; nitrogen species are produced between 294 nm to 300 nm. From 350 nm to 380 nm, the spectra also showed the presence of hydrogen lines and CO bands. The oxygen and nitrogen species overlapped the band from 390 to 950 nm 17–19. In Fig. 6 (i), OH group is present at 309 nm for the water plasma-treated Bambara groundnut. In contrast, this OH group is absent during oxygen plasma-treated chilli and papaya seeds, shown in Fig. 6 (ii, iii), respectively.
Firstly, the ageing behaviour is investigated based on the hydrophilicity of substrate surface and their hydrophobic recovery over time. The hydrophilicity of the plasma-treated seeds is associated with a reduction of water contact angle, as demonstrated in Fig. 1. The SEM micrographs in Fig. 4 also suggest the plasma-etched seeds have improved hydrophilicity because their corrugated and porous surface absorbs the water more rapidly than those not plasma-treated, illustrated in the water uptake section (Fig. 2). The rapid water uptake is an initial and essential factor for the germination and growth improvement of any crop because it provides an easy path for the transfer of nutrients. Those nutrients provide the energy needed to initiate germination.
An increase in electrical conductivity confirmed the leaching of nutrients, electrolytes, and salt contents from the seeds, indicating the seed vigour. However, water uptake and electrical conductivity are not always correlated linearly to seeds germination and growth improvement. For example, the higher dosage of plasma-treatment increases the water uptake and electrical conductivity values by damage of the seed surface. This damage hurts the seeds’ germination and growth; higher electrical conductivity is also associated with more dead seeds, as reported elsewhere 20. This latter result suggested that the seeds might have been damaged by the high ion dosage of the plasma-treatment. On the other hand, a moderate rise in electrical conductivity was associated with the positive interaction between water and active plasma species introduced on the seed surface by cold plasma-treatment. The leaching of nutrients and sugar contents was useful to provide the initial energy for germination.
From Table S1 and Fig. 5, the surface oxidation of the plasma-treated seeds leads to an increase of oxides-containing moieties, as exemplified by O/C ratios, known for promoting surface hydrophilicity.9
The mechanisms for the germination and growth of various crops are likely associated with increased hydrophilicity from the etched seed structure and subsequent water uptake by the etched seeds. The elevation of these factors is associated with the changes introduced by cold plasma-treatment. OES results from Fig. 6 describe the active species generated during water and oxygen plasma-treatment. These active plasma species produced the necessary physical-chemical changes in the seeds leading to the germination and growth improvement of various crops. However, herein, the effect of plasma-treatment on those factors induced by the physical-chemical changes was investigated for 60 days to determine the longevity and storage duration of the plasma-treated seeds.
The contact angle reduced from 114° to 44°for Bambara seeds, as shown in Fig. 1 and the recovery of contact angle reached 69° after 60 days. A similar reduction in contact angle was studied elsewhere, where the contact angle reduced from 115° to 0° for the wheat seeds after plasma-treatment 8. Our current findings were compared to those obtained from polymer surfaces with a large dataset for a better understanding of ageing phenomena, because crop seeds are essentially lignin and cellulose, composed of glucose and other small monomers. In the case of synthetic polymers, different degrees of hydrophobic recovery existed for different plasma polymers reported in the literature 21,22. Various factors have been discussed in the literature. The difference between our and published results for synthetic polymers or seeds crops (like wheat seeds) could be attributed to the different seeds having different inherent morphology and different plasma-treatment condition. Further studies with the water uptake tests and FE-SEM, which will be discussed next, supported this suggestion. Residual hydrophilicity in the Bambara seeds ensures that the plasma-treated seeds can be used later after 60 days of storage for observation (Fig. 1).
As mentioned earlier, the water uptake values increased significantly after plasma-treatment, but the water uptake reduced rapidly after 60 days of the ageing period, but the plasma-treated seeds’ water uptake values are still higher at day 60 than those reported for untreated seeds. Furthermore, the effect of vacuum was also investigated separately; while our results showed that vacuum also influences the water uptake value but its effectiveness subsided by day 60; showing identical water uptake values for untreated and vacuum treated seeds (Figure S2). Thus, the plasma-treatment was responsible for the water uptake for the plasma-treated seeds.
Plasma-treatment is useful to remove the wax layer from many seed surfaces to convert them to hydrophilic. The hydrophobic recovery may take part in the reduction of water uptake, but this recovery is insignificant until day 30. The water uptake results for plasma-treated seeds are significantly higher than untreated seeds on day 30. The increase and decrease in water uptake values were associated with physical and chemical changes in the seed's surface discussed in the following section.
A significant increase in electrical conductivity for solutions used to soak plasma-treated seeds is shown in Fig. 3; the observation was, again, carried out for 60 days. A slight decrease in the electrical conductivity was reported towards the end of the ageing period. This reduction in electrical conductivity could be attributed to the decrease of the reactive species on the seed surface, such as radicals with a short life span post-plasma-treatment.
In addition, the oxygen plasma produced ions that etched and oxidised the seed hilum and testa, as confirmed by the XPS measurements (Fig. 5). These oxidising and etching ions had a longer-lasting effect than the shorter-lived radicals that decayed over time. These ions were more likely to sustain the electrical conductivity effects reported in these studies. The lack of a vacuum influence on electrical conductivity (Figure S2) further demonstrates that the vacuum only increases the water uptake values, but not the chemical composition.
FE-SEM experiment was carried out immediately after plasma-treatment to investigate the physical changes in the surface of Bambara, chilli, and papaya seeds. The physical changes on the surface of Bambara, chilli, and papaya seeds after plasma-treatment are shown in Fig. 4(i-iii), respectively. Other researchers have reported similar surface changes as reported in the current investigation. For example, air plasma-treatment is used to modify the surface of wheat, beans, and lentil seeds in a similar way 8. Their FE-SEM investigation corroborated the physical changes during the post-plasma-treatment, but their study was not extended for 60 days to account for ageing. Since plasma oxidation caused the surface changes, ageing studies were carried out using XPS studies to examine these oxidation changes. The results for water uptake, contact angle, and electrical conductivity were also supported by this FE-SEM research.
The oxygen-related moieties are significantly enhanced after plasma-treatment, as indicated in Table S1 and Fig. 5. For both oxygen and water plasma-treatment, the O/C ratio increased, (from 0.2 to 0.4 for Bambara, 0.4 to 1.0 for papaya and 0.2 to 0.5 for chilli) whereas the N/C ratio increased for Bambara and chilli seeds (0.4 to 0.5 and 0.2 to 0.6 ) but the N/C ratios decreased for papaya seeds (0.107 to 0.106). However, this reduction was also reported elsewhere, when pepper and melon seeds were treated with a similar plasma source, but they differ in nitrogen concentration on the seeds 23. These findings suggested that the precursors used in the plasma-treatment impacted oxygen and nitrogen moieties found on the surfaces.
Due to their similarities with the seed surfaces, made up of a "cellulose-lignin" structure with cross-linked lignols and polysaccharides, we proposed a possible mechanism for this ageing phenomenon similar to those reported in synthetic polymers. The ageing of these polymeric structures is governed by two main mechanisms: post-plasma oxidation and surface adaptation. The initial increase in O/C ratios was caused by radicals interacting with in-diffusing oxygen from the environment. The interfacial chemistry influenced the surface adaption, and the plasma-treated seed surface uniquely interacted with its surroundings. Mobile and immobile polar groups on the polymeric substrate cause surface reorientation. These factors are classed as internal and external ageing factors. Internal factors contain interfacial enthalpy, entropy and cross-linking density, while external factors contain contaminant adsorption, oxidation and temperature 12. Internal factors influence polymer chain mobility, whereas external factors pertain to the environment's effect on ageing behaviour.
Although this surface reorientation is commonly observed in the synthetic polymer, this mechanism is unlikely to be operational for hard testa coats, which tend to have a relatively high glass transition temperature of more than 0 ℃.24 Hence, seed type and plasma modification type play a significant impact on the internal and exterior components of these ageing processes. Furthermore, the ageing of these materials frequently involved more than just oxidative chemical changes; over time, the physical process of surface remodelling also occurred, resulting in visible changes in contact angles. However, this study concluded that the influence of germination and growth-promoting factors is present during 60 days of ageing, but it is most significant within the first 30 days.