Since seed imbibition is a passive process that depends on the relationship between internal water potential of the seed and that of the surrounding medium (Hegarty, 1978), the imbibition rate of a dry seed decreases with the increase of the concentration of PEG solutions (Bewley et al., 2013; Vertucci, 1989; Moret-Fernández et al., under review). However, according to Fig. 4, under the absence of water supply restrictions between the seed and the surrounding medium, the imbibition process in PEG between 0 and − 0.01 MPa was not affected by the h of the germination medium. In addition, the significant relationship, with a slope value equal to one, between the barley and vetch seed weights measured with PEG at 0 MPa and the corresponding values measured with TG-loam and TG-sand at 0 MPa (Fig. 5) indicates that the imbibition process at 0 MPa is independent of the media where the seeds are placed. Taking these considerations into account, if the PEG imbibition curves within [0, -0.01] MPa are not affected by water potential (Fig. 4), and the PEG imbibition curve at 0 MPa is equal to the corresponding TG curve, then the equivalent PEG curves at -0.002 and − 0.006 MPa should be equal to the imbibition curve measured with TG at 0 MPa. This reasoning indicates that the imbibition curves obtained in the TG experiments (Fig. 3) can be used: (i) to compare the TG imbibition curves with each other, and (ii) to compare the TG curves at 0, -0.002 and − 0.006 MPa with the equivalent ones for PEG, where the equivalent PEG curves at 0, -0.002 and − 0.006 MPa corresponds to the TG curve at 0 MPa.
Taking these assumptions into account, results showed that although no differences were observed between TG-loam and PEG, the imbibition curves in TG-sand at h < 0 MPa were significantly different to the equivalent ones for PEG. Since seed imbibition curves in PEG depend only on h, these results indicate that the imbibition process should depend not only of h but also on the medium where the seed is placed. As reported by Williams and Shaykewich (1971), and Hadas and Russo (1974), these differences should be caused by the hydraulic properties of the porous substrate that surrounds the seeds, e.g. soil. Thus, while in PEG experiments the imbibition of completely wet seeds is only limited by the Δh between seed and the external solution, the seed imbibition process within porous substrates is controlled by both Δh (Eq. 3) and the K(h) (Eq. 2) of the substrate. All this information is summarized by the Darcy’s law for unsaturated media (Eq. 3), which can be applied to the seed-soil system. According to Eq. 3 the flow of water from the soil to a seed depends on the soil K(h) and the relationship between the distance and the Δh between a point of soil and the seed surface. In conclusion, unlike to PEG experiments, where there is no hydraulic restriction between the seed and the water solution, the imbibition curve of a seed buried in a soil will depend on both Δh and the K(h) of the soil. This theoretical description partially agrees with Camacho et al. (2021), who observed that K(h) rather than h, was a more informative variable to predict seed germination in soil.
Based on these general theoretical considerations, we can then discuss in detail the different behaviors observed between the seed imbibition curves obtained in the PEG and TG experiments:
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The significant relationship with slope close to one obtained in the two seed species for the comparison between PEG and TG-loam and TG-sand at 0 MPa (Fig. 5) is explained by the fact that Ks (Eq. 2) of the sand and loam soils was is not a limiting factor for the seed imbibition process. At h = 0 MPa, the high imbibition rate during the first steps of seed hydration is explained by the large Δh between the seed (around − 50 to -350 MPa) (Bewley et al., 2013) and the surrounding medium. However, as water is absorbed by seeds, the h of the seed becomes less negative, which decreases Δh and thus the rate of water absorption.
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The similar imbibition curves observed comparing TG-loam between 0 and − 0.006 MPa and the equivalent PEG curves, indicate that the K(h) of the loam soil for 0 > h > -0.006 MPa (Fig. 1) was not a limiting factor for seed imbibition either. Note that, as mentioned above, the equivalent PEG curves at -0.002 and − 0.006 MPa should be similar to the imbibition curves measured with TG at 0 MPa.
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The completely different seed imbibition behavior observed between the TG-sand curves measured at -0.006 MPa (Fig. 3) and the equivalent curves for PEG indicates that this porous media has a significant effect on seed imbibition. Since the PEG curve at -0.006 MPa is equivalent to the PEG one at 0 MPa (Fig. 4), and the latter should correspond to TG-sand curve at 0 MPa (Fig. 5), in principle, it could seem that there is a contradiction between the null seed imbibition in TG-sand at -0.006 MPa and the corresponding equivalent curve for PEG. By allusions, there would also be a contradiction between this null imbibition curve in TG-sand and the PEG curve at -2.5 MPa, since this latter shows an increasing trend despite the more negative h value. As suggested by Hadas and Russo (1974) and Camacho et al. (2021), the null imbibition curves found in sand should be related to the low unsaturated hydraulic conductivity of this material at -0.006 MPa (Fig. 2), which drastically restricted the water flow between the sand and the seeds. Note that this hydraulic limitation does not exist in the PEG experiments, where the seeds are in constant contact with a film of water. Thus, these results, which demonstrate that seed imbibition in soil depends on Δh and the soil K(h), clearly indicate that PEG experiments are not adequate to describe seed germination under soil conditions.
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The intermediate imbibition curve behavior observed in the TG-sand experiment at -0.002 MPa for both species, indicates that there was a moderate restriction of water flow between the sand and the seeds at this soil tension. Although this behavior could be explained by the low unsaturated hydraulic conductivity of the sand at -0.002 MPa (Fig. 2) (Hadas and Russo, 1974; Camacho et al. 2021), there seems to be a sort of contradiction between these results and those observed in TG-loam at -0.006 MPa, which, with lower unsaturated hydraulic conductivity (Fig. 2), the corresponding imbibition curve was less affected by h (Fig. 3). Thus, the higher water flow restriction between the seeds and sand at -0.002 MPa could be also explained by low water content of sand at that soil tension (Fig. 2) or a poor contact between seeds and the surrounding soil particles. Thus, these results suggest that seed imbibition could depend not only on the surrounding h and K(h), but also on other factors such as: (1) soil water content; while PEG experiments show that vetch can germinate at -2.5 MPa, in most real soils germination is not possible at this water potential because its soil moisture corresponds to the residual water content, or (2) the surface contact between seeds and soil, which determines the capacity of the soil to provide water to the seed independently of the soil K(h) and Δh between seed and soil. Thus, these results open the door to new applications of TG, which could be used, for instance, to study the role of seed shape or the mucilage as a strategy to increase the surface contact with the soil (Tsai et al., 2021). Another application of the tested equipment could be analysing the germination patterns of those weed species that especially thrive in minimum tillage or no-till compared to those adapted to ploughing. Results could shed light on understanding why certain species especially adapt to those two environments.
All these results indicate, as reported by Camacho et al. (2021), that PEG experiments are not appropriate to infer germination behavior under field conditions. This discussion, however, does not mean that PEG experiments are not useful. On the contrary, although PEG experiments cannot predict the interaction between soil and seed, they provide valuable information about the maximum |h| above which the seed stops absorbing water. Thus, if the soil factor is included in the germination process, then it is necessary to use alternative experimental designs, such as the TG, which considers the hydraulic properties of the seed and the surrounding medium. On the other hand, and as recently reported by Moret-Fernández et al. (under review), PEG experiments are an essential tool to characterize the hydraulic properties of the seeds.
The different shapes of imbibition curves obtained in TG for barley and vetch and 0 and − 0.002 MPa (Fig. 3) suggest that the imbibition process does not only depend on the hydraulic properties of the soil but also the intrinsic hydraulic characteristics of the seeds (Vertucci, 1989; Moret-Fernández et al., under review). Finally, compared to -0.002 MPa, the lower germination percentage obtained in TG-sand and TG-loam at 0 MPa (Table 2), may be due to the lack of soil aeration (Bewley et al., 2013). Note that the soil porosity at 0 MPa is completely filled with water, so the seeds do not germinate because they cannot breathe. However, this does not occur in PEG, because the wet seeds at 0 MPa of water potential are not submerged in water, but placed on a sheet of water.
Usually, wet thermal accumulation models to simulate seed germination are built using PEG to simulate soil water potential or to test osmotic stress on germination of certain weed species. Based on our results, this technique is not accurate enough. But these models are used with some success in predicting seed germination in field (e.g. 60% of successful predictions in Rawlins et al. 2012). This success could be related to the fact that over some potentials [0, -0.01], the PEG experiments present a similar behavior (Fig. 4). Hence, in these cases, PEG would be accurate enough to predict seed germinations. Thus, our findings have the potential to enhance the precision of wet thermal accumulation models. These models traditionally rely on the assumption of a soil water potential threshold below which germination is deemed impossible. This threshold is sometimes arbitrarily set, as using values such as the wilting point or others (Bullied et al. 2012 or Rawlins et al. 2012), or it is derived as the minimum potential required for germination in PEG germination experiments (Boddy et al. 2012, Watt et al. 2010, Patané & Tringali 2010). Our results challenge both of these assumptions. Firstly, we illustrate that the soil threshold potential, at which seeds can no longer imbibe, is contingent upon both seed and soil properties. Consequently, assuming a uniform threshold potential for all soil and seed combinations is not realistic. Secondly, we establish that the thresholds provided by PEG germination experiments are unrealistic because they do not account for soil properties, resulting in excessively negative thresholds that may not correspond to real soil water availability.
This new device presents a solution by facilitating the creation of more realistic thresholds. It considers that, for each unique combination of soil and seed, the threshold potential would vary. This approach acknowledges the inherent variability in seed and soil interactions, leading to a more accurate representation of germination potential under varying conditions. The maximum matric potential allowed by TG was limited by the maximum suction (-0.008 MPa) allowed by the 10 µm pore size nylon mesh used in the experiment. Above this suction, the rupture of the water film contained within the pores of the mesh breaks the vacuum inside the germinator. Although more negative suctions could be obtained by using meshes with smaller porous, this would imply lower permeabilities, which could restrict the replenishment of water lost by evaporation. However, although the employed nylon mesh allowed demonstrating that the hydraulic characteristics of the soils have a significant influence on seed germination, further studies would be necessary to test alternative meshes of smaller pore size, and to verify whether these meshes allow achieving more negative tensions without significantly affecting the mesh permeability. On the other hand, although this device is limited to relatively coarse textured soils, its applications in seed ecology are very broad. Since the unsaturated hydraulic conductivity of sand is very sensitive to h, small variation in h will result in larger changes of K(h) (Fig. 2). This large elasticity would allow, for instance, studying the influence of K(h) on the seed imbibition time or quantifying a hypothetical critical θ for seed germination. In addition, TG could be also used, for instance, to study the strategies of some seed species to improve the seed imbibition under soil hydraulic conductivity restrictions.
This work presents a new device, the tension germinator, which has proven to be a useful tool to experimentally demonstrate the important influence of the substrate properties on seed imbibition during germination. The device opens the possibility of testing the germination of seeds belonging to species with different seed shapes and sizes in different soils under several h. The device, which is inexpensive and easy to implement, present great stability and precision and does not require external electrical vacuum pumps, has proven to be effective in maintaining a constant tension on the soil layer. Although the applied tension is limited to -0.006 MPa, this range has been enough to demonstrate the importance of the substrate on seed germination. Data obtained from PEG experiments, although useful to detect tension thresholds for water absorption by seeds, have severe limitations for predicting seed germination in field conditions because they do not consider soil properties which are critical for seed imbibition.