## 4 Discussion  
This study investigated the effects of PEF parameters on the electrical conductivity of potato tissue using open-ended four-probe coaxial cable EIS. We have shown that untreated potato tissue can be considered electrically homogeneous (\(p>0.9\)). By covering a wide range of pulse parameters, our study gives indications for future studies in potato as a plant-based PEF model. Throughout four sets of PEF experiments that varied different  
Figure 7: Analysis of potato tissue electrical conductivity across 1 Hz to 5 MHz frequencies at pulse intervals from 50 to 500 ms (N = 6). Three-dimensional conductivity map with standard deviation (**a**) and mean conductivity plots (**b**) for different pulse intervals (N = 6). Three-dimensional plots of relative conductivity with standard deviation (**c**) and mean relative conductivity (**d**) compared to a control group (“CrIt”) for different pulse intervals. treatment parameters, 1000 Hz was found to be the optimal frequency decade in most cases, as it yielded the maximal measured relative conductivity changes. However, in some experiments, 10 Hz resulted in the maximum conductivity changes (Table 2). At 1000 Hz, significant differences between an untreated control region and the PEF-treated regions were found from PV = 200 V, NP = 10, PW = 10 \(\upmu\)s, and PI = 50 ms onwards, respectively, which comprised nearly all the investigated pulse parameter ranges. When varying the pulse voltage, the smallest \(p\)-value was found for PV = 700 V, corresponding to a mean conductivity increase from 0.025 \(\pm\) 0.004 to 0.337 \(\pm\) 0.060 S/m (\(p\) = 0.0007). When varying the number of pulses, the smallest \(p\)-value (at 1000 Hz) was found for NP = 70, resulting in a mean conductivity increase from 0.030 \(\pm\) 0.006 S/m to 0.491 \(\pm\) 0.083 S/m (\(p\) < 0.004). When varying the pulse width, the smallest \(p\)-value was found for PW = 70 \(\upmu\)s, corresponding to a mean conductivity increase from 0.031 \(\pm\) 0.004 to 0.427 \(\pm\) 0.04 S/m (\(p\) < 0.0001). Lastly, when varying the pulse interval, the smallest \(p\)-values were found for PI = 250 ms and PI = 500 ms, corresponding to a mean conductivity increase from 0.031 \(\pm\) 0.003 S/m to 0.459 \(\pm\) 0.044 S/m (\(p\) < 0.0001) and to 0.400 \(\pm\) 0.032 S/m (\(p\) < 0.0001), respectively. In this study, we selected pulse parameters around the most reported ones for clinical IRE treatments, which are 100 \(\upmu\)s pulse width and 1000 V/cm electric field strength [8; 68]. While we intentionally employed \"traditional\" pulse protocols, recent studies have achieved nanopore formation by using nanosecond pulses with electric field strengths of 15-30 kV/cm (NP = 800, PW = 300 ns) [69]. Studies showed that a single pulse, even with supercritical amplitude, may not cause irreversible membrane rupture, indicating that the number of pulses (NP) is one of several parameters influencing membrane response [1]. This well-known behaviour was replicated with our setup: our results did not show any significant conductivity changes for the smallest number of pulses (NP = 5). In this study, we used high-voltage unipolar square wave pulses in line with other PEF studies [70]. This study focused on pulse intervals greater than 50 ms, although previous studies suggest that shorter intervals (<1 ms) may further enhance PEF treatment efficacy [71]. We observed significant conductivity changes at all pulse intervals up to 500 ms. The stability of tissue temperature post-PEF treatment, as observed in Supplementary Figure S18, shows an advantage of irreversible electroporation (IRE) over thermal ablation methods such as radiofrequency ablation (RFA). Across varied PEF parameters, tissue temperatures remained close to room temperature, indicating minimal thermal effects. This supports the potential of IRE for applications where heat-induced damage is a concern. Plant-based models were chosen for this study to accommodate the need for a larger number of samples in line with the 3R policy on animal research [34; 35].