Introduction

Exposure of cell membranes to sufficiently high external electric field results in increase of conductivity and permeability of cells due to the creation of pores in the cell membrane (Neumann and Rosenhec 1972; Neumann et al. 1982; Zimmermann et al. 1974; Zimmermann and Vienken 1982). Depending on the severity of the applied parameters, this process can be irreversible (Phillips et al. 2011; Rowan et al. 2000) or reversible (Benz and Zimmermann 1981; Glaser et al. 1988). The number of applications of electroporation in different fields, including food processing, is rapidly expanding (Satkauskas and Saulis 2004; Saulis 2010). However, in food processing, pulse electric field (PEF) is mostly used in its irreversible form to inactivate microorganisms (Evrendilek et al. 1999; Qin et al. 1996, 1998) or to improve extraction yield (Bouzrara and Vorobiev 2000; Schultheiss et al. 2002; Vorobiev et al. 2005).

To the best of our knowledge, reversible electroporation in food processing has been poorly investigated even though promising applications have been reported, such as improvement of freezing tolerance of spinach leaves after PEF and vacuum impregnation treatment (Phoon et al. 2008). The response of plant tissues to PEF under reversible conditions is not well understood.

A number of parameters influence the electroporation and the reversibility of the process, and these have been investigated using different model organisms. Pulse polarity was reported to be one of the crucial parameters for the efficiency of electropermeabilization. Some studies on PEF in food processing have shown that bipolar pulses are more efficient in inactivation of microorganisms than monopolar pulses (Qin et al. 1994; Vorobiev and Lebovka 2009; Wouters and Smelt 1997) as bipolar pulses lead to structural fatigue of the membrane and hence easier electrical breakdown (Qin et al. 1994). However, other authors show that monopolar pulses are as effective as bipolar ones in inactivation of spoilage microorganisms occurring in alcoholic beverages (Beveridge et al. 2003). The effects of monopolar and bipolar pulses are also influenced by the material under investigation; apparently, there were no differences between mono- and bipolar treatment in the survival rate of Escherichia coli in apple juice, whereas in milk bipolar pulses were more efficient for the same purpose (Evrendilek and Zhang 2005). The effectiveness of PEF in the inactivation of spoilage microorganisms is influenced by the pulse duration of mono- or bipolar pulses; for short pulses (1, 2, or 3 μs), monopolar treatment is more efficient, while for longer (4 μs) pulses there is no significant difference in the inactivation of microorganisms between mono- or bipolar treatment. As the polarity changes in bipolar pulses, short pulse durations might not be enough for obtaining the desired disrupting effect on the membrane. However, for longer pulses, the duration of the pulse in any direction might be sufficient to obtain irreversible electroporation (Beveridge et al. 2005). Lower pulse amplitude was required for achieving the same number of permeabilized cells for monopolar pulses compared with bipolar pulses, but pulse polarity did not influence the survival of the cells (Kotnik et al. 2001). In other studies, Tekle et al. (1991) showed that the permeabilization of NIH 3T3 (mouse embryonic fibroblast cells) cells is comparable for mono- and bipolar treatments, while survival levels are higher for bipolar pulses.

Another parameter influencing the effectiveness of electropermeabilization of the membrane is the pulse duration compared at the same pulse amplitude. Some studies reported that inactivation of E. coli is more effective at longer pulses (Martín-Belloso et al. 1997), while others showed small differences in the survival of E. coli depending on different pulse durations (Mañas et al. 2001). Pulse duration also influences the uptake of macromolecules by mammalian cells and has an impact on their survival (Rols and Teissié 1998). Longer pulses provoked more efficient permeabilization of DC3F cells (Lebar and Miklavčič 2001).

Pulse frequency is another meaningful parameter in the electroporation process. Theoretical modeling demonstrated that the degree of electroporation decreases with increasing frequency (Bilska et al. 2000). The PEF-induced breakage in onion tissue caused by monopolar pulses at different frequencies was studied by Asavasanti et al. (2011) and the results showed that a larger number of cells are permeabilized irreversibly at low frequency.

To the best of our knowledge, studies investigating the influence of different parameters on permeabilization and survival were generally based on single cells. Plant tissues have been studied mostly in terms of tissue damage (Ben Ammar et al. 2011), and information on preservation of viability at different PEF protocols is lacking.

The plant tissue is characterized by a heterogeneous structure with a high degree of anisotropy. Cell size, cell size distribution, and cell orientation have an impact on the effectiveness of electropermeabilization (Ben Ammar et al. 2011; Chalermchat et al. 2010). Therefore, uniform electropermeabilization of such a morphologically differentiated structure of plant tissue represents a technological challenge. Among these heterogeneous plant materials, leaves consist of a lower and upper epidermis, which is the outer single cell layer tissue. The internal tissue is called mesophyll (parenchyma) and is divided into two types: palisade and spongy. Palisade mesophyll with closely packed cells is located under the upper epidermis and is followed by a layer of spongy mesophyll with large air spaces between cells (Hill et al. 1987).

The aim of this paper was to examine the influence of PEF parameters on the reversible electroporation of rucola leaves. Five different protocols were compared to study the influence of pulse polarity, intervals between pulses, and number of pulses on the reversible permeabilization of this plant tissue as indicated by the occurrence of electroporation and preservation of viability of the treated tissue. The survival of the leaves could not be predicted by measurements of electrical resistance or permeabilization levels of the leaf surface, suggesting that cells were killed due to metabolic consequences induced by the electric pulses, probably after pore resealing.

Materials and Methods

Raw Material

Rucola leaves were grown in the fields in the south of Sweden. Leaves were collected from the field and stored at 1–2 °C (90–95 % RH) before packaging. These are conditions routinely practiced by the producer. After delivery of the leaves to our laboratory, they were stored at 1–2 °C in closed plastic bags and used for the experiments until the expiration date established by the producer (9 days from the packaging date). Leaves of similar size, 6.0 ± 0.5 cm in length and 2.5 ± 0.3 cm in width, were chosen for use in the experiments.

Sample Preparation

Rectangular samples, 15 mm in length and 7 mm in width, were cut from rucola leaves using a sharp blade. Samples were cut from the main vein in the central part of the leaf towards the edge (Fig. 1).

Fig. 1
figure 1

Sampling of the rucola leaf

Full size image

Electrical Treatments

Rectangular samples were placed in an electroporation cuvette (Cell Projects, Harrietsham, UK; 4 mm gap, 22 mm in length, and 12 mm in width). The cuvette was filled, totally covering the electrodes, with deionized water with conductivity adjusted to 130 μS/cm with NaCl. The electrical conductivity of the solutions was measured with an Orion 150 conductivity meter (Orion Research Inc., Jacksonville, FL, USA). Electric pulses were delivered by a CythorLab electroporation unit (ADITUS AB, Lund, Sweden). Five different protocols were used and are presented in Fig. 2. Based on preliminary experiments establishing permeabilization conditions for rucola leaves, the electric field strength was set to 600 V/cm and the number of pulses used for the treatments was 25, 50, 75, 100, 250 and 500, respectively. Pulses were delivered in one train. The duration of a bipolar pulse is reported here as the duration of the positive or the negative part of the pulse (Fig. 2). The comparison between mono- and bipolar pulses was based on the total treatment duration (calculated by multiplying the number of pulses by their duration) instead of the total number of pulses.

Fig. 2
figure 2

Schematic representation of investigated pulsed electric field protocols. I monopolar square pulses with field width of 250 μs and 1-ms intervals between pulses, II bipolar square pulses with field width of 250 μs and 1-ms intervals between pulses, III bipolar square pulses of 125 μs with 1-ms intervals, IV bipolar square pulses of 125 μs with no intervals, V bipolar square pulses with field width of 125 μs and 2-ms intervals between pulses

Full size image

Analysis

Microscope Examination of Electroporation and Cell Survival

The effect of pulsed electric field treatments on electroporation and tissue survival was tested with fluorescent microscopy. Cell electroporation at the surface of the leaves was evaluated with propidium iodide (PI; Sigma-Aldrich, USA, λ ex = 535 nm, λ em = 617 nm), a commonly used test molecule for membrane electroporation that binds to DNA in the cell, increasing its fluorescence by 20–30 times (Pakhomov et al. 2010). The sample was placed in the electroporation cuvette, which was filled with 250 μM PI with conductivity of 130 μS/cm. After application of pulses, treated samples were rinsed with deionized water to remove the excess of the dye from the surface and immediately examined under the microscope. Micrographs were captured in three different areas of the cut sample, chosen randomly but avoiding the edges, with magnification of ×10. The number of PI focal binding sites in the pictures, assumed to be DNA in the nuclei, were counted using ImageJ (Wayne Rasband, MD, USA) software. Pictures were converted to 8-bit type and, after adjustment of contrast and brightness, the nuclei were counted using the “find maxima” option. The noise level was established individually for each picture depending on the picture resolution.

Cell viability was evaluated by fluorescein diacetate (FDA; Sigma-Aldrich, USA, λ ex = 492 nm, λ em = 517) in samples PEF-treated with 130 μS/cm deionized water as described in Phoon et al. (2008) with certain modifications. A stock solution of 12 mM FDA was prepared in acetone and stored at 4 °C. Before the experiments, the stock solution was diluted with deionized water to obtain a final concentration of 12 × 10−4 μM. At least 30 leaves from different batches were investigated in terms of survival for each tested protocol. Samples were incubated in the FDA solution for 30 min in darkness at room temperature. The incubated samples were rinsed with deionized water and examined under fluorescent light in a Nikon upright microscope (Elipse Ti-U, Nikon, Japan) equipped with a Nikon digital camera (digital sight DS-Qi1Mc, Nikon Co., Japan). The survival of samples is expressed as a percentage, which represents the number of samples that fully survived the applied treatments. Typical microscope pictures representing the alive and dead tissue are shown in Fig. 3a, b.

Fig. 3
figure 3

Survival investigation by fluorescent microscopy. a Typical micrograph presenting a living tissue stained with fluorescein diacetate. b Typical micrograph presenting a dead tissue stained with FDA, treated with protocol I, 250 pulses. The scale bar represents 100 μm

Full size image

Electrical Resistance Measurements

Electroporation was evaluated by measuring PEF-induced changes in the electrical resistance of the leaves. The rectangular samples were placed between two flat, parallel stainless steel electrodes (15 × 7 mm) with wet (NaCl solution with 130 μS/cm) filter paper (Qualitative filter paper, grade 5, Munktell Filter AB, Bärenstein, Germany) between the sample and the electrodes. The electrodes were slightly squeezed with a metal clamp to ensure that the entire area of the leaf sample was attached to the electrodes. The initial leaf resistance was measured at a frequency of 100 Hz 1 s before the delivery of pulses. Post-pulse resistance was measured 1 s after PEF treatment. As the resistance was measured through the entire leaf cross section, measurements represent a bulk response of electroporation throughout the leaf.

Statistical Analysis

Statistical analysis of the measured electrical resistance for different protocols was performed using two-way analysis of variance. Analysis was done in Excel (Microsoft Office, Redmond, WA, USA). Two sources of variation were analyzed: the applied PEF protocols and the number of pulses for each of the tested protocols. Tukey’s confidence intervals were used to evaluate true difference in treatment means. The variability of the quantification of PI binding sites in the figures by image analysis was expressed as the standard deviation of the mean.

Results

Reversible electroporation is indicated by the two following criteria: occurrence of electroporation itself and preservation of viability of the treated tissue.

Influence of Pulse Polarity

To examine the influence of pulse polarity on tissue permeabilization and survival, three protocols were investigated: monopolar pulses with field width of 250 μs were compared with bipolar pulses with field widths of 250 and 125 μs (Fig. 2, protocols I, II, and III). Figure 4a shows the changes in electrical resistance with increasing total treatment time in the three examined protocols. Overall, no statistically significant differences (p < 0.05) were found on the levels of permeabilization achieved by the tested protocols. Figure 4b–d shows the resistance and survival changes provoked by these protocols, plotted separately. Twenty-five monopolar pulses reduced the resistance of the leaf by 57 % (Fig. 4b). Resistance decreased slightly when the number of pulses was increased to 500, but survival decreased sharply when more than 25 pulses were applied, reaching 0 % at 250 pulses. Twenty-five bipolar pulses of 250-μs pulse width reduced the electrical resistance by 50 %, at which 100 % survival of the samples was preserved (Fig. 4c). The resistance continued to decrease until 250 pulses, where survival reached 0 %. Twenty-five bipolar pulses of 125-μs pulse width caused a drop of resistance by 50 %, preserving 100 % survival (Fig. 4d). Further increase in number of pulses reduced the resistance by 70 %, maintaining 100 % of sample survival when applying 75 pulses. No further decrease in resistance was observed as the number of pulses increased, but survival decreased, reaching 16 % at 500 pulses.

Fig. 4
figure 4

Influence of pulse polarity on the electrical resistance and survival of rucola leaves. a Decrease of the resistance with increasing number of pulses for three protocols compared: monopolar square pulses with field width of 250 μs with 1-ms intervals between pulses, bipolar square pulses with field width of 250 μs and 1-ms intervals between pulses, and bipolar square pulses with field width of 125 μs and 1-ms intervals between pulses. b Resistance and survival at increasing number of pulses for the protocol using monopolar square pulses with field width of 250 μs and 1-ms intervals between pulses. c Resistance and survival at increasing number of pulses for the protocol using bipolar square pulses with field width of 250 μs and 1-ms intervals between pulses. d Resistance and survival at increasing number of pulses for the protocol using bipolar square pulses with field width of 125 μs and 1-ms intervals between pulses. Each point, joined with a dotted line, represents the average of three electrical resistance measurements. The error bar represents the standard deviation of the mean. Survival, plotted with points joined with a dashed line, is reported as the percentage of samples that fully survived the treatments from at least 30 samples from different packages, treated on different days, as described in “Materials and methods”

Full size image

Intervals Between Pulses

Three different protocols were studied to compare the impact of the different intervals between bipolar pulses with field width of 125 μs on reversible electroporation: pulses without intervals (Fig. 2, protocol IV), 1-ms intervals (Fig. 2, protocol III), and 2-ms intervals between pulses (Fig. 2, protocol V). Figure 5a shows the decrease in electrical resistance with increasing number of pulses for the three protocols. Bipolar pulses without intervals showed statistically significant (p < 0.05) lower resistance levels than the protocols where intervals were introduced. The combined effect of the application of bipolar pulses with field widths of 125 μs without intervals and high number of pulses (250 and 500) resulted in a significant increase (p < 0.05) in the permeabilization level. Figure 5b, c shows the resistance and survival changes for the three tested protocols. Figure 5b shows that 100 % survival was obtained when 25 and 50 pulses were applied, while resistance was reduced by 43 and 44 %, respectively. Further decrease in resistance, up to 76 %, was obtained when applying 75 pulses, obtaining a survival rate of 83 %. Increasing the number of pulses provoked a slight decrease in resistance, while survival was reduced to 16 % with 500 pulses. The protocol with 1-ms intervals between pulses provoked 71 % reduction of the electrical resistance at 50 pulses, maintaining 100 % survival (Fig. 5c). The resistance showed no further decrease, while the survival decreased to 16 % at 500 pulses. The longest tested intervals between pulses, 2 ms, resulted in 100 % viability preservation at 25, 50, and 75 pulses (Fig. 5d) and, even at higher pulse counts, resulted in higher survival than the shorter interpulse intervals, while the electrical resistance was reduced by 59, 60 and 55 %, respectively. Furthermore, resistance decreased slightly, followed by a drastic decrease in survival to 50 % when 500 pulses were applied.

Fig. 5
figure 5

Influence of intervals between pulses on the electrical resistance and survival of rucola leaves. a Decrease of the resistance of rucola leaves with increasing number of pulses for three protocols: bipolar square pulses using continuous 125 μs width pulses, bipolar square pulses with field width 125 μs and 1-ms intervals between pulses, and bipolar square pulses with field width of 125 μs and 2-ms intervals between pulses. b Resistance and survival at increasing number of pulses for the protocol using bipolar square pulses with field width of 125 μs and no intervals between pulses. c Resistance and survival at increasing number of pulses for the protocol using bipolar square pulses with field width of 125 μs and 1-ms intervals between pulses. d Resistance and survival at increasing number of pulses for the protocol using bipolar square pulses with field width of 125 μs and 2-ms intervals between pulses. Each point, joined with a dotted line, represents the average of three electrical resistance measurements. The error bar represents the standard deviation of the mean. Survival, plotted with points joined with a dashed line, is reported as the percentage of samples that fully survived the treatments from at least 30 samples from different packages, treated in different days, as described in “Materials and methods”

Full size image

Microscope Observation of Surface Permeabilization

The electroporation experienced by the samples at the applied PEF conditions is demonstrated by the penetration of propidium iodide and the staining of the DNA in their nuclei, which can be clearly seen in the pictures as bright circles inside the cells (Figs. 6, 7, and 8). In each of the captured areas in Fig. 6 (1.3 × 10−6 μm2), there were approximately 487 ± 103 epidermal cells from the upper side of the leaf and approximately 500 ± 154 epidermal cells from the lower part, including the two guard cells of stomata. The microscope pictures shown in Fig. 6a demonstrate the typical pattern of nuclei staining occurring after bipolar treatment conditions where the upper and lower sides of the leaf were permeabilized. Figure 6b, c shows the results of the application of monopolar pulses where fluorescent nuclei were only detected on the side of the leaf facing the anode. More nuclei were observed for monopolar treatment than for bipolar treatment (Fig. 6b, c). Figures 7 and 8 show a more detailed description of the surface permeabilization, with the main differences observed between mono- and bipolar treatments. Figure 7a shows that the nuclei originating from round mesophyll cells (marked with closed arrows) and puzzle-shaped epidermal cells (marked with open arrows) were observed mainly for monopolar treatment, while for bipolar treatment mostly epidermal cells (marked with open arrows) were visually electroporated (Fig. 7b). Figure 8 shows microscope pictures where higher accumulation of nuclei is seen in the accessory cells around stoma (marked with arrows) for monopolar (right panel) than for bipolar treatment (left panel). Figures 7 and 8 are representative for all tested protocols using mono- and bipolar pulses.

Fig. 6
figure 6

Typical microscope pictures representing the electroporated cells. Electroporation is indicated by the penetration of propidium iodide into the cells and the staining of their nuclei, which can be clearly seen in the pictures as bright circles inside the cells. a Bipolar pulses of field width of 125 μs were applied. b Monopolar pulses were applied. The upper surface of the samples with no fluorescence (left panel) is facing the cathode. c Monopolar pulses were applied. The lower surface of the samples with no fluorescence (right panel) is facing the cathode. Scale bars represent 100 μm. All pictures presented here were taken after 250 pulses were applied; for further details, see “Materials and methods”

Full size image
Fig. 7
figure 7

Microscope pictures showing differences in surface electroporation caused by mono- and bipolar pulses. a Microscope picture showing large puzzle-shaped epidermal cell (marked with open arrows) and round mesophyll cell (marked with closed arrows), of which cell walls and nuclei were stained with propidium iodide after monopolar pulses were applied. b When bipolar pulses were applied, only epidermal cells presented fluorescent nuclei (marked with open arrows), while in mesophyll cells (marked with closed arrows) electroporation was not detected. The pictures shown are representative of all tested protocols using bipolar pulses. The scale bars represent 100 μm

Full size image
Fig. 8
figure 8

Microscope pictures showing higher density of nuclei accumulated around stoma in monopolar treatment (right panel) than in bipolar treatment (left panel). Stomata are marked with arrows. The picture in the left panel is representative of all tested protocols using bipolar pulses. The scale bars represent 10 μm

Full size image

The relationship between the number of nuclei counted in each picture and the electrical resistance for all tested protocols (Figs. 4 and 5) is shown in Fig. 9. For monopolar pulses (Fig. 9a), when the resistance drops to a constant value of around 0.4, the number of nuclei from the bottom and top of the leaf increases from 597 to 1051 from the upper and lower leaf surface. The influence of bipolar pulses with field width of 250 μs on the decrease in the resistance and the number of nuclei that appeared on the surface is shown in Fig. 9b. It can be seen that, for the bottom of the leaf, the resistance was first reduced by 50 % and the number of nuclei was 258. The further decrease in the resistance up to 78 % resulted in an increased number of nuclei, from 410 up to 528. On the top of the leaf, when the resistance was decreased from 50 to 78 %, the nuclei appeared in a narrow range, from 404 to 501.

Fig. 9
figure 9figure 9

Relationship between electrical resistance of the bulk tissue and number of nuclei on the leaf surface. a Monopolar square pulses with field width of 250 μs and 1-ms intervals between pulses. b Bipolar square pulses with field width of 250 μs and 1-ms intervals between pulses. c Bipolar square pulses with field width of 125 μs and 1-ms intervals between pulses. d Bipolar square pulses with field width of 125 μs and no intervals between pulses. e Bipolar square pulses with field width of 125 μs and 2-ms intervals between pulses. Each point represents the average of three electrical resistance measurements. The error bar represents the standard deviation of the mean

Full size image

The protocol using 125-μs-width bipolar pulses with 1-ms intervals between pulses (Fig. 9c) resulted in initial reduction of resistance of 50 %, and 402 nuclei were detected from the top of the leaf and 353 nuclei from the bottom. The resistance values decreased further to approximately 0.3 and the number of nuclei increased from 357 to 542 for the top and the bottom of the leaf. A similar tendency was observed for the protocol using 125-μs bipolar pulses with no intervals (Fig. 9d). The resistance decreased, first by approximately 40 % and then by around 80 %. When the resistance decreased by 40 %, there were none or only few nuclei appearing on the leaf surface (63 on the top and 117 on the bottom). When the resistance decreased by 80 %, the number of permeabilized cells increased from 141 to 483. The protocol using bipolar pulses with field width of 125 μs with 2-ms intervals (Fig. 9e) resulted in a decrease of resistance by 60 %, which was not initially followed by permeabilization of cells. The resistance values did not decrease further when nuclei were detected (from 149 up to 472 from the top and the bottom of the leaf). Comparing mono- with bipolar pulses, it can be seen that monopolar pulses resulted in higher numbers of nuclei, up to 1,051, on the upper and lower surface of the leaf. Bipolar pulses resulted in lower surface permeabilization, up to 542 nuclei at the surface.

The relationship between the number of nuclei appearing on the leaf surface and the survival of the samples subjected to different pulsed electric field protocols is shown in Fig. 10. The influence of monopolar pulses on survival and surface permeabilization is shown in Fig. 10a. Survival of 100 % was maintained when 597 nuclei appeared on the top of the leaf surface and 632 on the bottom. The number of nuclei increased to 997 for the top and 1,051 for the bottom, while survival sharply decreased to 0 %. The influence of bipolar pulses with field width of 250 μs on the survival and number of nuclei that appeared on the surface is shown in Fig. 10b. The number of nuclei increased from 259 to 528, while survival decreased sharply from 100 to 0 %. A similar tendency was observed for the protocol using 125-μs-width bipolar pulses with 1-ms intervals between pulses (Fig. 10c) where the number of nuclei increased in the range from 142 to 542 nuclei, and this increase in the number of permeabilized cells was followed by a sharp decrease in survival, down to 0 %. The protocol using 125-μs bipolar pulses with no intervals (Fig. 10d) resulted in a decrease in survival, while the number of nuclei on the surface increased from 0 up to 461 from the top of the leaf and 546 from the bottom. The protocol using bipolar pulses with field width of 125 μs and 2-ms intervals (Fig. 10e) resulted in preservation of 100 % viability when the number of permeabilized cells increased from 0 to 389 from the bottom and 430 from the top of the leaf. Further increase in the number of nuclei (up to 407 from the top and 472 from the bottom) resulted in a sharp decrease in survival, up to 66 %.

Fig. 10
figure 10figure 10

Relationship between survival of leaves and number of nuclei on the leaf surface. a Monopolar square pulses with field width of 250 μs and 1-ms intervals between pulses. b Bipolar square pulses with field width of 250 μs and 1-ms intervals between pulses. c Bipolar square pulses with field width of 125 μs and 1-ms intervals between pulses. d Bipolar square pulses with field width of 125 μs and no intervals between pulses. e Bipolar square pulses with field of 125 μs and 2-ms intervals between pulses. The error bar represents the standard deviation of the mean of the number of nuclei

Full size image

Discussion

Our results show that, for all tested protocols, there was a rapid decrease in resistance at a low number of applied pulses (Figs. 4 and 5). Interestingly, the decrease in survival was not associated with this initial decrease in resistance or with the permeabilization levels of the leaf surface (Fig. 10).

Two separate mechanisms may be responsible for the decrease in survival level: permanent damage to cell membrane (no resealing) which would prevent the buildup of cell potential difference and thereby life or metabolic consequences provoked by electric pulses, probably occurring after the resealing of pores. Complex metabolic responses of cells exposed to external electric field are not well understood (Gómez Galindo et al. 2008). After resealing, the recovery of cells depends on their ability to achieve biochemical balance after PEF-induced intense influx and efflux of ions and molecules have taken place. If that balance is not attained, cell death may occur (Weaver 2000). Other factors that may contribute to a reduced survival rate are the production of reactive-oxygen species (Gabriel and Teissié 1994) and/or the release of intracellular calcium from mitochondria (Berridge et al. 1998; Buescher and Schoenbach 2003). The survival of the samples may be influenced by the properties of the treated leaf even if leaves were grown under the same conditions in the field. Leaves within the same plant may be at different development stages (Coley 1980) and therefore have variations in thickness and fine structure of certain tissues, i.e., the cuticle (Riederer and Schönherr 1988). Consequently, they may respond differently in terms of electroporation and survival since sensitivity to electroporation depends inter alia on the physiological state of the cells (Hapala 1997). The air spaces expand during the development of the leaf (Psaras and Rhizopoulou 1995), and since air is non-conductive, it may influence the general conductivity, the response of the leaf upon application of PEF, and the capacity of the leaf to recover after stress. However, it was observed that samples either fully survived the treatment or were fully killed by the same applied parameters. We observed no samples with a combination of living and dead cells. It is premature to speculate about pathways that might be involved in the death or survival of cells in the tissue at certain levels of PEF-induced damage. However, the “all or none” nature of the phenomenon, together with the fact that survival did not correlate with resistance, suggests a more subtle mechanism than cell resealing, and this is an attractive subject for further research in the field to improve understanding of survival mechanisms of plant tissue upon PEF treatment.

The recovery of the cell membrane after electroporation is an important factor when investigating the influence of intervals between pulses on electroporation and survival (Chalermchat 2005). We have observed that, for longer intervals between pulses, the survival rate is higher (Fig. 5), suggesting that the longer time between pulses (2 ms) had a beneficial influence on the recovery of cells after electroporation. The distance between pulses may determine the way cells would be affected by a series of pulses (Asavasanti et al. (2011). The pulses could be perceived as separate events or as a long pulse. The dynamics of pores opening and resealing, both during the pulse and the interval, may influence the general recovery and survival after electroporation.

Our results show that microscope observations of the surface electroporation provide additional information not detected by bulk resistance measurements. Results from microscope pictures of samples subjected to monopolar treatment show that cells on the surface are further permeabilized even if the resistance does not decrease further (Fig. 9a). This would suggest that the intact surface layers contribute little to the observed resistance of the leaf and that resistance is dominated by the mesophyll tissue, probably the air-filled spongy mesophyll.

The nuclei density in the sample was significantly higher when monopolar pulses were applied (Fig. 6b, c). In search of an explanation for this high nuclei density, Fig. 8 shows that the stomatal complex may be one source of additional nuclei since higher nuclei accumulation was observed around stoma, mainly in accessory cells when monopolar pulses were applied (right panel) compared to application of bipolar pulses (left panel). The stomatal complex consists of the pore–stoma, guard cells, and accessory cells (Berger et al. 1998). The size of accessory cells is smaller than the ordinary epidermal cell, influencing the effect of the electric field since the electroporation of cells depends on cell size (Ben Ammar et al. 2011; Chalermchat et al. 2010), and larger cells are easier to electroporate (Vorobiev and Lebovka 2009). The higher number of nuclei when monopolar pulses were applied may also originate from the observation of nuclei in the next layer of cells, which could be either palisade or spongy mesophyll depending on the side of the leaf under examination. These results prompt speculation about a possible difference in the effect of monopolar pulses compared with bipolar ones, where monopolar pulses may have specific effects on the cells of certain characteristics and functions in the leaf. We cannot exclude the possibility that the internal organelles containing DNA such as mitochondria and chloroplast, which also contain other DNA than the nuclei (Charlene et al. 1978), may be influenced by monopolar pulses, increasing the number of propidium iodide binding sites in the pictures. The electroporation of internal organelles due to nanosecond PEF is well known (Khan and El-Hag 2011), but in theoretical studies, Esser et al. (2010) reported that with conventional PEF protocols (μs) the membrane of internal organelles can also be electroporated.

Monopolar pulses resulted in detectable permeabilization of the anode-facing surface of the leaf (Fig. 6b, c). The lack of detected nuclei on the cathode-facing side of the leaf may be due to the asymmetrical transport of propidium iodide. There are several reports showing that, under certain conditions, propidium iodide enters single cells through the anode-facing side (Gabriel and Teissié 1997, 1999; Golzio et al. 2002). Golzio et al. (2002) reported that, in Chinese hamster ovary cells, the asymmetrical appearance of propidium iodide was dependent on pulse duration, being visible from both sides of the cells at longer pulses. We have observed that in the tissue the permeabilization of the cathode-facing side was not detected at any of the tested conditions, in agreement with Gabriel and Teissié (1999) who, working with mammalian cells, reported that fluorescence was always present in the cell areas facing the anode. However, the diffusion time needed for propidium iodide to reach all the cells could have also influenced the results. If propidium enters the tissue from the anode-facing side of the leaf, it may not reach the cells at the other side while the pores are still open. Longer pulses could increase the time of propidium movement through electrophoretic forces and enable the propidium iodide to reach the cells at the opposite side of the leaf.

Conclusions

The results presented here provide information about the influence of PEF protocols on the reversible electroporation of rucola leaves. The following remarks summarize important findings:

  1. 1.

    Monopolar pulses resulted in more efficient surface permeabilization than bipolar pulses, but lower survival was obtained when more than 25 pulses were applied.

  2. 2.

    Longer intervals between bipolar pulses resulted in higher viability preservation rates, while surface permeabilization was maintained at the same level for all tested protocols.

  3. 3.

    For all tested protocols, the resistance, considered an indicator of electroporation of the bulk tissue, reached maximum values when 75 pulses were applied, while survival of the leaves was not impaired. An increase in the number of pulses (from 75 to 500) did not increase electroporation even though the survival of the leaves decreased drastically. A direct correlation between surface permeabilization, observed with microscopy, and survival was not found.