Electroporation is a physical phenomenon in which the exposure of cells to short but intense
electric pulses results in a transient or permanent increase in cell membrane permeability. When
combined with appropriate viability stains, this method enables a straightforward visual and
quantitative assessment of tissue damage. In this study, potato tubers (Solanum tuberosum L.)
were chosen as the model system due to their anatomical homogeneity, high water content,
turgidity, and strong color contrast after staining with TTC, making them ideal for evaluating
electroporation effects.
Experiments were performed using a BTX Gemini X2 generator, applying square-wave pulses
of 100 μs duration at a frequency of 1 Hz, with voltages of 200, 400, 600, 800, and 1000 V, and
two pulse counts (1 and 8). Following treatment, the samples were incubated in a solution of
2,3,5-triphenyltetrazolium chloride (TTC) with 0.75% citric acid at refrigerator temperature for
24 hours, enabling selective staining of metabolically active cells into the red pigment
triphenylformazan (TPF).
Captured images of the stained samples were analysed using a custom Python script involving
RGB-to-HSV color space conversion, red hue thresholding, segmentation, and quantification
of the proportion of viable tissue. In addition to red-stained regions, yellow-hued areas were
also considered, representing partially damaged yet metabolically active tissue, thus providing
a more comprehensive evaluation of electroporation effects. Furthermore, the coefficient of
variation of staining intensity was calculated as an indicator of the spatial uniformity of the
electroporation effect. Results demonstrated that increasing voltage and pulse number
systematically reduced the proportion of viable tissue, while increasing staining heterogeneity,
indicating uneven electric field distribution and progressive tissue damage.
The study confirms that the combination of electroporation and TTC staining provides a cost-
effective, rapid, and reproducible method for assessing plant tissue viability. The method is
well-suited for further optimization and adaptation to educational, research, and industrial
contexts.
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