Študija izvedljivosti zaznave elektroporacije rastlinskih tkiv z indikatorjem celične metabolne aktivnosti

Josifovska, Lina (avtor), Mahnič-Kalamiza, Samo (mentor)

Elektroporacija je fizikalni pojav, pri katerem izpostavljenost celic kratkotrajnim, a intenzivnim električnim pulzom povzroči prehodno ali trajno povečanje prepustnosti celične membrane. V kombinaciji z ustreznimi barvnimi indikatorji vitalnosti omogoča enostavno, vizualno in kvantitativno oceno obsega poškodb tkiva. V pričujoči raziskavi smo kot model uporabili krompir (Solanum tuberosum L.), ki zaradi svoje anatomske homogenosti, visoke vsebnosti vode, turgidnosti in izrazite barvne kontrastnosti po obarvanju s TTC predstavlja idealen sistem za preučevanje učinkov elektroporacije. Eksperimenti so bili izvedeni z napravo BTX Gemini X2, pri čemer smo uporabili pravokotne pulze dolžine 100 μs in frekvence 1 Hz, z napetostmi 200, 400, 600, 800 in 1000 V ter z dvema različnima številoma pulzov (1 in 8). Po tretiranju so bili vzorci inkubirani v raztopini 2,3,5- trifeniltetrazolijevega klorida (TTC) z dodatkom 0,75 % citronske kisline pri temperaturi hladilnika 4 °C, 24 ur, kar je omogočilo selektivno obarvanje metabolno aktivnih celic v rdečo barvo trifenilformazana (TPF). Zajete fotografije obarvanih vzorcev smo analizirali z lastno razvito Python skripto, ki je vključevala pretvorbo iz RGB v HSV barvni prostor, pragovno filtriranje rdečih odtenkov, segmentacijo in kvantifikacijo deleža vitalnega tkiva. Pri tem smo poleg rdeče obarvanih področij upoštevali tudi rumeno obarvana območja, ki predstavljajo delno poškodovano, a še vedno metabolno aktivno tkivo, kar omogoča celovitejšo oceno učinkov elektroporacije. . Poleg tega smo izračunali koeficient variacije intenzitete obarvanja kot kazalnik prostorske enakomernosti elektroporacijskega učinka. Rezultati so pokazali, da se z naraščanjem napetosti in številom pulzov delež vitalnega tkiva sistematično zmanjšuje, medtem ko se heterogenost obarvanja povečuje, kar kaže na neenakomerno porazdelitev električnega polja in progresivno poškodbo tkiva. Raziskava potrjuje, da kombinacija elektroporacije in TTC barvanja predstavlja stroškovno učinkovito, hitro in ponovljivo metodo za oceno vitalnosti rastlinskega tkiva. Metoda je primerna za nadaljnjo optimizacijo in prenos v izobraževalno, raziskovalno ter industrijsko okolje.

• Jezik: Slovenski jezik
• Ključne besede: elektroporacija, TTC, trifenilformazan, vitalnost tkiva, HSV analiza, heterogenost
• Vrsta gradiva: Diplomsko delo/naloga
• Organizacija: FE - Fakulteta za elektrotehniko
• Leto izida: 2025
• PID: 20.500.12556/RUL-172830
• Datum objave v RUL: 11.09.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Feasibility study of plant tissue electroporation detection using a cell metabolism indicator

Izvleček

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.

• Ključne besede: electroporation, TTC, triphenylformazan, tissue viability, HSV analysis, heterogeneity