Vzpostavitev metode izolacije in transformacije protoplastov konoplje (Cannabis sativa)

Krnc, Andraž (author), Murovec, Jana (mentor)

Preurejanje genoma protoplastov konoplje (C. sativa L.) je pomembno orodje za doseganje produkcije želenih sekundarnih metabolitov. Cilj naše raziskave je bil vzpostaviti učinkovit protokol izolacije in transformacije protoplastov konoplje, pri čemer smo želeli preveriti vpliv različnih dejavnikov. Primerjali smo uporabo različnih genotipov konoplje, načinov gojenja izbranega rastlinskega tkiva ter pogoje encimolize. Z optimizacijo teh parametrov smo uspeli iz 1 g rastlinskega materiala izolirati od 2,0*10⁶ do 1,2*10⁷ protoplastov z živostjo med 65 in 75 %, kar je več v primerjavi z rezultati drugih raziskovalcev, ki so protoplaste izolirali iz konoplje, in rezultati nekaterih drugih rastlinskih vrst. Pri protokolu transformacije smo preverjali vpliv koncentracije plazmida in PEG na živost protoplastov ter časa tretiranja protoplastov na uspešnost transformacije protoplastov. Ugotovili smo, da plazmid in PEG nimata negativnega vpliva na živost protoplastov. Zaradi pojava avtofluorescence protoplastov smo za ugotavljanje uspešnosti transformacije izvedli qPCR analizo izražanja gena gfp in ugotovili, da je bila transformacija uspešna, saj smo v vseh vzorcih zaznali povečano izražanje gena gfp v primerjavi s kontrolo, čas tretiranja pa na uspešnost transformacije ni imel vpliva.

• Language: Slovenian
• Keywords: Cannabis sativa, protoplasti, izolacija, transformacija, preurejanje genoma, kanabinoidi
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Year: 2026
• PID: 20.500.12556/RUL-187880
• COBISS.SI-ID: 291281667
• Publication date in RUL: 16.09.2026

Secondary language

• Language: English
• Title: Development of a protoplast isolation and transformation method for Cannabis sativa

Abstract

Genome editing of cannabis (C. sativa L.) protoplasts is an important tool for achieving the production of desired secondary metabolites. The aim of our study was to establish an efficient protocol for the isolation and transformation of cannabis protoplasts while evaluating the influence of various factors. We compared the use of different cannabis genotypes, cultivation methods of the selected plant tissue, and enzymolysis conditions. By optimizing these parameters, we successfully isolated between 2.0*10⁶ and 1.2*10⁷ protoplasts per 1 gram of plant material with a viability ranging from 65 % to 75 %, which is higher than that reported by other researchers who isolated protoplast from cannabis and by studies on some other plant species. In the transformation protocol, we investigated the effect of plasmid and PEG concentrations on protoplast viability, as well as the effect of protoplast treatment duration on transformation efficiency. We found that neither the plasmid nor PEG had a negative effect on protoplast viability. Due to the occurrence of protoplast autofluorescence, qPCR analysis of gfp gene expression was performed to determine transformation efficiency. The results showed that the transformation was successful, as increased expression of the gfp gene was detected in all samples compared to the control, while treatment duration had no effect on transformation efficiency.

• Keywords: Cannabis sativa, protoplasts, isolation, transformation, genom editing, canabinoids