Analiza metilacijskih profilov DNA navadne konoplje (Cannabis sativa L.) gojene in vivo in in vitro

Troha, Simon (author), Murovec, Jana (mentor)

Metoda, ki temelji na polimorfizmu metilacijsko specifičnih pomnoženih fragmentov (angl. methylation sensitive amplification polymorphism, MSAP) se uporablja za ugotavljanje metilacijskih sprememb pri organizmih. Sestoji iz encimske razgradnje DNA, selektivne pomnožitve fragmentov in elektroforeznega pregleda pomnožkov, kateri tvorijo vzorčno specifične metilacijske profile za medsebojno primerjavo. Metoda je uporabna za zagotavljanje kakovosti in vitro kultur, kjer je poleg genetskih sprememb potrebno spremljati spremembe tudi na epigenetskem nivoju. V magistrskem delu predstavljamo naš pristop izvedbe analize MSAP na rastlinskem materialu navadne konoplje (Cannabis sativa L.) gojene in vivo in in vitro. Primerjali smo metilacijske profile vzorcev C. sativa z namenom ugotavljanja epigenetske stabilnosti pri vegetativnem razmnoževanju z metodo zelenih potaknjencev v in vivo pogojih in pri različnih ciklih zaporednih subkultivacij na podlagi regeneracije stranskih brstov nodalnih izsečkov v in vitro pogojih. Pri analizi rastlin gojenih v in vivo pogojih smo zaznali statistično značilne razlike med posameznimi kloni. V drugem delu analize so rezultati pokazali statistično značilne razlike v metilacijskih profilih med vzorci donorskih rastlin gojenimi in vivo v rastlinjaku in prvo generacijo poganjkov in vitro, medtem ko med prvim in petim ciklom subkultivacije ni bilo zaznane statistično značilne razlike. Rezultati tako nakazujejo, da se razlike v fizikalnih pogojih rasti (in vivo vs. in vitro) in razvojne faze rastlin odražajo v metilacijskem profilu ter da vrsta C. sativa ohranja epigenetsko integriteto tekom mikropropagacije.

• Language: Slovenian
• Keywords: Cannabis sativa, metilacija, MSAP, subkultivacija, regeneracija, in vivo, in vitro
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Year: 2022
• PID: 20.500.12556/RUL-142009
• COBISS.SI-ID: 126012419
• Publication date in RUL: 15.10.2022

Secondary language

• Language: English
• Title: Analysis of DNA methylation profiles of Cannabis sativa L. cultured in vivo and in vitro

Abstract

Methylation Sensitive Amplification Polymorphism (MSAP) is a method for assessing DNA methylation changes in organisms. After restriction digestion, the method involves electrophoresis based visualization of PCR fragments from selectively amplified DNA and analysis of differences between sample specific methylation profiles. The method can be used for quality assurance of in vitro cultures where changes also need to be monitored at the epigenetic level and not only at the genetic level. In this master’s thesis, we present our approach to perform MSAP analyzes on in vivo and in vitro cultures of C. sativa L. We compared methylation profiles of C. sativa samples with the aim of evaluating their epigenetic stability during in vivo clonal propagation from stem cuttings and during successive in vitro subcultures based on shoot organogenesis from nodal explants. When analyzing the in vivo plants, we observed significant differences between methylation profiles of C. sativa clones. Regarding in vitro analysis there was an expected significant difference between methylation profiles of donor plants grown in vivo and subsequent in vitro cultures, due to different environmental conditions and plant developmental stages, while we did not detect significant differences in methylation profiles between plantlets from the 1st and the 5th subculture. These results indicate that C. sativa maintains its epigenetic integrity throughout micropropagation cycles.

• Keywords: Cannabis sativa, methylation, MSAP, subcultivation, regeneration, in vivo, in vitro