Indukcija organogeneze in genske transformacije konoplje (Cannabis sativa) in vitro

Arsov, Sasho (author), Murovec, Jana (mentor), Košmelj, Katarina (comentor)

Vzpostavili smo uspešen protokol za gensko transformacijo konoplje (Cannabis sativa L.). Za določitev protokola za adventivno regeneracijo konoplje smo izvedli več poskusov. Najprej smo preizkusili različne regenerativne sposobnosti treh tipov izsečkov (listnih pecljev, listnih izsečkov in internodijev) ter njihov potencial za tvorbo kalusa. Raziskan je bil tudi učinek osnovnih gojišč DKW (Driver in Kuniyuki, 1984) in MS (Murashige in Skoog, 1962). Predmet raziskave je bil tudi vpliv svetlobnega režima na regenerativni potencial listnih pecljev in organogenetski potencial šestih genotipov konoplje za uporabo v medicinske namene ter ene industrijske sorte konoplje. Proučevan je bil tudi vpliv različnih koncentracij fitohormonov, kot so tidiazuron (TDZ), 6-benzilaminopurin (BAP) in 1-naftalenocetna kislina (NAA), da bi našli najbolj obetavno kombinacijo za razvoj kalusa in poganjkov. Listni peclji genotipa BF.KEM1.2 so imeli večji potencial za razvoj kalusa in poganjkov v primerjavi z drugima dvema tipoma izsečkov. Vzpostavljena je bila adventivna regeneracija netransformiranih poganjkov s 5 % uspešnostjo iz listnih pecljev genotipa BF.KEM3 na gojišču DKW z 0,2 mg/l TDZ, 0,5 mg/l NAA in 0,1 μM/l peptida fitosulfokin (PSK). Prisotnost svetlobe je pozitivno vplivala na razvoj poganjkov in kalusa. Nadalje smo listne peclje iz genotipa BF.KEM3 transformirali s kokultivacijo z bakterijo Agrobacterium tumefaciens, ki je vsebovala plazmid pCAMBIA1302-ZsGreen, ki vsebuje v svoji regiji T-DNA selekcijski gen hptII in reporterski gen ZsGreen. Prisotnost reporterskega gena ZsGreen smo preverili z detekcijo zelene fluorescence in verižno reakcijo s polimerazo (PCR) ter ga potrdili v 95 % pregledanih transformiranih kalusnih skupkih, prisotnost gena hptII pa smo potrdili s PCR pri 85 % pregledanih transformiranih kalusnih skupkih.

• Language: Slovenian
• Keywords: Konoplja, tkivne kulture, kalus, adventivna regeneracija, genska tranformacija
• Work type: Master's thesis/paper
• Organization: BF - Biotechnical Faculty
• Year: 2024
• PID: 20.500.12556/RUL-161858
• Publication date in RUL: 15.09.2024

Secondary language

• Language: English
• Title: In vitro induction of organogenesis and genetic transformation of cannabis (Cannabis sativa)

Abstract

We have established a successful protocol for the genetic transformation of cannabis (C. sativa L.). More stages of research were performed to establish the protocol for adventitious regeneration of cannabis. We have first tested the different regenerative potentials of three explant types (petioles, leaf cuttings, and internodes), and their potential to produce callus. The effect of the DKW (Driver in Kuniyuki, 1984), and MS (Murashige in Skoog, 1962) basic media was also explored. The influence of the light regime on the regenerative potential of petioles, and the organogenetic potential of 6 medicinal genotypes one industrial cultivar of cannabis was also the subject of research. The different concentrations of phytohormones thidiazurone (TDZ), 6- benzylaminopurine (BAP), and 1-naphtaleneacetic acid (NAA), were also investigated to find the most promising combination for callus production and shoot development. Petioles from the BF.KEM1.2 genotype, showed the highest potential for callus and shoots development compared with the other two explant types. Adventitious regeneration of non-transformed shoots was also successfully established, with a 5% rate of success, from the petioles of the BF.KEM3 genotype, on the DKW medium supplemented with 0,2 mg/l of TDZ, 0,5 mg/l of NAA, and 0,1 μM/l of the phytosulfokine (PSK) peptide. The presence of light positively influenced the shoots and callus development. Furtherly, petioles from the genotype BF.KEM3 were transformed by co-cultivation with Agrobacterium tumefaciens, which was carrying the pCAMBIA1302-ZsGreen plasmid, which contains in its T-DNA region selection gene hptII and reporter gene ZsGreen. We have confirmed the presence of the ZsGreen reporter gene in 95 % of the examined transformed calli and the presence of the hptII gene was confirmed in 85 % of the examined transformed calli.

• Keywords: Cannabis, tissue culture, callus, genetic transformation, adventitious regeneration