Možnosti genske transformacije navadne konoplje (Cannabis sativa L.) z uporabo bakterije Agrobacterium tumefaciens

Bordon, Aleks (author), Murovec, Jana (mentor)

Razvili smo uspešno metodo za odpravo okužb semen navadne konoplje (Cannabis sativa L.) in hkrati uspešno transformacijo hipokotilov, s katero smo v celice vnesli selekcijski gen hptII in reporterski gen ZsGreen, ki preko proteina izraža zeleno fluorescenco. Genske transformacije so se izvajale s tri dnevno kokultivacijo hipokotilov s suspenzijo bakterije Agrobacterium tumefaciens. Adventivno regeneracijo hipokotilov smo dosegli z gojenjem izsečkov na MS gojišču z vitamini (Duchefa, M0222), 30 g/l saharoze, 8 g/l agarja, 0,2 mg/l hormona TDZ, 0,5 mg/l hormona NAA, 200µM acetosiringona in pH 5,8 (t.i. gojišče KON3). Uspešnost transformacije smo potrdili z uporabo epifluorescentne lupe, saj so regenerirani hipokotili svetili fluorescentno zeleno. V drugem poskusu, kjer smo narezane hipokotile inokulirali na KON3 gojišče, smo preverjali ali oddaljenost izsečka od koreninskega vratu hipkotilov vpliva na adventivno regeneracijo tkiva. Ugotovljeno je bilo, da razlik v regeneraciji ni bilo in, da je za uspeh poskusa ključna izbira nepoškodovanih semen, ki imajo značilno marmoriran vzorec in nerazpokano semensko ovojnico. V nasprotnem primeru pride do večjega števila bakterijskih in glivičnih okužb predvsem endogenega izvora, saj le-teh ni mogoče odpraviti s klasičnim površinskim razkuževanjem.

• Language: Slovenian
• Keywords: Cannabis sativa, industrijska konoplja, medicinska konoplja, genska transformacija, Agrobacterium tumefaciens
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [A. Bordon]
• Year: 2020
• PID: 20.500.12556/RUL-120652
• UDC: 602.6:582.630.1(043.2)
• COBISS.SI-ID: 30091523
• Publication date in RUL: 24.09.2020

BORDON, Aleks, 2020, Možnosti genske transformacije navadne konoplje (Cannabis sativa L.) z uporabo bakterije Agrobacterium tumefaciens [online]. Master’s thesis. A. Bordon. [Accessed 3 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=120652

Secondary language

• Language: English
• Title: Gene transformation experiments on cannabis (Cannabis sativa L.) using Agrobacterium tumefaciens

Abstract

We have developed a successful method for the elimination of cannabis seed infections (Cannabis sativa L.) and at the same time the successful transformation of hypocotyls, with which we introduced the hptII selection gene and the ZsGreen reporter gene into plant cells, which expresses green fluorescence via protein. Gene transformations were performed by three-day cocultivation of hypocotyls with Agrobacterium tumefaciens. Adventitious regeneration of callus was achieved by growing explants on MS medium supplemented with vitamins (Duchefa, M0222), 30 g/l sucrose, 8 g/l agar, 0.2 mg/l TDZ, 0.5 mg/l NAA, 200µM acetosiringone and pH 5.8 (the KON3 medium). The success of the transformation was confirmed using an epifluorescent macroscope, as the regenerated hypocotyls glowed fluorescent green. In another experiment, where sliced hypocotyls were placed on KON3 medium, we examined whether the position of explants on the hypocotyl influenced adventitious regeneration. It was found that there were no differences in regeneration. For the success of the experiment, we found that the key is to choose undamaged seeds with a characteristically marbled seed coat pattern. Otherwise, a larger number of bacterial and fungal infections, mainly of endogenous origin, occur, as these cannot be eliminated by classical sterilization.

• Keywords: Cannabis sativa, industrial hemp, medical hemp, gene transformation, Agrobacterium tumefaciens