magistrsko delo
Aleks Bordon (Author), Jana Murovec (Mentor)

Abstract

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.

Keywords

Cannabis sativa;industrijska konoplja;medicinska konoplja;genska transformacija;Agrobacterium tumefaciens;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [A. Bordon]
UDC: 602.6:582.630.1(043.2)
COBISS: 30091523 Link will open in a new window
Views: 605
Downloads: 210
Average score: 0 (0 votes)
Metadata: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Other data

Secondary language: English
Secondary title: Gene transformation experiments on cannabis (Cannabis sativa L.) using Agrobacterium tumefaciens
Secondary 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.
Secondary keywords: industrial hemp;medical hemp;gene transformation;
Type (COBISS): Master's thesis/paper
Study programme: 0
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Oddelek za agronomijo
Pages: VIII, 29 f.
ID: 12046359