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Alteration of flower color of Iris germanica L. through genetic modification of carotenoid biosynthetic pathway

dc.contributor.advisorRadović, Svetlana
dc.contributor.otherSimonović, Ana
dc.contributor.otherSubotić, Angelina
dc.contributor.otherGiba, Zlatko
dc.creatorJeknić, Zoran D.
dc.date.accessioned2016-01-05T11:48:26Z
dc.date.available2016-01-05T11:48:26Z
dc.date.available2020-07-03T08:11:07Z
dc.date.issued2013-07-10
dc.identifier.urihttp://eteze.bg.ac.rs/application/showtheses?thesesId=1086
dc.identifier.urihttp://nardus.mpn.gov.rs/handle/123456789/2156
dc.identifier.urihttps://fedorabg.bg.ac.rs/fedora/get/o:7789/bdef:Content/download
dc.identifier.urihttp://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=45059087
dc.description.abstractBoja cveta perunike (Iris germanica L.) je određena na osnovu dva odvojena biohemijska puta sinteze biljnih pigmenata. Na osnovu biosinteze karotenoida dobijaju se cvetovi žute, narandžaste i ružičaste boje, dok biosinteza antocijanina dovodi do plave, ljubičaste i bordo boje cvetova. Crvene perunike ne postoje u prirodi, i do danas nisu dobijeni kultivari sa ovom bojom primenom konvencionalnih načina oplemenjivanja. U ovoj disertaciji ispitana je mogućnost primene metoda genetičkog inženjeringa sa ciljem dobijanja perunika sa crvenim cvetovima. Za ostvarenje ovog osnovnog cilja razvijeni su protokoli za regeneraciju perunike (I. germanica L. ‘Skating Party’) iz ćelijskih suspenzija, protokoli za efikasnu genetičku transformaciju kao i genetičku transformaciju sa ciljem modifikacije biosintetskog puta karotenoida kod perunika. Za genetičku transformaciju perunika su korišćene dve nezavisne metode, jedna zasnovana na biolističkoj transformaciji (bombardovanje mikroprojektilima) pomoću PDS-1000/He (Particle Delivery System) i druga zasnovana na Agrobacterium-posredovanoj transformaciji. Ovo su prve poznate metode za genetičku transformaciju vrsta roda Iris, koji će omogućiti dalje oplemenjivanje ovih ukrasnih vrsta primenom savremenih molekularnih metoda. Protokol zasnovan na Agrobacterium-posredovanoj transformaciji se pokazao kao bolji od biolističke transformacije u smislu efikasnosti i jednostavnosti primene. Ispitana je osetljivost ćelija perunika na dejstvo nekoliko antibiotika i pokazano je da su higromicin i gentamicin (G418) najpogodniji za selekciju transformisanih ćelija. Soj A. tumefaciens LBA 4404 koji nosi superbinarni vektor pTOK233 je bio najefikasniji za transformaciju perunika. Više od 300 morfološki normalnih transgenih perunika je dobijeno posle 6 meseci. Oko 80% regeneranata je pokazalo pozitivnu reakciju za marker gen ß-glukuronidazu (GUS) i gen za neomicin fosfotransferazu II (NPTII) (paromomicin-rezistencija) koji se nalaze na plazmidu za transformaciju. Integracija transgena u genom jedra transformisanih perunika je potvrđena Southern blot-analizom. Sa ciljem razvoja metoda za modifikaciju karotenoidnog biosintetskog puta kod perunika, prvo je transformisan kultivar ‘Fire Bride,’ koji ima ružičaste cvetove sa bakterijskim genom za fitoen sintazu (crtB) iz Pantoea agglomerans pod kontrolom promotorskog regiona gena za kapsantin-kapsorubin sintazu iz Lilium lancifolium (PLlccs). Ovaj pristup imao je za cilj da poveća protok prekursora karotenoida i dovede do povećanja koncentracije likopena i na taj način izazove pojavu tamnijih nijansi ružičaste ili crvenih nijansi cvetova perunika. U kalusnim kulturama ovog kultivara došlo je do povećane ekspresije crtB gena i promene boje kalusa od žute u ružičasto-narandžastu i nijanse crvene usled povećane akumulacije likopena. Cvetovi transgenih biljka dobijenih iz transgenih kalusa ovog kultivara su pokazali značajniju promenu boje plodnika i cvetnih drški (od zelene ka narandžastoj) kao i antera (od bele ka ružičastoj). Nisu uočene značajnije promene u boji perigona cvetova (standardima i folovima) u odnosu na kontrolne biljke. Rezultati ukazuju da su ovi geni u zelenim delovima cvetova perunike pod drugačijom regulacijom od delova cveta koji nisu zeleni. Kao generalni zaključak dobijamo da se ektopijska ekspresija gena crtB može uspešno koristiti za delimično menjanje boje delova cvetova kultivara perunike ‘Fire Bride’ ali nije najpogodaniji metod za značajnije menjanje boje u standardima i folovima...sr
dc.description.abstractFlower color in irises (Iris germanica L.) is determined by two distinct biochemical pathways. The carotenoid biosynthetic pathway generates yellow, orange and pink flowers, while the anthocyanin biosynthetic pathway produces blue, violet and purple flowers. Red iris flowers do not exist naturally, and conventional breeding methods have thus far failed to produce them. This study examines genetic engineering approaches as a potential avenue for the development of a red iris that has not yet been explored. In the course of the work, protocols were developed for genetic transformation of I. germanica, and genetic modifications to the carotenoid biosynthetic pathway in irises. Two independent methods for the genetic transformation of irises were developed; one based on biolistic (microprojectile bombardment) using PDS-1000/He (Particle Delivery System), and the other using Agrobacterium-mediated transformation. These are the first known protocols for the genetic transformation of I. germanica, which will allow this ornamental crop to benefit from modern molecular applications. The Agrobacterium-mediated protocol demonstrated significant advantages in both efficiency and ease of use over the biolistic transformation protocol. In the course of development of the Agrobacterium-mediated protocol, a series of selection agents were tested. Hygromycin and geneticin (G418) were identified as the most suitable antibiotics for selecting transformed iris cells. A. tumefaciens strain LBA4404, carrying the superbinary vector pTOK233, was identified as the most efficient strain/vector combination for transformation of iris, of those tested. More than 300 morphologically normal transgenic iris plants were obtained in about 6 months. About 80% of the regenerants tested positive for the β-glucuronidase (GUS) marker gene and the neomycin phosphotransferase II (NPTII) (paromomycin-resistance) gene carried by the transformation plasmid. The integration of the transgenes into the nuclear genome of the transformed iris plants was confirmed by Southern blot analysis. With the goal of developing methods for modifying the carotenoid biosynthetic pathway in irises using genetic engineering a pink iris cultivar, ‘Fire Bride,’ was transformed with the bacterial phytoene synthase gene (crtB) from Pantoea agglomerans under control of the promoter region of a gene for capsanthin-capsorubin synthase from Lilium lancifolium (PLlccs). This approach aimed to increase the flux of carotenoid precursors into the carotenoid biosynthetic pathway, and ultimately lead to elevated levels of lycopene, thus generating shades of darker pink or red in the flowers. Iris callus tissue overexpressing the crtB gene showed a color change from yellow to pink-orange and red, which was shown to be due to an increased accumulation of lycopene. Transgenic plants regenerated from the calli showed prominent color changes in the ovaries (green to orange), flower stalk (green to orange), and anthers (white to pink). However, the color of the standards and falls showed no significant differences when compared to the control plants. This suggests that carotenogenesis in non-green parts of iris flowers is regulated differently compared to the green tissues. Altogether the results demonstrated that ectopic expression of a bacterial phytoene synthase gene can be used to successfully alter the color of some flower parts in I. germanica ‘Fire Bride’ and produce new flower traits...en
dc.formatapplication/pdf
dc.languagesr
dc.publisherУниверзитет у Београду, Биолошки факултетsr
dc.rightsopenAccessen
dc.sourceУниверзитет у Београдуsr
dc.subjectbiolistička transformacijasr
dc.subjectbiolistic transformationen
dc.subjectAgrobacterium-transformacijasr
dc.subjectCRT gensr
dc.subjectkapsantinsr
dc.subjectkapsorubinsr
dc.subjectLlccs gensr
dc.subjectAgrobacterium-transformationen
dc.subjectCRT genen
dc.subjectcapsantinen
dc.subjectcapsorubinen
dc.subjectLlccs geneen
dc.titlePromena boje cveta perunike (Iris germanica L.) genetičkom modifikacijom biosinteze karotenoidasr
dc.titleAlteration of flower color of Iris germanica L. through genetic modification of carotenoid biosynthetic pathwayen
dc.typedoctoralThesis
dc.rights.licenseBY-NC-ND
dcterms.abstractРадовић, Светлана; Гиба, Златко; Суботић, Aнгелина; Симоновић, Aна; Јекнић, Зоран Д.; Промена боје цвета перунике (Ирис германица Л.) генетичком модификацијом биосинтезе каротеноида; Промена боје цвета перунике (Ирис германица Л.) генетичком модификацијом биосинтезе каротеноида;
dc.identifier.fulltexthttp://nardus.mpn.gov.rs/bitstream/id/2454/Disertacija.pdf


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