Yaygın fiğ (Vicia sativa l.) çeşitlerinde mikroçoğaltım ve Agrobacterium tumefaciens aracılığıyla Cry1Ac, Cry2A ve bar genlerinin aktarılması

Protein, mineral maddeler ve vitaminler bakimindan oldukca zengin olan yaygin fig bitkisi (Vicia sativa L.)’de rejenerasyon ve genetik transformasyon buyuk bir sorun olarak gorulmektedir. Bu tez kapsaminda yaygin figin Cumhuriyet 99, Karaelci, Kubilay 82 ve Selcuk 99 cesitlerinde surgun rejenerasyonu ve A. tumefaciens araciligiyla transgenik bitki elde edilmesi hedeflenmistir. Yaygin figin dort cesidinde yuzey sterilizasyonda farkli oranlarda camasir suyu kullanilmistir. Daha sonra surgun rejenerasyon calismalarinda farkli oranlarda BAP + NAA iceren MS ortaminda, yarim ve tam kotiledon bogum eksplanti kullanilarak rejenerasyon sistemi gelistirilmistir. Adaksial ve abaksial sekilde kulture alinmis kotiledon eksplantlari arasinda surgun rejenersyonun bakimindan adaksial sekilde kulture alinmasinin daha uygun oldugu gorulmustur. Her dort yaygin fig cesidinde, BAP, SA, GA3 ve BAP + GA3’nin tohumlara priming olarak uygulanmasinin surgun olusumuna olumlu etkileri izlenmistir. Olgunlasmamis embriyo eksplantlarinda, farkli oranlarda BAP + NAA iceren MS ortamlarinda da surgun rejenerasyonu uzerine etkileri incelenmistir. Yukarida belirtilmis tum denemelerden elde edilen surgunler, 0.75 μg/ml IBA iceren MS ortamda koklendirilmis ve dis kosullara adaptasyonda basari saglanmistir. A. tumefaciens’in GV2260 GUS INT, LBA4404 pTF101 AopR1 Cry1Ac bar, LBA4404 Cry2A, GNA, C58C1 pGreen AopR1 Cry1Ac bar ve C58C1 pGreen 35S Cry1Ac bar hatlariyla embriyo, hipokotil ve koltuk alti meristem eksplanti kullanarak Selcuk 99 ve Kubilay 82 cesitlerine gen aktarim yapilmis, T0 ve T1 generasyonuna kadar tohum ve transgenik bitkiler elde edilmistir. Gen aktarim calismasindan elde edilen sonuclar, GUS tesi, PCR testi ve biyoassay ile de dogrulanmistir.AbstractProtein, minerals and vitamins rich common vetch (Vicia sativa L.), is very difficult to regenerate and transform genetically. This thesis aimed to develop protocols for shoot regeneration and Agrobacterium tumefaciens mediated transformation of the plants using common vetch cultivars Cumhuriyet 99, Karaelci, Kubilay 82 and Selcuk 99. These cultivars were surface sterilized using different concentrations of commercial bleach. Thereafter, a regeneration system from ½ and fully developed cotyledon node explants was developed using different concentrations of BAP + NAA in MS medium. Comparing shoot regenration system on adaxially and abaxially cultured cotyledon explants on different concentrations of BAP in MS medium, it seemed as adaxillay cultured explants were more productive. Priming of seeds with different concentrations of BAP, SA, GA3 and BAP + GA3 showed positve effects on regeneration. Similarly, different concentrations of BAP + NAA were used to regenerate immature embryo explants positively. Shoots regenerated on all of the above mentioned experiments were rooted on MS medium containing 0.75 μg/ml IBA and acclimetised to external environmental conditions. Successful genetic transformation was after treatment of embryo, hypocotyl and stem node explants from common vetch cv. Selcuk 99 and the Kubilay 82 with GV2260 GUS INT, LBA4404 pTF101 AopR1 Cry1Ac bar, LBA4404 Cry2A , GNA, C58C1 pGreen AopR1 Cry1Ac, bar and C58C1 pGreen 35S Cry1Ac bar lines of A. tumefaciens. Successfully transformed plants were used to obtain T0 and T1 generations, that floered and set seeds. The results were confirmed using GUS test, PCR test and bioassay.