Adventitious shoot regeneration and agrobacterium tumefaciens metiated gene transfer in rapeseed (brassicanapus L. ssp. oleifera)

m ADVENTITIOUS SHOOT REGENERATION and Agrobacterium tumefaciens-MEDIATED GENE TRANSFER in RAPESEED (Brassica napus L. ssp.oleifera) (Ph.D. Thesis) Semra MİRİCİ GAZI UNIVERSITY INSTITUTE OF SCIENCE AND TECHNOLOGY April 2000 ABSTRACT In this thesis, the studies related to adventitious shoot regeneration in some important Brassica napus L. cultivars and gene transfer via Agrobacterium tumefaciens have been carried out. In adventitious shoot regeneration studies hypocotyl, 2/3 cotyledons and whole cotyledons with petiol were used as explants. As a result of the experiments with various growth regulators (BAP, kinetin, 2/4 -D, TDZ, NAA) and their concentrations, it was observed that whole cotyledons with petiol was the explant to have the highest frequency in forming adventitious shoot regeneration. Besides, it was realized that Spok cultivar had the highest frequency in shoot regeneration with 43,33 % and MSO containing 2,00 mg/1 or 4,00 mg/1 BAP was the most suitable medium. In addition, for adventitious shoot regeneration in Spok and Hansen cultivars, immature embryo explants were treated with different combinations of BAP and NAA. In these treatmens, the highest percentage of explant producing adventitious shoots was obtained from MSO medium, supplemented with 0,50 mg/1 BAP and 2 mg/1 NAA in both Hansen (55,00 %) and Spok (66,67 %) cultivars. In both cultivars, immature embryo axes (55,47 %) developed more adventitious shoot regeneration than immature cotyledon explants (25,77 %). Some experiments were carried out in leaf disk explants of Samsun tobacco cultivar in order to define the most suitable doses of various antibiotics (hygromycin and kanamycin) and herbisites (methotrexate and phosphinothricin) to use in selection medium. As a result of the experiments, to select the transgenic shoots, the most suitable kanamycin dose was found asIV 50 mg/1, hygromycin was 40,00 mg/1, methotrexate was 0.05 mg/1 and phosphinothricin was 5,00 mg/1. Gene transfer to leaf disk explants of Samsun cultivar was carried out via various non-onkogenic AJumefaciens and A.rhizogenesis strains. In terms of kanamycin and hygromycin resistant shoot developing explant percentages, 2260355 GUS-INT, 15834 pRGGhpt, EHA 101 pRGGhpt were defined as the most successful strains with 100 %, and so were 2260 AoPRlNEOl with 95 %. The tumor production ability of the bacteria differed according to cultivars and explants as a result of the experiments in which the sensibility of whole cotyledon with petiol and hypokotil explants of Rapeseed cultivar to AJumefaciens A281 and C58 wild types. According to the tested cultivars, in the hypokotil explants that were inoculated with C58 strains, the tumor developing explant percentage varied between 20,00 % and 60,00 %, and so did the whole cotyledon with petiol explants 20,00 % and 73,00 %. In the hypokotil explants that were inoculated with A281 strain, the tumor developing explant percentage varied between 16,67 % and 63,33 % and so did the whole cotyledon with petiol explants 20,00% and 73,33 %. Gene transfer studies were conducted with pGV2260 35S GUS-INT strain to whole cotyledon with petiol explants of Westar as well as to Spok, Helios, Darmor, Quinta and Tarok cultivars, which were more successful than the other cultivars tested in the adventitious shoot regeneration studies. The percentage of explants producing kanamycin resistant shoots varied between 3,59% and 19,20% according to cultivars. Later on, canamycin resistant shoots were cut and planted in root medium containing kanamycin. Finally, the rooted shoots were grown in pots in the green house. In this way, 174 putative transgenic plants were obtained and among them only two plants that belong to Westar were found GUS positive. Transformation frequency was observed as 1,15 %. Science Code : 5001018 Key Words : Brassica napus L., rapeseed, Agrobacterium tumefaciens, adventitious shoot regeneration, gene transfer. Page Number : 153 Supervisor : Prof. Dr. Orhan ARSLAN Doç. Dr. Sebahattin ÖZCAN IC YÜKSEKÖ?RETİM KTOWi