In vitro regeneration genetic transformation and determination of cytological properties of some sexual and apomictic arabis species

ABSTRACT IN VITRO REGENERATION, GENETIC TRANSFORMATION AND DETERMINATION OF CYTOLOGICAL PROPERTDES OF SOME SEXUAL AND APOMICTIC ARABIS SPECD2S Kemal Melik TAŞKIN PhD in Field Crops Adviser: Prof. Dr. Kenan TURGUT June, 2003, 83 pages In this work, the reproductive biology, in vitro regeneration ability and genetic transformation of Arabis species (A. drummondii, A. holboellii and A. gunnisoniand) via Agrobacterium tumefaciens were investigated. Various explants (leaves, cotyledons, hypocotyls, roots and immature embryos) excised from seedlings that were either grown in sterile conditions or pots were tested in Murashige and Skoog (MS, 1962) medium supplemented with plant growth regulators (6-benzylaminopurine, Thidiazuron, a- naphtalen acetic acid and 2,4-diclorofenoxy acetic acid). Callus formation started in ten days and a high frequency of callus formation (up to 100%) was obtained. Shoots were obtained after 4 weeks in culture from hypocotyls, roots and immature embryos depending on species and plant growth regulators used. Regenerated shoots were rooted on half- strength MS basal medium supplemented with 1 % (w/v) sucrose, with or without NAA. This protocol then used to produce transformed Arabis gunnisoniana plants. A. gunnisoniana hypocotyl explants were co-cultivated with Agrobacterium tumefaciens strain GV3101 harbouring pBJ40. Transgenic shoots were selected on MS medium supplemented with 50 mg/1 kanamycin. PCR analysis verified the presence of the NPTII gene in the plant DNA isolated from kanamycin resistant shoots. In cytological works, the use of ovule- clearing technique to characterize embryo sac development and flow cytometry analysis to reveal seed formation in Arabis species were investigated. Apomeiotic embryo sac development was detected as Taraxacum type diplosporous dyad formation. Flow cytometric seed screen analyses revealed pseudogamous apomixis. KEY WORDS: Arabis, apomixis, in vitro regeneration, Agrobacterium, transformation, embryo sac development, flow cytometry. COMMITTEE Prof. Dr. Kenan TURGUT Prof. Dr. M. Emin TUGAY ProfDr. Rüştü HATİPO?LU Assoc.Prof.Dr. Hüseyin BASIM Assoc.Prof.Dr. A. Naci ONUS ıı