AGROBACTERIUM MEDIATED GENE TRANSFER IN TOBACCO

Mahmoudian, Mehrzad
Ms., Department of Biotechnology
Supervisor: Assoc. Prof. Hüseyin Avni Öktem
Co-Supervisor: Prof. Dr. Meral Yücel

January 1995, 88 pages

This work describes Agrobacterium mediated gene transfer in Samsun variety of tobacco plants. Transferred genes were the coding sequences of neomycin phosphotransferase-II, which is known to confer kanamycin resistance, and a bacterial originated gene, b-glucuronidase (GUS), which is commonly used as reporter gene.

Transfer of genes were achieved by "leaf disk transformation" method, by the use of Agrobacterium tumefaciens LBA4404 strain, carrying the binary vector pBI121. Binary vectors were mobilized in LBA4404 strains by a process known as "tri-parental mating". Success of gene transfer method was analysed by monitoring the resistance of leaf disks to kanamycin and by measuring the GUS activities of transformants.
Transformation frequency was over 80% as deduced by callus formation of transformed leaf disks on Murashige-Skoog (MSB) based growth media containing kanamycin at lethal doses. More than 90% of regenerated shoots from transformed plants were able to form roots on MSC selective media. Fully regenerated plants were potted out in soil and grown normally.

Leaf disks prepared from transgenic plants were fully potent to regenerate on MSB selective media. High levels of exogeneous GUS activity were recorded in transgenic plants where control plants exhibit no GUS activity. Seeds obtained from the self pollinated Fo transgenic plants were able to germinate in the presence of kanamycin and were able to express GUS enzyme, indicating the inheritance and functional expression of transferred genes in F1 progeny.

By employing these techniques we have obtained several lines of transgenic plants functionally expressing the transferred genes. Different lines of evidences clearly demonstrated transfer, stable integration and expression of the transferred genes in transgenic plants and their F1 progeny.

Key Words: Tobacco, Gene Transfer, Neomycin Phosphotransferase-II, GUS, Agrobacterium