AGROBACTERIUM MEDIATED GENE TRANSFER IN TOBACCO
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
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,
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