F1-F3 PROGENY ANALYSIS OF HERBICIDE RESISTANT TRANSGENIC TOBACCO PLANT

Arpat, Alaaddin Bulak

M.S., Department of Biotchnology
Supervisor: Assoc. Prof. Dr Hüseyin Avni Öktem
Co-supervisor: Prof. Dr. Haluk Hamamci

September 1999, 75 pages

In this study F1-F3 progenies of tobacco plant transformed with a chimeric cassette containing bar gene from Streptomyces hygroscopicus with CaMV 35S promoter were analysed in terms of their resistance against phosphinothricin (PPT) and the inheritance of the transgene during successive progenies was showed. For the propagation of the progenies, starting with the F1 seeds, plantlets were grown under sterile conditions and micropropagated in culture media. After the plants were transferred into soil pots they were self-pollinated to obtain self-crossed progenies.
All the germination and micropropagation procedures were carried under herbicide selection. After transfer to soil the plants were tested for their resistance against Basta®, a commercial herbicide containing PPT. Explants from those plants were also analysed on pH indicating selective medium with chlorophenol red. According to the results of all phenotypic analyses all three progenies were determined to be completely resistant to PPT and PPT based herbicides.
From the leaves of the propagated plants total protein isolation and genomic DNA isolation carried out. PCR amplification of genomic DNA samples with bar specific primers indicated the presence of the gene within the genome of the transgenic lines. Within the total protein profile of the transformed lines we have detected the presence of a 23 kD protein with SDS-PAGE analysis. Since the PAT protein, coded by bar gene, is also in the range of 21-23 kD, we have concluded that this protein corresponds to PAT.
Since the molecular data support the data obtained from phenotypic analyses we concluded that the bar gene is integrated within the genome of the transformed tobacco lines, and it is inherited and expressed successfully among successive progenies.
Keywords: Transgenic tobacco, Nicotiana tabacum, PPT, bialaphos, herbicide resistance, progeny analysis