GENETIC TRANSFORMATION OF TOBACCO WITH Hs1(pro-1) GENE AND EVALUATION OF NEMATODE RESISTANCE IN TRANSGENIC PLANTS

Altun, Cagla
Ms., Department of Biotechnology
Supervisor: Prof. Dr. Huseyin Avni Oktem
Co-Supervisor: Prof. Dr. Halil Elekcioglu

September, 2001, 82 pages

Plant-parasitic nematodes constitute 10% of all the nematodes and they are the major animal parasites of plants. They have a very broad host range and they cause an estimated 10 to 25% annual reduction in crop yield along with a global economic effect on crops of more than $100 billion annually.

This study aimed to genetically transform tobacco plant with a nematode resistance gene Hs1pro-1 via Agrobacterium mediated gene transfer technology, in order to develop transgenic tobacco plants conferring resistance to nematode infection.

For this purpose, an Agrobacterium strain, EHA105 has been transformed with the plasmid, pDNEM33 bearing the Hs1pro-1 gene via
electroporation. The newly formed bacterial culture, EHA105::pDNEM33 was used in the plant transformation studies.

After the transformation studies, putative transgenic plants were regenerated under the selection of Phosphinothricin (PPT) with varying concentrations and several types of analyses were carried out on the regenerated plants.

The leaf disc assay and colour viability test for the plants under selection of PPT (5mg/L) revealed positive results compared to the wild type control plants. The PCR analysis and the Southern Blot Hybridization results also indicated that the gene, Hs1pro-1 was stably inserted within the plant genome.

Finally, bioassays of the F1 seeds were performed with two different nematode species, Meloidogyne incognita and M. javanica. The roots of both the wild type and transgenic tobacco plants were examined 20-days post infection and evaluated according to 0-10 Gall Index Scale, the wild type and transgenic samples were compared by one-way ANOVA test and found out to be statistically different. The results indicated that, transgenic tobacco seedlings were less affected by nematode infection compared to the wild type seedlings.

Keywords: plant-parasitic nematodes, nematode resistance, Hs1pro-1 gene, transgenic tobacco, Agrobacterium