INTRODUCTION OF GENES USING AGROBACTERIUM

8. INTRODUCTION OF GENES USING Agrobacterium

This lecture dealt with ways of introducing foreign genes in higher plants with a focus on the Tobacco transformation protocol. The initial stages of the experiments were preparation of Agrobacterium culture and the culture medium for transformation. YEB liquid medium was prepared and inoculated with Agrobacterium which was previously stored at –80 degrees C. The culture medium for transformation was also prepared namely co-cultivation media and selection media.

Tobacco leaf explants were isolated and cut into small pieces, placed on filter paper on the co-cultivation plate. The agrobacterium suspension was added to the leaf explants and leaves immersed for 10 minutes, and were later transferred to sterilized filter paper in a petri dish to remove the excess agrobacterium suspension. The explants were transferred onto filter paper on the co-cultivation medium and cultured for 2 days at 20-25 degrees C.

After the co-cultivation period, the leaf explants were transferred to the selection media aseptically under the laminar flow chamber. The explants were periodically sub-cultured after 2-3 weeks, and later observed of the transformed cells. These included regenerated shoots and calli that formed on the explants.

Any red-colored tissues that were observed were considered to have expressed the gene that was inserted.

DNA was isolated from the tobacco leaf tissues and a gel electrophoresis was ran at 100 volts for 20 minutes, and subsequently stained in Ethidium bromide molecules for 10-15 minutes for visualization.

This photo shows Electrophoresis of DNA extraction from transformed and non-transformed tobacco plants.