Question

D. Using a molecular biology based approach; foreign pieces of DNA can be readily integrated into the nuclear genomes of a number of plant species via the use of the Agrobacterium tumefaciens (Agrobacterium) Tumour-inducing (Ti) plasmid and its encoded Transfer-DNA (T-DNA). For the Agrobacterium Ti plasmid and T-DNA to be used successfully for the introduction of foreign DNA to the plant nuclear genome, some gene sequences need to be retained while others must be removed. Please state; (1) which gene sequences are retained and which are removed; (2) the position (Ti plasmid-based or T-DNA-based) of the retained and removed sequences, and; (3) explain why some gene sequences must be retained while others require removal.

Answer 1

Ti-plasmid contains T-DNA (known as transfer DNA which codes for proteins involved in tumor formation), vir genes (required for transfer of T-DNA region to plant cells), T-DNA border sequences (left border and right border which are required for integration of the T-DNA region into the plant genome), ori (for replication), and opine gene (for opine catabolism).

(1) Retained sequences: vir genes, T-DNA border sequences, ori.

Removed sequences: T-DNA, opine genes.

(2) position of removed and retained genes can be inferred from the Ti-plasmid map.

(3) T-DNA region contains genes which cause excessive growth resulting in transformation of plant cells (tumor formation). T-DNA is removed and gene of interest is inserted between left and right border.

T-DNA border sequences are retained since they assist in integration of T-DNA region into host genome.

Opine genes are removed since they are not required in the experiments.

vir genes are retained since they are essential for transfer of T-DNA region into host plant.