Induction of Crown Gall on Carrot Slices

Abstract

Laboratory experiments on the transfer of a plasmid to a bacterium, whether by conjugation, transformation or transduction, are available for use in the teaching laboratory. However, the transfer of plasmid from a bacterium (prokaryote) to a plant cell (eukaryote) has received little attention. The following is an experiment for studying this latter type of genetic transformation. The Gram negative soil bacterium, Agrobacterium tumefaciens L., is the causative agent of crown gall disease, a malignant tumor, occurring on the stems and leaves of infected dicotyledonous plants. Induction of this tumor is mediated by the presence in the bacterium of the large (200 kbp in length) tumor-inducing (Ti) plasmid. Upon bacterial infection of a wounded plant, phenolic defense chemicals released from the plant activate virulence genes on the Ti plasmid, leading to the processing and transferring of a small (20-23 kbp) segment of the Ti plasmid into the host plant cell. The bacterium remains outside the plant cell, living in the intercellular spaces of the plant. By transference through a conjugation tube initiated by the bacterium, this small segment of the Ti plasmid, designated transferred DNA or T-DNA, penetrates the host plant cell and subsequently incorporates into the plant's genome. T-DNA contains genes for the syntheses of opines, chemicals used as sources of carbon and nitrogen by the infecting bacterium, and phytohormones. Overproduction of these phytohormones (i.e. auxin and cytokinin) results in uncontrolled plant cell growth and hence the development of a tumor (Gelvin & Karcher 1996; Russell 1996). In this exercise, we induce crown