Research Paper On Ti Plasmid Curing

3.18.2.1

Agrobacterium-Mediated Transformation

Two different species of Agrobacterium are commonly used for genetic transformation of plants: A. tumefaciens and A. rhizogenes. These are Gram-negative soil bacteria that belong to the family Rhizobiaceae and are able to infect different plant hosts, most often dicotyledons or less frequently monocotyledons,32 and even yeast and animal cells.33–35Agrobacterium tumefaciens and A. rhizogenes are the causal agents of the plant diseases crown gall and hairy root, respectively. Diseases are caused by the presence of bacterial DNA, the transferred DNA (T-DNA), within the plant cells. The T-DNA controls the synthesis of plant growth regulators, auxin and cytokinin, in the infected cells resulting in the induction of tumor or hairy roots, as well as the biosynthesis of other types of compounds, such as opines or octopines serving as nutrients for the infecting bacterium.36 These bacteria are considered natural metabolic engineers because of their ability to transfer genes into the target plant cells and other organisms (fungi, yeast, bacterium, animal cells) and thus are capable of genetically crossing kingdoms.37,38

Genetic transformation occurs after bacterial infection of the plant cells or tissues. Following infection, the T-DNA, which can be engineered to carry the genes of interest, gets inserted into the plant nuclear DNA. Furthermore, other elements of the bacterial plasmids, the Ti-plasmid from A. tumefaciens (tumor inducer) or the Ri-plasmid from A. rhizogenes (root inducer), also participate in the transformation process. Both plasmids show large functional homologies and appear to have evolved from a common ancestor.39 These plasmids also possess a virulence region, containing various silent vir genes, which do not penetrate the plant genome, but are indispensable for the T-DNA transfer. These genes are switched on by interacting with phenolic-type compounds (Figure 2), such as lignin precursors and acetosyringone, wound tissue metabolites, demonstrating the need of tissue wounding for efficient infection to take place.40 Furthermore, the rol- and onc-genes encode enzymes for the production of plant growth regulators by the infected plant cell, as well as other opine and octopine synthase genes that activate the synthesis and catabolism of different classes of opines and octopines (Figure 2). These are unique natural metabolites, pseudo amino acids, which serve as a nutrient source of carbon and nitrogen for the pathogenic bacteria.41

Transformation by Agrobacterium requires several conditions, that is, an acidic pH (5.0–6.0), the presence of phenolic compounds produced after tissue wounding, and, more recently, it has been described that light also increases the success of Agrobacterium transformation.42 Nonetheless, it is not fully understood how the T-DNA integrates into the plant nuclear genome, but it seems to resemble illegitimate recombination, proceeding analogously in dicot and monocot plants.36 Insertion of the T-DNA in the plant genome occurs at random positions, but showing preferences for transcriptionally active regions. Contrary to other gene insertion techniques such as particle bombardment, the plant transgenic cell lines generated via Agrobacterium often contain one copy or a low copy number of the T-DNA, although cell lines with multiple T-DNA copies can also be found.

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