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Transformation Techniques

Transformation using Agrobacterium tumefaciens

This technique builds on a natural transformation system. The Agrobacterium tumefaciens bacterium causes growths (galls) and tumours on dicotyledons or 'dicots' (plants whose seed contains two embryonic leaves) if the bacteria are able to enter the plant via injuries to the plant tissue. The associated genes lie in the bacteria within a special ring-shaped DNA molecule, the Ti plasmid (Ti stands for tumour-inducing) which contains the transfer DNA (T-DNA). In the case of infection, the T-DNA enters the plant's cells, becomes integrated into the plant genome and causes the plant to produce nutrients to feed the bacteria.

Genetic engineering uses this vector system and replaces the tumour-inducing and metabolism-changing genes with foreign DNA, usually selection and marker genes (for selection of the transformed plants) and the target gene together with regulatory sequences. Because many parts of the process take place in the Escherichia coli (E-coli) bacterium, another marker gene for selection of the transformed bacteria, usually an antibiotic-resistance gene (ARG), is needed which is introduced into the backbone DNA of the Ti plasmid. The agrobacteria equipped with the new plasmid are cultivated together with the leaf tissue. Individual plant cells take up the T-DNA and integrate it into their own genetic material. Finally, the bacteria are removed and the transformed plant cells are regenerated using phytohormones. The plant markers and selection media ensure that only transformed plant cells survive and regenerate into whole plants.

No definitive model of how the T-DNA is built into the plant genome is available as yet. Relocation and deletion of foreign DNA and of the plant's own DNA have been observed in nearly all transgenic plants.


Biolistic Transformation

With the exception of maize, cereals and other monocotyledons or 'monocots' (plants whose seeds contain a single embryonic leaf) cannot be transformed using agrobacteria. Sandford et al. (1987) provided help in the form of biolistic transformation (also known as particle gun bombardment), where foreign DNA attached to metal particles is fired into the cells. In this method, the foreign DNA also contains regulatory elements, the target genes and a marker gene, usually an antibiotic-resistant or herbicide-resistant gene. Compared with agrobacterial transformation, plant regeneration is easier in this case because the cell walls do not need to be removed. The method is also suited to use with plasmids, linear DNA and larger gene sequences. As more than one particle can hit a cell, biolistic transformation often results in multiple (up to 20) copies being incorporated at different places within the genome. The plant cells are only transformed with low efficiency and the DNA in the cell nucleus is not always integrated in a stable manner (transient gene expression). When integrating larger plasmids, fragments can occur and can become inserted into different places in the plant genome. The high number of copies and the occurrence of fragments increases the likelihood that biolistic transformation will produce unexpected effects.


Assessment

For both transformation methods, the foreign DNA must be integrated into the plant genome at random. This can alter the plant's own regulation mechanisms and can lead to undesired effects. Gene regulation is an extremely complex process and is controlled by a range of different mechanisms. Regulating segments are often far removed on the DNA molecule from the genes they regulate. Newly introduced DNA sequences can alter and disrupt regulatory relationships. Studies have shown that in the transfer of T-DNA, sequences from the plasmid backbone are also transferred and in some cases the agrobacteria are passed down through multiple generations. This is why the genes in the plasmid backbone (e.g. ARGs) must also be included in the risk assessment. Plant cell regeneration using phytohormones is also thought to cause mutations.

The transformation methods used in genetic engineering impact on the gene regulation system in the plant genome. In traditional breeding, however, the genes usually remain within their regulation complex.

Last Change: 06/07/2006

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