Plant transformation vector

Plant transformation vectors are plasmids that have been specifically designed to facilitate the generation of transgenic plants. The most commonly used plant transformation vectors are termed binary vectors because of their ability to replicate in both E. coli, a common lab bacterium, and Agrobacterium tumefaciens, a bacterium used to insert the recombinant (customized) DNA into plants. Plant Transformation vectors contain three key elements;

Propagate binary vector in E. coli

Isolate binary vector from E.coli and engineer (introduce a foreign gene)

Re-introduce engineered binary vector into E. coli to amplify

Isolate engineered binary vector and introduce into Agrobacteria containing a modified (relatively small) Ti plasmid

Infect plant tissue with engineered Agrobacteria (T-DNA containing the foreign gene gets inserted into a plant cell genome)

In each cell T-DNA gets integrated at a different site in the genome

Note: There are many variations to these steps. A custom DNA plasmid sequence can be created and replicated in more than one way.

Foreign DNA inserted

Insertional mutagenesis (but not lethal for the plant cell – as the organism is diploid)

We want to transform the whole organism, not just one cell. This is done by transforming plant cells in culture, selecting transformed cells and regenerating an entire plant from the transformed cell (e.g. tobacco)

When the bacteria with the desired, implanted gene are grown, they are made containing a selector. A selector is a way to isolate and distinguish the desired cells. A gene that makes the cells resistant to an antibiotic such as the antibiotics kanamycin, ampicillin, spectinomycin or tetracyclin, is an easy selector to use. The desired cells (along with any other organisms growing within the culture) can be treated with an antibiotic, allowing the desired cells to survive while other organisms cannot. The antibiotic gene is not usually transferred to the plant cell but remains within the bacterial cell.

Plasmids replicate to produce many plasmid molecules in each host bacterial cell. The number of copies of each plasmid in a bacterial cell is determined by the replication origin. This is the position within the plasmids molecule where DNA replication is initiated. Most binary vectors have a higher number of plasmids copies when they replicate in E. coli, the plasmid copy-number is usually less when the plasmid is resident within Agrobacterium tumefaciens. Plasmids can also be replicated in the polymerase chain reaction (PCR).

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Wikipedia