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Transformation 1 - Plant Tissue Culture

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Selectable Marker Genes

After callus cells have gone through the transformation process, it takes weeks of recovery and growth in a petri dish before they can develop into plants. Thousands of cells are growing on a single petri dish, but only a few may actually have received the new gene(s). It would be very cumbersome to grow up each cell into a plant to test for the presence of the transgene. It is much more efficient for geneticists to determine which cells have been transformed while still at the cellular level. They can then select those few transgenic cells out, and grow them into entire plants. Therefore, geneticists need a way to determine transgenic cells from non-transgenic cells.

What if you tried looking for the trait the gene encodes?
This may work for a few traits, but many transgenes encode traits that are not easily detected, especially at the cellular level. For example, you could not test a cell in tissue culture for the expression of a trait that influences grain protein content.

What if you extracted the DNA from each cell and searched for the gene?
You would have to separate each cell, allow the cell to multiply into a pile of callus, and sample a portion of each callus pile and extract DNA. Then tests would have to be perforemed to identify the presence of the gene. This would be just as cumbersome as trying to grow each cell into a plant.

What is the solution?

Usually the desired transgene and the marker gene insert in the chromosome at the same location.
Callus cells that have been transformed with an anitibiotic resistance marker gene grow on selection media while non-transgenic cells around them die.
Selecting out transgenic cells is done by co-transforming the cells with the transgene plus an additional gene called a selectable marker gene. Selectable marker genes encode a trait that is easily detectable, even at the cellular level. The two most commonly used selectable marker genes encode the traits of herbicide and antibiotic resistance.

To select out the transgenic cells, all of the cells are grown on media containing the herbicide or antibiotic. Only those cells transformed with the resistance transgene can survive. Thus growing the cells on selection media with the herbicide or antibiotic will allow the scientist to regenerate only plants that are transformed.

A selectable marker used in the development of some Bt corn lines is the BAR gene or PAT gene that confers resistance to the herbicide called Liberty. Thus these plants will have European corn borer resistance and Liberty resistance.

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