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Module 4: Nutrient Composition

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Techniques

This first section is intended to provide a general background about what techniques are used to make nutritional changes in plants.

When genetic changes are made in crop plants, there are associated changes that occur in the plants’proteins, which in turn produce the new phenotype. These proteins arise from translation of DNA into RNA into amino acid sequences of polypeptide chains. Often, the proteins that are changed are ones that act as enzymes in the plant. These enzymes direct the synthesis of cell components that then determine the cell’s characteristics (e.g. what type of cell it is). Cells then make up tissues, organs and finally, the plant itself.

In order to make these proteins, and thereby the desired plant characteristic, several techniques are used, with the end result being addition of the desired foreign DNA into the plant.

Pathogen

One technique for introducing DNA changes into a plant involves the use of a plant pathogen, Agrobacterium tumefaciens.

In nature, this bacterium invades tissue wounds in plants(1). During this process the bacterium introduces a portion of its own DNA into the cell nuclei of the tissue it has invaded. That DNA becomes a part of the plant cell’s DNA makeup. This incorporation usually results in tumor formation within the tissue.

In the laboratory setting, the bacteria is introduced to the plant cell with its tumor causing DNA inactivated. When it invades the plant cell, it doesn’t cause tumor formation, however foreign DNA can be spliced into the bacteria’s DNA. The foreign DNA will be carried into the plant’s cell nuclei and become part of the plant’s chromosomes (1).

As part of the chromosome, promoters can then be used to express the genetic information from the foreign DNA. An important step in alteration involving nutrients is then expressing the genetic information at the correct stage of the plant’s development (e.g. leaves, flowers, fruits or seeds)(1).
 

Agrobacterium that contain a plasmid recombinant with the gene of interest are added to a solution containing callus cells DNA, including the gene of interest, is inserted by the Agrobacterium into the nucleus of some of the callus cells where it may insert into a chromosome.


Particles

Not all plants are susceptible to the pathogen in the agrobacterium-based method of transformation. Therefore, for some plants another technique is used.

Microscopic gold or tungsten particles are used in this transformation method. The particles are accelerated toward the plant cells using an explosive charge, high-pressure helium, or electric discharge. A small number of the cells will be penetrated by the particles, resulting in transformation (1).

 

Gene gun transformation begins by growing cells in tissue culture, bombarding the cells with gene coated gold particles in the gene gun, selecting out transgenic cells on selection media, and regenerating the transgenic cells into plants. Once inside the nucleus of a cell, the genes dissolve off of the gold particle and can potentially insert into a chromosome. A major limitation of this method is that several copies of the transgene will often insert into a chromosomal position. These high copy insertion events can be detected as DNA that should not be expressed by the plant cell and the transgene copies are silenced.

 



Electroporation

The third transformation method uses cells. These cells have had their outer protection, the cell wall, removed by an enzyme. The foreign DNA is then introduced via electroporation or by the natural uptake of DNA into the nucleus of the cell(1).

Antisense RNA Technology

The final technique involves using genetic information within the plant to decrease gene expression.

 

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