For many years plant breeding entailed the selection of the finest plants to get the best crops. In those days, variation occurred through induced mutation or hybridization where two or more plants were crossed. Selection occurred through nature, using a “selection of the fittest” concept, where only the seeds best adapted to that environment succeeded. For example, farmers selected only the biggest seeds with non-shattering seed heads, assuming these to be the best. Today, scientists can not only select, but also create crops by inserting genes to make a seeds bare any trait desired.
In order to make a transgenic crop, there are five main steps: extracting DNA, cloning a gene of interest, designing the gene for plant infiltration, transformation, and finally plant breeding (see Figure 1).
To understand this process, one must first known a bit about DNA (deoxyribonucleic acids). DNA is the universal programming language of all cells and stores their genetic information. It contains thousands of genes, which are discrete segments of DNA that encode the information necessary to produce and assemble specific proteins. All genes require specific regions in order to be utilized (or expressed) by a cell. These regions include (see Figure 2):
1. A promoter region, which signals where a gene begins and it used to express the gene;
2. A termination sequence, which signals the end of a gene;
3. And the coding region, which contains the actual gene to be expressed.
All these regions together allow a gene to create a protein. Once a gene is transcribed into a protein, it can then function as an enzyme to catalyze biochemical reactions or as a structural unit of a cell, both of which will contribute to the appearance of a particular trait in that organism.
All species are capable of turning DNA into protein through a process known as translation. This capability makes it possible to artificially put genes from one organism into another-a process generally termed transgenics. But just isolating random DNA and inserting it into another organism is not practical. We must first know what particular segments of DNA, and in particular what genes, to insert. Unfortunately, with reference to producing new crops, not much is known about which genes are responsible for increased plant yield, tolerance to different stresses and insects, color, or various other plant characteristics. Much of the research in transgenics is now focused on how to identify and sequence genes contributing to these characteristics.
Genes that are determined to contribute to certain traits then need to be obtained in a significant amount before they can be inserted into another organism. In order to obtain the DNA comprising a gene, DNA is first extracted from cells and put into a bacterial plasmid. A plasmid is a molecular biological tool that allows any segment of DNA in be put into a carrier cell (usually a bacterial cell) and replicated to produce more of it. A bacterial cell (i.e. E. coli) that contains a plasmid can put aside and used over and over again to produce copies of the gene the researcher is interested in, a process that is generally referred to as “cloning” the gene. The word “cloning” referring to how many identical copies of the original gene can now be produced at will. Plasmids containing this gene can be used to modify the gene in any way the researcher sees fit, allowing novel effects on the gene trait to be produced (see Figure 1).
Once the gene of interest has been amplified, it is time to introduce it into the plant species we are interested in. The nucleus of the plant cell is the target for the new transgenic DNA. There are many methods of doing this but the two most common methods include the “Gene Gun” and Agrobacterium method.
The “Gene Gun” method, also known as the micro-projectile bombardment method, is most commonly used in species such as corn and rice. As its name implies, this procedure involves high velocity micro-projectiles to deliver DNA into living cells using a gun [1]. It involves sticking DNA to small micro-projectiles and then firing these into a cell. This technique is clean and safe. It enables scientists to transform organized tissue of plant species and has a universal delivery system common to many tissue types from many different species1. It can give rise to un-wanted side effects, such as the gene of interest being rearranged upon entry [1] or the target cell sustaining damage upon bombardment. Nevertheless, it has been quite useful for getting transgenes into organisms when no other options are available.
The Agrobacterium method involves the use of a soil-dwelling bacteria known as Agrobacterium tumefaciens, which has the ability to infect plant cells with a piece of its DNA. The piece of DNA that infects a plant is integrated into a plants chromosome through a tumor-inducing plasmid (Ti plasmid), which can take control of the plant’s cellular machinery and use it to make many copies of its own bacterial DNA. The Ti plasmid is a large circular DNA particle that replicates independently of the bacterial chromosome [1] (see Figure 3).
The importance of this plasmid is that it contains regions of transfer DNA (tDNA), where a researcher can insert a gene, which can be transferred to a plant cell through a process known as a floral dip. A floral dip involves dipping flowering plants into a solution of Agrobacterium carrying the gene of interest, followed by the transgenic seeds being collected directly from the plant [1]. This process is useful in that it is a natural method of transfer and therefore thought of as a more acceptable technique. In addition, Agrobacterium is capable of transferring large fragments of DNA very efficiently without substantial rearrangements, followed by maintaining high stability of the gene that was transferred [1]. One of the biggest limitations of Agrobacterium is that not all important food crops can be infected by this bacteria [1].
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