Figure 1. Overview of how transgenic crops are created.
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: (1)extracting DNA, (2)cloning a gene of interest, (3)designing the gene for plant infiltration, (4)transformation, and finally (5)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.
Figure 2. Gene Regions.
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.
Figure 3. Transfer DNA on a plasmid in Agrobacterium
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. 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 . One of the biggest limitations of Agrobacterium is that not all important food crops can be infected by this bacteria.
Other methods
Some of the techniques used to transfer foreign cells into animals and plants include:
• Bacterial carriers
• Biolistics
• Calcium phosphate precipitation
• Electroporation
• Gene silencing
• Gene splicing
• Lipofection
• Microinjection
• Viral carriers.
Bacterial carriers
The bacterium Agrobacterium can infect plants, which makes it a suitable carrier for delivering DNA. The bacterium is prepared in a special solution to make its cell walls more porous. The selected gene is inserted into a bacterium extra chromosomal DNA molecule (called a plasmid) and dropped into the solution. The solution is heated, which allows the plasmid to enter the bacterium and express the new gene. The genetically altered bacterium (or recombinant) is allowed to recover (is ‘rested’) and grow and, depending on the plasmid, make extra copies of the new gene. The bacterium is then allowed to infect the target plant so it can deliver the plasmid and the new gene.
Biolistics
The selected DNA is attached to microscopic particles of gold or the metal tungsten. Like firing a gun, these DNA-laden particles are shot into the target cells using a burst of gas under pressure.
Calcium phosphate precipitation
The selected DNA is exposed to calcium phosphate. This mixture creates tiny granules. Target cells respond to these granules by surrounding and ingesting them (endoocytosis), allowing the granules to release the DNA and deliver it to the host nuclei and chromosome(s).
Electroporation
The prepared target cells are immersed in a special solution with the selected DNA. A short but intense electric shock is then passed through the solution. The result is small tears in the cell walls, which allow the new genetic material access to the nuclei. Then, the cells are placed into another solution and encouraged to repair their breached walls, locking the ‘donor’ DNA inside the cell. The selected DNA is incorporated into the host chromosomes to provide the host with a new gene.
Gene silencing
The gene responsible for the organism’s undesirable trait is identified. One method of ‘silencing’ that particular gene is to attach a second copy of the gene the wrong way around. This technique is used to prevent plants like peanuts and wheat from producing the proteins (allergens) commonly responsible for human allergies.
Gene splicing
Bacteria contain restriction enzymes that form part of the bacterium’s ‘immune system’ against invasion by another organism or bacteriophage (a bacterial virus). The restriction enzymes attack the foreign DNA by cutting it into precise sections and preventing it from being inserted into the bacterium’s chromosome.
Different bacteria produce different restriction enzymes that cut any DNA at different places, making the DNA ‘sticky’ in some cases, which means they can be ‘pasted’ directly onto the target organism’s prepared DNA.
Using these restriction enzymes from bacteria, molecular biologists can ‘genetically engineer’ the DNA for ‘insertion’ into target (host) cells to modify gene traits. The molecular biologist then uses another enzyme (ligase) to fuse the new gene into the chromosome.
Alternatively, instead of ‘pasting’, the new gene may be inserted into a bacterium’s extra chromosomal DNA molecule (a plasmid), which carries invasion genes that allow it to invade the target cell and deliver the gene.
Lipofection
Small bubbles of fat called liposomes are used as the carriers of selected DNA. The target cells and the liposomes are placed into a special solution. The liposomes merge with the cell membrane, allowing the DNA into the cells for inclusion in the chromosome.
Microinjection
The selected DNA is injected into a fertilised ovum (female egg cell) through an extremely slender device called a glass capillary tube. The genetically modified egg is then transplanted into the prepared uterus of a receptive female and allowed to grow to term. This method ensures that almost every cell in the developing organism’s body contains the new DNA but not every progeny carries the transgene (is deemed a ‘transgenic’ animal).
Viral carriers
A virus that will invade the target cells but not cause damage or death is chosen. The selected DNA is added to the genetic makeup of the virus, and then the virus is allowed to infect the target. As the virus invades cells and replicates, the selected DNA is added to the target cells.
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