The Process of Plant Genetic Engineering
DNA and DNA Extraction
Gene Cloning
Gene Regions
Backcross breeding
Backcross Breeding

Some crop lines are genetically more equipped to handle the stresses of tissue culture. Since these lines are typically lower yielding, older lines, once a plant is successfully transformed it must go through backcrossing to move the transgene into a high yielding, elite line.

Gene cloning is the process in which a gene of interest is located and copied (cloned) out of all the DNA extracted from an organism. Since there is no way to locate a gene by visibly looking at all of the DNA, scientists make gene libraries to catalogue the organism's DNA and then select the gene he/she is looking for from this library.

Why is it necessary to take the time and effort to do backcrossing? Backcrossing is necessary because the lines that lend themselves well to tissue culture and transformation are typically older, low yielding lines. Plant breeders use backcrossing to transfer the transgene from these older lines into elite, high yielding lines. Also, plant breeders can screen for the presence of negative mutations that occurred during the transformation process.

How is backcross breeding done?

The backcross breeding method has been used by plant breeders for decades to incorporate specific traits into elite lines. This method works by crossing the transgenic inbred line with an elite inbred line of choice. In the following step, the breeder crosses the selected transgenic offspring back to the elite inbred again. This process of crossing back to the elite line (backcrossing) is repeated until the offspring has 99+% elite genes and the transgene. plant breeding method.

Identifying Transgenic Plants
How do breeders determine which plants express the trait encoded by the transgene?

One option is for the breeders to observe the plant for the trait of interest. However, if the trait is not easily detectable in the field (such as seed protein) then this method is inefficient.

Another option is to utilize the selectable marker that was inserted during transformation. However, this is not feasible if the selectable marker gene was an antibiotic resistance gene. Also, the selectable marker gene sometimes inserts into a different chromosome than the transgene. As a result, some offspring will have the transgene and not the selectable marker gene and vice versa.

Plant breeders often use an ELISA test, which detects the presence of the protein encoded by the transgene in a sample of plant tissue. This test can be quickly done in the field using a kit and a small sample of tissue and will give a simple positive or negative response.

Sometimes a more sophisticated test called PCR is used. The second method tests for the presence of the transgene itself. This laboratory method, called PCR (Polymerase Chain Reaction), is much more time consuming and expensive. However, it can be used to detect the presence of a transgene in tissues that are not expressing the gene.

A final option sometimes used by major companies is genetic fingerprinting. This technique can be used to not only identify the presence of the transgene, but find which plants, through the natural variation in breeding, have obtained a greater percentage of the elite inbred genes. This can potentially shorten the number of generations required for backcrossing. Athough very advantageous, this technique is also very time consuming and expensive, and could not be performed on a large number of lines.

All of these detection methods are important tools that help the plant breeder identify the plants they should be making crosses with.

It takes the breeder at least two or three years to derive a backcross line that is the genetic equivalent of the elite line plus the event. After that point the plant breeder can work with the genetically engineered line in the same manner they work with other parents in their breeding program. Many companies have taken advantage of genetic fingerprinting technology and year-round nurseries to maximize the efficiency and speed of backcross line development.

Yield Drag and Yield Lag
Yield drag is the negative effect on yield potential associated with crop plants that have a specific gene or trait. Yield drag can be caused by the transgene may inserting into a gene important for plant growth and yield disrupting its expression, or it can be caused by a drain in the limited pool of amino acids due to having to produce large quantities of new proteins. This limits the amount of amino acids available for the production of other proteins important to plant growth and yield. Genetically engineered crops are not necessarily more prone to yield drag or yield lag than non-genetically engineered crops. However, genetically engineered crops are unique in that an additional gene is being placed into the chromosome.

Yield lag is a difference in yield potential between a transgenic line and the newest elite lines. The difference is because there is no selection for increased yield during the 3-5 years of backcross breeding while non-transgenic lines have had selection for improved yield potential every year. Therefore the lines coming out of a backcrossing program have gone through a "lag period" in which the plant breeder has not imposed selection for yield. These lines would be expected to experience a yield lag.

Once a transgene has been backcrossed into an elite inbred background, it is no longer necessary for it to undergo backcrossing again. It can be used in the same manner as non-transgenic lines in breeding programs being mated to other lines and undergoing selection for improved qualities including yield. Thus, over time yield lag is no longer an issue in a particular transgenic event.

Gene Stacking
Gene stacking is a term that is used in the context of genetically engineered crops, but is not a new idea in plant breeding. Gene stacking is combining desired traits into one line. Plant breeders are always stacking genes by making crosses between parents that each have a desired trait and then identifying offspring that have both of these desired traits. This is the quickest and easiest way to stack genes. Another way to stack genes is by transferring two or more genes into the cell nucleus during transformation. The use of a selectable marker in addition to the gene of interest would be considered gene stacking.

The effect of the transgenes on the overall metabolism of the plant will probably be the major limitation to how many genes and what combinations of genes we can stack into a crop plant. Right now we are only asking the plant to make a few copies of one or two additional proteins. If the transgenes are designed to change protein synthesis and metabolism more dramatically, it is likely that the productivity of the plant will be compromised and yield drag will result. The loss of yield will need to be offset by cost savings in production or extra value for the grain.

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