Deathmatch: Conventional Breeding vs Transgenesis

“Mutation. It is the key to our evolution. It has enabled us to evolve from a single-celled organism into the dominant species on the planet. This process is slow and normally taking thousands and thousands of years. But every few hundred millennia, evolution leaps forward.” – Professor Xavier
I love that quote from X-Men.
Other than the last sentence, it’s true. Mutations happen at a fairly constant rate and can occur every time a cell divides. Although we tend to think of mutations as negative events associated with genetic diseases or cancer, some mutations are beneficial: in our species, mutations have allowed for adaptation to high altitude in Tibetans or have protected individuals from heart disease. The same is true in nature: mutations allow for plants to develop resistance to pests, or in the case of weeds, to pesticides.
However, as Professor Xavier points out in the opening credits of the movie, the process is slow. So how can we “force” beneficial mutations to occur quickly? In crop development, we’ve been forcing mutations to happen by a process known as “mutagenesis“, where chemicals or radiation are used to create random mutations generating new traits.
A few recent news articles have reported that plant breeders are turning more frequently to mutagenesis to create new strains because there are fewer regulations. Mutagenesis is considered to be “traditional breeding” and crops generated through mutagenesis are accepted under the USDA’s organic label. This stands in stark contrast to transgenic crops, which are heavily regulated and are excluded from the organic label.
So is traditional breeding, including mutagenesis, “better” than transgenesis? This post shall seek out the answer. Drumroll… It’s the battle of the methods, thunderdome-style!! Deathmatch: traditional breeding vs transgenesis. Two methods enter, one method leaves. Or maybe there will be a tie. Who knows?
There are a handful of papers that have done comparisons between methodologies, so each round will review an article.

Round 1. Mutagenesis
The first round is battled out in a 2008 paper from the Proceedings of the National Academy of Sciences, written by Batista and colleagues. The authors examined the expression of thousands of genes to find out if there were any unintended consequences in transgenic and mutagenic crops by examining gene expression. Gene expression refers to how much of a gene is turned on or turned off, and is measured by amounts of RNA. As you may know, DNA encodes for RNA, which is then translated into protein, and the protein is generally considered to be the final goal. Now, proteins generally do not work independently and often regulate one another. For example, if protein A and protein B work together in the cell and you change the amount of protein A, you might also affect protein B. That consequence is often easier to identify, particularly if you know that protein A and B work together. But sometimes, you see a change in protein C and then you scratch your head and try to think of how protein C could possibly be affected by protein A. So, in this study, the authors wanted to determine if there were any unintended changes in gene expression when you add a gene in a transgenic plant (i.e. GMO), and compare it to the unintended changes in gene expression when you create a plant by the more “traditional” mutagenesis route, such as by gamma-irradiation. Yes… Gamma-radiation is real and is not confined to creating the Hulk or other super-heroes.
The authors compared transgenic and mutagenic rice strains to their closest non-modified strains (i.e control). All plants were grown in the lab. Their methodology consisted of using microarrays, which can examine the expression of thousands of genes at a time. The authors found that in all the strains, there were unintended changes in the expression of genes that are related to plant stress or defense. There were also changes in gene expression in certain genes that might be related to the transgene or mutant gene itself (i.e. changes in protein B in my previous explanation). The authors draw several conclusions:

• Although there were unintended consequences in gene expression using both methods, transgenic strains had fewer changes.
• Changing a plant through mutagenesis or transgenesis creates stress in the plant and leads to changes in gene expression, which are carried through several generations.

Round 1 Results: advantage to transgenics
Round 2:  Marker Assisted Backcrossing
The second round is battled out in a 2013 paper published in the journal BMC Genomics. It consists of examining two strains of rice that have the same trait, but generated using different methodologies. Marker assisted backcrossing consists of crossing a strain that has a desired trait (donor line) to a recipient line. The resulting strain is backcrossed against the recipient line so that all other traits/genes are gradually cleansed with the exception of the single desired trait. The term “marker assisted” is used because researchers don’t have to wait till all the crops are grown to see if the trait is there or not: they can simply test the seeds to determine which one has the trait using genetic markers, similar to what’s done for human paternity testing or forensics analyses.
The paper used the gene that confers rice bacterial leaf blight resistance and incorporated it from one strain of rice into another using marker assisted backcrossing, as well as transgenesis. Then, similar to what was done in Round 1 of this Deathmatch, they examined gene expression levels in the new strains to determine which one was more similar to the original rice strain, i.e. which crop modification method generated more unintended consequences. Their methodology consisted of RNA sequencing, which can examine the expression of nearly all RNA molecules in a sample (also known as whole transcriptome sequencing). The rice plants were all grown in the same field and all three strains of rice (the two modified ones and the control) looked identical upon visual inspection, except for the fact that the control was susceptible to blight. Interestingly, the authors planted an unrelated variety of rice alongside these three, which proved to be an interesting control.
The results of the study are:

• Although both methods (marker assisted backcrossing and transgenesis) had differences in gene expression compared to the control, the transgenic rice strain had ~40% fewer differences in gene expression compared to marker assisted backcrossing.
• The number of differences in the expression of genes between the modified crops and the control was significantly fewer than between the control and the unrelated variety. The authors state “transgenesis and MAB [marker assisted backcrossing] breeding did not alter the transcriptome more significantly than another natural rice variety.”

This last point is an important one and I’ll explain in the next section. For now, the score is:
Round 2 results: advantage to transgenics
Reproducibility & Substantial Equivalence
It’s important to note that the two papers I’ve reviewed here are not the only papers that have compared methodologies to determine if they generate unintended consequences. Additional papers can be found here, here, here, here, here, here, and here, among others. These papers used different techniques and crops, and collectively they tell the same story: that a crop that generated through transgenesis generates few changes in the expression of genes and proteins, and that these changes are far fewer than the amount of variability that is generated through the environment as well as the variability between strains.
To explain, let’s imagine that there are three apple varieties: genetically-modified-green (GM-green), green, red. We plant GM-green, green, and red in my backyard in California. We also plant a green apple tree in Ontario, Canada. We do an analysis of the genes turned on/off in apples from all 4 trees. There will be differences between all four, but the differences between GM-green and green will be far, far, less than the differences observed between the green and red from my backyard, as well as the green from California and the green from Ontario, Canada. It is this principle, that a genetically modified crop has variation, but that the variation observed falls within the normal variation of the species, that is known as “substantial equivalence”.
Substantial equivalence is often confused for identicality, however, the Food and Agricultural Organization of the United Nations states that substantial equivalence “is established by a demonstration that the characteristics assessed for the genetically modified organism, or the specific food product derived therefrom, are equivalent to the same characteristics of the conventional comparator. The levels and variation for characteristics in the genetically modified organism must be within the natural range of variation for those characteristics considered in the comparator and be based upon an appropriate analysis of data” (emphasis has been added).
Activists are quick to pounce on the differences observed between transgenic crops and controls, and hold these differences up as evidence that the principle of substantial equivalence is false. However, they tend to ignore the significant number of differences observed through traditional breeding which the two rounds of our Deathmatch highlight. They also ignore differences observed from one generation to the next, which Professor Xavier highlighted at the beginning of this post. The natural mutation rate in humans is currently estimated at 30-75 variations/mutations per genome per generation (ASHG talks, 2014). As such, it is quite probable that every one of us is a mutant relative to our parents and that no two “identical twins” are truly identical. Similar estimates exist in plants, where researchers have been able to determine the “natural” mutation rate for Arabidopsis (see here and here), one of the more common plants used in genetic studies. However, it would be nonsense to decry that these plants are not substantially equivalent to their parents, as would stating that an apple grown in California is not equivalent to the same variety of apple grown in North Dakota.
Today’s Deathmatch had a clear outcome: that transgenesis can generate crops that have fewer unintended consequences than those generated through traditional methods.