2.6—Breeders produce genetically stable crops


Transgenic plants containing the Cauliflower mosaic virus 35S promoter are not unstable.

See Genetic Roulette’s False Claims at Bottom of Page

Analysis of Peer-Reviewed Research:

Most people do not spend their time thinking about what chromosomes do. They will likely not realize that numerous DNA rearrangements occur in chromosomes over the many generations of plant survival in the wild or in farmer’s fields. These numerous alterations to DNA structure do not occur uniformly or predictably. “Hotspots” – or places in the chromosome where rearrangement and recombination of DNA occurs as a relatively high- frequency change — are a natural phenomenon and frequently encountered.

In section 2.6 of Genetic Roulette, Jeffrey Smith discusses the possibility of change to plant DNA as if it necessarily means a catastrophe will occur. He provides little indication of the wide occurrence of DNA rearrangement in plants or the common appearance of chromosomal hotspots for DNA recombination and movement.

 

Genetic Roulette is accurate in mentioning that the structure of inserted transgenes can be different from that which is intended by genetic engineers. Nevertheless, these are changes that are anticipated by plant scientists, and they have developed precautionary procedures to ensure that predictable DNA structures are present in commercialized transgenic plants. Smith does not mention that the primary reason that transgenes can undergo some changes in structure during the insertion process is that they are inserted by a plant repair system, and that this natural repair system causes numerous changes to plant chromosomes wherever it acts to repair broken DNA. Many readers of Genetic Roulette will not realize that  such necessary and error-prone DNA repair is a daily occurrence in plants, which are constantly subjected to radiation and other sources of DNA damage in the field. The process of transgene insertion causes far fewer changes to chromosomes than many other treatments used in conventional breeding.

Smith seems to think that chromosomes never change during their natural history, but as far as evolution is concerned, chromosomal change on a grand scale is normal. Even if there was some basis to his assertion that some transgenic crops may be unstable (despite the fact that they are carefully checked to have reliable inheritance of a novel trait), this would not add significant extra variability beyond what we see already with many crop plants.

1. Speculation without evidence does not prove something happens. No evidence is presented by Smith to support his claim the common promoter 35S, used to drive transgene expression, is a site of genetic instability in transgenic plants. Practical experience with transgenic plants shows that stable inheritance of transgenic traits can generally be obtained by dilligent application of standard breeding procedures. For example, comprehensive analysis of disease resistant papaya grown in Hawaii, a commercialized transgenic food crop containing the 35S promoter, shows that the 35S promoter in these papaya plants undergoes no change whatsoever over multiple generations of propagation (Kohli and Christou 2008; Ming and others 2008; Suzuki and others, 2008). Although unstable traits are theoretically possible, the elimination of any plants showing unstable inheritance during greenhouse trials and field testing is able to insure that commercialized plants behave in a predictable fashion over several generations. The regulatory requirements for transgenic crops ensure that only crops with stable inheritance are commercialized.

The kind of evidence needed to prove genetic instability in a plant is provided by recent research paper (Sureshkumar and others 2009) where a gene in the mustard cress plant was demonstrated to be genetically unstable. The genetic instability was nothing to do with genetic engineering. It involved repetition of small intervals of DNA that was present many times at a genetic site. None of the behaviour seen with this naturally occuring, unstable DNA has been demonstrated for the 35S promoter.

2. Plants chromosomes have many “hotspots” that do not cause chromosome instability. Chromosomes are not inert unchanging structures, but neither are they blandly uniform in their ability to undergo change. They have hotspots and coldspots. And although positions on chromosomes that undergo relatively rapid change are called hotspots, the term hotspot does not necessarily imply instability in a practical sense. This is because the timescale over which hotpots for genetic exchanges operate is a long one, stretching out over evolutionary time scales of hundreds, if not thousands, of generations. Putting this differently, to find a hotspot one might have to search through millions of plants before one encounters a genetic alteration.

Studies of chromosomes that have evolved separately for hundreds of generations do, however, reveal that they have undergo many rearrangements, gene additions, gene deletions, and other complicated changes. The causes of most of this change are numerous DNA parasites hiding in chromosomes that trigger chromosomal rearrangement wherever they interact with chromosomes.  DNA parasites responsible for such chromosomal change include parasites are found in maize (Lal and Hannah 2005, Lai and others 2005, Morgante and others 2005), in rice (Lisch 2005), and soybeans (Zabala, Vodkin 2008). Over time, these cause much change in chromosomal structure, especially because they are present in such large numbers.

The presence of these parasite hotspots for change though, does not mean that chromosomes are particularly unstable over a single generation. As mentioned it needs long time periods or investigation of very large numbers of plants before their activities are detected. But in large plant populations such as total world population of a crop they are responsible for large amounts of genetic change. Hence mobile parasitic DNA’s active role in rearranging chromosomes exposes humans to a large amount of genetic novelty because of their wide distribution in food crops and high total numbers per plant.

Genetic Roulette makes no comparison of the genetic risks posed by the natural activities of mobile DNA in corn, rice, soybean and other food crops compared with the genetic risks posed by genetically engineered plants, but both pose similar risks of unintended genetic changes. The magnitude of the genetic novelty created by parasitic DNA is substantially larger than that posed by transgenic crops created by genetic engineering. The genetic risks of instability from the 35S promoter are miniscule compared to these existing potential genetic hazards.

3. Plants have hotspots for sex but these hotspots are not unstable. Another type of rearrangement that chromosomes can undergo is exchanging their DNA with homologous DNA in a sister chromosome during sexual reproduction.  Careful studies of this type of DNA swapping occurring during the sexual cycle have shown that regions of chromosomes without genes are relatively inactive –cold spots that is — compared to the genes themselves, or regions near the genes — which are hotspots. Thus genes are hotspots for exchange of DNA between sister chromosomes (Yandeau-Nelson and others 2005, Lichten and Goldman 1995, Petes 2001). For example, in at the beginning of the maize gene called, anthocyanin1, there is a recombination hotspot where the rate of sexual DNA recombination 20- to 60-fold more than the average rate seen for the entire chromosome (Xu and others 1995). These hotspots do not cause chromosomes to become unstable.

4. Introduction of a transgene promoter doesn’t cause GM plants to be unstable. British scientist Ajay Kohli and colleagues have reported some experiments indicating that the 35S promoter might act as a hotspot for DNA joining reactions between the incoming transgene and the chromosomal DNA site into which it is inserted (Kohli and others 1999). Jeffrey Smith claims that this hotspot means that transgenes will be genetically unstable when plants are growing in the field.

Smith’s argument that the 35S promoter used in transgenic crops generates instability seems to be based on his misunderstanding or misapplication of what the term “hotspot” means.  It does not refer to assessments of gene stability after it has been inserted into a plant chromosome. It actually refers to events that occur when DNA repair mechanisms of the plant are stimulated by the presence of fragmented DNA introduced in the plant during scientific experiments. Under these special conditions DNA repair enzymes are triggered into activity inside the plant and produce high rates of DNA rearrangement (Gorbunova and Levy  1999, Kohli, Christou 2008).

Smith ignores a scientific report that explicitly refutes his interpretation published by Hull and others in 2000, and which clearly makes the distinction between transgene DNA before it is inserted (integrated) into a chromosome, and transgene DNA after it is inserted.

It has this to say:

“There is uncertainty concerning the stage of transformation at which the recombination described by Kohli and others occurred. They did not distinguish between recombination taking place during the process of transformation and recombination taking place after the sequences have been integrated. There is accumulating evidence of rearrangements of DNA during transformation [i.e. before insertion]…  In most cases these rearrangements result in the non-functioning of the transgene and are selected out in the early stages of analysis of the properties of transformed lines. Furthermore, the construct [DNA] used for transformation by Kohli and others had three copies of the 35S promoter, one inverse orientation in relation to the other two. The presence of repeated sequences in transformed integrants [i.e. plants with transgene inserts], and especially inverse repeats, also tends to lead to gene silencing … a condition which would be selected against in the developing transgenic line.”

In other words, there is no reason to take the observations by Kohli and others as an indication of possible instability in transgenic plants carrying the 35S promoter when it is inserted in replicating chromosome during normal plant propagation. Additionally, the incoming transgenic DNA studied by Kohli and others had a very unusual structure which is not used in commercialized transgenic plants because it would lead to silencing of the very traits that seed companies seek to get expressed. The evidence for potential instability of the 35S promoter thus falls to bits under scientific scrutiny. Genetic Roulette carefully avoids letting the reader know about this published scientific scrutiny.

Besides being confused about what the Kohli and colleagues were investigating, Genetic Roulette does not provide any evidence to show that any commercialized genetically engineered plant is genetically unstable, and neither does it  mention that these crops are scrutinized by both regulatory bodies and seed company scientists to ensure that they are genetically stable for practical use by farmers. After all, it’s in the interests of both the companies that develop these crops and the farmers themselves that they retain their ability to give good performance during seed multiplication and in the field by being genetically stable.

References

Gorbunova V and Levy AA (1999) How plants make ends meet: DNA double-strand break repair. Trends in Plant Science 4(7):263-269. Plants have particularly error-prone mechanisms that join together bits of broken chromosomes. These repair mechanisms scramble the DNA at the site at which the chromosomes are joined together during their repair. Radiation is a common cause of broken chromosomes and triggers these processes which scramble plant DNA and cause mutations.

Hull R, Covey S & Dale P (2000). Genetically modified plants and the 35S promoter: assessing the risks and enhancing the debate. Microbial Ecology in Health and Disease 12: 1–5. An explanation why the 35S promoter is not a site of DNA instability in transgenic plants, plus a discussion of other worries about 35S.

Kohli A and others (1999) Molecular characterisation of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microbiology mediated recombination. The Plant Journal 17(6):561-601. Investigation of the changes to insert and target DNA that occur during transgene insertion into plants.

Kohli A, Christou P (2008) Stable transgenes bear fruit. Nature Biotechnology 26(6):653-654. Analysis of the transgenic papaya genome sequence suggests that transgenes generally stay put following integration and can achieve stable expression level from generation to generation.

Lal SK and L. Hannah LC (2005) Helitrons contribute to the lack of colinearity observed in modern maize inbreds. Proceedings of the National Academy of Sciences of the USA 102 (29): 9993–9994

Lai J Li Y, Messing J, Dooner HK. (2005) Gene movement by Helitron transposons contributes to the haplotype variability of maize. Proceedings of the National Academy of Sciences of the USA 102(25):9068-73.

Lewin B. Genes VIII. James and Bartlett. (Chapter 15 on recombination.)

Lichten and Goldman (1995). Meiotic recombination hotspots. Annual Review of Genetics 29:423-444

Ming R and others (2008). The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus).  Nature  252:991-997

Sureshkumar S and others (2009). A genetic defect caused by a triplet repeat expansion in Arabidopsis thaliana. Science 323(5917): 1060-1063. Published online 15 January 2009 [DOI: 10.1126/science.1164014] This article documents the rapid genetic changes that occur in a gene in the mustard cress plant Arabidopsis.They are caused by repeats of three bases in the DNA –the so-called triplet repeats — that lead to genetic instability. In this case the plant splits out progeny which are defective at a high rate. For more understanding about this kind of phenomenon, readers are urged to read Christopher Wills’ The Runaway brain: the evolution of human uniqueness. Flamingo 1993. In in chapter 9 of that book genetic instability affecting the Love Song of the Fruit Fly is explained in graphic detail .

Suzuki J and others (2008). Characterisation of insertion sites in Rainbow papaya, the first commercialized transgenic food crop.  Tropbical Plant Biology. Tropical Plant Biol. (2008) 1:293–309 DOI 10.1007/s12042-008-9023-0. “Detection of the same three inserts in samples representing transgenic generations five to eight (R5 to R8) suggests that the three inserts are stably inherited.”

Morgante M and others (2005) Gene duplication and exon shuffling by helitron-like transposons generate intraspecies diversity in maize.Nature Genetics 37(9):997-1002.

Petes TD (2001). Meiotic recombination hotspots and cold spots. Nature Reviews Genetics.2:360-369.

Yandeau-Nelson MD and others (2005). Genetics MuDR transposase increases the frequency of meiotic crossovers in the vicinity of a Mu insertion in the maize a1 gene. Genetics 169: 917–929.

Xu X and others (1995). Meiotic recombination break points resolve at high rates at the 5′ end of a maize coding sequence. Plant Cell 7: 2151–2161.

Zabala G, Vodkin L (2008). A putative autonomous 20.5 kb-CACTA transposon insertion in an F3′H allele identifies a new CACTA transposon subfamily in Glycine max. BMC Plant Biology. Research article Open Access www.biomedcentral.com/1471-2229/8/124 Soybeans harbour active mobile genes that insert disruptively and cause mutations.

Genetic Roulette Falsely Claims:

1. Evidence suggests that CaMV promoter, used in most GM foods, contains a recombinant hotspot.

2. If confirmed, this might result in breakup and recombination of the gene sequence.

3. This instability of the inserted gene material might create unpredicted defects.

Genetic Roulette discusses changes to structure of the first-generation 35S promoter used in transgenic plants that can occur when it is inserted into plant chromosomes. It then extrapolates from what can happen during laboratory manipulation of DNA to what might happen with transgenic plants when they are cropped on farms.

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