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Genome scrambled, myth or reality?

Genome scrambled, myth or reality?

By RALLT

GMOs and their products have been evaluated "case by case". This is an acknowledgment that genetic transformation methods do not generate risks related directly to technology, but that some transgenic crops can present some problems.

At the international level, biosafety regulations have focused on analyzing the problems that can arise from specific crops and their products, in relation to possible impacts on human health and the environment.

GMOs and their products have been evaluated "case by case". This is an acknowledgment that genetic transformation methods do not generate risks related directly to technology, but that some transgenic crops may eventually present some problems.


However, the genetic transformation of crops through genetic engineering carries inherent risks with this technology.

For example, to genetically transform a culture, a tissue culture technique is required, that is, a whole plant is regenerated from a single cell, which has been treated with hormones and antibiotics to force it to continue. an abnormal developmental pattern.

On the other hand, genetic transformation is done through the intermediation of infectious organisms such as Agrobacterium tumefaciens, a bacterium that infects the plant genome.

Another method of inserting the transgenes is through tungsten particle bombardment.

Both technologies unleash risks at the genetic level, which will be described later.

Both the tissue culture technique and the gene insertion methods have been used in science as mutagenic agents. Therefore, it is not surprising that a transformed plant faces genetic consequences, which are alien to the characteristics contained in the transgene to be transferred to the crop.

Mutations induced by genetic transformation

Theoretically, genetically transformed plants are the result of the insertion of a transgene in an exact place in the avocado genome, and in each transformation, only one gene is inserted.

In practice, this never happens. On the contrary, it has been shown that in addition to the transgene to be inserted into the plant, each transformed genome contains its own spectrum of mutations for each event, as a result of:

  • tissue culture processes
  • Genetic transformation methods using Agrobacterium, or through particle bombardment
  • Insertion of the transgene
  • The insertion of superfluous DNA. Superfluous DNA is any piece of DNA other than the transgene that you want to insert so that the plant acquires the desired characteristic. Superfluous DNA includes: the marker gene sequence (often antibiotic resistance), the Agrobacterium bacterial plasmid sequences, and additional or partial copies of the transgene.

Mutations due to insertion site

The researchers found that very little is known about mutations created in plant cultures due to insertion site, both by insertions made through Agrobacterium and by bombardment of tungsten particles. This lack of understanding is due to the lack of large-scale research.

Additionally, much of the relevant information comes from an investigation carried out on a plant that is not a crop, such as Arabidopsis thaliana, and it is not clear that the results found can be applied to crops.

Agrobacterium-mediated transformation

Transformations made through Agrobacterium have been used to create commercial crops for 10 years and this type of insertion is known to produce mutations.

Despite this, there is only one large-scale study to evaluate mutations related to transgene insertion using Agrobacterium as a vector. Mutations created by insertion events have been studied (1) containing inserts containing only copies of T-DNA (2).

In that study, 112 T-DNA insertion events were carried out in Arabidopsis thaliana, and it was found that exact T-DNA integration never occurred. Most T-DNA inserts resulted in deletions (3) small of the plant genome sequences at the insertion site (between 1 and 1000 base pairs).

However, a significant number of events underwent large-scale rearrangements of the plant genome at the insertion site. Two of these insertion events contained chromosomal translocations. The rest had rearrangements that could not be fully characterized.

Other researchers have found duplication and translocation of a DNA segment of at least 40 Kbp in size.

Insertion of superfluous DNA is also common in insertion events made through Agrobacterium. For example, in the aforementioned research with A. thaliana, it was found that 8 of the 112 insertion events mediated by Agrobacterium, had included long segments of superfluous plasmid DNA or by T-DNA sequences. Most of the other events had incorporated DNA of undefined origin.

The results of these studies suggest that the vast majority of T-DNA insertion events include changes in the plant genome, affecting both small segments of DNA as well as larger segments, and that there was the insertion of superfluous DNA.

Particle bombardment transformation

Particle bombardment has been used to create numerous commercial crops. Although it can result in large-scale alterations in the genome, there are few studies detailing the insertion site-related mutations resulting from particle bombardment. Also, there have been no large-scale studies of those mutations.

The scientific literature describes most of the particle bombardment insertion events as extremely complex. For example, it has been reported that along with the desired transgene, multiple copies of small or large pieces of DNA from the plant genome are included.

One publication even reported insertion of bacterial chromosome DNA at the DNA insertion site by particle bombardment.

The analyzes that have been made of mutations caused at the insertion site are incomplete, because they were done without the use of PCR (4) and other DNA sequencing methods. The researchers found only two studies where PCR was used to characterize mutations, created by insertion events, of DNA isolated from whole plants. In one of those publications, three insertion events were analyzed.

In the other, RR soy was used, in which the 40-3-2 event was analyzed. The mutations present in each of these "simple" insertion events, it was found that there were large-scale deletions and rearrangements of the genome.

For example, in event 40-3-2 of RR soybean, in addition to the EPSPS gene (which is the gene that confers glyphosate resistance to the plant), it was found:

A 254 bp EPSPS gene fragment
A 540 bp segment of unidentified DNA
A segment of plant DNA
A 72 bp segment of EPSPS
Alterations in the genome with specific.

These insertion event-related mutations were not disclosed until only after RR soy had been commercialized.

It is interesting that in an independent analysis made of another transgenic variety already commercialized, Yieldgard maize, with the Mon810 insertion event, it also included non-specific and previously unreported mutations, related to insertion site mutations.

No study could be found on successful particle bombardment insertion events, compared to the original site.

Mutations in the insertion site of transgenes by particle bombardment have never been reported in their entirety, neither in the scientific literature, nor in the applications made to regulators.

The data that exist on the sequences that describe the insertion events by particle bombardment are extremely limited, and the techniques used to sequence the inserted DNA are not the most adequate. However, these data suggest that the integration of transgenes by particle bombardment is always accompanied by substantial changes in the genome and by the insertion of superfluous DNA.

Mutations related to tissue culture and gene transfer

This study also examined what is known about mutations that are introduced as a result of tissue culture and gene transfer procedures, but are not associated with transgene insertion.

There are five studies in which the number of mutations produced during the transformation of plants has been investigated. The researchers compared the genome of the transformed plants with that of non-transformed control plants.

The results suggest that many hundreds of thousands of mutations are present in the transformed plants. Since the techniques used are not the most appropriate, it is possible that these results underestimate the size of the mutations.


The magnitude of the mutations (for example, whether they are large or small), their origin, or whether they were present in the functional genome were not studied.

It is very possible that these mutations are maintained in commercial transgenic plants, and that they are inherited

However, the regulators who have deregulated a large number of GM crops have not taken these factors into account.

Importance of transformation-induced mutations

Mutations produced by the insertion of the transgene can be dangerous if they are present in the functional part of the genome.

Mutations in the functional part of the genome, including sequences that encode proteins such as those that regulate them, can have implications in the agronomic behavior of the crop, in its interactions with the environment and in animal and human health.

For example, a mutation in the functional part of the genome related to the insertion site can interrupt the functioning of genes related to the regulation of the synthesis of compounds that can be toxic to humans.

Important metabolic changes can occur with unpredictable consequences, and are difficult to identify in the studies that are typically done when you want to approve a new transgenic crop.

Frequency of functional DNA disruption by transformation-induced mutations

There are few studies on this, but these show that transgenes are often inserted close to gene sequences.

In the few plants studied, it is shown that the DNA sequences analyzed to evaluate the insertion site of the T-DNA, between 35 and 58% interrupt gene sequences. This is important because the regulatory sequences of a particular gene are often far removed from the gene it controls. The presence of foreign DNA segments between the two can alter the regulation system of the expression of that gene.

This type of study has never been done in transgenic plants obtained by means of particle bombardment.

On the other hand, it is very difficult to know whether a part of the genome is functional or not.

Most applications to commercialize new lines of transgenic crops do not include the DNA sequences that surround the insertion site, nor the sequences of the genome that surround the transgene.

The tissue culture technique has been used very successfully to induce mutations both for research purposes and for "genetic improvement" purposes. It has also been possible to isolate mutants from populations of transformed plants and mutant phenotypes, which are not related to the insertion of the transgene. This means that mutations occur due to tissue culture in functional DNA.

Even when there is no functional DNA disruption, the superfluous DNA entering with the desired transgene as well as the transgene itself are necessarily inert or harmless. For example, promoter sequences can alter the expression of neighboring genes. Sequences of bacterial chromosomal DNA or plasmids can increase the probability of horizontal gene transfer.

Of the 8 commercial cultures and events analyzed in this report, 6 had both bacterial and viral superfluous DNA insertions at the insertion events.

Conclusions

The report identifies that the mutations created by the insertion site and by genetic transformation procedures and tissue culture are potentially very large. But it has been very little studied and not very well understood. These mutations can pose a danger especially if they occur in commercial crops.

The authors propose that the implications of this type of mutations should be taken into account in regulatory systems and in the decision-making process about transgenic crops and products.

Notes:

(1) A transgene insertion event consists of the transgene and its additional sequences.

(2) T-DNA is a segment of DNA tied by the edges of T-DNA that is transferred to the plant via Agrobacterium. The T-DNA contains the desired transgene and often a genetic tag. These are carried by the Ti plasmid, and sometimes the outermost part of the plasmid is also carried and inserted into the plant genome.

(3) deletion is a mutation where one or more pairs of nucleotides are deleted from a gene.

(4) PCR: Polymerase chain reaction. It is a method to sequence DNA

* Source: Genome Scerambling - Mite or Reality ?. Transformaction-Induced Mutations in Transgenic Crop Plants.
Allison Wilson, Jonathan Latham and Ricarda Steibrecher
Technical Report October 2004. Econux. http://www.econexus.info


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