ngin - Norfolk Genetic Information Network

10 February 2002


1. Watering Down Europe’s New Rules on GMOs - ISIS
2. Science in crisis as biotech lies surface - nlpwessex


1. Watering Down Europe’s New Rules on GMOs

Europe’s new rules governing releases of GMOs into the environment require molecular data on genetic stability as well as comprehensive environmental risk assessment, which, if strictly implemented, could sink all GMOs ( ISIS News 11/12 Unfortunately, the process of watering down has already begun in the United Kingdom, Europe’s most corporate-friendly nation. Dr. Mae-Wan Ho reports.

Europe’s New Rules on Releases of GMOs (Directive 2001/18) entered into force on 17 April 2001, but member states have until 17 October 2002 to take measures at national level. UK’s Advisory Committee for Releases to the Environment (ACRE) has issued a draft document "Guidance on best practice for the presentation of molecular data" to provide advice for companies applying for approval of release of GM crops. ACRE is the main scientific body that sets standards for the molecular data.

A careful reading of the document reveals that it effectively requires no molecular evidence of genetic stability.

The document begins by reminding the applicant of the new Directive, "An essential component of applications to release is molecular characterisation of the genetically modified plant. Applicants must provide information on what transgenes have been inserted during the genetic modification process, their copy number and stability, the expression of the transgene and the mode of action of any transgene products."

This message is repeated in different forms elsewhere throughout the document. But beneath the strict-sounding rhetoric, the company is given plenty of leeway. The first major loophole is under "differentiated procedures".

Even though the molecular data must be supplied for both part B (research & development) and part C (commercial) approval, companies often apply for the "First Simplified Procedure" to enter varieties into the National Seed testing programmes. "This procedure allows for an a priori programme of work and takes account of the fact that at the time of application it might not be known what will be the location of the other releases in the programme of work." In other words, companies are given permission to carry out an unspecified number of future tests whenever and wherever they like.

Genetically modified plants qualify for the First Simplified Procedure if:
"the taxonomy and biology of the recipient plant species are well known;
information is available about the interactions of the recipient plant species with the ecosystem in which the releases are intended to take place;
the inserted sequences and their expression products are safe for humans and the environment under the conditions of the released;
the inserted genetic material is well characterised and;
the inserted genetic material is integrated into the nuclear genome of the recipient plant."

These are extremely weak criteria compared to those required by the present Directive. Notably, no molecular characterisation is demanded, nor is molecular evidence of stability required, except for a derisory "inheritance patterns in one generation", or the transfer of transgene "through one cycle of tissue culture".

For part C approval, companies have to provide molecular data to ensure traceability and labelling.

The molecular data here should enable regulators to identify specific GM lines and distinguish among them. Each GM line results from random gene insertion events that took place in a single cell, out of which an entire plant was produced, and after several generations of propagation, a GM line is obtained. The molecular identification is unique to each GM line, and depends on the genetic stability of the GM line obtained.

Here too, we are treated to long admonitions of the detailed molecular information that must be supplied, only to discover at the end that ACRE does not ask for molecular data of genetic stability as such!

Genuine molecular data on genetic stability should consist of at least five successive generations of relevant molecular analyses, to show that the insert has remained stable in both its structure and its location in the plant genome.

Genetic stability is the single most important criterion in biosafety risk assessment in terms of concreteness and simplicity. Unfortunately, evidence is accumulating that GM lines are inherently unstable. Genetic instability not only compromises agronomic performance, it is a safety issue, as unstable transgenes could spread out of control by horizontal gene transfer and recombination (see "Mexican corn contaminated by horizontal gene transfer?", this issue).

Decades and billions of dollars of investments have already been wasted on GM, on account of the failure to take genetic stability seriously. This should now be put right under Europe’s new Directive.

Be sure to send comment before 28 February 2002 to The Secretary, ACRE, Floor 3/H11, Ashdown House, London SW1E 6DE, The document you need is:

Guidance on best practice for the presentation of molecular data in submissions to the advisory committee on releases to the environment. Advice for applicants seeking permission to deliberately release genetically modified organisms into the environment (under Directive


2.   Science in crisis as biotech lies surface - nlpwessex

The article below comes from the ISB News Report February 2002, a pro-biotech news bulletin for the scientific community.  Constantly we hear from the proponents of genetic engineering how scientific and 'precise' this technology is.

As you will see from this article these claims are ....ahem... a lie. To quote the article:

".... due to a lack of understanding of the underlying molecular mechanisms of transgene introduction and integration, plant transformation remains more an art than a science. All of the three main techniques used for plant transformation, Agrobacterium-mediated, protoplast, and particle bombardment transformation, result in unpredictable integration of transgenes. This has led to concerns that transformation might indirectly alter the expression of other genes, resulting in a toxic or allergenic phenotype.... Frequently, many transgenic plants will contain multiple copies of the transgene, either in the form of tandem repeats at a single locus, or scattered throughout the genome of the plant.... Currently, transgene integration into the host genome is essentially random, regardless of the method used to perform the transformation."

A previous ISB News Report further confirms this situation -

The fact that food related illnesses doubled in the period following the widespread introduction of GM foods in the US (, and the fact that the regulatory authorities in both the EU and the US have admitted that they do not know how to pre-market test for adverse effects (see:
and GOVERNMENT & POLICY - WHAT'S HIDING IN TRANSGENIC FOODS, Chemical & Engineering News, 80 (1), January 7, 2002
add further to a truly scandalous situation.

The admissions below of the lack of a scientific approach in genetic engineering coincide with a media report this week that confirms that scientists in the biotechnology sector have been putting their names to articles in scientific journals which are in fact ghost authored by industrial interests. This situation is reported in the Guardian in more detail 7 February

Science is truly in crisis when radical and potentially dangerous technologies are placed in the hands of people willing to lie or to avoid reporting on the science as it has been carried out.  The amount of money at stake in these matters is now so great that this crisis looks likely to deteriorate to the point where the scientific community totally loses the respect and confidence of the rest of society (fairly or unfairly the current row over MMR vaccines already demonstrates how this mistrust is beginning to express itself). This is a situation that the scientific community has failed to pre-empt.

Focusing in particular on psychiatric biotech products Fuller Torrey, executive director of the Stanley Foundation Research Programmes in Bethesda, Maryland, refers in the Guardian article to the damaging relationship between science and industry that has emerged: "Some of us believe that the present system is approaching a high-class form of professional prostitution."

Modern science is in danger of rapidly becoming implicated in Enrongate type fraud - that is, fraud for the sake of influencing financial performance and reporting. In fact it was clearly already there long before Enron took to it (as the 'scientific' defence of the tobacco industry amply illustrates).

No wonder the public don't want anything to do with GM foods.

Other reports confirming the lack of precision and scientific knowledge supporting genetic engineering can be viewed at:


"...GM techniques which in the precise and targeted way bring in a couple of genes that you know what they do and you know where they are is vastly safer, vast, vastly more controlled than this so-called conventional breeding...."
Sir Robert May, UK Government Chief  Scientist 1995 - 2000, and current President of the Royal Society, UK (BBC interview 9th March 2000)

"We should be on our guard not to overestimate science and scientific methods when it is a question of human problems; and we should not assume that experts are the only ones who have a right to express themselves on questions affecting the organization of society."
Albert Einstein May 1949


ISB News Report
February 2002



The current widespread application of genetic engineering to crop species is largely due to the ease of plant transformation. Plant transformation, the process of introducing a foreign or engineered DNA element into the native genome of a plant, has been successfully performed for almost 20 years. However, due to a lack of understanding of the underlying molecular mechanisms of transgene introduction and integration, plant transformation remains more an art than a science. All of the three main techniques used for plant transformation, Agrobacterium-mediated, protoplast, and particle bombardment transformation, result in unpredictable integration of transgenes. This has led to concerns that transformation might indirectly alter the expression of other genes, resulting in a toxic or allergenic phenotype. Fortunately, recent research is expanding our understanding of how introduced genes are integrated at the molecular level during transformation, suggesting strategies for controlling the location and expression of transgenes.

In any plant transformation experiment, the researcher knows that many independent transgenic lines will have to be screened before a line stably expressing a single copy of the transgene is isolated. Frequently, many transgenic plants will contain multiple copies of the transgene, either in the form of tandem repeats at a single locus, or scattered throughout the genome of the plant. This is a problem for two reasons. First, the integration of multiple copies of a transgene has been linked with gene silencing, a poorly understood phenomenon, where the expression of an introduced gene is somehow detected and "shut off" by the plant's cellular machinery. Second, overexpression of the transgene due to multiple copies can prove to be toxic to the plant, leading to poor growth or even no growth. Even if the plant contains only a single copy of the gene, there is no guarantee that it will be expressed correctly. The degree of expression of the transgene can also be determined by the site of insertion, otherwise known as the "positional effect."

The main techniques used for plant transformation can be loosely grouped under two headings: Agrobacterium-mediated transformation and methods that use direct DNA delivery for transformation. Protoplast transformation was the first plant transformation technique developed using direct DNA delivery. In this method, protoplasts derived either directly from plant tissues or from a plant cell suspension culture are induced to take up naked DNA through treatment with membrane permiabilization agents such as polyethylene glycol (PEG) or by electroporation. The method is useful, as it is genotype-independent, but the degree of finesse required for success, along with the high occurrence of spontaneous mutations caused by long periods in tissue culture, restrict the application of the technique to species recalcitrant to other methods of transformation.

A more commonly used method of direct DNA delivery transformation is a method known as microprojectile, particle bombardment, or biolistic transformation. In this method, tiny particles of tungsten or gold are coated with DNA containing the construct of interest. These particles are then "shot" into the plant tissue using gunpowder, gas, compressed air, or other methods of acceleration. The force of the acceleration drives the tiny particles through the wall and membrane of the plant cells, delivering the naked DNA directly into the cells' interiors. The exact mechanism of how the naked DNA then becomes integrated into the plant's genome is unknown, but multiple studies have shown that in the vast majority of cases microprojectile transformation results in the integration of multiple, often rearranged, copies of the transgene. One currently proposed theory suggests that the introduced transgenes are first spliced into arrays by the cells' endogenous machinery before integration into the plant's genome. This theory might partially explain the main drawback of this transformation method - the high occurrence of genetic rearrangements found in recovered transformants.

Another explanation may be found in the results of a study, reported in Theoretical and Applied Genetics, indicating that microprojectile transformation may involve chromosome breakage and re-ligation. In this study, Svitashev et al. characterized transgenic lines of hexaploid oat, using a combination of phenotype, genotype segregation, Southern blot, and fluorescence in situ hybridization  (FISH) analyses.(1) Six of the 25 transgene loci examined were associated with rearranged chromosomes. Through Southern blot analysis and FISH performed on metaphase chromosomes, evidence of both chromosomal rearrangement and breakage events could be detected. The authors theorize that this may be the result of physical breakage of the host cell's DNA during particle bombardment or, possibly, the integration event itself. However, these results conflict with the data described in a second, more recent paper in the same journal. Jackson et al. studied 13 independent transgenic wheat lines transformed using microprojectile bombardment.(2) The authors used a high-resolution form of FISH to physically map the location and structure of the integrated transgenes. Although the authors found evidence of large, tandem repeats of the transgenes integrated in the plant's genome, they were unable to detect any chromosomal rearrangements associated with the integrated transgenes. Regardless of the exact nature of the mechanism, it seems clear from the data described in these papers that microprojectile transformation often results in transgenic plants with a complex pattern of transgene integration.

Agrobacterium-mediated transformation, the most widely used method of plant transformation, utilizes the natural ability of the plant pathogen, Agrobacterium tumefaciens, to transfer DNA sequences from a particular segment of an endogenous plasmid within the bacterium to the nuclear genome of the plant. This segment, known as the T-DNA, usually includes one or two genes of interest, as well as a marker gene. This method is widely favored due to its ease of use and low cost. Unfortunately, the restricted host range of the bacterium meant that some dicot and most monocot species were, until recently, incompatible with the technique. However, the
development of new, supervirulent forms of the plasmid vector and species-specific pretreatments has led to a dramatic expansion in the number of species transformed using this technique. Another reason for the popularity of this method is that the T-DNA usually seems to integrate in transcriptionally active regions of the plant genome, increasing the likelihood that the transgene will be expressed.

However, it is not uncommon for Agrobacterium-mediated transformation to result in the integration of multiple copies of the transgene in the form of tandem repeats. Tandem repeats resulting from T-DNA insertion have been reported and investigated in a number of crop species. These types of repeats can be difficult to detect by Southern blot, since they tend to integrate at a single location in the plant genome. In a recent paper published in Molecular and General Genetics, Kumar and Fladung reported using rpPCR, a method that utilizes primer pairs oriented in opposite directions, to identify tandem repeats in 45 transgenic aspen and hybrid aspen lines transformed with six different constructs.(3) All the transgenic lines were generated through standard Agrobacterium-mediated transformation. In the lines examined, 21% contained multiple repeats of the transgene; however the organization of the repeats consisted of both direct and inverted repeats. Some of the lines were also found to contain "filler" DNA between the repeated T-DNAs, ranging from four to almost 300 base pairs. Interestingly, the authors found that all of the direct repeats contained identical residual right-border repeat sequences. They speculate that this sequence, combined with the mechanism of T-DNA insertion, is responsible for the formation of direct repeats. However, a single mechanism is unlikely to account for all the different repeat structures seen resulting from Agrobacterium-mediated transformation.

Currently, transgene integration into the host genome is essentially random, regardless of the method used to perform the transformation. As a result, attempts have been made to develop a system for targeted transgene insertion, either through the use of scaffold attachment sites or through the introduction of elements of a homologous recombination system, such as the Cre/lox system. To date, however, these efforts have yielded inconsistent results, making them unsuitable for commercial application. Nevertheless, these technologies are making great advances, and it is hoped that, combined with the increasing understanding of the mechanisms of transgene integration, it will soon be possible to precisely and consistently engineer plants to express a single copy of an introduced gene.


1.  Svitashev S, Ananiev E, Pawlowski WP, and Somers DA. 2000. Association of transgene integration sites with chromosome rearrangements in hexaploid oat. Theoretical and Applied Genetics 100: 872-880.

2.  Jackson SA, Zhang P, Chen WP, Phillips RL, Friebe B, Muthukrishnan S, and Gill BS. 2001. High-resolution structural analysis of biolistic transgene integration into the genome of wheat. Theoretical and Applied Genetics 103: 56-62.

3. Kumar S and Fladung M. 2000. Transgene repeats in aspen: molecular characterisation suggests simultaneous integration of independent T-DNAs into receptive hotspots in the host genome. Molecular and General Genetics 264: 20-28.

Claire Granger

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