permaculture

["Lawrence F. London, Jr." <venaurafarm@bellsouth.net>]

The Institute of Science in Society
Science Society Sustainability
http://www.i-sis.org.uk

This article can be found on the I-SIS website at
http://www.i-sis.org.uk/new_route_for_GM_gene_escape.php

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ISIS Report 02/03/11

Scientists Discover New Route for GM-gene 'Escape'

http://www.i-sis.org.uk/new_route_for_GM_gene_escape.php
ISIS Report 02/03/11
Scientists Discover New Route for GM-gene 'Escape'

Genetically modified genes can jump species via wounds, yes horizontal 
gene transfer happens, and at high frequencies; it is the greatest, most 
underestimated hazard from GMOs released into the environment Dr. Mae-Wan Ho

Please circulate widely and forward to your elected representatives
Gene “escape” a misnomer for horizontal gene transfer

Scientists at Bristol University in the UK announced the discovery of 
[1] “a previously unknown route” whereby “GM genes may escape into the 
natural environment.”  “Escape” is a misnomer. There is no need for the 
GM (genetically modified) genes to “escape”, when genetically modified 
organisms (GMOs) have been released in great abundance and with gay 
abandon into the environment over the past 17 years. At issue is how 
fast and how widely the GM genes can spread, and what dire consequences 
could arise.

The “escape” referred to is horizontal gene transfer – the spread of GM 
genes by infection and multiplication (literally like a virus) 
regardless of species barriers; hence the rate of spread is much more 
rapid, and the extent virtually unlimited.  New combinations of genetic 
material are created at unprecedented speed; affecting species the most 
that reproduce the fastest, i.e., bacteria and viruses that cause 
diseases. Horizontal gene transfer and recombination is indeed a main 
route for generating new strains of bacteria and viruses that cause 
diseases. Genetic modification and release of GMOs into the environment 
is nothing if not greatly facilitated horizontal gene transfer and 
recombination. It has created highways for gene trafficking in place of 
narrow by-ways and occasional footpaths that previously existed.

Some of us have long considered horizontal gene transfer to be the most 
serious hidden and underestimated hazard of genetic engineering, and 
have alerted regulators accordingly, time and again, since GMOs were 
first released (see for example [3, 4] (Gene Technology and Gene Ecology 
of Infectious Diseases, ISIS scientific publication; Genetic Engineering 
Dream or Nightmare, ISIS publication). The recent “emergency” warning 
sent by a senior US Department of Agriculture scientist to US Secretary 
of Agriculture on a suspected pathogen “new to science” associated with 
GM crops may prove to be a case in point [5] (Emergency! Pathogen New to 
Science Found in Roundup Ready GM Crops? SiS 50).
Plant wounds hotspots for gene trafficking

The researchers at Bristol University showed that plant wounds, that 
could be created by insect bites, abrasion and other mechanical damage, 
are hotspots for gene trafficking due to the wound hormones produced by 
the plant. Under such circumstances, the soil bacterium Agrobacterium 
tumefaciens, which causes crown gall disease in plants, could enlarge 
its host range to infect fungi, and insert foreign genes into the 
fungi’s genome [2]. This has large implications on the safety of GMOs 
already widely released into the environment.

A. tumefaciens is probably unique among natural plant pathogens in 
carrying out trans-Kingdom horizontal gene transfer during an infection, 
and it is this ability that has been widely exploited for creating GM 
crops, grown on an estimated 134 million hectares worldwide in 2009, and 
“jumped’ another 10 percent in 2010, according to industry-funded 
International Service for the Acquisition of Agri-biotech Applications 
(ISAAA) [6].

Research commissioned by the UK Department of the Environment, Food and 
Rural Affairs (DEFRA) in the 1990s had already revealed that it is very 
difficult, if not impossible to get rid of the Agrobacterium vector used 
in creating the transgenic plant [7], and the bacterium is likely to 
remain dormant even after the transgenic plants are transplanted into 
the soil. Hence, it is expected to facilitate horizontal gene transfer, 
in the first instance, to wild-type Agrobacterium in the soil, and 
further afield.

Disease-causing strains of A. tumefaciens have an extrachromosomal Ti 
(tumour-inducing) plasmid that enables the horizontal transfer of a 
segment of the Ti plasmid, the T-DNA, into the plant cell genome when 
the bacterium’s virulence (disease causing) system is activated by 
hormones produced by the wounded plant. This feature is exploited in 
creating genetically modified organisms (GMOs), by disarming the 
bacterium, and incorporating the virulence genes in a ‘binary’ vector 
that has to be used in conjunction with the disarmed Agrobacterium strain.

In the 1990s, it was shown that the range of organisms transformed by 
Agrobacterium could be extended if the wound hormone acetosyringone was 
used to induce the virulence system.

The researchers at Bristol University reasoned that as A. tumefaciens is 
a soil-dwelling pathogen that often infects plants through wounds, it is 
conceivable that the bacterium could encounter numerous species of 
microorganisms, including pathogenic fungi that the same method to gain 
entry into the plant. The wound sites are likely to be exuding wound 
hormones such as acetosyringone, so the bacteria are primed for T-DNA 
transfer.
Experiments confirmed their suspicion in full

They carried out their investigation using the wilt-causing fungus 
Verticillium albo-atrum, a strong candidate for encounters with 
Agrobacterium in the plant, as it has a similar wide host range in 
plants, infecting both root and crown. Previous lab experiments have 
shown that V. albo-atrum cannot be transformed by Agrobacterium in the 
absence of acetosyringone. So, if it is presented with Agrobacterium on 
plant tissue, and transformation does occur, it must be the plant that 
supplies the wound hormone.

Peeled and sliced potato tubers and carrots, leave- and stem-sections 
from tobacco plants were used as the plant tissues for testing. After 
sterilization, they were inoculated with both A. tumefaciens and V. 
albo-atrum and left at room temperature in a covered agar dish for a 
minimum of 8 days and a maximum of 42 days.

Successful transformants of V. albo-atrum were obtained from every kind 
of plant tissue. 2 out of 17 potato slices, 1 out of 15 carrot slices; 
14 out of 42 dishes each with 3-5 leaf pieces, and 10 out of 31 stem 
sections (without agar plate, so as to be as close to the natural 
condition as possible). These transformants were confirmed with 
molecular genetic analyses.
Implications on risk assessments of GMOs still understated

The researchers concluded [2]: “This work therefore raises interesting 
questions about whether the host range of A. tumefaciens in nature is 
greater than just plants. It is possible that evidence of such events 
could be looked for retrospectively in the increasing number of genome 
sequences becoming available....

“In addition, the result may well have implications for the risk 
assessment of GM plants generated via Agrobacterium-mediated 
transformation, as Agrobacterium can survive within plant tissue through 
transformation and tissue culture and can therefore be found within 
regenerated transgenic plants...”

This is an understatement of a serious risk that has been known almost 
since the first release of Agrobacterium-transformed GMOs into the 
environment.
The risks are far greater than admitted

We have repeatedly drawn attention to the possibility of facilitated 
horizontal gene transfer from GMOs created with Agrobacterium vector, 
which is even stronger than originally envisaged due to other 
discoveries made since then. I reproduce what we wrote in 2008 [8] 
(Horizontal Gene Transfer from GMOs Does Happen, SiS 38), which repeats 
an earlier account [9] (Living with the Fluid Genome , ISIS publication) 
(see Box).
Agrobacterium vector a vehicle for facilitated horizontal gene transfer 
[8, 9]

“We have ..provided evidence strongly suggesting that the most common 
method of creating transgenic plants may also serve as a ready route for 
horizontal gene transfer [9, 10].

“Agrobacterium tumefaciens, the soil bacterium that causes crown gall 
disease, has been developed as a major gene transfer vector for making 
transgenic plants. Foreign genes are typically spliced into the T-DNA - 
part of a plasmid of A. tumefaciens called Ti (tumour-inducing) – which 
ends up integrated into the genome of the plant cell that subsequently 
develops into a tumour.

“But further investigations revealed that the process whereby 
Agrobacterium injects T-DNA into plant cells strongly resembles 
conjugation, the mating process between bacterial cells.

Conjugation, mediated by certain bacterial plasmids requires a sequence 
called the origin of transfer (oriT) on the DNA that’s transferred. All 
the other functions can be supplied from unlinked sources, referred to 
as ‘trans-acting functions’ (or tra). Thus, ‘disabled’ plasmids, with no 
trans-acting functions, can nevertheless be transferred by ‘helper’ 
plasmids that carry genes coding for the trans-acting functions. And 
that’s the basis of a complicated vector system devised, involving 
Agrobacterium T-DNA, which has been used for creating numerous 
transgenic plants.

“It soon transpired that the left and right borders of the T-DNA are 
similar to oriT, and can be replaced by it. Furthermore, the disarmed 
T-DNA, lacking the trans-acting functions (virulence genes that 
contribute to disease), can be helped by similar genes belonging to many 
other pathogenic bacteria. It seems that the trans-kingdom gene transfer 
of Agrobacterium and the conjugative systems of bacteria are both 
involved in transporting macromolecules, not just DNA but also protein.

“That means transgenic plants created by the T-DNA vector system have a 
ready route for horizontal gene escape, via Agrobacterium, helped by the 
ordinary conjugative mechanisms of many other bacteria that cause 
diseases, which are present in the environment.

“In fact, the possibility that Agrobacterium can serve as a vehicle for 
horizontal gene escape was first raised in 1997 in a study sponsored by 
the UK Government [7, 12], which found it extremely difficult to get rid 
of the Agrobacterium in the vector system after transformation. 
Treatment with an armoury of antibiotics and repeated subculture of the 
transgenic plants over 13 months failed to get rid of the bacterium. 
Furthermore, 12.5 percent of the Agrobacterium remaining still contained 
the binary vector (T-DNA and helper plasmid), and were hence fully 
capable of transforming other plants.

“Agrobacterium not only transfers genes into plant cells; there is 
possibility for retrotransfer of DNA from the plant cell to 
Agrobacterium [13]. High rates of gene transfer are associated with the 
plant root system and the germinating seed, where conjugation is most 
likely [14]. There, Agrobacterium could multiply and transfer transgenic 
DNA to other bacteria, as well as to the next crop to be planted. These 
possibilities have yet to be investigated empirically.

“Finally, Agrobacterium attaches to and genetically transforms several 
human cell lines [15, 16] (Common plant vector injects genes into human 
cells ISIS News 11/12). In stably transformed HeLa cells (a human cell 
line derived originally from a cancer patient), the integration of T-DNA 
occurred at the right border, exactly as would happen when it is 
transferred into a plant cell genome. This suggests that Agrobacterium 
transforms human cells by a mechanism similar to that which it uses for 
transforming plants cells.

“The possibility that Agrobacterium is a vehicle for horizontal transfer 
of transgenic DNA remains unresolved to this day.”
Agrobacterium transfers genes into human cells

It is also worth reiterating our comment on the scientific paper [15] 
documenting that Agrobacterium can transfer genes into human cells [16].

“The paper shows that human cancer cells along with neurons and kidney 
cells were transformed with the Agrobacterium T-DNA. Such observations 
should raise alarm for those who use Agrobacterium in the laboratory.

“The integrated T-DNA will almost certainly act as a mutagen as it 
integrates into human chromosomes. Cancer can be triggered by activation 
of oncogenes (ie, cancer genes) or inactivation of cancer-suppressing 
genes. Furthermore, the sequences carried within the T-DNA in the 
transforming bacterium can be expressed in the transformed cells (the 
viral promoter CaMV has been found to be active in HeLa cells [17]) ….

“It is clear that little has been done to prevent environmental escape 
of the transforming bacteria or to quantify such releases. In 
conclusion, a study of cancer incidence among those exposed to 
Agrobacterium tumefaciens in the laboratory and in the field is needed. 
It would be worthwhile to screen workers for T-DNA sequences.”
To conclude

The discovery by the Bristol University researchers barely scratches the 
surface of the hidden hazards of GMOs from horizontal gene transfer. It 
is high time for a global ban to be imposed on further environmental 
releases of GMOs, and all those responsible for releasing them should be 
brought to book.
References

1. “Hazards of GMOS: Agrobacterium mediated 
transformation”http://www.bristol.ac.uk/news/2010/7279.html

2. Knight CJ, Bailey AM, Foster GD. Investigating Agrobacterium-mediated 
transformation of Verticillium albo-atrum on plant surfaces. PLOS ONE 
2010, 5(10): e13684. Doi:10.1371/journal.pone.0013684

3. Ho MW. Gene technology and gene ecology of infectious diseases. 
Microbial Ecology in Health and Disease 1998, 10, 33-59.

4. Ho MW. Genetic Engineering Dream of Nightmare? The Brave New World of 
Bad Science and Big Business, Third World Network, Gateway Books, 
MacMillan, Continuum, Penang, Malaysia, Bath, UK, Dublin, Ireland, New 
York, USA, 1998, 1999, 2007 (reprint with extended Introduction). 
http://www.i-sis.org.uk/genet.php

5. Ho MW. Emergency! Pathogen new to science found in Roundup Ready GM 
crops? Science in Society 50 (to appear).

6. Global Status of Commercialized Biotech/GM Crops, 2009, ISAAA, 
http://www.isaaa.org/

7. Mc Nicol MJ, Lyon GD, Chen MY, Barrett C and Cobb E. Scottish Crop 
Research Institute. Contract No RG 0202.The Possibility of Agrobacterium 
as a Vehicle for Gene Escape. MAFF. R&D and Surveillance Report: 395.

8. Ho MW and Cummins J. Horizontal gene transfer from GMOs does happen. 
Science in Society 38, 22-24, 2008

9. Ho MW. Living with the Fluid Genome, ISIS/TWN, London/Penanag, 2003. 
http://www.i-sis.org.uk/fluidGenome.php

10. Ferguson G and Heinemann J. Recent history of trans-kingdom 
conjugation . In Horizontal Gene Transfer 2nd ed., Syvanen M and Kado 
CI. (eds.) Academic Press, San Diego, 2002.

11. Ho MW. Horizontal gene transfer, book review. Heredity 2003, 90, 6-7.

12. Barrett C, Cobb E, MacNicol R and Lyon G. A risk assessment study of 
plant genetic transformation using Agrobacterium and implication for 
analysis of transgenic plants.Plant Cell Tissue and Organ Culture 1997, 
19,135-144.

13. Kado C. in Horizontal Gene Transfer 2nd ed., Syvanen M and Kado CI. 
(eds.) Academic Press, San Diego, 2002.

14. Sengelov G, Kristensen KJ, Sorensen AH, Kroer N, and Sorensen SJ. 
Effect of genomic location on horizontal transfer of a recombinant gene 
cassette between Pseudomonas strains in the rhizosphere and spermosphere 
of barley seedlings. Current Microbiology 2001, 42, 160-7.

15. Kunik T, Tzfira T, Kapulnik Y, Gafni Y, Dingwall C, and Citovsky V. 
Genetic transformation of HeLa cells by Agrobacterium. PNAS USA, 2001, 
98, 1871-87.

16. Cummins J.  “Common plant vector injects genes into human cells. 
ISIS News 2002, 11/12, p. 10.

17. Ho MW, Ryan A and Cummins J. CaMV 35S promoter fragmentation hotspot 
confirmed and it is active in animals. Microbial Ecology in Health and 
Disease, 2000, 12, 189.

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There are 1 comments on this article so far. Add your comment
Prof Keshava Nireshwalia Comment left 2nd March 2011 16:04:27
In the light of arguments, there is a strong case for banning GMOs 
unless a protective strategy against the horizontal transfers and the 
potential hazards are controlled. Thanks indeed for the enlightenment on 
the topic.