This technology cannot be contained and it does matter!
1. Selected papers on pollen dispersal over medium to long distances
2. Horizontal Gene Transfer - DNA in the Soil
---

1. Selected papers on pollen dispersal over medium to long distances (200m)
Compiled by The National Pollen Research Unit,
University College, Worcester ,WR2 6 AJ
8/3/01

The following selection of papers illustrate the fact that pollen from GM crops will spread far beyond the proposed separation distances. If commercial production takes place this will inevitably lead to an increase in plot size and number. It is reasonable to assume that the background concentrations of the pollen will rise and that the maximum 1% cross pollination guideline will be exceeded regionally.

Dispersal of pollen via Insects

The dynamics of insect foraging behaviour are vital in gaining an understanding of pollen dispersal and ultimately cross- pollination. Until recently it was thought that insects minimize the distance to obtain maximum of energy (optimal foraging) and consequently few studies have addressed the medium scale. Recent studies show that insects can follow complex foraging patterns at great distances.

Ramsey et al. (1999)

Honey bees and wild bumble bees are important pollinators of oilseed rape (OSR). Honey bees were found to leave the hive with OSR pollen from different sources indicating that either the individual bees forage on different OSR crops or simply pick up loose pollen in the hive. In either case this would lead to the possibility of cross pollination between crops and the authors conclude that With most honeybee colonies foraging up to 2km from the hive, some pollen transfer and fertilization up to 4km must be expected.

Osborne et al.(1999)

Individual Bumble bees have been tracked using harmonic radar. The foraging bees were fitted with lightweight radar trans- ponders and were found to far exceed their expected foraging range. Osborne et al.(1999) have shown, that most bees regularly fly over 200m (range 70-631 m) from the nest to forage. This was observed even when apparently plentiful food was available from an OSR crop adjacent to the bees nest. "The results support the hypothesis that Bumble bees do not necessarily forage close to the nest, and illustrate that studies on a landscape scale are required if we are to evaluate bee foraging ranges fully with respect to resource availability. Such evaluations are required to underpin assessments of gene flow in bee-pollinated crops and wild flowers."

Skogsmyr (1994)

The pollen beetle, can play a role in medium range cross- pollination of crops. In potatoes grown for seed the pollen beetle was considered to be responsible for very high levels of cross-pollination between a GM and non-GM potato fields. Gene dispersal from the GM crop was highest (72% ) in the near vicinity of the source, decreasing to 36% at 100m, while at 1000m it had only fallen to 31% (Skogsmyr, 1994). "Thus gene dispersal occurred both over large distances and to a higher extent than had previously been shown ".

Free and Williams, 1978

In oilseed rape the pollen beetle is often very common (Free and Williams, 1978), although its role in cross-pollination between OSR crops has, to our knowledge, not been studied.

Dispersal of pollen by wind

Thompson et al. (1999)

This study used male sterile bait oilseed rape plants spaced at 0-4000m from a pollen source to monitor gene flow on a regional basis. Seed set was observed at a rate of 5% at 4km. At one bait site the majority of the pollinated plants (<80%) were shown, by DNA profiling, to have been fertilised by pollen from the nearest crop, 900m distant. A smaller number were found to have been fertilised by pollen 4km from the nearest known source, probably by insects.

These results illustrate potential gene flow. The pollination levels for male fertile bait plants are likely to be much lower, although many modern varietal associations have a high proportion of male sterility increasing the likelihood of external pollination. " Despite the predominance of non GM OSR crops in the immediate locality, all sites were pollinated by a mixture of GM and non GM sources. The results suggest that farm to farm spread of OSR transgenes will be widepsread ".

Squire et al. (1999).

Many plants with pollen that becomes airborne show a very rapid decline in pollen concentrations near the source followed by a very slow decline with increasing distance. The absolute level of pollen in such a leptokurtic decline is a function of the scale of source emission. It would seem probable, therefore, that source scale is of more significance in cross pollination at the medium scale than is distance. The source scale is determined by size of source fields and also the interaction between such fields on a landscape level ( Squire et al., 1999). " The evidence indicates that large pollen sources, such as crop fields, interacted on a regional scale to increase gene flow".

Timmons et al. (1995)

Oil seed rape is commonly both insect and wind pollinated (the relative importance of each is dependant on environmental conditions). In a two year study of pollen dispersal from isolated Rape fields Timmons et al. (1995) used emasculated bait plants and a volumetric pollen trap up to 2.5km from the nearest Rape field.. They found a frequency of pollination of 0.8% at 2.5 km. The petals of the bait plants were removed making insect pollination less likely, possibly explaining the lower levels of cross-pollination compared to Thompson et al. (1999). Airborne pollen concentrations were found to be 10% of those at the field margin at a distance of 360m. This study also highlights the extensive distribution of feral populations of OSR which can act both as additional pollen sources and recipients. " Oil seed rape pollen has greater capacity for long range dispersal than has been suggested by small scale field trials"

Jones and Brooks (1950)

Pollination of Maize is by wind, although some insects, including bees, do collect pollen allowing for the possibility of inclusion in honey. According to MAAF (2000), the best body of data for estimating cross-pollination in maize is that of Jones and Brooks (1950). The 3-year means (and range) of cross- pollination at different separation distances are given below:

25m 14.2% (7 - 19.8%)
75m 5.8% (3.6 - 8.6%)
125m 2.3% (.8 - 3.7%)
200m 1.19% (0.4 - 2.5%)
300m 0.48% (0.15 - 0.99%)
400m 0.23% (0.15 - 0.32%)
500m 0.20% (0.12 - 0.32%)
Salamov (1940)

A study by Salamov (1940 cf Jones and Brooks, 1950) shows relatively high cross-pollination at 600m and above:

10m 3.3%
50m 0.33%
100m 0.36%
150m 0.25%
200m 0.54%
400m 0.02%
500m 0.08%
600m 0.79%
700m 0.18%
800m 0.21%

The significance of these results is further increased as the recipient field was much larger than the source (effectively leading to a dilution of source pollen) and was in a direction opposite to that of the prevailing wind.

References:

Free, J.B. and Williams, I.H. (1978) The responses of the pollen beetle, Meligethes aeneus, and the seed wevil, Ceuthorhychus assimilis, to oil seed rape, Brassica napus, and other plants. Journal of Applied Ecology. 15. 761-774

Jones, M.D. & Brooks, J.S. (1950) Effectiveness of distance and border rows in preventing outcrossing in corn. Oklahoma Agricultural Experimental Station. Bulletin no. T-38

Osborne, J.L., Clark, S.J., Morris, R., Williams, I., Riley, R., Smith, A., Reynolds, D. and A. Edwards. (1999) A landscape-scale study of bumble bee foraging range and constancy, using harmonic radar. Journal of Applied Ecology. 36, 519-533

Ramsey, G., Thompson, C.E., Neilson, S. and Mackay, G.R. (1999) Honeybees as vectors of GM oilseed rape pollen. In: Lutman, P.J.W. Gene flow and Agriculture: Relevance for Transgenic Crops. BCPC Symposium Proceedings no.72.

Skogsmyr, I. (1994) Gene dispersal from transgenic potatoes to conspecifics: A field trial. Theoretical and Applied Genetics. 88: 770-774

Squire, G.R., Crawford, J.W., Ramsey, G., Thompson, C. and Bown, J. (1999) Gene flow at the landscape level. In: Lutman, P.J.W. Gene flow and Agriculture: Relevance for Transgenic Crops. BCPC Symposium Proceedings no. 72.

Thompson, C.E., Squire, G., Mackay, G.R., Bradshaw, J.E., Crawford, J. and Ramsay, G. (1999) Regional patterns of gene flow and its consequence for GM oilseed rape. In: Lutman, P.J.W. Gene flow and Agriculture: Relevance for Transgenic Crops. BCPC Symposium Proceedings no. 72

Timmons, A.M., O'Brien, E.T., Charters, Y.M., Dubbels S.J. and Wilkinson, M.J. (1995) Assessing the risks of wind pollination from field of genetically modified Brassica napus ssp. oleifera. Euphytica 85 pp417-423

(To be released June 2001 in a Canadian scientific publication)
---

"Horizontal Gene Transfer - DNA in the Soil"
Ag BioView Post
http://www.biotech-info.net/soil_DNA.html
Kaare M. Nielsen, Ph.D. Hartl Lab. Dept. of Evolutionary and Organismic Biology Harvard University 16 Divinity Ave. Cambridge, MA 02138
May 15, 2001

I would like to post the following response to the Dr. Innes question: Horizontal gene transfer happens all the time?

--Recently (May 14) Dr. Innes raises the question; I am confused in that if this (horizontal gene transfer, my addition) happens all the time anyway, what is so unnatural about transgenics? The argument appears inconsistent.  

I have not seen the Greenpeace website referred to, so I address the above question only. having followed the field of horizontal gene transfer (HGT) for the last 10 years, I have noticed a remarkable shift has occurred in the argument for why HGT is of no concern for transgenes. Earlier, it was argued from data (or lack thereof) that HGT occurs so rarely, if at all, that it would be insignificant for the spread of transgenes. Today, it is argued (from accumulating data) that HGT occurs all the time so why worry about possible HGT of transgenes?  

Whereas the previous argument had some scientific basis, the latter is questionable. The process of horizontal gene transfer (like mutations) can occur frequently without leaving traces in a given bacterial population. This is because purifying selection will remove those individuals (either carrying mutations or horizontally acquired genes) from the populations since the genetic changes introduced do not provide a benefit to the carrier. Such (trans) genes will not reach fixation in the bacterial population. Secondly, horizontally acquired (native) genes from distantly related species are less likely to be retained and function in the recipient bacterium due to differences between the donor organism and the recipient i= n nucleotide sequence, gene expression, codon usage, and possibly posttranslational modification and protein interactions.  

Thus, assuming transgenes are equal to any other naturally occurring gene, it is reasonably to assume that transgenes will behave no differently than genes that are transferred from native donors.  

However, transgenes do often differ in several ways from native genes. This poses problems.

 1.Transgenes often contain DNA sequence homology to prokaryotes thereby increasing their likelihood ofintegration in bacteria significantly. Many studies have shown that DNA homology is the main barrier toHGT of chromosomal DNA (such as transgenes) in bacteria.  

 2.Transgenes are often modified to allow broad expression in a variety of hosts; they often lack introns,contain promoters active across a broad range of hosts (e.g. viral or bacterial of origin), and seldom requireextensive interactions with other proteins in the host cytoplasm for functionality. Thus, transgenes mayhave an increased likelihood of expression if horizontally transferred.  

 3.The transgenes may represent novel genetic variability due to the use of synthetic genes with new proteindomains or encoding novel biochemical pathways that have not been subject to natural selection in theirnew host environment. Therefore, they may or may not provide a selective advantage i= n the new host.Most likely they will not, but this cannot be assumed in all instances. Mechanisms providing geneticvariability in bacteria do not combine DNA sequences from several organisms into a compact functionalunit within the time scale done by genetic engineering. Thus, the argument that this is naturally occurring,cannot be used when th= e genetic novelty the transgenes extends beyond simple modifications.  

Thus, when compared to any native gene of a divergent organism, transgenes may differ both with respect to their likelihood of HGT, expression in the new host, and selection. The current debate on the likelihood of HGT has been much focused on the likelihood of transfer, whereas, as argued above, transfer does not generate an environmental impact. Selection would, if positive.  

I enclose some relevant references which also refers to an earlier request in the Agbioview list on fate of DNA in soil.  

Kind regards

References

 1.J. Maynard Smith et al., Population structure and evolutionary dynamics of pathogenic bacteria, Bioessays22 (2000) 1115-1122.

 2.J. P. Claverys et al., Adaptation to the environment: Streptococcus pneumoniae, a paradigm forrecombination-mediated genetic plasticity, Molecular Microbiology 35 (2000) 251-259.

 3.H. Ochman et al., Lateral gene transfer and the nature of bacterial innovation, Nature 405 (2000) 299-304.

 4.K. M. Nielsen et al., Horizontal gene transfer from transgenic plants t o terrestrial bacteria - a rare event?FEMS Microbiology Reviews 22 (1998) 79-103.

 5.G-H. Lee and G. Stotzky, Transformation and survival of donor, recipient, transformants of Bacillussubtilis in vitro and in soil, Soil Biology & Biochemistry 31 (1999) 1499-1508.

 6.K. M. Nielsen et al., Natural transformation of Acinetobacter sp. strai n BD413 with cell lysates ofAcinetobacter sp., Pseudomonas fluorescens and Burkholderia cepacia in soil microcosms, Applied andEnvironmental Microbiology 66 (2000) 206-212.

 7.M. DrF6ge et al., Horizontal gene transfer among bacteria in terrestrial and aquatic habitats as assessed bymicrocosms and field studies, Biology and Fertility of Soils 29 (1999) 221-245.

 8.J. Davison, Genetic exchange between bacteria in the environment, Plasmid 42 (1999) 73-91.

 9.F. Widmer et al., Sensitive detection of transgenic plant marker gene persistence in soil microcosms,Molecular Ecology 5 (1996) 603-13.

10.F. Widmer et al., Quantification of transgenic marker gene persistence in the field, Molecular Ecology 6(1997) 1-7.

11.E. Paget et al., The fate of recombinant plant DNA in soil, European Journal of Soil Biology 34 (1998)81-88.

12.F. Gebhard and K. Smalla, Monitoring field releases of genetically modified sugar beets for persistence oftransgenic plant DNA and Horizontal gene transfer, FEMS Microbiology Ecology 28 (1999) 261-272.

13.G. Ro manowski et al., Use of polymerase chain reaction and electroporation of Escherichia coli to monitorthe persistence of extracellular plasmid DNA introduced into natural soils, Applied and EnvironmentalMicrobiology 59 (1993) 3438-3446.

14.G. Recorbet et al., Kinetics of persistence of chromosomal DNA from genetically engineered Escherichiacoli introduced to soil, Applied and Environmental Microbiology 59 (1993) 4289-4294.

15.K. M. Nielsen et al., Natural transformation and availability of transforming DNA to Acinetobactercalcoaceticus in soil microcosms, Applied and Environmental Microbiology 63 (1997) 1945-1952.

16.K. M. Nielsen et al., Induced natural transformation of Acinetobacter calcoaceticus in soil microcosms.Applied and Environmental Microbiology 63 (1997) 3972-3977.

17.K. M. Nielsen et al., Transformation of Acinetobacter sp. BD413(pFG490nptII) with transgenic plant DNAin soil microcosms and effects of kanamycin on selection of transformants, Applied and EnvironmentalMicrobiology 66, (2000) 1237-42.

18.S. A. E. Blum et al., Mechanisms of retarded DNA degradation and prokaryotic origin of DNases innon-sterile soil, Systematic and Applied Microbiology 20 (1997) 513-521.

19.G. Romanowski et al., Adsorption of plasmid DNA to mineral surfaces and protection against DNase I,Applied and Environmental Microbiology 57 (1991 ) 1057-1061.

20.A. Ogram et al., Effects of DNA polymer length on its absorption to soils, Applied and EnvironmentalMicrobiology 60 (1994) 393-396.

21.M. Khanna and G. Stotzky, Transformation of Bacillus subtilis by DNA bound on montmorillonite andeffect of DNase on the availability of bound DNA, Applied and Environmental Microbiology 58 (1992)1930-1939.

22.E. Paget and P. Simonet, On the track of natural transformation in soil, FEMS Microbiology Ecology 15(1994) 109-118.

23.E . Gallori et al., Transformation of Bacillus subtilis by DNA bound on clay in non-sterile soil, FEMSMicrobiology Ecology 15 (1994) 119-126.

24.M. G. Lorenz and W. Wackernagel, Bacterial gene transfer by natural genetic transformation in the environment, Microbiology Reviews 58 (1994) 563-602.

25.M. Vulic et al., Molecular keys to speciation: DNA polymorphism and the control of genetic exchange in enterobacteria, Proceedings of the National Academy of Sciences USA 94 (1997) 9763-9767.

26.J. Majewski et al., Barriers to genetic exchange between bacterial species: Streptococcus pneumonia transformation, Journal of Bacteriology 18 2 (2000) 1016-1023.

27.K. M. Nielsen et al., Dynamics, horizontal transfer and selection of novel DNA in bacterial populations in the phytosphere of transgenic plants, Annals of Microbiology 51 (2001) (June issue, in press) [28] P. Shen and H. V. Huang, Homologous recombination in Escherichia coli: dependence on substrate length and homology, Genetics 112 (1986) 441-457

28.J. Majewski and F. M. Cohan, DNA sequence similarity requirements for interspecific recombination in Bacillus, Genetics 153 (1999) 1525-1533.

29.P. Zawadzki et al., The log-linear relationship between sexual isolation and sequence divergence in Bacillus transformation is robust, Genetics 140 (1995) 917-932.

30.F. Gebhard, and K. Smalla. Transformation of Acinetobacter sp. Strain BD413 by transgenic sugar beet DNA, Applied and Environmental Microbiology 64 (1998) 1550-1554.

31.J. De Vries, and W. Wackernagel, Detection of npt-II (kanamycin resistance) genes in genomes of transgene by marker-rescue transformation, Molecular and General Genetics 257 (1998) 606-613.

32.J. De Vries et al., The natural transformation of the soil bacteria Pseudomonas stutzeri and Acinetobacter sp. by transgenic plant DNA depends strictly on homologous sequences in the recipient cells, FEMS Microbiology Letters 195 (2001) 211-215.