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Transgenic DNA introgressed into traditional maize landraces in Oaxaca, Mexico

NATURE
November 29, 2001
Vol. 414, pp. 541-543

DAVID QUIST AND IGNACIO H. CHAPELA

Department of Environmental Science, Policy and Management, University of California, Berkeley, California
94720-3110, USA

Correspondence and requests for materials should be addressed to I.H.C. (e-mail: ichapela@nature.berkeley.edu).


Concerns have been raised about the potential effects of transgenic introductions on the
genetic diversity of crop landraces and wild relatives in areas of crop origin and diversification,
as this diversity is considered essential for global food security. Direct effects on non-target
species1, 2, and the possibility of unintentionally transferring traits of ecological relevance onto
landraces and wild relatives have also been sources of concern3, 4. The degree of genetic
connectivity between industrial crops and their progenitors in landraces and wild relatives is a
principal determinant of the evolutionary history of crops and agroecosystems throughout the
world5, 6. Recent introductions of transgenic DNA constructs into agricultural fields provide
unique markers to measure such connectivity. For these reasons, the detection of transgenic
DNA in crop landraces is of critical importance. Here we report the presence of introgressed
transgenic DNA constructs in native maize landraces grown in remote mountains in Oaxaca,
Mexico, part of the Mesoamerican centre of origin and diversification of this crop7-9.

In October and November 2000 we sampled whole cobs of native, or 'criollo', landraces of
maize from four standing fields in two locations of the Sierra Norte de Oaxaca in Southern
Mexico (samples A1*A3 and B1*B3), more than 20 km from the main mountain-crossing
road that connects the cities of Oaxaca and Tuxtepec in the Municipality of Ixtlán. As each
kernel results from ovule fertilization by individual pollen grains, each pooled criollo sample
represents a composite of 150*400 pollination events. One additional bulk grain sample
(K1) was obtained from the local stores of the Mexican governmental agency Diconsa
(formerly the National Commission for Popular Subsistence), which distributes subsidized
food throughout the country. Negative controls were cob samples of blue maize from the
Cuzco Valley in Peru (P1) and a 20-seed sample from an historical collection obtained in the
Sierra Norte de Oaxaca in 1971 (H1). Positive controls were bulk grain samples of Yieldgard
Bacillus thuringiensis (Bt)-maize (Bt1; Monsanto Corporation) and Roundup-Ready maize
(RR1; Monsanto Corporation) obtained from leftover stock for the 2000 planting season in
the United States. Using a polymerase chain reaction (PCR)-based approach, we first tested
for the presence of a common element in transgenic constructs currently on the market*the
35S promoter (p-35S) from the cauliflower mosaic virus (CMV). The high copy number and
widespread use of p-35S in synthetic vectors used to incorporate transgenic DNA during
plant transformation make it an ideal marker to detect transgenic constructs10-12.

We obtained positive PCR amplification using primers specific for p-35S in five of the seven
Mexican maize samples tested (Fig. 1). Four criollo samples showed weak albeit clear PCR
amplification, whereas the Diconsa sample yielded very strong amplification comparable in
intensity to transgenic-positive Bt1 and RR1 controls. The historical negative control (data not
shown) and the contemporary sample from Cuzco, Peru, were both invariably negative. Low
PCR amplification from landraces was due to low transgenic abundance (that is, a low
percentage of kernels in each cob), not to differential efficiency in the reaction, as
demonstrated by internal control amplification of the maize-specific alpha zein protein 1 gene
(Fig. 1, zp1). During the review period of this manuscript, the Mexican Government (National
Institute of Ecology, INE, and National Commission of Biodiversity, Conabio) established an
independent research effort. Their results, published through official government press
releases, confirm the presence of transgenic DNA in landrace genomes in two Mexican states,
including Oaxaca. Samples obtained by the Mexican research initiative from sites located near
our collection areas in the Sierra Norte de Oaxaca also confirm the relatively low abundance
of transgenic DNA in these remote areas. The governmental research effort analysed
individual kernels, making it possible for them to quantify abundances in the range of 3*10%.
Because we pooled all kernels in each cob, we cannot make such a quantitative statement,
although low PCR amplification signal from criollo samples is compatible with abundances in
this percentage range.

Using a nested primer system, we were able to amplify the weak bands from all
CMV-positive criollo samples (Fig. 1) sufficiently for nucleotide sequencing (GenBank
accession numbers AF434747*AF434750), which always showed at least 98% homology
with CMV p-35S constructs in commercially used vectors such as pMON273 (GenBank
accession number X04879.1) and the K1 sample (accession number AF434746).

Further PCR testing of the same samples showed the presence of the nopaline synthase
terminator sequence from Agrobacterium tumefasciens (T-NOS) in two of the six criollo
samples (A3 and B2; GenBank accession numbers AF434752 and AF434751, respectively)
and the Diconsa sample (K1; accession number AF434753). We detected the B.
thuringiensis toxin gene cryIAb in one criollo sample (B3) (data not shown). We confirmed
all of the PCR results through repeated testing.

We performed inverse PCR (iPCR) to reveal the various genomic contexts in which the CMV
construct was embedded in the Oaxacan criollo maize. This method enabled us to sequence
unknown DNA regions flanking the known p-35S sequence in each of the samples. For each
sample, iPCR yielded 1*4 DNA fragments differing in size. We isolated these fragments from
electrophoresis gels and attempted to sequence them individually, yielding sequences in eight
cases (GenBank accession numbers AF434754*AF434761; Fig. 2). Sequences adjacent to
the CMV p-35S DNA were diverse, suggesting that the promoter was inserted into the criollo
genome at multiple loci. When compared with GenBank (BLAST, February 2001), two
sequences were similar to synthetic constructs containing regions of the adh1 gene found in
transgenic maize currently on the market, such as Novartis Bt11 (Fig. 2, samples A3 and K1).
Notably, these two sequences had high homology with each other. Other sequences
represented maize-native genomic DNA, including retrotransposon regions, whereas others
showed no significant homology with any GenBank sequence (Fig. 2). The diversity of
transgenic DNA constructs present in criollo samples suggests the occurrence of multiple
introgression events, probably mediated by pollination. In some of these events, the
introgressed DNA appeared to have retained its integrity as an unaltered construct (as with
adh1 (ref. 10), whereas in others the transgenic DNA construct seemed to have become
re-assorted and introduced into different genomic backgrounds, possibly during transformation
or recombination13. The apparent predominance of re-assorted sequences obtained in our
study might be due to PCR bias for amplification of short fragments, as intact functional
constructs are expected to be much longer.

Our results demonstrate that there is a high level of gene flow from industrially produced maize
towards populations of progenitor landraces. As our samples originated from remote areas, it
is to be expected that more accessible regions will be exposed to higher rates of introgression.
Our discovery of a high frequency of transgene insertion into a diversity of genomic contexts
indicates that introgression events are relatively common, and that the transgenic DNA
constructs are probably maintained in the population from one generation to the next. The
diversity of introgressed DNA in landraces is particularly striking given the existence in Mexico
of a moratorium on the planting of transgenic maize since 1998. Whether the presence of these
transgenes in 2000 is due to loose implementation of this moratorium, or to introgression
before 1998 followed by the survival of transgenes in the population, remains to be
established. The intentional release of large amounts of commercial transgenic seed into the
environment since the mid-1990s represents a unique opportunity to trace the flow of genetic
material over biogeographical regions, as well as a major influence on the future genetics of the
global food system.

Further study of the impact of the gene flow from commercial hybrids to traditional landraces
in the centres of origin and diversity of crop plants needs to be carefully considered with
respect to the future of sustainable food production. Long-term studies should establish
whether, or for how long, the integrity of the transgenic construct is retained, and whether the
relatively low abundance of transgene introgression detected in the 2000 harvest cycle in
Oaxaca will increase, decrease, or remain stable over time.

References
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Losey, J. E., Raynor, L. S. & Carter, M. E. Transgenic pollen harms monarch larvae. Nature
399, 214 (1999). | Article | PubMed |
2.
Saxena, D., Flores, S. & Stotzky, G. Insecticidal toxin in root exudates from Bt corn.
Nature 402, 480 (1999). | Article | PubMed |
3.
Ellstrand, N. C. When transgenes wander, should we worry? Plant Physiol. 125, 1543-1545
(2001). | Article | PubMed |
4.
Doebley, J. Molecular evidence for gene flow among Zea species--genes transformed into
maize through genetic engineering could be transferred to its wild relatives, the Teosintes.
Bioscience 40, 443-448 (1990).
5.
Ellstrand, N. C., Prentice, H. C. & Hancock, J. F. Gene flow and introgression from
domesticated plants into their wild relatives. Annu. Rev. Ecol. Syst. 30, 539-563 (1999).
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White, S. & Doebley, J. Of genes and genomes and the origin of maize. Trends Genet. 14,
327-332 (1998). | Article | PubMed |
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maize domestication. Nature 398, 236-239 (1999). | Article | PubMed |
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Piperno, D. R. & Flannery, K. V. The earliest archaeological maize (Zea mays L.) from
highland Mexico: new accelerator mass spectrometry dates and their implications. Proc.
Natl Acad. Sci. USA 98, 2101-2103 (2001). | Article | PubMed |
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Iltis, H. From teosinte to maize: the catastrophic sexual transmutation. Science 222,
886-894 (1983).
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Matsuoka, T. et al. A method of detecting recombinant DNAs from four lines of genetically
modified maize. Shokuhin Eiseigaku Zasshi 41, 137-143 (2000).
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Gachet, E., Martin, G. G., Vigeau, F. & Meyer, G. Detection of genetically modified
organisms (GMOs) by PCR: a brief review of methodologies available. Trends Food Sci.
Technol. 9, 380-388 (1999). | Article |
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Anonymous Development of Methods to Identify Foods Produced by Means of Genetic
Engineering EU Project SMT4-CT96-2072 (Bundesinstitut für gesundheitlichen
Verbraucherschutz und Veterinärmedizin, Berlin, 1999).
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Pawlowski, W. P. & Somers, D. A. Transgenic DNA integrated into the oat genome is
frequently interspersed by host DNA. Proc. Natl Acad. Sci. USA 95, 12106-12110
(1998). | Article | PubMed |
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Hartl, D. L. & Ochman, H. in Methods in Molecular Biology (ed. Harwood, A.) 293-301
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Zimmermann, A., Lüthy, L. & Pauli, U. Event specific transgene detection in Bt11 corn by
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