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2-Plants: OECD 2002 publication: Evidence of gene flow from transgenic maize to local varieties in mexico



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   "These preliminary results present provocative evidence suggesting
    that the amplification of the 35S sequence and the T-NOS are due to
    the introgression of transgenic sequences into Mexican traditional
    maize populations. However, because our analysis was done through
    PCR amplification, the possibility of false positive results cannot
    be totally ruled out. If these results are corroborated by a series
    of other analyses currently in progress, the presence of transgenic
    elements planted in Mexico will be definitely confirmed in spite of
    a national policy that has put into place a standby moratorium on
    the planting and cultivation of transgenic maize in the country."
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-------------------------------- GENET-news -------------------------------

TITLE:  Evidence of Gene Flow from Transgenic Maize to Local Varieties in
        Mexico
SOURCE: OECD, LMOS and the Environment
        Proceedings of an International Conference, pages 289-295
        by Exequiel Ezcurra Sol Ortiz and Jorge Soberón Mainero
        http://www.oecd.org/document/18/
0,2340,en_2649_34385_2509330_1_1_1_1,00.html
        files attached: MexMaize_Table1.gif, MexMaize_Table2.gif
        extraced from the original pdf-file
DATE:   2002

------------------- archive: http://www.genet-info.org/ -------------------


LMOS and the Environment
Proceedings of an International Conference

27-30 November 2001
Raleigh--Durham
North Carolina
United States of America
Organized by
Organization for Economic Cooperation and Development (OECD)
In cooperation with the United States Department of Agriculture
and the Environmental Protection Agency
Editor
Craig R. Roseland
United States Department of Agriculture
APHIS, Policy and Program Development
Riverdale, Maryland
United States of America

Evidence of Gene Flow from Transgenic Maize to Local
Varieties in Mexico

Exequiel Ezcurra Sol Ortiz
Instituto Nacional de Ecología
SEMARNAT
Periférico Sur 5000
Col. Cuicuilco Insurgentes
México D. F.
Mexico

Jorge Soberón Mainero
Comisión Nacional para el Conocimiento y Uso de la Biodiversidad
(CONABIO)
México D. F.
Mexico

Abstract

Maize, originated and domesticated Mexico, is the basis of many food and
feed products. Its development is correlated with the development of
Mesoamerican civilizations. To date, traditional agricultural practices
in Mexico promote and maintain maize diversity. Following a communication
related to the presence of transgenic material in Mexican maize
landraces, the National Institute of Ecology and the National Commission
on Biodiversity started an investigation in collaboration with two
national institutions. Here we present preliminary results of the first
of a series of tests in progress. We obtained polymerase chain reaction
amplifications of the CaMV 35S promoter and the NOS terminator from DNA
extracted from maize seedlings grown from seeds collected in different
localities at Oaxaca and Puebla in Mexico. Our preliminary data suggest
that the frequency of transgenic constructs in the field might be low,
although the geographic dispersion seems to be widespread. Further
analyses will help to corroborate this pattern.

Introduction

Even though there is still some controversy on the origin and early
history of maize, in general agreement exists that the domestication of
Zea mays occurred in Central Mexico (Kato 1976, Mangelsdorf 1974, Dobley
and Goodman 1984, Doebley et al. 1987, Doebley 1990). The development and
improvement of maize are correlated with the development of cultural
complexity and the rise of highly organized civilizations in pre-hispanic
Mesoamerica. A recent analysis (Piperno and Flannery 2001) using
accelerator mass spectrometry to date maize cobs from the Guilá Naquitz
cave in the mountainous eastern part of the Valley of Oaxaca established
that this sample from about 6,250 calendar years ago represents the
oldest maize cobs known to date. Because of the absence of Zea
macrofossils in earlier sediments of the cave, these phytoliths, unlike
pollen grains, provide evidence of an early domestication process
somewhere else before settlement in this region.

A high diversity of maize populations is still present in many regions of
the Mexican territory, where more than 40 landraces of maize have been
described (Ortega 1980, Benz 1986, Sánchez 1989, Wellhausen 1987,
Hernández Xolocotzi 1998). Traditional agricultural practices in many
parts of the country promote and maintain maize diversity. In places with
high biological and landrace diversity, most of the land planted with
maize occurs in relatively small units and often in combination with
beans and squash. The small farmer and peasant communities are highly
open to seed exchange, and it can be observed that the traditional
management of varieties leads to a constant flow of genetic material
among communities over large areas (Louette 1997). Farmers continually
maintain cultivars through seed selection. Through the years Mexican
races of maize have been used by Mexican farmers to generate new varietal
mixtures as well as creolized materials, which are crosses between modern
improved varieties and hybrids with traditional landraces.

The wild relatives of maize, the teosintes, are present in many areas of
maize production. Because maize is primarily a wind cross-pollinating
species, the possibility of a low-frequency introgression cannot be
completely discounted. Maize and teosinte coexist sympatrically and form
fertile hybrids (originated from maize plants fertilized by teosinte
pollen) in many regions (Kato 1997). Although there are genetic barriers
that hinder the fertilization of teosintes by maize pollen (Evans and
Kermicle 2001), the risk of gene flow from the cultivated species into
its wild relatives cannot be totally ruled out.

In late 2000, researchers from the Zapotec-Chinantec Union (UZACHI) and
the University of California at Berkeley initiated a program to document
the absence of transgenic markers in traditional maize in the Sierra de
Juárez, Oaxaca, with the aim of opening a market for "transgenic-free
gourmet corn." However, during the process of setting up their
experimental protocols they found that some ears from criollo samples
gave positive results for the transgenic 35S promoter. Ignacio Chapela
from the University of California at Berkeley, the coordinator of this
research program, communicated his findings to the environmental
authorities in Mexico. On the basis of this communication, the National
Institute of Ecology (INE) from the Ministry of Environment and Natural
Resources (SEMARNAT) and the National Commission on Biodiversity
(CONABIO) started an investigation to corroborate the results and to
evaluate and quantify the levels of the gene flow from transgenic corn to
landraces from Oaxaca. The research performed by Chapela was published
last year (Quist and Chapela 2001).

Methods

We sampled 21 locations as well as two grain distribution centers.
Sampling of maize consisted of both complete ears and harvested seeds.
Most locations were small rural communities in the Sierra de Juárez in
the State of Oaxaca. Two localities were sampled in the State of Puebla
in Mexico (table 1).

Two random subsamples were taken and sent to two independent
laboratories: the Center of Research and Advanced Studies (CINVESTAV)
from the National Polytechnic Institute at Irapuato and the Institute of
Ecology at the National University of México (UNAM). The samples were
blind-coded, and the whole procedure was notarized by a Mexican public
notary. When the sample consisted of complete ears (i.e., seeds left
attached to the cobs), the seeds arising from each different ear were
tagged to preserve the maternal identity. The work at each laboratory
followed similar research protocols for better comparison of results.
Seeds were treated with a fungicide and planted in controlled conditions.
After germination the first leaf was used for DNA extraction. Subsequent
polymerase chain reaction (PCR) analyses followed standard protocols.

The DNA was extracted and purified from a total of 1,876 seedlings, which
included between 30 and 275 for each location. PCR analyses were
performed with primers for the 35S promoter from the cauliflower mosaic
virus (CMV) and the nopaline synthase terminator (T-NOS) sequence from
Agrobacterium tumefasciens. Two series of primers for each DNA sequence
were tested. T-NOS 118bp--GCA TGA CGT TAT TTA TGA GAT GGG; T-NOS 118bp--GAC
ACC GCG CGC GAT AAT TTA TCC; 35S 195pb--GCT CCT ACA AAT GCC ATC A; and 35S
195pb--GAT AGT GGG ATT GTG CGT CA. We also amplified the 16S nuclear
ribosomal gene to test DNA quality. Positive and negative controls were
included in each PCR run.

Once both laboratories finished their initial analyses we compared their
results for potential discrepancies and pooled them if they did not
differ significantly. In the few cases in which significant differences
were found in two subsamples, the analyses were repeated to discard
contamination and other technical artifacts.

Table 1. Localities sampled in the States of Oaxaca and Puebla. [attached]

Results

We found PCR evidence for the presence of the 35S promoter in 95-percent
of the localities sampled. For all different localities a total of 142
(7.6-percent) seedlings gave positive results for this sequence. All
(100-percent) seedlings gave positive results for ribosomal gene 16S.
Amplifications indicating the presence of the T-NOS sequences showed
consistently lower frequencies (see table 2). A small sample of the PCR
amplifications obtained with the 35S primers was cloned and sequenced and
compared with the sequence of the 35S CMV promoter. Most of them showed
sequence identity whereas one showed a single base pair difference.

In 15 localities we found that less than 10-percent of the seeds showed
evidence of transgenic markers. However there was considerable variation
in the frequencies found (from 1 to 35-percent). In the sample taken in a
grain store at Ixtlán de Juárez, where maize grains for tortillas
imported from outside the region are sold, 17-percent of the grains
showed amplifications of the 35S promoter, whereas the sample from the
local market, where we sampled locally grown pozole (stewed maize)
grains, showed no evidence of the presence of either marker used.

In five localities (mostly outside the core of the Sierra de Juárez,
Oaxaca) we found higher frequencies of transgenic introgression ranging
between 10 and 35-percent. These localities are found in the Central
Valleys of Oaxaca: in the Mixtec Region, in the southern portion of the
Sierra de Juárez, and in the Tehuacán Valley in Puebla. However, the high
frequencies observed in these last sites could also be caused by a sample
artifact: our sampling involved only a few, randomly selected maize ears
on which we arbitrarily sampled individual grains. Thus, there is a fixed
experimental maternal effect (what statisticians call "plantsnested-
within-sites") that could be driving these results. A logistic analysis
(Crawley 1993) of the data did detect significant maternal effects, but
these effects were always detected at low-frequency sites. Thus, we can
conclude (with some caution) that the sites showing high frequencies are
probably places where the frequency of transgenic constructs is
significantly higher.

Table 2. Observed frequency of PCR amplification products [attached]

Discussion

These preliminary results present provocative evidence suggesting that
the amplification of the 35S sequence and the T-NOS are due to the
introgression of transgenic sequences into Mexican traditional maize
populations. However, because our analysis was done through PCR
amplification, the possibility of false positive results cannot be
totally ruled out. If these results are corroborated by a series of other
analyses currently in progress, the presence of transgenic elements
planted in Mexico will be definitely confirmed in spite of a national
policy that has put into place a standby moratorium on the planting and
cultivation of transgenic maize in the country.

The ecological consequences of the possible flow of transgenic constructs
into traditional varieties are not well known, and more research is
clearly needed on the subject. Among other consequences, the possible
introduction of transgenic constructs into populations of the different
species and subspecies of teosintes (corresponding to all the wild
species of the genus Zea, including all the wild subspecies of Zea mays;
see Buckler and Holtsford 1996) needs to be studied in detail. Two of the
possible consequences that need to be addressed are (a) the potential
genetic erosion of the traditional landraces (e.g., Ortega Paczka 1999)
and (b) the possible increased weediness of teosinte plants if insect-
resistant or herbicide-tolerant transgenes were allowed to drift into the
wild populations. Effects on biodiversity in general should also be evaluated.

Our preliminary data suggest that the frequency of transgenic constructs
in the field might be low, although the geographic dispersion of the
presence of the transgenes seems to be widespread. Further analyses in
other parts of the country as well as monitoring and the sampling of
additional localities will provide a clearer picture of the situation.
However, we still need to know if enzyme-linked immunosorbent assay
(ELISA) tests, as well as BASTA resistance experiments and Southern blot
hybridization will further confirm this distribution pattern and rule out
the possibility of false positives in the PCR analysis. More extensive
sampling--including milpas (traditional maize fields) in many parts of
Mexico as well as wild populations of teosintes in successive planting
seasons--will allow us to define in a more precise manner the trends and
the risks involved for biodiversity.

Acknowledgments

We want to acknowledge Rafael Rivera from CINVESTAV and Elena Álvarez-
Buylla at UNAM for the molecular analyses presented in this first
communication. We also want to thank Ignacio Chapela and David Quist from
the University of California at Berkeley for sharing their results before
publication. Antonio Serratos and Fabián Islas from INIFAP-CIMMYT and
Elleli Huerta from CONABIO offered significant comments to a first
version of the presentation at the OECD meeting.

References

Benz, B.F. 1986. Racial systematics and the evolution of Mexican maize.
In: Manzanilla, L. (Ed). Studies in the Neolithic and Urban Revolutions.
B.A.R. International Series 349. pp. 121-136.

Buckler, E. S., IV and Holtsford, T. P. 1996. Zea systematics: ribosomal
ITS evidence. Mol. Biol. Evol. 13, 623-632.

Crawley, M. 1993. GLIM for Ecologists. Blackwell Scientific Publications.
Oxford. London.

Doebley, J. F., Goodman, M. M and Stuber, C. W. 1984. Isoenzymatic
variation in Zea (Gramineae). Syst. Bot. 9(2), 203- 218.

Doebley, J. F., Goodman, M. M and Stuber, C. W. 1987. Patterns of isozyme
variation between maize and Mexican annual teosinte. Econ. Bot. 41(2),
234- 246.

Doebley, J., Stec, A., Wendel, J. and Edwards, M. 1990. Genetic and
morphological analysis of a maize-teosinte F2 population: implications
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Hernández Xolocotzi, E. 1998. Aspectos de la domesticación de plantas en
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and Fa, J. (eds), Diversidad Biológica de México. Instituto de Biología,
Universidad Nacional Autónoma de México.

Kato Y., T. A. 1976. Cytological studies of maize (Zea mays L.) and
teosinte (Zea mexicana Schrader Kuntze) in relation to their origin and
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Kato Y., T. A. 1997. Review of introgression between maize and teosinte.
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Castillo, F. (Eds). CIMMYT, Mexico, D.F. pp. 44-53.

Louette, D. 1997. Seed exchange among farmers and gene flow among maize
varieties in traditional agricultural systems. In: Serratos, A., Willcox,
M.C. and Castillo, F. (Eds). "Gene flow among maize landraces, improved
maize and teosinte: Implications for transgenic maize". CIMMYT, Mexico,
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Mangelsdorf, P. C. 1974. Corn. Its origin, evolution and improvement.
Harvard University Press. 262 p.

Ortega Pazcka, R. 1980. Resultados preliminares del reestudio de las
razas mexicanas de maíz. Resúmenes del VIII Congreso Nacional de
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Ortega Pazcka, R. 1999. Genetic erosion in Mexico. Proceedings of the
Technical Meeting on the Methodology of the FAO World Information and
Early Warning System on Plant Genetic Resources. FAO, Prague, pp69-75.

Piperno, D. R. and Flannery, K. V. 2001. The earliest archeological maize
(Zea mays L.) from highland Mexico: New accelerator mass spectrometry
datas and their implications. Proc. Nat. Acad. Sci. USA 98, 2101-2103.

Quist, D. and Chapela, I.. 2002. Transgenic DNA introgressed into
traditional maize landraces in Oaxaca, Mexico. Nature 4141, 541-543.

Sánchez G., J.J. 1989. Relationships among the Mexican races of maize.
Ph.D. Dissertation, North Carolina State University, Department of Crop
Science. Raleigh, N.C. 187p.

Wellhausen, E. J., Roberts, L. M., Hernández, X.E. and Mangelsdorf, P. C.
1987. Razas de Maíz en México. Su origen, características y distribución.
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Geografía Agrícola México. Universidad Autónoma de Chapingo




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