ISIS Report, 17 April 2002
What Lurks Behind Triple Herbicide-Tolerant Oilseed Rape?
The surprising speed with which multiple herbicide tolerant oilseed rape appeared in Canada and the United States raises serious questions over horizontal gene transfer and transgenic instability. Dr. Mae-Wan Ho calls for definitive molecular characterisation of the volunteers to settle the current dispute over transgenic pollution of Mexican landraces.
Triple herbicide-tolerant oilseed rape volunteers were first discovered in Alberta, Canada, in 1998 . A year later, these multiple herbicide tolerant volunteers were found in a further 11 fields in Canada.
The United States only started growing herbicide-tolerant canola in 2001. Research in Idaho University showed that similar multiple gene-stacking had occurred over two years in experimental plots, and during the same period, weeds with two herbicide tolerant traits were found.
The speed with which triple-herbicide tolerant oilseed rape has evolved gives cause for concern. I decided to review the original paper in detail.
A farmer in Alberta planted three varieties of GM oilseed rape (B. napus) in the same or adjacent fields in 1997. The varieties were tolerant to herbicides glyphosate, glufosinate, and imidazolinone respectively. In field 1, the farmer started planting the glufosinate-tolerant variety, but, after 15 hectares, switched to the imidazolinone-tolerant for the remainder of the field. In field 2 located across a road, 22 m from the edge of the first field, he planted the glyphosate-tolerant variety.
In 1998, field 1 was left fallow, and part of field 2 was planted with the imidazolinone-tolerant variety. Weeds in the fallow field 1 were sprayed with glyphosate, but the farmer noticed that B. napus volunteers in this field were not being controlled by the herbicide.
At this point, the Canadian government research team came and collected the B. napus volunteers that had survived the glyphosate treatments - 9 from the part sown with glufosinate-tolerant variety and 25 from the part sown with imidazolinone - and grew them in the greenhouse to obtain seeds for further study.
The researchers considered two possibilities that might account for the presence of glyphosate-tolerant oilseed rape in field 1. It could be transport of glyphosate-tolerant seeds from field 2 across the road to field 1 (most likely by farm equipment), or it could be cross-pollination between the glyphosate-tolerant variety and either the glufosinate- or imidazolinone-tolerant varieties. The glyphosate- and glufosinate-tolerant varieties were transgenic, and "homozygous for the trait coded by a single insert", that is two copies of a single transgene were present. The imidazolinone variety was non-transgenic and was homozygous for two unlinked genes. Thus, both transgenic and non-transgenic traits are expected to behave according to classical, Mendelian, genetics.
If the volunteers were due to cross-pollination, then the plants must be heterozygous for the glyphosate-tolerance trait, as well as for the glufosinate- and imidazolinone- tolerance traits. The progeny of such plants are expected to show the typical 3:1 Mendelian ratio of tolerant to sensitive plants for one gene, and 15:1 for two genes in the case of imidazolinone.
For all 34 volunteers collected, four replicates of 25 seeds from each plant were grown in the greenhouse to test for tolerance to glyphosate as well as for glufosinate or imidazolinone.
In addition, four replicates of 25 seeds from 14 of the volunteers were grown and tested for tolerance to all three herbicides.
All nine volunteer plants from the part of field 1 sown with glufosinate-tolerant variety gave high percentages of glyphosate tolerance offspring, but only 7 out of the 9 showed the expected 3:1 Mendelian ratio, or more accurately, failed to deviate from it. The other two had an excess of glyphosate-tolerant offspring. Of the 25 volunteers from the part of field 1 sown with the imidazolinone-tolerant variety, 8 gave 0 to 3% glyphosate tolerant progeny, and were considered non-hybrids. Of the remainder, only 9 gave progeny that failed to depart from the expected 3:1 ratio, 8 of them gave too small a proportion of tolerant plants.
These results cannot be fully explained in terms of simple cross-pollination. It seems to me that additional processes such as horizontal gene transfer and/or transgenic instability might have been involved.
Two plants from field 1 each gave rise to a single progeny seedling tolerant to all three herbicides. This was attributed to sequential hybridization among the plants. It was not clear to me, however, whether the volunteers when collected were already in flower or in seed. To acquire two additional tolerance traits through cross-pollination requires a minimum of two complete generations. With horizontal gene transfer, however, this could have happened during one generation of growth.
Unfortunately, the molecular evidence is very scanty and uninformative, as it was not stated whether the progeny of the volunteers tested were tolerant to the herbicide for which the corresponding DNA fragment is present.
Different plants gave different patterns, even from the parental (nonhybrid) seed generation. That is indicative of non-uniformity of the transgenic lines and hence of transgenic instability. A better test would have been Southern hybridisation of genomic DNA, and/or inverse PCR to give event specific characterisation of the inserts.
In other words, the Canadian government scientists should have done some of the experiments that have been carried out on the Mexican maize by Berkeley researchers. Or are they afraid of what they would discover, given that the Berkeley scientists who did find evidence suggesting transgenic instability are being bitterly attacked (see "Astonishing denial of transgenic pollution", ISIS report, 8 April, 2002 <www.i-sis.org.uk>). We challenge the Canadian group to settle the dispute by analysing their samples properly.
According to the report, B. napus is capable of selfing and outcrossing, having an outcrossing frequency of 20-30%. In the field, the actual cross-hybridization rate is a function of distance, with percent out-crossing diminishing the farther the recipient is from the pollen source. But one of the triple-resistant plants was found over 550 m from the pollen source, greatly exceeding the 100-m buffer mandated for seed producers. Insects are known to transfer pollen over long distances. And insects that feed on plants could also have injected transgenic DNA through sharp mouth-parts directly into plant cells that subsequently became germ cells.
- Hall L, Topinka K, Huffman J, Davis L, and Good A. Pollen flow between herbicide-resistant Brassica napus is the cause of multiple-resistant B. napus volunteers. Weed Science 2000, 48, 688-94.