Henri Darmency & Marc Richard-Molard
Concern has grown in Europe over the agricultural and environmental
impacts of genetically engineered (GE) crops, especially about gene flow to
conventional varieties and wild relatives.
Concern has grown in Europe over the agricultural and environmental
impacts of genetically engineered (GE) crops, especially about gene flow to
conventional varieties and wild relatives. Contrary to most crops that are
grown for their seed or fruit, sugar beet (Beta vulgaris L.) is grown for
its root. Therefore, pollen-mediated gene flow in root production areas is
not a concern in the debate on GE and non-GE crop co-existence, including
table beet and chard in private gardens. However, pollen flow could be
responsible for the admixture of GE and non-GE materials in the seed lots
provided to farmers; but it can be easily prevented by breeding and
multiplicating GE and non-GE seeds in different regions. In contrast, pollen
flow to wild relatives could certainly generate agronomic trouble much more
easily and quickly than with any other crop.

There is always a small proportion of sugar beet plants that flower,
in spite of being a root crop, because of either their sensitivity to
vernalization or the presence of a dominant bolting gene. Since wild sea
beet, weed beet, and sugar beet are the same botanical species, and since
they are allogamous, they can easily produce hybrids and are completely
interfertile1. The progeny of crosses among these beet types is perfectly
adapted to field conditions, has no hybrid fitness cost, and therefore can
display the favorable traits encoded by the transgenes. In particular,
herbicide resistance, which is desired by farmers to significantly reduce
the number of herbicide sprays and working hours, could result in the spread
of herbicide-resistant weed beets. The weed beet is already a serious
problem to sugar beet growers since no herbicide available differentiates
between weed beet and sugar beet. Severe infestations can reduce to nothing
the sugar beet yield and lead the farmer to stop growing this crop.
Therefore, breeding transgenic herbicide-resistant varieties can solve this
particular problem, provided that gene flow is contained for a long time.

The question of pollen-mediated gene flow to weed beets in sugar beet
root production areas was addressed in a six-year farm-scale study. Since GE
sugar beet is not yet grown on commercial fields, this study is the only
documented background of field growth for that crop. It was a joint action
of governmental research institutes (INRA), professional associations (ITB,
for beet; CETIOM, for oilseed rape) and industry (Hilleshog and KWS, for
providing the GE beets). The program started in 1995 in two locations:
Châlons, in Champagne, and Dijon, in Burgundy, in the north-eastern part of
France. Besides the gene flow study, the trial confirmed that the number of
herbicide sprays was reduced from 4.1 to 2.5 thanks to the post-emergence,
non-selective herbicides used for the GE varieties, without any difference
in weed control or yield than with conventional herbicide programs2.

Field monitoring
In each location there were four 1-ha adjacent fields grown in
rotation with GE sugar beet, GE oilseed rape, conventional wheat and fallow.
The sugar beet field was divided in two parts, each sown with a heterozygous
herbicide-resistant line: a Roundup Ready glyphosate-resistant line from
Hilleshog, and a Liberty Link glufosinate-resistant line from KWS. Each
variety was sprayed with its respective herbicide, except for the central
lane of the field that was sprayed with various herbicides used on
conventional sugar beets in the region. Weed beets were transplanted into
the central lane of the field when no local weed beet emerged in the sugar
beet field. The monitoring took place between 1996 and 2001. More detail is
available in the full publication3.

The number of sugar beet bolters varied widely according to the
transgenic line and year, from 0 to 121 per ha, thus providing a good
opportunity to study the consequences of pollen flow under a wide range of
realistic conditions and amounts of pollen escape. Indeed, the rate of sugar
beet bolters of commercial varieties in France during the same period ranged
from 0.001 to 0.1%4. These flowering plants included vernalized
herbicide-resistant sugar beets and susceptible annual hybrids coming from
pollination of the seed mother plants by susceptible wild beets surrounding
the nursery. Susceptible hybrids and spontaneous weed beets could grow and
flower in the central lane of the field, which was treated with selective
herbicides. All the bolting plants were mapped, and their seeds were
carefully collected and then tested for herbicide resistance in the
greenhouse.

Production of herbicide-resistant seeds by sugar beet bolters
On average, 58.2% of seeds produced by resistant sugar beet bolters
were resistant. The deviation from the 75% expected from mating among
heterozygotes denoted both the contribution of pollen coming from
susceptible beets and probably the better viability and fertilizing ability
of the pollen of weed beets. This category of plant accounted for 84.8% of
the total resistant seed production over the years studied, but, as shown in
Figure 1, their importance decreased during the second round of the
rotation.

Resistant seedlings also appeared in the progeny of susceptible sugar
beet bolters at a mean percentage of 1.7%, and they accounted for 1.2% of
the total resistant seed production over the six years under study.

Production of herbicide-resistant seeds by weed beet
On average, 6.2% of the seeds produced by weed beets were resistant,
which accounted for 14% of the total resistant seed production over the six
years in the two locations. A more detailed analysis has been published3.
Some of these resistant seeds were not produced within the sugar beet field,
but rather in the fallow field, and their proportion decreased as the
distance between the fields increased. Fallow field production represented
0.2% of the total resistant seed production. The largest distance at which a
cross was recorded between the GE sugar beet bolters and a weed beet was 112
m. This showed that a foreign pollen grain entering a pollen cloud at low
frequency over a weed beet population that grows in a distant fallow field
has an effective fertilizing ability. Pollen flow monitoring using male
sterile plants within and around the farm scale experiments showed
fertilization at 277 m and up to 1172 m3,5. The distribution curve of the
number of fertilized seeds in terms of distance from the pollen source had
generally a negative power shape3,5,6. For instance, the number of resistant
seeds recorded in different groups of plants in 1999 in Châlons, up to 120 m
away from the resistant bolters, followed the equation N = 14.4 d-0 ,75, R2
= 0.89.

Thus, in spite of the low density of GE bolters (8 in one ha in 1999),
pollen flow can reach adjacent as well as distant weed beet populations and
transfer the herbicide-resistance gene. Within the sugar beet field, there
was an average of 3.5% resistant seeds in the seed produced by the
susceptible weed beet, accounting for 7.4% of the total resistant seed
production over the six years and the two locations. The weed plants that
produced resistant offspring were not located at shorter distances from the
resistant bolters or at farther distances from susceptible plants than the
other weed beets. They flowered simultaneously with other weed plants, but
they produced more flowers and 2.4 times more viable seeds than plants that
did not produce resistant offspring. Higher production of flowers and higher
seed sets could partly explain the ability of those plants to catch more
numerous pollen grains and mature more embryos, thus simply having a higher
probability of producing resistant offspring. However, if the number of
viable seeds per plant somewhat depended on a genetic factor, the
consequence would be the propagation of herbicide resistance together with
the most reproductive individuals, which would be a very unfavorable
conjunction of factors with respect to weed control.

Finally, resistant seeds could also originate from resistant weed
beets. In 2000 and 2001, a few resistant weed beets emerged, either
spontaneously from the soil seed bank containing seeds left to shed in the
same field in 1999, or sowed in the field in order to simulate the creation
of a soil seed bank containing herbicide-resistant seeds, as would have
occurred if the seeds had not totally been harvested in former years. All
these resistant weed beets were heterozygotes and produced, on average, 74%
resistant seedlings. This category of plants accounted for 6.4% of the total
resistant seed production, but unlike resistant seeds produced by sugar beet
bolters, it was concentrated in the last two years, during the second crop
rotation (Fig. 1).

Management
At the end of the first round of crop rotation, the cumulated number
of seeds released on the farm-scale trial (4 ha x 2 locations) was 222,000,
of which 22.3% were herbicide-resistant, representing 0.6 resistant seed per
m2. However, there was a large variability among fields. Most resistant
seeds were produced by resistant sugar beet bolters (see Fig. 1). This
result strengthens the urgent need to eradicate all transgenic bolters. On
one hand, eradication can be achieved through production of high quality
certified seeds of varieties that are not sensitive to vernalization and
free of annual hybrids. On the other hand, destruction of bolters should
also be pointed out as a compulsory task among farmers' good agronomic
practices. Clearly, some of the bad results reported above belong to a
worst-case scenario, because most farmers would have reduced the risk of
seed release and pollen flow by destroying bolters when they were too
numerous.

However, if bolting still occurs, even at very low frequency, and is
not destroyed by farmers, or if transgenic volunteer roots grow and flower
in crops subjected to the same herbicide or in fallow fields, the transgenes
will unavoidably be transmitted to weed beets within a short period of time.
Pollen flow from resistant sugar beets to susceptible ones and to weed beets
outside the field accounted for 0.2% of resistant seeds in the farm-scale
study. Subsequent multiplication of resistant weed beets in the second round
of crop rotation accounted for 13.6% of resistant seeds. These seeds would
be the source of further multiplication of herbicide resistant weed beets.
Therefore, farmers must prevent the constitution of a soil seed bank
containing herbicide-resistant seeds. Useful practices, besides destruction
of bolters, could include management of fallow fields to control weed beets
and change of crop rotation. The effect of various farming systems on gene
escape from GE crops to volunteers and weed beets could be anticipated by
simulation models fed with basic data on weed beet biology, such as those
collected in the farm scale study7,8.


References

1. Boudry P, Mörchen M, Saumitou-Laprade P, Vernet Ph and Van Dijk H
(1993) The origin and evolution of weed beets: consequences for the breeding
and release of herbicide-resistant transgenic sugar beets. Theor Appl
Genet87, 471-478

2. Gestat de Garambé T and Richard-Molard M (1999) Produire des
betteraves OGM tolérantes ? un herbicide non sélectif: conséquences sur les
syst?mes de culture. Rev Ind Aliment Agric, juillet/Aout.

3. Darmency H, Vigouroux Y, Gestat de Garambé T, Richard-Molard M and
Muchembled C (2007) Transgene escape in sugar beet production fields: data
from six years farm scale monitoring. Environ Biosafety Res6, 197-206

4. Perarnaud V, Souverain F, Prats S, Dequiedt B, Fauchere J and
Richard-Molard M (2001) Influence du climat sur le phénom?ne de montée ?
graine de la betterave: synth?se http ://www.itbfr.org (accessed January,
2008)

5. Darmency H, Klein E, Gestat de Garambé T, Gouyon PH, Richard-Molard
M and Muchembled C (2008) Pollen dispersal in sugar beet production fields,
submitted

6. Bateman AJ (1947) Contamination of seed crops II. Wind pollination
Heredity 1, 235-246

7. Tricault Y, Sester M, Darmency H, Angevin F, and Colbach N (2007)
La gestion des betteraves adventices résistantes ? un herbicide: une
approche par simulation. In AFPP, 20th Conf. COLUMA, Dijon, December,
213-222

8. Sester M, Tricault Y, Darmency H, and Colbach N (2008)
GENESYS-BEET: a model of the effects of cropping systems on gene flow
between sugar beet and weed beet. Field Crops Res in press


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