CROP MANAGEMENT OF TRANSGENIC RAPESEED :

RISK ASSESSMENT OF GENE FLOW

J Champolivier ⁽¹⁾, J Gasquez ⁽²⁾, A Messéan ⁽¹⁾

(1) CETIOM, Centre de grignon, B.P. 4, 78850 Thiverval-Grignon, France. (j.champolivier@cetiom.fr, messean@cetiom.fr)

(2) INRA, Laboratoire de malherbologie, rue Sully, BV 1540, 21034 Dijon, France. (gasquez@dijon.inra.fr)

ABSTRACT

With the development of Genetically Modified Organisms (GMO) in agriculture, new concerns about crop management have been addressed. Apart from the evaluation carried out with the regulation process before marketing, observations under current agricultural practices are required in order to build suitable agronomic management and design a monitoring system. A multi-year and multi-crop monitoring study has been carrying out in France since 1995 and preliminary results suggest that a more integrated crop management should be required.

INTRODUCTION

After about 15 years of transgenic research carried out by public research teams as well as private companies, the first marketing releases occurred in North America in 1995, while in Europe the first applications are still under discussion. Tobacco resistant to bromoxynil, imports of glyphosate resistant soybean, insect resistant corn through the Bt strategy and some herbicide tolerant rapeseed sum up the current European status for GMO marketing. Several other applications for marketing clearance have been submitted.

Corn, sugar beet and rapeseed are the main crops for which genetic modification has been applied. While several traits have been introduced (oil quality, disease and insect resistance) and are under development, herbicide resistance has been developed extensively and three systems are near marketing : glyphosate and glufosinate resistance for the three crops and bromoxynil resistance for rapeseed.

Development of transgenic plants raises several questions, most of them are not specific to recombinant DNA techniques : ethical concerns, relationship between science, society and organization of collective expertise, marketing of transgenic plants with new rules, protection of biotechnology and patent policy, food and feed safety of these novel plants, environmental and agronomic concerns. With respect to these last concerns, the evaluation has to be performed on a case-by-case basis. The risk assessment of gene flow must take into account the specific trait introduced (e.g. herbicide resistance vs oil quality), the biology of the plant (open vs self pollination, seed dormancy) and the agricultural context (cropping systems, spatial organization of the crops, agricultural practices, ...).

Herbicide resistance is not only one of the first traits for which marketing clearance has been applied but it is also an adequate model to carry out the risk assessment of crop management of transgenic plants. In this paper the main criteria for consideration in herbicide tolerant crops and the effect of their use in cropping systems will be reviewed. Rapeseed and sugar beet provide a good example of the principles involved (Richard-Molard & Gestat de Garambe, 1998).

RISK ASSESSMENT

For several years, the main question with respect to modified rapeseed and sugar beet was : will the transgene be disseminated outside the field and be transferred to other plants and, especially, to weeds ? From many studies carried out by different scientific teams, it can be concluded that transgenes will disseminate and can lead to outcrossing with weeds. Although interspecific crosses between rapeseed and related wild species lead to less fertile plants, they can produce a small quantity of seeds (Kerlan et al., 1992).

As we know that transgenes will disseminate, the question is now : So what ? Could the consequences of such a dissemination be managed ? With respect to long-term effects, no experiments are available for assessing the transgene behaviour. In order to estimate gene flow, simulations using genetic models are performed. These models generally represent the gene transfer from a field towards the wild species located at field edges and take into account various parameters such as the gene migration rate, its dominance level or the competitivity of the hybrid. Long-term behaviour appears to be difficult to predict as the model is highly dependent on specific events. It is thus necessary to take into account the spatial and temporal variability.

On the other hand, we can look for markers already introduced into rapeseed in the past and to survey their behaviour in the non-cultivated areas. Such a survey is being performed in various regions of France, where we are intending to detect the introgression of traits like "low-erucic" in wild species.

Gene flow

In the case of rapeseed, gene flow can occur through two different ways :

*    the pollen, either towards rapeseed plants (intraspecific crosses) or towards wild relatives which are quite numerous (interspecific crosses) ;

*    the seeds, through volunteers in subsequent crops or seed dissemination during transportation.

The long-term effect of such phenomena on farmers' crop management  of transgenic plants   and the design of adequate agricultural practices are assessed by carrying out several types of studies :

*    Modeling the gene flow. Models of gene flow between two adjacent fields have been designed (Reboud, 1992 ; Lavigne et al., 1994) and are being improved by taking into account crop rotations, spatial patterns of crops and agricultural practices.

*    Specific studies about outcrossing have been performed in order to estimate pollination distances and interspecific crosses  (Jorgensen and  Andersen, 1996 ; Kerlan et al., 1992 ; Eber et al., 1994 ; Baranger et al., 1995). Spontaneous hybridization of rape with wild mustard, wild hoary mustard and wild radish has been demonstrated to occur when using a male-sterile oilseed rape cultivar as the pollen recipient, i.e. without pollen competition (Chèvre et al., 1996). Lefol et al. (1996b) have showed that the reciprocal cross can occur in the field with hoary mustard, but it was not been observed with wild mustard to date (Lefol et al., 1996a).

      Recently, and for the first time, the possibility of producing interspecific hybrids in the field between oilseed rape and wild radish as the seed parent  have been reported (Darmency et al., 1998). Two hybrids were obtained from 59 wild radish plants grown at low density in the field, but none when wild radish was grown at high density. The germination rate of these hybrids is low and their fitness is reduced.

Other studies have been performed in North America and first large scale releases already took place there. However, climatic and agricultural conditions are quite different in Europe : shorter rotations (every two years in some European regions), winter sown  type rather than spring sown types, different kinds of wild relatives. Thus, it appears to be rather difficult to extrapolate data from North America for assessing the gene flow and agronomic impact of herbicide tolerant crops.

A multi-crop and multi-year monitoring study

In order to assess the effect of such outcrossing under agricultural conditions, in 1995 the French technical institutes CETIOM, AGPM, ITB and ITCF, designed and implemented  a monitoring study for various transgenic crops on three platforms  located in different regions of France : Champagne,  Burgundy and Midi-Pyrénées (South-West). Each platform consists of a 6 ha field where transgenic corn, rapeseed and sugar beet are cropped with the usual local cropping system (see Figure 1).

Figure 1. Example of a cropping system in Burgundy (1996-97) using transgenic traits.

	Monitoring area
		500 m

Example of cropping system burgundy

Year 1995-1996

Set-aside land

Rapeseed

Wheat

Sugar-beet

Maize

Fixed set-aside land (trefoil)

Year 1996-1997

Rapeseed

Wheat

Sugar-beet

Wheat

Maize

Fixed set-aside land (trefoil)

Year 1997-1998

Set-aside land

Sugar-beet

Set-aside land

Rapeseed

Maize

Fixed set-aside land (trefoil)

The transgenic traits are as follows :

* glufosinate and glyphosate resistance in corn, rapeseed and sugar beet ;

* bromoxynil resistance in rapeseed and corn borer tolerance (using the Bt system) in corn. 

A 500 meter area around the field was defined and  monitored in order to assess the spatial impact of transgenic crops.

This multi-year experiment aimed mainly at  :

* assessing the impact of transgenic crops when cultivated together in the same field area ;

* designing the weed control of volunteers in subsequent crops which are resistant to the same herbicide (e.g. glyphosate-resistant rapeseed volunteers in the subsequent sugar beet resistant to glyphosate) ;

* evaluating the multiple resistance rate when cropping two adjacent rapeseed fields with two different herbicide resistances ;

* estimating the interspecific outcrossing towards the wild relatives under real and local conditions and

* estimating the cost-benefit of herbicide resistance technology with respect to conventional techniques.

Outcrossing with wild relatives

Within the monitoring area, each wild relative plant of rapeseed was located and surveyed until seed maturity. The flowering period was observed and compared with the flowering periods of the transgenic rapeseed crops. Seeds were sampled for assessing the herbicide resistance which was checked by spraying herbicides after re-sowing. Table 1 gives the occurrence of wild relatives observed during three years of the study (1996-1998) : a plot represents one or several plant(-s) located at the same place.

Table 1.       Identification of wild relatives within the monitoring area from 1996 to 1998.
			Weed species and number of samples
Location		Rapeseed plot		Other crops and
				survey zone
Midi-Pyrénées		Sinapis arvensis - 1		Sinapis arvensis - 6
		Rapistrum rugosum - 77		Rapistrum rugosum - 1
		Brassica nigra - 5		Brassica nigra - 77
		Rapessed volunteers - 13		Sinapis alba - 51
	Total	96 samples		135 samples
Burgundy		Sinapis arvensis - 54		Sinapis arvensis - 58
				Rapeseed volunteers - 14
					Arabidopsis thaliana - 1
					Capsella bursa pastoris - 13
					Sisymbrium officinale - 1
					Thlaspi arvense - 1
					Barbarea intermedia - 1
					Alliara petiolata - 18
	Total	54 samples			107 samples
Champagne-Ardennes		Sisymbrium officinale - 20			Sinapis arvensis - 28
		Capsella bursa pastoris - 15			Sinapis alba - 4
		Calepina irregularis - 9			Raphanus raphanistrum - 1
		Rapeseed voulunteers - 38		Capsella bursa pastoris - 13
		Sinapis arvensis - 6			Rapeseed volunteers - 22
					Sisymbrium officinale - 2
					Calepina irregularis - 7
					Thlaspi arvense - 4
					Cardamine hirsuta - 4
					Alliara petiolata - 20
	Total	88 samples			105 samples
Total		238 samples			347 samples

RESULTS

Preliminary results indicated that no herbicide resistance with wild mustard and other mustard species occurred during the first two years. Unfortunately, wild radish was not present in our situations and a specific location site has been implemented in 1998 in the South-West region. frequency of outcrossing will continue to be studied in the subsequent years and will allow us to increase the precision of this frequency.

Multiple resistance and dispersal of pollen

The three herbicide resistant rapeseed varieties were cropped in adjacent fields and double resistant plants were detected in two different ways :

* by applying the herbicides on volunteers whose emergence occurred after harvesting ;

* by sampling seeds and re-sowing using a specific design of experiments and direct application.

Both methods gave similar results with respect to the rate of double resistance. Although the results were dependent upon the variety, the average rate of double  resistance can be estimated under our specific conditions  : about 2 % at a one meter distance, 0.2 % at 20 meters and less than 0.01 % at 65 meters. (Figure 2)

Figure 2.         Average dispersal curves for the three location sites

CONCLUSIONS

Although further data are still required, these results seem to indicate that multiple resistance should probably be the major concern for  farmers rather than  interspecific crosses. Suitable agronomic practices should be proposed in order to provide a sustainable use for transgenic crops such as herbicide tolerant oilseed rape. 

Preliminary results obtained during the first years of our project confirmed what was expected from previous studies. Thus results have been obtained under current farmer practices and provided data which will be used to fit simulation models for gene flow. While further data will be obtained for three more years, adequate crop management practices can already be discussed and recommendations provided for public decision-making.

REFERENCES

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