ABSTRACT

Sinapis arvensis is a common weed species and a relative of Brassica napus. There is concern that herbicide tolerance genes may escape from genetically modified B. napus crops into weed populations.

We have crossed S. arvensis plants from populations across the UK and France with herbicide tolerant B. napus, without assistance from embryo rescue or ovule culture. Hybrids were identified by molecular markers, morphology and chromosome counts.

The S. arvensis x B. napus cross was unsuccessful. Out of 4000 pollinations, one hybrid was formed from the B. napus x S. arvensis cross.

INTRODUCTION

Sinapis arvensis is frequently found growing in or around Brassica crops and was a serious weed in the UK and Canada before control by herbicides was achieved (Fogg, 1950; Mulligan and Bailey, 1975; Edwards, 1980). S. arvensis belongs to the same tribe (the Brassicae) as Brassica napus (Rich, 1991) and herbicide tolerant oilseed rape may be one of the first GM crops to be released on a commercial scale in the UK.

For these reasons it is important to clarify the ability of S. arvensis to hybridise with oilseed rape. Many studies have concluded that the two species are sexually incompatible (Bing et al., 1995; Bing et al., 1996a; Bing et al., 1996b; Brown and Brown, 1996; Kerlan et al., 1992). However, Chevre et al. (1996) and Lefol et al. (1996) have found hybrids can be produced under open pollination conditions.

We collected seed from populations of S. arvensis populations across the UK and from France where hybrids were previously found (Chevre et al., 1996). We have found that the production of interspecific hybrids is possible without embryo rescue or ovule culture. However, the cross was unsuccessful when S. arvensis was the maternal parent.

METHODS AND MATERIALS

Seed were collected from populations of S. arvensis around the UK in September/October 1997. Details of their locations are given in Moyes et al. (1999) Seed from French populations were supplied by A. Chevre (INRA, Rennes).

The oilseed rape line used as a pollen donor was the cultivar Westar 10, transformed with a construct containing the BAR and NPTII genes conferring resistance to glufosinate ammonia and kanamycin, respectively. The plants were hemizygous for the transgene. Oilseed rape plants used as maternal parents were from the same line backcrossed to untransformed Westar 10. Therefore, half of these plants contained the transgene.

Seed from 102 S. arvensis populations were grown and two plants from each population were used for the pollinations. For each plant, 30 buds were emasculated just before opening and pollinated with rape pollen. Each plant was also used to pollinate 20 emasculated rape buds. In addition, 16 plants were pollinated with other plants of the same species to give a figure for the maximum number of seed obtainable under the experimental conditions. The pods were allowed to develop naturally in the glasshouse and seed harvested after 8 weeks for S. arvensis and 6 weeks for rape.

The seed were sown and the resulting plants were tested for presence of the transgenes by PCR using primers for BAR and NPTII. Leaf discs were cultured in the presence of the herbicide to test for glufosinate tolerance and to back up the PCR results.

Sourthern blots of DNA digested with EcoR1, from each parent and the potential hybrids, were probed with a range of molecular markers.

RESULTS

Crosses involving 20 S. arvensis populations produced seed when S. arvensis was the maternal parent. Crosses involving 28 S. arvensis populations produced seed when S. arvensis was the paternal parent. For each successful cross the seed number produced was very low (Table 1). The mean number of potential hybrids resulting from the S. arvensis x B. napus crosses was 0.008/bud and from the B. napus x S. arvensis crosses 0.011/bud. The control crosses gave a mean of 5.1 seed/bud for S. arvensis crosses and 16.7 seed/bud for B. napus crosses. This means that the interspecific crosses yielded 0.05% and 0.2% of the total potential seed output, respectively.

The results of the PCR and BAR colour assay found that none of the potential hybrids produced from crosses with S. arvensis as the maternal parent contained the transgene. The B. napus parent line was hemizygous for the transgene and so this result did not eliminate the possibility that these progeny were true hybrids.

One potential hybrid (Hx67B) resulting from a B. napus x S. arvensis cross had an intermediate morphology. Despite the fact it was produced from a cross with B. napus as the maternal plant, H67B had a moderate number of leaf/stem hairs, had no clasping auricles on the upper stem leaves and was not glabrous in colour. All of the other potential hybrids had an identical morphology to that of the maternal parent.

A number of molecular markers have been used to identify hybrids. Marker pw141 (Campos de Quiroz and Mithen, 1996) specific bands for B. napus and S. arvensis between 7kbp and 9kbp. Plant H67B had bands from both species. Probe cs1 (unpublished data) gave S. arvensis-specific bands between 500bp and 3kbp which were also present in Hx67B.

Chromosome counts have been completed on half of the potential hybrids to date. All of these plants had the same chromosome complement as their maternal parents. The results from plant H67B are not yet in.

Table 1. The number of seed produced in the interspecific crosses. The mean seed number is given for crosses involving two S. arvensis plants with 30 S. arvensis buds and 20 B. napus buds pollinated per plant. The number of the resulting seed that successfully germinated is given. Figures in brackets give the number of seeds germinating and immediately dying.

DISCUSSION

A low number of seed were produced in the reciprocal crosses between S. arvensis and oilseed rape. These seed may have resulted from a number of processes other than hybridisation. They may be the result of contamination during the hand pollinations or matromorphy induced by intergeneric pollen as has been reported to occur in the Cruciferae (Banga, 1986). From our results so far it appears that only one of these plants is a true hybrid. This plant was obtained from a B. napus x S. arvensis cross using S. arvensis seed collected in Lincolnshire, England.

If the initial results are confirmed then the S. arvensis x B. napus cross has been unsuccessful. We hand pollinated 6000 buds in this cross and these buds had a potential total output of 30 780 seed. No study has reported any success with this cross. Lefol et al. (1996) screened 2.9 million seed from S. arvensis plants grown in the presence of a herbicide-tolerant rape cultivar and found no evidence of hybridisation. Therefore we conclude that this cross in this direction is incompatible.

In the reciprocal cross, B. napus x S. arvensis, we obtained one hybrid from 4000 buds pollinated (=0.025/100 buds). Chevre et al. (1996) obtained 0.18 hybrids/100 flowers of male sterile rape under field conditions. Lefol et al. (1996) obtained 6 hybrids/ 50 000 flowers of male sterile rape under field conditions. This equates to 0.012 hybrids/100 flowers. Therefore, we can conclude that this cross consistently yields a low number of hybrids.

This work was carried out using hand pollinations so it is not possible to predict numbers of hybrids arising in the field. However, it is clear that the possibility cannot be eliminated although if they do occur we would expect the frequency to be extremely low. This work will form the next part of our study.

If hybrids are formed in the field, only hybrids with B. napus as the maternal parent will be obtained. The majority of seed produced on the B. napus plants will be harvested. However some seed will be lost at harvest and dropped during transportation. A proportion of this seed will establish as feral or volunteer plants. Although the additive probability of a hybrid being formed and then being lost from the harvest and then successfully establishing as a reproductive plant is extremely low, about 500 000 ha of B. napus are grown every year in the UK alone. S. arvensis occurs in the same habitats as feral, volunteer and crop B. napus so it is important to consider the possibility of backcrossses between a hybrid and S. arvensis. This work is currently in progress.

ACKNOWLEDGEMENTS

We acknowledge the contribution of BBSRC and direct financial support for this project from MAFF, project code RG0218.

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