Abstract

Keywords

Introduction

One of the main concerns linked to the commercial release of transgenic crops is the likelihood of the gene spread from the cultivated species to wild relatives. To be efficient, gene transfer from one species to an other involves the following steps: (1) production of viable hybrids from crosses between the two species, (2) occurrence of fertile plants in the successive generations, (3) gene transmission through the different generations, (4) effective gene establishment within the natural populations and maintenance of the new trait. We assessed the hybridization rates between oilseed rape and three crucifer weeds common under our French conditions having the same flowering period as the crop, wild mustard (Sinapis arvensis, SarSar, 2n=18), hoary mustard (Hirschfeldia incana, AdAd, 2n=14) and wild radish (Raphanus raphanistrum, RrRr, 2n=18). Under optimal conditions, using male sterile oilseed rape as female and the same ratio of crop and weed, the highest rate of interspecific hybrids was obtained with wild radish as pollinator (Eber et al. 1994 ; Chèvre et al. 1996). Additionally, using wild radish as female parent, a low frequency of interspecific hybrid production was seen (Darmency et al. 1998). This model has been chosen for further analyses. The comparison of hybrid production under optimal and under agronomical conditions as well as the limiting factors at the first and successive generations are presented in this paper.

Production of interspecific hybrids

Using different male sterile oilseed rape varieties containing the bar gene at the heterozygous stage and wild radish as pollinator, we observed that the F1 interspecific hybrid rates depends on the female parent ; they ranged from 1.4 to 100.4 hybrid seeds/100 flowers and from 5.3 to 1213.3 hybrid seeds/plant (Baranger et al., 1995).

Under normal agronomic conditions, a one hectare field of ‘Synergy’ variety tolerant to Basta^® (80% of male sterile hybrid with the Ogu-INRA cytoplasm and two copies at the hemizygous stage + 20% a pure line with two copies at the homozygous stage) was sown. Wild radish plants were transplanted at different densities :  

• within the field, either as isolated plants (1plant/20m²) or in clusters (2plants/m²),
• in the border and in the margin of the field, either as isolated plants (1 plant each 10 meters) or in clusters (40 plants).

Three fields were grown : one in Rennes and two in Dijon during two years.

A good overlapping of the flowering period was observed.

Wild radish was harvested plant by plant. The mean number of seeds per plant was lower from plants harvested in the middle of the field than from plants harvested in the border or the margin of the field : in Rennes, on average 340 and 5400 seeds /plant, respectively and in Dijon 3 and 310 seeds/plant, respectively. From 951,622 seeds, 189,420 seedlings were obtained in greenhouse or in field and herbicide treated. Only one hybrid at 2n=37 (ACRrRr), Basta^® resistant, harvested on an isolated wild radish plant growing in the margin of the field, was detected . With 95% confidence limit, the frequency of hybrids obtained from wild radish plants growing in the same isolated situation was assessed to range from 4.10^-4 to 9.10^-2 but, from the total harvest, whatever the location of the wild radish mother plants, from 10^-7 to 3.10^-5.

Oilseed rape seeds were harvested only in the trial performed in Rennes, around the wild radish plants developed within the field or as clusters in the border or the margin of the field. They were sieved and only the smallest seeds (diameter<1.6mm) were analysed as it was established, from our previous data, that these latter contained the interspecific hybrids (Eber et al. 1994). From 67.10⁶ of the smallest seeds, 73,847 plants were obtained either in the greenhouse or in the field. In this latter, the criteria used to detect interspecific hybrids was the duration of the flowering period, as interspecific hybrids flower longer than oilseed rape because of their female sterility. Among the 650 plants obtained in greenhouse plus 1240 plants still flowering in the field and analysed by flow cytometry, 23 interspecific hybrids were detected ; 78% of them had 28 chromosomes (ACRr) but four amphidiploids (2n=56, AACCRrRr) and one ‘BC1’ like plant (2n=37, ACRrRr) were also obtained. Among the 21 hybrids more precisely studied, 13 contained the pat gene conferring herbicide resistance and 20 had an Ogu-INRA cytoplasm detected by PCR (Tinchant et al. 1997). The remaining hybrid with an oilseed rape cytoplasm was an amphidiploid (AACCRrRr, 2n=56), herbicide resistant. Interspecific hybrids were found from oilseed rape mother plants whatever their location in the field. So, from the smallest seeds harvested, the expected frequency of hybrid plants ranged from 2 10^-5 to 5 10^-4, with 95% confidence limit but these seeds represented 7.3 to 12.3% of the total harvest according to the different locations of the oilseed rape plants around wild radish plants.

Analysis of the limiting factors during the successive generations

Analysis of the first generations

Six interspecific hybrid plants, herbicide tolerant, obtained from the trial performed under normal agronomic conditions, were selected. Their cytoplasm, chromosome number, meiotic behavior and male fertility are reported in Table 1. The female fertility, assessed by the number of seeds per 100 flowers or per plant, was established from cuttings transplanted in field with a 1 :1 ratio of hybrids and of wild radish as pollinators (Table 1). Plants of « BC1 » progeny were analysed for their chromosome number assessed by flow cytometry (Eber et al. 1997) and for the presence of the transgene checked by herbicide spraying (Table 1).

Table 1 : Characterisation of F1 interspecific hybrids and of their « BC1 » progeny obtained under field conditions

F1 interspecific hybrids								« BC1 » hybrids
Cyto-plasm	Nb of plants	2n	Cells	Mean meiotic behavior	% male fert.*	Seeds /100 flowers	Seeds/ plant	Nb of pl.	2n ¤	% of Basta resist.plants
Rr	1	37	12	3.58I+15.42II+0.42III+0.33IV	6.5	0.07	0.15	0	-	-
Ogu-INRA	3	28	54	12.19I+7.80II+0.05IV	0	0.2	1.6	15	37 [21-57]	87
	1	56	16	6.38I+24.44II+0.25III	0	100.9	420.0	79	52 [44-57]	100
Bn	1	56	30	7.30I+23.37II+0.57III+0.07IV	85	455.9	4074.3	80	56 [48-69]	100

* Male fertility was assessed from the percentage of pollen grains stained by a 1% aceto-carmine solution

¤ chromosome number assessed by flow cytometry and range of the chromosome number

Rr Raphanus raphanistrum cytoplasm

Ogu-INRA : Ogu-INRA cytoplasm

Bn : B. napus cytoplasm

The meiotic behavior of the 2n=37 hybrid (ACRrRr) revealed a high frequency of chromosome pairing indicating that chromosome exchanges can occur at this stage. However, the male fertility was low and none of the seeds produced germinated.

The 2n=28 hybrids (ACRr) had a behavior similar to the one already described in our previous studies (Chèvre et al. 1997) : possibilities of gene exchanges through chromosome pairing, a poor male and female fertility, a progeny obtained mainly from unreduced gametes and so a high frequency of transgene transmission.

Whereas the two amphidiploid plants analysed (2n=56, AACCRrRr) had the 2 parental genomes at the diploid stage, the frequency of multivalents indicated that chromosome rearrangements can occur. Their male and female fertility depended on their cytoplasm : the amphidiploid on the Ogu-INRA cytoplasm was poorly fertile compared to the one on the oilseed rape cytoplasm. A decrease of the chromosome number was observed in the progeny of the sterile amphidiploid whereas most of the plants obtained from the fertile amphidiploid had the same chromosome number as the mother plant i.e. 2n=56. For this latter, it is likely that mainly self fertilization occurred.

Analysis of the following generations

From F1 interspecific hybrids (ACRr, 2n=28) (generation G1) obtained under optimal conditions using male sterile oilseed rape (Ogu-INRA cytoplasm) and wild radish as pollinator (Baranger et al. 1995), four successive generations were analysed. Each of them was produced under field conditions with the same ratio of hybrids and of wild radish and seeds were harvested on hybrids. We observed at the following G2 generation an increase of the chromosome number and of the bar gene transmission because of the efficiency of unreduced gametes (Chèvre et al. 1997 ; 1998). However, the female fertility remained very low. During the following G3, G4 (Chèvre et al., 1997) and G5 generations, the chromosome number decreased and at the G5 generation, 91% of the herbicide tolerant hybrids had less than 23 chromosomes. Similarly, the percentage of Basta^® tolerant plants decreased whereas the male and female fertility increased. Some herbicide tolerant plants had a female fertility equivalent to the one of wild radish but none of them had 18 chromosomes as wild radish. This result indicated that the transgene was not inserted through recombination into the wild radish genome. On another hand, their vegetative development was slow down because of a chlorophyll deficiency. Reciprocal hybrids obtained by hand pollination between fertile hybrids and wild radish as female parent, were dark green with a good vigour. This result revealed that this chlorophyll deficiency is due to an incompatibility between the radish nucleus and the oilseed rape chloroplasts. The reciprocal phenomena was already described by Pelletier et al. (1983).

Discussion

We showed that under optimal conditions, using male sterile oilseed rape as female, the frequency of F1 interspecific hybrids production is high and depends on the oilseed rape genotype. However, whatever the female parent, it is very low under normal agronomic conditions. Among the seeds harvested on wild radish, the only hybrid was obtained from an isolated plant growing in the margin of the field. It is likely that the situation of isolated plant is the most favorable because of the self-incompatibility present in this wild species. On the contrary, interspecific hybrids were obtained on oilseed plants whatever their location in the field. As the oilseed rape variety used for this experiment was male sterile for 80% of the plants, it is likely that we underestimated hybrid production on wild radish plants but overestimated it on oilseed rape. New experiments are in progress from a field of a fertile, herbicide resistant line in which wild radish have grown spontaneously.

At the first generation, we observed that the male and female fertility depends on the genomic structure of the plants as triploid (ACRr) or BC1 like (ACRrRr) hybrids had a poor fertility compared to the amphidiploid plants (AACCRrRr). Additionally, the cytoplasm influences the production of seeds and pollen. The possibility of maintenance of such fertile amphidiploid plants under natural conditions will be assessed.

The analysis of the following generations from triploid oilseed rape-wild radish F1 interspecific hybrids on Ogu-INRA cytoplasm revealed that, at the fifth generation, none of the herbicide tolerant plants had the same chromosome number as the weed. So, the transgene was not inserted in the genome of the weed (Chèvre et al., 1997). However, only one insertion transformation event was used to produce this material and our analysis of different specific oilseed rape loci indicated that their transmission rates are different according to the locus (Chèvre et al., 1998). Further analyses are needed to investigate the effect of the initial location of the transgene on gene introgression into the genome of a close related species. Additionally, we observed that if the hybrids kept along the different generations an oilseed rape cytoplasm, they became chlorophyll deficient.

From this study, we can conclude that F1 interspecific hybrids can be produced at a very low frequency but that their ability to produce a progeny depends on their genomic structure and on their ability to be either self-fertilized or crossed to oilseed rape or wild radish. In the following generations produced by pollination with wild radish, the vigor of the plants is determined by their initial cytoplasm, oilseed rape or wild radish. Transgene maintenance could occur either by self-fertilization of amphidiploid or by recombination, if possible, within the wild radish genome. Fitness analyses have to be performed from this material.

References

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