Henri Darmency

The impact of hybrids between genetically modified crop plants and their related species: introgression and weediness

Assessing the impact of hybrids between transgenic plants and nontarget wild species involves answering several questions such as: (i) what are the hybridization and introgression rates; (ii) what is the behaviour of a transgene in a wild population; and (iii) what will be the consequences of the expression of a transgene in a wild population? These issues are discussed using results from experiments on oilseed rape and wild related Brassiceae. Evidence is given of large variations in the estimates of cross‐fertilization probabilities. The first stage of introgression into wild populations is demonstrated to occur spontaneously through back‐crossing. Population analysis may also be valuable to detect traces of past introgression. Data from the literature on weed biology, and especially herbicide resistance, are used to illustrate the behaviour of a new gene in weed populations. The need for computer models simulating the introgression process is stressed.

Predicting hybridization between transgenic oilseed rape and wild mustard

Abstract Overlap between flowering of oilseed rape ( Brassica napus var. oleifera Metzger) and wild mustard ( Sinapis arvensis L.), artificial hybridization between the two species, spontaneous crosses, and backcrossing were assessed to estimate the risk of escape of genes from transgenic crops towards the wild species. In the Burgundy region of France, wild mustard flowers later than oilseed rape. Exposure to cross pollination was two to five times greater with late-flowering cultivars than with early cultivars. Artificial hybridizations using in vitro ovary culture produced up to 1 seed per 100 pollinated flowers. No hybrid was found among 2.9 million seeds produced by wild mustard grown in a garden in presence of a herbicide-resistant transgenic cultivar. No more than six hybrids were obtained from 50 000 flowers of a male-sterile oilseed rape grown in presence of wild mustard. Artificial hybrids grown in presence of wild mustard, or hand-crossed, produced a few aborted seeds. Thus, in similar “normal conditions”, it may be concluded that a flower of these two species has a probability smaller than 10 −10 of having an interspecific hybrid progeny.

Gene dispersal from transgenic crops : II. Hybridization between oilseed rape and the wild hoary mustard

The risk of release of genetically modified oilseed rape (Brassica napus) was investigated in relation to interspecific gene flow with hoary mustard (Hirschfeldia incana). Microscopic studies showed polymorphism within the population of hoary mustard for pollen germination on oilseed rape flowers. The transgenic herbicide-resistant and a commercial cultivar of oilseed rape were not different for pollen behaviour and ovule fertilization. Pollen tube growth was slow and erratic in interspecific crosses. Fertilization efficiency of oilseed rape and hoary mustard pollen in interspecific crosses was 15% and 1.3%, respectively, of that in intraspecific crosses. This unequal efficiency in reciprocal crosses was confirmed by hybrid seed set in pods. There was no post-zygotic barrier to the development of hybrid embryos in hoary mustard pods. Up to 26 spontaneous hybrids per male sterile oilseed rape plant, and one per hoary mustard plant, were obtained in field experiments. Hybrids were identified by isozyme electrophoresis, morphology and cytology. All hybrids were triploid with 26 chromosomes, and had low fertility. They produced 0.5 seeds per plant after spontaneous backcrossing with hoary mustard. Some of these descendants were produced from unreduced gametes. Our results suggest that gene flow is likely to occur, but its actual frequency under crop growing conditions remains to be estimated.

Assessment of interspecific hybridization between transgenic oilseed rape and wild radish under normal agronomic conditions.

Abstract In order to assess the hybridization rate between oilseed rape and wild radish under normal agronomic conditions, three 1-ha field experiments were performed. In each case, wild radish plants were transplanted at different densities in the middle, the border, or the margin of the herbicide-tolerant oilseed rape field. Among the 189084 seedlings obtained from seeds harvested on wild radish plants, only one herbicide-tolerant interspecific hybrid (RrRrAC, 2n = 37) was characterized from seeds harvested on an isolated plant growing in the margin of the field. Thus, for the wild radish total harvest, with a 95% confidence limit, the frequency of interspecific hybrids was assessed to range from 10–7 to 3.10–5. Interspecific hybrids were detected in all cases among the smallest seeds with a diameter less than 1.6 mm harvested on oilseed rape, but the highest frequency was obtained from oilseed rape close to wild radish plants growing as clusters in the border or the margin of the field. Most hybrids had the expected triploid genomic structure (ACRr, 2n = 28) except for four amphidiploids (AACCRrRr, 2n = 56) and one hybrid from a wild radish unreduced gamete (ACRrRr, 2n = 37). Among the 73847 seedlings observed on the oilseed rape total harvest, the frequency of interspecific hybrids was assessed to range from 2.10–5to 5.10–4, with a 95% confidence limit. The results are discussed with regard to the type of oilseed rape variety used and the characteristics of the interspecific hybrids.

Spontaneous hybridizations between oilseed rape and wild radish

The occurence of spontaneous hybridization between Brassica napus (oilseed rape) and Raphanus raphanistrum (wild radish) was investigated under different density conditions in cages and open‐field experiments. Hybrids with wild radish as the seed parent were identified by screening for herbicide resistance belonging to rape. Small seed size and intermediate morphology were used to screen for hybrids with rape as the seed parent. Leaf isozyme patterns and flow cytometry provided confirmation of hybrids. Wild radish in an oilseed rape field produced as many as three interspecific hybrids per 100 plants. This is the first report of such a spontaneous event. The frequency of hybrids is expected to range from 0.006 to 0.2% of the total seed produced, at P = 0.05. Male‐sterile oilseed rape plants surrounded by wild radish can produce up to 37 hybrids per plant. Seed production of the F1 hybrids and their F2 descendants was up to 0.4% and 2%, respectively, of that of wild radish. Gene escape from transgenic oilseed rape to wild related species is discussed.

Fitness of backcross six of hybrids between transgenic oilseed rape ( Brassica napus ) and wild radish ( Raphanus raphanistrum )

The process of introgression between a transgenic crop modified for better agronomic characters and a wild relative could lead potentially to increased weediness and adaptation to the environment of the wild species. However, the formation of hybrid and hybrid progeny could be associated with functional imbalance and low fitness, which reduces the risk of gene escape and establishment of the wild species in the field. Our work compares the fitness components of parents and different types of backcross in the sixth generation of hybrids between transgenic oilseed rape (Brassica napus, AACC, 2n = 38) resistant to the herbicide glufosinate and wild radish (Raphanus raphanistrum, RrRr, 2n = 18). The backcross with oilseed rape cytoplasm (OBC) has a fitness value 100 times lower than that of the backcross with wild radish cytoplasm (RBC). The herbicide‐resistant RBC has similar growth to the susceptible RBC, but final male and female fitness values are two times lower. In turn, susceptible RBC exhibit similar fitness to the control wild radishes. The relative fitnesses of the different types are the same whether or not they grow under competitive conditions. The consequence on fitness of the chromosome location of the transgene conferring resistance and the relevance of these results to the impact of gene flow on the environment are discussed.

Low frequency transmission of a plastid-encoded trait in Setaria italica

It has been claimed that engineering traits into the chloroplast will prevent transgene transmission by pollen, precluding transgene flow from crops. A Setaria italica (foxtail or birdseed millet) with chloroplast-inherited atrazine resistance (bearing a nuclear dominant red-leaf base marker) was crossed with five male-sterile yellow- or green-leafed herbicide susceptible lines. Chloroplast-inherited resistance was consistently pollen transmitted at a 3×10−4 frequency in >780,000 hybrid offspring. The nuclear marker segregated in the F2, but resistance did not segregate, as expected. Pollen transmission of plastome traits can only be detected using both large samples and selectable genetic markers. The risk of pollen transmission at this frequency would be several orders of magnitude greater than spontaneous nuclear-genome mutation-rates. Chloroplast transformation may be an unacceptable means of preventing transgene outflow, unless stacked with additional mechanisms such as mitigating genes and/or male sterility.

Molecular Bases for Sensitivity to Tubulin-Binding Herbicides in Green Foxtail

We investigated the molecular bases for resistance to several classes of herbicides that bind tubulins in green foxtail (Setaria viridis L. Beauv.). We identified two α- and two β-tubulin genes in green foxtail. Sequence comparison between resistant and sensitive plants revealed two mutations, a leucine-to-phenylalanine change at position 136 and a threonine-to-isoleucine change at position 239, in the gene encoding α2-tubulin. Association of mutation at position 239 with herbicide resistance was demonstrated using near-isogenic lines derived from interspecific pairings between green foxtail and foxtail millet (Setaria italica L. Beauv.), and herbicide sensitivity bioassays combined with allele-specific PCR-mediated genotyping. Association of mutation at position 136 with herbicide resistance was demonstrated using herbicide sensitivity bioassays combined with allele-specific PCR-mediated genotyping. Both mutations were associated with recessive cross resistance to dinitroanilines and benzoic acids, no change in sensitivity to benzamides, and hypersensitivity to carbamates. Using three-dimensional modeling, we found that the two mutations are adjacent and located into a region involved in tubulin dimer-dimer contact. Comparison of three-dimensional α-tubulin models for organisms with contrasted sensitivity to tubulin-binding herbicides enabled us to propose that residue 253 and the vicinity of the side chain of residue 251 are critical determinants for the differences in herbicide sensitivity observed between organisms, and that positions 16, 24, 136, 239, 252, and 268 are involved in modulating sensitivity to these herbicides.

Phylogenetic and genomic relationships in Setaria italica and its close relatives based on the molecular diversity and chromosomal organization of 5S and 18S-5.8S-25S rDNA genes.

Abstract We have analyzed the phylogenetic and genomic relationships in the genus Setaria Beauv. including diploid and tetraploid species, by means of the molecular diversity of the 5S rDNA spacer and chromosomal organization of the 5S and 18S-5.8S-25S rDNA genes. PCR amplification of the 5S rDNA sequences gave specific patterns. All the species studied here share a common band of about 340 bp. An additional band of an approximately 300-bp repeat unit was found for Setaria verticillata and the Chinese accessions of Setaria italica and Setaria viridis. An additional band of 450 bp was found in the sole species Setaria faberii. Fluorescent in situ hybridization was used for physical mapping of the 5S and 18S-5.8S-25S rDNA genes and showed that they are localized at two separate loci with no polymorphism of chromosome location among species. Two chromosome pairs carrying the 5S and 18S-5.8S-25S rDNA clusters can now be unambiguously identified using FISH. Phylogenetic trees based on the variation of the amplified 5S rDNA sequences showed a clear separation into four groups. The clustering was dependent on the genomic composition (genome A versus genome B) and confirmed the closest relationship of S. italica and S. viridis accessions from the same geographical region. Our results confirm previous hypotheses on the domestication centers of S. italica. They also show the wide difference between the A and B genomes, and even clarify the taxonomic position of S. verticillata.

Survival of buried seeds of interspecific hybrids between oilseed rape, hoary mustard and wild radish

Abstract Two experiments were designed to assess seed survival in the soil of rape (Brassica napus L.), hoary mustard, interspecific hybrids between rape and hoary mustard (Hirschfeldia incana (L.) Lagreze-Fossat) as well as between rape and wild radish (Raphanus raphanistrum L.). The first experiment dealt with seed survival in undisturbed soil over 41 months. It showed a slow decrease in viability of the seeds of hoary mustard, a quick drop to a constant 1% viability of rape, and intermediary behaviour of hybrids. The second experiment was conducted at two field sites in eastern and southern France, with normal cultivation and crop growth during 3 years. Quantitative differences were observed between the two sites, although the relative behaviour of each seed type was similar. Hoary mustard had the greatest seed viability, on average 14% after 3 years, while total seedling emergence in the field was 1.9% after 3 years. The decrease of the viability of rape seed was slower than in the undisturbed experiment, with more than 1% viable seeds remaining after 3 years. Total emergence of rape was up to 4%. Hybrids showed lower seed viability than rape. Total seedling emergence over 3 years was less than 1%. These results are discussed together with the consequences of commercial release of transgenic crops. Transgenes can escape via seeds and volunteer rape, and seeds of interspecific hybrids between rape and wild relatives can survive and germinate after several years, ensuring genetic and spatial spread of transgenes.