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.     

Hybridisation between Brassica napus L. and Raphanus raphanistrum L. under agronomic field conditions

The frequency of hybridisation between Brassica napus L. and Raphanus raphanistrum L. under agronomic conditions was assessed in field experiments, where R. raphanistrum were randomly planted at two different densities into large plots of B. napus. An acetolacate synthase (ALS)-inhibiting herbicide-resistant trait was used to detect potential hybrid individuals. No hybrids were detected amongst 25,000 seedlings grown from seed collected from R. raphanistrum plants. Two hybrids were obtained from more than 52-million B. napus seedlings. Both hybrids were characterised as amphidiploids (AACCRrRr, 2n = 56) and were fertile. The frequency of hybridisation into B. napus in this experiment using male-fertile B. napus was 4 × 10^–8. Received: 31 August 2000 / Accepted: 23 January 2001     

Characterization of backcross generations obtained under field conditions from oilseed rape-wild radish F1 interspecific hybrids: an assessment of transgene dispersal

Gene flow from glufosinate-resistant transgenic oilseed rape to wild radish was studied over two backcross generations. Under field conditions,?seed production from oilseed rape-wild radish F₁ hybrids due to pollination by wild radish was always low: on average 0.12 and 0.78 seeds per 100 flowers and per plant, respectively. The cytogenetics of the resulting «BC₁» plants can be explained in the main by three different genomic constitutions: either ACRrRr, 2n=37, ACRr, 2n=28 (the same chromosome number as the mother plant), or by the amphidiploid AACCRrRr, 2n=56. The probability of gene exchange through chromosome pairing was high only in plants with 2n=28 or 37 chromosomes. Due to the viability of unreduced or partially reduced female gametes, most of the «BC₁» plants (81.9%) were Basta resistant whereas the analysis of oilseed rape specific loci indicated that their transmission varied with the locus. In spite of low male fertility (8.7%), an improvement of the female fertility over the F₁ hybrids was observed with an average production of 1.4 and 11 seeds per 100 flowers and per plant, respectively. At the following «BC₂» generation, the bar gene transmission (57.2% of Basta-resistant plants) decreased as did the chromosome number, with a majority of plants having between 24 and 27 chromosomes, with 10.5% similar to wild radish (2n=18). The lower the chromosome number, the better the fertility of the «BC₂» plants. On average, 7.9 and 229.3 seeds per 100 flowers and per plant were produced. Gene-flow assessment is discussed based on these data.     

Persistence of oilseed rape (Brassica napus L.) outside of cultivated fields

It is widely assumed that most cultivated plants cannot persist in natural or semi-natural habitats. Most people thus assume that the plants growing outside of fields (in particular oilseed rape along roadsides) find their origins in the current or previous year’s cultivation of that crop. One consequence of this assumption is that the identity of plants growing on road verges is thought to reflect one of the cultivars currently or recently cultivated, while another consequence is the widespread belief that transgenic plants can be simply managed and controlled by stopping their cultivation. Our work shows that this assumption is false. We identify relict plants of a now unmarketable cultivar type of oilseed rape which have persisted in a semi-natural habitat (road verges) for at least 8 years according to farmer surveys in the studied area. More generally, we confirm that the dynamics of the feral oilseed rape plants of road verges is more complex than those resulting from spillage from agricultural machines or from neighbouring arable fields cultivated the previous year. Within the scope of transgenic oilseed rape cultivation, these results suggest that more studies on the dynamics of feral oilseed rape are needed in order to assess more precisely the risks of its invasiveness and its potential impact on genetic pollution between GM fields and non-GM fields. Received: 15 June 2000 / Accepted 31 July 2000     

Polymorphism for interspecific hybridisation within a population of wild radish (Raphanus raphanistrum) pollinated by oilseed rape (Brassica napus)

The within-population polymorphism of wild radish (Raphanus raphanistrum) for interspecific hybridisation with two cultivars of oilseed rape (Brassica napus) was investigated by hand crossing experiments and fluorescence microscopy. Wide variability among plants was observed in the ability of oilseed rape pollen to germinate on the wild radish stigma; the frequency of pistils showing pollen tubes ranged from 0 to 1, depending on the female plant. The ratio of fertilised ovules to the total number of ovules in ovaries where pollen tubes arrived ranged from 0.02 to 0.51. Overall, the results provide evidence for the presence of different phenotypes. In 40% of the plants, pistils had no or very few pollen tubes and few fertilised ovules. In 23%, the foreign pollen tubes grew through the style towards the ovary, but had low ovule fertilisation efficiency. The remaining 37% showed a large number of pollen tubes in the style and frequent ovule fertilisation, and two plants showed no difference between foreign and conspecific pollen. With regard to post-zygotic barriers, pollen germination and ovule fertilisation represent minor barriers to interspecific hybridisation between oilseed rape and wild radish. It is suggested that the effectiveness of these barriers could be improved through plant breeding; this could reduce the risk of gene flow from transgenic oilseed rape to populations of wild relatives. Received: 15 April 2001 / Accepted: 24 May 2001     

Frequency and distance of pollen dispersal from transgenic oilseed rape (Brassica napus)

The objective of this study was to evaluate pollen dispersal inBrassica napus (oilseed rape). The selectable marker, used to follow pollen movement, was a dominant transgene (bar) conferring resistance to the herbicide glufosinate-ammonium. Transgenic and non-transgenic plants of the cultivar Westar were planted in a 1.1 ha field trial, with the transgenic plants in a 9 m diameter circle at the centre, surrounded by non-transgenic plants to a distance of at least 47 m in all directions. A 1 m circle of non-transgenic plants was sown in the centre of the transgenic area to allow estimation of the level of pollen dispersal when plants were in close contact. Honeybee hives were placed at the trial site to optimize the opportunity for cross-pollination. During the flowering period, regular observations were made of the number of plants flowering and the number and type of insects present in 60 1 m² areas. These areas were located uniformly around the plot at distances of 1, 3, 6, 12, 24, 36 and 47 m from the edge of the 9 m circle of transgenic plants. Seed samples were harvested from each of the 7 distances so that approximately 20% of the circumference of the plot was sampled at each distance. The centre non-transgenic circle was also sampled. Plants were grown from the seed samples and sprayed with glufosinate to estimate the frequency of pollen dispersal at each distance. In order to screen enough samples to detect low frequency cross-pollination events, seed samples were tested in the greenhouse and on a larger scale in the field. Results were confirmed by testing progeny for glufosinate resistance and by Southern blot analysis. The estimated percentage of pollen dispersal in the non-transgenic centre circle was 4.8%. The frequency was estimated to be 1.5% at a distance of 1 m and 0.4% at 3 m. The frequency decreased sharply to 0.02% at 12 m and was only 0.00033% at 47 m. No obvious directional effects were detected that could be ascribed to wind or insect activity.     

Somaclonal variation in the progeny of transgenic barley

 Somaclonal variation (SCV) in transgenic plants may slow the incorporation of introduced genes into commercially competitive cultivars. Somaclonal variation in transgenic barley (Hordeum vulgare L.) was assessed in one experiment by comparing the agronomic characteristics of 44 segregating transgenic lines in the T₂ generation to their non-transformed parent (‘Golden Promise’). A second experiment examined the agronomic characteristics of seven transgenic-derived, null (non-transgenic) segregant lines in the T₂ and T₄ generations. Compared to their uncultured parent, Golden Promise, most of these lines were shorter, lower yielding, and had smaller seed, and the variability among individual plants was higher. The frequency and severity of the observed SCV was unexpectedly high, and the transformation procedure appeared to induce greater SCV than tissue culture in the absence of transformation. Attempts to understand the sources of SCV, and to modify transformation procedures to reduce the generation of SCV, should be made. Received: 26 June 1997 / Accepted: 31 October 1997     

Alfin1 transcription factor overexpression enhances plant root growth under normal and saline conditions and improves salt tolerance in alfalfa

Plant root development is an essential determinant of plant growth and crop yield that could be enhanced by induced changes in the expression of root-specific regulatory factors. We reported previously that Alfin1 binds DNA in a sequence-specific manner and that Alfin1 overexpression in transgenic alfalfa (Medicago sativa L.) enhances expression of the salt-inducible MsPRP2 gene in roots, suggesting that Alfin1 functions to regulate gene expression in roots. Here we show that Alfin1 is an essential gene for root growth and that its overexpression in transgenic plants confers a many-fold increase in root growth under normal and saline conditions. Alfin1-binding sites occur in promoters of genes expressed in roots of a wide variety of plant species and we propose that it is a general root growth regulator. Even though Alfin1 overexpression was under the control of the CaMV 35S promoter, plant shoot growth was not adversely affected. We show further that introduction of the Alfin1 transgene in plants confers a dominant characteristic that significantly increases plant growth and salt tolerance.     

A pollen-dispersal experiment with transgenic oilseed rape. Estimation of the average pollen dispersal of an individual plant within a field

 In order to help establish a basis for the assessment of gene flow associated with the large-scale release of transgenic oilseed rape, we previously designed a method which makes it possible to retrieve the average pollen dispersal of a single plant from that of a large source plot. The ‘individual’ pollen distribution thus obtained is less dependent on the experimental design than pollen distributions usually published and could therefore be used to model the possible escape of a transgene from commercial transgenic crops. In this study we report on a field experiment set up to study the pollen dispersal from an herbicide-resistant transgenic variety of oilseed rape and to test the applicability of the method on the experimental data. Two techniques were used to determine the individual pollen dispersal, and their outcomes are compared. The results suggest that approximately half of the pollen produced by an individual plant fell within 3 m and that the probability of fertilisation afterwards decreased slowly along a negative exponential of the distance. Comparison with the global pollen distribution from the source plot indicates that pollen-dispersal distributions based on dispersal from whole plots instead of individual plants would have underestimated the proportion of pollen that was dispersed over average or long distances. Received: 20 September 1997 / Accepted: 28 October 1997     

Localization of introduced genes on the chromosomes of transgenic barley, wheat and triticale by fluorescence in situ hybridization

 Using fluorescence in situ hybridization (FISH) we localized introduced genes on metaphase chromosomes of barley, wheat, and triticale transformed by microprojectile bombardment of microspores and scutellar tissue with the pDB1 plasmid containing the uidA and bar genes. Thirteen integration sites were detected in the nine lines analysed. Southern analysis showed that three or more copies of the plasmid were present in the lines. In a triticale line containing four copies three different integration sites were identified, indicating that the method described is sensitive enough for the detection of single-copy integrations. There was a slight tendency towards the localization of transgenes in distal chromosome regions. Using the GAA-satellite sequence for chromosome banding, the chromosomes containing the inserted genes were identified in most cases. Two barley lines derived from the same transformant showed a totally different integration pattern. Southern analysis confirmed that the inserted genes were segregating independently, resulting in different integration patterns among the progeny lines. The application of the FISH technique for the analysis of transgenic plants is discussed. Received: 28 October 1996/Accepted: 15 November 1996     

Quantification of ammonia exchange between agricultural cropland and the atmosphere: Measurements over two complete growth cycles of oilseed rape,wheat, barley and pea

The exchange of ammonia between the atmosphere and the canopy of barley, wheat, oilseed rape and pea crops was studied over two growing seasons by use of a modified aerodynamic gradient technique in which passive horizontal flux samplers were applied with a wind profile in gradient configuration. The crop foliage was a net source of NH₃ to the atmosphere, with NH₃ emissions on a seasonal basis between 1 and 5 kg NH₃–N ha^–1. The amount of NH₃ lost constituted between 1 and 4% of the applied nitrogen and between 1 and 4% of the actual amount of nitrogen present in the mature shoots. The volatile NH₃ losses depended on seasonal variations in climatic conditions affecting the growth and nitrogen economy of the crops and increased under conditions with excessive N absorption by roots and a high N concentration in the foliage. The accumulated NH₃ loss was positively correlated with the above-ground crop N content at anthesis, but not with that at final maturity. There were no indications that NH₃ emissions were larger under conditions unfavourable for nitrogen remobilization from vegetative plant parts (low N harvest index). Nevertheless, a distinct peak in NH₃ emission occurred during senescence. It is concluded that crops in many areas will represent a significant input of ammonia to the atmosphere and that NH₃ losses may become large enough to significantly affect crop N budgets.     

Cottonwood hybridization affects tannin and nitrogen content of leaf litter and alters decomposition

Cottonwoods are dominant riparian trees of the western United States and are known for their propensity to hybridize. We compared the decomposition of leaf litter from two species (Populus angustifolia and P. fremontii) and their hybrids. Three patterns were found. First, in one terrestrial and two aquatic experiments, decomposition varied twofold among tree types. Second, backcross hybrid leaves decomposed more slowly than those of either parent. Third, the variation in decomposition between F₁ and backcross hybrids was as great as the variation between species. These results show significant differences in decomposition in a low-diversity system, where >80% of the leaf litter comes from just two species and their hybrids. Mechanistically, high concentrations of condensed tannins in leaves appear to inhibit decomposition (r ²=0.63). The initial condensed tannin concentration was high in narrowleaf leaves, low or undetectable in Fremont leaves, and intermediate in F₁ hybrid leaves (additive inheritance). Backcross hybrids were high in condensed tannins and were not different from narrowleaf (dominant inheritance). Neither nitrogen (N) concentration nor the ratio of ash-free dry weight to N (a surrogate for carbon:nitrogen ratio) were significantly correlated with decomposition. The N content of leaf material at the end of each year’s experiment was inversely correlated with rates of litter mass loss and varied 1.6- to 2.1-fold among tree classes. This result suggests that hybrids and their parental species are used differently by the microbial community. Received: 7 April 1999 / Accepted: 2 November 1999     

Genetic improvement of anther culture response in maize: relationships with molecular, Mendelian and agronomic traits

 Two cycles of androgenetic in vitro doubled haploid (DH) plant production and intermating were implemented in an experimental synthetic population of maize. In vitro traits, including androgenetic embryo production, the regeneration potential and the frequency of spontaneous chromosome doubling, were studied. The success of the regenerated plants to self pollinate was also observed. Impressive genetic progress is reported for all the steps of the androgenetic process including seed set. Differential genetic progress is recorded according to the trait measured. Using a set of Mendelian and molecular markers that mapped to the different maize chromosomes, we were able to characterize the variation in the genetic variability in the population. Progress in the in vitro response was not found to be associated with any noticeable decline in global genetic variability. In addition, the QTL chromosomic regions tested, which were involved in androgenetic response, were not found to be subjected to a strong variation during the breeding experiment. Some phenotypical and morphological traits were also evaluated, and these showed that there was no depreciation effect in the agronomic value of the population. DH plant production and intermating the regenerated plants may be considered for the introduction and use of androgenesis in material which responds poorly. Received: 3 October 1997 / Accepted: 25 November 1997     

Assessment of genetic relationships between Setaria italica and its wild relative S. viridis using AFLP markers

AFLP markers were used to assess genetic diversity and patterns of geographic variation among 39 accessions of foxtail millet (Setaria italica) and 22 accessions of green foxtail millet (S. viridis), its putative wild progenitor. A high level of polymorphism was revealed. Dendrograms based on Nei and Li distances from a neighbour joining procedure were constructed using 160 polymorphic bands. Bootstrap values revealed that no specific geographic structure can be extracted from these data. The high level of diversity among Chinese accessions was consistent with the hypothesis of a centre of domestication in China. The results also showed that accessions from Eastern Europe and Africa form two distinct clusters. The narrow genetic basis of these two gene pools may be the result of local-adaptation. Received: 1 June 1999 / Accepted: 16 September 1999     

Characterisation of resistance to turnip mosaic virus in oilseed rape (Brassica napus) and genetic mapping of TuRB01

Turnip mosaic virus (TuMV) is the major virus infecting Brassica crops. A dominant gene, TuRB01, that confers extreme resistance to some isolates of TuMV on Brassica napus (oilseed rape), has been mapped genetically. The mapping employed a set of doubled-haploid lines extracted from a population used previously to develop a reference RFLP map of the B. napus genome. The positioning of TuRB01 on linkage group N6 of the B. napus A–genome indicated that the gene probably originated from Brassica rapa. Resistance phenotypes were confirmed by indirect plate-trapped antigen ELISA using a monoclonal antibody raised against TuMV. The specificity of TuRB01 was determined using a wide range of TuMV isolates, including representatives of the European and American/Taiwanese pathotyping systems. Some isolates of TuMV that did not normally infect B. napus plants possessing TuRB01 produced mutant viruses able to overcome the action of the resistance gene. TuRB01 is the first gene for host resistance to TuMV to be mapped in a Brassica crop. A second locus, TuRB02, that appeared to control the degree of susceptibility to the TuMV isolate CHN 1 in a quantitative manner, was identified on the C-genome linkage group N14. The mapping of other complementary genes and the selective combining of such genes, using marker-assisted breeding, will make durable resistance to TuMV a realisable breeding objective. Received: 14 December 1998 / Accepted: 10 April 1999     

Decrease of phosphoribulokinase activity by antisense RNA in transgenic tobacco: definition of the light environment under which phosphoribulokinase is not in large excess

 To test the hypothesis that the contribution of phosphoribulokinase (PRK) to the control of photosynthesis changes depending on the light environment of the plant, the response of transgenic tobacco (Nicotiana tabacum L.) transformed with antisense PRK constructs to irradiance was determined. In plants grown under low irradiance (330 μmol m⁻² s⁻¹) steady-state photosynthesis was limited in plants with decreased PRK activity upon exposure to higher irradiance, with a control coefficient of PRK for CO₂ assimilation of 0.25 at and above 800 μmol m⁻² s⁻¹. The flux control coefficient of PRK for steady-state CO₂ assimilation was zero, however, at all irradiances in plant material grown at 800 μmol m⁻² s⁻¹ and in plants grown in a glasshouse during mid-summer (alternating shade and sun 300–1600 μmol m⁻² s⁻¹). To explain these differences between plants grown under low and high irradiances, Calvin cycle enzyme activities and metabolite content were determined. Activities of PRK and other non-equilibrium Calvin cycle enzymes fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase and ribulose-1,5-bisphosphate carboxylase-oxygenase were twofold higher in plants grown at 800 μmol m⁻² s⁻¹ or in the glasshouse than in plants grown at 330 μmol m⁻² s⁻¹. Activities of equilibrium enzymes transketolase, aldolase, ribulose-5-phosphate epimerase and isomerase were very similar under all growth irradiances. The flux control coefficient of 0.25 in plants grown at 330 μmol m⁻² s⁻¹ can be explained because low ribulose-5-phosphate content in combination with low PRK activity limits the synthesis of ribulose-1,5-bisphosphate. This limitation is overcome in high-light-grown plants because of the large relative increase in activities of sedoheptulose-1,7-bisphosphatase and fructose-1,6-bisphosphatase under these conditions, which facilitates the synthesis of larger amounts of ribulose-5-phosphate. This potential limitation will have maintained evolutionary selection pressure for high concentrations of PRK within the chloroplast. Received: 15 November 1999 / Accepted: 27 January 2000     

Genetic diversity and gene flow between wild, cultivated and weedy forms of Beta vulgaris L. (Chenopodiaceae), assessed by RFLP and microsatellite markers

 Beets belonging to the species Beta vulgaris L. can be found in crop, wild and weedy forms, all of which are interfertile. We studied the intra-specific genetic relationships of about 300 individuals from 54 populations of various French geographic origins using nuclear molecular markers (five single-copy RFLP loci and one microsatellite locus). The patterns of diversity were congruent for both types of markers. Genetic diversity in wild beets appeared to be high, both in term of allele number and observed heterozygosity, whereas the narrowness of the cultivated-beet gene pool was confirmed. Genetic distances between all forms showed that weed beets in northern France are intermediates between sugar beet and inland wild beets in south-western France. This analysis allowed us to infer the paternal origin of weed beets and furthermore, is in agreement with a previous study which focused on their maternal origin: weed beet infesting sugar-beet fields originated from accidental and recurrent hybridization between cultivated lines and ruderal inland wild beets during the production of commercial seeds in south-western France. Inland wild beets are genetically close to Mediterranean coastal wild beets, but differ from other coastal forms (from Biscay, Brittany and northern France). The study of gene flow in the beet complex contributes to the risk assessment of transgenic beets. Received: 8 June 1998 / Accepted: 8 October 1998