Rikke Bagger Jørgensen

Costs of transgenic herbicide resistance introgressed from Brassica napus into weedy B. rapa

Wild relatives of genetically engineered crops can acquire transgenic traits such as herbicide resistance via spontaneous crop–wild hybridization. In agricultural weeds, resistance to herbicides is often a beneficial trait, but little is known about possible costs that could affect the persistence of this trait when herbicides are not used. We tested for costs associated with transgenic resistance to glufosinate when introgressed into weedy Brassica rapa. Crosses were made between transgenic B. napus and wild B. rapa from Denmark. F1 progeny were backcrossed to B. rapa and BC1 plants were selected for chromosome numbers similar to B. rapa. Further backcrossing resulted in a BC2 generation that was hemizygous for herbicide resistance. We quantified the reproductive success of 457 BC3 progeny representing six full‐sib families raised in growth rooms (plants were pollinated by captive bumblebees). Pollen fertility and seed production of BC3 plants were as great as those of B. rapa raised in the same growth rooms. Segregation for herbicide resistance in BC3 plants was 1:1 overall, but the frequency of resistant progeny was lower than expected in one family and higher than expected in another. There were no significant differences between transgenic and nontransgenic plants in survival or the number of seeds per plant, indicating that costs associated with the transgene are probably negligible. Results from this growth‐chamber study suggest that transgenic resistance to glufosinate is capable of introgressing into populations of B. rapa and persisting, even in the absence of selection due to herbicide application.

Fitness of backcross and F2 hybrids between weedy Brassica rapa and oilseed rape (B. napus)

With the cultivation of genetically modified crops, transgenes may spread by introgression from crops into weedy and wild populations of related species. The likelihood of this depends in part on the fitness of first and later generation hybrids. We here present results on the fitness of F2 and backcross hybrids between oilseed rape (Brassica napus) and weedy B. rapa. Two populations of B. rapa, two varieties of B. napus, and their F1 hybrids were used for controlled crosses, and seed development, survival in the field, pollen viability, pod- and seed-set were estimated for the offspring. Offspring from F2 and backcrosses had a reduced fitness relative to their parents for most of the fitness components and for a combined estimate of fitness, with F2 offspring suffering the lowest fitness. Despite their lower fitness on average, some of the hybrids were as fit as the parents. Significant fitness differences were detected between backcross and F2 offspring from different B. rapa populations, B. napus varieties, and parental plants. Our results suggest that introgression of transgenes from oilseed rape to B. rapa will be slowed down, but not hindered, by the low fitness of second generation hybrids.

Frequency-dependent fitness of hybrids between oilseed rape (Brassica napus) and weedy B. rapa (Brassicaceae)

Fitness of interspecific hybrids is sometimes high relative to their parents, despite the conventional belief that they are mostly unfit. F(1) hybrids between oilseed rape (Brassica napus) and weedy B. rapa can be significantly more fit than their weedy parents under some conditions; however, under other conditions they are less fit. To understand the reasons, we measured the seed production of B. napus, B. rapa, and different generations of hybrid plants at three different densities and in mixtures of different frequencies (including pure stands). Brassica napus, B. rapa, and backcross plants (F(1) ♀ × B. rapa) produced many more seeds per plant in pure plots than in mixtures and more seeds in plots when each was present at high frequency. The opposite was true for F(1) plants that produced many more seeds than B. rapa in mixtures, but fewer in pure stands. Both vegetative and reproductive interactions may be responsible for these effects. Our results show that the fitness of both parents and hybrids is strongly frequency-dependent and that the likelihood of introgression of genes between the species thus may depend on the numbers and densities of parents and their various hybrid offspring in the population.

Male fitness of oilseed rape ( Brassica napus ), weedy B. rapa and their F 1 hybrids when pollinating B. rapa seeds

The likelihood that two species hybridise and backcross may depend strongly on environmental conditions, and possibly on competitive interactions between parents and hybrids. We studied the paternity of seeds produced by weedy Brassica rapa growing in mixtures with oilseed rape (B. napus) and their F1 hybrids at different frequencies and densities. Paternity was determined by the presence of a transgene, morphology, and AFLP markers. In addition, observations of flower and pollen production, and published data on pollen fertilisation success, zygote survival, and seed germination, allowed us to estimate an expected paternity. The frequency and density of B. napus, B. rapa, and F1 plants had a strong influence on flower, pollen, and seed production, and on the paternity of B. rapa seeds. Hybridisation and backcrossing mostly occurred at low densities and at high frequencies of B. napus and F1, respectively. F1 and backcross offspring were produced mainly by a few B. rapa mother plants. The observed hybridisation and backcrossing frequencies were much lower than expected from our compilation of fitness components. Our results show that the male fitness of B. rapa, B. napus, and F1 hybrids is strongly influenced by their local frequencies, and that male fitness of F1hybrids, when pollinating B. rapa seeds, is low even when their female fitness (seed set) is high.

Preferential exclusion of hybrids in mixed pollinations between oilseed rape (Brassica napus) and weedy B. campestris (Brassicaceae).

In most experimental hybridizations between oilseed rape (Brassica napus) and weedy B. campestris, either intra- or interspecific pollen has been applied to individual flowers. Under field conditions, however, stigmas will often receive a mixture of the two types of pollen, thereby allowing for competition between male gametophytes and/or seeds within pods. To test whether competition influences the success of hybridization, pollen from the two species was mixed in different proportions and applied to stigmas of both species. The resulting seeds were scored for paternity by isozyme and randomly amplified polymorphic DNA analysis. Using data on the proportion of fully developed seeds and the proportion of these seeds that were hybrids, a statistical model was constructed to estimate the fitness of conspecific and heterospecific pollen and the survival of conspecific and heterospecific zygotes to seeds. B. campestris pollen in B. napus styles had a significantly lower fitness than the conspecific pollen, whereas no difference between pollen types was found in B. campestris styles. Hybrid zygotes survived to significantly lower proportions than conspecific zygotes in both species, with the lowest survival of hybrid zygotes in B. napus pods. This is in contrast to the higher survival of hybrid seeds in B. napus than in B. campestris pods when pollinations are made with pure pollen. Altogether, the likelihood of a foreign pollen grain producing a seed was much lower on B. napus than on B. campestris. In addition, pods on B. napus developed to a lower extent the more heterospecific pollen was in the mix, whereas this had no effect on B. campestris.

Adventitious presence of other varieties in oilseed rape (Brassica napus) from seed banks and certified seed

To obtain information on possible sources of contamination of the seed harvest of oilseed rape (Brassica napus L., spp. napus) by other varieties (adventitious presence), we investigated the purity of certified seed lots; the abundance and origin of volunteers; and longevity and origin of seeds in the soil seed-bank. This information was acquired through DNA analysis of volunteers collected in the field and seedlings derived from the soil seed-bank. DNA profiles of the volunteers and seedlings were obtained using Inter Simple Sequence Repeat (ISSR) markers, and the profiles were compared with ISSR profiles from an assortment of 14 of the most commonly cultivated oilseed rape varieties from 1985 to 2004. This comparison was performed using the assignment program, AFLPOP. The age of the seed bank germinating to become volunteers was assumed from information on previously cultivated oilseed rape varieties at the site and the year of cultivation. The results showed that plants or seeds belonged to varieties cultivated at the site 4‐17 years earlier, suggesting a long persistence of volunteer populations. High volunteer frequencies (6%, 29% and 32%) were found in the three oilseed rape fields analysed and, fromsoil cores, their soil seed-bank was estimated to be 50‐100 seeds m 22 . The ISSR analysis of the 14 reference varieties showed that three of the certified seed lots contained other varieties above the allowed threshold. Our findings are discussed in the context of the coexistence between GM (genetically modified) and non-GM oilseed rape, and future levels of seed purity.

Morphological and molecular diversity of Nordic oat through one hundred years of breeding

SummaryGenetic diversity in microsatellites and development of agronomical characters in Nordic oat cultivars (Avena sativa) from the 20th century, ranging from landraces to new cultivars, were studied. A clear development in agronomical characters has taken place in this period: Straw length has been reduced, harvest index has increased and heading date has declined. The persistent oat breeding effort in the northern part of the region was indicated by the data, since cultivars from this region showed higher harvest index. Also adaptation to shorter summers was apparent in cultivars from the most northern part of the area. When comparing cultivars released after 1940 to the landraces, the loss of diversity revealed for the agronomical characters was also indicated by the molecular data. This indicates that a more general loss of diversity has taken place in the period, possibly due to random factors during the breeding process (bottleneck effect). The reduction in diversity revealed by recent cultivars at an agronomical as well as a molecular level emphasises the importance of implementing a conservation strategy for older material in order to secure genetic diversity for future oat breeding efforts.

Haploid barley from the intergeneric cross Hordeum vulgare x Psathyrostachys fragilis

SummaryThe intergeneric hybrid Hordeum vulgare x Psathyrostachys fragilis was fairly easily obtained. During each growing season the intermediate, perennial hybrid yielded haploid tillers of H. vulgare. Late in one season few, hybrid tillers headed. The morphology, cytology and enzymatic patterns of hybrid and haploid tillers were investigated.

Transfer of engineered genes from crop to wild plants

The escape of engineered genes — genes inserted using recombinant DNA techniques — from cultivated plants to wild or weedy relatives has raised concern about possible risks to the environment or to health. The media have added considerably to public concern by suggesting that such gene escape is a new and rather unexpected phenomenon. However, transfer of engineered genes between plants is not at all surprising, because it is mediated by exactly the same mechanisms as those responsible for transferring endogenous plant genes: it takes place by sexual crosses, with pollen as the carrier. Such sexual reproduction has been the basis for breeding almost all crops.

The variability of processes involved in transgene dispersal-case studies from Brassica and related genera

Background, aim, and scopeWe strive to predict consequences of genetically modified plants (GMPs) being cultivated openly in the environment, as human and animal health, biodiversity, agricultural practise and farmers’ economy could be affected. Therefore, it is unfortunate that the risk assessment of GMPs is burdened by uncertainty. One of the reasons for the uncertainty is that the GMPs are interacting with the ecosystems at the release site thereby creating variability. This variability, e.g. in gene flow, makes consequence analysis difficult. The review illustrates the great uncertainty of results from gene-flow analysis.Main featuresMany independent experiments were performed on the individual processes in gene flow. The results comprise information both from laboratory, growth chambers and field trials, and they were generated using molecular or phenotypic markers and analysis of fitness parameters. Monitoring of the extent of spontaneous introgression in natural populations was also performed. Modelling was used as an additional tool to identify key parameters in gene flow.ResultsThe GM plant may affect the environment directly or indirectly by dispersal of the transgene. Magnitude of the transgene dispersal will depend on the GM crop, the agricultural practise and the environment of the release site. From case-to-case these three factors provide a variability that is reflected in widely different likelihoods of transgene dispersal and fitness of introgressed plants. In the present review, this is illustrated through a bunch of examples mostly from our own research on oilseed rape, Brassica napus. In the Brassica cases, the variability affected all five main steps in the process of gene dispersal. The modelling performed suggests that in Brassica, differences in fitness among plant genome classes could be a dominant factor in the establishment and survival of introgressed populations.DiscussionUp to now, experimental analyses have mainly focused on studying the many individual processes of gene flow. This can be criticised, as these experiments are normally carried out in widely different environments and with different genotypes, and thus providing bits and pieces difficult to assemble. Only few gene-flow studies have been performed in natural populations and over several plant generations, though this could give a more coherent and holistic view.ConclusionThe variability inherent in the processes of gene flow in Brassica is apparent and remedies are wished for. One possibility is to expose the study species to additional experiments and monitoring, but this is costly and will likely not cover all possible scenarios. Another remedy is modelling gene flow. Modelling is a valuable tool in identifying key factors in the gene-flow process for which more knowledge is needed, and identifying parameters and processes which are relatively insensitive to change and therefore require less attention in future collections of data. But the interdependence between models and experimental data is extensive, as models depend on experimental data for their development or testing.RecommendationsMore and more transgenic varieties are being grown worldwide harbouring genes that might potentially affect the environment (e.g. drought tolerance, salt tolerance, disease tolerance, pharmaceutical genes). This calls for a thorough risk assessment. However, in Brassica, the limited and uncertain knowledge on gene flow is an obstacle to this. Modelling of gene flow should be optimised, and modelling outputs verified in targeted field studies and at the landscape level. Last but not least, it is important to remember that transgene flow in itself is not necessarily a thread, but it is the consequences of gene flow that may jeopardise the ecosystems and the agricultural production. This emphasises the importance of consequence analysis of genetically modified plants.