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Stable Bacillus thuringiensis transgene introgression from Brassica napus to wild mustard B. juncea
Abstract
Transgenic canola (Brassica napus) with a Bacillus thuringiensis cry1Ac gene and a green fluorescent protein (GFP) marker gene was used in hybridization experiments with wild B. juncea. Hybrid F1 and successive five backcross generations were obtained.
The pod-set frequency on backcrossed B. juncea plants was over 66%, which suggested relatively high crossing compatibility between the hybrids and wild species. The seed setting in BC1 was the least of all generations tested, and then increased at the BC2 generation for which the thousand-seed weight was the highest of all generations. Seed size in backcrossed generations eventually approached that of the wild parent. The plants in all backcrossed generations were consistent with the expected 1:1 segregation ratio of the transgenes. The Bt Cry1Ac protein concentrations at bolting and flowering stages was higher compared to the 4-5-leaf and pod-formation stages. Nonetheless, the Bt toxin in the fifth backcrossing generation (BC5) was sufficient to kill both polyphagous (Helicoverpa armigera) and oligophagous (Plutella xylostella) Lepidoptera.
As a consequence, the subsequent generations harboring the transgene from F1 to BC5 could have selection advantage against insect pests. The result is useful in understanding gene flow from transgenic crops and the followed transgene introgression into wild.
... This relatively high number of compatible wild relatives might facilitate the introgression of transgenes in their progenies, and indeed there is evidence of this occurring. For example, the Bt Cry1Ac gene has been shown to be introgressed into wild brown mustard from Bt OSR with the expected Mendelian ratio (Cao et al., 2014). In addition, the Bt toxin in backcrossed generations was sufficient to kill a Bt-susceptible insect species, Plutella xylostella (diamondback moth) in a greenhouse assay (Cao et al., 2014). ...
... For example, the Bt Cry1Ac gene has been shown to be introgressed into wild brown mustard from Bt OSR with the expected Mendelian ratio (Cao et al., 2014). In addition, the Bt toxin in backcrossed generations was sufficient to kill a Bt-susceptible insect species, Plutella xylostella (diamondback moth) in a greenhouse assay (Cao et al., 2014). In another example, Warwick et al. (2008) found stable introgression of a glyphosate-resistant transgene into a B. rapalike plant from 2001 to 2005 at two sites in Quebec, Canada. ...
... In addition, the Brassica group is prone to producing unreduced gametes, so that many chromosome formulae are possible with viable introgressed progeny (Chèvre et al., 1997). Advanced backcrosses of B. napus × B. juncea were generated and research showed that plant production increased from BC 1 to BC 6 populations (Cao et al., 2014). Gene flow from glyphosate-resistant B. napus to its weedy relative B. rapa induced herbicide-resistant hybrids and backcross volunteers in fields (Warwick et al., 2008). ...
Background and aims:
Since pollen flow or seed dispersal can contribute to transgene persistence in the environment, the sympatric presence of transgenic crops with their wild relatives is an ecological concern. In this study, we tested the hypothesis that proximate growth of a herbivore-resistant Bt crop and wild relatives coupled with the presence of herbivores can increase relative frequency of crop-to-wild transgene flow persistence outside of cultivation.
Methods:
We conducted a field experiment using insect enclosures with and without herbivores with cultivated Bt-transgenic Brassica napus (Bt OSR) and wild brown mustard (Brassica juncea) in pure and mixed stands. Low-density diamondback moth (Plutella xylostella) caterpillar infestation treatments were applied and transgene flow and reproductive organs were measured.
Key results:
Bt-transgenic B. napus produced more ovules and pollen than wild mustard, but the pollen to ovule (P/O) ratio in the two species was not significantly different. Low-level herbivory had no effects on fitness parameters of Bt OSR or wild brown mustard or on the transgene flow frequency. All progeny from wild brown mustard containing the Bt transgene came from mixed stands, with a gene flow frequency of 0.66 %. In mixed stands, wild brown mustard produced less pollen and more ovules than in pure stands of brown mustard. This indicates a decreased P/O ratio in a mixed population scenario.
Conclusions:
Since a lower P/O ratio indicates a shift in sex allocation towards relatively greater female investment and a higher pollen transfer efficiency, the presence of transgenic plants in wild populations may further increase the potential transgene flow by altering reproductive allocation of wild species.
... At present, herbicide-tolerant transgenic oilseed rape has been commercialized in the United States, Canada, Australia and other countries [12]. Although insect-resistant Bt oilseed rape has not been commercialized, it has served as a model biotech crop for studying the effects of transgenic plants (e.g., Cao et al. 2014; [13,14]. Owing to current methods of transgene insertion, transgenic plants may exhibit random genomic side effects such as variable gene expression, mutations to endogenous loci at the points of insertion, homologous gene expression inhibition effects (i.e., silencing), activation effects and other unanticipated changes in crop phenotypes. ...
... At present, herbicide-tolerant transgenic oilseed rape has been commercialized in the United States, Canada, Australia and other countries [12]. Although insect-resistant Bt oilseed rape has not been commercialized, it has served as a model biotech crop for studying the effects of transgenic plants (e.g., Cao et al. 2014; [13,14]. Owing to current methods of transgene insertion, transgenic plants may exhibit random genomic side effects such as variable gene expression, mutations to endogenous loci at the points of insertion, homologous gene expression inhibition effects (i.e., silencing), activation effects and other unanticipated changes in crop phenotypes. ...
Oilseed rape (Brassica napus L.) is an important cash crop, but transgenic oilseed rape has not been grown on a commercial scale in China. It is necessary to analyze the characteristics of transgenic oilseed rape before commercial cultivation. In our study, differential expression of total protein from the leaves in two transgenic lines of oilseed rape expressing foreign Bt Cry1Ac insecticidal toxin and their non-transgenic parent plant was analyzed using a proteomic approach. Only shared changes in both of the two transgenic lines were calculated. Fourteen differential protein spots were analyzed and identified, namely, eleven upregulated expressed protein spots and three downregulated protein spots. These proteins are involved in photosynthesis, transporter function, metabolism, protein synthesis, and cell growth and differentiation. The changes of these protein spots in transgenic oilseed rape may be attributable to the insertion of the foreign transgenes. However, the transgenic manipulation might not necessarily cause significant change in proteomes of the oilseed rape.
... In those studies, the bolting and flowering stages, from F1 hybrid to BC5, showed statistically lower Cry1Ac protein concentrations compared to their GM parental plants. However, in the pod formation stage, from F1 hybrid to BC5, the plants showed statistically similar level of Cry1Ac concentration compared to GM parental plant [37]. In rice, other researchers also found that the concentration of Bt protein in F1 hybrid plants was expressed at similar concentrations as in their parental plants, with even higher concentrations of Bt toxin detected in some plants of the crop-wild hybrids and F2 progeny [38]. ...
... Halfhill et al. [35] revealed that the cry1Ac transgene reduced target pest herbivores when back-crossed onto wild oilseed rape populations. Another study involving transgenic B. napus also showed that all crosses, from F1 to BC5, had similar insecticidal efficacy as their parents against the insect pest species Helicoverpa armigera and Plutella xylostella [37]. ese findings are critical for risk assessment as O. nubilalis, or other lepidopteran species considered "pests" on maize, would functionally serve as "biocontrol agents" on weedy teosinte. ...
Background
In 2009, Spanish farmers reported a novel weed, now identified as a relative of maize’s ancestor, teosinte, in their maize fields. Introgression of the insect resistance transgene cry1Ab from genetically modified (GM) maize into populations of this weedy Spanish teosinte could endow it with additional defense mechanisms. The aims of this study were: (1) to test if hybridization between GM maize and weedy plants from Spain is possible; (2) to understand the relationship between transgene transcription activity, concentrations of the expected transgene product (Cry1Ab protein) and the bioactivity of the latter on target insect pests following transgene flow from GM maize into Spanish teosinte plants.
Results
We demonstrated that hybridization between GM maize and the weedy Spanish teosinte is possible, with no observable barrier to the formation of crop/weed hybrids when teosinte served as pollen donor. When GM maize plants were used as pollen donors, significant crossing incompatibility was observed: hybrid plants produced only few “normal” seeds. Nevertheless, viable F1 seeds from GM pollen crossed onto teosinte were indeed obtained. The cry1Ab transgene was stably expressed as mRNA in all crossings and backgrounds. Similarly, toxicity on neonate Ostrinia nubilalis , presumably due to Cry1Ab protein, was consistently expressed in teosinte hybrids, with mortality rates 95% or higher after only 4 days exposure, similar to rates on parental GM maize plants. Nevertheless, no strong correlations were observed between transgene transcription levels and Cry1Ab concentrations, nor between Cry1Ab concentrations and insect mortality rates across all of the different genetic backgrounds.
Conclusions
Our results establish fundamental parameters for environmental risk assessments in the European context: first, we show that crop/weed hybridization in fields where maize and teosinte exist sympatrically can lead to potentially catastrophic transfer of resistance traits into an already noxious weed; second, our results question the viability of using gene dosage to model and predict ecological performance in either the intended crop plant or the undesired teosinte weed. Significant questions remain that should be addressed in order to provide a scientific, sound approach to the management of this novel weed.
... In certain hosts, transgenes could be transferred into hybrids through outcrossing of GM crops with crop varieties or wild relatives, and in some cases, introgressed transgenic advanced generations could occur [21][22][23][24][25] . Gene flow from GM crops to their wild relatives is one main environmental regulatory issue 22,24,26 , with the concern of increasing risks, such as increased weediness 27 . ...
... juncea), an allotetraploid wild relative species of B. napus, is a widespread weed in agricultural fields in China and elsewhere. Hybrids between B. napus and wild B. juncea are successfully obtained by open pollination 21,30 . The aim of this study was, for the first time, to perform a proteomic study in a transgenic B. napus and B. napus/B. ...
Transgene insertions might have unintended side effects on the transgenic host, both crop and hybrids with wild relatives that harbor transgenes. We employed proteomic approaches to assess protein abundance changes in seeds from Bt-transgenic oilseed rape (Brassica napus) and its hybrids with wild mustard (B. juncea). A total of 24, 15 and 34 protein spots matching to 23, 13 and 31 unique genes were identified that changed at least 1.5 fold (p<0.05, Student's t-test) in abundance between transgenic (tBN) and non-transgenic (BN) oilseed rape, between hybrids of B. juncea (BJ)×tBN (BJtBN) and BJ×BN (BJBN) and between BJBN and BJ, respectively. Eight proteins had higher abundance in tBN than in BN. None of these proteins was toxic or nutritionally harmful to human health, which is not surprising since the seeds are not known to produce toxic proteins. Protein spots varying in abundance between BJtBN and BJBN seeds were the same or homologous to those in the respective parents. None of the differentially-accumulated proteins between BJtBN and BJBN were identical to those between tBN and BN. Results indicated that unintended effects resulted from transgene flow fell within the range of natural variability of hybridization and those found in the native host proteomes.
... Several studies into crop-wild hybrids have considered aboveground stages (e.g. Hooftman et al., 2007;Vacher et al., 2011;Yang et al., 2011;Cao et al., 2014), but none has included simultaneous measures of seed bank survival. ...
... Those studies show that Brassica hybrids have highly variable fitness (Devos et al., 2009;Jørgensen et al., 2009). Furthermore, several studies suggest that F 1 hybrids might have reduced fitness compared with later backcrosses (Burke and Arnold, 2001;Snow et al., 2010;Yang et al., 2011) and fitness could bounce back in later generations towards that of the wild parent (Liu et al., 2013;Cao et al., 2014). Therefore, our fitness estimates of Brassica hybrids are likely at the lower end of what might happen in reality. ...
Gene flow from crops to their wild relatives has the potential to alter population growth rates and demography of hybrid populations, especially when a new crop has been genetically modified (GM). This study introduces a comprehensive approach to assess this potential for altered population fitness, and uses a combination of demographic data in two habitat types and mathematical (matrix) models that include crop rotations and outcrossing between parental species.
Full life-cycle demographic rates, including seed bank survival, of non-GM Brassica rapa × B. napus F1 hybrids and their parent species were estimated from experiments in both agricultural and semi-natural habitats. Altered fitness potential was modelled using periodic matrices including crop rotations and outcrossing between parent species.
The demographic vital rates (i.e. for major stage transitions) of the hybrid population were intermediate between or lower than both parental species. The population growth rate (λ) of hybrids indicated decreases in both habitat types, and in a semi-natural habitat hybrids became extinct at two sites. Elasticity analyses indicated that seed bank survival was the greatest contributor to λ. In agricultural habitats, hybrid populations were projected to decline, but with persistence times up to 20 years. The seed bank survival rate was the main driver determining persistence. It was found that λ of the hybrids was largely determined by parental seed bank survival and subsequent replenishment of the hybrid population through outcrossing of B. rapa with B. napus.
Hybrid persistence was found to be highly dependent on the seed bank, suggesting that targeting hybrid seed survival could be an important management option in controlling hybrid persistence. For local risk mitigation, an increased focus on the wild parent is suggested. Management actions, such as control of B. rapa, could indirectly reduce hybrid populations by blocking hybrid replenishment.
© The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company.
... flow involved an engineered gene that has a fitness advantage, this gene could persist in nature and compete with other species, possibly developing weediness that may be difficult to manage (Cao et al., 2014;Jhala et al., 2021). Further, GE crops have the potential to affect target and nontarget organisms. ...
Societal Impact Statement
A diverse portfolio of genetically engineered food crops, as well as animal livestock and fish, are currently being developed and commercialized. To ensure their contributions to long‐term sustainability, a broad range of environmental, health, ethical, and societal parameters should be used in their evaluations. This paper proposes a set of parameters to evaluate the sustainability of genetically engineered food and agriculture products and discusses mechanisms to improve their governance and oversight. With such holistic evaluations, genetic engineering applications that are deemed beneficial to sustainable agriculture could be identified in an effort to foster sustainability.
Summary
To achieve international sustainable development goals, food and agricultural production need to rely on sustainable and resilient practices. Traditional breeding as well as the use of new agricultural technologies, including genetic engineering and gene editing, have the potential to help achieve sustainable agrifood production. Although numerous oversight mechanisms exist to guarantee the secure and sustainable advancement and utilization of genetically engineered agrifoods, the majority of these mechanisms heavily depend on a narrow set of parameters to assess risks and safety concerning human health and nontarget organisms. However, a more comprehensive range of parameters should be considered to promote environmental and social sustainability in a more holistic manner. This Opinion article argues that to achieve a more sustainable agrifood production that relies on genetic engineering, governance systems related to new agrifood biotechnologies should incorporate a broader array of environmental, health, ethical, and societal factors to ensure their sustainability in the long‐term. To facilitate this process, we propose a set of parameters to help evaluate the sustainability of agrifoods that rely on genetic engineering. We then discuss major challenges and opportunities for formalizing sustainability parameters in US governance policy and decision‐making systems. Overall, this work contributes to further developing a more comprehensive assessment framework that aims to minimize potential risks and maximize potential benefits of agrifood biotechnology while also fostering sustainability.
... The current studies have found that there was transgene silencing in the backcross progenies of transgenic Brassica napus and wild Brassica juncea which transgene was located on the C-chromosome, and the resistant-gene-expressing and resistant-gene-silencing progenies have been segregated from the self-pollinated progenies of the plants with resistance gene silencing. Although the progenies of these two different phenotypes showed similar fitness-associated traits and composite fitness without herbicide selection, plants that cannot normally express the transgene will be killed under the pressure of herbicide selection, while plants that can stably express the transgene will survive and reproduce stably, and have the possibility of becoming new species in nature, for the selection pressure is one of the driving forces of species evolution [29]. ...
The commercialization of transgenic herbicide-resistant (HR) crops may cause gene flow risk. If a transgene in progenies of transgenic crops and wild relatives is silencing, these progenies should be killed by the target herbicide, thus, the gene flow risk could be decreased. We obtained the progenies of backcross generations between wild Brassca juncea (AABB, 2n = 36) and glufosinate-resistant transgenic Brassica napus (AACC, 2n = 38, PAT gene located on the C-chromosome). They carried the HR gene but did not express it normally, i.e., gene silencing occurred. Meanwhile, six to nine methylation sites were found on the promoter of PAT in transgene-silencing progenies, while no methylation sites occurred on that in transgene-expressing progenies. In addition, transgene expressing and silencing backcross progenies showed similar fitness with wild Brassica juncea. In conclusion, we elaborate on the occurrence of transgene-silencing event in backcross progenies between transgenic crop utilizing alien chromosomes and their wild relatives, and the DNA methylation of the transgene promoter was an important factor leading to gene silencing. The insertion site of the transgene could be considered a strategy to reduce the ecological risk of transgenic crops, and applied to cultivate lower gene flow HR crops in the future.
... It is known to have the highest potential for gene transfer from B. napus after B. rapa [4]. It has previously been reported that conventional and transgenic B. napus hybridize with B. juncea spontaneously or by hand pollination [5][6][7][8]. Recently, Tang Processes 2022, 10, 240 2 of 10 et al. [9] found the estimated frequencies of natural gene flow from the genetically modified (GM) B. napus to 10 different B. juncea cultivars in the field experiment varied from 0.08 to 0.93%. ...
The rapid advancement of genetically modified (GM) technology over the years has raised concerns about the safety of GM crops and foods for human health and the environment. Gene flow from GM crops may be a threat to the environment. Therefore, it is critical to develop reliable, rapid, and low-cost technologies for detecting and monitoring the presence of GM crops and crop products. Here, we used visible near-infrared (Vis-NIR) spectroscopy to distinguish between GM and non-GM Brassica napus, B. juncea, and F1 hybrids (B. juncea X GM B. napus). The Vis-NIR spectra were preprocessed with different preprocessing methods, namely normalization, standard normal variate, and Savitzky–Golay. Both raw and preprocessed spectra were used in combination with eight different chemometric methods for the effective discrimination of GM and non-GM plants. The standard normal variate and support vector machine combination was determined to be the most accurate model in the discrimination of GM, non-GM, and hybrid plants among the many combinations (99.4%). The use of deep learning in combination with Savitzky–Golay resulted in 99.1% classification accuracy. According to the findings, it is concluded that handheld Vis-NIR spectroscopy combined with chemometric analyses could be used to distinguish between GM and non-GM B. napus, B. juncea, and F1 hybrids.
... By keeping pest populations at extremely low levels with insecticides, B. thuringiensis plants can completely destroy their enemies, while they only need small amounts of food to survive in agroecosystems. The possibility that B. thuringiensis toxins move into the Arthropod's food chain has serious implications for the agroecosystem equilibrium (Cao et al. 2014). ...
Biopesticides or biological pesticides refer to the use of living biological organisms or their metabolites for pests, pathogens, weeds, nematodes, rodents, etc. Regarding the field of biopesticides, there is no precise and unified definition. In accordance with the United Nations Food and Agriculture Organization standards, biopesticides are generally natural compounds or genetic modification agents, including biochemical pesticides (pheromones, hormones, plant regulators, insect growth regulators) and microbial pesticides (fungi, bacteria, or genetically modified microorganisms), which do not include antibiotic agricultural preparations. According to their composition and sources, biopesticides can be divided into microbial biopesticides, microbial pesticides, botanical pesticides, and animal pesticides. According to the control object, they can be divided into pesticides, fungicides, herbicides, acaricides, rodenticides, and plant growth regulators. In terms of their use, biopesticides are generally used either directly, in the case of living biological organisms, or indirectly, when using the bioactive substances from organisms. The first category includes bacteria, fungi, nematodes, viruses, and antagonist microorganisms. The latter includes agricultural antibiotics, growth regulators, sex pheromones, feeding inhibitors, juvenile hormones, and herbal physiologically active substances. However, in the practical application of agricultural production, biopesticides generally refer mainly to the large-scale industrial production of microbial pesticides.
... In addition, both the 445 bp band that indicated the amplification of bar and the 738 bp band that indicated the amplification of the partial 35S promoter and BrAGL20 were detected in the PCR amplification products of all of the herbicide-resistant BC 1 progeny (S1 Fig). [14,1,19]. However, the acreage of transgenic B. napus increases each year, owing to its economic benefits, and the increase is expected to continue. ...
A number of studies have been conducted on hybridization between transgenic Brassica napus and B. rapa or backcross of F1 hybrid to their parents. However, trait changes must be analyzed to evaluate hybrid sustainability in nature. In the present study, B. rapa and transgenic (BrAGL20) B. napus were hybridized to verify the early flowering phenomenon of F1 hybrids, and F1 hybrid traits were analyzed to predict their impact on sustainability. Flowering of F1 hybrid has been induced slightly later than that of the transgenic B. napus, but flowering was available in the greenhouse without low temperature treatment to young plant, similar to the transgenic B. napus. It is because the BrAGL20 gene has been transferred from transgenic B. napus to F1 hybrid. The size of F1 hybrid seeds was intermediate between those of B. rapa and transgenic B. napus, and ~40% of F1 pollen exhibited abnormal size and morphology. The form of the F1 stomata was also intermediate between that of B. rapa and transgenic B. napus, and the number of stomata was close to the parental mean. Among various fatty acids, the content of erucic acid exhibited the greatest change, owing to the polymorphism of parental FATTY ACID ELONGASE 1 alleles. Furthermore, F2 hybrids could not be obtained. However, BC1 progeny were obtained by hand pollination of B. rapa with F1 hybrid pollen, with an outcrossing rate of 50%.
Integration of a transgene into chromosomes of the C-genomes of oilseed rape (AACC, 2n = 38) may affect their gene flow to wild relatives, particularly Brassica juncea (AABB, 2n = 36). However, no empiric evidence exists in favor of the C-genome as a safer candidate for transformation. In the presence of herbicide selections, the first- to fourth-generation progenies of a B. juncea × glyphosate-tolerant oilseed rape cross [EPSPS gene insertion in the A-genome (Roundup Ready, event RT73)] showed more fitness than a B. juncea × glufosinate-tolerant oilseed rape cross [PAT gene insertion in the C-genome (Liberty Link, event HCN28)]. Karyotyping and fluorescence in situ hybridization–bacterial artificial chromosome (BAC-FISH) analyses showed that crossed progenies from the cultivars with transgenes located on either A- or C- chromosome were mixoploids, and their genomes converged over four generations to 2n = 36 (AABB) and 2n = 37 (AABB + C), respectively. Chromosome pairing of pollen mother cells was more irregular in the progenies from cultivar whose transgene located on C- than on A-chromosome, and the latter lost their C-genome-specific markers faster. Thus, transgene insertion into the different genomes of B. napus affects introgression under herbicide selection. This suggests that gene flow from transgenic crops to wild relatives could be mitigated by breeding transgenic allopolyploid crops, where the transgene is inserted into an alien chromosome.
Transgene flow from engineered Brassica napus to wild weed relatives could potentially have an environmental effect. To evaluate the introgression of transgenic B. napus into wild Brassica juncea, the hybrid F1 and backcross progenies derived from B. juncea (genome constitution AABB) and transgenic B. napus (AACC) crosses were investigated. C‐genome‐specific simple sequence repeat (SSR) markers corresponding to linkage groups N11–N19 in B. napus were screened and used to estimate the marker frequency in hybrid F1 and backcross progenies. C‐genome‐specific markers could be stably detected in hybrid F1 and backcross BC1 plants, but were only rarely found in the BC2–BC5 generations. For example, a specific SSR marker for linkage group N12 segregated in BC2 generation but were completely lost in BC3–BC5, while a specific SSR marker of linkage group N15 segregated in BC1, BC2 and BC3 generations and was absent in more advanced backcrossed generations (BC4 and BC5). The results indicate that a certain gene regions in Brassica napus plants are transmitted at a relatively lower frequency to wild relatives, and more rapidly disappeared in subsequent backcross generations. We propose that a foreign gene or transgene that is integrated in the C‐chromosome of Brassica napus could reduce the risk of introgression in nature.
Transgene introgression from transgenic rapeseed (Brassica napus) to different varieties of B. juncea was assessed in this study. Crossability between a transgenic rapeseed line Z7B10 (pollen donor) and 80 cultivars of 16 B. juncea varieties (including two wild accessions) was estimated by artificial pollination in a greenhouse. As a result, interspecific crossability between the transgenic Z7B10 line and the 80 B. juncea cultivars varied considerably, with seeds per flower from 0.00–10.67. Seed germination rates of the interspecific F1 hybrids ranged from 49.0%–89.3%. The estimated frequencies of natural gene flow from the transgenic Z7B10 line to 10 B. juncea cultivars with different uses in the experiment field varied from 0.08% to 0.93%. The natural F1 hybrids were highly sterile, with seeds per silique ranging from 0.27 to 1.03. In addition, seeds per flower of hybrid descendants varied from 0.02 to 0.22 when F1 hybrids were self-pollinated, and those ranged from 0.03 to 0.30 when F1 hybrids were backcrossed with their corresponding B. juncea parents. Results of this study suggest a low level of transgene introgression from transgenic rapeseed to different B. juncea varieties, which provides a sound scientific basis for the safety management of coexisting transgenic B. napus and B. juncea varieties in China.
New functionalized CuO hollow nanospheres on acetylene black (CuO/AB) and on charcoal (CuO/C) have been found to be effective catalysts for C-S bond formation under microwave irradiation. CuO catalysts showed high catalytic activity with a wide variety of substituents which include electron-rich and electron-poor aryl iodides with thiophenols by the addition of two equivalents of K2CO3 as base in the absence of ligands.
Chloroplast molecular markers can provide useful information for high-resolution analysis of inter- and intra-specific variation in Brassicaceae and for differentiation between its species. Combining data generated from nuclear and chloroplast markers enables the study of seed and pollen movement, and assists in the assessment of gene-flow from genetically modified (GM) plants through hybridization studies. To develop chloroplast DNA markers for monitoring of transgene introgression in Brassica napus L., we searched for sequence variations in the chloroplast (cp) genome, and developed a simple cpDNA marker that is reliable, time-saving, and easily discriminates among 4 species (B. napus, B. rapa, Raphanus sativus, and Sinapis alba) based on PCR-product length polymorphism. This marker will be useful to identify maternal lineages and to estimate transgene movement of GM canola.
Insecticides derived from Bacillus thuringiensis have become important for pest management, but recently resistance has been reported from field populations of diamondback moth Plutella xylostella, and laboratory populations of a number of species of Lepidoptera, Coleoptera and Diptera have shown similar characteristics. In this context, the author examines laboratory selection for resistance, resistance risk assessment, variation among conspecific populations, mechanisms, cross-resistance, genetics, stability, fitness costs, and management. -P.J.Jarvis
Introgression between transgenic, insect-resistant crops and their wild relatives could lead to a progressive increase of the frequency of resistant plants in wild populations. However, few studies help predict the impact on the population dynamics. To simulate the performance of introgressed insect-resistant plants of wild Brassica juncea, independently from the interspecific hybridization cost, healthy plants were cultivated in pure and mixed stands with damaged plants through cutting leaves in field experiments over two field seasons. As expected, resistant (healthy) plants held a competitive advantage when in competition with susceptible (damaged) plants. Individual biomass and seed production of both types of plants decreased as the percentage of resistant plants increased, so that the relative advantage of resistant plants increased. The combined effects of defoliation and competition on the individual performance of B. juncea were additive. Replacement series experiments confirmed this trend but did not show different seed output in pure stand of susceptible versus resistant plots. The total vegetative and reproductive production of mixed populations was not significantly different of that of pure populations. These results suggest that if a transgene for insect-resistance were to colonize wild populations, high herbivory of susceptible plant and low resource availability would facilitate the spread of resistant individuals. However, at the population level, the shift from an insect-susceptible to a predominantly resistant population would not result in exacerbated habitat colonization.
Leaf bioassays and oviposition choice tests were used to investigate the effects of transgenic broccoli expressing Cry1Ac toxin of Bacillus thuringiensis (Berliner) on susceptible and resistant Plutella xylostella (L.) larvae. Survival of susceptible 2nd instars on Cry1Ac-expressing broccoli declined from 99.1 to 19.2% at 24 and 72 h, respectively, and larvae exhibited an average weight loss of 0.2 mg/10 larvae at 24 h. Larvae that evolved resistance to foliar sprays of B. thuringiensis subsp. kurstaki in the field, however, showed no debilitating effects from the Cry1Ac broccoli. Survival of resistant larvae at 24 and 72 h was 98.6 and 90.8%, respectively, and weight gain at 24 h was 1.7 mg/10 larvae, none of which was significantly different from survival or weight gain on control plants. In oviposition choice tests, both susceptible and resistant females were unable to discriminate between Cry1Ac and normal broccoli, laying ≈38-41 eggs per plant per 2 females. Comparing mortality of susceptible larvae on 2 lines of transgenic broccoli (J1R and K20) derived from independent transformation events, we found that the majority of the variance (43.2%) in toxin expression was caused by transformation. Depending upon the transformation, plant could be a significant source of variation but toxin expression within plant was always fairly uniform. Our data indicate that resistance to sprays of B. thuringiensis can confer resistance to plants when similar toxins are involved.
There is strong evidence indicating that gene flow from transgenic B. napus into weedy wild relatives is inevitable following commercial release. Research should now focus on the transmission, stability,
and impact of transgene expression after the initial hybridization event. The present study investigated the transfer of a
phosphinothricin-tolerance transgene by inter-specific hybridization between B. rapa and two transgenic B. napus lines. The expression of the transgene was monitored in the F1 hybrids and in subsequent backcross generations. The transgene was transmitted relatively easily into the F1 hybrids and retained activity. Large differences in the transmission frequency of the transgene were noted between offspring
of the two transgenic lines during backcrossing. The most plausible explanation of these results is that the line showing
least transmission during backcrossing contains a transgene integrated into a C-genome chromosome. Approximately 10% of offspring
retained the tolerant trait in the BC3 and BC4 generations. The implications of these findings for the stable introgression of transgenes carried on one of the chromosomes
of the C-genome from B. napus and into B. rapa are briefly discussed.
To investigate association between glutathione S-transferases (GSTs) and cervical cancer.
Published literature from PubMed, EMBASE, and other databases were retrieved. All studies evaluating the association between GSTM1/GSTT1 polymorphisms and cervical were included. Pooled odds ratio (OR) and 95% confidence interval (CI) were calculated using fixed- or random-effects model.
A total of 15 case-control studies were included in the meta-analysis of GSTM1 genotypes (1,825 cases and 2,104 controls). The overall result showed that the association between GSTM1 null genotype and risk for cervical cancer was statistically significant (OR=1.53, 95%CI=1.18-2.00). Great heterogeneity was found between studies. Subgroup analysises were performed based on smoking and ethnicity. Our results showed that smokers with null GSTM1 genotype had higher risk of cervical cancer (OR=1.56, 95%CI=1.01-2.41). For the ethnicity stratification, significant increased risk of null GSTM1 genotype was found in Chinese and Indian population, but no increased risk in other population.
This meta-analysis provides strong evidence that the GSTM1 null genotype is associated with the development of cervical cancer, and especially in Chinese and Indian population, and smoking shows a modification on the association between GSTM1 null genotype and cervical cancer.
Introgression between genetically modified (GM) crops and wild relatives is considered to potentially modify the genetic background of the wild species. The emergence of volunteer-like feral populations through backcross of hybrids to the crop is also a concern. The progeny of spontaneous hybrids between mutant herbicide-resistant oilseed rape (Brassica napus) and wild B. juncea was obtained. Parents, F(2) and BC(1) to B. napus were planted together in the field so as to study their performance. The chromosome number of BC(1) followed a Normal distribution. Mendelian ratio of the herbicide-resistance gene was found. The F(2) produced less seeds than B. napus, and BC(1) had intermediate production. Herbicide-resistant BC(1) were not different of their susceptible counterparts for plant weight, seed weight and seed number, but most of them exhibited B. napus morphology and larger flowers than the susceptible BC(1). They displayed an important genetic variability allowing further adaptation and propagation of the herbicide-resistance gene. Pollen flow to susceptible plants within the mixed stand was observed. As a consequence, the resistant BC(1) produced with B. napus pollen could frequently occur and easily establish as a false feral crop population within fields and along roadsides.
Four successive reciprocal backcrosses between F(1) (obtained from wild Brassica juncea as maternal plants and transgenic glyphosate- or glufosinate-tolerant oilseed rape, B. napus, as paternal plants) or subsequent herbicide-tolerant backcross progenies and wild B. juncea were achieved by hand pollination to assess potential transgene flow. The third and forth reciprocal backcrosses produced a number of seeds per silique similar to that of self-pollinated wild B. juncea, except in plants with glufosinate-tolerant backcross progeny used as maternal plants and wild B. juncea as paternal plants, which produced fewer seeds per silique than did self-pollinated wild B. juncea. Germination percentages of reciprocal backcross progenies were high and equivalent to those of wild B. juncea. The herbicide-tolerant first reciprocal backcross progenies produced fewer siliques per plant than did wild B. juncea, but the herbicide-tolerant second or third reciprocal backcross progenies did not differ from the wild B. juncea in siliques per plant. The herbicide-tolerant second and third reciprocal backcross progenies produced an amount of seeds per silique similar to that of wild B. juncea except for with the glufosinate-tolerant first and second backcross progeny used as maternal plants and wild B. juncea as paternal plants. In the presence of herbicide selection pressure, inheritance of the glyphosate-tolerant transgene was stable across the second and third backcross generation, whereas the glufosinate-tolerant transgene was maintained, despite a lack of stabilized introgression. The occurrence of fertile, transgenic weed-like plants after only three crosses (F(1), first backcross, second backcross) suggests a potential rapid spread of transgenes from oilseed rape into its wild relative wild B. juncea. Transgene flow from glyphosate-tolerant oilseed rape might be easier than that from glufosinate-tolerant oilseed rape to wild B. juncea. The original insertion site of the transgene could affect introgression.
Green fluorescent protein (GFP) is increasingly being used in plant biology from the cellular level to whole plant level. At the cellular level, GFP is being used as an in vivo reporter to assess frequency of transient and stable transformation. GFP has also proven to be an invaluable tool in monitoring trafficking and subcellular localization of protein. At the organ level and up, many exciting applications are rapidly emerging. The development of brighter GFP mutants with more robust folding properties has enabled better macroscopic visualization of GFP in whole leaves and plants. One interesting example has been the use of GFP to monitor virus movement in and among whole plants. GFP is also emerging as a powerful tool to monitor transgene movement and transgenic plants in the field. In a proof-of-concept study, tobacco was transformed with a modified version of the GFP gene controlled by a constitutive (35S) promoter. GFP expression in progeny plants ranged from 0% to 0.5%, and approximately 0.1% GFP was the minimal amount needed for unambiguous macroscopic detection. GFP is the first truly in vivo reporter system useful in whole plants, and we project its usefulness will increase even further as better forms of GFP genes become available.
There is considerable public and scientific debate for and against genetically modified (GM) crops. One of the first GM crops, Brassica napus (oilseed rape or canola) is now widely grown in North America, with proposed commercial release into Australia and Europe. Among concerns of opponents to these crops are claims that pollen movement will cause unacceptable levels of gene flow from GM to non-GM crops or to related weedy species, resulting in genetic pollution of the environment. Therefore, quantifying pollen-mediated gene flow is vital for assessing the environmental impact of GM crops. This study quantifies at a landscape level the gene flow that occurs from herbicide-resistant canola crops to nearby crops not containing herbicide resistance genes.
The frequency of gene flow from Brassica napus L. (canola) to four wild relatives, Brassica rapa L., Raphanus raphanistrum L., Sinapis arvensis L. and Erucastrum gallicum (Willd.) O.E. Schulz, was assessed in greenhouse and/or field experiments, and actual rates measured in commercial fields in Canada. Various marker systems were used to detect hybrid individuals: herbicide resistance traits (HR), green fluorescent protein marker (GFP), species-specific amplified fragment length polymorphisms (AFLPs) and ploidy level. Hybridization between B. rapa and B. napus occurred in two field experiments (frequency approximately 7%) and in wild populations in commercial fields (approximately 13.6%). The higher frequency in commercial fields was most likely due to greater distance between B. rapa plants. All F1 hybrids were morphologically similar to B. rapa, had B. napus- and B. rapa-specific AFLP markers and were triploid (AAC, 2n = 29 chromosomes). They had reduced pollen viability (about 55%) and segregated for both self-incompatible and self-compatible individuals (the latter being a B. napus trait). In contrast, gene flow between R. raphanistrum and B. napus was very rare. A single R. raphanistrum × B. napus F1 hybrid was detected in 32,821 seedlings from the HR B. napus field experiment. The hybrid was morphologically similar to R. raphanistrum except for the presence of valves, a B. napus trait, in the distorted seed pods. It had a genomic structure consistent with the fusion of an unreduced gamete of R. raphanistrum and a reduced gamete of B. napus (RrRrAC, 2n = 37), both B. napus- and R. raphanistrum-specific AFLP markers, and had
The inevitable escape of transgenic pollen from cultivated fields will lead to the emergence of transgenic crop-wild plant hybrids in natural patches of wild plants. The fate of these hybrids and that of the transgene depend on their ability to compete with their wild relatives. Here we study ecological factors that may enhance the fitness of genetically modified hybrids relative to wild plants for a Bacillus thuringiensis ( Bt) transgene conferring resistance to insects. Mixed stands of wild plants and first-generation hybrids were grown under different conditions of herbivore pressure and density, with Bt oilseed rape ( Brassica napus) as the crop and B. rapa as the wild recipient. Biomass and fitness components were measured from plant germination to the germination of their offspring. The frequency of transgenic seedlings in the offspring generation was estimated using the green fluorescent protein marker. The biomass of F(1) Bt-transgenic hybrids relative to that of wild-type plants was found to be sensitive to both plant density and herbivore pressure, but herbivore pressure appeared as the major factor enhancing their relative fitnesses. In the absence of herbivore pressure, Bt hybrids produced 6.2-fold fewer seeds than their wild neighbors, and Bt plant frequency fell from 50% to 16% within a single generation. Under high herbivore pressure, Bt hybrids produced 1.4-fold more seeds, and Bt plant frequency was 42% in the offspring generation. We conclude that high-density patches of highly damaged wild plants are the most vulnerable to Bt-transgene invasion. They should be monitored early to detect potential transgene spread.
Transgenes from transgenic oilseed rape, Brassica napus (AACC genome), can introgress into populations of wild B. rapa (AA genome), but little is known about the long-term persistence of transgenes from different transformation events. For example, transgenes that are located on the crop's C chromosomes may be lost during the process of introgression. We investigated the genetic behavior of transgenes in backcross generations of wild B. rapa after nine GFP (green fluorescent protein)-Bt (Bacillus thuringiensis) B. napus lines, named GT lines, were hybridized with three wild B. rapa accessions, respectively. Each backcross generation involved crosses between hemizygous GT plants and non-GT B. rapa pollen recipients. In some cases, sample sizes were too small to allow the detection of major deviations from Mendelian segregation ratios, but the segregation of GT:non-GT was consistent with an expected ratio of 1:1 in all crosses in the BC1 generation. Starting with the BC2 generation, significantly different genetic behavior of the transgenes was observed among the nine GT B. napus lines. In some lines, the segregation of GT:non-GT showed a ratio of 1:1 in the BC2, BC3, and BC4 generations. However, in other GT B. napus lines the segregation ratio of GT:non-GT significantly deviated from 1:1 in the BC2 and BC3 generations, which had fewer transgenic progeny than expected, but not in the BC4 generation. Most importantly, in two GT B. napus lines the segregation of GT:non-GT did not fit into a ratio of 1:1 in the BC2, BC3 or BC4 generations due to a deficiency of transgenic progeny. For these lines, a strong reduction of transgene introgression was observed in all three B. rapa accessions. These findings imply that the genomic location of transgenes in B. napus may affect the long-term persistence of transgenes in B. rapa after hybridization has occurred.
Release of transgenic insect-resistant crops creates the potential not only for the insect pest to evolve resistance but for the escape of transgenes that may confer novel or enhanced fitness-related traits through hybridization with their wild relatives. The differential response of diamondback moth (Plutella xylostella) populations in eastern and western Canada to Bt-producing (GT) Brassica napus and the potential for enhanced fitness of GT B. napus and weedy GT Brassica rapa x B. napus hybrid populations (F1, BC1, BC2) were studied. Comparative bioassays using neonates and 4th instars showed that GT B. napus and GT B. rapa x B. napus hybrids are lethal to larvae from both populations. No measurable plant fitness advantage (reproductive dry weight) was observed for GT B. napus (crop) and GT B. rapa x B. napus hybrid populations at low insect pressure (1 larva per leaf). At high insect densities (>10 larvae per leaf), vegetative plant weight was not significantly different for GT B. napus and non-GT B. napus, whereas reproductive plant weight and proportion of reproductive material were significantly higher in GT B. napus. Establishment of the Bt trait in wild B. rapa populations may also increase its competitive advantage under high insect pressure.
The movement of transgenes from crops to weeds and the resulting consequences are concerns of modern agriculture. The possible generation of "superweeds" from the escape of fitness-enhancing transgenes into wild populations is a risk that is often discussed, but rarely studied. Oilseed rape, Brassica napus (L.), is a crop with sexually compatible weedy relatives, such as birdseed rape (Brassica rapa (L.)). Hybridization of this crop with weedy relatives is an extant risk and an excellent interspecific gene flow model system. In laboratory crosses, T3 lines of seven independent transformation events of Bacillus thuringiensis (Bt) oilseed rape were hybridized with two weedy accessions of B. rapa. Transgenic hybrids were generated from six of these oilseed rape lines, and the hybrids exhibited an intermediate morphology between the parental species. The Bt transgene was present in the hybrids, and the protein was synthesized at similar levels to the corresponding independent oilseed rape lines. Insect bioassays were performed and confirmed that the hybrid material was insecticidal. The hybrids were backcrossed with the weedy parent, and only half the oilseed rape lines were able to produce transgenic backcrosses. After two backcrosses, the ploidy level and morphology of the resultant plants were indistinguishable from B. rapa. Hybridization was monitored under field conditions (Tifton, GA, USA) with four independent lines of Bt oilseed rape with a crop to wild relative ratio of 1200:1. When B. rapa was used as the female parent, hybridization frequency varied among oilseed rape lines and ranged from 16.9% to 0.7%.
As consequence of the concern about the biosafety of genetically modified plants, biological and ecological studies are considered crucial for environmental risk assessment. Laboratory experiments were carried out in order to evaluate the transfer of the Cry1Ac Bt-toxin from a transgenic Bt-oilseed rape to a non-target pest, Myzus persicae Sulzer. Cry1Ac protein levels in plants and aphids were determined using a double sandwich enzyme-linked immunosorbent assay. Phloem sap from (Bt+) and (Bt-) oilseed rape plants was collected from leaves using a standard method of extraction in an EDTA buffer. Bt-toxin was present in phloem sap, with a mean concentration of 2.7 +/- 1.46 ppb, corresponding to a 24-fold lower level than in oilseed rape leaves. Toxin was also detected in aphid samples, with a mean concentration in the positive samples of 2.0 +/- 0.8 ppb. The evidence that Bt-toxin remains in herbivores, in this case an aphid, could be useful to clarify functional aspects linked to possible consequences of Bt-crops on food chains involving herbivore-natural enemy trophic systems. Further studies are needed in order to improve the knowledge on the functional aspects linked to the transfer of the Cry1Ac Bt-toxin from GM-oilseed rape to aphids and their possible consequence.
Norman C. Ellstrand's disagreement with aspects of our review¹ reflects genuine differences of opinion among scientists in the field of biosafety.
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.
We have examined the inheritance of 20 rapeseed (Brassica napus)-specific RAPD (randomly amplified polymorphic DNA) markers from transgenic, herbicide-tolerant rapeseed in 54 plants of the BC1 generation from the cross B. junceax(B. junceaxB. napus). Hybridization between B. juncea and B. napus, with B. juncea as the female parent, was successful both in controlled crosses and spontaneously in the field. The controlled backcrossing of selected hybrids to B. juncea, again with B. juncea as the female parent, also resulted in many seeds. The BC1 plants contained from 0 to 20 of the rapeseed RAPD markers, and the frequency of inheritance of individual RAPD markers ranged from 19% to 93%. The transgene was found in 52% of the plants analyzed. Five synteny groups of RAPD markers were identified. In the hybrids pollen fertility was 0-28%. The hybrids with the highest pollen fertility were selected as male parents for backcrossing, and pollen fertility in the BC1 plants was improved (24-90%) compared to that of the hybrids.
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.Keywords: backcrosses, Brassicaceae, controlled crosses, F2, interspecific hybrids, introgression
Recent concern about gene flow from transgenic plants to weedy species has attracted much research on the fitness of their hybrids. However, no studies have been reported on the very early effects of the seed size of hybrids compared with parental plants for germination, seedling establishment and plant growth. We produced hybrids between male sterile Brassica napus L. (oilseed rape) and five weedy relatives, including Brassica juncea (L.) Czern, B. nigra (L.) Koch, B. rapa L., Hirschfeldia incana (L.) Lagrèze-Fossat and Raphanus raphanistrum L. The hybrid seeds formed between B. napus and B. rapa varied in size, while all the hybrid seeds formed with the other species were small. In a direct-seeded field experiment, small seeds of both parents and hybrids had a lower frequency of emergence and a lower seedling survival rate than large seeds, and resulted in later flowering with less biomass. However, no difference was recorded in a transplant experiment, indicating that growth in the juvenile period was sensitive to the small seed class in field conditions only. The optimum environmental conditions in the greenhouse probably homogenized the developmental differences observed at the early stage, and thus reduced the variation during subsequent growth in the field. This point has not been, but should be, considered in risk assessment of transgenic plants. The lower seedling establishment of small-seeded hybrids could hamper further gene flow.
The persistence and stability of a transgene encoding a Bacillus thuringiensis (Bt) Cry1Ac insecticidal protein was investigated in hybrids between crop Brassica napus and a recurrent wild Brassica juncea population. Interspecific hybrids (F1) and backcross progenies (BC1, BC2) containing green fluorescent protein (GFP) and Bt genes were successfully produced in the greenhouse. Stable Bt toxin levels were found in hybrid and advanced backcross progenies formed in wild B. juncea. Bt Cry1Ac concentration was significantly lower in BC2 plants than in transgenic B. napus, F1, BC1, while no significant differences were detected among the latter three plant genotypes. A GFP marker gene was used as a scorable marker and indicator of Bt transgene expression. GFP fluorescence intensity was significantly correlated with Bt Cry1Ac concentration at the flowering stage and the pod formation stage in both transgenic oilseed rape hybrids and backcrossed progenies (BC1, BC2). It was demonstrated that GFP was a suitable marker for Bt protein in the backcross of B. juncea, which could facilitate the detection of gene flow and is useful in biosafety management.
The expression of a synthetic Bacillus thuringiensis (Bt) cry1Ac gene in oilseed rape (OSR, Brassica napus) was monitored under field conditions in China, and performance against Helicoverpa armigera larvae was compared in intraspecific hybrids with a Chinese OSR variety. Leaf samples from transgenic OSR were collected at various developmental stages in two separate field experiments. The Bt Cry1Ac concentrations in the third uppermost leaves increased before pod formation stage and either increased or decreased after pod formation stage whereas the total soluble protein increased before and decreased after pod-fill in the later growing season. Spontaneously formed intraspecific hybrids between transgenic OSR and a Chinese conventional OSR were obtained in the field and transgenic status was confirmed by a green fluorescent protein (GFP) phenotype and polymerase chain reaction. A bioassay on the neonate larvae of a susceptible strain of H. armigera was performed to test the efficacy of Bt Cry1Ac toxin in hybrid OSR plants. Both the original transgenic OSR and hybrid plants had a negative effect on body-weight gain of insect larvae. It was assumed that Bt Cry1Ac toxin concentration was similar in hybrids compared to the original transgenic OSR at the investigated developmental stages. The frequency of hybrid production and volunteerism could potentially enhance the evolution of insect pest tolerance in the field.
Many crops transformed with insecticidal genes isolated from Bacillus thuringiensis (Bt) show resistance to targeted insect pests. The concentration of Bt endotoxin proteins in plants is very important in transgenic crop efficacy and risk assessment. In the present study, changes in levels of Cry1Ac protein in the leaves of transgenic Bt oilseed rape (Brassica napus) carrying a Bt cry1Ac gene under the control of the cauliflower mosaic virus 35S promoter were quantified during vegetative growth by enzyme-linked immunosorbent assay. Plants were grown in a glasshouse, sampled at 2, 4, 5 and 6 weeks, and the concentration of Cry1Ac was quantified in basal, top and previous top leaves. The mean concentration differed between sowing dates when Cry1Ac concentration was expressed as ng g−1 fresh leaf weight but not when expressed as ng mg−1 total soluble protein. It was demonstrated that Cry1Ac concentration increased significantly as the leaf aged, while the total soluble plant protein decreased significantly. Levels of Cry1Ac were therefore higher in leaves at the base of the plants than in leaves close to the growing point. However, even young leaves with very low Cry1Ac concentrations caused high mortality in the larvae of a Cry1Ac-susceptible laboratory strain of the diamondback moth. The feeding area of leaves consumed by larvae in vivo and in situ was similar. Leaf damage caused by sampling (i.e. artificially) or by feeding of larvae did not affect the levels of Cry1Ac in the leaves under the experimental conditions in this study.
Before novel transgenic plant genotypes are grown outside containment facilities and evaluated under field conditions, it is necessary to complete a risk assessment to consider the possible consequences of that release. An important aspect of risk assessment is to consider the likelihood and consequences of the transgene being transferred by cross-pollination to related species, including other crops, weeds and ruderal populations. The purpose of this report is to review the literature to assess the ease with whichBrassica napus can hybridize with related species. The evidence for hybridization is considered at three levels: a) by open pollination, b) by hand pollination and c) by the use ofin vitro ovule and embryo rescue techniques; and also examines the fertility and vigour of the F1, F2 and backcross generations. Four species are reported to hybridize withB. napus by open pollination:B. rapa andB. juncea using fully fertile parents; andB. adpressa andR. raphanistrum using a male-sterileB. napus parent. Seventeen species are reported to form hybrids (including the four species above) withB. napus when pollination is carried out manually. At least 12 of these species were unable to form F2 progeny, and eight were unable to produce progeny when the F1 was backcrossed to one of the parental species. Many factors will influence the success of hybridization under field conditions, including: distance between the parents, synchrony of flowering, method of pollen spread, specific parental genotypes used, direction of the cross and the environmental conditions. Even where there is a possibility of hybridization betweenB. napus and a related species growing in the vicinity of a release, poor vigour and high sterility in the hybrids will generally mean that hybrids and their progeny will not survive in either an agricultural or natural habitat.
It is possible to monitor the movement of transgenes by tagging them with green fluorescent protein (GFP). In order to develop
a model to study transgene flow, canola (Brassica napus cv Westar) was transformed with two GFP constructs, mGFP5er (GFP only) and pSAM 12 [GFP linked to a synthetic Bacillus thuringiensis (Bt) cry1Ac endotoxin gene]. Transformed callus sectors that fluoresced green were preferentially selected in the tissue culture process.
Four independent GFP canola events and 12 events of GFP/Bt canola were regenerated through tissue culture. GFP fluorescence
was macroscopically detectable throughout the entire life cycle of canola. The GFP/Bt events were insecticidal to neonate
corn earworm (Helicoverpa zea) larvae and prevented herbivory damage. Fluorescence intensity at 508 nm varied between the independent transformation events,
and ranged from 7.6×105 to 13.8×105 (counts per second) in contrast with the wild-type at 5.3×105 cps. Nine GFP/Bt and three GFP events were hybridized with three wild accessions of B. rapa. The resultant hybrids fluoresced green and were insecticidal to neonate corn earworm larvae to the same degree as the transgenic
canola parents. However, fluorescence intensities of the hemizygous F1 hybrid lines were lower than the respective original homozygous canola parents. Each F1 hybrid line was backcrossed by hand onto the B. rapa parent, and transgenic backcrosses were produced at rates ranging from 15% to 34%. These data suggest that GFP can be used
as a tool to monitor transgene flow from crop species to wild relatives.
Employing in vitro culture of ovaries, ovules and embryos, interspecific hybrids have been obtained amongst two important oilseed crops, Brassica napus x B. juncea and their reciprocal. The test-tube hybrid plants have been transferred to the field, and reared to maturity. The F1 seeds obtained from the hybrid ovaries showed normal germination, and the hybrid plants exhibited a range of variation of characters.
This paper reviews the most important biological processes that determine the yield of winter oilseed rape (Brassica napus L.). Biological yield is the product of growth rate and duration of the growing period, both of which indicate the potential for improvement in yield. Likewise, a greater harvest index leads to a higher seed yield. A brief survey of five recently published rapeseed crop models is given. Most of these models are poor predictors of biomass and yield; there is a lack of information about key physiological processes involved in establishment of the stand, the production of biomass and formation of yield, cessation of growth in winter, flowering and post-anthesis growth.During flowering and pod set, the relation between source and sink regulates the availability of assimilates necessary for seed filling. The most source-limiting process is related to small photosynthetically active area, caused by a drastic decline in the leaf area index from the start of flowering despite a slow increase in the assimilating pod area. To analyse yield, it is necessary to understand the structure of the yield and the primary and secondary components, which determine seed yield. Plant density governs the components of yield and, thus, the yield of individual plants. A uniform distribution of plants per unit area is a prerequisite for yield stability. The number of pods per plant is decisive for seed yield; this trait is ultimately determined by the survival of branches, buds, flowers and young pods rather than by the potential number of flowers and pods. Seed number per pod is correlated with pod length. It is, therefore, concluded that pod length is a suitable trait for indirect selection in plant breeding.
Gene flow between crops and weedy relatives depends on the survivorship and reproduction of early-generation hybrids in a field environment. The primary aim of this study was to compare the fitness of transgenic crop x wild hybrids with their parental types and a non-transgenic crop type in the field under enhanced temperature and humidity.
Transgenic insect-resistant oilseed rape (Brassica napus L.), wild brown mustard [B. juncea (L.) Czern et Coss.], their hybrids and non-transgenic B. napus were grown in such a way as to mimic field conditions after harvest under which volunteer plants might appear in agricultural settings. Factor analysis revealed that vegetative growth characteristics explained most of the observed differences among plant types. Wild brown mustard had the highest fitness during its entire life history. Hybrids had intermediate composite fitness and lowest reproductive fitness. The hybrid and the wild weed shared similar vegetative growth characteristics and seed dormancy in their respective progenies.
These data indicate that there might be enhanced persistence of the transgene in warmer climates. The absence of fitness cost of the transgenes might allow transgenes to persist in ecosystems. These data will contribute to risk assessments of transgene persistence and weed management against the backdrop of global climate change.
Transgenic cotton that has been engineered to produce insecticidal toxins from Bacillus thuringiensis (Bt) and so to resist the pest cotton bollworm (Helicoverpa armigera) has been widely planted in Asia. Analysis of the population dynamics of H. armigera from 1992 to 2007 in China indicated that a marked decrease in regional outbreaks of this pest in multiple crops was associated with the planting of Bt cotton. The study area included six provinces in northern China with an annual total of 3 million hectares of cotton and 22 million hectares of other crops (corn, peanuts, soybeans, and vegetables) grown by more than 10 million resource-poor farmers. Our data suggest that Bt cotton not only controls H. armigera on transgenic cotton designed to resist this pest but also may reduce its presence on other host crops and may decrease the need for insecticide sprays in general.
The level of transgene expression in crop x weed hybrids and the degree to which crop-specific genes are integrated into hybrid populations are important factors in assessing the potential ecological and agricultural risks of gene flow associated with genetic engineering. The average transgene zygosity and genetic structure of transgenic hybrid populations change with the progression of generations, and the green fluorescent protein (GFP) transgene is an ideal marker to quantify transgene expression in advancing populations. The homozygous T(1) single-locus insert GFP/ Bacillus thuringiensis (Bt) transgenic canola ( Brassica napus, cv Westar) with two copies of the transgene fluoresced twice as much as hemizygous individuals with only one copy of the transgene. These data indicate that the expression of the GFP gene was additive, and fluorescence could be used to determine zygosity status. Several hybrid generations (BC(1)F(1), BC(2)F(1)) were produced by backcrossing various GFP/Bt transgenic canola ( B. napus, cv Westar) and birdseed rape ( Brassica rapa) hybrid generations onto B. rapa. Intercrossed generations (BC(2)F(2) Bulk) were generated by crossing BC(2)F(1) individuals in the presence of a pollinating insect ( Musca domestica L.). The ploidy of plants in the BC(2)F(2) Bulk hybrid generation was identical to the weedy parental species, B. rapa. AFLP analysis was used to quantify the degree of B. napus introgression into multiple backcross hybrid generations with B. rapa. The F(1) hybrid generations contained 95-97% of the B. napus-specific AFLP markers, and each successive backcross generation demonstrated a reduction of markers resulting in the 15-29% presence in the BC(2)F(2) Bulk population. Average fluorescence of each successive hybrid generation was analyzed, and homozygous canola lines and hybrid populations that contained individuals homozygous for GFP (BC(2)F(2) Bulk) demonstrated significantly higher fluorescence than hemizygous hybrid generations (F(1), BC(1)F(1) and BC(2)F(1)). These data demonstrate that the formation of homozygous individuals within hybrid populations increases the average level of transgene expression as generations progress. This phenomenon must be considered in the development of risk-management strategies.
Transgenes engineered into annual crops could be unintentionally introduced into the genomes of their free-living wild relatives. The fear is that these transgenes might persist in the environment and have negative ecological consequences. Are some crops or transgenic traits of more concern than others? Are there natural genetic barriers to minimize gene escape? Can the genetic transformation process be exploited to produce new barriers to gene flow? Questions abound, but luckily so do answers.
Stable expression of a transgene may lead to increased fitness for wild plants after acquiring the transgene via crop-weed hybridization. Here, we investigate the stability of Bt toxin content in wild Brassica rapa acquiring the Bt gene from Bt Brassica napus. The Bt toxin content in nine Bt-expressing B. napus lines was 0.80-1.70 micro g/g leaf tissue throughout the growing season. These nine lines were crossed with three accessions of wild B. rapa and the Bt gene was successfully transferred to interspecific hybrids (F1) and successive backcross generations (BC1 to BC4). The Bt toxin level in F1 and BC progenies containing the Bt gene remained at 0.90-3.10 micro g/g leaf tissue. This study indicates that the Bt gene can persist and be stably expressed in wild B. rapa.
Oilseed rape (Brassica napus) is sexually compatible with its wild and weedy relative B. rapa, and introgression of genes from B. napus has been found to occur over a few generations. We simulated the early stages of transgene escape by producing F1 hybrids and the first backcross generation between two lines of transgenic B. napus and two populations of weedy B. rapa. Transgene expression and the fitness of the hybrids were examined under different environmental conditions. Expression of the transgenes was analyzed at the mRNA level by quantitative PCR and found to be stable in the hybrids, regardless of the genetic background and the environment, and equal to the level of transcription in the parental B. napus lines. Vigor of the hybrids was measured as the photosynthetic capability; pollen viability and seed set per silique. Photosynthetic capability of first generation hybrids was found to be at the same level, or higher, than that of the parental species, whereas the reproductive fitness was significantly lower. The first backcross generation had a significantly lower photosynthetic capability and reproductive fitness compared to the parental species. This is the first study that examines transgene expression at the mRNA level in transgenic hybrids of B. napus of different genetic background exposed to different environmental conditions. The data presented clarify important details of the overall risk assessment of growing transgenic oilseed rape.
Fitness of hybrids between genetically modified (GM) crops and wild relatives influences the likelihood of ecological harm. We measured fitness components in spontaneous (non-GM) rapeseed x Brassica rapa hybrids in natural populations. The F1 hybrids yielded 46.9% seed output of B. rapa, were 16.9% as effective as males on B. rapa and exhibited increased self-pollination. Assuming 100% GM rapeseed cultivation, we conservatively predict < 7000 second-generation transgenic hybrids annually in the United Kingdom (i.e. approximately 20% of F1 hybrids). Conversely, whilst reduced hybrid fitness improves feasibility of bio-containment, stage projection matrices suggests broad scope for some transgenes to offset this effect by enhancing fitness.
Gene flow from transgenic oilseed rape (BRASSICA NAPUS) might not be avoidable, thus, it is important to detect and quantify hybridization events with its relatives in real time. Data are presented showing the correlation between genetically linked green fluorescent protein (GFP) with BACILLUS THURINGIENSIS (Bt) CRY1AC gene expression in hybrids formed between transgenic B. NAPUS "Westar" and a wild Chinese accession of wild mustard (B. JUNCEA) and hybridization between transgenic B. NAPUS and a conspecific Chinese landrace oilseed rape. Hybrids were obtained either by spontaneous hybridization in the field or by hand-crossing in a greenhouse. In all cases, transgenic hybrids were selected by GFP fluorescence among seedlings originating from seeds harvested from B. JUNCEA and the Chinese oilseed rape plants. Transgenicity was confirmed by PCR detection of transgenes. GFP fluorescence was easily and rapidly detected in the hybrids under greenhouse and field conditions. Results showed that both GFP fluorescence and Bt protein synthesis decreased as either plant or leaf aged, and GFP fluorescence intensity was closely correlated with Bt protein concentration during the entire vegetative lifetime in hybrids. These findings allow the use of GFP fluorescence as an accurate tool to detect gene-flow in time in the field and to conveniently estimate BT CRY1AC expression in hybrids on-the-plant.
Wild Brassica juncea (L.) is a troublesome arable land weed and ruderal. It is critical to understand the responses of this weed to herbicides, because the assessment of its susceptibility profile has important ecological and evolutionary consequences for future cultivation of herbicide-tolerant oilseed rape. The response of 31 wild populations from different geographic origins in China to glyphosate was evaluated with two bioassay methods, and variable responses were found in initial studies. Dose-response assays were conducted to characterize the extreme populations further, and shikimate accumulation in vivo was determined using a spectrophotometric method.
On the basis of ID(50) values, the resistance ratios (R/S) were 5.85 and 4.19 for two glyphosate-resistant B. juncea populations in germination tests, whereas they were 4 times more resistant to glyphosate in spray tests. There were differences in shikimate accumulation patterns between the two biotypes. Shikimate concentrations in resistant populations began to decline from 6 days after treatment (DAT), while they increased continually in susceptible populations.
The results obtained suggest that the populations responded differentially to glyphosate, and this variability may provide the genetic basis for evolution of individuals with increased resistance to glyphosate, with important implications for herbicide resistance management, especially in the context of risk assessment of glyphosate-tolerant crops.
The existence of transgenic hybrids resulting from transgene escape from genetically modified (GM) crops to wild or weedy relatives is well documented but the fate of the transgene over time in recipient wild species populations is still relatively unknown. This is the first report of the persistence and apparent introgression, i.e. stable incorporation of genes from one differentiated gene pool into another, of an herbicide resistance transgene from Brassica napus into the gene pool of its weedy relative, Brassica rapa, monitored under natural commercial field conditions. Hybridization between glyphosate-resistant [herbicide resistance (HR)]B. napus and B. rapa was first observed at two Québec sites, Ste Agathe and St Henri, in 2001. B. rapa populations at these two locations were monitored in 2002, 2003 and 2005 for the presence of hybrids and transgene persistence. Hybrid numbers decreased over the 3-year period, from 85 out of approximately 200 plants surveyed in 2002 to only five out of 200 plants in 2005 (St Henri site). Most hybrids had the HR trait, reduced male fertility, intermediate genome structure, and presence of both species-specific amplified fragment length polymorphism markers. Both F(1) and backcross hybrid generations were detected. One introgressed individual, i.e. with the HR trait and diploid ploidy level of B. rapa, was observed in 2005. The latter had reduced pollen viability but produced approximately 480 seeds. Forty-eight of the 50 progeny grown from this plant were diploid with high pollen viability and 22 had the transgene (1:1 segregation). These observations confirm the persistence of the HR trait over time. Persistence occurred over a 6-year period, in the absence of herbicide selection pressure (with the exception of possible exposure to glyphosate in 2002), and in spite of the fitness cost associated with hybridization.
Fitness and maternal effects in hybrids formed between transgenic oilseed rape
- K Di
- C N Stewart
- W Wei
- B C Shen
- Z X Tang
- K P Ma
K. Di, C.N. Stewart, W. Wei, B.c. Shen, Z.X. Tang, K.P. Ma, Fitness and maternal
effects in hybrids formed between transgenic oilseed rape (Brassica napus L.)
and wild brown mustard [B. juncea (L.) Czern et Coss.] in the field, Pest Manag.
Sci. 65 (2009) 753–760.
- T P Hauser
- R B Jørgensen
T.P. Hauser, R.B. Jørgensen, Fitness of backcross and F2 hybrids between weedy
Brassica rapa and oilseed rape (B. napus), Heredity 81 (1998) 436-443.