Effect of herbivore stress on transgene behaviour in maize crosses with different genetic backgrounds: cry1Ab transgene transcription, insecticidal protein expression and bioactivity against insect pests.

Background: Decades after their first commercial release, many theoretical assumptions are still taken for granted in the deployment of genetically modified (GM) crops. Theoretically, in the case of maize, active transcription of the cry1Ab transgene would result in dose-dependent production of the insecticidal Cry1Ab protein, which would in turn induce dose-dependent mortality on lepidopteran pests. We produced data to realistically approach this question by using a model that includes two genetic background contexts from two geographical provenances in Brazil and South Africa, and two lepidopteran pests (Helicoverpa armigera and Spodoptera littoralis). However, in this study, the effect of insect herbivory was superimposed to investigate possible stress-induced effects in transgene expression at three levels: mRNA, protein and bioactivity. Results: Overall, we found that herbivore damage by H. armigera was reflected only at the translational level, with a higher level of Cry1Ab protein measured in the Brazilian crosses under herbivore stress. On the other hand, compared to non-stress growing conditions, the herbivore damage by S. littoralis was not directly reflected in mRNA, protein or bioactivity in the South African crosses. Conclusions: The differences between South African and Brazilian genetic backgrounds, and between the stressor effect of the two herbivores used, highlight the complexity of transgene expression at the agroecological level. Supplementary Information: The online version contains supplementary material available at 10.1186/s12302-023-00815-3.

Confirmation of 'Pollen- and Seed-Specific Gene Deletor' System Efficiency for Transgene Excision from Transgenic Nicotiana tabacum under Field Conditions.

The commercial application of genetically modified plants has been seriously impeded by public concern surrounding the potential risks posed by such plants to the ecosystem and human health. Previously, we have developed a 'pollen- and seed-specific Gene Deletor' system that automatically excised all transgenes from the pollen and seeds of greenhouse-grown transgenic Nicotiana tabacum. In this study, we conducted seven field experiments over three consecutive years to evaluate the stability of transgene excision under field conditions. Our results showed that transgenes were stably excised from transgenic Nicotiana tabacum under field conditions with 100% efficiency. The stability of transgene excision was confirmed based on PCR, as well as the GUS staining patterns of various organs (roots, leaves, petiole, stem, flower, fruit, and seeds) from transgenic N. tabacum. In six transgenic lines (D4, D10, D31, D56, and D43), the transgenes were stably deleted in the T0 and T1 generations. Thus, the 'Gene Deletor' system is an efficient and reliable method to reduce pollen- and seed-mediated unintentional gene flow. This system might help to alleviate the food safety concerns associated with transgenic crops.

Increased Longevity and Dormancy of Soil-Buried Seeds from Advanced Crop-Wild Rice Hybrids Overexpressing the EPSPS Transgene.

Estimating the fitness effect conferred by a transgene introgressed into populations of wild relative species from a genetically engineered (GE) crop plays an important role in assessing the potential environmental risks caused by transgene flow. Such estimation has essentially focused on the survival and fecundity-related characteristics measured above the ground, but with little attention to the fate of GE seeds shattered in the soil seed banks after maturation. To explore the survival and longevity of GE seeds in soil, we examined the germination behaviors of crop-wild hybrid seeds (F4-F6) from the lineages of a GE herbicide-tolerant rice (Oryzasativa) line that contains an endogenous EPSPS transgene hybridized with two wild O. rufipogon populations after the seeds were buried in soil. The results showed significantly increased germination of the GE crop-wild hybrid seeds after soil burial, compared with that of the non-GE hybrid seeds. Additionally, the proportion of dormant seeds and the content of the growth hormone auxin (indole-3-acetic acid, IAA) in the GE crop-wild hybrid seeds significantly increased. Evidently, the EPSPS transgene enhances the survival and longevity of GE crop-wild rice seeds in the soil seed banks. The enhanced survival and longevity of the GE hybrid seeds is likely associated with the increases in seed dormancy and auxin (IAA) by overexpressing the rice endogenous EPSPS transgene. Thus, the fate of GE seeds in the soil seed banks should be earnestly considered when assessing the environmental risks caused by transgene flow.

Environmental risk assessment of glufosinate-resistant soybean by pollen-mediated gene flow under field conditions in the region of the genetic origin.

Pollen-mediated gene flow of genetically modified crops to their wild relatives can facilitate the spread of transgenes into the ecosystem and alter the fitness of the consequential progeny. A two-year field study was conducted to quantify the gene flow from glufosinate-ammonium resistant (GR) soybean (Glycinemax) to its wild relative, wild soybean (G. soja), and assess the potential weed risk of hybrids resulting from the gene flow during their entire life cycle under field conditions in Korea, where wild soybean is the natural inhabitant. Pollen-mediated gene flow from GR soybeans to wild soybeans ranged from 0.292% (mixed planting) to 0.027% at 8 m distance. The log-logistic model described the gene flow rate with increasing distance from GR soybean to wild soybean; the estimated effective isolation distance for 0.01% gene flow between GR and wild soybeans was 37.7 m. The F1 and F2 hybrids exhibited the intermediate characteristics of their parental soybeans in their vegetative and reproductive stages. Canopy height and stem length of hybrids were close to those of wild soybean, which shows an indeterminate growth; the numbers of flowers, pods, and seeds per hybrid plant were close to those of wild soybean and significantly higher than those of GR soybean. Seed longevity of F2 hybrid plants was also intermediate but significantly greater than that of GR soybean due to high seed dormancy. Our results suggest that transgenes of the GR soybean might disperse into wild populations and persist in the agroecosystem of the genetic origin regions due to the pollen-mediated gene flow and the relatively high fitness of the hybrid progeny.

The application of gene splitting technique for controlling transgene flow in rice.

Controlling transgene flow in China is important, as this country is part of the center of origin of rice. A gene-splitting technique based on intein-mediated trans-splicing represents a new strategy for controlling transgene flow via biological measures. In this study, the G2-aroA gene which provides glyphosate tolerance was split into an N-terminal and a C-terminal region, which were then fused to intein N and intein C of the Ssp DnaE intein, ultimately forming EPSPSn:In and Ic:EPSPSc fusion genes, respectively. These fusion genes were subsequently transformed into the rice cultivar Zhonghua 11 via the Agrobacterium-mediated method. The two split gene fragments were then introduced into the same rice genome by genetic crossings. Glyphosate tolerance analysis revealed that the functional target protein was reconstituted by Ssp DnaE intein-mediated trans-splicing and that the resultant hybrid rice was glyphosate tolerant. The reassembly efficiency of the split gene fragments ranged from 67 to 91% at the molecular level, and 100% of the hybrid F1 progeny were glyphosate tolerant. Transgene flow experiments showed that when the split gene fragments are inserted into homologous chromosomes, the gene-splitting technique can completely avoid the escape of the target trait to the environment. This report is the first on the reassembly efficiency and effectiveness of transgene flow containment via gene splitting in rice. This study provides not only a new biological strategy for controlling rice transgene flow but also a new method for cultivating hybrid transgenic rice.

Assessment of potential environmental risks of transgene flow in smallholder farming systems in Asia: Brassica napus as a case study in Korea.

The cultivation of genetically modified (GM) crops has raised many questions regarding their environmental risks, particularly about their ecological impact on non-target organisms, such as their closely-related relative species. Although evaluations of transgene flow from GM crops to their conventional crops has been conducted under large-scale farming system worldwide, in particular in North America and Australia, few studies have been conducted under smallholder farming systems in Asia with diverse crops in co-existence. A two-year field study was conducted to assess the potential environmental risks of gene flow from glufosinate-ammonium resistant (GR) Brassica napus to its conventional relatives, B. napus, B. juncea, and Raphanus sativus under simulated smallholder field conditions in Korea. Herbicide resistance and simple sequence repeat (SSR) markers were used to identify the hybrids. Hybridization frequency of B. napus × GR B. napus was 2.33% at a 2 m distance, which decreased to 0.007% at 75 m. For B. juncea, it was 0.076% at 2 m and decreased to 0.025% at 16 m. No gene flow was observed to R. sativus. The log-logistic model described hybridization frequency with increasing distance from GR B. napus to B. napus and B. juncea and predicted that the effective isolation distances for 0.01% gene flow from GR B. napus to B. napus and B. juncea were 122.5 and 23.7 m, respectively. Results suggest that long-distance gene flow from GR B. napus to B. napus and B. juncea is unlikely, but gene flow can potentially occur between adjacent fields where the smallholder farming systems exist.

Transgene flow in Mexican maize revisited: Socio-biological analysis across two contrasting farmer communities and seed management systems.

The flow of transgenes into landraces and wild relatives is an important biosafety concern. The case of transgene flow into local maize varieties in Mexico (the center of origin of maize) has been intensively debated over the past 15 years, including legal, political, and environmental disputes fanned by the existence of a significant scientific controversy over the methods used for the detection of transgenes. The use of diverse approaches and a lack of harmonized methods specific to the detection and monitoring of transgenes in landraces have generated both positive and negative results regarding contamination of Mexican maize with genetically modified material over the years. In this paper, we revisit the case of transgene contamination in Mexican maize and present a novel research approach based on socio-biological analysis of contrasting communities and seed management systems. Two communities were used to investigate how different social and biological factors can affect transgene flow and impact transgene spread in Mexico. Our results show the presence of transgenes in one community and thus support the position that transgenes are highly likely to be present in Mexican maize landraces. However, our work also demonstrates that the extent and frequency with which transgenes can be found will significantly depend on the societal characteristics and seed management systems of the local communities. Therefore, we argue that future analysis of transgene presence should include social research on the seed management practices in the sampling area so that more robust and comprehensive understandings and conclusions can be drawn.

An AGAMOUS intron-driven cytotoxin leads to flowerless tobacco and produces no detrimental effects on vegetative growth of either tobacco or poplar.

Flowerless trait is highly desirable for poplar because it can prevent pollen- and seed-mediated transgene flow. We have isolated the second intron of PTAG2, an AGAMOUS (AG) orthologue from Populus trichocarpa. By fusing this intron sequence to a minimal 35S promoter sequence, we created two artificial promoters, fPTAG2I (forward orientation of the PTAG2 intron sequence) and rPTAG2I (reverse orientation of the PTAG2 intron sequence). In tobacco, expression of the ß-glucuronidase gene (uidA) demonstrates that the fPTAG2I promoter is non-floral-specific, while the rPTAG2I promoter is active in floral buds but with no detectable vegetative activity. Under glasshouse conditions, transgenic tobacco plants expressing the Diphtheria toxin A (DT-A) gene driven by the rPTAG2I promoter produced three floral ablation phenotypes: flowerless, neuter (stamenless and carpel-less) and carpel-less. Further, the vegetative growth of these transgenic lines was similar to that of the wild-type plants. In field trials during 2014 and 2015, the flowerless transgenic tobacco stably maintained its flowerless phenotype, and also produced more shoot and root biomass when compared to wild-type plants. In poplar, the rPTAG2I::GUS gene exhibited no detectable activity in vegetative organs. Under field conditions over two growing seasons (2014 to the end of 2015), vegetative growth of the rPTAG2I::DT-A transgenic poplar plants was similar to that of the wild-type plants. Our results demonstrate that the rPTAG2I artificial promoter has no detectable activities in vegetative tissues and organs, and the rPTAG2I::DT-A gene may be useful for producing flowerless poplar that retains normal vegetative growth.

Dealing with transgene flow of crop protection traits from crops to their relatives.

Genes regularly move within species, to/from crops, as well as to their con- specific progenitors, feral and weedy forms ('vertical' gene flow). Genes occasionally move to/from crops and their distantly related, hardly sexually interbreeding relatives, within a genus or among closely related genera (diagonal gene flow). Regulators have singled out transgene flow as an issue, yet non-transgenic herbicide resistance traits pose equal problems, which cannot be mitigated. The risks are quite different from genes flowing to natural (wild) ecosystems versus ruderal and agroecosystems. Transgenic herbicide resistance poses a major risk if introgressed into weedy relatives; disease and insect resistance less so. Technologies have been proposed to contain genes within crops (chloroplast transformation, male sterility) that imperfectly prevent gene flow by pollen to the wild. Containment does not prevent related weeds from pollinating crops. Repeated backcrossing with weeds as pollen parents results in gene establishment in the weeds. Transgenic mitigation relies on coupling crop protection traits in a tandem construct with traits that lower the fitness of the related weeds. Mitigation traits can be morphological (dwarfing, no seed shatter) or chemical (sensitivity to a chemical used later in a rotation). Tandem mitigation traits are genetically linked and will move together. Mitigation traits can also be spread by inserting them in multicopy transposons which disperse faster than the crop protection genes in related weeds. Thus, there are gene flow risks mainly to weeds from some crop protection traits; risks that can and should be dealt with.

Transgene flow: facts, speculations and possible countermeasures.

Convincing evidence has accumulated that unintended transgene escape occurs in oilseed rape, maize, cotton and creeping bentgrass. The escaped transgenes are found in variant cultivars, in wild type plants as well as in hybrids of sexually compatible species. The fact that in some cases stacked events are present that have not been planted commercially, implies unintended recombination of transgenic traits. As the consequences of this continuous transgene escape for the ecosystem cannot be reliably predicted, I propose to use more sophisticated approaches of gene technology in future. If possible GM plants should be constructed using either site-directed mutagenesis or cisgenic strategies to avoid the problem of transgene escape. In cases where a transgenic trait is needed, efficient containment should be the standard approach. Various strategies available or in development are discussed. Such a cautious approach in developing novel types of GM crops will enhance the sustainable potential of GM crops and thus increase the public trust in green gene technology.

Rice transgene flow: its patterns, model and risk management.

Progress has been made in a 12 year's systemic study on the rice transgene flow including (i) with experiments conducted at multiple locations and years using up to 21 pollen recipients, we have elucidated the patterns of transgene flow to different types of rice. The frequency to male sterile lines is 10(1) and 10(3) higher than that to O. rufipogon and rice cultivars. Wind speed and direction are the key meteorological factors affecting rice transgene flow. (ii) A regional applicable rice gene flow model is established and used to predict the maximum threshold distances (MTDs) of gene flow during 30 years in 993 major rice producing counties of southern China. The MTD0.1% for rice cultivars is basically ≤5 m in the whole region, despite climate differs significantly at diverse locations and years. This figure is particularly valuable for the commercialization and regulation of transgenic rice. (iii) The long-term fate of transgene integrated into common wild rice was investigated. Results demonstrated that the F1 hybrids of transgenic rice/O. rufipogon gradually disappeared within 3-5 years, and the Bt or bar gene was not detectable in the mixed population, suggesting the O. rufipogon may possess a strong mechanism of exclusiveness for self-protection. (iv) The flowering time isolation and a 2-m-high cloth-screen protection were proved to be effective in reducing transgene flow. We have proposed to use a principle of classification and threshold management for different types of rice.

Gene flow from transgenic to nontransgenic soybean plants in the Cerrado region of Brazil

Evaluation of transgenic crops under field conditions is a fundamental step for the production of genetically engineered varieties. In order to determine if there is pollen dispersal from transgenic to nontransgenic soybean plants, a field release experiment was conducted in the Cerrado region of Brazil. Nontransgenic plants were cultivated in plots surrounding Roundup Ready transgenic plants carrying the cp4 epsps gene, which confers herbicide tolerance against glyphosate herbicide, and pollen dispersal was evaluated by checking for the dominant gene. The percentage of cross-pollination was calculated as a fraction of herbicide-tolerant and -nontolerant plants. The greatest amount of transgenic pollen dispersion was observed in the first row, located at one meter from the central (transgenic) plot, with a 0.52% average frequency. The frequency of pollen dispersion decreased to 0.12% in row 2, reaching 0% when the plants were up to 10 m distance from the central plot. Under these conditions pollen flow was higher for a short distance. This fact suggests that the management necessary to avoid cross-pollination from transgenic to nontransgenic plants in the seed production fields should be similar to the procedures currently utilized to produce commercial seeds.