A built-in mechanism to mitigate the spread of insect-resistance and herbicide-tolerance transgenes into weedy rice populations

Background

The major challenge of cultivating genetically modified (GM) rice (Oryza sativa) at the commercial scale is to prevent the spread of transgenes from GM cultivated rice to its coexisting weedy rice (O. sativa f. spontanea). The strategic development of GM rice with a built-in control mechanism can mitigate transgene spread in weedy rice populations.

Methodology/Principal Findings

An RNAi cassette suppressing the expression of the bentazon detoxifying enzyme CYP81A6 was constructed into the T-DNA which contained two tightly linked transgenes expressing the Bt insecticidal protein Cry1Ab and the glyphosate tolerant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), respectively. GM rice plants developed from this T-DNA were resistant to lepidopteran pests and tolerant to glyphosate, but sensitive to bentazon. The application of bentazon of 2000 mg/L at the rate of 40 mL/m², which is approximately the recommended dose for the field application to control common rice weeds, killed all F₂ plants containing the transgenes generated from the Crop-weed hybrids between a GM rice line (CGH-13) and two weedy rice strains (PI-63 and PI-1401).

Conclusions/Significance

Weedy rice plants containing transgenes from GM rice through gene flow can be selectively killed by the spray of bentazon when a non-GM rice variety is cultivated alternately in a few-year interval. The built-in control mechanism in combination of cropping management is likely to mitigate the spread of transgenes into weedy rice populations.     

A built-in strategy for containment of transgenic plants: creation of selectively terminable transgenic rice

Plant transgenic technology has been widely utilized for engineering crops for trait improvements and for production of high value proteins such as pharmaceuticals. However, the unintended spreading of commercial transgenic crops by pollination and seed dispersal is a major concern for environmental and food safety. Simple and reliable containment strategies for transgenes are highly desirable. Here we report a novel method for creating selectively terminable transgenic rice. In this method, the gene(s) of interest is tagged with a RNA interference cassette, which specifically suppresses the expression of the bentazon detoxification enzyme CYP81A6 and thus renders transgenic rice to be sensitive to bentazon, a herbicide used for rice weed control. We generated transgenic rice plants by this method using a new glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Pesudomonas putida as the gene of interest, and demonstrated that these transgenic rice plants were highly sensitive to bentazon but tolerant to glyphosate, which is exactly the opposite of conventional rice. Field trial of these transgenic rice plants further confirmed that they can be selectively killed at 100% by one spray of bentazon at a regular dose used for conventional rice weed control. Furthermore, we found that the terminable transgenic rice created in this study shows no difference in growth, development and yield compared to its non-transgenic control. Therefore, this method of creating transgenic rice constitutes a novel strategy of transgene containment, which appears simple, reliable and inexpensive for implementation.     

Establishment of transgenic herbicide-resistant creeping bentgrass (Agrostis stolonifera L.) in nonagronomic habitats

Concerns about genetically modified (GM) crops include transgene flow to compatible wild species and unintended ecological consequences of potential transgene introgression. However, there has been little empirical documentation of establishment and distribution of transgenic plants in wild populations. We present herein the first evidence for escape of transgenes into wild plant populations within the USA; glyphosate-resistant creeping bentgrass (Agrostis stolonifera L.) plants expressing CP4 EPSPS transgenes were found outside of cultivation area in central Oregon. Resident populations of three compatible Agrostis species were sampled in nonagronomic habitats outside the Oregon Department of Agriculture control area designated for test production of glyphosate-resistant creeping bentgrass. CP4 EPSPS protein and the corresponding transgene were found in nine A. stolonifera plants screened from 20,400 samples (0.04 +/- 0.01% SE). CP4 EPSPS-positive plants were located predominantly in mesic habitats downwind and up to 3.8 km beyond the control area perimeter; two plants were found within the USDA Crooked River National Grassland. Spatial distribution and parentage of transgenic plants (as confirmed by analyses of nuclear ITS and chloroplast matK gene trees) suggest that establishment resulted from both pollen-mediated intraspecific hybridizations and from crop seed dispersal. These results demonstrate that transgene flow from short-term production can result in establishment of transgenic plants at multi-kilometre distances from GM source fields or plants. Selective pressure from direct application or drift of glyphosate herbicide could enhance introgression of CP4 EPSPS transgenes and additional establishment. Obligatory outcrossing and vegetative spread could further contribute to persistence of CP4 EPSPS transgenes in wild Agrostis populations, both in the presence or absence of herbicide selection.     

Letting the gene out of the bottle: the population genetics of genetically modified crops

Genetically modified (GM) plants are rapidly becoming a common feature of modern agriculture. This transition to engineered crops has been driven by a variety of potential benefits, both economic and ecological. The increase in the use of GM crops has, however, been accompanied by growing concerns regarding their potential impact on the environment. Here, we focus on the escape of transgenes from cultivation via crop x wild hybridization. We begin by reviewing the literature on natural hybridization, with particular reference to gene flow between crop plants and their wild relatives. We further show that natural selection, and not the overall rate of gene flow, is the most important factor governing the spread of favorable alleles. Hence, much of this review focuses on the likely effects of transgenes once they escape. Finally, we consider strategies for transgene containment.     

Transgene Containment Using Cytokinin-Reversible Male Sterility in Constitutive, Gibberellic Acid–Insensitive (Δgai) Transgenic Tobacco

Mechanisms are needed to prevent gene flow from transgenic crops, and the later establishment of these transgenes in populations of other varieties, weeds, or wild relatives. Such prevention can be achieved by containing the transgene within a crop, and then mitigating the effects of the inherent leakage and unidirectionality of containment systems. Mitigation lowers the fitness of recipients below that of the wild-type so that transgenes cannot spread. Transplastomic and male-sterility systems suppress transgene outflow, but not the influx of pollen from relatives, requiring mitigation. The Arabidopsis thaliana Δgai (gibberellic acid–insensitive) gene, driven by its own promoter, induced male sterility in transgenic tobacco (Nicotiana tabacum), which is chemically reversible by kinetin applications. Female reproduction was not affected. Kinetin-treated sterile hemizygous and homozygous dwarf tobacco produced viable pollen, becoming self-fertile with copious viable seed, restoring the small amount of seed production needed for such a crop. Thus, Δgai, under its endogenous promoter, can be used as a containment mechanism to prevent transgene outflow. This application is in addition to the previously described highly effective role of Δgai as a dwarfing mitigator gene, which renders the rare transgenic tobacco hybrids unfit and unable to compete with the wild-type in the mixed cultures. Δgai is unique in that it can be used both to prevent transgene outflow and to mitigate the flow should containment fail or should gene influx occur, a dual role for the gene, not previously reported.     

Assessing the risk of gene flow from genetically modified trees carrying mitigation transgenes

The increasing use of genetically modified (GM) plants has raised concerns about the escape of transgenes to conventional populations. To counteract possible fitness advantages of GM plants, an advantageous ‘primary transgene’ may be linked to a ‘mitigation’ transgene that is selectively disfavored. A risk related to this technique is the possible break-up of the transgenes. This may lead to the establishment of genotypes that only express the primary transgenic trait and are therefore selectively favored over both conventional and GM genotypes. We study here how the establishment of break-up genotypes in tree populations depends on life history, pollen dispersal kernels, and the linkage between transgenes. To this end, we model the dynamics of an initially homozygous GM population and a conventional population growing next to each other. The simulations are based on a spatially explicit simulation model for population-genotype dynamics. Pollen and seed dispersal are described by fat-tailed dispersal kernels. Break-up of transgenes is due subsequent crossing of GM and conventional genotypes. The spatial pattern of pollen and seed dispersal causes break-up and establishment of new invasive genotypes to occur mostly in the conventional population. The amount of individuals of these genotypes established in the conventional population depends on the interaction between life-history traits, pollen dispersal distances and the linkage of the transgenes. Thus, risk assessment of transgene break-up requires information on the genetics, local dynamics, and dispersal of GM and conventional varieties. The approach presented here provides a tool for combining these different sources of information, and it contributes to the formulation of general rules for the management of GM tree populations.     

Marker-free transgenic corn plant production through co-bombardment

The use of particle gun for the production of marker-free plants is scant in published literature. Perhaps this is a reflection of the widely held notion that the events generated through bombardment tend to have multiple copies of transgenes, usually integrated at a single locus, features which precludes segregating away the selectable marker gene. However, our previous studies have shown that single-copy integrants are obtained at a high frequency if limited quantity of DNA is used for bombardment. Also, the concatemerized insertion of transgenes has been demonstrated to be greatly reduced if “cassette DNA” is employed in place of whole plasmid DNA for bombardment. Based on the above findings, in the present study the feasibility of co-bombardment was evaluated for the production of marker-free plants in corn, employing a combination of limited quantity DNA and cassette DNA approaches for bombardment. Transgenic events were generated after co-bombardment of a selectable marker cassette containing the nptII gene (2.5 ng per shot) and a GUS gene cassette (15 ng per shot). Among these events single-copy integrants for nptII gene occurred at an average frequency of 68% within which the co-expression frequency of GUS and nptII genes ranged from 41% to 80%. Marker-free corn plants could be identified from the progeny of 28 out of the 103 R0 co-expressing events screened. The results demonstrate that by using cassette DNA and low quantities of DNA for bombardment, marker-free plants are produced at efficiencies comparable to that of Agrobacterium-based co-transformation methods.     

Detection of Transgenes in Local Maize Varieties of Small-Scale Farmers in Eastern Cape,South Africa

Small-scale subsistence farmers in South Africa have been introduced to genetically modified (GM) crops for more than a decade. Little is known about i) the extent of transgene introgression into locally recycled seed, ii) what short and long-term ecological and socioeconomic impacts such mixing of seeds might have, iii) how the farmers perceive GM crops, and iv) to what degree approval conditions are followed and controlled. This study conducted in the Eastern Cape, South Africa, aims primarily at addressing the first of these issues. We analysed for transgenes in 796 individual maize plants (leaves) and 20 seed batches collected in a village where GM insect resistant maize was previously promoted and grown as part of an governmental agricultural development program over a seven year period (2001–2008). Additionally, we surveyed the varieties of maize grown and the farmers’ practices of recycling and sharing of seed in the same community (26 farmers were interviewed). Recycling and sharing of seeds were common in the community and may contribute to spread and persistence of transgenes in maize on a local or regional level. By analysing DNA we found that the commonly used transgene promoter p35s occurred in one of the 796 leaf samples (0.0013%) and in five of the 20 seed samples (25%). Three of the 20 seed samples (15%) included herbicide tolerant maize (NK603) intentionally grown by the farmers from seed bought from local seed retailers or acquired through a currently running agricultural development program. The two remaining positive seed samples (10%) included genes for insect resistance (from MON810). In both cases the farmers were unaware of the transgenes present. In conclusion, we demonstrate that transgenes are mixed into seed storages of small-scale farming communities where recycling and sharing of seeds are common, i.e. spread beyond the control of the formal seed system.     

Directed microspore-specific recombination of transgenic alleles to prevent pollen-mediated transmission of transgenes

A major challenge for future genetically modified (GM) crops is to prevent undesired gene flow of transgenes to plant material intended for another use. Recombinase-mediated auto excision of transgenes directed by a tightly controlled microspore-specific promoter allows efficient removal of either the selectable marker gene or of all introduced transgenes during microsporogenesis. This way, transgene removal becomes an integral part of the biology of pollen maturation, not requiring any external stimulus such as chemical induction by spraying. We here show the feasibility of engineering transgenic plants to produce pollen devoid of any transgene. Highly efficient excision of transgenes from tobacco pollen was achieved with a potential failure rate of at most two out of 16 800 seeds (0.024%). No evidence for either premature activation or absence of activation of the recombinase system was observed under stress conditions in the laboratory. This approach can prevent adventitious presence of transgenes in non-GM crops or related wild species by gene flow. Such biological containment may help the deployment and management of coexistence practices to support consumer choice and will promote clean molecular farming for the production of high-value compounds in plants.     

Gene flow from genetically modified rice to its wild relatives: Assessing potential ecological consequences

Pollen-mediated gene flow is the major pathway for transgene escape from GM rice to its wild relatives. Transgene escape to wild Oryza species having AA-genome will occur if GM rice is released to environments with these wild Oryza species. Transgenes may persist to and spread in wild populations after gene flow, resulting unwanted ecological consequences. For assessing the potential consequences caused by transgene escape, it is important to understand the actual gene flow frequencies from GM rice to wild relatives, transgene expression and inheritance in the wild relatives, as well as fitness changes that brought to wild relatives by the transgenes. This article reviews studies on transgene escape from rice to its wild relatives via gene flow and its ecological consequences. A framework for assessing potential ecological consequences caused by transgene escape from GM rice to its wild relatives is discussed based on studies of gene flow and fitness changes.     

Occurrence of Transgenic Feral Alfalfa (Medicago sativa subsp. sativa L.) in Alfalfa Seed Production Areas in the United States

The potential environmental risks of transgene exposure are not clear for alfalfa (Medicago sativa subsp. sativa), a perennial crop that is cross-pollinated by insects. We gathered data on feral alfalfa in major alfalfa seed-production areas in the western United States to (1) evaluate evidence that feral transgenic plants spread transgenes and (2) determine environmental and agricultural production factors influencing the location of feral alfalfa, especially transgenic plants. Road verges in Fresno, California; Canyon, Idaho; and Walla Walla, Washington were surveyed in 2011 and 2012 for feral plants, and samples were tested for the CP4 EPSPS protein that conveys resistance to glyphosate. Of 4580 sites surveyed, feral plants were observed at 404 sites. Twenty-seven percent of these sites had transgenic plants. The frequency of sites having transgenic feral plants varied among our study areas. Transgenic plants were found in 32.7%, 21.4.7% and 8.3% of feral plant sites in Fresno, Canyon and Walla Walla, respectively. Spatial analysis suggested that feral populations started independently and tended to cluster in seed and hay production areas, places where seed tended to drop. Significant but low spatial auto correlation suggested that in some instances, plants colonized nearby locations. Neighboring feral plants were frequently within pollinator foraging range; however, further research is needed to confirm transgene flow. Locations of feral plant clusters were not well predicted by environmental and production variables. However, the likelihood of seed spillage during production and transport had predictive value in explaining the occurrence of transgenic feral populations. Our study confirms that genetically engineered alfalfa has dispersed into the environment, and suggests that minimizing seed spillage and eradicating feral alfalfa along road sides would be effective strategies to minimize transgene dispersal.     

Herbicidal inhibitors of amino acid biosynthesis and herbicide-tolerant crops

Summary. Acetohydroxyacid synthase (AHAS) inhibitors interfere with branched-chain amino acid biosynthesis by inhibiting AHAS. Glyphosate affects aromatic amino acid biosynthesis by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Glufosinate inhibits glutamine synthetase and blocks biosynthesis of glutamine. AHAS gene variants that confer tolerance to AHAS inhibitors have been discovered in plants through selection or mutagenesis. Imidazolinone-tolerant crops have been commercialized based on these AHAS gene variants. A modified maize EPSPS gene and CP4-EPSPS gene from Agrobacterium sp. have been used to transform plants for target-based tolerance to glyphosate. A gox gene isolated from Ochrobactrum anthropi has also been employed to encode glyphosate oxidoreductase to detoxify glyphosate in plants. Glyphosate-tolerant crops with EPSPS transgene alone or both EPSPS and gox transgenes have been commercialized. Similarly, bar and pat genes isolated from Streptomyces hygroscopicus and S. viridochromogenes, respectively, have been inserted into plants to encode phosphinothricin N-acetyltransferase to detoxify glufosinate. Glufosinate-tolerant crops have been commercialized using one of these two transgenes.     

Limited fitness advantages of crop-weed hybrid progeny containing insect-resistant transgenes (Bt/CpTI) in transgenic rice field

Background

The spread of insect-resistance transgenes from genetically engineered (GE) rice to its coexisting weedy rice (O. sativa f. spontanea) populations via gene flow creates a major concern for commercial GE rice cultivation. Transgene flow to weedy rice seems unavoidable. Therefore, characterization of potential fitness effect brought by the transgenes is essential to assess environmental consequences caused by crop-weed transgene flow.

Methodology/Principal Findings

Field performance of fitness-related traits was assessed in advanced hybrid progeny of F₄ generation derived from a cross between an insect-resistant transgenic (Bt/CpTI) rice line and a weedy strain. The performance of transgene-positive hybrid progeny was compared with the transgene-negative progeny and weedy parent in pure and mixed planting of transgenic and nontransgenic plants under environmental conditions with natural vs. low insect pressure. Results showed that under natural insect pressure the insect-resistant transgenes could effectively suppress target insects and bring significantly increased fitness to transgenic plants in pure planting, compared with nontransgenic plants (including weedy parent). In contrast, no significant differences in fitness were detected under low insect pressure. However, such increase in fitness was not detected in the mixed planting of transgenic and nontransgenic plants due to significantly reduced insect pressure.

Conclusions/Significance

Insect-resistance transgenes may have limited fitness advantages to hybrid progeny resulted from crop-weed transgene flow owning to the significantly reduced ambient target insect pressure when an insect-resistant GE crop is grown. Given that the extensive cultivation of an insect-resistant GE crop will ultimately reduce the target insect pressure, the rapid spread of insect-resistance transgenes in weedy populations in commercial GE crop fields may be not likely to happen.     

Targeted Integration of Single-Copy Transgenes in Drosophila melanogaster Tissue-Culture Cells Using Recombination-Mediated Cassette Exchange

Transfection of transgenes into Drosophila cultured cells is a standard approach for studying gene function. However, the number of transgenes present in the cell following transient transfection or stable random integration varies, and the resulting differences in expression level affect interpretation. Here we developed a system for Drosophila cell lines that allows selection of cells with a single-copy transgene inserted at a specific genomic site using recombination-mediated cassette exchange (RMCE). We used the φC31 integrase and its target sites attP and attB for RMCE. Cell lines with an attP-flanked genomic cassette were transfected with donor plasmids containing a transgene of interest (UAS-x), a dihydrofolate reductase (UAS-DHFR) gene flanked by attB sequences, and a thymidine kinase (UAS-TK) gene in the plasmid backbone outside the attB sequences. In cells undergoing RMCE, UAS-x and UAS-DHFR were exchanged for the attP-flanked genomic cassette, and UAS-TK was excluded. These cells were selected using methotrexate, which requires DHFR expression, and ganciclovir, which causes death in cells expressing TK. Pure populations of cells with one copy of a stably integrated transgene were efficiently selected by cloning or mass culture in ∼6 weeks. Our results show that RMCE avoids the problems associated with current methods, where transgene number is not controlled, and facilitates the rapid generation of Drosophila cell lines in which expression from a single transgene can be studied.     

Structure and function of selectable and non-selectable transgenes in maize after introduction by particle bombardment

Zea mays transformants produced by particle bombardment of embryogenic suspension culture cells of the genotype A188 × B73 and selected on kanamycin or bialaphos were characterized with respect to transgene integration, expression, and inheritance. Selection on bialaphos, mediated by thebar orpat genes, was more efficient than selection on kanamycin, mediated by thenptII gene. Most transformants contained multicopy, single locus, transgene insertion events. A transgene expression cassette was more likely to be rearranged if expression of that gene was not selected for during callus growth. Not all plants regenerated from calli representing single transformation events expressed the transgenes, and a non-selectable gene (uidA) was expressed in fewer plants than was the selectable transgene. Mendelian inheritance of transgenes consistent with transgene insertion at a single locus was observed for approximately two thirds of the transformants assessed. Transgene expression was typically, but not always, predictable in progeny plants-transgene silencing, as well as poor transgene transmission to progeny, was observed in some plant lines in which the parent plants had expressed the transgene.     

Transgene stability and dispersal in forest trees

Transgenics from several forest tree species, carrying a number of commercially important recombinant genes, have been produced, and are undergoing confined field trials in a number of countries. However, there are questions and issues regarding stability of transgene expression and transgene dispersal that need to be addressed in long-lived forest trees. Variation in transgene expression is not uncommon in the primary transformants in plants, and is undesirable as it requires screening a large number of transformants in order to select transgenic lines with acceptable levels of transgene expression. Therefore, the current focus of plant transformation is toward fine tuning of transgene expression and stability in the transgenic forest trees. Although a number of studies have reported a relatively stable transgene expression for several target traits, including herbicide resistance, insect resistance, and lignin modification, there was also some unintended transgene instability in the genetically modified (GM) forest trees. Transgene dispersal from GM trees to feral forest populations and their containment remain important biological and regulatory issues facing commercial release of GM trees. Containment of transgenes must be in place to effectively prevent escape of transgenic pollen, seed, and vegetative propagules in economically important GM forest trees before their commercialization. Therefore, it is important to devise innovative technologies in genetic engineering that lead to genetically stable transgenic trees not only for qualitative traits (herbicide resistance, insect resistance), but also for quantitative traits (accelerated growth, increased height, increased wood density), and also prevent escape of transgenes in the forest trees.     

Expression of a transgene exchanged by the recombinase-mediated cassette exchange (RMCE) method in plants

We developed a site-directed integration (SDI) system for Agrobacterium-mediated transformation to precisely integrate a single copy of a desired gene into a predefined target locus by recombinase-mediated cassette exchange (RMCE). We produced site-specific transgenic tobacco plants from four target lines and examined expression of the transgene in T1 site-specific transgenic tobacco plants, which were obtained by backcrossing. We found that site-specific transgenic plants from the same target lines showed approximately the same level of expression of the transgene. Moreover, we demonstrated that site-specific transgenic plants showed much less variability of transgene expression than random-integration transgenic plants. Interestingly, transgenes in the same direction at the same target locus showed the same level of activity, but transgenes in different directions showed different levels of activity. The expression levels of transgene did not correlate with those of the target gene. Our results showed that the SDI system could benefit the precise comparisons between different gene constructs, the characterization of different chromosomal regions and the cost-effective screening of reliable transgenic plants.     

Transgene × Environment Interactions in Genetically Modified Wheat

Background

The introduction of transgenes into plants may cause unintended phenotypic effects which could have an impact on the plant itself and the environment. Little is published in the scientific literature about the interrelation of environmental factors and possible unintended effects in genetically modified (GM) plants.

Methods and Findings

We studied transgenic bread wheat Triticum aestivum lines expressing the wheat Pm3b gene against the fungus powdery mildew Blumeria graminis f.sp. tritici. Four independent offspring pairs, each consisting of a GM line and its corresponding non-GM control line, were grown under different soil nutrient conditions and with and without fungicide treatment in the glasshouse. Furthermore, we performed a field experiment with a similar design to validate our glasshouse results.The transgene increased the resistance to powdery mildew in all environments. However, GM plants reacted sensitive to fungicide spraying in the glasshouse. Without fungicide treatment, in the glasshouse GM lines had increased vegetative biomass and seed number and a twofold yield compared with control lines. In the field these results were reversed. Fertilization generally increased GM/control differences in the glasshouse but not in the field. Two of four GM lines showed up to 56% yield reduction and a 40-fold increase of infection with ergot disease Claviceps purpurea compared with their control lines in the field experiment; one GM line was very similar to its control.

Conclusions

Our results demonstrate that, depending on the insertion event, a particular transgene can have large effects on the entire phenotype of a plant and that these effects can sometimes be reversed when plants are moved from the glasshouse to the field. However, it remains unclear which mechanisms underlie these effects and how they may affect concepts in molecular plant breeding and plant evolutionary ecology.     

Integration and inheritance of transgenes in crop plants and trees

Transgene integration and inheritance have been investigated in a number of crop plants and few tree species. Transgene integration is predominantly a random process, whether mediated by Agrobacterium or particle bombardment. Depending on the genomic position of the integrated transgene and structure of the integration site as well as copy number of the transgene in the genome, its expression may be stable or variable. Therefore, integration patterns would affect the mode of transgene inheritance in plants, regardless of the method of gene transfer. So far, both Mendelian and non-Mendelian inheritance of transgenes has been reported across several generations (T1–T3) of crop plants. In few tree species (apple, poplar, plum, and American chestnut), mostly Mendelian inheritance of the transgenes has been observed in the T1 or BC1 generations. However, detailed studies in the transgenic papaya trees showed Mendelian segregation of the transgene in the T1 generation but non-Mendelian inheritance in the T2 generation. Variation in transgene inheritance was also detected in transgenic apple and plum trees. Long generation cycles in many economically important tree species preclude investigation of inheritance of transgenes in the tree progeny. Production of early flowering trees, either by genetic modification or by environmental modulation, would facilitate the study of transgene inheritance across generations of transgenic trees. In order to overcome problems of randomness of transgene integration, targeted transgene insertions by homologous or site-specific recombination or by designer recombinases or nucleases offer prospects for stable integration of transgenes in predetermined locations in the plant genome. And perhaps, that might provide a platform for stable expression and Mendelian inheritance of transgenes in plants.     

phiC31 Integrase-Mediated Site-Specific Recombination in Barley

The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP), which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F₁, even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity.