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.

Assessing environmental impact of pollen-mediated transgene flow.

Potential environmental impact caused by pollen-mediated transgene flow from commercially cultivated genetically engineered (GE) crops to their non-GE crop counterparts and to their wild and weedy relatives has aroused tremendous biosafety concerns worldwide. This chapter provides information on the concept and classification of gene flow, the framework of the environmental biosafety assessment caused by pollen-mediated gene flow, and relevant case studies about transgene flow and its environmental impact. In general, gene flow refers to the movement of genes or genetic materials from a plant population to other populations. Crop-to- crop transgene flow at a considerable frequency may result in transgene 'contamination' of non-GE crops, causing potential food/feed biosafety problems and regional or international trade disputes. Crop-to- wild/weedy transgene flow may bring about environmental impacts, such as creating more invasive weeds, threatening local populations of wild relative species, or affecting genetic diversity of wild relatives, if the incorporated transgene can normally express in the recipient wild/weedy plants and significantly alter the fitness of the wild/weedy plants and populations. It is therefore necessary to establish a proper protocol to assess the potential environmental impacts caused by transgene flow. Three steps are important for assessing potential environment impacts of transgene flow to wild/weedy relatives: (i) to accurately measure the frequencies of transgene flow: (ii) to determine the expression level of a transgene incorporated in wild/weedy populations; and (iii) to estimate the fitness effect (benefit or cost) conferred by expression of a transgene in wild/weedy populations. The recently reported case of non-random allele transmission into GE and non-GE hybrid lineages or experimental populations challenges the traditional method of estimating the fitness effect for the assessment of environmental impacts of transgene flow. Furthermore, case studies of transgenic mitigation (TM) strategies illustrate ways that may reduce the impacts of a transgene on wild/weedy populations if crop-to- wild/weedy transgene flow is not preventable, such as in the case of gene flow from crop rice to its co-occurring weedy rice.

Controlling transgene flow from engineered crops to unintended hosts by molecular approaches.

For most transgenic crops, the purported ecological risk from transgenic-host hybridization and introgression to unintended host species is negligible. Nonetheless, there remains a risk-associated focus on the potential for gene flow in the governance and regulation of crop biotechnology. Because of uncertainties in the large world of biology as well as regulatory certainties (regulations will likely not diminish), researchers and stakeholders have a great interest in eliminating or substantially decreasing gene flow from transgenic crops. To that end, numerous approaches have been investigated for limiting transgene flow via hybridization and introgression to unintended hosts. While such bioconfinement may be accomplished by ecological and management strategies as discussed elsewhere in this book, this chapter focuses on mitigating unintended gene flow from engineered crops by way of genetic engineering itself. The chapter will mainly discuss the manipulation of relatively simple means to alter plant sexual reproduction and plant growth and development to control transgene flow, with the desired outcome being the prevention of transgenes from moving and/or introgression into free-living unintended hosts. These approaches include: (i) decreasing or delaying flowering; (ii) eliminating pollen production via male sterility or selective male sterility; (iii) removing transgenes from pollen or eggs by gene use restriction technologies; and (iv) kill switches. Emerging synthetic biology approaches that may be used for transgene bioconfinement are explored. Taken together, the same molecular biology strategies that are used to improve crops can also help assure their biosafety.

Gene flow from transgenic soybean, developed to obtain recombinant proteins for use in the skin care industry, to non-transgenic soybean

Soybean has been recognized as a useful platform for heterologous protein production. This study compared the pollen characteristics of transgenic and non-transgenic soybean and investigated the rate of gene flow from transgenic soybean events, developed to obtain recombinant proteins (such as human epidermal growth factor, insulin-like growth factor 1, or thioredoxin) for use in the skin care industry, to non-transgenic soybean under field conditions, and determined the distance at which gene flow could occur. The lack of significant differences in pollen grain size, viability and pollen germination rates between transgenic and non-transgenic cultivars indicates that the overexpression of transgenes did not alter pollen characteristics in soybean. The highest rates of gene flow from the three transgenic soybean events to non-transgenic soybean ranged from 0.22 to 0.46% at the closest distance (0.5 m). Gene flow was observed up to 13.1 m from the transgenic plots. Our data fell within the ranges reported in the literature and indicate that an isolation distance greater than at least 13 m from transgenic soybean is required to prevent within-crop gene flow in soybean. As the potential markets for transgenic crops as a recombinant protein factory increase, gene flow from transgenic to non-transgenic conventional crops will become a key decision factor for policy makers during the approval process of transgenic crops. Our study may provide useful baseline data for the prevention of transgenic soybean seed contamination caused by transgene flow.

Simultaneous TALEN-mediated knockout of chrysanthemum DMC1 genes confers male and female sterility

Genome editing has become one of the key technologies for plant breeding. However, in polyploid species such as chrysanthemum, knockout of all loci of multiple genes is needed to eliminate functional redundancies. We identified six cDNAs for the CmDMC1 genes involved in meiotic homologous recombination in chrysanthemum. Since all six cDNAs harbored a homologous core region, simultaneous knockout via TALEN-mediated genome editing should be possible. We isolated the CmDMC1 loci corresponding to the six cDNAs and constructed a TALEN-expression vector bearing a CmDMC1 target site containing the homologous core region. After transforming two chrysanthemum cultivars with the TALEN-expression vector, seven lines exhibited disruption of all six CmDMC1 loci at the target site as well as stable male and female sterility at 10–30 °C. This strategy to produce completely sterile plants could be widely applicable to prevent the risk of transgene flow from transgenic plants to their wild relatives.