Relevance of RNA interference (RNAi) as a new mode of action for plant protection with high specificity

Aufgrund von verschiedenen Herausforderungen und Restriktionen sind im Bereich Pflanzenschutz neue Lösungsansätze gefordert, um zukünftigen Schädlingsproblemen zu begegnen. Eine mögliche Alternative zu konventionellen chemischen Pflanzenschutzmitteln sind Produkte, die auf dem natürlicherweise in Tieren und Pflanzen vorhandenen Kontroll- und Abwehrmechanismus der RNA-Interferenz (RNAi) beruhen. Praktische Verwendung fand RNAi bisher unter anderem bei der Erzeu­gung gentechnisch veränderter Pflanzen mit Virus­resistenz und mit eingebautem Schutz gegen Insekten. In den letzten Jahren hat aber auch der Anwendungs­bereich von extern applizierten RNA-basierten Pflanzenschutzmitteln zunehmend an Bedeutung gewonnen, und einige derartige Produkte sind schon weit in der Entwicklung. Um den sicheren und nachhaltigen Einsatz dieses neuen, durch hohe Spezifität gekennzeichneten Wirkprinzips im Pflanzenschutz zu ermöglichen, sind geeignete Regulierungsstandards und angepasste Testmethoden zur Risikoanalyse notwendig.Different challenges and restrictions require new solutions for plant protection in order to meet future pest and pathogen problems. Products based on the mechanism of RNA interference (RNAi), which is naturally present in animals and plants, offer a possible alternative to conventional chemical pesticides. Up to now, RNAi has been the basis of genetically modified plants with virus resistance and with plant incorporated protection against insects. In recent years the range of applications has been extended to externally applied RNA-based products, some of which are already quite advanced in development. In order to enable the safe and sustainable use of this new highly specific mode of action for plant protection, appropriate regulations and adapted test methods for risk analysis are required

Biosafety of GM Crop Plants Expressing dsRNA:Data Requirements and EU Regulatory Considerations

The use of RNA interference (RNAi) enables the silencing of target genes in plants or plant-dwelling organisms, through the production of double stranded RNA (dsRNA) resulting in altered plant characteristics. Expression of properly synthesized dsRNAs in plants can lead to improved crop quality characteristics or exploit new mechanisms with activity against plant pests and pathogens. Genetically modified (GM) crops exhibiting resistance to viruses or insectsviaexpression of dsRNA have received authorization for cultivation outside Europe. Some products derived from RNAi plants have received a favourable opinion from the European Food Safety Authority (EFSA) for import and processing in the European Union (EU). The authorization process in the EU requires applicants to produce a risk assessment considering food/feed and environmental safety aspects of living organisms or their derived food and feed products. The present paper discusses the main aspects of the safety assessment (comparative assessment, molecular characterization, toxicological assessment, nutritional assessment, gene transfer, interaction with target and non-target organisms) for GM plants expressing dsRNA, according to the guidelines of EFSA. Food/feed safety assessment of products from RNAi plants is expected to be simplified, in the light of the consideration that no novel proteins are produced. Therefore, some of the data requirements for risk assessment do not apply to these cases, and the comparative compositional analysis becomes the main source of evidence for food/feed safety of RNAi plants. During environmental risk assessment, the analysis of dsRNA expression levels of the GM trait, and the data concerning the observable effects on non-target organisms (NTO) will provide the necessary evidence for ensuring safety of species exposed to RNAi plants. Bioinformatics may provide support to risk assessment by selecting target gene sequences with low similarity to the genome of NTOs possibly exposed to dsRNA. The analysis of these topics in risk assessment indicates that the science-based regulatory process in Europe is considered to be applicable to GM RNAi plants, therefore the evaluation of their safety can be effectively conducted without further modifications. Outcomes from the present paper offer suggestions for consideration in future updates of the EFSA Guidance documents on risk assessment of GM organisms

RNA-based biocontrol compounds:Current status and perspectives to reach the market

Facing current climate challenges and drastically reduced chemical options for plant protection, the exploitation of RNA interference (RNAi) as an agricultural biotechnology tool has unveiled possible new solutions to the global problems of agricultural losses caused by pests and other biotic and abiotic stresses. While the use of RNAi as a tool in agriculture is still limited to a few transgenic crops, and only adopted in restricted parts of the world, scientists and industry are already seeking innovations in leveraging and exploiting the potential of RNAi in the form of RNA-based biocontrol compounds for external applications. Here, we highlight the expanding research and development pipeline, commercial landscape and regulatory environment surrounding the pursuit of RNA-based biocontrol compounds with improved environmental profiles. The commitments of well-established agrochemical companies to invest in research endeavours and the role of start-up companies are crucial for the successful development of practical applications for these compounds. Additionally, the availability of standardized guidelines to tackle regulatory ambiguities surrounding RNA-based biocontrol compounds will help to facilitate the entire commercialization process. Finally, communication to create awareness and public acceptance will be key to the deployment of these compounds. © 2019 Society of Chemical Industry

RNAi:What is its position in agriculture?

RNA interference (RNAi) is being developed and exploited to improve plants by modifying endogenous gene expression as well as to target pest and pathogen genes both within plants (i.e. host-induced gene silencing) and/or as topical applications (e.g. spray-induced gene silencing). RNAi is a natural mechanism which can be exploited to make a major contribution towards integrated pest management and sustainable agricultural strategies needed worldwide to secure current and future food production. RNAi plants are being assessed and regulated using existing regulatory frameworks for GMO. However, there is an urgent need to develop appropriate science-based risk assessment procedures for topical RNAi applications within existing plant protection products legislation

Marker-assisted identification of oilseed rape volunteers in oilseed rape (Brassica napus L.) fields

Seed losses of oilseed rape (OSR) occur before and during harvest. Seeds can persist in soils for several years and often appear as volunteers in successive crops. Oilseed rape volunteers (OSRV) can harm the product quality if they emerge in subsequent OSR crops differing in fatty acid profile or other quality traits. Several factors can affect volunteer abundance. Important factors identified under controlled conditions are OSR post-harvest management (stubble tillage) and OSR variety selection in terms of the genotypic secondary dormancy potential. In the growing seasons 2009/10 and 2010/11, OSR volunteer abundance was surveyed on agricultural OSR fields in Germany. The main objective was to assess factors affecting volunteer abundance under on-farm conditions by a prediction model. Volunteer numbers were determined by two different approaches: 1. Cultivation of a semi-dwarf hybrid OSR variety, and 2. Survey of OSR volunteers between wide rows in OSR crops. Data analysis taking into account farmers´ questionnaires revealed that the factor “variety selection” was not feasible for the prediction model. As an alternative approach to assess the impact of variety selection on volunteer abundance, the genotypic origin of volunteers was investigated by DNA fingerprints using ISSR (Inter Simple Sequence Repeat)-PCR. Molecular marker analysis confirmed that OSR volunteers were to a large extent identified correctly. In four of seven selected fields, plants could be assigned to open pollinating varieties cultivated previously. In two of these fields, a high dormancy (HD) variety was found to account for a large proportion of the volunteers. In contrast, low dormancy varieties appeared only rarely as volunteers. Hybrid varieties could never be identified, due to genetic segregation in the F2 generation. Taken together, the results indicate that HD varieties substantially contribute to high OSR volunteer abundance in agricultural fields, although more data are needed to confirm this

In-field frequencies and characteristics of oilseed rape with double herbicide resistance

When growing different transgenic herbicide-resistant oilseed rape cultivars side by side, seeds with multiple herbicide resistance can arise, possibly causing problems for the management of volunteer plants. Large-scale field experiments were performed in the years 1999/2000 and 2000/2001 in order to investigate the frequencies and the consequences of the transfer of herbicide resistance genes from transgenic oilseed rape to cultivars grown on neighboring agricultural fields. Transgenic oilseed rape with resistance to glufosinate-ammonium (LibertyLink, LL) and with glyphosate resistance (RoundupReady, RR), respectively, was sown in adjacent 0.5 ha plots, surrounded by about 8 ha non-transgenic oilseed rape. The plots and the field were either in direct contact (0.5 m gap width) or they were separated by 10 m of fallow land. Seed samples taken during harvest in the transgenic plots at different distances were investigated for progeny with resistance to the respective other herbicide. It was found that outcrossing frequencies were reduced to different extents by a 10 m isolation distance. In addition to pollen-mediated transgene flow as a result of outcrossing, we found considerable seed-mediated gene flow by adventitious dispersal of transgenic seeds through the harvesting machine. Volunteer plants with double herbicide resistance emerging in the transgenic plots after harvest were selected by suitable applications of the complementary herbicides Basta® and Roundup Ultra®. In both years, double-resistant volunteers were largely restricted to the inner edges of the plots. Expression analysis under controlled laboratory conditions of double-resistant plants generated by manual crosses revealed stability of transgene expression even at elevated temperatures. Greenhouse tests with double-resistant oilseed rape plants gave no indication that the sensitivity to a range of different herbicides is changed as compared to non-transgenic oilseed rape

Pollen-mediated intraspecific gene flow from herbicide resistant oilseed rape (Brassica napus L.)

Abstract The cultivation of genetically modified (GM) herbicide resistant oilseed rape (Brassica napus) has increased over the past few years. The transfer of herbicide resistance genes via pollen (gene flow) from GM crops to non-GM crops is of relevance for the realisation of co-existence of different agricultural cultivation forms as well as for weed management. Therefore the likelihood of pollen-mediated gene flow has been investigated in numerous studies. Despite the difficulty to compare different experiments with varying levels of outcrossing, we performed a literature search for worldwide studies on cross-fertilisation in fully fertile oilseed rape. The occurrence and frequency of pollenmediated intraspecific gene flow (outcrossing rate) can vary according to cultivar, experimental design, local topography and environmental conditions. The outcrossing rate from one field to another depends also on the size and arrangement of donor and recipient populations and on the ratio between donor and recipient plot size. The outcrossing levels specified in the presented studies are derived mostly from experiments where the recipient field is either surrounding the donor field (continuous design) or is located as a patch at different distances from the donor field (discontinuous design). Reports of gene flow in Brassica napus generally show that the amount of cross-fertilisation decreases as the distance from the pollen source increases. The evidence given in various studies reveals that the bulk of GM crossfertilisation occurs within the first 10 m of the recipient field. The removal of the first 10 m of a nontransgenic field facing a GM crop might therefore be more efficient for reducing the total level of crossfertilisation in a recipient sink population than to recommend separation distances. Future experiments should investigate cross-fertilisation with multiple adjacent donor fields at the landscape level under different spatial distributions of rapeseed cultivars and different cropping systems. The level of crossfertilisation occurring over the whole field is mainly important for co-existence and has not been investigated in agricultural scale experiments until now. Potential problems with herbicide resistant oilseed rape volunteers arising from intraspecific gene flow can be largely solved by the choice of suitable cultivars and herbicides as well as by soil management