Environmental Influences on the Growing Season Duration and Ripening of Diverse Miscanthus Germplasm Grown in Six Countries.

The development of models to predict yield potential and quality of a Miscanthus crop must consider climatic limitations and the duration of growing season. As a biomass crop, yield and quality are impacted by the timing of plant developmental transitions such as flowering and senescence. Growth models are available for the commercially grown clone Miscanthus x giganteus (Mxg), but breeding programs have been working to expand the germplasm available, including development of interspecies hybrids. The aim of this study was to assess the performance of diverse germplasm beyond the range of environments considered suitable for a Miscanthus crop to be grown. To achieve this, six field sites were planted as part of the EU OPTIMISC project in 2012 in a longitudinal gradient from West to East: Wales-Aberystwyth, Netherlands-Wageningen, Stuttgart-Germany, Ukraine-Potash, Turkey-Adana, and Russia-Moscow. Each field trial contained three replicated plots of the same 15 Miscanthus germplasm types. Through the 2014 growing season, phenotypic traits were measured to determine the timing of developmental stages key to ripening; the tradeoff between growth (yield) and quality (biomass ash and moisture content). The hottest site (Adana) showed an accelerated growing season, with emergence, flowering and senescence occurring before the other sites. However, the highest yields were produced at Potash, where emergence was delayed by frost and the growing season was shortest. Flowering triggers varied with species and only in Mxg was strongly linked to accumulated thermal time. Our results show that a prolonged growing season is not essential to achieve high yields if climatic conditions are favorable and in regions where the growing season is bordered by frost, delaying harvest can improve quality of the harvested biomass.

Extending Miscanthus Cultivation with Novel Germplasm at Six Contrasting Sites.

Miscanthus is a genus of perennial rhizomatous grasses with C4 photosynthesis which is indigenous in a wide geographic range of Asian climates. The sterile clone, Miscanthus × giganteus (M. × giganteus), is a naturally occurring interspecific hybrid that has been used commercially in Europe for biomass production for over a decade. Although, M. × giganteus has many outstanding performance characteristics including high yields and low nutrient offtakes, commercial expansion is limited by cloning rates, slow establishment to a mature yield, frost, and drought resistance. In this paper, we evaluate the performance of 13 novel germplasm types alongside M. × giganteus and horticultural "Goliath" in trials in six sites (in Germany, Russia, The Netherlands, Turkey, UK, and Ukraine). Mean annual yields across all the sites and genotypes increased from 2.3 ± 0.2 t dry matter ha-1 following the first year of growth, to 7.3 ± 0.3, 9.5 ± 0.3, and 10.5 ± 0.2 t dry matter ha-1 following the second, third, and fourth years, respectively. The highest average annual yields across locations and four growth seasons were observed for M. × giganteus (9.9 ± 0.7 t dry matter ha-1) and interspecies hybrid OPM-6 (9.4 ± 0.6 t dry matter ha-1). The best of the new hybrid genotypes yielded similarly to M. × giganteus at most of the locations. Significant effects of the year of growth, location, species, genotype, and interplay between these factors have been observed demonstrating strong genotype × environment interactions. The highest yields were recorded in Ukraine. Time needed for the crop establishment varied depending on climate: in colder climates such as Russia the crop has not achieved its peak yield by the fourth year, whereas in the hot climate of Turkey and under irrigation the yields were already high in the first growing season. We have identified several alternatives to M. × giganteus which have provided stable yields across wide climatic ranges, mostly interspecies hybrids, and also Miscanthus genotypes providing high biomass yields at specific geographic locations. Seed-propagated interspecific and intraspecific hybrids, with high stable yields and cheaper reliable scalable establishment remain a key strategic objective for breeders.

Harvest Time Optimization for Combustion Quality of Different Miscanthus Genotypes across Europe.

Delayed harvest can improve the quality of miscanthus biomass for combustion and enhance the long-term sustainability of the crop, despite accompanying yield losses. The aim of this study is to identify the optimal harvesting time, which can deliver improved biomass quality for combustion of novel miscanthus genotypes at various sites across Europe, without high yield losses and without compromising their environmental performance. The relevant field trials were established as part of the European project OPTIMISC with 15 genotypes at six sites across Europe. For this study, the five highest yielding genotypes from each germplasm group and three sites with contrasting climatic conditions (Stuttgart, Germany; Adana, Turkey; and Moscow, Russia) were selected for assessment. The biomass samples were collected between August and March (depending on site) and subjected to mineral and ash content analysis. At Stuttgart, the delay in harvesting time led to a significant variation in combustion quality characteristics, such as N content (0.64-0.21%), ash content (5.15-2.60%), and ash sintering index (1.30-0.20). At Adana, the delay in harvesting time decreased the N content from 0.62 to 0.23%, ash content from 10.63 to 3.84%, and sintering index from 0.54 to 0.07. At Moscow, the impact of delay in harvesting was not significant, except for N, Mg, and ash sintering index. Overall, a delay in harvesting time improved the combustion quality characteristics of each genotype, but at the expense of yield. Yield losses of up to 49% in Stuttgart and Adana and 21% for Moscow were recorded, with variations between genotypes and sites. The harvesting time also affected nutrient offtake, which in turn influences the long-term environmental performance of the crop. The highest N, P, and K offtakes were recorded at Stuttgart for each harvesting time except for final harvest (March), where Moscow had the highest N offtake. This study describes the three criteria (biomass quality, yield losses, nutrient offtake) for determining the ideal harvesting time, which gives the best compromise between dry matter yields and biomass quality characteristics without negatively affecting the environmental performance of the crop.

Site-Specific Management of Miscanthus Genotypes for Combustion and Anaerobic Digestion: A Comparison of Energy Yields.

In Europe, the perennial C4 grass miscanthus is currently mainly cultivated for energy generation via combustion. In recent years, anaerobic digestion has been identified as a promising alternative utilization pathway. Anaerobic digestion produces a higher-value intermediate (biogas), which can be upgraded to biomethane, stored in the existing natural gas infrastructure and further utilized as a transport fuel or in combined heat and power plants. However, the upgrading of the solid biomass into gaseous fuel leads to conversion-related energy losses, the level of which depends on the cultivation parameters genotype, location, and harvest date. Thus, site-specific crop management needs to be adapted to the intended utilization pathway. The objectives of this paper are to quantify (i) the impact of genotype, location and harvest date on energy yields of anaerobic digestion and combustion and (ii) the conversion losses of upgrading solid biomass into biogas. For this purpose, five miscanthus genotypes (OPM 3, 6, 9, 11, 14), three cultivation locations (Adana, Moscow, Stuttgart), and up to six harvest dates (August-March) were assessed. Anaerobic digestion yielded, on average, 35% less energy than combustion. Genotype, location, and harvest date all had significant impacts on the energy yield. For both, this is determined by dry matter yield and ash content and additionally by substrate-specific methane yield for anaerobic digestion and moisture content for combustion. Averaged over all locations and genotypes, an early harvest in August led to 25% and a late harvest to 45% conversion losses. However, each utilization option has its own optimal harvest date, determined by biomass yield, biomass quality, and cutting tolerance. By applying an autumn green harvest for anaerobic digestion and a delayed harvest for combustion, the conversion-related energy loss was reduced to an average of 18%. This clearly shows that the delayed harvest required to maintain biomass quality for combustion is accompanied by high energy losses through yield reduction over winter. The pre-winter harvest applied in the biogas utilization pathway avoids these yield losses and largely compensates for the conversion-related energy losses of anaerobic digestion.

Progress on Optimizing Miscanthus Biomass Production for the European Bioeconomy: Results of the EU FP7 Project OPTIMISC.

This paper describes the complete findings of the EU-funded research project OPTIMISC, which investigated methods to optimize the production and use of miscanthus biomass. Miscanthus bioenergy and bioproduct chains were investigated by trialing 15 diverse germplasm types in a range of climatic and soil environments across central Europe, Ukraine, Russia, and China. The abiotic stress tolerances of a wider panel of 100 germplasm types to drought, salinity, and low temperatures were measured in the laboratory and a field trial in Belgium. A small selection of germplasm types was evaluated for performance in grasslands on marginal sites in Germany and the UK. The growth traits underlying biomass yield and quality were measured to improve regional estimates of feedstock availability. Several potential high-value bioproducts were identified. The combined results provide recommendations to policymakers, growers and industry. The major technical advances in miscanthus production achieved by OPTIMISC include: (1) demonstration that novel hybrids can out-yield the standard commercially grown genotype Miscanthus x giganteus; (2) characterization of the interactions of physiological growth responses with environmental variation within and between sites; (3) quantification of biomass-quality-relevant traits; (4) abiotic stress tolerances of miscanthus genotypes; (5) selections suitable for production on marginal land; (6) field establishment methods for seeds using plugs; (7) evaluation of harvesting methods; and (8) quantification of energy used in densification (pellet) technologies with a range of hybrids with differences in stem wall properties. End-user needs were addressed by demonstrating the potential of optimizing miscanthus biomass composition for the production of ethanol and biogas as well as for combustion. The costs and life-cycle assessment of seven miscanthus-based value chains, including small- and large-scale heat and power, ethanol, biogas, and insulation material production, revealed GHG-emission- and fossil-energy-saving potentials of up to 30.6 t CO2eq C ha-1y-1 and 429 GJ ha-1y-1, respectively. Transport distance was identified as an important cost factor. Negative carbon mitigation costs of -78€ t-1 CO2eq C were recorded for local biomass use. The OPTIMISC results demonstrate the potential of miscanthus as a crop for marginal sites and provide information and technologies for the commercial implementation of miscanthus-based value chains.

Does wheat genetically modified for disease resistance affect root-colonizing pseudomonads and arbuscular mycorrhizal fungi?

This study aimed to evaluate the impact of genetically modified (GM) wheat with introduced pm3b mildew resistance transgene, on two types of root-colonizing microorganisms, namely pseudomonads and arbuscular mycorrhizal fungi (AMF). Our investigations were carried out in field trials over three field seasons and at two locations. Serial dilution in selective King's B medium and microscopy were used to assess the abundance of cultivable pseudomonads and AMF, respectively. We developed a denaturing gradient gel electrophoresis (DGGE) method to characterize the diversity of the pqqC gene, which is involved in Pseudomonas phosphate solubilization. A major result was that in the first field season Pseudomonas abundances and diversity on roots of GM pm3b lines, but also on non-GM sister lines were different from those of the parental lines and conventional wheat cultivars. This indicates a strong effect of the procedures by which these plants were created, as GM and sister lines were generated via tissue cultures and propagated in the greenhouse. Moreover, Pseudomonas population sizes and DGGE profiles varied considerably between individual GM lines with different genomic locations of the pm3b transgene. At individual time points, differences in Pseudomonas and AMF accumulation between GM and control lines were detected, but they were not consistent and much less pronounced than differences detected between young and old plants, different conventional wheat cultivars or at different locations and field seasons. Thus, we conclude that impacts of GM wheat on plant-beneficial root-colonizing microorganisms are minor and not of ecological importance. The cultivation-independent pqqC-DGGE approach proved to be a useful tool for monitoring the dynamics of Pseudomonas populations in a wheat field and even sensitive enough for detecting population responses to altered plant physiology.

Mixtures of genetically modified wheat lines outperform monocultures.

Biodiversity research shows that diverse plant communities are more stable and productive than monocultures. Similarly, populations in which genotypes with different pathogen resistance are mixed may have lower pathogen levels and thus higher productivity than genetically uniform populations. We used genetically modified (GM) wheat as a model system to test this prediction, because it allowed us to use genotypes that differed only in the trait pathogen resistance but were otherwise identical. We grew three such genotypes or lines in monocultures or two-line mixtures. Phenotypic measurements were taken at the level of individual plants and of entire plots (population level). We found that resistance to mildew increased with both GM richness (0, 1, or 2 Pm3 transgenes with different resistance specificities per plot) and GM concentration (0%, 50%, or 100% of all plants in a plot with a Pm3 transgene). Plots with two transgenes had 34.6% less mildew infection and as a consequence 7.3% higher seed yield than plots with one transgene. We conclude that combining genetic modification with mixed cropping techniques could be a promising approach to increase sustainability and productivity in agricultural systems, as the fitness cost of stacking transgenes within individuals may thus be avoided.

Competitive performance of transgenic wheat resistant to powdery mildew.

Genetically modified (GM) plants offer an ideal model system to study the influence of single genes that confer constitutive resistance to pathogens on the ecological behaviour of plants. We used phytometers to study competitive interactions between GM lines of spring wheat Triticum aestivum carrying such genes and control lines. We hypothesized that competitive performance of GM lines would be reduced due to enhanced transgene expression under pathogen levels typically encountered in the field. The transgenes pm3b from wheat (resistance against powdery mildew Blumeria graminis) or chitinase and glucanase genes from barley (resistance against fungi in general) were introduced with the ubiquitin promoter from maize (pm3b and chitinase genes) or the actin promoter from rice (glucanase gene). Phytometers of 15 transgenic and non-transgenic wheat lines were transplanted as seedlings into plots sown with the same 15 lines as competitive environments and subject to two soil nutrient levels. Pm3b lines had reduced mildew incidence compared with control lines. Chitinase and chitinase/glucanase lines showed the same high resistance to mildew as their control in low-nutrient treatment and slightly lower mildew rates than the control in high-nutrient environment. Pm3b lines were weaker competitors than control lines. This resulted in reduced yield and seed number. The Pm3b line with the highest transgene expression had 53.2% lower yield than the control whereas the Pm3b line which segregated in resistance and had higher mildew rates showed only minor costs under competition. The line expressing both chitinase and glucanase genes also showed reduced yield and seed number under competition compared with its control. Our results suggest that single transgenes conferring constitutive resistance to pathogens can have ecological costs and can weaken plant competitiveness even in the presence of the pathogen. The magnitude of these costs appears related to the degree of expression of the transgenes.

Transgenic Pm3b wheat lines show resistance to powdery mildew in the field.

Plant resistance (R) genes are highly effective in protecting plants against diseases, but pathogens can overcome such genes relatively easily by adaptation. Consequently, in many cases R genes do not confer durable resistance in agricultural environments. One possible strategy to make the use of R genes more sustainable depends on the modification of R genes followed by transformation. To test a possible transgenic use of R genes, we overexpressed in wheat the Pm3b resistance gene against powdery mildew under control of the maize ubiquitin promoter. Four independent transgenic lines were tested in the greenhouse and the field during 3 years. The four lines showed a five- to 600-fold transgene overexpression compared with the expression of the endogenous Pm3b gene in the landrace 'Chul'. Powdery mildew resistance was significantly improved in all lines in the greenhouse and the field, both with naturally occurring infection or after artificial inoculation. Under controlled environmental conditions, the line with the strongest overexpression of the Pm3b gene showed a dramatic increase in resistance to powdery mildew isolates that are virulent on the endogenous Pm3b. Under a variety of field conditions, but never in the greenhouse, three of the four transgenic lines showed pleiotropic effects on spike and leaf morphology. The highest overexpressing line had the strongest side effects, suggesting a correlation between expression level and phenotypic changes. These results demonstrate that the successful transgenic use of R genes critically depends on achieving an optimal level of their expression, possibly in a tissue-specific way.

Gene flow in genetically modified wheat.

Understanding gene flow in genetically modified (GM) crops is critical to answering questions regarding risk-assessment and the coexistence of GM and non-GM crops. In two field experiments, we tested whether rates of cross-pollination differed between GM and non-GM lines of the predominantly self-pollinating wheat Triticum aestivum. In the first experiment, outcrossing was studied within the field by planting "phytometers" of one line into stands of another line. In the second experiment, outcrossing was studied over distances of 0.5-2.5 m from a central patch of pollen donors to adjacent patches of pollen recipients. Cross-pollination and outcrossing was detected when offspring of a pollen recipient without a particular transgene contained this transgene in heterozygous condition. The GM lines had been produced from the varieties Bobwhite or Frisal and contained Pm3b or chitinase/glucanase transgenes, respectively, in homozygous condition. These transgenes increase plant resistance against pathogenic fungi. Although the overall outcrossing rate in the first experiment was only 3.4%, Bobwhite GM lines containing the Pm3b transgene were six times more likely than non-GM control lines to produce outcrossed offspring. There was additional variation in outcrossing rate among the four GM-lines, presumably due to the different transgene insertion events. Among the pollen donors, the Frisal GM line expressing a chitinase transgene caused more outcrossing than the GM line expressing both a chitinase and a glucanase transgene. In the second experiment, outcrossing after cross-pollination declined from 0.7-0.03% over the test distances of 0.5-2.5 m. Our results suggest that pollen-mediated gene flow between GM and non-GM wheat might only be a concern if it occurs within fields, e.g. due to seed contamination. Methodologically our study demonstrates that outcrossing rates between transgenic and other lines within crops can be assessed using a phytometer approach and that gene-flow distances can be efficiently estimated with population-level PCR analyses.

Transgene x 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.