Development of improved perennial non-food biomass and bioproduct crops for water stressed environments

Informacje na temat projektu

WATBIO

Identyfikator umowy o grant: 311929

Projekt zamknięty

Data rozpoczęcia 1 Listopada 2012

Data zakończenia 31 Października 2017

Finansowanie w ramach

• Specific Programme "Cooperation": Food, Agriculture and Biotechnology

Koszt całkowity € 11 660 401,40

Wkład UE € 8 999 997,00

Koordynowany przez

UNIVERSITY OF SOUTHAMPTON
United Kingdom

Final Report Summary - WATBIO (Development of improved perennial non-food biomass and bioproduct crops for water stressed environments)

Executive Summary:
The over-arching aim of WATBIO was to develop new and improved germplasm for non-food perennial feedstock crops suitable for the emerging bioeconomy in Europe. Three perennial crops were chosen: Populus, Miscanthus and Arundo, and their ability to tolerate droughted environments and to maintain biomass production in water-scarce and droughted conditions was investigated. Excellent progress was made to deliver all aspects of the project and in several areas the project has moved significantly beyond the original workplan. This includes in particular, the extended development of genomic resources where a reduction in sequencing costs enabled additional research on (i) RNAseq analysis of WATBIO RNAi lines, (ii) a new innovative ‘stress memory’ experiment in Populus to be completed and (iii) additional sequencing and assembly of the Arundo and Miscanthus genomes be achieved. The collaboration between SMEs and academia has been significant and this has enabled long-term partnership and integration that will continue beyond the life of the project.
The project brought together fundamental plant cell and crop physiology, molecular biology, quantitative and high throughput phenomics and genomics alongside the skills of plant breeders and seven SMEs that spanned the whole chain of life science and agro-ecological expertise in academia. In particular, we wished to harness the powers of next generation sequencing, the project would have been impossible to propose five years before it started. These technologies were used to full effect in the project.
WATBIO identified a core set of common germplasm for each of the three species that was widely shared across partners and across the contrasting geo-climatic zones of the EU. This common germplasm was used to elucidate the cellular, molecular and physiological ideotype determining drought tolerance, defined by the consortium as ‘the ability to maintain biomass yield under moderate drought stress’. Using this germplasm, physiology was linked to the transcriptome using RNASeq and this was used, alongside extensive high throughput phenotypic data obtained in world-leading platforms at three partner sites, to determine regulatory networks underpinning the genetic architecture of drought tolerance in all three species. In both Populus and Miscanthus this resulted in the identification of a set of candidate genes for drought tolerance. In Populus these have been used to develop RNAi and over-expressed lines to test functionality of genes In Miscanthus it has resulted in the identification of a new line that performs significantly better than current commercial material in droughted conditions. In Arundo, the least developed of our target crops, a different approach was taken to develop new germplasm using mutagenesis that produced over 1,000 unique new lines that were screened to identify six promising drought tolerant and high yield lines that has resulted in two new genotypes that have an improved performance in droughted conditions compared to the current commercial material.
A significant number of activities in WATBIO involved joint activities between academia and SMEs including delivering assembled genomes, developing biotechnological routes to new germplasm development, bulking and supply of new germplasm commercially, protection of IP, sensing and monitoring drought stress in plants during field and controlled environment experiments and working along scientists to develop the long-term impact of WATBIO and in understanding the socio-economic barriers to these crops across Europe and how these might be overcome. Novel technologies developed in the project include a partnership between academia and SME that helped to test a new technology for Genotyping by Sequencing (GBS) in Populus that has enabling over 100K of SNPs markers to be identified for this crop.
WATBIO has delivered a significant amount of new knowledge and technology including 1,000 new genotypes of Arundo, 30 new transgenic lines of Populus with potentially improved drought tolerance, new drought tolerant lines of Miscanthus, the first publicly available transcriptome of Arundo, a new draft assembly of the Miscanthus genome, a significant set of field site plantings with over 10,000 Populus trees at the Alasia site in Italy, 4.1 billion DNA/RNA reads, with an average read length over 9.5 million bases.
Taken together, the WATBIO project has contributed significantly to the development of these new crops for Europe and provides an excellent starting point for future widescale deployment and demonstration of the material for the future bioeconomy of Europe.

Project Context and Objectives:
The concept of WATBIO was to develop crop plants suitable for the bio-based industries (for bioenergy, biofuel and bioproducts) that are able to maintain biomass production and quality in water-limited, marginal environments unsuitable for high yielding food crops (Allwright and Taylor, 2016). Currently, such land types would require abstracted water for irrigation in order for them to be productive. Our project moves away from high water-use, intensive agricultural systems for food to low input systems for biomass and bio-products. With the development of considerable EU activity in biorefineries, WATBIO worked to ensure that improved feedstocks would become available for these emerging bio-industries, from within Europe, thus reducing imports where sustainability criteria are harder to certify.

Our three crop types are perennial and not annual – a tree (Populus), a C4 grass - Miscanthus and a C3 grass –Arundo, and therefore likely to have a lower carbon footprint than many food crops (Rowe et al., 2011; Holland et al., 2016; Milner et al., 2016), but breeding, improvement and selection in such future crops is in its infancy with only limited progress made, despite the fact that there will be increased demand for such feedstock crops in future in the EU. WATBIO is helping to meet this requirement, in the face of a changing climate and more frequent droughts.

The project brought together a multi-disciplinary group, as necessary to tackle this problem, that links academic and SME partners and covers the whole value chain for biomass crop production from DNA and biotechnology through to the commercial production and sale of improved material. The central approach was to use new technologies, not available even five years ago at the start of the project – that of next generation DNA and RNA sequencing, to accelerate the breeding pipeline, in an innovative way, leap-frogging traditional breeding timelines, making new material available through industrial partners in the consortium. The use of next generation sequencing technologies was significant because, until recently, very few resources for breeding and improvement were available in two of these three crops (Populus and Miscanthus) and none in Arundo which is a significant barrier to fast molecular breeding (mostly informed by the allelic architecture of traits and development of molecular markers) and this was addressed in WATBIO. Given the innovative nature of the project, training a new generation of multi-disciplinary technologists was viewed as imperative and the project focussed significant effort on workshops, seminars and summer schools to address this and ensure future European innovation and competitiveness in this area.

Water scarcity (when long-term water demand outstrips water supply) and periodic droughts (changes in hydrology and precipitation that lead to soil moisture defcitis) are a growing and serious problem across many European Union Member States. The number of areas and people affected by water scarcity in the EU has increased from 6 % (1976-1990) to 13% (1991-2006) and is likely to rise further in future. The cost of drought across Europe over the past 30 years across Europe is estimated at €100 billion (EC, 2012).
WATBIO contributes to water security by moving away from irrigation to rainfed agriculture in marginal environments, making best-use of the European land resource. Deployment of perennial crops will improve the hydrology of soil systems, increasing soil re-charge and retention and minimising soil water losses and the development of salinized soils
Food, Energy and Water Demand are increasing – The Perfect Storm
Alongside water limitations, there is increased pressure on land to produce up to 50% more food by 2030 and to contribute to the growing energy gap, estimated as an increase in demand of 50% by 2030, through the development of renewable energy streams, including the use of green plants as feedstocks for heat, power and liquid biofuels and more widely in the biorefineries of the future. This ‘perfect storm’ of land required for food, energy and bio-based chemicals, in a water scarce environment where climate change is an overriding driver, means that there is an urgency to develop a range of options for future land use where food, energy and chemical requirements can be met with a sustainable use of water.

Here we proposed to develop dedicated non-food crops that can be grown on marginal land unsuitable for food crops. Green plants can offer considerable benefits over fossil fuels when used as a renewable source of energy. Globally, significant biomass resources exist. It has been estimated that between 2-22 EJ y-1 of energy could be supplied as a conservative estimate from biomass by 2025, from a technical potential of available bioenergy of 2,900 EJ (Sims et al. 2006), but only a tiny fraction of global biomass is currently utilised. Clearly, there remains significant potential to capture more energy from biomass in the future, which will be necessary within the EU, since the Renewable Energy Directive (RED, 2009) requires an increase in renewables of 20% by 2020 and for transport fuels a commitment of 10% by 2020. Meeting these ambitious targets will require considerable development of new and novel bioenergy feedstock crops and in order to meet RED, total lignocellulosic resources from trees and dedicated crops such as grasses must increase. Within this increase, GHG savings relative to fossil fuels of 35% must be met, biodiversity preserved and pristine high carbon soils avoided. With such stringent requirements, feedstocks grown within the EU are required, but these must be grown on non-agricultural soils, with limited inputs to ensure a positive LCA (life cycle analysis) and GHG balance (Whitaker, et al. 2010; Rowe et al., 2011).
WATBIO addresses the ‘food, fuel water’ dilemma by developing crops that can maintain biomass production in droughted, marginal lands unsuitable for food production, enabling EU Member States to develop home-grown feedstocks for the future bio-based industries where sustainability and certification is more easily assured
Climate Change will impose further water limitations across Europe
The IPCC has already identified water availability and quality as the main pressure on societies as they adapt to global climate change over the coming decades (IPCC, 2007). Climate change will cause significant alterations in the quality and availability of water resources, affecting many sectors including crop production, where water plays a crucial role in determining yield. Limited water availability already poses a problem in many parts of Europe and the situation is likely to deteriorate further due to climate change, with Europe’s high water stress areas expected to increase from 19% today to 35% by the 2070s (Comm. EC, 2009). Agricultural systems rely on both rain-fed soil moisture and irrigation water and both will be threatened by future climates across large areas of Europe. Not only is crop yield highly sensitive to water supply, but several ecosystem services are linked either directly or indirectly to the way in which water is managed in agricultural systems. This includes the protection of provisioning ecosystem services (food, energy and water supply), but also regulating services related to soil organic matter protection, to preservation of biodiversity and which is likely to be impacted by . Similarly, poorly managed irrigation systems in the face of reduced resource may result in salinization of soil resources. Sustainable crop production in a future European climate will require a multi-faceted approach that considers whole ecosystems and the protection of ecosystem services. WATBIO provides new strategic resources to fulfil Europe’s ambitions for adaptation to climate change in the three following ways

-By developing crops that are perennial, rather than annual, with the potential to protect soil resources in the face of climate change, particularly soil organic matter and consequent GHG emissions, that are protected in perennial systems
-By developing crops that maintain biomass production in droughted environments thus reducing reliance on irrigation agriculture
-By indirectly protecting other ecosystems services that may be threatened in future, intensive annual cropping systems in water-scarce areas of Europe, including regulating services from water quality, soil biodiversity, soil formation and water retention capacity.

Provision of new, drought tolerant biomass non-food crops, forms a critical part of the multidisciplinary strategy in Europe for wide adaptation of societies to limited water resources which will be exacerbated in the face of climate change.
WATBIO contributes to the strategic necessity across Europe for adaptation to the changing climate, producing new crops suited to water limited environments and with other benefits for ecosystem service preservation
New germplasm with improved drought tolerance for novel biomass crops
It is under these circumstances that we aim to develop new improved germplasm that can fulfill the EU requirements for increasing bioenergy and bio-based feedstocks that are in line with RED and WFD, and suited to water scarce and drought-prone conditions of growth in current and future climates. In recent years considerable progress has been made in improving our fundamental understanding of plant response to drought stress, at molecular and physiological level and this knowledge is now being deployed to develop drought tolerant food crops such as wheat. The Australian wheat breeding programme has utilized physiological information on transpiration efficiency to develop wheat lines that not only maintain growth in droughted conditions, but are able to increase yield relative to standard lines. This suggests that large advances are still possible, if we can understand in detail, the complex physiology underpinning drought tolerance, we can then interrogate this physiology and identify routes to produce better crops. Such advances are however, being completely eclipsed but another step-change in our ability to generate new, improved crops that is now underway with the advent of next generation sequencing technologies.
WATBIO links underpinning understanding of plant response to drought, to next generation sequencing technologies in three, largely understudied crops, to accelerate the production of new biomass genotypes for wide deployment in Europe
Biorefineries for the future require sustainably produced EU-grown feedstocks
Biorefineries in the future will make an important contribution to the development of a low carbon economy across Europe. Analogous to a petroleum refinery, feedstock is used to gain a multiplicity of fuel and chemical outputs from a single input of raw material, the feedstock. Biorefinering in Europe is still largely at the research and development phase, with limited commercial activity but technologies are developing fast in this area and for both thermo-chemical and biochemical routes to deployment fuel and chemical options are likely for the feedstocks crops in WATBIO. There is a recognition that the use of first generation food crops in biorefineries is a mature technology with limited scope for further development and that such crops perform poorly when considering sustainability metrics – including lifecycle GHG assessments, impacts on water and biodiversity resources and also conflicts with the production of food. This will make their continued use difficult as the Renewable Transport Fuel Obligation and RED place greater emphasis of sustainably sourced and certified biofuel feedstocks, beyond 2012.
WATBIO will produce new dedicated lignocellulosic crops suitable as sustainable EU-derived feedstocks for themo-chemical and biochemical processing routes into the future and beyond 2030
The overall objective of WATBIO was to enable accelerated breeding for drought tolerance in three novel non-food crops for Europe. Our route to achieve this was to use an innovative approach to harness the power of next generation sequencing technologies to identify both single gene-trait associations for drought tolerance alongside the capture of natural genetic variation and allelic diversity, identifying potential targets for marker assisted selection and breeding (MAS). We worked together as a twenty three partner multi-disciplinary consortium where the inclusion of 7 SMEs that span the whole chain from fundamental science and molecular sequencing, to traditional and molecular plant breeding, biotechnology, plant propagation, commercial sales of plant material and an expert in dissemination and delivery, ensured that the project achieved the overall aim of rapid deployment of new, improved plant material for non-food use suited to water stressed environments of Europe. We wish to develop more efficient breeding and genetic optimisation of these crops on unproductive land not utilised for food.

The project objectives of WATBIO were:
1. Fundamental understanding of drought signalling. To provide a better understanding of the fundamental mechanisms determining drought tolerance in Populus, Miscanthus and Arundo for the control of growth and transpiration efficiency in response to water stress (WP1, WP7)
2. A database of water-stress traits. To provide a database of ‘water stress response’ information, from laboratory to phenotyping platform to field for three novel biomass crops and quantifying these responses for a wide range of new germplasm developed in the project (WP2, WP7)
3. Innovative molecular breeding/genetic optimisation. To deploy next generation sequencing data, in 3 different, innovative approaches (RNASeq, GWAS and genetical genomics) for genetic optimisation and supply of new material in breeding programmes of partners within the consortium
(WP3, WP4)
4. Innovative genetic optimisation using GM. To deploy GM technologies to test importance of
single genes for drought tolerance, (WP3,WP5)
5. Environmental assessments of crops. To assess field performance of the new crop plants, in a range of environments, on marginal soils, spanning the European climatic envelope, to determine selection of superior genotypes with biomass production and biomass quality maintained (WP6)
6. To train the next generation of multidisciplinary non-food crop bio scientists through workshops, seminars and science exchanges and to ensure spreading of excellence between academics and SMEs (WP8)
7. To disseminate the project through 6 stakeholder workshops and additional ‘Local Fora’ meetings, both with appropriate literature and handouts with all partners involved. To accelerate dissemination through the 23 partner critical mass developed in the project, using industrial partners for spreading excellence activities (WP9/10)
8. Interaction with SWEETFUEL, DROPS, OPTIMA, OPTIMISK, and a number of other EU projects. Achieve interaction, communication and cross-fertilization of ideas with current projects on sweet sorghum, maize, perennial grasses, Miscanthus and others, including organising a joint workshop (WP9)
References
Allwright MR and Taylor G (2016) Molecular breeding for improved second generation bioenergy crops. Trends in Plant Science. 21: 43–54
Commission of The European Communities (2009) Adapting to climate change: Towards a European framework for action – White Paper. URL: http://ec.europa.eu/health/ph_threats/climate/docs/com_2009_147_en.pdf
European Commission (2012) Water Scarcity and Droughts – Policy Review. URL: http://ec.europa.eu/environment/water/quantity/pdf/non-paper.pdf
IPCC (2007) Climate Change 2007: Synthesis Report. Contribution of Working Groups I, II and III to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change [Core Writing Team, Pachauri, R.K. and Reisinger, A. (eds.)]. IPCC, Geneva, Switzerland.
Holland, RA et al. (2016) Bridging the gap between energy and the environment. Energy Policy. 92: 181–189
Milner, S et al. (2016) Potential impacts on ecosystem services of land use transitions to second-generation bioenergy crops in GB. GCB Bioenergy. 8 (2): 317–333
Renewable Energy Directive (2009) Directive 2009/28/EC of the European Parliament and of the Council of 23 April 2009 on the promotion of the use of energy from renewable sources and amending and subsequently repealing Directives 2001/77/EC and 2003/30/EC. URL: http://eur-lex.europa.eu/legal-content/en/ALL/?uri=CELEX:32009L0028
Rowe, RL et al. (2011) Potential benefits of commercial willow Short Rotation Coppice (SRC) for farm-scale plant and invertebrate communities in the agri-environment. Biomass and Bioenergy. 35 (1): 325–336
Sims, RH et al. (2006) Energy crops: current status and future prospects. Global Change Biology. 12: 2054–2076
Whitaker J et al. (2010) Sources of variability in greenhouse gas and energy balances for biofuel production: a systematic review. Global Change Biology Bioenergy. 2: 99–112

Project Results:
Overview
WATBIO brought together 23 partners, including 7 SMEs that worked together in an integrated way, using common plant material and experiments to deliver a project of high quality that has significantly advanced the knowledge and understanding of three important novel bioenergy crops. We have also identified new germplasm for the emerging bioeconomy of Europe and embedded long-term impact of the project through interactions with industry and policy makers. Our major achievements include:

- Description of the ideotype for drought tolerance in three contrasting biomass crops for Europe, that will underpin future breeding efforts for drought-prone environments
- Development of an extensive RNA transcriptomic database for all three bioenergy species
- Generation of over 1000 new lines of Arundo and identification of two genotypes with better drought tolerance than current commercial material
- Identification of two new genotpyes of Miscanthus with improved water use efficiency compared to current commercial material
- Production of 30 new genotypes of Populus using RNAi and over-expression technologies
- Production of the first publicly available transcriptome of Arundo
- Production of draft genome assemblies of Miscanthus and Arundo
- Development of a novel genotyping by sequencing method for Genome Wide Association in Populus and identification of more than 60,000 informative SNPs.
- Development of an new drought stress index as a tool for screening
- An understanding of barriers to biomass crop deployment in contrasting climatic zones of Europe


Science and Technology Results for the three biomass crops

Poplar
Trees of the genus Populus (commonly known as poplars, aspens or cottonwoods) are forest angiosperms (hardwoods), widespread throughout the northern hemisphere; with around 30 separate species (comprising six distinct sections) residing under a range of environmental conditions (Eckenwalder, 1996). Poplar’s rapid growth; extensive geographical and environmental range; wide genetic diversity; ready transformation and ultimate adoption as a model tree (Brunner et al., 2004) bestow it with great potential as a commercial crop. Industrial plantations and poplar breeding programmes for the enhancement of desirable traits exist in a number of countries where poplar wood may be used in the manufacture of paper and plywood and as a construction material (International Poplar Commission, 2012). Perhaps most excitingly however; poplar became considered an attractive prospect as a bioenergy tree; a source of lignocellulosic biomass for second generation bioethanol production (Gray et al., 2006; Sannigrahi et al., 2010). The research of WATBIO was focused on developing the native European black poplar, Populus nigra L., as a bioenergy feedstock since this is native across Europe and is also known to grow in a variety of climatic conditions from very wet to very dry, ranging from 300 to more than 1500 mm annual rainfall (Viger et al. 2016).

Transcriptomic studies elucidated the plastic response of Populus to droughtand candidate genes for drought tolerance. Extensive use in WATBIO was made of three core contrasting core genotypes of P. nigra. originating from individual trees of natural populations in France (Drôme 6; FR-6), Italy (La Zelata; IT1) and Spain (Ebro 2; SP-2; see DeWoody et al. 2015). The cuttings were planted in 10 l plastic pots filled with a 1:1 (v/v) mixture of peat and sand, amended with a slow-release fertilizer (4 g l−1 of Nutricote T100, 13:13:13 NPK and micronutrients; FERTIL S.A.S. Boulogne Billancourt, France) and 1 g l−1 CaMg(CO3)2. Plants were grown in two chambers of a fully automated glasshouse for phenotyping located at Champenoux, France (48°45'09.3'N 6°20'27.6", under natural light conditions with daily maxima of irradiance ranging from 154 to 1011 μmol m−2 s−1photosynthetically active radiation.
After 6 weeks of growth, 32 plants of each genotype were randomly assigned to either a control or a drought treatment. The plants were randomized across the two greenhouse chambers, so that each chamber contained balanced proportions of each genotype and treatment. The position of plants was changed automatically. The experimental set-up was used as a core facility that brought together more than twenty WATBIO scientists to characterize the drought ideotype for trees, leaves, wood and roots. Extensive use was made of this shared material. Wood is a renewable resource that can be employed for the production of second generation biofuels by enzymatic saccharification and subsequent fermentation. Knowledge on how the saccharification potential is affected by genotype-related variation of wood traits and drought is scarce. Here, we use populus to (i) investigate the relationships between wood anatomy, lignin content and saccharification and (ii) identify genes and co-expressed gene clusters related to genotype and drought-induced variation in wood traits and saccharification potential. The three poplar genotypes differed in wood anatomy, lignin content and saccharification potential. Drought resulted in reduced cambial activity, decreased vessel and fiber lumina, and increased the saccharification potential. The saccharification potential was unrelated to lignin content as well as to most wood anatomical traits. RNA sequencing of the developing xylem revealed that 1.5% of the analyzed genes were differentially expressed in response to drought, while 67% differed among the genotypes. Weighted gene correlation network analysis identified modules of co-expressed genes correlated with saccharification potential. These modules were enriched in gene ontology terms related to cell wall polysaccharide biosynthesis and modification and vesicle transport, but not to lignin biosynthesis. Among the most strongly saccharification-correlated genes, those with regulatory functions, especially kinases, were prominent. We further identified transcription factors whose transcript abundances differed among genotypes, and which were co-regulated with genes for biosynthesis and modifications of hemicelluloses and pectin. Overall, our study suggests that the regulation of pectin and hemicellulose metabolism is a promising target for improving wood quality of second generation bioenergy crops.

Alongside these studies on the wood transcriptome, additional analysis was undertaken together of leaf, root and woody tissue that employed network modelling to identify regulatory hubs, alongside phenotypic data. In WATBIO we built upon the array-based approaches which have been employed previously in Populus to link plant gene networks and expression profiles to key phenotypes (Street et al., 2006; Bogeat-Triboulot et al., 2007; He et al., 2014; Chefdor et al., 2006; Cohen et al. 2010; Hamanishi et al. 2010; Hamanishi et al. 2015), by using RNAseq to identify gene-phenotype networks and key regulatory gene hubs determining leaf development under drought.

As a first step analyse the RNA-seq data we performed a PCA across all samples. The plot of the first two PCs revealed a strong clustering according to tissue type, suggesting strong variation of transcript abundance among tissues. This finding prompted us to continue analysing the drought effect separated according to tissue. In all three tissues, more than half of the tested genes showed a significant (FDR = 0.05) genotype main effect. The number of DDEGs (drought related differentially expressed genes) differed among the tissues. In the developing xylem 347 genes were DE in response to drought, while in fine roots 3927 genes, and in leaf tissue 1313 genes were significantly affected by drought. There was no gene identified as DE in response to drought in all three tissue, but there were intersections of DDEGS in pairs of two of the analysed tissues. The number of genes significantly affected by a genotype-drought interaction effect was small and differed among tissues. The intersection between genes affected by a genotype-drought interaction effect and a drought main effect was one gene for fine roots and four genes for leaf tissue. While significant drought-induced physiological responses were limited to gs and WUEi, there was a clear drought effect on growth and biomass. Significant genotype-drought-interaction effects were not detected for any of these traits, and also the drought response on transcriptome profiles were highly conserved among the genotypes. These findings provide an ideal basis to focus on conserved transcriptomic responses underlying a reduction in growth and biomass accumulation under moderate drought. In order to relate the transcriptomic data to growth traits, we re-constructed transcriptional networks of DDEGs within each tissue. Within the sets of genes showing a drought main effect, three modules of co-expressed genes were identified for the developing xylem, 15 modules of co-expressed genes were identified for fine roots, and six modules were identified for leaf tissue. This approach was used to identify sets of candidate genes that determined drought response in all three tissue types and in constrating genotypes.

The physiological and growth responses were conserved across the three P. nigra genotypes studied here. The reduction in growth was accompanied by a highly tissue-specific remodelling of the transcriptome. Also the extent of the drought effect in terms of the number of DE genes differed among tissues, with fine roots being most strongly affected, while drought effects were moderate or small in the leaf and developing xylem, respectively. Similar results regarding the extent of the drought effect on roots as compared to fully-expanded leaves were reported earlier for poplar (Bogeat-Triboulot et al., 2007; Cohen et al., 2010) and willow, and it was suggested that this difference is related to the fact that fine roots as an actively growing tissue are more sensitive to water limitation. In our experiment, stem diameter growth was reduced but not abolished under drought, suggesting that the developing xylem constituted an actively growing tissue. Considering this, the stronger drought effect on fine roots might reflect that fine roots are the first organ that perceive soil water deprivation and are the place were molecular and physiological responses acting on whole-plant metabolism are initiated and coordinated (Janiak et al., 2015). Along with the conserved phenotypic responses to drought, also the transcriptomic response to drought was highly conserved across the three genotypes studied here. While Wilkins et al (2009) reported a highly species-dependent transcriptional drought response in leaves of two hybrid poplar clones, other studies on inter- (Street et al., 2006) or intra-specific (Hamanishi et al., 2010) variation of transcriptional drought responses reported evidence for conserved drought-induced changes. The conserved phenotypic and transcriptional response to drought found in our study, together with reduced but not abolished growth, provides an ideal background, and high statistical power to identify candidate genes involved in growth adjustments in response to drought in Populus.

Gene correlation network analysis revealed that stem dry matter was significantly correlated with five top genes - two unknown function. In a study on drought-related transcriptome changes in cambial and young developing xylem cells of P. alba, (Berta et al., 2010) also detected a high fraction of genes coding for protein of unknown function within the set of DE genes. The eigengene of co-expression module R-D8 was significantly negatively correlated with root dry matter and significantly positively correlated with root/shoot ratio. The homologue of the Arabidopsis gene encoding the MEI2-like protein 1 (AML1, AT5G61960) was among the genes of module R-D8 most strongly correlated to root/shoot ratio. AML1 has RNA-binding activity and was originally identified as a meiosis-signalling molecule. Another gene which was down-regulated under drought and highly negatively correlated with root/shoot ratio is annotated as a protein SOYBEAN GENE REGULATED BY COLD-2 (SRC2; homolog of Potri.010G024200). The protein encoded by the Arabidopsis best hit of this gene (AT1G09070) interacts with the NADPH oxidase RbohF and was shown to increase its Ca2+ -dependent ROS-producing activity (Kawarazaki et al., 2013). ROS producted by NADPH oxidases act as signalling molecules (Miller et al., 2010) in root growth (Monshausen et al., 2007), programmed cell death (Torres et al., 2005), and cell wall lignification (Denness et al., 2011; Hamann et al., 2009), and cell wall loosening (Janiak et al., 2015). Regulation of ROS-production via SRC2 might thus be part of a signalling network adjusting root growth under drought stress in Populus. Among the genes highly correlated to root/shoot ratio are also some down-regulated genes coding for cytochrome P450 enzymes, suggesting an altered brassinosteroid and cytokinin metabolism in the developing xylem. Moreover a gene encoding for a cellulose synthase like G3 (Potri.003G142400) protein was down-regulated under drought and highly correlated to root/shoot ratio. The eigengene of module L-D2 was significantly correlated to leaf dry matter and height increment. GO term enrichment analysis of genes assigned to module L-D2 revealed a significant over-representation of DDEGs associated with signalling, such as protein kinases, leucine-rich repeat family proteins and transcription factors. In comparison to the biomass-related modules of the developing xylem and fine roots, DDEGs of module L-D2 showed a high number of inter-modular correlations. Many results emerged from this analysis and to our knowledge this is the first report on drought-induced changes in transcriptome profiles in all major plant compartments of a woody biomass plant. One very remarkable results of our study on three genetically differentiated genotypes of Populus originating from contrasting habitats is the absence of genotype–drought interaction effects on physiological and growth traits, underpinned by absent of small interaction effects on whole transcriptome gene expression. This suggests that when exposed to a realistic, gradual progression of drought, acclimation processes lead to a conserved drought-related steady-state of metabolism and growth. We took advantage of this common response to extract novel candidate genes underlying the regulation of growth processes in Populus. Functional validation of these candidates is on-going.

Genome wide association studies (GWAS) identified the genetic control of complex traits, including drought tolerance. The phenotypic variation present within the Populus genus provides potential for selective, marker-assisted breeding to produce optimised pedigrees for bioenergy without the need for genetic engineering. This requires mapped genetic polymorphisms known to be linked with traits of interest. Within WATBIO we utilised a wide population that was developed in three previous EU Framework projects – POPYOMICS, EVOLTREE and ENERGY POPLAR to bring together nearly 1,000 unique genotypes of Populus from across Europe and to place them in a field experiment with a controlled drought treatment. The site was located in Savigliano, Italy (44.6˚N, 7.6˚E) and followed a randomised, block design with 8 blocks each containing 1 replicate of each genotype. A selection of 661 genotypes from the natural population were established however; subsequent genotyping with a 12K Illumina array (Faivre-Rampant et al., 2016) revealed 67 genotypes to be clonal replicates leaving 594 suitable for analysis of which 485 were selected for GBS (see below). The site was planted in April, 2013 and is still in place for future research on the epigenetics of drought tolerance in Populus. Drip irrigation was installed to develop well-watered and moderately droughted treatments in 2014 and 2015.

Phenotypic analysis at this site was completed intensively for a diversity panel of twenty genotypes (Smith et al., in press) and extensive, less intense phenotyping was completed on the whole set of > 600 genotypes in replicate blocks. In total more than 50,000 phenotypic datapoints were collected in this part of the project. Two main drought response strategies were considered for Populus and these can be defined as tolerance and avoidance (Marron et al., 2003; Monclus et al., 2006; Giovannelli et al., 2007). The former relies on maintaining stomatal conductance and photosynthesis, while limiting tissue dehydration through osmotic adjustment (Gebre et al., 1994; Marron et al., 2002; Barchet et al., 2013). In contrast, the latter minimises water loss through rapid stomatal closure, leaf growth inhibition and/or leaf abscission (Couso and Fernandez, 2012). The combination of traits which underlie drought responses are often adaptive and develop over generations as a consequence of environmental conditions of the native origin of a genotype, with populations such as ours exhibiting wide natural variation (Viger et al., 2016). However, alongside adaptive drought responses, plants often exhibit plasticity in their responses. Phenotypic plasticity occurs in response to changing environmental conditions (Bradshaw, 1965) and is of critical importance to plants due to their sessile nature. The extent of plasticity in response to environment is variable; dependent on both genotype and trait, and high plasticity could enable populations or individuals to survive, or maintain performance, in a rapidly changing climate (Benito Garzόn et al., 2011). To make genetic gains in Populus breeding for a future, water-scarce environment, these target traits and their plasticity must be thoroughly understood. Elucidating plastic and adaptive mechanisms of drought tolerance was the focus of the research on the diversity panel in Italy. We identified the underpinning physiological and morphological traits for drought tolerance, whereby high yields can be maintained under water deficits, in this key bioenergy species. This approach has highlighted the importance of examining the yield stability index (YSI) over the drought resistance index (DRI) to assess genotypes for performance under moderate drought. In this way, we found genotypes with high hydraulic capacity, and large leaves made up of many cells to be best suited to the multiple drought and non-stressed environments across Europe. Moreover, although water use efficiency and saccharification potential are less heritable breeding targets, we identified genotypes which combine yield, water use efficiency and saccharification potential and these are an important resource for future breeding programmes. This provides powerful evidence of the ideotype for drought tolerance in Populus and enabled our genetic research to be tightly linked to a detailed physiological understanding.

In a novel approach, we significantly extended our GWAS analysis in Populus by developing a new genotyping by sequencing methodology. Early research in WATBIO utilized a Populus genotyping chip, where we report the first coppiced biomass bioenergy study for a wide population of Populus nigra. A 12K Illumina genotyping array highlighted significant population genetic structure with pairwise FST showing strong differentiation between the Spanish and Italian subpopulations. Robust associations reaching genome-wide significance were reported for main stem height and cell number per leaf; two traits tightly linked to biomass yield. The 3 associations identified include a transcription factor; a putative stress response gene and a gene of unknown function. None of them have been previously linked to bioenergy yield and they represent exciting, novel candidates for further study in a bioenergy tree native to Europe and Euro-Asia (Allwright et al., 2015). Following this we used a new sequence capture genotyping by sequencing (GBS) using novel, single primer enrichment (SPET) technology targeting the gene space the wide P. nigra population. 57,098 markers with less than 10% missing calls were employed in population genetic analyses using principal component analysis and FST and XTX outliers to identify genes under adaptive selective pressure to geoclimate variables. 471 genotyped individuals were eventually used from the cultivated short rotation coppice (SRC) in Savigliano, Italy. Phenotyping for biomass yield and leaf development traits over 2 years in 2014 and 2015 showed wide diversity within the population. In summer 2015 drought was again imposed and the response to water stress was quantified for the first time in this population. These phenotypic and genotypic data were employed in the largest GWAS to date in P. nigra with more than 130,000 SNPs with less than 20% missing calls. Genome wide significant (p <0.05) associations were identified in 48 genes for traits related to drought tolerance, biomass yield and leaf development. Marker effect sizes ranged from 1.8 – 10.0% of phenotypic variance explained and many candidate genes were associated with more than one trait and proved robust across years and / or drought treatments. Several candidates have been previously linked with drought or stress tolerance in the literature and these are good targets for further study and advanced molecular breeding for the development of bioenergy poplar. This population had not been previously cultivated under drought conditions in the field however, Viger et al. (2016) did quantify Δ C13 in wood sampled from the Belgium site as a measure of water-use efficiency (WUE). They found that genotypes from northern Italy, Germany and the Netherlands showed the lowest values of Δ C13, suggesting higher WUE, despite originating from wetter, cooler climates than those genotypes from Spain and the south of France. In this experiment, the Spanish genotypes showed a more effective response to drought than the Italian or Dutch genotypes, able to alter their WUE under stress by more rapidly closing their stomata thus limiting leaf loss. This was consistent with field research demonstrating that WUE showed plasticity in response to seasonal variations in soil water availability within a poplar bioenergy plantation (Broeckxa et al. 2014) with a plastic response observed during an uncharacteristically dry spring as WUE increased and gross primary productivity decreased as a result of stomatal closure (Broeckxb et al. 2014). This novel GWAS analysis provided several strong targets for functional testing in RNAi and OE approaches and also identified a significant number of genes associated with climatic variable that may also be of value for introgression into future breeding programmes.

Functional analysis of Populus candidate genes using transgenic approaches In order to elucidate candidate genes we utilised a three-fold bioinformatic strategy. Firstly, RNA sequencing data (RNA-seq) generated from analysis of differential drought-tolerant lines of Populus (Grönlund et al., 2009) were used to select regulatory genes underlying the drought response. Secondly, we employed prior knowledge of drought responses in the model species Arabidopsis to select additional candidate genes especially those involved in ABA signalling (Nakashima et al., 2009). Finally, further interrogated available data gained from comparing a wide association population of P. nigra genotypes known to differ in drought tolerance (Viger et al., 2016; Allwright et al., 2016). Taken together, these three approaches were used to select candidate genes for drought tolerance in Populus. Subsequently, the expression of the target genes was modulated in transgenic plants in order to analyse the modified lines for potential improvement of drought response, and simultaneously ensure that improved drought response does not have negative impact on growth performance and wood properties.

Of these transgenic genotypes, from which expression levels of the candidate gene was analysed by quantitative PCR (qPCR), three lines (in triplicate) per construct, representing the variation in expression levels of the genotype, were tested in drought stress experiments between August and November 2016 at STT and SLU in Umeå, Sweden. These experiments screened the lines for drought responses and improvements to growth and wood properties. To this end, plants were grown in batches of 72, including wild type reference plants (Populus tremula x tremuloides, Clone T89), in a glasshouse where they were watered manually to target pot weights, which were monitored several times per day, and were tightly linked to soil moisture (RH%). Plant morphology and physiology was assessed both prior to drought and during/after the stress. Stem height, diameter and volume were measured alongside specific leaf area (SLA), leaf area and number of fully expanded leaves as used as indicators of productivity. Additionally, at the end of the experiment dry weight of the stem and leaves was assessed, as was wood density. Plant water use was measured as changes in pot weight and water use, as well as through stomatal conductance measurements and wood carbon isotope discrimination. Furthermore, plant drought response was estimated through a drought stress score system from 0-4 where 0 was normal and 4 represented a dead apical meristem. Candidate genes were tested in two plant constructs under control of two different promoters, constitutive p35s (Construct W01) and drought inducible pRD29 (Construct W11). Significant phenotypes were seen is this drought screen. For example, for one of the 11 lines, improved growth properties, i.e. improved stem volume index +26%, stem dry weight +23%, leaf dry weight +14%, wood density +8% and improved water use parameters i.e. significantly reduced carbon isotope discrimination (13C discrimination) were measured. However these responses were not always consistent across further screen and additional analysis is on-going to assess the commercial potential of this material. In summary, WATBIO has produced a significant amount of new germplasm material for Populus which in initial trials across the 11 new lines has provided several interesting leads for further analysis that is currently on-going between STT, SOTON and UGOE.

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Miscanthus
Dedicated perennial energy crops, produced on marginal land offer a sustainable alternative to fossil fuels (Cherubini et al., 2009; Crutzen et al., 2008; Dondini et al., 2009; Hastings et al., 2008; Richter et al., 2015; Zatta et.al. 2014). One such perennial energy crop is Miscanthus which exhibits a number of desirable characteristics, including high yields from low inputs (McCalmont et al., 2015). The main Miscanthus crop grown today is of a single type, M. x giganteus which is a sterile triploid hybrid of M. sacchariflorus and M. sinensis (Hodkinson et al., 2002). It has been reported that M. x giganteus exhibits poor water use efficiency (WUE) compared with some genotypes of the parental species (Clifton-Brown & Lewandowski, 2000) and that drought stress negatively impacts on its yield (Maughan et al., 2012; Price et al.,2004). In the WATBIO project we sought to understand the genetic diversity for drought tolerance and WUE in Miscanthus, and then identify potential mechanisms for drought tolerance and how they differed between genotypes.

High yielding Miscanthus genotypes were selected to examine water-stress responses.
Miscanthus is capable of producing high biomass yields but tolerance to water stress may be associated with slow and low biomass accumulation. We therefore attempted to break this link by selecting Miscanthus that produced high yield under well-watered treatments and examined how these performed under water stress. We identified two high yielding exemplars of both M. sacchariflorus and M. sinensis, which were grown in comparisons with M. x giganteus. The genotypes were in common with another EU-funded project OPTIMISC in which Miscanthus was grown at different locations throughout Europe so that it would be possible to relate a detailed understanding of drought responses under controlled conditions in WATBIO to performance in the field under different and variable natural rainfall conditions across Europe in OPTIMISC.

Different Miscanthus species exhibited different sensitivities to water stress. Two experiments were completed at different water stress levels 20% (mild) and 15% (moderate) field capacity. The lower field capacity produced a more significant treatment effect and was the focus of further analysis. High yielding exemplars of Miscanthus species differed in yield response to moderate water stress with one M. sacchariflorus and M. x giganteus showing significant treatment effects for biomass accumulation. This allowed us to compare a responsive and non-responsive biomass phenotype under different treatments.

Biomass accumulation was associated with different ideotypes under well-watered and water stress treatments. The highest correlates with biomass accumulation in Miscanthus are traits that describe growth of stems. We sought to identify a high yielding ideotype under drought. One strategy to study the maintenance of yield under water stress, was to focus on the same traits that describe the ideotype for high yield in control conditions and then ask if and how this changed under water-stress conditions. Leaf and stem development (growth rates, leaf dimensions and stem numbers), photophysiology (stomatal conductance and chlorophyll fluorescence) and biomass accumulation were examined. Under well-watered conditions biomass was adequately explained (modelled) by stem traits alone but under water stress a more complicated model was required which included leaf development and stomatal conductance, and suggested a reduction in the importance of stem traits alone. In other words, additional traits need to be optimized for achieving high biomass yields under water stress.

Cell wall composition was weakly affected and sugar ratios significantly affected by water stress treatment. Lignin content is responsive to changes in abiotic and biotic stress in many plants (Moura et al., 2010); however, we did not detect significant changes in lignin in the genotypes used and between the treatments applied in our experiment. There was a weakly significant difference in lignin content in the leaf tissue under water stress treatment in the M. sacchariflorus genotypes and the genotype showing the strongest treatment affect for biomass was also most effected. Sugar content including arabinose, fructose, galactose, glucose, mannose, xylose showed weak significance; however the ratio of arabinose and galactose was significantly affected by treatment in genotypes of both species and M. x giganteus.
Carbon isotope (13C) discrimination could not be used as a proxy for water use efficiency in Miscanthus. The differential discrimination of different carbon radioisotopes has been successfully used as a proxy for water use efficiency in C3 plants and we were interested to determine if it could be similarly used in the C4 grass Miscanthus. There was a significant effect of treatment on Δ13C across all genotypes; however, this result was not as expected for C3 plants. The results may be related to the “Leak" parameter in the C4 model but whatever the mechanism it is concluded that the 13C cannot be associated to WUE, as it is in C3 plants.

Differentially expressed transcripts were identified that were strongly associated with leaf length in water stress. Transcript expression was clustered into 8 modules which were associated with traits. The trait that had the highest association with one of the modules was leaf length. By defining the correlation between the gene and the trait the quantification of the association of the individual genes (in the cluster) with the given trait of interest was made (gene significance). One expression module was highly correlated with the leaf length (coefficient of correlation = 0.71 p < 3e-16), there was also very high positive correlation between gene significance and membership of one module. Among the 29 genes with the strongest significance for the trait and module membership, genes with known function were selected. These genes were exclusively differentially expressed in the leaves of two genotypes with a significant treatment effect for biomass accumulation under drought. The genes have putative roles in regulation of abiotic stress, cell wall synthesis and the control of phase change and represent possible target genes for further study and manipulation in Miscanthus.

High throughput phenotyping. Conventional screening for plants with improved traits is time-consuming, labour intensive and very often destructive. Automated phenotyping platforms can make genotype-phenotype studies more effective and reliable, and are capable of screening many genotypes simultaneously. Other studies have focused on grain crops; however, the maintenance of biomass yield under stress is particularly important for biomass crops, e.g. to identify genotypes that have different yield accumulation kinetics under stress. Our study combined the use of high-throughput phenomics with a diverse population of Miscanthus genotypes. The Miscanthus genus has a wide distribution in East-Asia and genotypes arising from different climates included in the study are hypothesised to differ in their response to water deficit.

Phenomics was suitable to assess drought response in a diversity panel of Miscanthus, and genotypes were identified that were not responsive to mild drought. Miscanthus genotypes were selected based on genotype, species and geographical origin. Plants were grown under well-watered and water-stressed conditions using an automated phenomics system that provided gravimetrically controlled water and functional measurement of growth via RGB images. Pixel area correlated well with actual biomass R=0.951 (fresh weight) and 0.916 (dry weight). All genotypes responded to terminal drought (as would be expected) but some genotypes were not responsive to mild drought. Digital biomass accumulation in well-watered and mild drought correlated strongly (R = 0.916) but correlations with terminal drought were lower but significant (R = 0.613) suggesting that for some genotypes performance in well-watered conditions could predict performance in extreme drought conditions. Similar comparisons replacing actual biomass for accumulated digital biomass showed no correlation between genotypes under well-watered and extreme drought (R = -0.073) suggesting that the continuous phenomics assessment provided different information to the more conventional end of experiment assessment.

WUE in a diversity population of Miscanthus increased in drought treatments, the typical correlation between low biomass and high WUE was weak in some genotypes of interest.
WUE as a ratio of yield to input of water (g kg-1) was compared across different treatments. Many genotypes varied for WUE across treatments; however some genotypes were identified where WUE did not appear to be affected by water availability. Genotypes displaying the largest variations in WUE tended to have higher accumulated biomass and genotypes displaying stable WUE across treatments tended to have lower accumulated biomass. Genotypes were also identified that maintained both WUE and reasonable biomass accumulation across all treatment conditions. Comparisons of species differences showed that M. sacchariflorus types produced above ground biomass with greater WUE.

Genotypes were identified that equaled the biomass accumulation of the commercial Miscanthus type and outperformed it under moderate water stress. The 5 core Miscanthus genotypes were grown in a large substrate volume soil-plant-air (SPA) facility that allowed controlled treatments in an experiment more similar to field growth than glasshouse trials. The genotypes selected scored in the top 10% in the phenomics analysis. The final yield from the 5 selected genotypes was analysed in detail to determine if the selected genotypes had the potential to fix carbon under drought and maintain a commercially realistic growth rate. Biomass was harvested and the design of the SPA experiment allowed total above, and below, ground biomass to be harvested as rhizome and root separately, something not previously attempted.
When grown in control conditions the two M. sacchariflorus genotypes (WAT3 and WAT4), accumulated equivalent above and below ground biomass to the commercial genotype M. x giganteus and significantly more than did the two M. sinensis genotypes. Above ground biomass measured (wet or dry weight) was significantly affected by water treatment in all genotypes except WAT3 which also maintained similar belowground biomass. For most plants the rhizome biomass, (wet or dry weight), was more significantly affected by treatment than was root biomass. It was notable that the M. sacchariflorus and M. x giganteus genotypes accumulated more biomass in rhizome than in roots and the M. sinensis genotypes accumulated more biomass in roots than in rhizome which may explain the relative tolerance to drought measured in M. sinensis. All genotypes allocated a greater proportion of biomass to roots when growing under water stress treatments but this treatment effect was only significant in M. x giganteus.

WUE was increased by treatment and negatively affected by high canopy duration in one genotype. There was a significant treatment effect on WUE for all biomass produced either aboveground, belowground, rhizome or root, from all genotypes except WAT3 which was tolerant to moderate water stress. The 3 highest yielding genotypes produced similar aboveground dry weight biomass under well-watered treatments; however, harvested biomass was produced at a lower WUE in WAT4 which reflected the high amount of water applied. This poor WUE reflected high water extraction and was probably because the canopy of WAT4 had the largest number of green leaves and this high total transpiration and low WUE suggested that the stay-green phenotype (eg as identified by phenomics) increased water use.

In summary the functional nature of the phenomics data allowed different senescence profiles to be compared, but the main advantage of the technology was to screen a large number of genotypes under controlled water stress conditions in detail which identified extremes of biomass, senescence and WUE associations. The diverse nature of the genotypes screened and the complexity of drought tolerance meant that simple correlations were difficult to identify; however, the phenomics analysis provided a rapid comparative screen to identify potential breeding candidates and responsive genotypes for further study. The experiments utilizing the new SPA facility, suggested that the M. sacchariflorus genotype WAT3 may be as productive as M. x giganteus in environments with sufficient water but will outperform it under drought.

Identifying global regulators of drought responses for genetic manipulation. There are few reports of successful transgenic manipulation in Miscanthus; however, such technology is a powerful tool to both understand basic biological processes. We used transgenesis to improve our understanding of the responses to water stress in Miscanthus. We aimed to identify potential pathways and gene products that would most likely allow us to target significant global regulators such as hormones. In addition we aimed to manipulate the cell wall structure as a potential site to produce a multifunctional product effecting both biomass processing and response to water stress.
We identified 4 gene targets for transgenic manipulation: isopentenyl transferase (IPT), squalene synthase, 9-cis-epoxycarotenoid dioxygenase (NCED1) and ferulic acid esterase A (FAE A). Manipulating the selected gene products had previously resulted in promising increases in drought tolerance (Bhatti et al., 2013, Iuchi et al., 2001; Manavalan et al., 2012; Qin & Zeevaart, 2002; Reguera et al., 2013 ; Thompson et al., 2000). Two constructs were engineered as overexpression vectors in a standard high copy number plasmid vector (IPT and FAE A), and two constructs using the pANIC system (VuNCED1 and squalene synthase) created for monocots.

Improved tissue culture for embryogenesis. An amenable embryogenic genotype of M. sinensis was identified; however, the growth and regeneration of callus was significantly less in Miscanthus when compared with other grass transformation systems, therefore to increase the number of putative transformants for screening we initially focussed on improving callus tissue culture. A series of 15 callus growth experiments were performed testing four compounds known to effect growth in the callus culture medium and then three were used in combinations. The most successful combination was assessed further in the regeneration medium. The callus and regeneration media have been further modified to take into account the additional minerals and several amino acids also evaluated. Miscanthus callus can nearly double in weight in one week on the improved callus medium which produced 4.5x as much root and shoot tissue as the same weight of control callus grown and regenerated without additives. The improved media have been successfully tested with five other amenable Miscanthus genotypes of different species.

Alternative vectors and selection were critical for transformation. Despite the improved tissue culture media the transformation rate remained low. We engineered equivalent vectors for Agrobacterium-mediated transformation as well as particle bombardment. We have determined that the selection was likely the critical factor. Previously we have successfully used bialophos in maize transformation as this was considered more reliable. By comparisons in Brachypodium transformation no positive transformants were achieved unless PPT was used. We also compared different vector backbones in Brachypodium, and the pANIC vector did not result in any positive regenerants; however, an alternative vector was successful. We had much greater success using hygromycin resistance selection and thus we have engineered a hygromycin selection marker in to the PSEE1::IPT vector and this is being transformed into Miscanthus using biolistics.

Manipulation of cell wall cross linking under drought. We were able to successfully engineer Miscanthus to overexpress the FAE A gene and two independent transformation events were bulked in the glasshouse during 2016 to to allow sufficient replicates for physiological studies in 2017. FAE activity was analysed by HPLC and identified high and low ferulic acid esterase transgenic events. Growth of the two transgenic events was compared with a non-transgenic control that had been through concurrent tissue culture. During the initial growth, stem elongation previously shown to be highly sensitive to water stress in Miscanthus (Ings et al., 2013), was measured and growth curves analysed using non-parametric characteristics calculated from the gradient of the curve and the integral (Hurtado et al., 2011). Genotypes were grown under well-watered and water stressed conditions and there was a decrease in stem elongation under drought in the control and one of the transgenics. The second transgenic event, that expressed the highest concentrations of FAE, had a more complex growth habit and plants exhibiting this event differed pre-drought. We concluded that the differences in response to moderate drought were not significantly affected by the FAE A transgene. This may not be the case when exposed to more severe treatment conditions and the transgene may improve saccharification potential and experiments to determine both are ongoing.

Comparison of experiments at different scales. In general the more controlled the experiment, the more likely a reliable and consistent treatment effect will be achieved. However, such experiments may approximate poorly to field performance where soil volume and light and temperature may be very variable. The use of the same 5 core genotypes across all experiments in WATBIO allowed an assessment of consistency across scales. One approach compared drought indices across experimental scales. A drought index relating yield under control and mild drought treatment was calculated that normalised data using the population yield (Fisher & Maurer 1978). Using simple correlations of drought indices most correlations were considered moderate to high but calculated p-values showed that the significance was low at up to 0.15. Overall the comparisons suggested that if a treatment effect was significant, such as the comparisons between field data and the phenomics experiments, the more rapid experiment correlated well with field performance. However in more complex environments in which a treatment effect was less significant, or in more established trials with older plants the correlations were less reliable. We next tested a mixed model which compared the contributions of experimental variables and showed that pot size was the most significant contributor to variation between experiments. This may be expected since we earlier showed that all core genotypes responded to water stress by increasing relative root production and the effectiveness of this response was correlated with the volume of soil available to the roots.

Growth curves represent a useful measure of yield development in biomass crops. Unlike annual or seed based crops, grown for a short period sufficient to generate an optimal harvest index, perennial biomass crops represent the integral of seasonal available resource capture and the ability to grow for longer has been demonstrated to correlate with improved yield (Robson et al., 2013). The functional nature of growth curves allows equations to be fit to data to facilitate statistical comparisons. To allow a more consistent fit, non-parametric methods were applied to datasets that have a sigmoid shape. This method has allowed the identification of many characteristics including peak growth rates, inflection points corresponding to differentials and summary statistics such as area under the curve (Hurtado et al., 2011). Stem elongation was measured as the length of stem from the base (at soil level) to the uppermost differentiated ligule and was measured across the entire growing season from early emergence to late senescence. The same measurements were applied to a single plant field growth experiment and a more controlled experiment in the SPA facility. Visual inspection of the growth curves indicated that the treatment effect was more significant in the SPA facility than in the field. Responsive and non-responsive genotypes were consistent between the two experiments: early growth characteristics did not differ between plants, but diverged after treatment and later in the growth analysis. Growth curves from one M. sacchariflorus genotype in the SPA facility indicated that the regulation of start and end of growth was not affected by treatment nor was the maximum growth rate recorded but the time at which this was achieved (ipoint) and later characteristics of the decline in growth were significantly different (P = 0.061) and this was not refleected in the second M. sacchariflorus genotype. In comparing growth curves across different experiments (field vs SPA) most of the significant differences in characteristics reflected the difference in stem growth and the cessation in stem growth but indicated that initiation of stem growth was not significantly different between genotypes. In all species, the timing of the maximum growth rate was delayed in well-watered treatments.

To model yield and performance in different environments and across potential future climate scenarios we need to improve growth models and produce genotype specific response surfaces across parameters that contribute to yield accumulation. An existing growth model for Miscanthus (Davey et al., 2017) includes components such as the interception of light over the season and the efficiency with which that light is turned into harvested biomass but these components of the model had not previously been parameterised with empirical data but were inferred from yield and correlations with meteorological data. We used field data of light interception by plots combined with and SPA data of more controlled and varied treatment effects to produce genotype specific parameterisation of the model. We detected small differences in the development of leaf area index between treatments but no significant effect of treatment on radiation use efficiency although the relationship between radiation intercepted and biomass accumulated was different between genotypes. The lack of treatment effect may reflect the wet summer conditions in the location of the field trial and therefore that rainfed and irrigated plots were only marginally different during the early season but were significantly different late season. We improved the accuracy of the modelled relationships by using non-linear equations rather than following previous approaches which used a fit of two linear equations, this was particularly important when water stress occurred late in the season when the more complex equations provided a more accurate fit. In the more controlled conditions available in the SPA we were able to empirically model the relationships between photophysiological parameters and leaf water potential and showed for example that the cessation of maximum photosynthetic rate when measured across different water potentials occurred at the lowest water potential in a genotype that rapidly senesced in response to water stress.

References
• Bhatti et al. (2013) Transgenic tobacco with rice FAE gene shows enhanced resistance to drought stress. Pak. J. Bot. 45: 321-326.
• Cherubini et. al. (2009). Energy- and greenhouse gas-based LCA of biofuel and bioenergy systems: Key issues, ranges and recommendations. Resources, Conservation and Recycling 53: 434-447.
• Clifton-Brown & Lewandowski (2000). Water use efficiency and biomass partitioning of three different Miscanthus genotypes with limited and unlimited water supply. Annals of Botany 86: 191-200.
• Crutzen et al. (2008). N2O release from agro-biofuel production negates global warming reduction by replacing fossil fuels. Atmospheric Chemistry and Physics 8: 389-395.
• Davey et al. (2017) Radiation capture and conversion efficiencies of Miscanthus sacchariflorus, M. sinensis and their naturally occurring hybrid M. x giganteus. GCB Bioenergy 9: 385-399.
• Dondini et al. (2009). The potential of Miscanthus to sequester carbon in soils: comparing field measurements in Carlow, Ireland to model predictions. GCB Bioenergy 1: 413-425.
• Fischer & Maurer (1978) Drought resistance in spring wheat cultivars. I. Grain yield response. Aust. J. Agric. Res. 29: 897-907.
• Hastings et al. (2008). Potential of Miscanthus grasses to provide energy and hence reduce greenhouse gas emissions. Agronomy for Sustainable Development 28: 465-472.
• Hodkinson et al. (2002). Characterization of a genetic resource collection for Miscanthus (Saccharinae, Andropogoneae, Poaceae) using AFLP and ISSR PCR. Annals of Botany 89: 627-636.
• Hurtado et al. (2011). Dynamics of senescence-related QTLs in potato. Euphytica 183: 289-302.
• Ings et al. (2013) Physiological and growth responses to water deficit in the bioenergy crop Miscanthus x giganteus. Frontiers in Plant Sci. 4: 468.
• Iuchi et al. (2001) Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis. Plant J. 27, 325-333
• Li et al. (2000). Drought responses of Eucalyptus microtheca provenances depend on seasonality of rainfall in their place of origin Chunyang. Aust. J. Plant Physiol. 27: 231-238.
• Lloyd & Farquhar (1994). C13 discrimination during CO2 assimilation by the terrestrial biosphere. Oecologia 99: 201-215.
• Mäkelä et al. (1996). Optimal control of gas exchange during drought: Theoretical analysis. Annals Of Botany 77: 461-467.
• Malinowska et al. (2017). Phenomics analysis of drought responses in Miscanthus collected from different geographical locations. GCB Bioenergy 9: 78–91.
• Manavalan et al. (2012) RNAi-mediated disruption of squalene synthase improves
drought tolerance and yield in rice J. Exp. Bot. 63 : 163-175.
• Maughan et al. (2012). Miscanthus × giganteus productivity: The effects of management in different environments. GCB Bioenergy 4: 253-265.
• McCalmont et al. (2015). Environmental costs and benefits of growing Miscanthus for bioenergy in the UK. GCB Bioenergy 9: 489-507.
• Moura et al. (2010) Abiotic and biotic stresses and changes in the lignin content and composition in plants. J. Integr. Plant Biol. 52: 360-376.
• Munné-Bosch et al. (2001) Drought-induced senescence is characterized by a loss of antioxidant defences in chloroplasts. Plant, Cell and Environment 24: 1319-1327.
• Price et al. (2004). Identifying the yield potential of Miscanthus x giganteus: an assessment of the spatial and temporal variability of M-x giganteus biomass productivity across England and Wales. Biomass & Bioenergy 26: 3-13.
• Qin & Zeevaart (2002) Overexpression of a 9-cis-epoxycarotenoid dioxygenase gene in Nicotiana plumbagenifolia increases abscisic acid and phaseic acid levels and enhances drought tolerance. Plant Physiol 128 : 544-551.
• Reguera et al. (2013) Stress-induced cytokinin synthesis increases drought tolerance through the coordinated regulation of carbon and nitrogen assimilation in Rice. Plant Physiol. 163 : 1609-1622.
• Richter et al. (2015). Sequestration of C in soils under Miscanthus can be marginal and is affected by genotype-specific root distribution. Agriculture, Ecosystems & Environment 200: 169-177.
• Robson et al. (2013). Variation in canopy duration in the perennial biofuel crop Miscanthus reveals complex associations with yield. J. Exp. Bot. 64: 2373–83.
• Thomas (2012) Senescence, ageing and death of the whole plant. New Phytologist 197: 696-711.
• Thompson et al. (2000) Ectopic expression of a tomato 9-cis-epoxycarotenoid dioxygenase gene causes over-production of abscisic acid. Plant J. 23 : 363-374.
• Zatta et al. (2014). Land use change from C3 grassland to C4 Miscanthus: effects on soil carbon content and estimated mitigation benefit after six years. GCB Bioenergy 6: 360-370.

Arundo

Arundo donax is a highly promising biomass crop for areas with warm to hot climates such as the Southern Mediterranean. However, at the initiation of the WATBIO, Arundo was the least developed of the WATBIO crops, with very limited genomic data, no RNAseq, no genome assembly and few pre-breeding resources. There was also limited information on physiological performance and genetic diversity.

WATBIO discovered that like many biomass crops, the rapid growth of A. donax is sustained by high photosynthesis (PN) alongside elevated rates of transpiration. This high water demand may render A. donax unsuited to growth in drought-prone rain-fed marginal lands. A central aim of WATBIO was “to investigate the complex interactions between molecular pathways of signalling drought stress with those controlling cell and organ growth and to compare the responses of selected genotypes at different levels of complexity from the cell to tissue quality and plant architecture”. To this end, WATBIO has made great progress in elucidating the genetic, biochemical, physiological and morphological responses of A. donax to growth under drought stressed conditions. WATBIO members have conducted pot, rhizotron and field-based studies of the effect of drought stress on A. donax. These studies have confirmed previous observations regarding the potential rapid growth, and high rates of PN and stomatal conductance (Gs) of A. donax. Moreover, this work has demonstrated that A. donax still produces high levels of biomass under rain-fed conditions, making it a viable biomass crop for semi-arid warm to hot regions.

Physiological Assessment in Arundo. Prior to WATBIO, comparatively little in-depth research had been undertaken into the physiological adaptation of A. donax to drought stress. To establish the degree of variation in drought response that could be found in A. donax, two ecotypes were collected from contrasting habitats: a semi-arid pre-desert in Morocco and a warm-humid sub-Mediterranean region of Central Italy. The Moroccan and Italian ecotypes showed identical rates of PN, Gs and emission of the protective volatile organic compound isoprene on a leaf area basis [1]. Analysis of 40 ecotypes of A. donax collected from diverse habitats in Southern Italy and grown in common garden conditions also suggested a distinct lack of variability in photosynthetic and photo-protective physiology measured using gas exchange or chlorophyll fluorescence techniques [2]. These findings are consistent with genetic analyses suggesting low diversity in A. donax due to its sterility and clonal reproduction [3].

The greater biomass yield of A. donax in comparison to other biomass crops [4] is likely associated with high photosynthetic capacity. To prevent excessive transpiration resulting in desiccation, A. donax exhibits a high degree of stomatal control. Moreover, the physiological stomatal behaviour of A. donax is modified under drought stress, becoming more sensitive to [CO2] and light [5]. The degree of stomatal control in A. donax that was characterised by WATBIO may be useful in increasing water use efficiency and capacity for gas exchange in crops such as rice (Oryza sativa) with low leaf-level PN and Gs [6].

Biochemical Assessment in Arundo. The rapid growth and tolerance of drought observed in A. donax are also associated with biochemical adaptations. Under drought-stress, the concentration of abscisic acid (ABA) increases in the leaves of many plants, resulting in reduced Gs and transpirative water-loss [7]. In A. donax grown under rain-fed and irrigated field conditions, the foliar concentrations of inactive fixed glycosylated-ABA, biologically active free-ABA and ABA-breakdown products (phaseic and dihydrophaseic acid) rises [2]. The increase in foliar free-[ABA] modified stomatal physiological behaviour in A. donax allowing for improved water use efficiency. Modification of stomatal ABA sensitivity may enable production of more drought tolerant and productive varieties of A. donax [5].

The emission of isoprene stabilises and protects plant photosynthetic membranes [8]. Under well-watered conditions, isoprene is derived from sugars recently formed by PN. Emission rates of isoprene are usually maintained under drought-stress, but the isoprene formed is derived from stored carbohydrates [9]. A similar stability of isoprene emission rates under control and severe drought stress was found in A. donax [2]. However, under less severe moderate drought-stress, which may be more representative of conditions likely to occur in the field [10], the emission of isoprene in A. donax was stimulated [11]. This also coincided with increased synthesis of dimethylsulphoniopropionate (DMSP) in the leaves of A. donax. DMSP is the pre-cursor to the volatile organic compound dimethyl sulphide (DMS), a major component in the global sulphur cycle. The observation of increased DMSP and isoprene formation in A. donax is counter to previous suggestions that the methionine and methyl-erythriol pathways (respectively responsible for the synthesis of DMSP and isoprene) operate in antagonism to one another [12]. The work of WATBIO has provided valuable insights into the operation of the methionine pathway in regulating plant response to abiotic stress.

Improved Genetics and Genomics in Arundo. The genetics of A. donax are extremely complex due to its hexaploid genome. One of the major breakthroughs associated with WATBIO is the first characterisation of the genome of A. donax by IGA Technology Services (IGATS). The genome of A. donax is composed of three ancestral sub-genomes allowing an understanding of the origins of the plant, its success as an invasive non-fertile species and the potential to modify A. donax to enhance growth and drought tolerance. Analysis of RNA expression in A. donax under drought stress has also elucidated the adaption of A. donax to water deficit and the biochemical regulation of growth and protective mechanisms such as isoprene synthesis [13].

The lack of genetic/ecotypic variation in A. donax may limit the potential to exploit genetic variability to enhance yield and/or drought tolerance using traditional breeding approaches. Exposure of A. donax cell cultures to γ-radiation resulted in the production of ~1000 A. donax mutants [14]. One of the mutations (UniBO3) was associated with increased numbers of stems – a key attribute in the biomass yield of A. donax [15]. Comparison of the mutant to a wild-type A. donax under well-watered and drought stressed conditions suggested no difference in leaf-level PN, but greater number and height of stems in the UniBO3 mutant. The collection of A. donax mutants produced for WATBIO will serve as a major resource in the commercial development of A. donax as a viable biomass crop.

Morphology of Arundo. Field trials of A. donax in Catania, Sicily (a collaboration between the EU FP7 projects WATBIO and OPTIMA) suggest that A. donax produces high yield under both rain-fed and irrigated conditions. Indeed, in 2016 the mean detriment to yield incurred by plants grown without supplementary irrigation was 14%. Later in the year when soil water content rose as precipitation increased, the plants grown under rain-fed conditions maintained photosynthetic capacity, while the plants from the irrigated treatment exhibited drown-regulation of photosynthetic capacity. In effect, the rain-fed plants experienced a longer growing season, allowing them to ‘catch-up’ with their irrigated counterparts. This suggests that the physiological adaptations of A. donax to drought make it a highly suitable species for growth in drought-prone rain-fed marginal lands.

Taken together, WATBIO has produced a significant amount of new pre-breeding information on Arundo. This information is highly novel and relevant to the commercial development of this specia as a bioenergy crop. At the same time, the project has identified a new genotype of relevance for commercialisation that produced more biomass than the current commercial genotype under droughted conditions.

References
1. Haworth M, Centritto M, Giovannelli A, Marino G, Proietti N, Capitani D, et al. Xylem morphology determines the drought response of two Arundo donax ecotypes from contrasting habitats. GCB Bioenergy. 2017;9:119-31. doi: 10.1111/gcbb.12322.
2. Haworth M, Cosentino SL, Marino G, Brunetti C, Scordia D, Testa G, et al. Physiological responses of Arundo donax ecotypes to drought: a common garden study. GCB Bioenergy. 2017;9:132-43. doi: 10.1111/gcbb.12348.
3. Pilu R, Cassani E, Landoni M, Badone FC, Passera A, Cantaluppi E, et al. Genetic characterization of an Italian giant reed (Arundo donax L.) clones collection: exploiting clonal selection. Euphytica. 2014;196(2):169-81.
4. Mantineo M, D’Agosta GM, Copani V, Patanè C, Cosentino SL. Biomass yield and energy balance of three perennial crops for energy use in the semi-arid Mediterranean environment. Field Crops Res. 2009;114(2):204-13. doi: http://dx.doi.org/10.1016/j.fcr.2009.07.020.
5. Haworth M, Cosentino SL, Marino G, Brunetti C, Riggi E, Avola G, et al. Increased free abscisic acid during drought enhances stomatal sensitivity and modifies stomatal behaviour in fast growing giant reed (Arundo donax L.). Environ Exp Bot. 2017:In Press.
6. Lauteri M, Haworth M, Serraj R, Monteverdi MC, Centritto M. Photosynthetic diffusional constraints affect yield in drought stressed rice cultivars during flowering. PloS One. 2014;9(10):e109054.
7. Zhang J, Davies W. Changes in the concentration of ABA in xylem sap as a function of changing soil water status can account for changes in leaf conductance and growth. Plant, Cell and Environment. 1990;13(3):277-85.
8. Velikova V, Loreto F. On the relationship between isoprene emission and thermotolerance in Phragmites australis leaves exposed to high temperatures and during the recovery from a heat stress. Plant Cell and Environment. 2005;28(3):318-27. PubMed PMID: ISI:000227087600005.
9. Brilli F, Barta C, Fortunati A, Lerdau M, Loreto F, Centritto M. Response of isoprene emission and carbon metabolism to drought in white Poplar (Populus alba) saplings. New Phytol. 2007;175(2):244-54.
10. Yan W, Zhong Y, Shangguan Z. A meta-analysis of leaf gas exchange and water status responses to drought. Scientific reports. 2016;6.
11. Haworth M, Catola S, Marino G, Brunetti C, Michelozzi M, Riggi E, et al. Moderate drought stress induces increased foliar dimethylsulphoniopropionate (DMSP) concentration and isoprene emission in two contrasting ecotypes of Arundo donax. Frontiers in Plant Science. 2017;8:1016. doi: 10.3389/fpls.2017.01016. PubMed PMID: WOS:000403308100002.
12. Dani KGS, Loreto F. Trade-Off Between Dimethyl Sulfide and Isoprene Emissions from Marine Phytoplankton. Trends Plant Sci. 2017;22(5):361-72. doi: 10.1016/j.tplants.2017.01.006.
13. Evangelistella C, Valentini A, Ludovisi R, Firrincieli A, Fabbrini F, Scalabrin S, et al. De novo assembly, functional annotation, and analysis of the giant reed (Arundo donax L.) leaf transcriptome provide tools for the development of a biofuel feedstock. Biotechnology for Biofuels. 2017;10(1):138.
14. Valli F, Trebbi D, Zegada-Lizarazu W, Monti A, Tuberosa R, Salvi S. In vitro physical mutagenesis of giant reed (Arundo donax L.). GCB Bioenergy. 2017;9(8):1380-9. doi: 10.1111/gcbb.12458.
15. Cosentino SL, Copani V, D’Agosta GM, Sanzone E, Mantineo M. First results on evaluation of Arundo donax L. clones collected in Southern Italy. Industrial Crops and Products. 2006;23(2):212-22.


WATBIO interactions between academia and industry

The European Union has a significant commitment to SMEs, (small-medium enterprises), where they are recognised as determining a large share of employment and GDP in European economies, emerging or mature and because all great things start small, especially in present times when flexibility is a must. The EU also wants science and innovation to be a pulsing component of industry, since industry will strongly benefit from applied research, even more when science is supplied side-by-side, when academia sits in the same room, within the same project, with industry.

The advancement of knowledge in the sustainable management, production and use of biological resources will provide the basis for safer, eco-efficient and competitive products and services for agriculture, food, health, forest-based and related industries. Important contributions to the implementation of existing and prospective policies and regulations in public, animal and plant health and consumer protection are anticipated. New renewable energy sources will be supported under the concept of a European knowledge-based bio-economy. Within this framework, and in particular the plant sector to which the EU project WATBIO applies, we see that while the private sector primarily focuses on classical cash crops where markets are large, the public sector is often left to develop biotechnologies foremerging new crops such as those that were the subject of study in WATBIO, or for crops that are developing for a new market. Within WATBIO, i, a mix of different plants, from the highly valuable Poplar to the almost unknown Arundo donax, were chosen to search for candidates to produce biofuels putting directly in contact academia with seven different SMEs.

The seven SMEs within WATBIO cover the whole value scope of the project and include:

• two biotechnology companies engaged in the rapid propagation and supply of enhanced plant material to grow bioenergy crops in marginal environments (STT, GeneticLab) ;
• one company developing sensor technologies to sense and monitor plant performance (YARA ZIM Plant technology) ;
• one sequencing company that has developed out of academia, but now supplies the most advanced sequencing capacity and can compete in an global market (IGATS) ;
• one independent scientist providing input on agri-environmental policy and communications (DMB) ; and
• two companies for the breeding and supply of improved bioenergy feedstocks (Alasia, Kai-Uwe Schwarz)

The development of a European bio-based industry sector is expected to open the way for innovations and effective technology transfer, aiming to include all industries and economic sectors that produce, manage and otherwise exploit biological resources as well as related services from the supply or consumer industries. These activities are in line with the European strategy on life sciences and biotechnology and is expected to promote competitiveness of European agriculture and biotechnology, seed and food companies and in particular high-tech SMEs, while improving social welfare and well-being. WATBIO was well-represented by this sector and the WATBIO SMEs benefitted from immediate access to the latest findings of the group.

Several aspects of WATBIO involved the collaborative working between industry and academia. These included:

• Collaboration of IGATS across the whole academic consortium in the supply of sequencing data, technology and skills for analysis of complex next generation sequencing data on non- model crops. Eight partners from WATBIO worked extensively with IGATS on DNA sequencing, RNA sequencing, genome assembly, bisulphite DNA sequencing, RNAseq transcriptome analysis, genotype by sequencing, eQTL and genetical genomics approaches. IGATS also shared data quickly with external partners to ensure rapid success for WATBIO, for example in developing a draft assembly of Miscanthus in collaboration with UK Earlham Institute.

• Collaboration with YARA ZIM – ZIM probes to asses plant water status were deployed in several WATBIO experiments where ZIM employees worked alongside researchers. These included: twice at the Italian field site on Populus, in the UK on Miscanthus and in France and Germany on Populus.

• Collaboration between UNIBO and GeneticLab on the development of mutagenesis and the propagation of Arundo germplasm – these two partners worked extensively together.

• Collaboration between AU-IBERs and SCHWARTZ in the supply of bulked plant material by SCHWARTZ to Au-IBERS, in joint field research and the development of the SPA system to underpin modelling and in the execution of joint field research and assessment of new germplasm.

• SweTree Technologies STT collaborated extensively with UGOE and SOTON in determining the candidate genes for testing and development of RNAi and OE lines of Populus. This collaboration is on-going.

• ALASIA collaborated intensively with several partners of the project in the supply of a field site for extensive drought tolerance research where more than 10,000 Populus and several hundred Arundo were planted. This collaboration will have long-term impact since ALASIA, SOTON, IGATS and UNITUS will continue to work together at this site on adaptive genotypes for a drought-prone Europe.

• DMB as an independent scientist and research policy specialist worked extensively with all partners to ensure scientific opportunities were developed in the context of commercial drivers and to ensure the long-term impact of the project scientific outcomes.

The delivery of the research in relation to SME participants’ needs and the exploitation of the outputs by SMEs, including Intellectual Property, was also well coordinated and facilitated in WATBIO. In collaboration with another EU project, BIOINNO, WATBIO offered a course entitled “A Knowledge Alliance for Biotech Entrepreneurship Education” to link students and early career researchers (ECRs) to industry providing also the necessary skills and education needed to work along with industry.
An important aim of the project was to ensure that these exploitation activities were complementary, mutually supporting, and supported by a common set of communication materials. As set out in the Consortium Agreement, WATBIO research has been published in the academic press to assure the quality of the research as a whole and to ensure the project team’s work in characterised by the rigour and depth of analysis this involves.

One of the project objectives was to train the next generation of multidisciplinary non-food crop bio-scientists through workshops, seminars and science exchanges and to ensure spreading of excellence between academics and SMEs. Large complex problems of global significance and wide societal concern, including the energy gap, climate change, and increasing population all require multidisciplinary approaches if we are to find solutions. However, training in many universities and institutes remains strongly focussed upon a single or a few disciplines – e.g. genetics, plant science and computing. Researchers and developers who understand the socio-political context of their work are relatively rare. Such understanding is essential to successfully linking research to practice and to the future leadership of research of high socio-political impact. Within WATBIO we aimed at ensuring that our researchers had a full exposure to the whole range of technologies that was required for future plant breeding and deployment of biomass crops, using advanced biotechnological approaches and vast sets of sequence data. The work also sought to ensure that the researchers were fully aware of the wider context of their research and how their research can deliver wider impact. How this links to the field implementation of crops and difficulties associated with cost supply chain, protection of Intellectual Property and Systems Biology approaches was integrated within WATBIO.

The WATBIO partner SweTree Technologies is a world leading provider of biotechnology products for forestry and offers an opportunity to commercialise outputs from the reverse genetic approaches. These include through the breeding of eucalyptus. The production of more than thirty original transgenic lines in the reverse genetics approach taken within WATBIO is highly novel and there is potential for the direct commercialisation of this material from WATBIO.

The WATBIO project has provided a supportive environment for translational research and genuine and long-lasting collaborative activities are now fully embedded in our activities. For example, the YARA ZIM probes have been used in three separate experimental campaigns in Poplar droughted conditions. Valuable insight has been gained into the performance of these sensors on Poplar from the project and adjustments to protocols made accordingly.

Finally, one of the main results of integrating science with academia and industry working together was already designed in the project appointing to a SME, IGA Technology Services, the responsibility of one of the crucial Workpackages of the project, WP3, the Next Generation Sequencing WP. Leading the WP gave this SME the possibility to monitor the whole process from experimental design, to samples shipment, to sequencing, to data handling and shipping and results delivery. Moreover, this SME got the possibility to get in contact with several partners of the project interested in its activity, demonstrating its products and skills and finally creating new business with them. Similar situations arose also for some of the other SMEs involved in the project.

WATBIO contribution to skills and education

WATBIO brought together a knowledge and innovation community through partnership across Europe. This community that connects academia with business was instrumental in developing and delivering an Innovative Learning and Training Model (ILTM) and creating an effective route to education to ready the next generation of talents to advance research and innovation and to develop scientific, technical and entrepreneurial capacities and mindsets in biotechnology, bioenergy and the future bio-based sector in Europe. WATBIO’s ILTM relied on structured programmes to increase knowledge sharing through ‘skills workshops’ and ‘intensive training summer schools’ linked to other European networks specifically designed for graduates and postgraduate students as well as early career researchers (ECRs) both from Academia and Industry, and to better benefit both Universities and SMEs. It provided also the educational prerequisites needed to enable researchers to develop their own-ideas and build self-confidence, and to foster the development of entrepreneurship initiatives in Europe. This was achieved during the sixty months through elite scientific, technical and entrepreneurial programs, crash courses, summer schools and information days involving eight EU countries. As part of its mandate to educate its students, the WATBIO’s ILTM paired an enriching educational experience with the skills and relationships of some of the EU successful SMEs in next-generation genotyping and phenotyping technologies, new breeding targets (new varieties, water use efficiency, yield/input, and new crops), and new biomass-based products.

The skills and education programme’s outputs ranged from activities, to events, and services that reach people. For example, this model has been successfully implemented through six ‘skills workshops’ and two ‘training summer/winter schools’, for which researchers were awarded a certificate of completion upon their successful fulfilment of training and attendance requirements.

By complementing the supply of scientific ideas from the academic community with demand signalled from users of research, we focused training on key research outcomes relevant to improving bioenergy crop breeding to accelerate the development of new crops for non-food biomass for water stressed environments, and to empowering Europe’s young bioinnovators in plant biotechnology and the bioeconomy. Throughput the life of the project, the consortium worked together effectively to ensure a smooth operation and to accomplish the program’s objectives by delivering six ‘skills workshops’ (D8.1) across Europe. These were:
• A first skills workshop on ‘Improving Water Stress Tolerance of Crop Plants’, University of Southampton, United Kingdom;
• A second skills workshop on ‘Design and Analysis of Experiments in Plant Stress Biology’, Wageningen University and Research Centre, The Netherlands;
• A third skills Workshop on ‘Next Generation Sequencing Training – Data Crunching: from hell to heaven’, IGA Technology Services/University of Udine, Italy;
• A fourth skills workshop WATBIO/EPPN on ‘Phenomics and Metabolomics’, UK National Plant Phenomics Centre, Institute of Biological, Environmental & Rural Sciences (IBERS), Aberystwyth University, United Kingdom;
• A fifth skills workshop WATBIO/BIOINNO on ‘Innovation and Entrepreneurship in Biotechnology, Bioenergy and Bioeconomy’, Brussels;
• A sixth skills workshop SUPERGEN Bioenergy Hub/WATBIO on ‘Leaders Consultation in the Bioenergy Research and Development’, University of Oxford, United Kingdom.

And two ‘intensive training summer schools’ (D8.2). These were:
• A first summer school WATBIO/PEPG on ‘Environmental Field Techniques for Scaling Molecular Physiology to Leaf and Crop Canopy’, Lisbon, Portugal.
• A second summer school WATBIO/pwllpeiran/BEACON/GRACE on ‘Miscanthus Development - Miscanthus Safari’, Aberystwyth University, United Kingdom.

Skills and education program outcomes and results. The objectives of the WATBIO skills and education program were achieved through intensive activities that demonstrated specifically how the alliances of knowledge within and outside the consortium could be realized in education in genomics- and phenomics-based biotechnology research for bioenergy crops, a key area in the development of Europe‟s innovative bioeconomy potential.

We focused on the “what we do” and “who we reach”. We were highly active in training our postgraduate students and early career researchers in what it is that we do, the education services we provide, and how we are unique. Here are just the main outputs of the program:

We developed and successfully delivered six skills workshops that span the whole value chain of the multidisciplinary research of the project. We were also able to develop and deliver two training summer schools in key areas of technological and translational research. These activities are summarized as follows:

(i) We conducted, a one-day intensive starter seminar on educating on “‘Improving Water Stress Tolerance of Crop Plants’” at the University of Southampton; 31 students, early career researchers and senior scientists from 22 Academia and Industry partners as well as the European Commission representing the whole consortium attended and participated to the first skills workshop. The workshop set the stage for the future of the project. It provided a comprehensive background to water deficit, and how best to phenotype and genotype for drought tolerance of the three bioenergy crops in the genomics era, to develop innovative germplasm and design drought ideotypes, rationalizing why the collective effort was created, and then explaining the workstreams of activity and relating that work to overall project goals.

(ii) We conducted a two-day intensive training on ‘Design and Analysis of Experiments in Plant Stress Biology’, at the Wageningen University and Research Centre. The workshop was attended by 26 students, early career researchers and scientists form 11 university and SME partners. The training sessions were aimed at facilitating the design and analysis of experiments to systematically and strategically generate and analyze data. The workshop helped to bridge the gap between biologists and statisticians and foster interdisciplinary collaborations among all WATBIO partners. Students and early career researchers were involved in brainstorming sessions on field and greenhouse trial designs and data analysis in an interdisciplinary way, where the best results could only be obtained by joint efforts. The workshop also identified and synthesized critical research issues in experimental design in plant response to drought stress and stimulated future dedication to these key research activities.

(iii) We conducted, a three-day intensive skills workshop on training on ‘Next Generation Sequencing Training – Data Crunching: from hell to heaven’, at the IGA Technology Services and the University of Udine. The workshop was attended by 27 students, early career researchers and senior scientists form 23 university, research centres and SME partners. The training sessions were aimed at breaking the ice between our early career researchers and NGS data analysis by offering practical hands-on training. These training session enabled WATBIO postgraduate students and early career researchers to familiarize with the tools and the UNIX environment by simulating a real analysis on data generated by an Illumina platform. They also became self-sufficient in basic NGS data analysis. This skills workshop contributed to developing skills in the analysis of NGS data in particular so to allow WATBIO scientists who are working with the target bioenergy crops to analyse the NGS data from the samples they provide.

(iv) We conducted, a two-day full-immersion skills workshops on training on ‘Phenomics and Metabolomics’, at the UK National Plant Phenomics Centre at Aberystwyth University jointly with the International Plant Phenotyping Network. The workshop was attended by 29 students, early career researchers and senior scientists form 14 university and SME partners. These training sessions also offered university-industry knowledge exchanges. The practical programme included hands-on practical sessions and data analysis and pre-market technologies demonstration including laser imaging and fluorescence technologies as well as a tour of the UK National Plant Phenomics Centre. Such activities have led to increased cohesiveness and deeper understanding of phenotyping drought stress in plants using the latest high-throughput phenotyping technologies, as well as phenomics data analysis. Moreover, early carer researchers were exposed to the challenges of integrating data from imaging sensors and environmental sensors to predict the responses of plants to environmental stressors, and how data collected under controlled conditions in the greenhouse may help to predict the growth patterns of plants in the field. Altogether, these training activities have provided an invaluable input for the application of phenomics for the advancement of the project by explaining how to measure the onset and progression of stress.

(v) Nearing the end of the project we conducted, a one-day intensive skills workshop on educating on ‘Innovation and Entrepreneurship in Biotechnology, Bioenergy and Bioeconomy’ in Brussels. The workshop hosted over 80 students, successful entrepreneurs, patent attorneys, Technology Transfer experts and academics, from numerous countries (Belgium, Bulgaria, England, France, Ireland, Italy, Kosovo, Scotland and United States of America). The training sessions were aimed at facilitating innovation and entrepreneurship education with an emphasis on creativity, IP and TT, business canvas models and pitching to investors by creating a safe environment where the students and participants have the chance to stretch their imagination and participate in the group to learn from each’s other experience and by providing counselling to participants and preparing them to get out of their chair and use their brain, because creativity and innovation in biotech take practice. We urgently need more innovation and entrepreneurship education. Young researchers need to be better aware of/interested in innovation and entrepreneurship (I&E). Currently I&E are mostly absent from education programmes. Through this skills workshops WATBIO improved learning/teaching in innovation-related skills for early career researchers through the design and delivery of new innovative ways of skills education, including technologies, processes and relations. Empowering the young through skills for innovation and entrepreneurship, including plant biotechnology and bioenergy, is particularly important to building more green societies giving opportunities to all.

(vi) Last but not least, the project skills and education program concluded with a remarkable half-day intensive training on ‘Leaders Consultation in the Bioenergy Research and Development’ at the University of Oxford. The workshop was attended by almost 30 people amongst 5 from the WATBIO consortium representing 3 different universities. This skills workshop was timely and important, and it was jointly run with the SUPERGEN Bioenergy Hub Leaders Consultation 2017 (Prof. Patricia Thornley, University of Manchester, United Kingdom). The training sessions were aimed at “establishing the research priorities and identifying appropriate research partners and stakeholders in Bioenergy”. WATBIO and SUPERGEN Bioenergy Hub joined forces to support the development of research and innovation skills as part of WATBIO training and early stage researcher support program. This connection was successfully used to develop project development training in WATBIO and help to foster the bioentrepreneurial business skill sets for the bioenergy industry. Participants learned by doing through brainstorming sessions on the ‘Grand Challenges’ , shaping the questions, refining the questions, and building a modelst o address the large research questions for bioenergy. The skills workshop witnessed as well a pitching session featuring key projects, where the pitches were made by ECRs.

Moreover, the WATBIO consortium has been brought together to form a cohesive partnership complementing each other in terms of skills and expertise as well as research and innovation diversity. While each partner delivered and hosted a skills workshops, all partners contributed to all education activities, since each was able to make a unique contribution based on solid experience and taking into consideration their research and innovation field of expertise. For the purpose of advancing knowledge, encouraging positive actions, or changing conditions, or in other words “what difference does WATBIO skills and education make” we also conduced summer/winter training schools to reach partners, postgraduate students and early career researchers. WATBIO’s skills and education program through training schools reached a number of meaningful milestones that are summarized as follows:

(i) We conducted in collaboration with the Plant Environmental Physiology Group (PEPG – a special interest group of Society of Experimental Biology & British Ecological Society) the summer school on ‘Environmental Field Techniques for Scaling Molecular Physiology to Leaf and Crop Canopy’ in Portugal. This provided a first-class summer school-type opportunity to the WATBIO early career researchers by uniquely concentrating on scientific and technical topics that are of immediate interest to our consortium. More than 80 persons attended the summer school from within and outside the consortium. Five WATBIO early career researchers from three university partners pitched their results during the poster session. A senior WATBIO scientist trained on the recent advances in plant physiology. The summer school ensured exchange of information and technical knowledge between partners and leaders from both education and industry with a very wide range of skills ranging from physiology to high-throughput field phenotyping so students could establish a common understanding of the range of research and technical activities. This ultimately helped WATBIO postgraduate students and early career researchers to capitalize on the recent advances in environmental physiology and integrate it into their breeding programs for the three target bioenergy crops. It also helped to advance and promote the science and practice of plant environmental physiology and genomics, integrate the plant environmental physiology and genomics community and research opportunities within and outside the consortium and support, train and liaise with young plant physiologists, genomicists and biotechnologists.

(ii) We also conducted a second summer school on ‘Miscanthus Development – Miscanthus Safari’ in Wales. This provided a first-class summer school-type opportunity to the WATBIO researchers by uniquely concentrating on scientific, technical and commercial topics that are of immediate interest to our consortium. More than 50 persons have attended the summer school from within and outside the consortium. WATBIO partners from three different universities attended the discussion during the various sessions. WATBIO partners were exposed to the various Technology Transfer strategies to move the WATBIO research results from the laboratory to the market. These training activities have led to increased cohesiveness and deeper understanding of commercial and research scale Miscanthus plantations and commercial supply chains.

Overall, we produced seven flyers and seven news releases for all skills workshops and training schools for public dissemination of the skills and education program results to a wide range of stakeholders. We designed and customized a bespoke curriculum for each skills workshop and training school consisting of numerous lectures and active learning by integrating practical hands-on and laboratory experiments.

In conclusion, the WATBIO project had a strong commitment to education that ensured the design and delivery of a high quality learning environment to post-graduate students and early career researchers. The multidisciplinary approach that captured the whole value chain provided a rich learning environment. Students and early career researchers contributed enthusiastically to all skills workshops and training schools where in addition to reporting their research activities they were able to increase their awareness of the various technological advances and novel methodologies, acquire the necessary knowledge, and participate in discussions about project management, research strategy and commercial exploitation. They were also supported in interacting with other students and early career researchers as well as role models from outside the consortium, either from related EU projects or from outside Europe. The education program has successfully contributed to both the post-graduate experience and the planning and execution of the research.

WATBIO partners and associated partners made every effort to ensure each skills workshop and training school succeeded and produced quantifiable benefits no matter how complex the organization, development and delivery. This program highlights the importance of training to WATBIO research and researchers success. As the project continued to rely on advanced genotyping and phenotyping technologies, we invested in continuous training and education so that early career researchers were well versed in the latest biotechnological solutions. WATBIO on the whole offered a model of teaching and training that is both impactful and resource efficient. Its educational program has demonstrated that our better-educated researchers performed their jobs with great success leading to achievement of its significant goals and ability to meet major milestones. Focusing on the skills of the project team was an effective way to help achieve the project’s goals.


Potential Impact:
Potential Impact (including the socio-economic impact and the wider societal implications of the project so far) and the main dissemination activities and the exploitation of results. By Donal Murphy-Bokern

In WATBIO, delivering impact started with embedding an awareness of the importance of wider impact in the research process. By complementing the supply of scientific ideas from the academic community with demand signals from users of research, we sought to focus research on key technical outcomes. The development of molecular tools and breeding techniques drawing on basic research on model plant species lies at the heart of WATBIO. Complementing this, there is emphasis on education and training to boost relevant developments in the long term, and there were also complementary investigations of the environmental impacts and implications for public policy. Early in the work, the consortium identified seven areas of impact as the foundation of an Impact Strategy (D9.2). These are:
• the breeding of Populus, Miscanthus and Arundo (3 areas of impact);
• the support of decision-making on crop development;
• education and training;
• scientific impact; and
• impact on the European Research Area.
For the first five impact areas, the flows results from and between tasks was mapped to the point where they deliver technical outputs to the two sets of key primary users: breeders and policy-makers. These maps are presented in WATBIO Report D9.6.(WATBIO 2017) For the first three impact areas (breeding), the approach to the development of impact is species-specific. This is because the primary users (breeders), the key technical outputs, and the research activities leading to these are distinct for each species. Against this background, the report of impact here is arranged according to the seven categories of impact used by the European Commission:

1. Scientific and technological impact
2. Impacts on innovation
3. Economic and social impacts
4. Environmental impacts
5. Impact of EU and other policies
6. Structural impacts on the European Research Area
7. European added value
1. Scientific and technological impact
WATBIO has a particularly high scientific and technological impact potential due to the interdisciplinary systems biology approach as already described. The research has particularly high potential in terms of scientific impact. Overall we plan to deliver over 100 peer-reviewed academic publications and 20 doctoral theses. The following describes the scientific and technological impacts making reference to the project’s existing and planned scientific and technical publications.

For a conceptual framework to guide the work, we have built on the ideotype concept (Donald, 1968) drawing on earlier EU Framework Projects such as DROPS and EURoot, and work at the CGIAR (Thiry et al., 2016) Results of WATBIO experimental work will by synthesised to develop ‘drought ideotypes’. The consortium plans to publish this research in a special issue of Annals of Botany.

Populus
WATBIO built directly on three past Framework Programme projects – POPYOMICS, EVOLTREE and ENERGY POPULUS to examine the genetic control of responses to drought in three contrasting genotypes from Spain, France and Italy (Viger et al., 2013).

Drought responses in the shoot meristematic zone - shoots: In biomass species such as Populus, the entire shoot is the harvested component. Early work revealed that osmotic stress interfered with shoot growth by changing hormonal status and activating regulatory proteins rather than by repressing the underlying machinery of expansive growth (Jia et al., 2016; Viger et al., 2016; Royer et al., 2016). Transcriptional remodelling under drought differed among tissues. By correlating the RNA-seq data sets with phenotypic data, distinct drought adaptation strategies and the molecular level were determined (Smitha et al., in press; Smithb et al., in press).

Drought responses in the shoot meristematic zone - roots: The structure and extent of the root system is a key factor in how plants respond to sub-optimal soil conditions in scavenging in deficient soils. A novel method to analyse root growth kinetics of Populus was defined and successfully applied in an experiment controlled osmotic stress (Puertolas, 2015; Bizet et al., 2014).Transcriptome profiling revealed that the gene expression responses in roots to drought was strongly time-dependent. Among all differentially expressed genes, annotations related to hormone signalling and cell wall modifications were over-represented.

The genetic architecture of drought tolerance (Harfouche, 2015): Drought is the main challenge in Europe to increasing biomass productivity. WATBIO has advanced our understanding of the genetic architecture of drought tolerance in Populus. This includes the identification of several areas of the Populus genome that underpin the drought tolerance phenotypes, determine the regulatory transcriptomic networks and their plasticity in different genetic backgrounds and the investigation of these findings using functional, reverse genetic approaches.

Genome wide association studies (GWAS): Following on from the development of a DNA genotyping chip in black Populus (Faivre-Rampant et al., 2016), a new cost-effective system to perform targeted genotyping-by-sequencing (GBS) was developed in WATBIO (Allwright et al., 2016; Allwright, 2015).GBS is a huge opportunity in all biology disciplines, most notably exploited in GWAS. The main three new associations identified in WATBIO include a transcription factor, a putative stress response gene, and a gene of unknown function. A further 26 markers (representing 22 gene models) related to biomass yield, leaf area, epidermal cell expansion and stomatal patterning were found. We were able to develop several million SNPs of Populus using a novel Genotype-by-Sequencing (GBS) approach and over 60K informative SNPs that were used to undertake a new Genome-Wide Association Study. Thus, a significant improvement in genotyping in Populus was provided in this project
Reverse genetic approaches: ABA is the key drought stress hormone that has a role in regulating all of the plants key drought responses. Potential candidate genes involved in ABA signalling were identified and screened. More than 10 ABA signalling related genes were introduced into Populus and experiments to test their ability to respond to drought are underway. In addition to this, a list of candidate genes was supplied to STT for the development of RNAi and over-expression (OE) lines in hybrid aspen, chosen because of its high efficiency and ease of transformation. 11 candidate genes and several constructs are being used to test these gene loci. These were identified from transcriptomic studies in the project, early microarray studies in previous projects and from literature searches.

The technical impacts of the research on Populus is through five areas of development:
1. Conventional breeding of Populus
2. Breeding of other tree species, especially using gene transfer approaches
3. Methodological developments
4. Technology
5. Field experiment facilities

Conventional breeding of Populus: Our partner breeding organisation (Franco Alasia Vivai) remains one of the few Populus breeders in Europe and is actively engaged in large-scale planting of Populus for bioenergy in Poland with the global forest resource company Greenwood Resources. Thus, Franco Alasia Vivai is the best company in Europe place in future to exploit the findings of WATBIO. Through a programme of active crossing and selection, it is hoped in future that this can be the test-bed site to assess the potential of genomic selection as a breeding tool in Populus to accelerate tree breeding for the bio-based industries. With that in mind, SOTON has committed a further three years of funding to maintain the wide population and drought treatment in the field at Franco Alasia, for future testing. Through the project’s Research Users Forum mechanism, Alasia has had the opportunity to influence the direction of the research and now has direct access to all the pre-breeding material at the main field trial site at Savigliano in Italy. This is described in WATBIO Innovation Note 1 (Fabbrini, 2016). The generic tools developed in WATBIO, such as transcriptomic data and functional and structural genomic resources will be made more widely available to other breeders and academic groups, delivering to the sector as a whole. These data will be placed in public databases – indeed the transcriptomic data are already in the NCBI database and other data will be made publicly available in appropriate databases.

Breeding of other tree species, especially using gene transfer approaches: The principles established in WATBIO are applicable to all plant species, especially perennial plant species. The WATBIO partner SweTree Technologies (STT) is a world leading provider of biotechnology products for forestry and offers an opportunity to commercialise outputs from the reverse genetic approaches. These include through the breeding of eucalyptus. The production of more than thirty original transgenic lines in the reverse genetics approach described above is highly novel and there is potential for the direct commercialisation of this material from WATBIO. In addition, the candidate genes identified for drought tolerance are highly likely to have wider significance for other species.

Methodological developments: A new genotyping-by-sequencing method was developed in WATBIO. This modifies the NuGEN technology levering the “second-in-pair” read, being juxtaposed to the probe oligonucleotide, specifically designed to be close enough to a known SNP to maximize the sequencing output to re-call selected SNP sites. This improved the overall power in the genome-wide association study enabling genotyping with NGS technologies with retrieval of extra information outside the target (Scaglione and Scalabrin, 2017).

Phenotyping technology: WATBIO work on Populus contributed especially to two areas of sensor development: the in-field/on-plant sensing of the water status of plants, and remote sensing using drones (Ludovisi et al., 2017). Lancaster constructed a greenhouse-based phenotyping facility with 200 balances and used this to phenotype Populus. The platform has also been made available to other researchers (including DROPS). This complements the work on phenotyping Miscanthus described below.

Field experimental facilities: The establishment of the WATBIO field fully instrumented at Savigliano (Italy) with a total of 12,000 Populus trees (over 600 unrelated genotypes) and several hundred Arundo plants is one of the project’s most significant practical achievements. The site’s combination of environmental manipulation, the huge range of genotypes tested, and the instrumentation is novel. This unique field research facility is now available to researchers outside the consortium. Payment to maintain the SOTON Populus has been found by SOTON.

Automated phenotyping in Miscanthus: The National Plant Phenotyping Centre at Aberystwyth, was new at the start of the project and therefore part of the work was to commission the state of the art facility for drought analyses. A total of 100 diverse Miscanthus genotypes were screened and the facility was used to identify new ways of scoring for drought tolerance. The work showed that there was significant potential to accelerate phenotyping. High throughput phenotyping facilities collect large quantities of data points and images, and so an important part of the project was to develop, test and use software that was able to process the data generated and present it in the form that biologists and plant breeders can utilise. Assessments of relating actual measurements, e.g. final fresh weight, to biomass scores from images were successful and pixel colour showed promise as a predictor of the impact of drought severity. Digitally determined biomass accumulation was therefore used to identify differences between Miscanthus genotypes in water use efficiency and ability to maintain growth under drought stress (Malinowska et al., 2016).

Miscanthus
The primary impact of the breeding tools and materials will be in the partner breeding organisation (AU-IBERS) who will exploit the results in breeding programmes. Germplasm identification will make new sources of drought tolerant material available to breeders, as has been the case in the past, while the selection tools and modelling (D6.2) developed will make the selection process more effective and more economical. Broadening the germplasm base through the use of novel materials identified should result in greater durability of stress tolerance and greater potential yield gains from resilient plants. Parallel to the WATBIO research at AU-IBERS, the ongoing breeding programme has resulted in the identification of a Miscanthus hybrid clone that is remarkably well adapted to dry conditions.

Two new genotypes that produced more biomass in droughted conditions compared with commercially available genotypes were identified. Transcriptomic studies determined four candidate gene targets correlated with this genotypic finding. These candidate genes are of high relevance to developing future molecular breeding and selection of commercial genotypes for future deployment. At the same time, some ‘well-tried’ traits for drought tolerance were found to have limited value for Miscanthus breeding – carbon isotope analysis in particular. This is an important finding for future drought tolerance breeding.

Transgenic Miscanthus is not widely available and limited success has been achieved on a transformation protocol with high enough efficiency of transformation to be useful. However in WATBIO, AU-IBERS are leading the way in this approach and now have an improved tissue culture system running and now have a hygromycin resistance selection marker working and this is part of on-going research for future transformed Miscanthus.

Taken together, the partner AU-IBERS is leading in Europe for the breeding and release of commercial Miscanthus germplasm and the research of WATBIO has moved significantly to provide new and novel pre-breeding information for on-going activity at AU-IBERS.

Arundo
Arundo donax is a promising biomass crop for areas with warm to hot climates such as the Southern Mediterranean, but at the start of WATBIO, this crop was limited by a lack of genomic information and resources and germplasm from which to develop a new breeding programme. Significant progress was made in WATBIO in this largely understudied crop. Detailed investigations of the physiology of Arundo under drought stress showed that Arundo combines exceptionally high growth potential under well-watered conditions with good adaptation to drought with the ability to maintain relatively high levels of productivity (Haworth et al., 2016). Following consideration of potential for synergy between EU projects (WATBIO Deliverable Report 9.5) an analysis of 40 genotypes was performed in late summer 2014 in Sicily in collaboration with the EU ‘Optima’ project. The research showed that selection of A. donax ecotypes on the basis of xylem responses to drought may facilitate the development of varieties suited to drought (Haworth et al., 2016). A new compartmentalised rhizotron system that attempts to integrate some positive features of conventional methods for assessing root patterns at field and laboratory scale was developed that enabled us to perform a root phenotyping study within distinct and independent soil portions (Sartoni et al., 2015).

A first pilot experiment of genotyping-by-sequencing was carried on Arundo on 17 different samples using 100bp paired reads totalling 95 million reads equivalent to 9.5Gb of RAD (restriction-associated DNA) tags. The method proved to be informative in collecting polymorphic SNP sites across a panel of Arundo donax and Arundo plinii (a smaller-genome phylogenetically species related to A. donax) genotypes. Genotyping-by-sequencing can provided information in Arundo as a largely unstudied species that later supports crop improvement. Moreover, we are testing our GBS protocol to retrieve information on highly mutagenized Arundo plants.

Physical mutagenesis of Arundo: UNIBO and Geneticlab have established a protocol for efficient physical mutagenesis of Arundo based on both gamma-ray and fast-neutron. 1,114 A. donax clones have been regenerated from in-vitro cultures. These have been tested in the field for major plant architectural traits affecting biomass production. This enabled the identification of 93 putative mutants strongly affected for shoot vegetative traits. Additionally, several clones showing positive quantitative alterations in agronomically important traits (early vigor, erect habitus, high tillering). These clones have been tested in replicated drought experiment and one of these has consistently out-performed the commercial genotype and is a target for future testing and commercialisation.

The transcriptome of Arundo donax: WATBIO has resulted in the first publicly available leaf transcriptome for the A. donax bioenergy crop (Evangelistella et al., 2017). The functional annotation and characterization of the transcriptome will be highly useful for providing insight into the molecular mechanisms underlying its extreme adaptability. The identification of homologous transcripts offers a platform for directing future efforts in genetic improvement of this species. Finally, the identified SSRs will facilitate the harnessing of untapped genetic diversity.
Innovation
Much of the impact on innovation has already been described under science and technological impacts. Further information on exploitation is presented here.

The Research Users Forum (RUF) provided commercial partners with a mechanisms to engage in the research and have their specific needs are met. This was supported by partners’ Local User Fora whereby a wider circle of commercial interests were considered in the project. This linkage with wider commercial interests was fostered particularly in Arundo and Miscanthus. The WATBIO project has provided a supportive environment for translational research and genuine and long-lasting collaborative activities are now fully embedded in our activities. For example, the YARA ZIM probes have been tested in three separate experimental campaigns in droughted conditions and adjustments to protocols made accordingly. Significant progress has been made for our Populus breeding companies, Alasia and STT. A number of two way agreements are being prepared between STT and partners for IP protection of gene loci underpinning yield that will enable further investigation and data gathering on these gene targets for pre-breeding beyond the life of WATBIO.

The exploitation of WATBIO research in the development of Miscanthus is embedded in the Miscanthus breeding and development programme at AU-IBERS (Clifton-Brown et al., 2016). Plant material has been bulked and supplied to the project and also used in the development of unique crosses and a QTL mapping population that is now being subjected to drought stress for the genetical genomic approach to elucidate candidate genes. An especially drought tolerant genotype has already been identified and this has now been established at two sites, in the UK and Germany for further study. The commercialization pipeline developed for Miscanthus by AU-IBERS using hybridisation (Clifton-Brown et al., 2017) is an exemplar for the commercialisation of Arundo being planned by project partners and UNIBO and AU-IBERS will meet so that those at UNIBO can benefit from the experience with Miscanthus in order to accelerate developments in Arundo. For Arundo, a two way agreement between Geneticlab and UNIBO has been signed to jointly own 1,000 unique genotypes generated by the WATBIO project, as they become tested and commercialised as appropriate.

Of relevance to all species is the work done in WATBIO on implementing the Lancaster stress index. The general paper (Thiry et al., 2016) has been used by 5 groups in the WATBIO project to assess the basis of stress tolerance and test the usefulness of this index.

Economic and social impacts
The additional ‘up-front’ investment that such perennial crops require due to establishment and additional machinery are a very tangible barrier to adoption. A combination of farmers’ general interest in on-farm energy security (or autarky) and local interest in local energy sources increase confidence. A more stable and coherent policy with simpler institutional frameworks environment is required.

Along with these tangible conditions, a number of less tangible observations of the social conditions influencing farmers decisions were observed. Farmers non-financial motivations play a role such as ‘pride in being a farmer’ in the UK, urban perceptions and environmental expectations of farming in Germany, and the lack of social activism amongst farmers in Greece.
Policy making needs to adopt a more systemic approach to designing and implementing energy policies. Several, economic, environmental, and cultural concerns need to be addressed simultaneously. Farm sustainability, a central concern for farmers needs to be understood and interpreted broadly, over and above strict economic and financial considerations. Issues of identity, dignity and autonomy at the individual, the collective and the communal level should also be respected and taken into consideration. At the same time, the design of policy strategies necessitates the active engagement of farmers and stakeholders directly influenced by the adoption of bioenergy crops.

Relevant to and of the debate on new breeding technologies
WATBIO is relevant to the development of transgenic approaches in all three crops. Most outputs support conventional breeding through for example marker-assisted selection. However, public acceptance and European policy currently operates on the precautionary principle about the breeding technology used rather than the particular genotype being released. The policy on new nucleotide technologies, for example CRISPR-Cas9 and Genome Editing (GE), is still emerging but early indications from the European Court of Justice suggest that for point mutations at least, CRISPR-Cas9 edits will be deemed ‘non-GM’, even in Europe (Abbott, 2018). If this is the way forward for Europe, then the data collected by WATBIO has an even higher value as we have identified several gene targets for editing in Populus and Miscanthus that could be tested with immediate effect.

Education
WATBIO dedicated one Workpackage to education and this work has already been described. The project was designed and conducted to give a high quality learning environment to post-graduate students and early-career researchers (ECRs). The multidisciplinary approach that captures the whole value chain provided a rich learning environment. Students and early-stage researchers contributed to all consortium meetings where in addition to reporting their research they were able to participate in discussions about project management, research strategy, commercial exploitation and policy development. They were also supported in interacting with other students and early stage researchers in related projects through a special workshop at the Plant Biology Europe (FESPB/EPSO) meeting in Dublin in June 2014 (www.epsoweb.org/plant-biology-europe-fespbepso-congress-2014-dublin-ireland-22-26-june-2014). At that meeting, a special workshop was held for all early-stage researchers in WATBIO, OPIMA, OPTIMISC and GRASS MARGINS to facilitate exchange of experiences. This was followed up by the joint conference of the four project consortia in Hohenheim in 2015 (www.biomass2015.eu) which supported the participation on post-graduate and early-stage researchers in particular. The project supported a total of 20 post-graduate students producing 16 doctoral theses and 4 masters theses. Three further early-stage researchers were supported.
Environmental impacts
The environmental impact of WATBIO depends ultimately on the expansion of the production of perennial crops for biomass, especially bioenergy, but also biomass to underpin the wider bio-based sector. The ultimate high-level environmental impact of this work arises from greenhouse-gas emission savings from the replacement of fossil carbon-based resources using the ligno-cellulose produced. This impact on a per unit area basis varies depending on yield and other factors, but is typically in excess of 10 t CO2 equivalent per hectare per year.

In the case of Miscanthus, WATBIO has resulted in insight into drought tolerance in potential hybrid parents used in the hybrid seed system developed by AU-IBERS in other parts of the plant breeding programme there. The AU-hybrid seed system reduces crop establishment costs and the seed-based hybrids now being developed (e.g. the drought tolerant WAT 8) has the potential to account for nearly 500,000 hectares of biomass cropping in the UK. The IBERS-based breeding programme is the world-leading generator of new Miscanthus cultivars which will be essential to increased Miscanthus cropping in Europe. These hybrid cultivars are sterile meaning that seed propagation is combined with the low risk of Miscanthus becoming invasive. A further outcome of the project is the selection of genotypes which might be well suited for wetlands, although again, the overall environmental footprint of this land use would need to be determined relative to the counter-factual use of land in such wetland areas that may have a high conservation value

The WATBIO research on Populus has a strong enabling character that is relevant to other tree species, especially eucalyptus. A report of the International Populus Commission indicates that the total area of short rotation coppice (SRC) Populus across Europe is about 23,502 ha. However, the WATBIO partner Alasia Franco Vivai estimates that the current surface area planted with SRC Populus in the EU is about 45,000 ha taking into account the recent establishment of thousands ha in recent years in Poland. However, SRC-based production is only a very small proportion of the area. Estimates made in 2011 based on FAO indicate that the area of single-stem Populus in Europe (including Turkey) was nearly 1 million ha at that time, of which about a quarter is in France. For this, Populus enotypes are based on interspecific F1 hybridization, whereby black Populus (Populus nigra) is a key source of parental genetic variation. The expansion of Populus production is subject to complex regulation in both the forestry and agricultural policy areas (Nervo et al., 2011).

The research has demonstrated that while Arundo is remarkably productive in well-watered conditions, it is also well adapted to drought and adopts water conserving strategies under dry conditions. This means that Arundo can be developed as a cultivated biomass crop confident that it its production will not increase pressure on scarce water resources. The perennial habit can make a very significant contribution to reducing soil erosion which is a serious problem in Mediterranean areas.
Impact on EU and other policies
Partners DMB and ULANC in particular embedded an awareness in the consortium of the role of public policy. A review of policy impacts in Germany, UK and Ireland (Murphy-Bokern, 2016) was complemented by social science investigations of farmers’ views of the development of bioenergy cropping in Greece, UK and Germany (Petropoulou et al., in press; Petropoulou et al., 2017). Overall, these investigations confirm the complexity of developing bioenergy policy. It supports the incremental approach to policy that is now becoming more widely advocated (e.g. Slade et al., 2017).

Structural impacts on the European Research Area and European added value

WATBIO was designed to bring together a wide diversity of skills not available in any single country, support collaboration across Europe and to transfer expertise from one crop to another and also between academia and industry. In particular, at the outset, the project aimed to integrate physiologists and cell biologists, with those working on molecular biology and quantitative molecular genetics, alongside modellers and breeders. We also wished to integrate these life science activities with socio-economic expertise for land-use change and bioenergy deployment. We have achieved this at the very highest level since many of the individual partners of WATBIO have produced ground-breaking research in their own specialism that has then been integrated more widely and utilised quickly in the project. For example the fundamental research findings of Grill (TUM) on ABA signalling in Arabidopsis are ground-breaking and Grill showed in 2016 that these could be manipulated to improve the water use efficiency in Arabidopsis (Yang et al., 2016). We have taken these recent findings in WATBIO, identified these ABA receptors in Populus and modified the plants within the project. 14 ABA receptors have been found in Populus and reported (Papacek et al., 2017), providing very strong targets for improved water use efficiency in this bioenergy crop. They are being investigated in WATIO in Sweden and Germany and the UK. Many other such examples exists in the project that required European integration and have significantly enhanced the European Research Area. For example, the development of Arundo and Miscanthus has benefitted from the RNA-seq and other approaches already being applied in Populus. Systems for for Populus transformation are in Sweden at STT and SLU, and these have now been transferred to Germany (UGOE, TUM) and the UK (SOTON), whilst a world-class modelling platform for linking data is in The Netherlands. This has been transferred to AU-IBERS, SOTON, UNITUS and UGOE. In addition, there are many shared resources in this project. Common core germplasm of Populus, Miscanthus a and Arundo has been extensively shared across the consortium, whilst at the beginning of WATBIO it was generally held by only one partner. This is now available for future research as other research questions emerge and for accelerated testing and commercialisation. The study of genotype responses to drought relies on experiments undertaken in a world-class facility in INRA France, but plant material sourced from UK, Italy and Germany. Similarly Miscanthus was shared across the UK and Germany, whilst partners from Italy, the Netherlands and the UK worked on Arundo. The project was designed to build on previous European-funded or on-going research that provides shared tools, expertise, shared learning and resources to be developed further in WATBIO including from EVOLTREE, POPYOMICS, ENERGYPOPULUS, DROPS, OPTIMISTIC, SWEETFUEL, GRASS MARGINS and OPTIMA.

Given the relevance of past and other on-going projects, the potential for synergy across projects was the subject of a special WATBIO internal report (D9.5). This work on synergy led directly to close interaction between the main relevant and (then) ongoing FP7 projects: Optima, Optimisc and GrassMargins, with some shared experiments and expertise. The four projects worked closely together on the Bioenergy 2015 Conference held in September 2015 (wwww.biomass2015.eu). In addition to a Book of Abstracts (Murphy-Bokern et al., 2015), a special issue of Global Change Biology Bioenergy and a Springer book of articles based on presentations, WATBIO worked with the other projects to produce a summary of the state of the development of biomass crops (Lewandowski et al., 2016).

WATBIO’s main field site is open to Europe’s Populus and Arundo researchers
The establishment of the WATBIO field research site at Savigliano (Italy) with a total of 12,000 Populus trees and several hundred Arundo plants planted in 2013 is one of the project’s most significant practical achievements. The site is equipped with extensive drip irrigation facilities, sensors and a weather station. This unique field research facility is now available to researchers outside the consortium. The site holds three field experiments. The first was designed to examine approximately 600 contrasting Populus genotypes to support genome wide association mapping (GWAS). An eight block design is used and the trees which were planted in 2013, have been the subject of an intensive programme of measurements and sampling under rain-fed and irrigated conditions. The second experiment is a mapping population of 650 genotypes that are the siblings of a cross between two contrasting grandparents selected in northern and southern Italy is growing in eight replicates. As mentioned above, SOTON has acted to secure the use of the site for a further three years.

Global links
Finally, WATBIO placed special emphasis in fostering relevant global links with events designed specifically to network leading scientists from the USA, Australia and North Africa (CGIAR-ICARDA). In particular the four new USA DOE Bioenergy Centres that were announced in July 2017, have strong links with WATBIO scientists. For the Berkeley Center, Gail Taylor (SOTON) in her new role at UC Davis, has colleagues working on cell walls and feedstock optimization. Gail Taylor is also a member of the scientific advisory group for the center based at Oak Ridge, with significant collaboration on Populus. Iain Donnison and colleagues at AU-IBERS have long-term collaborations with the center at Illinois, working extensively on optimisation of Miscanthus as an energy feedstock. Given the considerable amount of funding for each of these centres – each to receive $25M pa, it is critical that the EU maintains strategic links with these large-scale bioenergy developments.

Project website
In terms of the technology used, the project website (www.watbio.eu) was provided by Pageworks on behalf of SOTON. It was designed by DMB and SOTON and maintained by DMB and SOTON as set out in D9.3 in conjunction with Pageworks. SOTON develop the twitter feed for the website @watbioeu that was maintained and updated throughout the project by SOTON and DMB and provided the coverage of the live streaming of the Oxford meeting, September 2017. All project logos, videos and other materials are freely available on the website. The website will be maintained by DMB for at least two years after the project ends to provide full access to the projects results as they emerge in the post-project period.


References
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List of Websites:
Website: www.watbio.eu
All project logos, videos and other materials are freely available on the website. The website will be maintained by DMB for at least two years after the project ends to provide full access to the projects results as they emerge in the post-project period.

Coordinator:
Professor Gail Taylor, BSc, PhD, FSB
Director of Research for Biological Sciences
Chair, Energy Multidisciplinary Research
University of Southampton
Life Sciences Building
Southampton, SO17 1BJ
Tel: [phone]

Project manager:
Lian Lomax-Hamster
INRA Transfert
3, rue de Pondichéry
F-75015 Paris
FRANCE
Tel: [phone]