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SID 5 Research Project Final Report

SID 5 (Rev. 3/06) Page 1 of 28

NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

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Project identification

1. Defra Project code NF0426

2. Project title

The genetic improvement of Miscanthus for Biomass

3. Contractororganisation(s)

Institute of Biological, Environmental and Rural Sciences (IBERS)Aberystwyth UniversityPlas GogerddanCeredigion, UK SY23 3EB

Plant Research International (PRI)P.O. Box 166700 AA Wageningenvisitors address: Building no. 107Droevendaalsesteeg 1

6708 PB Wageningen, Netherlands.               

54. Total Defra project costs £ 1216288(agreed fixed price)

5. Project: start date................ 01 April 2004

end date................. 01 April 2009

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

The work has built on (a) the DEFRA investment on the yield potential, agronomy, taxonomy and genetic resources of Miscanthus (b) state-of-the-art expertise and parental material developed in the EU EMI project captured through the involvement of John Clifton-Brown, who has 17 years experience in Miscanthus and in the EU BIOMIS project through the involvement of Oene Dolstra at PRI (c) the unique expertise of IBERS and PRI in the breeding of perennial/ high ploidy grasses (such as tetraploid ryegrasses and oats), in cytogenetics and tissue culture.

IBERS (formally IGER) at Aberystwyth co-ordinated the work programme with PRI in Wageningen in the Netherlands. IBERS was lead partner for the assessment of genetic resources, both partners were responsible for hybridisation and selection, and PRI was the lead partner for assessment of plant progenies for general combining ability.

The work will build on (a) the unique expertise of IGER in the breeding of perennial/ high ploidy grasses (such as tetraploid ryegrasses and oats), and in cytogenetics and tissue culture, (b) the DEFRA investment on the yield potential, agronomy, taxonomy and genetic resources of Miscanthus (c) PRI’s state-of-the-art expertise and parental material developed in the BIOMIS project (d) links to genomics work at IGER, RRes, Bristol and Wageningen (e) involvement of both IGER and RRes in the recently funded EPSRC funded SuperGen Biomass and Bioenergy Consortium project, relevant objectives of which are to establish how biomass chemical composition affects thermal conversion properties and emissions and to identify quality traits in grasses for improved biofuel processing.

Phenotypic evaluation of 249 accessions have been made for 14 traits [flowering time, leaf length, leaf width, basal diameter, stem density, stem diameter, senescence, yield, moisture content, NDF, ADF, Lignin, Cellulose, and Hemicellulose]. Valuable variation has been identified which has been used in the breeding programme. The yield from the industry standard M. x giganteus was exceeded in several accessions demonstrating the potential for breeding in this crop. Some traits such as canopy height, flowering and stem diameter are important yield components. Others such as lignin and mineral content are important determinants of combustion quality. Other end uses such as lignocellulosic fermentation or production of bio-oil by fast pyrolysis will most likely demand varieties of Miscanthus with much lower levels of lignin and these will be bred using genotypes with low levels of aromatic cell wall components. Low moisture content is desirable in the harvested crop to reduce transport costs and improve fuel characteristics (Lewandowski and Kicherer, 1997) and this characteristic is usually correlated with early senescence. Delayed senescence may be beneficial to extend the productive growth season and in some

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accessions this characteristic has been correlated with low moisture content at harvest; these accessions may provide the genetic basis for extending the growing season by delaying senescence while maintaining low moisture in the crop at harvest.

1202 paired, polycrosses and open crosses were made at IBERS and PRI over the five years. Poor seed set in some paired crosses was attributed to self-incompatibility genes. Over 15,000 seedlings have been planted in field trials from which 333 elite selections have been made to date on the basis of high biomass potential and other characters. In setting up the project, we noted the high cost of planting Miscanthus from rhizomes, and we proposed to devote a relatively small effort to testing the feasibility of developing seed-propagated M. sinensis. Reduced establishment costs would make a large difference to the economics of establishment and reduce the necessity for a planting grant.

To this end, we have measured the general combining ability of genotypes using polycrosses and test-crosses, essential steps to the generation of synthetic varieties that are necessary in order to maintain heterozygosity. The minimum temperature for germination was found to be in the range of 8-10OC. This is relatively high, so that seed-propagated Miscanthus would either have to sown relatively late but not too late as to jeopardise winter-survival, be established under polythene, or young plants raised and transplanted.

Early-morphometric selection in a narrow genetic base was more reliable than in trials with a broader genetic base. In the broader genetic base it has some value, but there are a number of important outliers which would have been screened out if selections had been made after a single growing season.

It is likely that selection systems that combine the use of early-morphometric and marker assisted selection will be highly effective in filtering down the quantity of germplasm that needs to be held in single spaced plant trials, and expedite the selection of seedlings that can be promoted to plot trials.

In future work, firstly we believe it is important to continue the characterisation of novel Miscanthus germplasm with outstanding agronomic traits and combining ability. It will be essential to join this with progeny testing from wide and interspecific crosses under several diverse climates to discover the heritabilities of complex traits such as drought tolerance. If the IPCC predictions for climate change in Europe are correct, we expect summer droughts to become frequent and extreme. Biomass production, like hydro-power, is very sensitive to water supply. We believe therefore that the traits associated with drought tolerance will be key to the future development of a bioenergy industry based on Miscanthus.

Secondly, further development of efficient breeding and selection methods are crucial. Genome-based marker-assisted selection strategies for the Miscanthus breeding programme need to be developed in order to accelerate breeding.

Thirdly, as the urgency for low carbon economies increases, it will be important that the international effort from both the public and private sectors is co-ordinated to deliver solutions to the market as fast as possible.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

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Introduction

The work has built on the unique expertise of IBERS and PRI in the breeding of (a) perennial/ high ploidy grasses (such as tetraploid ryegrasses and oats), and in cytogenetics and tissue culture, (b) the DEFRA investment on the yield potential, agronomy, taxonomy and genetic resources of Miscanthus (c) state-of-the-art expertise and parental material developed in the EU EMI project captured through the involvement of John Clifton-Brown, who has 17 years experience in Miscanthus and in the EU BIOMIS project through the involvement of Oene Dolstra at PRI.

IBERS (formally IGER) at Aberystwyth co-ordinated the work programme with PRI in Wageningen in the Netherlands. The funding for genetic improvement of Miscanthus for the UK has based on roughly 75% expenditure in IBERS and 25% of costs in PRI. IBERS was lead partner for the assessment of genetic resources, both partners were responsible for hybridisation and selection, and PRI was the lead partner for assessment of plant progenies for general combining ability.

Scientific Objectives

1. Assessment of genetic resources available in UK and elsewhere for yield, canopy development, flowering time, over-wintering, and combustion quality.

2. Hybridisation and selection based on general and specific combining ability of diploid sinensis x sinensis accessions using a priori knowledge and information from 1. The hypothesis is that diploid clones can be identified which are superior to the triploid 'X giganteus' in N. Europe.

3. Hybridisation of diploid and tetraploid accessions to produce new sterile triploid hybrids.

4. Improvement of breeding efficiency based on early morpho-physiological prediction of productivity and persistence and the indirect measurement of chemical composition using FTIR.

5. The production of large trait mapping populations and the identification of a realistic cost-effective roadmap to more efficient breeding through the development of marker-assisted selection.

6. Identification with Defra of the exploitation route in UK and Europe, taking into account expertise in large-scale production and marketing.

Detailed reporting on the scientific objectives

1. Assessment of genetic resources available in UK and elsewhere for yield, canopy development, flowering time, over-wintering, and combustion quality.

1.1 Genetic resources

Between 2004 and 2006, 336 Miscanthus accessions were assembled from UK and European sources at IBERS. The largest single source of Miscanthus diversity in the UK was the National Collection held at ADAS Arthur Rickwood that was assembled with financial support from DEFRA projects (MAFF QA3580, NF0404 and NF0411). In April 2004 a material transfer agreement (MTA) was drawn up between IBERS and ADAS. This agreement also received the approval of Kew and embodies the Convention on Biological Diversity (CBD). In July 2004, 111 genotypes from collections made in Japan and Korea were supplied by ADAS to IBERS. In Feb 2005, a further 90 accessions were dug directly from the National Miscanthus field plots at Arthur Rickwood and transferred to IBERS. 130 further accessions were obtained from a wide variety of sources including a commercial company and various institutes, botanic gardens and horticultural suppliers.

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A subsidiary project (NF0436) to collect new Miscanthus accessions from the wild in Eastern Asia was approved for funding in 2006. 120 accessions were added to the breeding programme from China, Japan and Taiwan. Quarantine work was completed in October 2008.

Phenotypic characterisation

To allow phenotypic characterisation, one plant of each of 249 accessions, collected from these various sources, was cloned in 2004 by rhizome splitting to produce four propagules of a defined genotype. These were planted at Aberystwyth, IBERS in 2005 in a phenotyping trial on a south west facing slope 30m above sea level, termed the 2TT trial. To ensure good early establishment, weed control was achieved using RoundUp® and Atrazine. Herbicides were applied with a knapsack sprayer to destroy the grass sward over a 1.5 m diameter circle and each genotype was planted into a treated circle in each of four randomised blocks. As part of the statistical design one clone (M.x giganteus) was split to produce eight propagules and planted twice in each block.

Soil taxonomy classifies the soil as a Cambic stagnogley (FAO system). The stone fraction (particles >2mm) was estimated as approximately 50% of the soil mass in the 0-40 cm layer. This challenging growing environment provides significant selection pressure to the clones during dry periods.

14 traits were assessed in 2007 : flowering time, leaf length, leaf width, basal diameter, stem density, stem diameter, senescence, yield, moisture content, Neutral Detergent Fibre (NDF) which estimates cellulose, hemicellulose and lignin content (Anon, 1998a), Acid Detergent Fibre (ADF) which estimates cellulose and lignin (Anon, 1998b), and acid detergent lignin (van Soest, 1963, van Soest and Wine, 1967, Kitcherside et al., 2000 and cellulose and hemicellulose by calculation from NDF, ADF and lignin contents. For some of these traits, more sophisticated methods were used than originally planned with the aid of BBSRC and EPSRC (SuperGen) funding. An account of the methods and the considerable variation found was given by Clifton-Brown et al (2008).

The phenolic compounds ferulic acid and p-coumarate, important determinants of the accessibility of cell wall polymers, are currently being assessed in 2TT as part of the Biorenewables and Environmental Change Strategic Programme Grant at IBERS.

At PRI a trait trial was used to evaluate phenotypic variation in progeny from the relatively narrow genetic base of the BIOMIS population for flowering time, plant height, stem number, leafiness, and stem diameter. Stem samples were taken in winter 2009 for analyses of mineral contents.

Results

Table 1.1 shows the range of variation together with the average standard error of difference for each trait in the 2TT trial in 2007. Some variates were analysed on a transformed scale.  All means are on the original scale with the relevant transformation indicated by the superscript on the corresponding s.e.d. The data was analysed by REML rather than ANOVA which would have been inappropriate.

Table 1.1. The range of variation together with the average standard error of difference for each trait in the 2TT trial.

Variate Accession mean Average s.e.d.Minimum MaximumCanopy height (cm) 40.2 331.8 9.81Leaf length (cm) 24.5 95.2 6.37Leaf width (cm) 0.24 2.89 0.0731

Basal diameter (mm) 102.2 958.1 1.512

Stems density (stems/m) 13.7 123.2 0.283

Stem diameter (mm) 2.2 16.8 0.454

Yield (g DM/plant) 12.7 1271.6 3.071

NDF (% in DM) 76.0 91.8 1.55ADF (% in DM) 43.1 62.6 1.67Lignin (% in DM) 6.7 13.0 0.50Cellulose (% in DM) 35.5 50.0 1.31Hemicellulose (% in DM) 27.8 36.5 0.67

1; Applicable to square root transformed data2; Applicable to data Box-Cox transformed with λ=0.43; Applicable to data Box-Cox transformed with λ=0.14; Applicable to data Box-Cox transformed with λ=0.6

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Autumn canopy height varied from 30 to 303 cm with the majority (63%) of plants reaching between 100 to 150 cm by the end of the 2007 growing season (Figure 1a). The degree of variation in stem density (0.2 - 1.4 stems cm-1, Figure 1b), reflects the considerable variation of form observed in the Miscanthus collection which includes both clump forming M. sinensis and M. sacchariflorus with running rhizomes.

Figure 1.1 Frequency distribution for a) canopy height b) stem density in the 2TT trial (249 genotypes) in the third year (2007) after planting at IBERS. Each category on the x axis is labelled with the maximum possible value of that category. Vertical broken line indicates mean value for Giganteus.

The variation in the onset of flowering time in the trial is shown in Figure 2. In 2007 and 2008 flowering was first observed in June. Over 70% of genotypes had flowered by the first frost in autumn. In 2006 flowering time was significantly later than in 2007 and 2008. Further analysis of the delay in flowering time is made later in this report.

Figure 1.2 Accumulated flowering (percent of 249 genotypes) in three consecutive years in 2TT at IBERS (data in 2007 and 2008 collected by Dr. E. Jensen, funded by the BBSRC responsive mode grant on flowering time).

Figure 1.3 shows the variation in above ground harvestable yield. In 2007, 66% percent of genotypes produced between 200 and 600 g DM plant-1 and a small proportion of genotypes produced more than twice the average yield of 409 g DM plant-1. In 2008 average yields were about double those in 2007 (854 g DM plant-1). In 2007 the spaced plant yield from M. x giganteus was exceeded by more than 50% in the leading accession, but in 2008, a M. x giganteus type (triploid) was the highest yielder. Moisture content of harvested material varied from 12 to 52% in 2007 and from 17 to 48 % in 2008 (Figure 1.4). 55% of genotypes had moisture contents between 20 and 40 %. Some of the highest yielding genotypes also have low moisture contents (data not shown). Moisture

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content correlates well with crop senescence (Figure 1.5); however, a number of genotypes display low moisture content and reduced or delayed senescence.

The increased mean and range of yield of 2008 compared to 2007 probably reflects increased maturity and recovery from the drought conditions of 2006.

Figure 1.3. Frequency distribution for harvestable biomass in 249 genotypes in the third year (2007 growing season, open bars) and the fourth year (2008 growing season filled bars) after planting at IBERS. Each category on the x axis is labelled with the maximum possible value of that category. Arrows indicates mean values for M x giganteus for each year.

Figure 1.4. Frequency distribution for moisture content in 249 genotypes in the third year (2007 growing season, open bars) and the fourth year (2008 growing season filled bars) after planting at IBERS. Each category on the x axis is labelled with the maximum possible value of that category.

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y = -5.0x + 61.0R2 = 0.6

0

10

20

30

40

50

60

1 2 3 4 5 6 7 8 9 10

Senescence (score)

Moi

stur

e co

nten

t (%

)

Figure 1.5. Average senescence score from 3 months prior to harvest and moisture content in February 2008. The linear regression, its fit and coefficients are shown in the inserted box. Some accessions display reduced senescence while maintaining low moisture content (circled). Senescence score 1= fully green; score 10 = fully senesced.

Lignin content was determined as % dry matter (%DM) from a representative sample of ground stem. Values were approximately normally distributed (Figure 1.6) and ranged from 5.76 %DM to 13.25 %DM (mean of 9.26 %DM). The amount of coumarate (Figure 1.7) in the stems is higher than that in the literature for other species (Gordon Allison, personal communication) and could have implications to the accessibility of cell wall polymers.

Figure 1.6. Frequency distribution for acid detergent lignin (% in dry matter) in 249 genotypes following the third year after planting at IBERS. Each category on the x axis is labelled with the maximum possible value of that category.

Sample Compound n mean median max min sdstems p-coumarate 65 23.75 24.84 34.46 5.42 6.31leaves p-coumarate 127 8.29 8.11 14.94 3.16 2.28stems Ferulic acid 55 7.65 7.98 11.93 1.49 2.46leaves Ferulic acid 16 4.70 4.58 7.46 3.06 1.08

Figure 1.7 Table of preliminary analysis of phenolic concentrations in ground samples of Miscanthus taken from 2TT in October 2008. All concentrations are given as mg/g dry weight whole tissue.

An analysis of traits which co-vary has begun, and this has revealed a number of positive and inverse relationships between the measured ‘one off’ traits (Figure 1.7). These relationships are key to further

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improvements to the strategic crossing programme. The statistical significance of these relationships are given in Figure 1.8. Information on the measurement of lignin, hemicellulose and cellulose is given under objective 4.

Figure 1.7. Pre-correlation analysis of selected phenotypic traits in 249 genotypes at IBERS following the third year after planting. Traits 1-6, 7 and 8-12 were collected with funding from BBSRC (Responsive mode and fellowship to Dr. E. Jensen; Central Science Grant funding to Dr. Paul Robson), DEFRA (NFO426) & BBSRC and EPSRC (Supergen) respectively.

1 1

2  0.364*** 1

3  0.512***  0.531*** 1

4  0.447***  0.077  0.374*** 1

5  -0.302***  -0.270***  -0.602***  -0.466*** 1

6  0.578***  0.409***  0.521***  0.137*  -0.491*** 1

7  0.658***  0.295***  0.212**  0.262***  0.160*  0.419*** 1

8  0.421***  -0.233***  0.042  0.349***  -0.073  0.113  0.136* 1

9  0.482***  -0.166**  0.154*  0.446***  -0.185**  0.155*  0.132*  0.925*** 1

10  0.323***  0.038  0.240***  0.378***  -0.226***  -0.002  0.011  0.485***  0.657*** 1

11  0.452***  -0.240***  0.085  0.383***  -0.144*  0.181**  0.144*  0.941***  0.963***  0.466*** 1

12  -0.239***  -0.154*  -0.321***  -0.287***  0.333***  -0.116  0.011  0.024

 -0.316***

 -0.585***

 -0.187**

1 2 3 4 5 6 7 8 9 10 11 12Figure 1.8 Spearman rank correlation coefficients relating to Figure 1.7. *=P<0.05; **= P<0.01; P=<0.001.

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Stem samples of 23 genotypes from the BIOMIS mapping populations (OD0307) as well as half-sib families from most of them (OD0401) harvested in winter 2006 were used to study the variation in the mineral content (Table 1.2). The mineral contents in general were low as expected for perennial crop like Miscanthus, making it a suitable feedstock for combined heat and power production. Nevertheless the set of samples showed considerable variation for the minerals relevant in relation to combustion quality, i.e. contents of potassium and chlorine. The correlation between the performance of parents and offspring for these traits was positive but rather weak (r=0.46 and 0.25, respectively).

Table 1.2 Mineral contents of accessions in PRI’s phenotyping trials.

Trait Unit Mean CV(%) n Range Mean CV(%) n RangeTot-N mg/100 g DM 115.5 25 23 135.0 146.6 16 20 85.0P mg/100 g DM 47.4 44 23 77.6 50.6 20 20 37.0K mg/100 g DM 109.0 61 23 298.2 158.5 30 20 196.1Ca mg/100 g DM 102.9 13 23 52.5 91.3 8 20 26.4Mg mg/100 g DM 69.6 16 23 45.5 61.7 8 20 14.3Fe mg/100 g DM 1.7 35 23 2.7 2.8 23 20 2.9Mn mg/100 g DM 3.1 26 23 3.6 4.1 20 20 3.2B mg/100 g DM 0.4 7 23 0.1 0.3 22 20 0.2Zn mg/100 g DM 1.4 25 23 1.4 1.9 24 20 1.8Na mg/100 g DM 32.5 10 23 13.0 34.9 8 20 10.8Cu mg/100 g DM 0.4 29 23 0.5 0.3 32 20 0.4S mg/100 g DM 0.3 23 23 0.4 0.3 8 20 0.1Cl mg/100 g DM 10.6 41 23 15.1 12.5 33 20 16.7Stem weight g DM/stem 4.9 38 23 9.7 6.6 15 20 4.2

Accessions HS families

2. Hybridisation and selection based on general and specific combining ability of diploid sinensis x sinensis accessions using a priori knowledge and information from 1. The hypothesis is that diploid clones can be identified which are superior to the triploid 'X giganteus' in N. Europe.

Work at IBERS involved a wide genetic base while that in PRI investigated the hypothesis that high progress in an outbreeding crop can be obtained by selection within a relatively narrow base, as for maize where continual genetic advance in oil content, for example, has been obtained through selection from within a population.

Emphasis in choosing parents for crossing has been on the basis of superior phenotype, complementary traits (i.e. for two yield components high/ low x low/high) and reinforcement of traits (high autumn greenness x high autumn greenness). High emphasis was placed on high yield given the importance of this character to the economics and sustainability of growing Miscanthus.

M. sinensis has better over-winter frost tolerance and lower nutrient off takes than M. x giganteus. With breeding and appropriate agronomy, it is considered that the yield of new M. sinensis hybrids could exceed M. x giganteus in the UK. These M. sinensis hybrids will be day neutral, and thus flowering time can be readily predicted by thermal time. Many M. sinensis accessions flower within the growing season at IBERS and PRI and set seed. As part of this objective, we proposed to devote a relatively small effort to testing the feasibility of developing seed-propagated M.sinensis.

Breeding activities may finally result in outstanding genotypes to be multiplied in vitro or even as seed-propagated varieties (hybrids, synthetics). All types require rapid identification of genotypes of a beneficial breeding value to facilitate the breeding process, in particular population improvement and combination crosses. Progeny testing used to be the most rewarding way to improve quantitatively inherited agronomic traits of M. sinensis.

Pair-wise crosses and open pollinationA summary of all crosses performed is presented in Table 2.1. At the start of the project, choice of parents was limited to JCB and OD’s knowledge based on the EMI and BIOMIS European projects, with no information being available other than geographical origin and pot phenotype with regards to other material including the National Collection. As the project proceeded, more information was obtained on genetic sources and crosses became more targeted. Paired crosses were obtained either by putting pairs of plants together in isolation i.e. in separate isolation chamber compartments, or by putting inflorescences together in large polyester pollination bags.

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However, a considerable proportion of these paired crosses failed to set seed. 75% of all crosses yielded one or more seeds in PRI and 31% at IBERS. The disparity could be attributable to differences in environmental conditions, plant condition, synchronisation of flowering,some variation in genetic load of genotypes used at IBERS/PR. In addition, it is likely that a proportion of crosses did not set seed because of incompatibility. The number of seeds obtained per cross turned out to be quite variable (Figure 2.1), but was nearly always very low in comparison to the potential number. In Wageningen the crosses yielded on average 60 seeds, the numbers ranged from 0 to 982 seeds per cross. In contrast, open pollinated inflorescences in the field produced many more seeds (at PRI these averaged about 100 seeds per inflorescence (Table 2.2)). The reasons for the fairly poor setting of seeds observed in pair crosses are not clear. From the field observations, it is more likely that the action of self-incompatibility genes rather than lack of viable pollen and/or egg cells is the major cause of poor seed set.

Table 2.1 Crosses performed at IBERS and PRI in 5 successive years.

Year Paired crosses Poly crosses Open crosses Paired crosses Poly crosses Open crosses Total2004 16 0 81 52 19 121 972005 39 0 111 110 2602006 59 0 138 26 187 4102007 82 5 17 1042008 50 14 26 88 61 92 331

Totals 246 19 373 276 80 400 1202

IBERS PRI

Generation of half-sib and top-cross families

A few M. sinensis populations have been established at PRI in order to use intra- and inter-population improvement methods to combine favourable plant characteristics. The progenies involved are half-sib families and top cross families, respectively.

Half-sib families, in which the female parent is subject to open-pollination from a number of male parents, have also been undertaken. Pollen contributing to the generation of such a family may be controlled to a certain level, varying from no control at all (= open pollination) to full control (pollination in a specialised isolation glasshouse) by a restricted and a well-defined set of pollen parents. At PRI selected genotypes were allowed to pollinate each other several times to produce a synthetic. Alternatively, female parents can be exposed to pollen clouds from a populations (in Hardy Weinberg equilibrium) grown in isolation. In 2008, the same field in PRI was used as tester to produce so-called ‘top crosses’ of novel accessions and some selected genotypes- a procedure commonly used in maize breeding to test the combining ability of the female parents under study. The female parents were put so far apart that no or only little cross pollination between females was expected to occur. The top cross procedure is possible in M. sinensis because of the high degree of self-incompatibility present. The numbers of seeds produced on the top crossed plants is shown in Figure 2.1. The numbers were better than those obtained through pair-wise crosses, but there still is scope for improvement.

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Fig. 2.1 Histograms of numbers of seeds obtained from pair-wise crosses (left) and top crosses (right).

Sibmating

Sibmating has been used to develop partially inbred lines. This approach of inbreeding was chosen to generate parental lines to be used for breeding uniform and hybrid varieties as an alternative to self pollination. Ten full-sib families derived from crosses among genotypes of the BIOMIS mapping population were germinated followed by transplanting the resulting seedlings in pots and were allowed to flower as separate family in separate glasshouse compartments. The families already have in comparison to the two grandparents of the mapping population a certain level of inbreeding due to two cycles of full-sib mating. The third cycle of sibmating turned out to be totally unsuccessful. This implies that it is not possible to generate genetically highly identical hybrid seeds but rather breeding methods which preserve heterozygosity must be used. The offspring of these crosses therefore will always show a certain degree of segregation. This is not that important since uniformity is not an important issue in breeding energy crops. Nevertheless to fully fix heterosis it would be interesting to generate fully homozygous genotypes of M. sinensis. Possible ways to achieve this goal would be microspore culture and screening for self-incompatible within the M. sinensis gene pool.

Seed harvesting

The bagged seed generated from the above crossing strategies was collected approximately two months after the inflorescences had been bagged together for paired crosses. For open crosses and those undertaken in isolation chambers, the seed was collected when the panicles were at the latter stages of development before seed shatter had occurred. Identifying the optimum time for harvesting the inflorescences is however difficult because of lack of synchronicity of flowering between shoots. Harvested inflorescences need to be dried thoroughly before seed threshing. The seeds could be rubbed out of the inflorescences quite readily by hand in a large tray with a ribbed rubber-lining and a stamp with a similar lining and cleaned with appropriate sieves and help of air suction.

Seed storage, seed germination and generation of seedlings for transplantation

There was little information on the storability of Miscanthus seeds at the start of the project. The 2008 tests at PRI of the germination rate of seed samples from crosses made in previous years have shown that the prevailing opinion that seed could be stored for a very short period is fortunately untrue when seeds were stored in the dark at 13°C and 30% relative humidity. This is exemplified in Figure 2.2 showing a summary of the proportion of seedlings obtained from 35 seeds per entry when sown in trays with potting soil. The percentage of seeds giving rise to a seedling was 93% with a range from ca 65 to 100%. The materials used in this study mainly consisted of one- and two-year old seeds but also five-years old seeds with a germination rate of 100%. Accordingly, it is fair to assume that seeds can be stored five years or longer provided they are stored in a proper way. The germination study was carried out in a greenhouse at temperatures around 20°C.

SID 5 (Rev. 3/06) Page 13 of 28

Figure 2.2 Germination rate of seed from M. sinensis crosses

A separate study was carried out at IBERS on the influence of temperature on the rate of germination. To this end a thermogradient table was used with temperatures ranging from 0 to 30°C. Figure 2.3 shows for each temperature a sigmoid time curve with a plateau above 90% for germination in temperature range 16-26°C. At lower temperatures the maximum was lower or nil. At temperatures above 16°C germination took between 2 to 7 days. The minimum temperature for germination of seeds of different Miscanthus accessions was observed to be in the range between 6 and 10°C and was relative high in comparison to switch grass, perennial ryegrass, reed canary grass and maize (Figure 2.4).

AC

DB

Figure 2.4 Cumulative germination of seed from one M. sinensis cross at each temperature (A), proportion of viable seeds in each treatment (B), the time in days to reach 50% germination at different temperatures (C) and a plot to estimate the base temperature for germination (D).

SID 5 (Rev. 3/06) Page 14 of 28

Figure 2.4 Minimum temperature for germination with 95% Confidence Interval based on curve fitting for Miscanthus (M. sinensis seedlots derived from crosses made at IBERS), SW-TB (Switchgrass cv Trailblazer), AVISO (maize cv Aviso), Lol p (Lolium perenne cv. Aber Dart), Phalaris (reed canary grass type 10, EU RCG programme). The implications of these results in terms of whether seed-propagated Miscanthus is a viable option are considered in the General Discussion.

Selection

Seed from crosses made at both PRI and IBERS were sown in modular trays in the glasshouse, before being planted out in field trials. Table 2.2 provides a summary of all the trials established from crosses made with the breeding programme. In total 15 000 seedlings have been planted in field trials, derived from 1061 successful crosses.

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Table 2.2 Miscanthus field trials derived from crosses at IBERS and PRI

Trial name Location Objective Design Planting date Plants Families Notes and selections

4S IBERSField evaluation of crosses

made in 2004 Spaced plants on a 1m grid Jun-2005 1823 88

Used for Early-morphometric analysis up to Feb 2008. Outstanding plants were selected in Aug 07 [47 plants],

Feb 08 [29 plants] and Sept 09 [~30]. Plants were split and promoted to plots

in 9MP.

5S IBERS Field evaluation of crosses made in 2005 Spaced plants on a 1m grid Aug-2006 593 51

5S was sown orginally in August 2005, but plants failed to overwinter. Crosses re-sown in May 2006, were planted in

July 2006. Establishment was slow and hampered by poor weed control. 20

selections made for plot assessment in March 2009

7S IBERS Field evaluation of crosses made in 2005 Spaced plants on a 1m grid Jun-2006 1428 74

Excellent establishment in the warm summer of 2006 with irrigation. 94

selections made for plot assessment in March 2008

8S IBERS Field evaluation of crosses made in 2006

Spaced plants on a 1m grid Jul-2007 2070 195No selections made to date, plants still

too immature due to cold growing seasons in 2007 and 2008

9MP IBERSPromotion of outstanding

seedlings to plot trials

Selected plants from 4S, 7S and 5S were planted in miniplots (12 plants per plot) at a density of 2

plants m-2

Mar-2008192

selections for plots

n.a.

Plant splitting and a dry spell fol lowing establishment in early 2008 resulted in poor establishment. Work continues in

March 2009 to replace missing plants to complete the plots. New selections from 5S were included to replace

selections made in 2008 that did not survive splitting and transplanting.

11S IBERS Field evaluation of crosses made in 2007

Spaced plants on a 1m grid, with controls Jul-2008 1568 159

Establishment from micro-modules (as used in Germany) combined with a cool

wet summer 2008 ment slow establishment. Winter survival will be

asessed in May 2009.

OD0401 PRI Observation of seedlings from crosses

one-row plots; plant spacing 75 cm x 75 cm; one row per family,

if sufficient plants available;Aug-2003 980 105 50 plants selected and used in trial

AV0701

OD0402 PRI Observation of seedlings from crosses

setup as in OD0401;variable number of plants per cross; Aug-2003 380 45 53 plants selected and used in trial

AV0702

OD0606A PRIObservation of seedlings from crosses:Testing FS

families

50 families; one-row plots of 10 plants; spacing 75x75cm; 2 replicate randomised blocks

Aug-2006 1000 50

16 plants were selected in Jan 2008 and a sample of rhizome from each

was transferred to a pot for use in 2008 crosses

OD0606B PRI Observation of seedlings from crosses: Single plants

setup as in OD0401;variable number of plants per cross; Aug-2006 260 31

24 plants were selected in Jan 2008 and a sample of rhizome from each

was transferred to a pot for use in 2008 crosses: in addition panicles from 15 selections were gathered in autumn

2008 (open pollination crosses)

OD0607 PRIObservation of seedlings from crosses: tested by

family

setup as in OD0401;variable number of plants per cross;

Aug-2006 480 29

A sample of rhizome from 4 crosses was transferred to a pot for use in 2008

crosses: in addition panicles from 15 selections were gathered in autumn

2008 (open pollination crosses)

AV0701 PRIObservation of seedlings from crosses: tested by

family

Polycross isolation;50 HS families;plant spacing

75x75cm;grids; 9 plants/familyAug-2007 450 50

Selections from OD0401; plants surviving winter/Linuron treatment

grouped;small isolation

AV0702 PRIObservation of seedlings from crosses: tested by

family

Polycross isolation;53 HS families;plant spacing

75x75cm;grids; 9 plants/familyAug-2007 477 53

Selections from OD0402; plants surviving winter/Linuron treatment

grouped; small isolation

AV0703 PRIObservation of seedlings from crosses: tested by

family

isolation; setup as in OD0606A; 89 famil ies Aug-2007 1780 89

Crosses and OP famil ies previous year; plants surviving winter/Linuron

treatment grouped;small isolation

SID 5 (Rev. 3/06) Page 16 of 28

At IBERS plants from crosses are planted into ‘S’ selection nurseries planted at 1 plant per m2 . The progress of crosses made from 2004 to 2007 are shown in the first seven rows of Table 2.2. (Crosses made in 2008 will be planted in summer 2009). The 4S nursery from crosses made in 2004 was kept for three years not two as originally planned (agreed change of milestone) so that early morphometric analyses could be completed. The 5S nursery from crosses made in 2005 had to be replanted. The 7S nursery from crosses made in 2005 established well. The 4S, 5S and 7S nurseries were all therefore selected in 2008, with 106, 20 and 94 superior plants being selected in each nursery respectively. This resulted in a total of 220 selected plants representing an overall selection rate of 5.7%. The 220 selected plants were cut down to 192 on the basis of phenotype and cloned (12 plants per selected plant). These plants were planted (12 plants per plot at 1 plant per 1m2) in a miniplot trial (9MP) originally planted in early 2008. The design of this involved unreplicated entries plus 19 replicated plots each of M. x Giganteus and 19 M.sinensis Goliath. Plant splitting and a dry spell after planting resulted in poor establishment and missing plants have been re-established in March 2009. The first assessments will be made in 2010.

The typical selection cycle is requiring 2 to 3 years, depending on the climatic conditions within the growing season. The cold wet growing seasons of 2007 and 2008 at IBERS have meant that plants in the trial 8S were not mature enough to select in spring 2009.

At PRI the first two seed based trials were OD0401 and OD0402 in which (1) half-sib families obtained from most of the BIOMIS population and (2) offspring of crosses among selected genotypes from the BIOMIS population were tested respectively. The two adjacent trials were isolated from other unrelated pollen sources. If amounts of seed did permit one single row of ten plants was used for testing, otherwise less plants were used. In each of these two trials a set of about 50 genotypes were selected at end of second evaluation year (2005) using some plant data (biomass rating, height, drought score) collected in 2005 and 2006. These two groups of selected plants were subsequently assessed in more detail for a variety of plant characteristics, i.e. plant dry matter yield, number of stems, stem diameter, plant height, tuft diameter, stem density and seed weight. The performance of the selected plants from OD0401 is shown in Table 2.3. So far these plants represent the only single seed-derived individuals followed over a period of period of three full growing seasons. Plant dry matter yields in the second growing season, 2006 were already substantial and that in the third year excellent. The 2006 yields were adversely affected by severe drought in July 2006. The selected plants from OD0402 showed similar results (not shown). The interrelationship between the two successive years for the traits: plant yield, plant height, number of stems, and stem diameter is depicted in Figure 2.5. The plants showed a large plant-to-plant variation for these traits in both years and a fairly strong year-to-year correlation for all traits, except stem diameter. The plant yields in both years were quite good, considering the fact that the plant density was 2/m2,. The yield in year 2007, however, was better in 2006. The performance of plants in year 2006 was a fairly good predictor of the performance in 2007, at least for the traits number of stems, plant height and plant yield. The heritability of variation among these plants will be studied in AV0701 and AV0702, respectively (a parent –offspring approach).

Table 2.3 Variation in a set of 50 selected genotypes from full sib crosses (OD0401) for different of plant characteristics the first and second growing seasons at PRI.

Trait Season Unit Mean SD Range CV(%)dry weight 2006 g/plant 676 212 1069 31.3 2007 g/plant 1133 419 2498 36.9plant height 2006 cm 263 11 43 4.1 2007 cm 267 17 75 6.3stem diameter 2006 mm 4.8 0.8 3.7 16.2 2007 mm 6.1 0.7 3 11.2tuft diameter 2007 cm 34.7 4.2 20 12.1stems/plant 2006 # 61 17 71 27.5 2007 # 85 24 120 28.8stem density 2007 #/dm2 3.9 1 4.4 25.4seed weight 2006 mg/infl 88 61 266 70

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Figure 2.5 Relation between performance of plants from population (OD0401) and crosses (OD0402) in two successive years for dry matter yield, number of stems, plant height and stem diameter.

Additional analyses of the mineral contents of dry matter samples of plants from OD0401 were made by ion chromatography using an ion chromatograph device with auto sampler from Metrohm (Table 2.3). To this end the starting material for the analysis is obtained by weighing 100 mg of fine dry powder of each biomass sample (particle size < 0.5 mm) in heat resistant glass tubes with cap. The open tubes are subsequently placed in a metal frame and transferred to an oven held at 575°C for 5 hours to ash them. After cooling, the ash is dissolved in 1 ml 0.5 M formic acid and heated at 100°C for 15 minutes followed by the addition of 7.5 ml milli-Q water of 80°C and held at this temperature and dilute the hot sample as possible to 25 ml (using a top weigher). One ml is subsequently dilute 10 times in Metrohm sample tubes. These tubes are put in an autosampler of the chromatograph for the automatic analyses of the contents of several minerals relevant in relation to combustion quality such as Cl and K. Table 2.4 shows the plant to plant variation in mineral contents observed in the OD0401. The chlorine content was in both years low and the correlation between plant performance in 2006 and 2007 was practically zero. The other major combustion trait, the K content also was on average low with a correlation between years that was moderate but significant (r=0.36).

Table 2.4 Statistics describing the variation in contents for different minerals in biomass (OD0401).

Trait Season Unit Mean SD Range CV(%)[K] 2006 mg/g 0.82 0.34 1.39 41 2007 mg/g 0.48 0.16 0.90 34[Na] 2006 mg/g 0.40 0.20 0.86 50 2007 mg/g 0.21 0.12 0.36 56[Ca] 2006 mg/g 0.96 0.20 0.87 21 2007 mg/g 1.24 0.25 0.92 20[Mg] 2006 mg/g 0.70 0.14 0.65 20 2007 mg/g 0.72 0.14 0.60 20[Cl] 2006 mg/g 0.04 0.07 0.34 194 2007 mg/g 0.01 0.01 0.04 196[PO4] 2006 mg/g 1.20 0.40 1.77 34 2007 mg/g 1.43 0.31 1.19 22[SO4] 2006 mg/g 0.51 0.11 0.59 22 2007 mg/g 0.49 0.11 0.44 23

SID 5 (Rev. 3/06) Page 18 of 28

M x giganteus was not included in the trial because this species differs a lot in plant habit and is propagated through rhizomes. We note however that average values found for M. sinensis are low in comparison to M. x giganteus. No comparable data exists for 2TT.

Family selectionInitially the crossing programme of PRI was focussed on generation of offspring of crosses between 20 selected genotypes from the BIOMIS population and accessions from the collection. The resulting full-sib families with sufficient seeds were tested in a field test with randomized block design, two replicates, and one-row plots of ten plants with plant density of about 2 plants per m2. The trial was established in August 2006 (OD0606A) and in winter 2008/09 the second crop was harvested. An overview of the results is given in Table 2.5. The crops yields in 2008/09, on average ca over 11 t dm ha-1, were a lot higher than those found in winter 2007/08. The results show considerable heritable variation for each of traits measured. The best-performing full-sib families yielded over 20 t dm ha-1. A correlation and path analysis was performed to find those plant characteristics having a substantial contribution to the plant yield (year 2). The results were depicted in Fig 2.5. The path coefficients (Pxy) are measures for the direct influence of these traits on the variation for yield; the higher the absolute values the higher the direct effect. The path diagram indicates that the variation in yield is predominantly determined by phenotypic traits collected in year 2: biomass in May/June, the heading date, and the tuft diameter (Fig. 2.5). All traits together determine about 60% of the total variation for yield. It is thought that a third evaluation year is needed to come to the final selection of individual plants and/or families for a next cycle of full-sib selection.

Table 2.5 Performance of 50 full-sib families in two successive years (OD0606A).

Trait Growing season Mean SED h 2Minimum Maximum

Survival rate 2008 0.6 1 0.94Biomass rating (1-9) 2007 3.2 7.5 5.8 0.8 0.55 2008 3.4 7.4 5.8 0.6 0.65Heading date (Julian days) 2007 197.9 256.2 227.6 5.4 0.87 2008 187.3 250.9 214.3 4.5 0.88Tuft diameter (cm) 2008 11.1 27.7 18.8 1.5 0.79Plant height (cm) 2008 48.8 95.6 76.1 5.9 0.72 2008 167.4 237.5 201.8 9.9 0.76Drymatter content biomass (%) 2007 58.5 86.7 78.1 4.5 0.55 2008 71.7 77.3 74.7 1.4 0.23Biomass yield (g/plot) 2007 443 2871 1232 380 0.55 2008 1385 11596 5680 1944 0.45

Range

SID 5 (Rev. 3/06) Page 19 of 28

Figure 2.5 Path analyses of interrelationships between phenotypic traits and yield (OD0606A). Path and correlation coefficients are indicated by P and r, respectively.

In this trial to speed-up the breeding process, a few plants from vigorous superior families were selected and a small plant piece of each was potted in March to be used in the crossing programme of 2008. The selection activities at PRI yielded so far 147 genotypes to be used for further breeding.

3. Hybridisation of diploid and tetraploid accessions to produce new sterile triploid hybrids (IGER).

M. x giganteus is the best known Miscanthus genotype after being identified as a potential bioenergy crop in the 1960’s at the Danish Institute of Landscape Plants. M. x giganteus was collected from the wild by the Danish plant collector Askel Olsen in 1930. It is thought to be the product of an interspecific cross between a diploid M. sinensis and a tetraploid M. sacchariflorus. M. x giganteus is triploid and is therefore sterile. In this objective, we aimed to use diploid M. sinensis (from genetic resources and from approach 2) and tetraploid M. sacchariflorus and also tetraploid M. sinensis and a diploid M. sacchariflorus to produce new triploid hybrids with improved yield, and concurrent drought and frost resistance. Interspecific hybrids may offer the best opportunities for the fixation of heterosis (as exemplified by wheat and hybrid perennial x Italian ryegrasses). In addition, triploid hybrids are sterile by virtue of having only set of chromosomes from the diploid parent of the cross.

In order to make such crosses information is required in relation to (i) ploidy of germplasm available in order to use diploid and tetraploid parents (ii) a knowledge of the conditions required for flowering to enable flowering synchronisation (iii) where partial incompatibility occurs that embryos can be rescued (iv) once rescued, the ability of the seedling to survive transfer to soil.

Determination of ploidy level by Flow Cytometry2 x 2cm long leaf pieces were added to a Petri dish containing 2 ml of buffer (1 litre contains 5ml Tween 20 and 10.5g citric acid monohydrate). The leaf pieces were chopped with a razor blade before 0.5ml of another buffer was added (1 litre contains 50g Na2HPO4 and 2mg DAPI). The resulting solution was thoroughly mixed and pipetted through a filter into a tube. The tube was placed into a Partec PA ploidy analyser and a run initiated.

Miscanthus has a basic chromosome number of 19 and as such a diploid genotype would contain 38 chromosomes, a triploid 57 chromosomes and a tetraploid 76 chromosomes.Table 1 summarises the ploidy of all accessions in 2TT. The naturally occurring 4x plants are all M. sacchariflorus types. It is possible that some of the triploids are duplicate accessions of the same clone from different source names.

SID 5 (Rev. 3/06) Page 20 of 28

Table 3.1 Ploidy of 249 genotypes in the 2TT trial at Aberystwyth.

Species 2x 3x 4xM. sinensis 201 4 0M. sacchariflorus 13 1 20M. floridulus 2 0 0M. transmorrisonensis 1 0 0M. x giganteus 0 7 0

Creation of tetraploid M. sinensis

Several approaches were attempted to double the ploidy of diploid M. sinensis. 1) Stems. Stems sections were exposed to different colchicine treatments through a triangular shaped incision. Following washing these stems were transplanted into shallow boxes containing potting compost. A duplicate control set was also prepared and treated similarly. 2). In vitro. Seeds from two open crosses were sterilised and germinated before exposure to 0.2% colchicine solution. Following washing the meristems were then transferred to MSZ agar plates (Murashige and Skoog medium, 3% sucrose and 0.1 mg/l zeatin) and allowed to grow up. 3) Seed. Germinating seeds suspended over solutions containing 0.03%, 0.06% and 0.1% aqueous solution of colchicine with DMSO 0.5% (by volume) for 6, 12 and 24 hours at room temperature. Seeds were then thoroughly washed with distilled water and sown into trays containing John Innes number 3 compost. Seeds were left to grow up in a glasshouse with daily watering and the resulting plants were then subjected to flow cytometry in order to determine their ploidy level.

Protocols 1 and 2 were unsuccessful with Sin 11. 900 seeds were subjected to protocol 3. One was found to be a tetraploid (0.03% colchicine for 24 hours) and has been allowed to grow on.

In 2006 we received five polyploid M. sinensis from colleagues in Sweden which we believed were 4x. Flow cytometry in 2008 showed these were 5x, thus explaining the absence of seed set in crosses attempted with these plants.

Obvious improvements in polypolidisation technique are needed and it is quite possible that there is genetic variation in the amenability to polyploidisation which would facilitate tetraploid production.

Flowering synchronisation

In order to allow successful crossing, it is obviously necessary to ensure that flowering time of both parents are synchronised so that pollen can meet receptive stigmas. A M. sinensis (Sin-11, 2x) and a M. sacchariflorus (Sac-5, 4x) were selected to attempt crosses for the following reasons: 1) ploidy levels were known from chromosome counts; 2) flowering under field conditions was observed at trials where daily meteorological conditions were available 3) proven excellent fertility of EMI-11 (a grandparent of BIOMIS mapping population); 4) sufficient clonal material in pots from EMI trials to stagger growth stages of plants.

Environmental requirements for flowering of Sac-5 and Sin-11 were derived from field observations in the EMI programme and some additional experimentation in the glasshouse. The Sac-5 flowered on the 1 Dec, and the Sin 11 about 3 weeks later. Staggering the M. sinensis side of the cross by holding plants back in the cold room was performed each year. During the project alignment of flowering time between M. sacchariflorus and M. sinensis (not always Sin 11) was achieved a number of times, but we found flowering time of plants grown in small pots difficult to predict with the precision needed. Paired crosses were made under bags. Fifteen paired inter-specific crosses were performed. It was found that open pollination within the glasshouse was more likely to produce embryos than when the cross was made in bags.

From 2007 onwards mature phenotype data is becoming available from 2TT. We found that none of the 20 4x M. sacchariflorus flowered in the field before the first frosts in autumn, so that sophisticated manipulation of articial environmental conditions will be needed in order to match additional M. sacchariflorus and M. sinensis parental combinations.

Embryo rescue

Initial crosses undertaken between M. sinensis and M. sacchariflorus had failed to produce viable seed. This is common in wide crosses usually as a result of degeneration of the seed endosperm (for a review, see Sharma,

SID 5 (Rev. 3/06) Page 21 of 28

Kaur and Kumar, 1995). In some cases it is still possible to obtain hybrids after pollination by excising the embryo formed and culturing it aseptically under controlled conditions. This method has proved to be very successful in a wide range of species.

The process of embryo rescue was attempted on these inter-specific crosses. The florets were harvested 14 days after pollination and the immature seeds collected from them. These were then sterilised (6% sodium hypochloride) and the embryos dissected out and placed onto agar slopes (Gamborg and Miller B5 media, plus 30g/l sucrose) to grow up at 22 °C in the dark. In vitro seedlings were transferred to soil based media after 1 to 6 months depending on the growth rate.

As indicated earlier, it was found that open pollination within the glasshouse was more likely to produce embryos than when the cross was made in bags.

Of the 50 embryos rescued, 15 have showed signs of growth. This is a reasonable success rate in relation to the range of results found in the literature. Extreme care has to be taken to damaging the embryo due to the extremely small size of the Miscanthus ovule. As indicated below, the main problem would seem to be in the ability of the seedling to transfer from aseptic culture to soil.

Only one grew vigorously, and this was transferred successfully to soil. The other 14 did not grow beyond the 1-2 cm height stage before dying this was probably due to their incompatible genetic make up. Flow cytometry showed that a triploid was produced. This has been clonally propagated in pots and has been added to the 9MP plot trial for yield evaluation.

4. Improvement of breeding efficiency based on early morpho-physiological prediction of productivity and persistence and the indirect measurement of chemical composition using FTIR.

Introduction

Miscanthus, unlike many other crop species, is a perennial. Perenniality has the advantage of reducing the inputs required to grow the crop, and is an essential component in raising the overall energy balance. In the UK climate Miscanthus requires at least 3 years before the mature phenotype is reached. This slow rate of establishment means that breeding cycles are slower than in annual crops.

In this objective, we sought to develop techniques to develop early-morphometric selection as a method to accelerate yield selection and other traits. It is based on the concept that an outstanding seedling develops into an outstanding mature plant. Concurrent with yield is the need to raise biomass quality since it is an important component in determining the yield of usable energy per hectare. Previous studies have shown considerable phenotypic variation in mineral composition (Atienza et al., 2003a, b, c, d, 2002; Clifton-Brown et al., 2001; Clifton-Brown and Lewandowski, 2002 and Lewandowski et al., 2003) using wet chemistry. There are few published studies quantifying the variation in structural carbohydrates and lignin. Wet chemistry of these cell wall components are very costly and time consuming. In this project techniques were developed to (1) evaluate the value of early-morphometric selection and (2) to develop equations to predict structural carbohydrates and lignin from infra-red scanning.

Early-morphometric selectionThe field trials most intensively used for early morphometric selection at IBERS and PRI were 4S and OD404A respectively. Some early morphometric analysis of OD404A was reported in section 2 (Fig. 2.4). 64-74% of the variation between DM yield in 2006 and that in 2007 is accounted for.

At IBERS, seed from crosses made in 2004 was germinated in modules in Spring 2005, and planted in the 4S field trial in June 2005 (further details in Table 2.1). The crosses made in 2004 have a wide genetic base with accessions from DEFRA, EMI and BIOMIS projects.

Harvests were made in the spring following each growing season. Following the first and second growing seasons all surviving seedlings were harvested to estimate fresh weight yield. A significant correlation coefficient between the fresh weight per plant in the first two years was found (Fig 4.1a). For logistical reasons dry weight per plant was only determined on a selection of 66 plants following three consecutive growing seasons. Fig. 4.1b shows that there was a significant correlation (r2 =0.51 and 0.49 respectively) between first year and second year dry weight yields and on average there was a 7 fold increase in the plant biomass. Yield increase from the second to the third growing season was much smaller with a 3.5 fold increase (Fig. 4.1c). These proportions of variation (49-51%) are much less than those obtained at PRI (64-74%).

More significantly, when year 1 and year 3 were regressed, a rather weak correlation was revealed (R2 of 0.18 (Fig. 4.1d)).

SID 5 (Rev. 3/06) Page 22 of 28

This shows that selection after the first growing season is not sufficient to make very reliable selections of the mature phenotype. Selections after 2 years of growth appear reliable enough to make selections. Waiting until the third year for the ‘mature’ yield will ensure high quality selections go into plot trial assessments, but we believe from this data that two years is a reasonable compromise ensuring the breeding programme can proceed at a pace which is required to ensure that products are brought in a reasonable time frame to the market.It is important to note that these analyses of yield in sequential years assume that the environmental gradients in the field are negligible. As shown in Table 2.2, the 11S nursery has been established with standard clones acting as controls at regular intervals to take better account of environmental variation.

Figure 4.1. Yield (g fresh weight plant-1 A) and (g DW plant-1 B, C, D) following three growing seasons (2006, 2007 and 2008) at Aberystwyth. Best fit regression and correlation co-efficient are shown in each panel. NB Ordinary least squares regression equations relating 2007 data to 2006 are not wholly appropriate since errors on both y and x axes and the x variate is not independent. Type II regressions are being undertaken.

Composition influencing combustion quality

MineralsOne of the outstanding properties of Miscanthus biomass is a low content of some minerals adversely affecting the combustion quality. This was found in the evaluation of stems of plants harvested in the winters following the growing season 2006 and 2007, respectively (Figure 4.2). Monovalent ions, such as Na, K, and Cl were low in content with no significant correlation between years except for K (Figure 4.2 A to C). In contrast, the contents found for bivalent ions, like Ca, SO4, PO4 measured in both successive years were fairly similar and positively correlated (Figure 4.2 D to F). First year plants were found to have higher mineral contents than at harvest following the second year. This is consistent with observations that were made previously in the EMI trials (Clifton-Brown et al. 2002). Experiments are in progress to get insight in to the relationships between the performance of these plants and their offspring.

SID 5 (Rev. 3/06) Page 23 of 28

y = 6.0644xR2 = 0.5528

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Figure 4.2. Mineral contents (mg per g dry matter) in year 1 (2006) and year 2 (2007) plotted against each other for 47 individuals in the trial OD 606A. Slope and correlation co-efficient (r2) are given where significant relationships were found. As for Fig. 4.1, ordinary least squares regression equations relating 2007 data to 2006 are not wholly appropriate since errors on both y and x axes and the x variate is not independent.

Structural carbohydrates

Variation in cell wall structural carbohydrates influence calorific value and the ease with which the biomass can be converted into different fuels (e.g. ethanol).This work has been carried out through a collaboration with the EPSRC SUPERGEN project by Dr. Ed Hodgson and Dr. Gordon Allison. A calibration data set for lignin, cellulose and hemi-cellulose has been derived from archive EMI. An example for lignin is shown in Fig. 4.3.

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y = 0.17x + 0.34R2 = 0.13

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Figure 4.3. Lignin (% in dry matter) of 73 Miscanthus samples selected from the EMI sample archive (366 samples) analysed by wet chemistry and use to generate a calibration data set for NIR. Fitted line and equation are shown, together with the 1:1 line.

The calibration between NIR and wet chemical extractions was applied to all the plants in the 2TT experiment to derive the estimates which are shown Fig. 1.6 and 1.7. These methods have yet to be applied to seedling and selection trials.

5. The production of large trait mapping populations and the identification of a realistic cost-effective roadmap to more efficient breeding through the development of marker-assisted selection.

In the original project application, we considered that marker-assisted selection would be an extremely worthwhile breeding tool but recognised that the project would not be able to develop or use molecular markers. The development and use of molecular markers work would need to be funded separately and to follow on from the identification of mapping populations and the development of large sets of markers that can be associated with traits.

Rather, we proposed

a) to identify suitable parents and generate up to 10 larger trait mapping populations each of approximately 200 progenies. We would seek to identify parents with contrasting leaf extension, low-temperature chlorosis, intra-row canopy enclosure and erectness of leaves, shoot numbers, flowering time, senescence patterns, winter-hardiness, DM yield and components of combustion quality. These populations would provide considerable choice for a limited number (in a separate project if this materialised) to be screened with molecular markers, assessed in the field and analysed to identify QTLs.

b) to sift the many ideas that different organisations have and develop a plan involving sound workable proposals which would best cost-effectively and usefully underpin the genetic improvement of Miscanthus. We would also collectively explore the possibility of BBSRC or joint Defra-BBSRC support of genomic research in Miscanthus. This could even include the full sequencing of the species. The plan will need to consider the conversion of candidate gene markers identified in maize in the Supergen project at IGER, the conversion of maize microsatellite markers (University of Bristol), the use of these markers to construct a useful Miscanthus map and the identification of associations between markers and QTLs through phenotypic assessments of mapping progenies at IGER, RRes and/or PRI.

In relation to a., we identified eight mapping populations involving segregation for key traits. These showed parental variation in key traits including biomass yield, autumn senescence, canopy height, stem angle, leaf angle and area, time of flowering, flowering intensity, spread, moisture content and lignin content. The total and average number of progeny produced per population were 1449 and 207 respectively, with the range being 114 – 304. From these populations, one consisting of 204 progeny has been identified as suitable for future Defra work showing high parental variation for the key traits biomass yield, autumn senescence, moisture content and leaf width and area. Seedlings of ~200 plants have been raised in modules and will be grown in larger pots to produce clonal material that can be planted in the field in 2010 in replicated trials. Molecular markers developed the BBSRC Strategic Programme Grant, BSBEC and Industry (subject to a licensing and IP agreement) will be used in due course.

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In relation to b., a limited number of markers will be available from BBSRC Strategic Programme Grant and from BSBEC.

In addition, high numbers of SNP markers have and are being developed by Industry but we will not be able to use these in the Defra project at present due to Industry’s need to be certain that its IP, which has taken millions of pounds to generate, can be protected In order to justify its investment, and earn royalties when brought into Miscanthus breeding.

Scope also exists for developing DaRT markers. These would require additional Defra funding. Associations will be sought in due course between molecular markers and key sustainability traits.

6. Identification with DEFRA of the exploitation route in UK and Europe, taking into account expertise in large-scale production and marketing.

A way forward for the commercialisation of new Miscanthus is being discussed with Defra. This will need to consider

A. Past experience in the provision of energy from biomass crops:B. Ability to form an effective research partnershipC. Direct contributions to the researchD. Market delivery

General Discussion

Valuable variation was identified for yield and other characters of value to the breeding programme. Further trials in miniplots are needed to establish the yields of selections relative to M. x giganteus. Some traits such as canopy height, flowering and stem diameter are important yield components. We avoid setting an ideotype however as there may be several pathways to maximum yield. Others such as lignin and mineral content are important determinants of combustion quality. Other end uses e.g. lignocellulosic fermentation or production of bio-oil by fast pyrolysis will most likely demand varieties of Miscanthus with much lower levels of lignin and these will be bred using genotypes with low levels of aromatic cell wall components. Low moisture content is desirable in the harvested crop to reduce transport costs and improve fuel characteristics (Lewandowski and Kicherer, 1997) and this characteristic is usually correlated with early senescence. Delayed senescence may be beneficial to extend the productive growth season and in some accessions this characteristic has been correlated with low moisture content at harvest; these accessions may provide the genetic basis for extending the growing season by delaying senescence while maintaining low moisture in the crop at harvest. Figure 1.3 demonstrates the high variation between genotypes in moisture content.

1202 successful M. sinensis crosses were at IBERS and PRI over the five years. Of these 522 were paired, 80 polycrosses and 773 open crosses. Poor seed set in some paired crosses was attributed to self-incompatibility genes. Over 15,000 seedlings have been planted in field trials from which 333 elite selections have been made to date on the basis of high biomass potential and other characters. Although further testing of selections in plots is needed, preliminary visual observations suggest that these selections represent a major advance over M. x giganteus achieved since the inception of the project in 2004.

In setting up the project, we noted the high cost of planting Miscanthus from rhizomes, and we proposed to devote a relatively small effort to testing the feasibility of developing seed-propagated M. sinensis. Reduced establishment costs would make a large difference to the economics of establishment and reduce the necessity for a planting grant. To this end, we have measured the general combining ability of genotypes using polycrosses and test-crosses, essential steps to the generation of synthetic varieties that are necessary in order to maintain heterozygosity. The minimum temperature for germination was found to be in the range of 8-10OC. This is relatively high, so that seed-propagated Miscanthus would either have to be sown relatively late but not too late as to jeopardise winter-survival, be established under polythene, or young plants raised and transplanted. Further

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work would be needed to establish the practicalities of seed-propagated Miscanthus. From the variation apparent in Figure 2.4, there would seem little scope for selection for lower minimum germination temperature.

Interspecific hybrids may offer the best opportunities for the fixation of heterosis (as exemplified by wheat and hybrid perennial x Italian ryegrasses). In addition, triploid hybrids are sterile by virtue of having only set of chromosomes from the diploid parent of the cross. However, the production of triploid interspecific hybrids has proved difficult and complex.

Considerable information is required relating to (i) ploidy of germplasm (ii) a knowledge of the conditions required for flowering to enable flowering synchronisation (iii) where partial incompatibility occurs (e.g. when no viable seeds are obtained from a cross) so that embryos can be rescued (v) once rescued, the ability of the seedling to survive transfer to soil. Triploid intraspecific hybrids should be easier. The embryo rescue technique showed a 30% success rate, of those successfully recovered one was genetically compatible and thus able to grow into an adult plant. The milestone ‘produce 2500 seedlings annually from 2008’ is unrealistic and despite much effort could not be met. Producing triploids through inter-specific hybridisation has proved much more difficult than previously considered. Only one triploid has been produced. There are further approaches to the production of triploids and which have not yet been attempted, but will be the subject of future funding proposals. It would appear that a limited number of triploid embryos can be cultured in aseptic conditions but that the ability of the seedling to survive transfer to soil may be a more difficult, though not insurmountable, step. The benefits of triploid varieties still make this a very worthwhile research theme for future work, but it must be seen as high risk.

Polyploidisation has proven to more difficult than first thought. Several different techniques were tried which proved to be unsuccessful but one technique was found to work at a relatively low rate. This technique now requires further fine tuning in order to increase the success rate. It may become apparent that some genotypes are more susceptible to chromosome doubling than others and so further work is required to find which if any are more able to yield positive results.

Early-morphometric selection in the narrow genetic base in PRI was more reliable (R2 =0.65-0.74) than in trials with a broader genetic base at IBERS (R2 =0.49-51) when successive years were considered but the association between year 1 and year 3 was inadequate. This emphasises the need for marker-assisted selection to filter down the quantity of germplasm that needs to be held in single spaced plant trials, and expedite the selection of seedlings that can be promoted to plot trials.

Future Work

Further work is necessary in order to improve the sustainability of Miscanthus as a key biomass feedstock for energy production through substitution of fossil fuels, thereby reducing greenhouse gas emissions. High yielding varieties are necessary in order to minimise land-use requirements, enable economic development of varieties adapted for climate change and reduce genetic vulnerability. Renewable biomass supply chains also offer major opportunities in rural areas to enhance rural employment, biodiversity and localised flood reduction.

Research is required to:

Assess novel genetic material in replicated single plant nurseries for genetic variation in traits, particularly the 120 accessions collected from China, Japan and Taiwan through a subsidiary project (NF0436) which completed quarantine in October 2008.

Assess novel genetic material obtained from replicated single plant nurseries for genetic variation in bio- conversion quality using high throughput technologies.

Combine this knowledge to generate breeding material for a range of high-yielding varieties of suitable quality suited for sustainable production under differing conditions on marginal land.

Develop genome-based marker-assisted selection strategies for Miscanthus breeding programme in order to accelerate breeding, by obtaining sequence-based molecular markers developed by the BBSRC Strategic Programme Grant and Industry, developing novel mapping populations and identifying associations between molecular markers and key sustainability traits.

References to published material

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9. This section should be used to record links (hypertext links where possible) or references to other published material generated by, or relating to this project.Anon. 1998a. Neutral detergent fibre and NCGD. Gerhardt Guideline Methodology, Method No. GMFC2.20th Oct.,1998.Anon. 1998b. Acid Detergent Fibre. Gerhardt Guideline Methodology. Method No. GMFC3, 20th Oct.,1998.Atienza SG, Satovic Z, Petersen KK, Dolstra O, Martin A. 2003a. Identification of QTLs influencing combustion quality in Miscanthus sinensis Anderss. II. Chlorine and potassium content. Theoretical and Applied Genetics 107, 857-863.Atienza SG, Satovic Z, Petersen KK, Dolstra O, Martin A. 2003b. Identification of QTLs associated with yield and its components in Miscanthus sinensis Anderss. Euphytica 132, 353-361.Atienza SG, Satovic Z, Petersen KK, Dolstra O, Martin A. 2003c. Identification of QTLs influencing agronomic traits in Miscanthus sinensis Anderss. I. Total height, flag-leaf height and stem diameter. Theoretical and Applied Genetics 107, 123-129.Atienza SG, Satovic Z, Petersen KK, Dolstra O, Martin A. 2003d. Influencing combustion quality in Miscanthus sinensis Anderss.: identification of QTLs for calcium, phosphorus and sulphur content. Plant Breeding 122, 141-145.Atienza SG, Satovic Z, Petersen KK, Dolstra O, Martin A. 2002. Preliminary genetic linkage map of Miscanthus sinensis with RAPD markers. Theoretical and Applied Genetics 105, 946-952.Clifton-Brown JC, Lewandowski I, Andersson B, Basch G, Christian DG, Bonderup-Kjeldsen J, Jørgensen U, Mortensen J, Riche AB, Schwarz K-U, Tayebi K, Teixeira F. 2001. Performance of 15 Miscanthus genotypes at five sites in Europe. Agronomy Journal 93, 1013-1019.Clifton-Brown JC, Lewandowski I. 2002. Screening Miscanthus genotypes in field trials to optimise biomass yield and quality in Southern Germany. European Journal of Agronomy 16, 97-110.Clifton-Brown, J. C., Robson, P. R. H., Allison, G. G., Lister, S. J., Sanderson, R., Morris, C., Hodgson, E., Farrar, K., Hawkins, S., Jensen, E. S., Jones, S. T., Huang, L., Roberts, P. C., Youell, S. J., Jones, B. R., Wright, A., Valentine, J., Donnison, I. S. (2008). Miscanthus: breeding our way to a better future. Biomass and Energy Crops III, Aspects of Applied Biology, 90, 199-206. Lewandowski I, Clifton-Brown JC, Andersson B, Basch G, Christian DG, Jørgensen U, Jones MB, Riche AB, Schwarz K-U, Tayebi K, Teixeira F. 2003. Environment and harvest time affects the combustion qualities of Miscanthus genotypes. Agronomy Journal 95, 1274-1280.Hodgson EM, Lister SJ, Bridgwater AV, Clifton-Brown J, Donnison. IS. 2009. Genotypic and environmentally derived variation in the cell wall composition of Miscanthus in relation to its use as a biomass feedstock. Biomass and Bioenergy In review.Kitcherside MA., Glen EF, and Webster AJF. 2000. Fibrecap: an improved method for the rapid analysis of fibre in feeding stuffs. Animal Food Science and Technology 86:125 – 132.Lewandowski I, Kicherer A. 1997. Combustion quality of biomass: practical relevance and experiments to modify the biomass quality of Miscanthus x giganteus. European Journal of Agronomy 6, 163-177.Sharma, DR, Kaur R and Kumar K. (1996) Embryo rescue in plants – a review. Euphytica 89, 325-337. Van Soest, PJ. 1963. Use of detergents in the analysis of fibrous feeds. 1. Preparation of fibre residues of low nitrogen content. Journal of the Association of Official Analytical Chemists 46: 825-829.Van Soest PJ, and Wine RH. 1967. Use of detergents in the analysis of feeds. IV. Determination of plant cell wall constituents Journal of the Association of Official Analytical Chemists, 50: 50-55.

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