Early selection of novel triploid hybrids of shrub willow with improved biomass yield relative to diploids

BACKGROUND: Genetic improvement of shrub willow (Salix), a perennial energy crop common to temperate climates, has led to the development of new cultivars with improved biomass yield, pest and disease resistance, and biomass composition suitable for bioenergy applications. These improvements have largely been associated with species hybridization, yet little is known about the genetic mechanisms responsible for improved yield and performance of certain willow species hybrids. RESULTS: The top performing genotypes in this study, representing advanced pedigrees compared with those in previous studies, were mostly triploid in nature and outperformed current commercial cultivars. Of the genotypes studied, the diploids had the lowest mean yield of 8.29 oven dry Mg ha(−1) yr(−1), while triploids yielded 12.65 Mg ha(−1) yr(−1), with the top five producing over 16 Mg ha(−1) yr(−1). Triploids had high stem area and height across all three years of growth in addition to greatest specific gravity. The lowest specific gravity was observed among the tetraploid genotypes. Height was the early trait most correlated with and the best predictor of third-year yield. CONCLUSIONS: These results establish a paradigm for future breeding and improvement of Salix bioenergy crops based on the development of triploid species hybrids. Stem height and total stem area are effective traits for early prediction of relative yield performance

Biological conversion assay using Clostridium phytofermentans to estimate plant feedstock quality

BACKGROUND: There is currently considerable interest in developing renewable sources of energy. One strategy is the biological conversion of plant biomass to liquid transportation fuel. Several technical hurdles impinge upon the economic feasibility of this strategy, including the development of energy crops amenable to facile deconstruction. Reliable assays to characterize feedstock quality are needed to measure the effects of pre-treatment and processing and of the plant and microbial genetic diversity that influence bioconversion efficiency. RESULTS: We used the anaerobic bacterium Clostridium phytofermentans to develop a robust assay for biomass digestibility and conversion to biofuels. The assay utilizes the ability of the microbe to convert biomass directly into ethanol with little or no pre-treatment. Plant samples were added to an anaerobic minimal medium and inoculated with C. phytofermentans, incubated for 3 days, after which the culture supernatant was analyzed for ethanol concentration. The assay detected significant differences in the supernatant ethanol from wildtype sorghum compared with brown midrib sorghum mutants previously shown to be highly digestible. Compositional analysis of the biomass before and after inoculation suggested that differences in xylan metabolism were partly responsible for the differences in ethanol yields. Additionally, we characterized the natural genetic variation for conversion efficiency in Brachypodium distachyon and shrub willow (Salix spp.). CONCLUSION: Our results agree with those from previous studies of lignin mutants using enzymatic saccharification-based approaches. However, the use of C. phytofermentans takes into consideration specific organismal interactions, which will be crucial for simultaneous saccharification fermentation or consolidated bioprocessing. The ability to detect such phenotypic variation facilitates the genetic analysis of mechanisms underlying plant feedstock quality

Overexpression of \u3ci\u3eSbMyb60\u3c/i\u3e impacts phenylpropanoid biosynthesis and alters secondary cell wall composition in \u3ci\u3eSorghum bicolor\u3c/i\u3e

The phenylpropanoid biosynthetic pathway that generates lignin subunits represents a significant target for altering the abundance and composition of lignin. The global regulators of phenylpropanoid metabolism may include MYB transcription factors, whose expression levels have been correlated with changes in secondary cell wall composition and the levels of several other aromatic compounds, including anthocyanins and flavonoids. While transcription factors correlated with downregulation of the phenylpropanoid biosynthesis pathway have been identified in several grass species, few transcription factors linked to activation of this pathway have been identified in C4 grasses, some of which are being developed as dedicated bioenergy feedstocks. In this study we investigated the role of SbMyb60 in lignin biosynthesis in sorghum (Sorghum bicolor), which is a drought-tolerant, high-yielding biomass crop. Ectopic expression of this transcription factor in sorghum was associated with higher expression levels of genes involved in monolignol biosynthesis, and led to higher abundances of syringyl lignin, significant compositional changes to the lignin polymer and increased lignin concentration in biomass. Moreover, transgenic plants constitutively overexpressing SbMyb60 also displayed ectopic lignification in leaf midribs and elevated concentrations of soluble phenolic compounds in biomass. Results indicate that overexpression of SbMyb60 is associated with activation of monolignol biosynthesis in sorghum. SbMyb60 represents a target for modification of plant cell wall composition, with the potential to improve biomass for renewable uses

Changes in polyamines, inorganic ions and glutamine synthetase activity in response to nitrogen availability and form in red spruce (Picea rubens)

We analyzed effects of nitrogen availability and form on growth rates, concentrations of polyamines and inorganic ions and glutamine synthetase activity in in-vitro-cultured red spruce (Picea rubens Sarg.) cells. Growth rates, concentrations of polyamines and glutamine synthetase activity declined when either the amount of nitrate or the total amount of N in the culture medium was reduced. When total N in the medium was increased, cell mass increased without significant changes in glutamine synthetase activity or polyamine concentration. Reductions in the amount of nitrate or total N in the culture medium resulted in increased accumulations of Ca, Mn and Zn in the cells, and K accumulation decreased in response to decreasing nitrate:ammonium ratios. The data indicate that changes in total N availability as well as the forms of N play important roles in the physiological responses of in-vitro-grown red spruce cells that mimic the observed responses of forest trees to soil N deficiency and N fertilization

Differential expression of genes encoding phosphate transporters contributes to arsenic tolerance and accumulation in shrub willow (Salix spp.)

Studies of arsenate and phosphate uptake by plants in hydroponic and soil systems indicate a common transport mechanism via the phosphate transporters (PHTs) due to structural similarity of the anions. Typically, the presence of phosphate decreases plant uptake and translocation of arsenate in hydroponic solution. This study quantified arsenic (As) uptake related to the presence of phosphorus in an As-tolerant willow (Salix viminalis×. Salix miyabeana) and an As-sensitive willow (Salix eriocephala). Addition of phosphate resulted in greater As accumulation than in treatments without phosphate in both genotypes, although the tolerant genotype accumulated more As than the sensitive one. Expression of genes for two putative high-affinity phosphate transporters, PHT1;3 and PHT1;12, were up-regulated in both willow genotypes upon addition of As, but to a greater extent in the As-sensitive genotype. Expression of a third putative transporter, PHT1;4, was greater in the As-sensitive genotype but was not up-regulated as a result of As addition in either genotype. Leaves of the As-tolerant willow genotype contained greater concentrations of γ-glutamylcysteine (γEC) than the sensitive genotype, although this was not due to differential expression of the γ-glutamylcysteine synthetase (γECS) gene. The results suggest that increased expression of PHT1 upon exposure to As in an As-sensitive genotype contributes to rapid toxicity. Our data suggest that although detoxification capacity may be different between genotypes, the differences are not due to up-regulation of γECS or phytochelatin synthase. © 2011 Elsevier B.V

Ploidy Level Affects Important Biomass Traits of Novel Shrub Willow ( Salix ) Hybrids

Polyploidy is a common observation in the genus Salix, including some of the shrub willow species currently being bred as a potential bioenergy feedstock. Breeding of shrub willow has produced new species hybrids, among which a disproportionate number of high-yielding genotypes are triploid, produced from crosses between diploid and tetraploid parents. These novel hybrids display significant variation in biomass compositional quality, including differences according to ploidy. The triploid and tetraploid genotypes possess lower lignin content than diploid genotypes. Biomass composition was also significantly different across the 3-year growth cycle typical of bioenergy plantings. There were differences in syringyl/guaiacyl (S:G) lignin ratios among the 75 genotypes examined, in addition to significant correlations with willow growth traits, yield, and composition. These differences suggest that a long-term strategy of breeding for triploid progeny will generate cultivars with improved growth traits and wood composition for conversion to biofuels

Effect of Particle Size on Low-Temperature Pyrolysis of Woody Biomass

When biomass is thermochemically processed, the size of the biomass particles affects processing time requirements and yields. This study investigates the effects of particle size at the centimeter scale on pyrolysis through both experimental and modeling approaches, with three types of woody biomass; poplar, pine sapwood, and pine heartwood. Large (<i>D</i> = 3.81 cm) and small (<i>D</i> = 2.54 cm) wood spheres were pyrolyzed under thermally thick conditions at three final pyrolysis temperatures in a reactor with turbulent gas flow; the same wood materials were also pyrolyzed in a thermogravimetric analyzer (TGA), under kinetic control. The experiments were simulated using a previously published 1-dimensional pyrolysis model, which includes transport and kinetics of solid to vapor reactions for biomass components. Particle size had a strong effect on devolatilization timing and also affected the yields of some species. The model was successful at predicting the qualitative features and approximate magnitudes of quantities such as temperature overshoot, product yields for thermally thick particles, and devolatilization timing in both TGA and thermally thick particles. However, the dependence of yields and timing on wood type and particle size were not reproduced by the model

Enzymatic Saccharification of Shrub Willow Genotypes with Differing Biomass Composition for Biofuel Production

In the conversion of woody biomass feedstocks into liquid fuel ethanol, the pretreatment process is the most critical and costly step. Variations in biomass composition based on genetic differences or environmental effects have a significant impact on the degree of accessibility accomplished by pretreatment and subsequent sugar release by enzymatic hydrolysis. To evaluate this, biomass from 10 genetically diverse, genotypes of shrub willow (Salix spp.) was pretreated with a hot-water process at two levels of severity, hydrolyzed using a combination of two commercial enzyme cocktails, and the release of hexose and pentose monomers was quantified by HPLC. Among the genotypes selected for analysis, cellulose content ranged from 39 to 45% (w/w) and lignin content ranged from 20 to 23% (w/w) at harvest. Differences in the effectiveness of the pretreatment process were observed among the various willow genotypes. Correlations were identified between total sugar release and % cellulose and % lignin content. There was a significant effect of pretreatment severity on polysaccharide accessibility, but the response to pretreatments was different among the genotypes. At the high severity pretreatment ‘SV1’ was the least recalcitrant with sugar release representing as much as 60% of total biomass. These results suggest that structural, as well as chemical characteristics of the biomass may influence pretreatment and hydrolytic efficiency

Pyrolysis of Centimeter-Scale Woody Biomass Particles: Kinetic Modeling and Experimental Validation

Pyrolysis of centimeter-scale wood particles is of practical interest and provides a sensitive test of pyrolysis models, especially their thermochemistry. In this paper we present an updated comprehensive pyrolysis model including chemical reactions and transport of heat and species, implemented independently in two different software environments. Results of the model are compared to experimental results of three independent sets of centimeter-scale experiments. Temperatures, mass losses, and rate of production of several gaseous and light tar species are included in the comparisons. Predictions and experiments agree qualitatively and in most cases have reasonable quantitative agreement. We also report comparisons of model predictions to literature data obtained in other regimes (thermogravimetric analysis and omogeneous tar cracking) in order to demonstrate that predictive capabilities of the model have not been compromised by the modifications presented here

Overexpression of \u3ci\u3eSbMyb60\u3c/i\u3e impacts phenylpropanoid biosynthesis and alters secondary cell wall composition in \u3ci\u3eSorghum bicolor\u3c/i\u3e

The phenylpropanoid biosynthetic pathway that generates lignin subunits represents a significant target for altering the abundance and composition of lignin. The global regulators of phenylpropanoid metabolism may include MYB transcription factors, whose expression levels have been correlated with changes in secondary cell wall composition and the levels of several other aromatic compounds, including anthocyanins and flavonoids. While transcription factors correlated with downregulation of the phenylpropanoid biosynthesis pathway have been identified in several grass species, few transcription factors linked to activation of this pathway have been identified in C4 grasses, some of which are being developed as dedicated bioenergy feedstocks. In this study we investigated the role of SbMyb60 in lignin biosynthesis in sorghum (Sorghum bicolor), which is a drought-tolerant, high-yielding biomass crop. Ectopic expression of this transcription factor in sorghum was associated with higher expression levels of genes involved in monolignol biosynthesis, and led to higher abundances of syringyl lignin, significant compositional changes to the lignin polymer and increased lignin concentration in biomass. Moreover, transgenic plants constitutively overexpressing SbMyb60 also displayed ectopic lignification in leaf midribs and elevated concentrations of soluble phenolic compounds in biomass. Results indicate that overexpression of SbMyb60 is associated with activation of monolignol biosynthesis in sorghum. SbMyb60 represents a target for modification of plant cell wall composition, with the potential to improve biomass for renewable uses