Analysis of lignocelluloses and lignins from Arundo donax L. and Miscanthus sinensis Anderss., and hydroliquefaction of Miscanthus

In a comparative investigation of the chemical composition of Arundo donax L. and Miscanthus sinensis Anderss. the following experiments were performed: ash determination and ash characterization by energy dispersive X-ray analysis; determination of solubility in cyclohexane/ ethanol, hot water, 1% hydrochloric acid and sodium hydroxide; C, H and N determination; determination of Klason lignin and acid soluble lignin content; sulfuric acid hydrolysis followed by borate complex ion exchange chromatography of the monomeric sugars; isolation of milled wood lignins (MWL) and dioxane lignins (DIL) and their analysis by C, H, and OMe determination; quantitative FTIR spectroscopy of MWL; recording the molecular weight distribution curves using high performance size exclusion chromatography (HPSEC); calculation of average molecular weights, such as M_w and M_n; calculation of the heating values of the lignocellulosics and their components. The quantitative composition of the lignin from the three basic phenylpropane units is presented.M. sinensis was submitted to hydroliquefaction. The conversion process yielded 35% of a net product oil (NPO) with low oxygen content (11%), low viscosity (10⁻² NS m⁻²), low asphaltene content (3·5%) low molecular weight (M_w 200) and with a specific gravity of c. 0·93 g cm⁻³. The NPO has a heating value of 39·4 MJ kg⁻¹ and contains 55% of the carbon of the starting material and 59% of the combined heating value from the biomass and the hydrogen used for hydroliquefaction. The process yields 28% water which contains 58% of the original oxygen of the biomass. The process gives rise to 9 g solids and 32–35 g gases, whose energy content can easily be recovered.     

Cadmium effects on growth and antioxidant enzymes activities in Miscanthus sinensis

Plants of Miscanthus sinensis (cv. Giganteus) were grown in hydroponics for three months in nutrient solution with 0, 2.2, 4.4 and 6.6 μM CdNO₃. Growth parameters, catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX) and superoxide dismutase (SOD) activities were analysed in leaves and roots collected after 1-and 3-month exposure. Dry biomass of all miscanthus organs was affected by Cd concentration both after 1-and 3-month exposure. No visible symptoms of Cd toxicity were observed in shoots and rhizomes of plants grown in presence of Cd. In contrast, roots became shorter and thicker and the whole root system more dense and compact already after one month of treatment with 6.6 μM Cd. The lower Cd concentration increased the enzymes activities after 3 months in leaves and only after 1-month in roots, while a decrease in activity was observed at higher Cd concentrations.     

Synthetic polyploid production of Miscanthus sacchariflorus,Miscanthus sinensis,and Miscanthus x giganteus

Plants from the genus Miscanthus are potential renewable sources of lignocellulosic biomass for energy production. A potential strategy for Miscanthus crop improvement involves interspecific manipulation of ploidy levels to generate superior germplasm and to circumvent reproductive barriers for the introduction of new genetic variation into core germplasm. Synthetic autotetraploid lines of Miscanthus sacchariflorus and Miscanthus sinensis, and autoallohexaploid Miscanthus x giganteus were produced in tissue culture from oryzalin treatments to seed‐ and immature inflorescence‐derived callus lines. This is the first report of the genome doubling of diploid M. sacchariflorus. Genome doubling of diploid M. sinensis, M. sacchariflorus, and triploid M. x giganteus to generate tetraploid and hexaploid lines was confirmed by stomata size, nuclear DNA content, and chromosome counts. A putative pentaploid line was also identified among the M. x giganteus synthetic polyploid lines by nuclear DNA content and chromosome counts. Comparisons of phenotypic performance of synthetic polyploid lines with their diploid and triploid progenitors in the greenhouse found species‐specific differences in plant tiller number, height, and flowering time among the doubled lines. Stem diameter tended to increase after polyploidization but there were no significant improvements in biomass traits. Under field conditions, M. x giganteus synthetic hexaploid lines showed greater phenotypic variation, in terms of plant height, stem diameter, and tiller number, than their progenitor lines. Production of synthetic autopolyploid lines displaying significant phenotypic variation suggests that ploidy manipulation can introduce useful genetic diversity in the limited Miscanthus germplasm currently available in the United States. The role of polyploidization in the evolution and breeding of the genus Miscanthus is discussed.     

芒AFLP反应体系的建立

以芒DNA为材料,对AFLP分子标记分析中的基因组酶切体系选择性扩增中Mg2+、dNTP和Taq酶浓度等4个因素进行了比较。结果表明20μL基因组双酶切体系中,使用1UEcoRⅠ和1UM seⅠ酶切3 h能够实现完全酶切;选择性扩增的PCR 10μL反应体系中1.4 mmol.L-1Mg2+,0.4 mmol.L-1dNTP及0.6 U Taq酶是进行芒AFLP分析的最佳反应条件,能够得到丰富稳定的带纹。该体系的构建为AFLP技术在芒相关研究中的应用奠定了基础。     

Genetic diversity of Miscanthus sinensis in US naturalized populations

Miscanthus is increasingly gaining popularity as a bioenergy grass because of its extremely high biomass productivity. Many clones of this grass were introduced into United States over the past century from East Asia where it originated, and planted for ornamental and landscaping purposes. An understanding of the genetic diversity among these naturalized populations may help in the efficient selection of potential parents in the Miscanthus breeding program. Here, we report our study analyzing the genetic diversity of 228 MiscanthusDNA samples selected from seven sites in six states (Ohio, North Carolina, Washington D.C., Kentucky, Pennsylvania, and Virginia) across the eastern United States. Ten transferable DNA markers from other plant species were employed to amplify genomic DNA of Miscanthus because of the paucity of molecular markers in Miscanthus. There were significant genetic variations observed within and among US naturalized populations. The highest genetic diversity (0.3738) was found among the North Carolina genotypes taken from Biltmore Deer Park and Biltmore, Madison County, Cody Rd. The lowest genetic diversity (0.2776) was observed among Virginia genotypes that were diverged from those from other states, suggesting Virginia genotypes might be independently introduced into the United States from the different origin. By the cluster and structure analysis, 228 genotypes were categorized into two major groups that were further divided into six subgroups at the DNA level and the groups were generally consistent with geographic region.     

基于RNA-seq的能源植物芒转录组分析

芒(Miscanthus sinensis Anderss)是多年生C4草本植物,可为能量和纤维素产品生产提供高品质的木质纤维素材料,是一种理想的能源植物。采用Illumina Hi Seq?2000高通量测序技术,对芒花芽和叶芽进行转录组分析。经拼接组装共获得98 326个Unigene,序列平均长度822 bp,N50为1 337 bp。将Unigene序列与NR、NT、Swiss-Prot、KEGG、GO和COG数据库进行比对(Evalue1e-5),共有74 134条Unigene获得了基因注释,占总Unigene的75.40%。其中,通过GO功能分类,45 507个Unigene映射到GO不同的功能节点上;通过KEGG pathways分析,共有36 710个Unigene参与了128个代谢通路;比对到同源序列比例最高的物种分别为高粱(37 731,60.86%)、玉米(16 258,26.22%)、水稻(3 065,4.94%),共占所有同源序列的92.02%。此外,获得了芒C4关键酶相关基因24个。这些注释信息的完成为芒功能基因及相关候选基因的发掘提供了重要依据。     

Preliminary genetic linkage map of Miscanthus sinensis with RAPD markers

We have used an "offspring cross" mapping strategy in combination with the random amplified polymorphic DNA (RAPD) assay to construct the first genetic map of the species Miscanthus sinensis (2 n = 2 x = 38). This map is based on an outbred population of 89 individuals resulting from the cross between two genotypes from a previously designed cross. Consequently, both parents are fullsibs. The same proportion of bi-parental markers (heterozygotic in both parents) and pseudo-testcross markers (heterozygotic in one parent and null in the other), mono-parental markers, have been obtained. A total of 383 RAPD markers were analysed within the 89 F1 plants. Out of these markers, 257 were mapped into 28 linkage groups which spanned a total map length of around 1,074.5 cM with an average density of 4.2 cM per marker. Out of 257 mapped markers, 62 were inherited from F1.1 (P1), 63 from F1.7 (P7) and 132 were bi-parental markers. The contribution to the map was equal from both parents. This map provides a useful tool for genetic analyses of agronomically interesting characters in M. sinensis such as flowering, yield, plant height, stem diameter and mineral constitution. The offspring cross mapping strategy is proposed to obtain a higher efficiency in developing integrated maps including both parents.     

Micropropagation and plant regeneration from embryogenic callus of Miscanthus sinensis

Miscanthus sinensis (Poaceae) is a typical perennial giant grass of East Asia. Due to its high photosynthetic efficiency, low input requirements, and high biomass production, M. sinensis shows outstanding potential as a biofuel feedstock. However, the lack of an efficient tissue culture system may limit its utilization potential. Different explants of M. sinensis were evaluated to develop an efficient tissue culture system. Shoot apices from in vitro-germinated seedling explants were tested for adventitious bud proliferation. The highest level of proliferation (multiplication coefficient 6.69) was obtained when shoot apices were cultured on Murashige and Skoog (MS) medium supplemented with 1.0 mg L⁻¹ 6-benzyladenine (BA), 2.0 mg L⁻¹ kinetin, 0.05 mg L⁻¹ α-naphthalene acetic acid (NAA), 3% sucrose, and 0.8% agar. The highest rooting percentage (95.4%) was obtained when adventitious buds were cultured on half-strength MS medium supplemented with 0.2 mg L⁻¹ NAA, 3% sucrose, and 0.8% agar. Significant differences were found in the formation of embryogenic callus among different explant types. The embryogenic callus derived from epicotyls had the highest regeneration capacity when cultured on a medium supplemented with 2.0 mg L⁻¹ 2,4-dichlorophenoxyacetic acid, 0.5 mg L⁻¹ BA, and 0.1 mg L⁻¹ thiamine. Under these conditions, the callus induction percentage was 82%.     

Environmental Tolerances of Miscanthus sinensis in Invasive and Native Populations

Miscanthus sinensis is a moderately invasive ornamental grass species being considered as a bioenergy species in the USA and elsewhere. In this study, we show the range of environmental conditions tolerated by this species in wild populations in the USA and in Japan. Six naturalized populations in the USA and five native populations in Japan were sampled in summer 2009. In each population, environmental factors (canopy cover and soil fertility) were measured, along with measurements of size and morphology for 30 plants. Relationships between M. sinensis size and environmental variables in the two countries were determined using linear mixed effects models. Results indicated that M. sinensis can tolerate extremely wide variation in soil and climate conditions in the populations we sampled across both ranges, suggesting that it could be successfully grown across a wide distribution in the USA, both intentionally as a bioenergy crop and unintentionally as an escaped invader. Plant size generally responded to different environmental conditions in both ranges, with USA plants being negatively influenced by canopy cover and Japanese plants being positively influenced by soil fertility measures. We recommend caution in growing M. sinensis for bioenergy or ornamental purposes to minimize escape outside of its native range.     

Mapping the genome of Miscanthus sinensis for QTL associated with biomass productivity

In light of rising energy costs, lignocellulosic ethanol has been identified as a renewable alternative to petroleum-based transportation fuels. In an attempt to reach government mandated ethanol production levels, potential plant biofeedstock candidates have been investigated, and cold-tolerant, perennial accessions within the C₄ grass genus Miscanthus have been identified as leading contenders in the Midwestern US. To facilitate the development of improved cultivars through marker-assisted breeding, a quantitative trait locus (QTL) study was conducted on a full-sib, F₁ mapping population segregating for flowering time, height, leaf width, and yield using a genetic map consisting of 846 segregating SNP and SSR markers. This was a 3 year study investigating the genetic architecture underlying traits important to biomass production in a population of 221 progeny from a cross between M. sinensis ‘Grosse Fountaine’ and M. sinensis ‘Undine’ established in the spring of 2010; 72 QTLs with LOD scores above the genome-wide, permuted threshold equivalent to a P-value of 0.05 were identified across 13 traits. Of the 36 QTLs identified in 2011, 22 were detected again the following year. Both the use of spring emergence and vigor rating as a covariate to account for variation related to differences in establishment increased the power to detect QTLs in the 2 year establishment period. Finally, a dry period in the middle of the 2012 growing season suggested that yield declines were due to a decrease in tiller diameter.     

Comparative antioxidant and cytotoxic effects of lignins from different sources

The potential antioxidant activity of industrial lignins obtained from different sources and their potential cytotoxic effect on two immortalized cell lines is studied. In addition the stability of aqueous preparations of these lignins is studied together with the effect on them of ultraviolet irradiation. The lignins studied show a high antioxidant capacity over a range of concentrations that are not harmful to normal human cells. The stability of the lignin solutions when they are exposed to UVA light is confirmed. These findings suggest new uses for lignins in cosmetic and topical medical formulations. This study aims to demonstrate the antioxidant activity and stability of several lignins from different sources. It assesses their suitability for new commercial applications, mainly in cosmetics and pharmaceuticals, by discarding any possible cytotoxic properties.     

Genetic Diversity and Population Structure of Miscanthus sinensis Germplasm in China

Miscanthus is a perennial rhizomatous C4 grass native to East Asia. Endowed with great biomass yield, high ligno-cellulose composition, efficient use of radiation, nutrient and water, as well as tolerance to stress, Miscanthus has great potential as an excellent bioenergy crop. Despite of the high potential for biomass production of the allotriploid hybrid M. ×giganteus, derived from M. sacchariflorus and M. sinensis, other options need to be explored to improve the narrow genetic base of M. ×giganteus, and also to exploit other Miscanthus species, including M. sinensis (2n = 2x = 38), as bioenergy crops. In the present study, a large number of 459 M. sinensis accessions, collected from the wide geographical distribution regions in China, were genotyped using 23 SSR markers transferable from Brachypodium distachyon. Genetic diversity and population structure were assessed. High genetic diversity and differentiation of the germplasm were observed, with 115 alleles in total, a polymorphic rate of 0.77, Nei’s genetic diversity index (He) of 0.32 and polymorphism information content (PIC) of 0.26. Clustering of germplasm accessions was primarily in agreement with the natural geographic distribution. AMOVA and genetic distance analyses confirmed the genetic differentiation in the M. sinensis germplasm and it was grouped into five clusters or subpopulations. Significant genetic variation among subpopulations indicated obvious genetic differentiation in the collections, but within-subpopulation variation (83%) was substantially greater than the between-subpopulation variation (17%). Considerable phenotypic variation was observed for multiple traits among 300 M. sinensis accessions. Nine SSR markers were found to be associated with heading date and biomass yield. The diverse Chinese M. sinensis germplasm and newly identified SSR markers were proved to be valuable for breeding Miscanthus varieties with desired bioenergy traits.     

芒和五节芒在中国的潜在分布

利用最大熵模型, 将我国现有芒(Miscanthus sinensis)和五节芒(M. floridulus)的地理分布信息与19个降水及温度等气候因子相拟合, 预测了芒和五节芒在我国的潜在分布区域, 并推测出芒和五节芒的基本生态位。结果显示: 芒的潜在适生区包括四川西部、陕西北部、宁夏、内蒙古中部、黑龙江、吉林西部、辽宁西部、青海东南部等地区, 其基本生态位参数为: 最暖季节降水量为400-1 000 mm, 平均8月降水量为100-350 mm, 7月平均最低气温为15 ℃, 平均7月降水量为100-350 mm, 11月平均最高气温为-10-22 ℃, 最干月平均气温为-15-20 ℃, 平均12月降水量为100 mm以下; 五节芒的潜在适生区为云南、陕西、山西、宁夏、河南、山东、吉林、辽宁以及四川西部、甘肃南部和内蒙古东部等地区, 其基本生态位参数为: 最暖季节降水量至少在400 mm以上, 平均6月降水量为150-550 mm, 7月平均最低气温在15-30 ℃之间, 6月平均最低气温为10 ℃, 平均4月降水量为50-100 mm。结果表明, 在进行遗传改良的前提下, 我国有丰富的适合栽植芒和五节芒的土地资源。     

Contrasting selection modes at the Adh1 locus in outcrossing Miscanthus sinensis vs. inbreeding Miscanthus condensatus (Poaceae)

We estimated DNA sequence variation of the Adh1 locus in the outcrossing Miscanthus sinensis (Poaceae) and its close selfing relative, M. condensatus. Tajima's test of selection is significantly negative for both overall exons and replacement sites in M. sinensis. Among its entire sample, nucleotide diversity of nonsynonymous sites is higher than that of synonymous sites. A McDonald and Kreitman test of neutrality indicates an excess of intraspecific replacement polymorphisms, suggesting possible directional selection toward advantageous mutants. However, frequent intragenic recombination suggests both purifying and positive selection is unlikely. Recent demographic expansions coupled with relaxation of purifying selection may have resulted in elevated genetic diversity at the Adh1 locus as well as the trnL-trnF intergenic spacer of cpDNA in this outcrossing species. In contrast, low levels of genetic diversity were detected at both the Adh1 locus and the cpDNA spacer in M. condensatus, consistent with bottlenecks associated with selfing in all populations. While Tajima's D and Fu and Li's F statistics did not reveal deviation from neutrality at the Adh1 locus in M. condensatus, 12 replacements vs. 10 synonymous changes were detected. Based on pairwise comparisons of the d(N)/d(S) ratio, lineages of closely related populations of the species distributed along saline habitats appeared to be under directional selection.     

First Report of Pseudomonas lurida Causing Bacterial Leaf Spot on Miscanthus sinensis

Miscanthus spp. are large perennial wetland grasses that are receiving considerable attention as bioenergy crops. In late summer 2011, leaf spot symptoms were observed in a field of Miscanthus sinensis in Jeongseon, Gangwon province, Korea. Bacterial strains that belonged to the γ‐Proteobacteria genus Pseudomonas were isolated from the affected leaves. By phylogenetic analysis and phenotypic characterization, the representative strain MDM‐03 was identified as Pseudomonas lurida. Healthy M. sinensis leaves inoculated with MDM‐03 developed leaf spots similar to those observed in field. Bacteria re‐isolated from the leaf lesions were identical to the original strain MDM‐03 based on their cultural characteristics and 16S rDNA sequencing. This is the first report of bacterial leaf spot in Miscanthus sinensis.     

Chloroplast DNA variation and genetic structure of Miscanthus sinensis in southwest China

Miscanthus sinensis is a dominant perennial C4 grass with the potential to being a feedstock crop in North America, Europe, and China. Variation in chloroplast DNA sequence was used to obtain information regarding the genetic diversity and structure of populations of M. sinensis in southwest China. Chloroplast DNA, trnL-F and rpl20-rps12 sequences from seventy-five individuals representing 14 populations of M. sinensis were used to study the sequence variation. Seven haplotypes and 16 polymorphic sites (2.7%) were identified. Five substitutions, 6 indels, and 5 existing substitutions and indels sites, were detected through splicing these two gene segments. The genetic diversity within the studied populations (diversity of haploids, h = 0.561, nucleotide diversity, π = 0.00504) was low, this may be affected by the relatively larger effect of genetic drift on the chloroplast DNA, reflecting smaller effective populations than nuclear DNA. Genetic variance within the populations was higher than that between the populations, suggesting that higher gene flow may exist within these populations. The results of parsimony network in seven haplotypes indicated that H1 and H2 may be ancient haplotypes, which may help guide future research on the origin of M. sinensis. Our results provide information on the genetic diversity and structure of M. sinensis and may assist future studies on the phylogeography of M. sinensis.     

Invasiveness potential of Miscanthus sinensis: implications for bioenergy production in the United States

Miscanthus sinensis (Anderss.) is a perennial grass species that has been grown widely as an ornamental since the late 1800s and is now being considered for bioenergy production in the United States. With its ability to be grown from seed and tolerate cold climates, this species offers practical advantages over current cultivars of the higher‐yielding hybrid species, M.×giganteus. Yet a large‐scale release of M. sinensis for bioenergy production in colder northern regions could result in new invasions into natural areas. We show, with reference to historical records and data collected in six wild US populations of M. sinensis in 2009, that ornamental varieties of this species have a long history of localized escape in the Eastern United States, primarily within the Appalachian region. To prevent further escape and gene flow, we recommend the development of sterile or functionally sterile varieties of M. sinensis or the restriction of its usage as a donor of genetic material to new sterile cultivars of M. ×giganteus. Other appropriate precautions for new biomass varieties include experimental demonstration of low invasiveness in the target region ahead of commercial production, along with postintroduction stewardship programs.     

Colchicine and oryzalin mediated chromosome doubling in different genotypes of Miscanthus sinensis

Different explant materials were treated with antimitotic agents to induce chromosome doubling in several Miscanthus sinensis clones. In vitro propagated plants established in soil, in vitro shoots, embryogenic callus, shoot apices and leaf explants were treated with different concentrations of colchicine or oryzalin. No tetraploids were obtained after antimitotic treatment of plants established in soil. The percentage of chromosome doubled plants after antimitotic treatment of single in vitro shoots was genotype dependent. Rooted in vitro plantlets were not a suitable target for antimitotic treatment, due to a high frequency of ploidy chimeras. Many tetraploid plants were regenerated after antimitotic treatment at the callus and explant level, but the efficiency was genotype dependent, primarily due to differences in the ability to form regenerable callus and to regenerate plants from embryogenic callus. Treatment of shoot apices with colchicine was the most efficient and reproducible system in the four genotypes tested. It was possible to repeatedly use the same colchicine-containing medium without any reduction in the induction of regenerable callus or in the percentage of tetraploids, thereby minimising the handling of this very toxic compound.