Switchgrass is a leading dedicated bioenergy feedstock in the United States because it is a native, high-yielding, perennial prairie grass with a broad cultivation range and low agronomic input requirements. Biomass conversion research has developed processes for production of ethanol and other biofuels, but they remain costly primarily because of the intrinsic recalcitrance of biomass. We show here that genetic modification of switchgrass can produce phenotypically normal plants that have reduced thermal-chemical (≤180°C), enzymatic, and microbial recalcitrance. Down-regulation of the switchgrass caffeic acid O-methyltransferase gene decreases lignin content modestly, reduces the syringyl:guaiacyl lignin monomer ratio, improves forage quality, and, most importantly, increases the ethanol yield by up to 38% using conventional biomass fermentation processes. The down-regulated lines require less severe pretreatment and 300-400% lower cellulase dosages for equivalent product yields using simultaneous saccharification and fermentation with yeast. Furthermore, fermentation of diluted acid-pretreated transgenic switchgrass using Clostridium thermocellum with no added enzymes showed better product yields than obtained with unmodified switchgrass. Therefore, this apparent reduction in the recalcitrance of transgenic switchgrass has the potential to lower processing costs for biomass fermentation-derived fuels and chemicals significantly. Alternatively, such modified transgenic switchgrass lines should yield significantly more fermentation chemicals per hectare under identical process conditions.ignocellulosic biomass is an abundant, domestic, renewable feedstock source that can be converted to liquid transportation fuels and other chemicals by fermentation. Cellulosic ethanol is a promising near-term technological option to reduce transportation sector greenhouse gas emissions (1). Because lignocellulosic biomass is made up of the complex structures of cellulose, hemicellulose, and lignin, such feedstock is highly recalcitrant to bioconversion of its carbohydrates into ethanol compared with starch (2, 3). Current biomass fermentation processes for fuels and chemicals have a relatively high cost primarily because of this recalcitrance, which in turn has limited commercialization of biomass ethanol (4). To achieve sustainable energy production, it is necessary to overcome the chemical and structural properties of biomass that inhibit its deconstruction in dedicated bioenergy crops (5).The conversion of lignocellulosic biomass to ethanol is a threestep process that involves pretreatment followed by polysaccharide hydrolysis to simple sugars followed by sugar fermentation to ethanol (6). The presence of lignin in cell walls negatively impacts these conversion steps (7,8). Examination of natural variation in alfalfa, switchgrass, canarygrass, and sorghum has shown that decreased lignin levels improve in vitro enzyme hydrolysis (9, 10). Lignin pathway modification in alfalfa generated transgenic lines with increased enzymati...

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The bioconversion of carbohydrates in the herbaceous bioenergy crop, switchgrass (Panicum virgatum L.), is limited by the associated lignins in the biomass. The cinnamyl alcohol dehydrogenase (CAD) gene encodes a key enzyme which catalyzes the last step of lignin monomer biosynthesis. Transgenic switchgrass plants were produced with a CAD RNAi gene construct under the control of the maize ubiquitin promoter. The transgenic lines showed reduced CAD expression levels, reduced enzyme activities, reduced lignin content, and altered lignin composition. The modification of lignin biosynthesis resulted in improved sugar release and forage digestibility. Significant increases of saccharification efficiency were obtained in most of the transgenic lines with or without acid pretreatment. A negative correlation between lignin content and sugar release was found among these transgenic switchgrass lines. The transgenic materials have the potential to allow for improved efficiency of cellulosic ethanol production.

The white clover ( Trifolium repens) nuclear genome (n = 2x = 16) is an important yet under-characterised genetic environment. We have developed simple sequence repeat (SSR) genetic markers for the white clover genome by mining an expressed sequence tag (EST) database and by isolation from enriched genomic libraries. A total of 2,086 EST-derived SSRs (EST-SSRs) were identified among 26,480 database accessions. Evaluation of 792 EST-SSR primer pairs resulted in 566 usable EST-SSRs. Of these, 335 polymorphic EST-SSRs, used in concert with 30 genomic SSRs, detected 493 loci in the white clover genome using 92 F1 progeny from a pair cross between two highly heterozygous genotypes--364/7 and 6525/5. Map length, as estimated using the joinmap algorithm, was 1,144 cM and spanned all 16 homologues. The R (red leaf) locus was mapped to linkage group B1 and is tightly linked to the microsatellite locus prs318c. The eight homoeologous pairs of linkage groups within the white clover genome were identified using 96 homoeologous loci. Segregation distortion was detected in four areas (groups A1, D1, D2 and H2). Marker locus density varied among and within linkage groups. This is the first time EST-SSRs have been used to build a whole-genome functional map and to describe subgenome organisation in an allopolyploid species, and T. repens is the only Trifolieae species to date to be mapped exclusively with SSRs. This gene-based microsatellite map will enable the resolution of quantitative traits into Mendelian characters, the characterisation of syntenic relationships with other genomes and acceleration of white clover improvement programmes.

Medicago truncatula has been developed into a model legume. Its close relative alfalfa (Medicago sativa) is the most widely grown forage legume crop in the United States. By screening a large population of M. truncatula mutants tagged with the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified a mutant line (NF2089) that maintained green leaves and showed green anthers, central carpels, mature pods, and seeds during senescence. Genetic and molecular analyses revealed that the mutation was caused by Tnt1 insertion in a STAY-GREEN (MtSGR) gene. Transcript profiling analysis of the mutant showed that loss of the MtSGR function affected the expression of a large number of genes involved in different biological processes. Further analyses revealed that SGR is implicated in nodule development and senescence. MtSGR expression was detected across all nodule developmental zones and was higher in the senescence zone. The number of young nodules on the mutant roots was higher than in the wild type. Expression levels of several nodule senescence markers were reduced in the sgr mutant. Based on the MtSGR sequence, an alfalfa SGR gene (MsSGR) was cloned, and transgenic alfalfa lines were produced by RNA interference. Silencing of MsSGR led to the production of stay-green transgenic alfalfa. This beneficial trait offers the opportunity to produce premium alfalfa hay with a more greenish appearance. In addition, most of the transgenic alfalfa lines retained more than 50% of chlorophylls during senescence and had increased crude protein content. This study illustrates the effective use of knowledge gained from a model system for the genetic improvement of an important commercial crop.