Dedicated to

My beloved wife and colleague,
Phullara

Preface

While facing the global fossil energy crisis, switchgrass, a warm-season C4 perennial species has been identified as one of the most important candidate grasses for development into an herbaceous energy crop providing cellulosic feedstock for bioenergy production. A better understanding of this bioenergy crop will greatly facilitate efforts in managing large-scale cultivation, developing novel agronomic, genetic, genomic and molecular as well as chemical and bioengineering tools for enhanced biomass production and bioconversion. This book contains the most comprehensive reviews on the latest development of switchgrass research including the agronomy of the plant, use of endophytes and mycorrhizae for biomass production, genetics and breeding of bioenergy related traits, molecular genetics and molecular breeding, genomics, transgenics, processing, bioconversion, biosystem and chemical engineering, biomass production modeling, and economics of switchgrass feedstock production.

The Chapter 1 by Rob Mitchell and Marty Schmer provides an excellent overview of switchgrass and its development into a biomass energy crop. This is followed by Chapter 2 by John Fike and co-authors discussing the agronomic considerations and potential associated with switchgrass use as a bioenergy crop. In Chapter 3, Chuansheng Mei and co-authors describe beneficial plant-microbe interactions that offer practical ways to improve plant growth and disease resistance, pointing out the great potential of endophytes and mycorrhizae for use in the development of a low-input and sustainable switchgrass production system. The Chapter 4 by Laura Bartley and co-authors gives a thorough and in-depth review of switchgrass biomass content, synthesis and biochemical conversion to biofuels. This chapter provides information about primary and secondary cell wall compositions, and biomass content variation with environment and genotype, discussions on switchgrass cell wall synthesis, regulation and molecular genetics approaches for cell wall modifications as well as biochemical conversion of biomass to biofuels including pretreatment, enzymatic digestion and fuel synthesis. In Chapter 5, Yanqi Wu describes and discusses the progress in switchgrass improvement using classic genetics and breeding, focusing on target bioenergy traits, basic information of inheritance and cytogenetics,

germplasm pools and collections, and breeding and selection methods and the potential to develop hybrid cultivars in switchgrass. In parallel to this, Chapter 6 by Linglong Liu and Yanqi Wu focuses on switchgrass molecular genetics including the development of molecular markers, construction of linkage maps, and application of molecular breeding. Chapter 7 by Christopher Saski and Hong Luo summarizes the recent advances in switchgrass genomics research focusing on structural genomics resources development and their important applications. This is followed by Chapter 8, in which Dayong Li and co-authors provide an overview of the research on switchgrass small RNA molecules, microRNAs, and discuss their potential applications in switchgrass genetic improvement. Chapter 9 by Bingyu Zhao and co-authors is a thorough review on research on tissue culture, genetic transformation, trait modifications using transgenic approaches in switchgrass. The chapter also discusses strategies for future switchgrass improvement. In Chapter 10, Ajay Kumar and Raymond Huhnke provide an overview of major thermochemical conversion processes for conversion of biomass into fuels, chemicals and power, which is followed by Chapter 11 from Terry Walker and co-authors reviewing biological and biosystems engineering for switchgrass feedstocks processing and biofuel production. In Chapter 12, Kathrine D. Behrman and co-authors highlight five applications of process-oriented models of switchgrass growth and show how they can be used to generate a better understanding of large-scale switchgrass biomass production, pointing out the effectiveness of crop simulation models for assessing the sustainability and long-term impacts of converting land to bioenergy crops in a timely and cost-effective manner. Chapter 13 by Francis M. Epplin and co-authors discusses economics of switchgrass feedstock production for the emerging cellulosic biofuel industry, focusing on identifying practical issues related to the economics of developing switchgrass as a dedicated energy crop and providing estimates of the price for delivered switchgrass biomass that would be required to compensate for the cost of inputs used to produce and deliver it to a biorefinery. Chapter 14 by Charles Kwit and co-authors highlights advantages, concerns, and future prospects of developing switchgrass as a bioenergy crop, discussing the role of switchgrass in avoiding biomass for energy and biomass for food dilemma, the impact of switchgrass on climate change and the effects of switchgrass on environmental sustainability and pointing out that improvement efforts in the bioenergy crop switchgrass on multiple fronts present numerous challenges.

The 14 chapters of this book volume contributed by 39 internationally reputed scientists will be of interest and great value to the bioenergy research communities in academia and industry as well as government agencies. It would be an important handbook for agronomists, geneticists, breeders, molecular biologists, physiologists, biosystems engineers and chemical engineers.

We would like to thank all the contributing authors for their excellent work and enthusiastic support and cooperation during the preparation of this volume. Financial support from the USDA National Institute of Food and Agriculture as well as the USDA-CSREES for research on genetic improvement of perennial grasses at Clemson University is gratefully acknowledged.

Hong Luo, Ph. D.

Clemson University

Yanqi Wu, Ph. D.

Oklahoma State University

Chittaranjan Kole

Bidhan Chandra Agricultural University

Switchgrass has an extensive perennial root system which protects soil from erosion and sequesters carbon (C) in the soil profile (Liebig et al.

2005) . Soil organic carbon (SOC) typically increases rapidly when annual cropland is converted to switchgrass (Schmer et al. 2011; Mitchell et al. 2012). Switchgrass grown and managed for bioenergy on three cropland sites that qualified for CRP in Nebraska resulted in an average SOC increase of 2.9 Mg C ha-1 yr-1 in the top 1.2 m of soil in just 5 years (Liebig et al. 2008). In South Dakota, switchgrass grown in former cropland enrolled in CRP stored SOC at a rate of 2.4 to 4.0 Mg ha-1 yr-1 at the 0 to 90 cm depth (Lee et al. 2007). Switchgrass managed for bioenergy on multiple soil types in the Northern Plains stored 4.42 Mg C ha-1 yr-1 into the soil profile (Frank et al. 2004), whereas switchgrass stored an average of 1.7 Mg C ha-1 yr-1 sequestered in the Southeast USA (McLaughlin et al. 2002).

Bioenergy production will become increasingly important in the future to relieve dependence on fossil fuels and lower greenhouse gas emissions because fossil-based energy is limited and its demand is continually increasing due to economic and population growth around the world. Switchgrass is one of the most promising bioenergy crops due to persistent high yields and its ability to grow on marginal land. Development of a low input and sustainable switchgrass feedstock production system is imperative as the use of chemical fertilizers causes deleterious environmental effects, such as water pollution and N2O release to atmosphere, a potential greenhouse gas. Endophytes and AM fungi have the potential to help address these challenges due to their enhancement of nutrient acquisition, including nitrogen fixation and mobilization of mineral nutrients as well as increased biotic and abiotic stress tolerance, which together will reduce the amount of fertilizer application and/or pesticide and fungicide use. It will also open a door to growing potential bioenergy crops, such as switchgrass on marginal land or achieving the same yield while reducing fertilizer use, resulting in lower cost and contributing to sustainable rural development.

Plants live in complex environmental conditions containing various microorganisms, both beneficial and detrimental. Although endophytes and AM fungi could benefit plant growth, other microorganisms may have negative effects, and different endophytes and AM fungi may not be compatible, therefore the specific functional compatibility of endophytes and AM fungi needs to be further investigated to develop multi-functional bio-inoculants (Podile and Kishore 2007) in switchgrass production. Additionally, while studies with endophytes as well as other plant growth promoting microorganisms in laboratories have been encouraging, there have also been reports of a general decrease in performance from the laboratory to the field (Riggs et al. 2001; Gyaneshwar et al. 2002). As with any ecosystem, the variables of field conditions and native microbial populations will have to be addressed to maximize the beneficial effects of bacteria and fungi. Therefore, screening endophytes having a broad spectrum of growth promotion that continues throughout the life of the plant will be another topic for endophyte application.

Genotype specific responses of host plants to endophytes are also a large barrier in application. For example, in poplar, different cultivars had different responses to different endophytes (Taghavi et al. 2009). One of the most studied plant growth promoting bacterium, B. phytofirmans strain PsJN, has a beneficial effect on many species, such as potato, tomato, and grape. However, PsJN is also genotype specific. In switchgrass, PsJN promoted growth of the lowland cultivar Alamo but not the upland cultivar Cave-in-Rock (Kim et al. 2012). Understanding these differences will also help in developing a more reliable, stable, and broad spectrum of growth promotion in plants.

Complete understanding of the mechanisms of various beneficial symbioses is the foundation for effectively applying these microorganisms in a sustainable switchgrass feedstock production and to achieve their synergistic activities (Podile and Kishore 2007). As more is learned from functional genomics of endophytic microorganisms in growth promotion, it may be possible to share these important genes between similar microorganisms through horizontal gene transfer via transformation, conjugation, or transduction, all common occurrences in the bacterial world. Researchers first reported in planta horizontal gene transfer in the bioenergy crop hybrid poplar when they found Burkholderia cepacia VM1468 transferred its toluene degradation gene to other endophytes (Taghavi et al.

2005) . This suggests that such transfer may be used to modify and improve the growth-promoting effects of other endophytes via gene sharing. The phenomenon of horizontal gene transfer may also occur in nature between different genera as the gene encoding the anti-fungal agent pyrrolnitrin in Burkholderia was likely horizontally transferred from Pseudomonas (de Souza and Raaijmakers 2003). Since AM fungi are coenocytic (many nuclei coexist in a common cytoplasm), genetic exchange was recently reported in different AM fungus Glomus intraradices strains (Colard et al. 2011), which could be beneficial for host plant growth. Generating novel AM fungus genotypes through genetic exchange will be a powerful tool in developing AM fungi that are more beneficial in bioenergy crop production.

Compared with plant genetic engineering, it is much easier for microorganisms to be genetically modified. One could easily transform some useful foreign genes into bacteria or fungi. For instance, the Bacillus thuringiensis cry1Ac7 and Serratia marcescens chiA genes were transformed to sugarcane-associated endophytic bacteria, which helped increase the tolerance of sugarcane plant to the sugarcane borer Eldana saccharina (Downing et al. 2000). These applications indicate that we may be able to genetically engineer endophytes with useful genes, such as the Bacillus thuringiensis toxin gene, to protect host plants against herbivorous insects, herbicide resistance genes to impart host plant resistance to herbicides, and genes related to abiotic stress tolerance to enhance host plant tolerance to abiotic stresses. An efficient endophyte transformation method by Agrobacterium was developed by Abello et al. (2008), which will help in the transfer and expression of agronomically important genes in host plants via endophytes. As functional genomics research is continually advanced, scientists will better understand the mechanisms under which beneficial microorganisms promote host plant growth and enhance stress tolerance to effectively utilize these microbes in bioenergy crop production. For example, endophytes having the ability to fix atmospheric nitrogen could be combined with endophytes having the ability to enhance host plant tolerance to abiotic stresses or endophytes inhibiting pathogen growth or with an AM fungus to improve nutrient uptake or, possibly, all could be combined.

Since 1999, over 15 new patents have been registered for microbial endophytes (Mei and Flinn 2010). The worldwide market for microbial inoculants is experiencing an annual growth rate of approximately 10% (Berg 2009). As world population demand for food is continually increasing, bioenergy crops should be grown on poor or marginal lands or contaminated soil, not competing with food crops for fertile lands. The use of endophytes and AM fungi may help bioenergy crops, such as switchgrass, grow on these lands via their normal mechanisms of action or genetic modification by introducing nitrogen fixation genes, heavy metal accumulation genes, or contaminated compound degradation genes.

Acknowledgements

This work was funded through Special Grants (2003-38891-02112, 2008­38891-19353 and 2009-38891-20092) and HATCH funds (Project No. VA — 135816) from the United States Department of Agriculture, the Office of Science (BER), U. S. Department of Energy for Plant Feedstock Genomics for Bioenergy Program (DE-SC0004951), and operating funds from the Commonwealth of Virginia to the Institute for Advanced Learning and Research.

Linglong Liu1’2[9] [10] and Yanqi Wu1

Introduction

Switgrass, a C4 perennial grass widely distributed in North America, has become one of the main bioenergy crops for production of cellulosic biofuels. Due to its relatively short history in research, the advance of molecular genetics and breeding of switchgrass is lagging behind that of the other major row crops (such as rice, wheat, and corn). However, with the increase of investment from public agencies and private organizations, many achievements have been attained, especially in recent years (Bouton

2007) . This chapter will focus on switchgrass classical molecular genetics including development of molecular markers, construction of linkage groups, and application of molecular breeding. Transgenic researches are not presented here, but are addressed in a separate chapter of this book.

As described above, bioinformatics predictions for switchgrass miRNAs might be limited by the lack of large genomic data and the number of available ESTs. Direct cloning is a possible experimental approach to discover not only conserved, but also novel switchgrass-specific miRNAs. Several groups have used this method to identify plant miRNAs in different plant species (Sunkar and Zhu 2004; Sunkar et al. 2005; Yao et al. 2007; Sunkar et al. 2008; Zhao et al. 2010; Kulcheski et al. 2011; Li et al. 2012).

The cloning methods involve in small RNA library construction (including isolation of small RNAs, ligation of adaptor oligonucleotides, reverse transcription, and amplification) and sequencing. Matts et al.

(2010) pooled equal molar amounts of total RNA from three-month-old switchgrass seedlings and inflorescences for small RNA library construction. The amplification products were then subjected to pyrosequencing following Sunkar et al. (Sunkar et al. 2008). A total of 21,999 raw sequences was generated and subjected to further analysis to discard duplicates, degradation products from ribosomal RNAs, transfer RNAs, small nuclear RNAs and mRNAs (Sunkar et al. 2008; Matts et al. 2010). The remaining small RNAs were subsequently analyzed for distinguishing miRNAs from siRNAs, and conserved miRNAs were identified by searching against miRBase (Matts et al. 2010).

Seedbed preparation is the next step following site selection. The goal prior to seeding is to have an environment that optimizes seed germination and seedling establishment. Ideal seedbeds are very firm below planting depth, have friable surface soil, and are free from competition with resident vegetation and weed seeds (Vallentine 1989). These ends can be achieved with both conventionally tilled and no-till systems.

Having clean fields with minimal weed competition can be a major factor for successful switchgrass establishment and the amount of residue also can be a factor in timing of planting. Zarnstorff (1990) reported greater stand success with later seeding dates when sowing into rye (Secale cereale L.) stubble and that shorter stubble heights supported increased seedling numbers. Excess herbage residue on fields can hinder seed placement, thus preventing proper soil-seed contact (Wolf and Fiske 2009). As seen with other crops, residues can also provide safe haven for slugs and other pests that prey on the emerging seedlings (Hammond 1996; Luna and Staben 2002; Vernava et al. 2004).

Tillage can be an effective method of seedbed preparation and residue removal, although tilling can be more expensive and has potential to expose susceptible sites to erosion. Because firm seedbeds are essential for switchgrass establishment, it is imperative that tilled fields be firmly packed at or just prior to seeding. Tillage also has the potential both to kill weed seedlings and to free weed seeds for germination; thus, weed conditions and management must be carefully considered with tillage.

No-till methods can be quite effective for switchgrass establishment. No-till systems conserve soil moisture, minimize soil erosion, require less fuel, and allow earlier entry of equipment into fields following precipitation events (Parrish and Fike 2005; Douglas et al. 2009). While no-till establishment has several advantages, residue management can be a prime concern given the issues of seed placement and pest habitat mentioned above.

Strategies of residue removal include harvest, or chemical burn followed by sufficient time to degrade the residue. Growing glyphosate-resistant crops such as soybeans on the site prior to switchgrass establishment can be an effective strategy for seedbed preparation. Glyphosate applications can decrease weed burdens, and, following harvest, the resultant stubble makes a suitable seedbed for planting switchgrass. However, caution should be used if prior production practices have utilized persistent herbicides that can prevent seed germination and growth (Douglas et al. 2009).

Burning field residues can also be an effective residue removal method for both tilled and no-till systems. In addition to removing crop residues, burns can kill small weeds and pests and reduce the size of the soil weed seed bank, thus decreasing competition for new seedlings (Wolf and Fiske

2009) . Burning may be especially useful for converting old pastures or abandoned field sites with large weed burdens, but, success is predicated on the temperature and speed of the burn.

At planting, an ideal seedbed—whether prepared with or without tillage—will enable placement of the seed at the proper depth (discussed in the following section) and in firm contact with the soil. This requires appropriate levels of soil compaction, which ensures rapid movement of water from the soil to the seed/seedling by improving capillary water flow. Increased moisture availability increases the likelihood of rapid, uniform germination, early seedling growth, and successful stand establishment (Bartholomew 2005). Too much compaction, however, can restrict the ability of seedlings and their roots to penetrate through the soil. Hudspeth and Taylor (1961) reported that switchgrass was able to germinate and emerge from 8 cm depth in loose soil, but only 10% of seeds emerged when compaction was 6.9 kPa and no seedlings emerged with pressure of 69 kPa. Too much compaction also affects oxygen diffusion, soil temperature, and light penetration, all of which influence germination and emergence (Hudspeth and Taylor 1961).

In contrast to over compacted soils, poor soil contact resulting from cloddy or loose soil or from excess residue can slow seed germination creating conditions for uneven emergence and subsequent seedling desiccation. Loose soils can also contribute to too great a seed depth when rains cause seed depths to be greater than ideal (Fig. 2). Such effects limit emergence and can lead to problems such as weed competition during the early establishment phase (Hall and Vough 2007).

Planting Depths

Proper planting depth is critical to successful switchgrass establishment, and many stand failures have occurred because seed placement was too deep. Appropriate depth maximizes emergence and seedling growth, and as a general recommendation, proper seed placement is difficult to regulate unless the seedbed is firm enough to prevent placing the seeds too deep (Masters et al. 2004), either directly or by soil washing into the planting furrow (Fig. 2). Typically seed should be covered with enough soil to maintain moist conditions for germination, but not so deep that the shoot cannot reach the surface (Zhang and Maun 1990; Roundy et al. 1993; Cosgrove and Collins 2003).

Although 1.5 cm is a common lower limit for planting depth, emergence from greater depth is possible (Zhang and Maun 1990). The ideal depth depends on soil texture and other soil physical properties (Aiken and Springer 1995; Evers and Parsons 2003), and deeper plantings are recommended for arid environments or on sandy soils where moisture limitations can slow imbibition, germination, and emergence (Newman and Moser 1988; Evers and Butler 2000; Evers and Parsons 2003).

Just as planting too deep is problematic, shallow plantings also can be the cause of stand failure. Shallow seed placement under drying conditions can cause seedlings to desiccate and die before they become established (Cosgrove and Collins 2003). This is especially so with bare soils which lose water more rapidly than those protected by litter (Winkel et al. 1991). Adventitious root formation may be compromised with shallow plantings —and the adventitious roots are the only roots that matter for the plant’s long-term survival (Parrish and Fike 2005).

Seed size can affect the appropriate seeding depth for many species, but the data on switchgrass seed size and germination, while suggestive of greater success with larger seeds, are not definitive. Larger seeds support more rapid germination and emergence (Aiken and Springer 1995) and more rapid adventitious root development (Smart and Moser 1999). However, these early advantages appear to be lost over time (Zhang and Maun 1990). Whether seed size affects competitive responses with weeds has not been definitively tested.

Endophytes, including bacteria and fungi, and arbuscular mycorrhizal (AM) fungi, directly or indirectly affect plant growth. In general, these microorganisms promote host plant growth, enhance nutrient uptake and stress tolerance, and inhibit plant pathogen growth. These three plant growth-promoting microorganisms have been studied in a broad range of plants including switchgrass, as will be detailed below.

Rather than targeting a particular cell wall synthesis enzyme, another promising direction for improving biomass quality is to modulate the expression of suites of enzymes by altering regulators of cell wall synthesis, especially transcription factors. Indeed, population genetic analyses have found that markers in known cell wall synthesis genes associate with only a few percentage points of cell wall quality variation (Wegrzyn et al.

2010) . As discussed in recent reviews, a growing network of transcription factors regulates secondary cell wall synthesis (Zhao et al. 2011; Gray et al. 2012; Handakumbura et al. 2012) (Fig. 4). As with all other aspects of cell wall biology, knowledge of cell wall transcriptional regulation networks specifically in grasses lags behind that in dicots. Still, where factors have been studied in both grass and dicot systems, it seems likely that many aspects of regulation are conserved (Handakumbura et al. 2012). Here, we will give an overview of the emerging cell wall regulatory hierarchy in Arabidopsis and grasses.

Many transcription factors implicated in secondary cell wall regulation belong to the NAC and the MYB R2R3 protein families. The relevant NAC-domain transcription factors known are known simply as NACs, or Secondary Wall NACs (SWNs), and include SND1 (SECONDARY WALL- ASSOCIATED NAC-DOMAIN PROTEIN 1, also known as NST3), NST1 (NAC SECONDARY WALL THICKENING FACTOR1), NST2, VND6 (VASCULATURE-RELATED NAC-DOMAIN 6) and VND7 (Handakumbura et al. 2012). Different secondary cell wall regulatory pathways appear to function in different cell types in different organs.

Figure 4. Schematic model of the regulatory network of secondary cell wall biosynthesis based primarily on studies in Arabidopsis. Peach circles represent transcription factors known to function in Arabidopsis. The red circles demarcate the transcription factors whose function has been studied in grasses. The yellow octagons represent enzymes. The grey squares represent secondary cell wall polymers. The green triangle represents a property of the cell wall, saccharification. Arrows signify positive regulation; whereas, dashed edges with T ends indicate negative regulation. Cis-elements are labeled on the edges as follows: Secondary wall NAC Binding Element (SNBE), Tracheary Element Responsive Element (TERE), Secondary wall MYB Responsive Element (SMRE), and the AC-rich elements found in lignin biosynthesis gene promoters (AC). See text for references and further discussion. For simplicity, not all known or suspected interactions are shown. Abbreviations are as follows: Lig Bios Enz, lignin biosynthesis enzymes; SCW Enz, secondary cell wall biosynthesis enzymes the specific identity of which has not been specified; PAL1, Phenylalanine Ammonium Lyase 1; 4CL1, 4-Coumaroyl Ligase 1; COMT, Caffeic acid O-MethylTransferase; C4H, Cinnamate 4-Hydroxylase; CESA, Cellulose Synthase A; SHN, shine/wax inducer 1; VND, Vasculature-related NAC-Domain; SND, Secondary wall-associated NAC-Domain protein; NST, NAC Secondary wall Thickening factor; VNI2, VND-interacting 2 NAC protein 2.

BSA is firstly developed by Michelmore et al. (1991) and has becomes a rapid mapping strategy suitable for monogenic or major qualitative traits. Two bulked DNA samples are generated from a segregating population that originated from a single cross. Each pool, or bulk, contains individuals (the number of individuals in each bulk varied between 10 and 20 plants) that are identical for a particular trait (e. g., disease resistant or susceptible) or genomic region but arbitrary at all unlinked regions. The two bulks are therefore genetically dissimilar in the selected region but seemingly heterozygous at all other regions. Thus, the two bulks can be made for any genomic region and from any segregating population, and they can be screened for differences the same way as near isogenic lines (NILs) (Michelmore et al. 1991). This approach is applicable both in those species where selfing is possible and in those that are obligatorily outbreeding like switchgrass. Since its invention, BSA technique has been widely used in different species for single gene and QTL analysis (Van Leeuwen et al. 2012).

Diseases had been reported to affect switchgrass biomass yields in southern Iowa (Gravert and Munkvold 2002). Thomsen et al. (2008) conducted a study in naturally infected condition caused by smut, and found biomass yield loss of an upland cultivar ‘Cave-in-Rock’ varying from 0.6% to 40.1% among fields. Other major diseases affecting biomass yields are rust (Gustafson et al. 2003) and leaf spots (Krupinsky et al. 2004). Little information is available regarding disease resistance of switchgrass cultivars. Gustafson et al. (2003) used four switchgrass populations (two were derived from cultivars ‘Summer’ and ‘Sunburst’, and the other two developed by breeder of Nebraska and Oklahoma, respectively), and evaluated rust resistance at two locations (Aurora and Kimball, SD) in two years. Significant variations for rust resistance were observed among and within populations, suggesting genetic improvement in rust resistance may be effective through selection (Gustafson et al. 2003). In an ongoing project, we found obvious differences for leaf rust resistance in a segregated population derived from selfing of ‘NL94 LYE 16Х13’. Considering that bulks screened method had been utilized for mapping resistance gene (s) initially in lettuce (Michelmore et al. 1991), and then successfully expanded in other plant species (Michelmore 1995), it can be expectedly moved to switchgrass to identify markers linked with major resistant genes. A gene mapping project of switchgrass rust resistance is on the way (Y. Q. Wu, unpublished).

Desirable feedstock qualities are largely dependent on the nature of processing technologies (Carroll and Somerville 2009). Currently, the two major biomass processing technologies are: thermal conversion and biological conversion. In thermal conversion (e. g., direct combustion or pyrolysis), it is more desirable to have feedstock with a lower amount of mineral residues and a higher energy content, which often correlates with a high lignin content of the biomass (Boateng et al. 2008). In biological conversion for biofuel production, the feedstock with lower lignin content has higher saccharification efficiency through enzyme hydrolysis, resulting in an increase in enzymatic fermentation efficiency (Fu et al. 2011a). Other cell wall components, such as hemicellulose (Lee et al. 2009) and pectin (Lionetti et al. 2010), also have a negative impact on bioenergy production using biochemical conversion technologies.

About 80 percent of the dry plant biomass is comprised of plant cell walls, which stores most of the biomass energy (Vogel and Jung 2001). Cellulose, hemicellulose, and pectin are the polysaccharide components of plant cell walls, of which cellulose is the primary component for biofuel (ethanol) production via fermentation (Carroll and Somerville 2009). Cell walls, especially secondary cell walls, are strengthened by lignin, a phenolic polymer derived from hydroxycinnamyl alcohols and produced by means of combinatorial radical coupling reactions (Boudet 2007). Lignin deposition reinforces plant cell walls to enable water transport, provide mechanical support and a barrier to pathogens, and help convey abiotic tolerance (Halpin 2004; Boudet 2007). However, high lignin content is not desirable for bioconversion of the lignocellulosic feedstock to biofuel for three reasons: 1) it prevents access of the hydrolytic enzymes to the polysaccharides, 2) it absorbs the hydrolytic enzymes, and 3) it inhibits the activities of the hydrolytic and fermentable enzymes used in the biofuel conversion process (Halpin 2004; Endo et al. 2008; Abramson et al. 2010). Studies using different alfalfa transgenic lines, with variable reduced lignin content, proved the negative correlation between lignin content and fermentable sugar release efficiency (Chen and Dixon 2007). Therefore, there is a strong interest in developing low-lignin content switchgrass cultivars for biofuel production.

The grass lignin polymer is usually composed of three monolignols [hydroxyphenyl (H), guaiacyl (G), and syringyl (S)] (Hatfield et al. 1999). Monolignols are derived from the amino acid phenylalanine through the monolignol biosynthesis pathway. The pathway has about ten key enzymes that catalyze the reaction steps and the pathway is evolutionarily conserved across angiosperms (Rastogi and Dwivedi 2008). Gene families encoding these key enzymes went through a rapid expansion after the divergence of monocots and dicots (Xu et al. 2009). By BLASTing against the switchgrass EST database, and utilizing phylogenetic analysis, we can find switchgrass homologs of all monolignol biosynthesis genes in model plant species. Through gene-expression patterns, in vitro enzymatic assays, and the generation of stable RNAi transgenic plants, a few switchgrass genes [4 Coumarate:Coenzyme A Ligase (4CL1,2), Cinnamyl Alcohol Dehydrogenase (CAD1,2), Catechol-O-methyltransferase (COMT)] in the monolignol biosynthesis pathway have been identified. RNAi: PvCOMT, RNAi: PvCAD2 and RNAi: Pv4CL1 transgenic plants have significantly less lignin content than wild type plants (Fu et al. 2011a, b; Saathoff et al. 2011a, b; Xu et al. 2011b).

Fu et al. (2011a) cloned a COMT cDNA and down-regulated its expression in cv. Alamo. Up to 90% of the COMT transcript was reduced and over 70% reduction in COMT enzyme activity was observed. Lignin content was reduced by 6 to 15% while the S/G ratio of the lignin was reduced from 0.69-0.71 in control plants to 0.37-0.40 in transgenics, mainly by reduction of S lignin content. The growth and development of transgenic plants appeared normal and the height and fresh and dry weight were similar to the controls under greenhouse conditions. Transgenic lines increased the ethanol yield by up to 38% using conventional biomass fermentation processes. The down-regulated lines required 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 (Fu et al. 2011a).

Saathoff et al. (2011b) demonstrated that switchgrass cv. Kanlow has at least two functional CAD genes. RNAi approach using 575 bp of PviCAD2 coding fragment (96% identical to the same region of PviCAD1 gene) was employed in an attempt to silence both CAD genes (Saathoff et al. 2011a). The CAD transcripts, protein amount and enzyme activities were substantially reduced in most transgenic lines. Four transgenic lines with single transgene copy were further analyzed. The lowest CAD activity found in these lines was less than 10% of that in the vector controls. The total lignin and cutin amount in these lines were reduced by 23% in average compared to the vector controls. Two transgenic lines had significantly higher glucose release after alkaline pretreatment and enzymatic saccharification. In a similar approach, Fu et al. (2011b) also cloned a CAD (PvCAD) cDNA from swithcgrass, which has 98-99% identity at amino acid level with the predicted proteins encoded by the PviCAD1 and 2 genes. Phylogenetic analysis suggests the gene is involved in lignin biosynthesis. For the eight RNAi transgenic plants analyzed, the extractable CAD activities were only 17-39% of that in control plants (using coniferaldehyde as a substrate). The transgenic plants grew normally in the greenhouse. Total lignin content in transgenic plants was 14-22% lower than in controls as determined by the acetyl bromide method, and both S and G lignins were reduced. In addition, chlorogenic acid, a soluble phenolic compound, was substantially increased in transgenic plants. Without acid pretreatment, transgenic plants released 28-59% more glucose with enzymatic hydrolysis than did the controls while 15-35% more glucose release was observed with pretreatment. Similarly, saccharification efficiency (total sugar release) increased by 19-89% without pretreatment, and by 19-44% with pretreatment. Sugar release was negatively correlated to lignin content but not to the S/G ratio, indicating reduced lignin content is the main reason for the improved sugar release.

Xu et al. (2011b) identified two 4CL genes in switchgrass. Phylogenetic and gene-expression pattern and enzymatic activity analyses suggest that Pv4CL1, but not Pv4CL2, is involved in monolignol biosynthesis. The RNAi:Pv4CL1 T0 transgenic plants downregulated Pv4CL1 expression to 0.05-0.73 fold of the WT controls. The 4CL enzyme activity was reduced by 80% on average as measured in T transgenic plants. The above-ground biomass yield of the transgenic plants was comparable to WT controls grown in the greenhouse conditions while brown color was seen in midvein, internodes, and mature roots of some transgenic plants. Pooled Tx transgenic plants had 22% reduction of the acid-insoluble lignin as well as total lignin. Lignin composition was also changed. T1 transgenic plants had 47% less G lignin and 45% more H lignin, than non-transgenic T segregates. Dilute acid-pretreated samples enhanced enzymatic hydrolysis of glucan but not xylan. With pretreatment, transgenic plant materials yielded 57.2% more fermentable sugar than the WT plants. In a similar effort, two highly homologous 4CL cDNAs were isolated and an RNAi construct attempting to suppress both genes were introduced into switchgrass. Up to 90% of the transcripts of both genes were suppressed. Although the total lignin content was not changed or only modestly reduced (up to 5.8%), the structure and composition of lignin appeared to have altered. That was reflected by significant reduction of acid insoluble lignin (up to 8.5) and increase of the ratio of acid soluble lignin vs. acid insoluble lignin (ASL/AIL increase by 21.4-64.3%), and by the increase of S/G ratio (11.8-164.5% higher in transgenic plants). Consequently, with alkaline pretreatment, glucan and xylan conversion efficiency of the best transgenic plant was increased by 16% and 18%, respectively (Wang et al. 2012).

The lignin biosynthesis pathway is regulated by complex transcription networks that involve many transcriptional activators and repressors. The transcriptional repressors could simultaneously inhibit the expression of several genes in the monolignol pathway, which could be another way to reduce lignin synthesis. Very recently, a transcription factor gene involved in lignin biosynthesis, PvMYB4, was cloned and characterized (Shen et al. 2012). PvMYB4 is an R2R3-MYB transcriptional repressor. PvMYB4 binds to the AC-rich AC-I, AC-II, and AC-III elements of monolignol pathway genes in vitro in EMSA assays, and down-regulates these genes in vivo. Ectopic overexpression of PvMYB4 in transgenic switchgrass, under control of the ZmUbi1 promoter, reduces lignin content by at least 40 to 50 percent; however, the S/G monolignol ratio remains unchanged. Additionally, the ester-linked p-CA: FA ratio in these plants is reduced by approximately 50 percent. Monosaccharide release after enzymatic saccharification, without acid pretreatment, is threefold higher in these transgenic plants.

However, total sugar release from cell wall residues remained the same. The morphology of the transgenic switchgrass was affected. The plant height was reduced by an average of 40 percent, but tiller numbers could be increased as much as 2.5 fold. Whether or not the total biomass was affected remains to be investigated (Shen et al. 2012).

Maize Corngrass1 (Cg1) gene encodes a grass-specific tandem repeat of miR156 gene, which "promotes juvenile cell wall identities and morphology" (Chuck et al. 2011). Overexpression of the gene, like in the maize mutant, increases biomass due to continuous initiation of tillers and leaves, and had less lignin and more glucose and other sugars in the leaves. Chuck et al. (2011) overexpressed Cg1 cDNA in switchgrass in an attempt to improve its biomass yield and the feedstock quality. In a field test, high and moderate expressers were dwarfed and had smaller leaves and lower yield of biomass whereas yield of low expressers were comparable to WT controls while producing four time more branches. The transgene affects flowering: none of the transgenic plants ever flowered after being grown in the field for two summers and a winter. This may be a favorite trait to prevent transgene flow. Total lignin content was moderately reduced in transgenic plants. Interestingly, the low expressers accumulated more than 250% starch in their stems compared to the WT controls. Consequently, saccharification using a mix of amyloglucosidase and a-amylase without pretreatment released 3-4 times more glucose from stems of the low expressers, which was similar to the amount released by dilute acid pretreatment, indicating that pretreatment could be reduced or completely eliminated in saccharification. In a similar approach, Fu et al. (2012) overexpressed a rice OsmiR156b precursor gene in switchgrass. Low expressers flowered normally, moderate expressers had reduced height and did not flower, while high expressers’ growth was severely stunted. Both low and moderate expressers had improved biomass yield: 58-101% more than the controls in greenhouse condition, mainly attributing to increase in tiller number. Solubilized sugar production (g/plant) after acid pretreatment and enzymatic hydrolysis increased by 40-72%.