Lifecycle assessments suggest that production of lignocellulosic biofuels, especially from high-yielding biomass crops such as switchgrass can be achieved with net energy production and substantial greenhouse gas reduction (Farrell et al. 2006). For example, the study of Schmer et al. of 10 multi-hectare fields in the northern midwestern U. S., measured the on-farm energy balance for switchgrass production and then used the literature to estimate the energy balance for conversion to ethanol. The average results were a yield of 2800 L per ha, a net energy yield (energy produced-input) of 80 GJ/ha, and a green house gas displacement of ~80% for use of the bioethanol compared with gasoline (Schmer et al. 2008). This study assumed an ethanol yield of 0.38 L per kg of biomass, which is typical of what is commonly found in other assessments and of laboratory saccharification yields (Fu et al. 2011). Based on the percentage of switchgrass biomass that is sugar (Vogel et al. 2010, Fig. 1), this is ~65% of the theoretical yield of conversion of all polysaccharide to ethanol (0.58 L per kg).

As we have described, substantial progress has been made via a panoply of approaches to improve plant biomass to close the gap between the typical and theoretical saccharification yields. These improvements in yield now await translation from the laboratory into the field. Of course, such efforts require substantial time and money, not to mention regulatory approvals if transgenic methods are employed. Furthermore, we can expect different phenotypes in the field since the range of biotic and abiotic stress conditions under which published studies have been conducted is limited. This is especially important for biofuel crops because they need to be produced on degraded or abandoned crop lands that do not displace substantial food production in order to avoid indirect increases in greenhouse gas production due to land clearing (Fargione et al. 2008; Youngs et al. 2012). No doubt, an even more thorough understanding of cell wall biosynthesis and regulation will be necessary to anticipate and mitigate pleiotropic effects of manipulating the major components of plant biomass. In addition, since the vast majority of studies have not been conducted in switchgrass or other biofuel species, there remains substantial work to be done in testing genes in biofuel species. As thus far only single genes have been examined, when selecting genes for testing in a species such as switchgrass one wonders about whether additive or synergistic effects might be achieved from simultaneously manipulating multiple cell wall synthesis or regulatory pathways. Modeling and informatics studies will certainly facilitate the transfer of information from model species and selection of engineering targets in bioenergy crops (e. g., Ruprecht and Persson 2012).

On the bioprocessing side, the baseline 65% of theoretical ethanol yield is typically achieved with harsh, i. e., expensive, dilute acid pretreatment (0.5% at 180°C for 8 min) and cellulase loading of approximately 15 active units/g biomass (Fu et al. 2011). More efficient catalysts that can function with milder pretreatments at lower concentrations would also facilitate attainment of near maximal yields. Indeed, by combining optimized feedstocks with improved enzymes and bioprocessing methods, we may have already achieved complete and efficient saccharification. Again, the bottleneck seems to be in translating current progress to the industrial scale. As for work with plants, translation to industrial-scale microbiology requires substantial additional understanding and process tuning. Effective scale-up will be facilitated by continued accumulation of additional options in terms of enzymes, strains, and organisms, and understanding at a both detailed biochemical and systems-wide levels. Platforms that reduce the capital requirements of biofuel production will be especially helpful for establishing a second-generation biofuel industry.

Acknowledgements

Thanks to Dr. M. Peck and K. Zhao for helpful comments on the manuscript. This work was supported by the National Science Foundation EPSCoR program under Grant No. EPS-0814361. Any opinions, findings, conclusions, or recommendations expressed are those of the authors and do not necessarily reflect the views of the National Science Foundation.

Biogenesis of MiRNAs

Plant miRNAs are typically encoded by miRNA genes (or MIRNA locus). In most cases, they exist in inter-regions of protein-coding genes and transcribe independently (Voinnet 2009). Biogenesis of miRNA begins with transcription at a MIRNA locus by RNA Polymerase II (Pol II) and produce 5′-capped and 3′-polyadenylated primary microRNA transcripts (pri-miRNAs) (Aukerman and Sakai 2003; Kurihara and Watanabe 2004; Voinnet 2009).

The pri-miRNAs contain imperfect stem-loop structure from which precursors (pre-miRNAs) are excised through the RNase III enzyme DICER LIKE 1 (DCL1) and several other proteins (Park et al. 2002; Reinhart et al. 2002; Voinnet 2009). The factors involved in the initial processing of pri — miRNAs in model plant Arabidopsis thanlina include subunits of the nuclear cap-binding complex (CBC), CBP20 and CBP80; SERRATE (SE), a C2H2- type zinc finger domain-containing protein; HYPONASTIC LEAVES 1 (HYL1), a member of dsRNA-binding protein family, and other components (Bartel 2009; Chen 2009; Chuck et al. 2009; Poethig 2009; Voinnet 2009; Zhu et al. 2009). A key feature of a pri-miRNA is that it is self-complementary and capable of forming the characteristic fold-back hairpin-like structure recognized by DCL1. Processing of a pri-miRNA by the DCL1 complex releases the precursor miRNAs (pre-miRNAs) which contain the stem-loop structure (Voinnet 2009; Zhu et al. 2009).

The pre-miRNAs are further processed by the DCL1 protein complex to generate a small RNA duplex consisting of a miRNA and its passenger strand (miRNA*). The duplex is exported into the cytoplasm by HASTY and methylated at the 3′ end by HEN1 (Park et al. 2002; Yu et al. 2005). One strand functions as the mature miRNA and is incorporated into the RNA — Induced Silencing Complex (RISC) to target mRNAs. The other strand, miRNA* is usually degraded, although some miRNA*s have been reported to be functional under certain conditions (Zhang et al. 2011).

Plant miRNAs recognize their targets through near-perfect complementarity to direct RISC-mediated cleavage, although in some cases translational inhibition and DNA methylation can be the mode of action of miRNA-mediated gene silencing (Chen 2004; Brodersen et al. 2008; Wu et al. 2010).

Most information on switchgrass plant structure and development is for plants from native prairies or cultivars released for forage. However, more botanical and anatomical information is forthcoming for bioenergy specific switchgrass strains. Switchgrass plants are strongly rhizomatous perennials that often form large clumps (Sutherland 1986). Tiller density is generally greatest in the spring and declines as the growing season progresses (Mitchell et al. 1998). The number of leaves per tiller varies by growth stage, genotype, and environment (Redfearn et al. 1997), with the maximum number of collared leaves present prior to advancing to the elongation stage for Trailblazer switchgrass ranging from 3 to 4 (Mitchell and Moser 1995). Ligules are 1.5 to 4 mm long consisting of a fringe of hairs from a membranous base (Sutherland 1986; Casler et al. 2012). Roots of established switchgrass plants have reached depths of 3 m (Weaver 1954).

Switchgrass reproduces sexually by seeds and asexually by rhizomes. The panicle inflorescence is diffuse and 15 to 55 cm long (Sutherland 1986; Casler et al. 2012). Spikelets are 3 to 5 mm long, are two-flowered with the upper floret perfect and the lower floret empty or staminate, and disarticulate below the glumes (Hitchcock 1951; Gould 1975; Casler et al. 2012). Florets are glabrous and awnless and the lemma of the fertile floret is slick and smooth (Casler et al. 2012). The seed is comprised of a smooth lemma and palea that hold tightly to the caryopsis (Casler et al. 2012). Glumes are unequal, with the first shorter than the second (Stubbendieck et al. 1997), and both are mostly removed by combining and cleaning (Casler et al. 2012). Switchgrass has been reported to contain 850 seeds g-1 (Wheeler and Hill 1957). However, Mitchell and Vogel (2012) demonstrated that differences in switchgrass seed weight exist within and among cultivars, reporting the seed number for 19 seed lots of four cultivars across two years ranged from 473 to 702 seeds g-1. Advantages to switchgrass seed are that it is easily threshed and cleaned (Casler et al. 2012) and commercial planting equipment can be calibrated easily to account for differences in seed lot seed number (Mitchell and Vogel 2012a). Unlike other native warm-season grasses, switchgrass seed is smooth and flows readily through grass drills. Additionally, a switchgrass seed industry has existed for over 50 years and numerous private companies and public crop improvement associations are involved in seed production, distribution, and marketing (Casler et al. 2012). The recommended seeding rate for switchgrass in the Great Plains is 300 to 400 PLS m-2 (Mitchell et al. 2010b), but successful stands can be established with seeding rates as low as 100 PLS m-2 if quality seed is used (Vogel 1987).

Switchgrass, a warm-season grass native to North America, has received much attention in recent years given its high yield potential, broad adaptability, and tolerance of marginal sites. The more robust lowland switchgrass ecotypes typically are preferred for bioenergy production systems given their greater yield potential, but lowlands have less cold tolerance, hence lower survivability, at greater latitude.

Switchgrass can be established both with conventional tillage and no-till planting systems. For either planting method, it is critical to have a clean, firm seedbed and to minimize weed competition after planting. Seeding depth is a critical issue for establishment, and can be greatly dependent on the firmness of the seedbed. Seed generally should not be planted more than about one centimeter deep for best establishment, although deeper plantings may be needed on drier sites.

Seeding rates as low as 1 kg ha-1 have been successful, but recommendations typically range from 4 to 8 kg ha-1. Seed often have high levels of dormancy, and this can cause stand failures in late-spring plantings if this is not accounted for. Several methods have been used to break dormancy successfully, but aging seed—holding for a year or so—may be the simplest method for doing so. Some have used planting timing to naturally break dormancy, and planting sometime from late fall to early spring can work well for this. Recommendations to this effect will be unlikely in regions where weed control issues are a challenge, however.

Switchgrass is known to benefit from mycorrhizal associations, and new research is showing a role for bacterial interactions that increase seedling growth. Many of the microbes produce plant-active hormones, and some may play a role in low N requirements due to biological N fixation. This is likely to be an important and growing realm of research in the future.

Little fertility is required for new switchgrass stands, and N as a general rule should not be applied in the year of seeding if the planting faces strong weed competition. Although mature, productive stands can benefit from N fertilization, input requirements will vary based on soils and harvest management, among other factors. As a rule of thumb, matching nutrient inputs to removal is likely to be closest to optimum in terms of meeting economic considerations and minimizing environmental impacts.

Weeds are a major issue in switchgrass establishment and there are very few herbicides currently labeled for use during switchgrass establishment. A number of herbicides have been evaluated in pre — and post-emerge applications to new seedings, and experimental results often vary by region —and even by switchgrass strain. For example, pre-emergence applications of atrazine have been successful in many cases but have been observed to harm certain lowland ecotypes. Safener treatments that protect seedlings from herbicide damage have proven less successful for switchgrass to date, but this work is ongoing.

One of the most successful ways to reduce weed problems at establishment is to plant switchgrass into the stubble of a glyphosate- resistant crop (especially soybeans) and follow good agronomic practices. Use of companion crops, grazing, mowing, or tillage may all provide some positive effect in controlling weeds—or at least, holding back the competition — in the establishment year. Once this first-year hurdle is overcome, there is typically little need for herbicides in established stands.

Diseases and insect pests may be a growing problem for switchgrass in the future as planted acres increase. The plant is host to a number of fungi, but pathogenicity is low for most species, and viral diseases may be an issue on the horizon. Accounts of yield reductions due to disease have increased in recent years, and breeding and selection for switchgrass as a biofuel crop needs to account for disease susceptibility to avoid potential disease vulnerability. Similarly, few insect pests have been a serious issue for switchgrass in the past, but several potential "species of interest" that could cause economic losses have been identified in recent years.

Harvest practices have potential to affect many parts of the supply chain in terms of fertility inputs, energy and CO2 balance, storage needs, and feedstock quality for processing. While most recommendations call for a single, end-of-season harvest, this may not account for the numerous feedbacks to the overall system. Although it makes great sense for minimizing costs on farm (i. e., by reducing nutrient losses), single, end-of — season harvests in a short harvest window may not account for the demands to the system in terms of meeting year-round processing needs. Rather, such a framework is likely to add to the equipment required to harvest, handle and move the material, as well as increase the demands for storage capacity. Thus, to be most effective, the development of switchgrass for biomass — to-bioenergy systems will need to consider the function of the system as a whole, and this is a different paradigm for much of agriculture.

Thermochemical conversion of biomass to biofuels is a highly accelerated form of the geologic processes that created petroleum fossil fuels. This mode of conversion consists of passing biomass through a heated reactor in the absence of oxygen at or above ambient pressures. Residence times within the reactor are dictated by the system type and vary from tenths of a second to up to an hour (NSF 2008). Within the reactor, most of the biomass is pyrolyzed into small molecules that flow out of the reactor as gasses. Depending on the severity of the reactor conditions, the major products are liquids or gasses, plus non-fuel by-products such as tars and mineral-rich char.

When the reactor temperatures are low (~100-750°C), most of the pyrolysis products condense when cooled to produce a liquid referred to as bio-oil. Bio-oil is a complex hydrocarbon mixture that includes water (~25%); 1- to 4-carbon alcohols, acids, and aldehydes (total ~45%); carbohydrates (~10%); and phenolics and other lignin derivatives (~20%) (NSF 2008). Additional heating in the presence of chemical catalysts upgrades and distills these products to form higher chain-length, less oxygenated hydrocarbons. Attractively, these upgraded mixtures are suitable for use in conventional combustion engines with or without blending with petroleum-derived fuels.

At higher reactor temperatures (750-1200°C) and in the presence of some oxygen, the most useful pyrolysis products are CO and H2, which are referred to as syngas. CO2, H2O, H2S, and other impurities also form (NSF

2008) . Of course, H2 in itself is a high-energy fuel molecule, though use as a transportation fuel is not yet technically feasible. For immediate needs, after a cleaning step, the CO and H2 can be recombined with heating and catalysts to form alkanes, via Fischer-Tropsch synthesis, or alcohols, especially

methanol. Another method of upgrading syngas to transportations fuel is known as indirect fermentation. In this process, anaerobic bacteria can utilize the CO to form ethanol, and in limited cases, butanol (Mohammadi et al. 2011). Bacteria can also couple the oxidation of CO to CO2 to produce H2 (Oelgeschlager et al. 2008).

Relative to current biochemical conversion, the short residence times within the reactor bed provide the possibility for distributed production of thermo-converted fuels, reducing the distance that low density biomass must be transported for biofuel production (NSF 2008). On the other hand, a major challenge for thermochemical processes is optimizing the energy efficiency and the fraction of carbon from the biomass that is incorporated into the final, useable fuel. A wide diversity of feedstocks, including switchgrass (Boateng et al. 2006), can be used for thermochemical conversion, and this technology has been seen as having the advantage of being largely feedstock-independent. However, scientists have recently begun to explore possible correlations between feedstock content and syngas and bio-oil formation (Boateng et al. 2006; Gan et al. 2012). Desirable biomass qualities for thermochemical biofuel production may rely on the details of the method of conversion. These qualities include higher content of reduced compounds (i. e., lignin) to maximize the starting material energy potential, lower nitrogen and mineral content to prevent these molecules from catalyzing oil degradation, and reduction of crosslinks between biomass components to allow staged conversion for the different biomass content fractions.

Genetic maps serve many practical biological purposes and are a key tool in both classical genetic and modern genomic research. Generally, two factors need to be considered in selecting appreciate parental lines to begin the development of mapping populations: 1) DNA polymorphism; 2) hybrid fertility (Xu 2010). A good segregating population is required for the construction of a genetic map. Being a species of wind-facilitated cross-pollination and strong genetic self-incompatibility, switchgrass is an allogamous species (Taliaferro and Hopkins 1996; Casler et al. 2011). Theoretically, every plant in switchgrass is heterozygous in many loci and homozygous in other loci. Switchgrass homozygous inbreds are unavailable in nature. Therefore, crossing of two heterozygous parents would produce a pseudo-F1 population, which often displays substantial segregations. Some loci may have four different alleles between the crossing parents, generating four genotype classes in the progeny. Many others may either follow the F2 pattern in a 1:2:1 ratio (called intercross loci) or the backcross pattern in a 1:1 ratio (called testcross loci) (Lu et al. 2004). Using the testcross markers, i. e., those that are segregating in one parent but not in the other, a

so-called "pseudo-testcross" strategy was proposed for linkage mapping in a controlled cross between two outbred parents (Grattapaglia and Sederoff 1994).

Although it only makes use of a portion of markers from the genome, "pseudo-testcross" strategy provides a simple way for genetic mapping of outcrossing species and has been utilized in practical mapping projects for switchgrass (Missaoui et al. 2005b; Okada et al. 2010). The population of Missaoui et al. (2005b) was composed of 85 full-sib progeny from a cross of ‘Alamo’ genotype AP13 (seed parent) and ‘Summer’ VS 16, whereas the population of Okada et al. (2010) consisted of 238 full-sib plants derived from crossing one genotype (seed parent) of ‘Kanlow’ with a selection of ‘Alamo’. Because male and female meioses in the full-sib populations were distinct and independent processes, two separate parental maps were constructed, one map for the male parent and another for the female parent (Okada et al. 2010).

Recently a self-compatible lowland switchgrass genotype ‘NL 94 LYE 16 x 13’ was identified. A first (S1) generation inbred population from selfing ‘NL 94 LYE 16 x 13’ was developed with the assistance of marker — based identification (Liu and Wu 2012a). This S1 population is similar to an F2 population derived from selfing a F1 hybrid of a cross between two different inbred lines; therefore, only one map was constructed instead of two separate (male and female) maps (Liu et al. 2012).

Although genetic manipulation of miRNAs in transgenic plants for switchgrass improvement is still in its infancy, there have already been excellent examples demonstrating the effectiveness of this approach in genetically modifying switchgrass (Chuck et al. 2011; Fu et al. 2012). The miR156 family is one of the most ancient miRNA families found in a large number of plants, from moss to flowering plants (Zhang et al. 2006a; Xie et al. 2006; Axtell and Brown 2008). The miR156 has been reported to target SQUAMOSA promoter-binding-like (SPL) genes, which encode plant-specific transcription factors (Klein et al. 1996; Cardon et al. 1999; Xie et al. 2006; Schwarz et al. 2008; Yang et al. 2008; Yamaguchi et al. 2009). Several studies have suggested the important roles the miR156 genes and their targets play in various plant developmental processes, especially in the transition from juvenile to adult development and floral induction (Schwab et al. 2005; Xie et al. 2006; Chuck et al. 2007; Poethig 2009; Wang et al. 2009; Wu et al. 2009; Yamaguchi et al. 2009). The Arabidopsis miR156, when overexpressed in transgenic plants, dramatically impacted plant morphology, resulting in accelerated leaf growth, greatly enhanced branching and biomass, and delayed flowering (Schwab et al. 2005; Wu and Poethig 2006; Wang et al. 2009; Wu et al. 2009). In maize (Zea mays L.), a classic dominant mutant Corngrass1 (Cg1) which was found about 80 years ago, exhibits phenotypes of dwarfism, multiple-tillers and prolonged vegetative phase (Chuck et al. 2007). In 2007, Chuck et al. successfully cloned this gene and found it encodes two tandem miR156 precursors (zma-miR156b and zma-miR156c) (Chuck et al. 2007). The phenotypes of Cg1 result from the overexpression of miR156s, which impacts the expression of a couple of targets of the SPL family of transcription factors, and the level of miR172 whose targets are involved in juvenile development (Chuck et al. 2007). Interaction of miR156 and its targets also has been studied extensively in rice (Xie et al. 2006, 2012). MiR156s play important roles in rice development (Xie et al. 2006, 2012; Jiao et al. 2010; Miura et al. 2010). Xie et al. (2006) reported that overexpression of two different rice miR156 precursors (stem-loop structures) in transgenic rice plants resulted in reduced plant height, delayed flowering and increased tiller number (Xie et al. 2006), similar to that observed in the maize Cg1 mutant (Chuck et al. 2007) and transgenic Arabidopsis plants overexpressing miR156 (Schwab et al. 2005).

The facts that overexpression of the miR156 genes could increase biomass and "hold plants in the juvenile phase of development" in many plant species (Schwab et al. 2005; Xie et al. 2006; Chuck et al. 2007, 2011; Li et al. unpublished) suggest that they would be potential candidates to improve biomass yield and feedstock quality in switchgrass. To test this hypothesis, two research groups overexpressed the miR156 gene in switchgrass, independently (Chuck et al. 2011; Fu et al. 2012). Chuck et al.

(2011) introduced the Cg1 cDNA fused to the maize Ubiquitin (Ubi) promoter into switchgrass. As expected, overexpression of Cg1 causes pleiotropic morphological and developmental changes in transgenic plants. The vegetative phase was prolonged and flowering time was delayed. In this case, flowering was not observed in transgenic plants, even after having been grown for two years both in the field and in the greenhouse. Total sterility with no flowering is a favorable trait for preventing transgene escape. Although in this case, the production of biomass, one of the important target traits for switchgrass breeding, was not improved in transgenics compared to wild type controls, the amounts of lignin were decreased and the levels of glucose and other sugars were increased in transgenic plants relative to wild type controls. Later, Fu et al. (2012) reported results in overexpressing the precursor of rice Osa-miR156b gene, also driven by the maize Ubi promoter, in transgenic switchgrass. The authors found that biomass yield was related to the expression levels of the exogenous rice miR156. Higher levels of miR156 in transgenic plants resulted in severely stunted plant growth, whereas moderate levels of miR156 expression led to improved biomass production and loss of the ability in flowering. Transgenic plants with low levels of rice miR156 expression flowered normally and their biomass yield was increased. The latter two groups of transgenic plants produced 58-101% more biomass than wild type controls. The authors also found that overexpression of miR156 could improve the solubilized sugar yield and forage digestibility in transgenic plants (Fu et al. 2012).

Recently, we found that overexpression of rice miR156b/c and miR156d genes led to enhanced drought tolerance in transgenic creeping bentgrass (Agrostis stolonifera L.), which is associated with less water consumption and increased water retention capacity (Li et al. unpublished data). These results point to the potential of manipulating miR156 genes in transgenic switchgrass for enhanced stress tolerance. Moreover, Sun et al. (2012) found that the expression level of miR156 increased in switchgrass when subjected to drought stress, suggesting that miR156 may be involved in plant stress responses in this bioenergy crop, and could be a good candidate for manipulation using transgenic approach to produce new switchgrass cultivars with enhanced stress tolerance.

Seed dormancy and slow seedling development often have been contributing factors to poor switchgrass establishment (Zarnstorff et al. 1994). Switchgrass seed display high levels of dormancy immediately following harvest (Knapp 2000; Madakadze et al. 2000; Teel et al. 2003). Sautter (1962) reported that seeds tested within 33 d of harvest were only 10% germinable, and rates approaching 5% are not uncommon (Parish and Fike 2005).

Dormancy is a natural mechanism that serves to prevent premature seed germination, and ultimately, seedling death. Under natural conditions, switchgrass seed will not germinate until stratified—i. e., until exposed to a period of cool, moist conditions as occur during late fall to early spring. Stratification can be imposed artificially (Zhang and Maun 1989; Beckman et al. 1993; Zarnstorff et al. 1994; Haynes et al.1997; Shen et al. 1999; Wolf and Fiske 2009) by allowing seed to imbibe moisture and keeping chilled for several weeks, and a similar process is used in seed testing methods. Herein lies the potential problem surrounding the planting of switchgrass based on a seed tag’s statement of PLS. Under official testing methods, seeds are exposed to a period of moist chilling before immediately incubating at appropriate germination temperatures (AOSA 1993). The conditions for seed testing often can break dormancy; with highly dormant seed lots, this could mask the low level of germination that would occur when they are planted in the field.

When sowing switchgrass, particularly in late spring, it is important to know the level of dormancy in a seed lot because planting rates should be based on the amount of germinable seed, not simply on PLS percentages.

We use the "late spring" qualifier, because some producers and researchers have had success with late fall/early spring plantings that break dormancy by allowing the seed to naturally stratify, and we discuss planting timing strategies in a subsequent section.

Several methods to artificially break dormancy have been explored, including after-ripening, stratification, seed priming, acid or mechanical scarification, and hormonal treatments (Jensen and Boe 1991; Beckman et al. 1993; Zarnstorff et al. 1994; Haynes et al. 1997; Shen et al. 1999; Madakadze et al. 2000; George 2009; Ghimire et al. 2009). These techniques work with various degrees of success—and success can vary by cultivar and growing conditions, among other factors. We note here, too, that any effective seed priming or dormancy abatement technique must face the greater question of whether it can be practically applied at a commercial scale.

Seed priming, an osmotic process in which seeds are hydrated to a level where metabolic activity begins but radicle emergence does not occur, may enhance switchgrass germination (Beckman et al. 1993). Some chemical treatments such as hydrogen peroxide treatment can increase seed germination and emergence and provide more uniform seedling development for non-dormant seeds (Sarath et al. 2006). Karrikinolide [3-methyl-2H-furo[2,3-c]pyran-2-one], a compound isolated from smoke that promotes germination and seedling establishment in several native species did not increase switchgrass germination or seedling vigor (George

2009) . Some biological agents such as mycorrhizae and bacteria are also known to benefit switchgrass germination and seedling vigor, but their role in breaking dormancy is, unexplored. This topic will be discussed in the section on switchgrass fertilization.

One of the simplest methods for reducing dormancy is merely to hold the seed at moderate temperatures for an adequate period of time. Holding seed at 23°C for 90 to 180 d is adequate for overcoming most short-term seed dormancy (Zarnstorff 1994). Elevated storage temperatures also can break dormancy and can reduce the storage time required for after-ripening (Shen et al. 1999). Both of these after-ripening techniques must be managed carefully, however, as aging, and especially accelerated aging (with heat) can reduce seed viability (Zarnstorff et al. 1994; Shen et al. 1999).

As a production issue affecting establishment success, dormancy may be beginning to fade in importance for growers. Some seed purveyors already market pretreated, ready-to-plant seed that do not require additional stratification or after-ripening. Dormancy also may be eliminated altogether as switchgrass use increases, because low-dormancy switchgrass lines could become standard for cultivars of the future. Sanderson et al. (1996) reported that by collecting and growing plants from non-dormant, neoteric (newly harvested) seeds, dormancy rates could be significantly reduced. Similarly, Burson et al. (2009), also have selected plants from non-dormant neoteric seeds for subsequent breeding and improvement. The advances made from these efforts suggest that dormancy-related stand failures could soon be a thing of the past and may simply be one of the birthing pains associated with bringing to market a new crop with a short history of plant improvement.

AM fungi can enhance a plant’s ability to acquire nutrients like phosphorus and nitrogen (Clark 2002; Parrish and Fike 2005; Leigh et al. 2009; Schroeder- Moreno et al. 2011), phytoremediate contaminated soil (Entry et al. 1999), and withstand acidic soil (Clark 2002). AM hyphae have the ability to extend beyond the usual nutrient absorption zone of plant roots, therefore reaching additional essential nutrients and transporting them to the plant (Clark 2002).

Mycorrhizal fungi and other rhizosphere microflora have played significant roles in switchgrass growth in nature (Parrish and Fike 2005). In field conditions, switchgrass plants are commonly associated with AM fungi and have shown growth stimulation under different conditions (Brejda et al. 1998; Parrish and Fike 2005; Schroeder-Moreno et al. 2011). Under acidic soil conditions, the inoculation of AM fungi (Glomus, Gigaspora and Acaulospora) increased the root length of switchgrass plants, as well as the uptake of minerals such as phosphorus, nitrogen, sulfur, potassium, calcium, magnesium, zinc, and copper but reduced the uptake of manganese, iron, boron, and aluminum (Clark 2002). Inoculation with the AM fungi Gisgospora margarita, Gi. Rosea, Glomus clarum, and Scutellospora heterogama significantly increased nitrogen in shoots (Schroeder-Moreno et al. 2011), which implies AM fungi play an important role in N cycling from the soil to switchgrass plants.

Microorganism diversities affect plant growth promotion because plants exist in a community of bacteria, fungi, algae and/or viruses (Rodriguez and Redman 2008), and plants could be associated with more than one microorganism. Inoculation of switchgrass seedlings with multiple types of rhizosphere microflora increased the yield of shoots and roots up to 15-fold and also increased nitrogen uptake 6-fold and phosphorus uptake 37-fold, compared with the control plants infected with rhizosphere bacteria only (Brejda et al. 1998). Environmental factors, such as nutrients and stress, also influence symbiosis between host plants and endophytes as well as AM fungi. Under high nutrient availability, symbiotic Neotyphodium occultans — Lolium multiflorum association showed higher seed weight than that of non-symbiotic plants (Gundel et al. 2012). Under greenhouse conditions, the combination of AM fungus and the fungal endophyte Epichloe elymi on growth promotion in the grass Elymus hystrix was found to be additive (Larimer et al. 2012). However, the presence and specificity of the fungal endophyte altered the interaction of AM fungus with the host plant as endophyte infection increased Glomus mosseae colonization while decreasing G. claroideums colonization (Bibi et al. 2012).

Pretreatments are procedures applied prior to the major depolymerization of the covalent bonds of biomass polysaccharides. Pretreatments are intended to make biomass saccharification proceed toward greater completion, at a higher rate, and with lower enzyme loading. The broad pretreatment classifications are physical, chemical, and biological procedures, with many utilized approaches consisting of a combination of these classes. Here, we provide a brief overview of some of the most commonly employed pretreatment methods. The topic has also been extensively reviewed (Galbe et al. 2007; Hendriks et al. 2009; Agbor et al. 2011).

Physical approaches to pretreatment include chopping, shredding, grinding, and other macroscopic methods to increase biomass surface area and accessibility. A recent review of optimal milling sizes concluded that for herbaceous crops, such as switchgrass, commonly used particle sizes below ~3 mm produce no further saccharification benefits (Vidal et al. 2011). Those authors also noted that results have been variable and that particle sizes have not often been systematically varied (Vidal et al. 2011). Physical approaches also generally include treatments such as heating and increased pressure, which are typically used along with chemical pretreatments. Experiments have also been conducted with various forms of irradiation, such as treatment with microwaves and gamma waves (Agbor et al. 2011).

After chopping, chemical and physiochemical pretreatments are the most common. They can be subdivided based on pH into acidic, basic, and neutral methods (Galbe et al. 2007). These methods include dilute acid, lime, steam, ammonia fiber expansion, and the use of ionic liquids (Agbor et al. 2011). Dilute acid pretreatment is typically carried out with heated sulfuric acid at <4%. To improve cellulose access, dilute acid primarily solubilizes matrix polysaccharides; a downside is the creation of sulfuric acid waste. Lime [Ca(OH)2] treatment achieves dilute base conditions and can proceed effectively at <120°C. This method can remove acetyl groups and partially depolymerize lignin, but requires high amounts of water. Steam pretreatement, also known as steam explosion or autohydrolysis, has been the most commonly studied. The process entails exposing the biomass to pressurized steam for several seconds or minutes at 160 to 240°C between

0. 5 and 5 MPa (Agbor et al. 2011). A related approach is to use liquid hot water under similar conditions. Both of these are mostly targeting increased solubilization of matrix polysaccharides to expose cellulose.

Two promising neutral treatment methods are ammonia fiber expansion (AFEX) and ionic liquid pretreatments. These both have the advantage of being highly effective and requiring relatively low energy inputs. In AFEX, biomass is mixed with liquid ammonia at an approximately 1:1 ratio at 50 to 100°C and elevated (4 MPa) pressure. Upon rapid pressure release the ammonia volatilizes and the biomass, especially of grasses, can be converted to sugars at approaching 90% efficiency (Balan et al. 2009). Drawbacks of AFEX include the cost of ammonia and production of gaseous ammonia as a pollutant. Ionic liquid pretreatment is another highly effective method. Ionic liquids are salts that are liquids below 100°C. Ionic liquids, such as 1-ethyl-3-methylimidazolium acetate, dissolve the cellulose from biomass and also allow very high saccharification rates. Though recyclable, one major drawback is that ionic liquids are relatively costly.

Biological pretreatment refers to the use of lignocellulolytic organisms, such as white rot fungi, to initiate the cell wall depolymerization process. In planta expression of enzymes to create fully or partially "self-pretreating" plants also falls under this heading. These biological approaches will be discussed further below in the context of consolidation of bioprocessing.