It has been reported that miRNAs play an important part in regulating some key genes in their own biogenesis (Xie et al. 2003; Vaucheret et al. 2004; Vaucheret 2006). For example, DCL1 is the target of miR162, and AGO1 is the target of miR168, indicating a negative role miRNAs play in their own biogenesis (Xie et al. 2003; Vaucheret et al. 2004; Liu et al. 2005; Vaucheret 2006). Recent studies also discoverd that the primary transcripts of trans-acting siRNA (tasiRNA) TAS1, TAS2, TAS3 and TAS4 are cleaved by miR173-AGO1, miR390-AGO7 and miR828-AGO1, demonstrating that miRNAs are also involved in the regulation of siRNAs (Allen et al. 2005; Montgomery et al. 2008a, b; Cuperus et al. 2011).

Switchgrass MiRNAs

Conditioning of syngas can be accomplished through hot or cold gas cleaning technologies. The cost of syngas conditioning can be a significant portion of the cost of syngas production, hence reducing cost of syngas conditioning is critical. Hot gas cleaning technologies use high temperature and catalysts to crack tar molecules into gaseous compounds, which can also improve the energy content and composition of the syngas. Cold gas technologies use solvents such as water, acetone, isopropanol, and oil to scrub tar and other contaminants from the gas.

Filamentous growth morphology of T. reesei results in a viscous broth rheology that affects oxygen mass transfer rate and changes the broth from a Newtonian mixture to a non-Newtonian mixture over periods of cellulolytic enzyme production. With increasing viscosity, power input requirements increase to achieve the same level of mixing. The increase in the viscosity increases the bubble size and hence reduces the bubble residence time in the fermenter and decreases the mass transfer coefficient. Enzyme and extracellular protein levels were significantly affected at lower (0.5 vvm) and higher (1.5 vvm) oxygen saturation levels and at lower (130 rpm) and higher (400 rpm) agitation levels (Schaffner and Toledo 1992). The change in morphology of T. reesei affects xylanase production at lower aeration (below 10% oxygen saturation level). In addition, xylanase production is sensitive to shear stress at power agitation (Weber and Agblevor 2005).

The purpose of this chapter is to identify practical issues related to the economics of developing switchgrass as a dedicated energy crop and to provide 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. As noted in the introduction, the potential for switchgrass biomass depends on (a) its production cost relative to alternative sources of feedstock and (b) a system to convert lignocellulosic biomass into economically competitive products.

The estimated breakeven price for switchgrass biomass delivered to a biorefinery ranges from $60 to $120/Mg. For the base estimates obtained from the programming model, 27 percent of the delivered cost of $60/Mg is for transportation from the field to the biorefinery; 26 percent is for harvest (windrowing, raking, baling, stacking) costs; 20 percent is for land rental; 14 percent is for fertilizer; and 13 percent is for establishment. Increasing yield could reduce most but not all the costs on a per unit basis. As modeled, harvest costs per Mg are found to be very similar across a wide range of yields per hectare. Given the rather substantial cost economies associated with harvest machines, and given that a biorefinery is expected to require a continuous flow of feedstock, if switchgrass is established on millions of hectares, a highly coordinated harvest system would be more economical than a haphazard system.

Switchgrass harvest would extend over as many months as permitted by feedstock quality requirements, weather, and policy. Given the quantity of biomass required, and the lack of an existing infrastructure to harvest a continuous flow of massive quantities of biomass, a harvest system would likely develop that exploits the economies of size associated with harvest machines. Whether or not independent companies develop, such as those that exist for grain harvest in the Great Plains, remains to be seen. Alternatively, harvest crews and harvest machines could be managed as wholly owned subsidiaries of biorefineries.

Rational land owners would not enter into switchgrass biomass feedstock production until a market is available. A rational investor would not invest in a biorefinery that did not have a reasonable plan for obtaining a flow of feedstock. One alternative would be for the biorefinery to engage in long-term leases with land owners to acquire the rights to a sufficient quantity of land to produce feedstock to meet its needs.

The U. S. Energy Independence and Security Act of 2007 mandated the production of 61 billion liters of cellulosic biofuels by 2022. But, no commercial sized facilities were operating in 2011. Hence, it seems reasonable to conclude the development of a commercially viable system for production of liquid biofuels has not progressed as rapidly as anticipated. Desirable feedstock properties, the biomass to biofuel conversion rate, and the investment required in plant and equipment differ depending on which one of several competing technologies is used. Determination of the most efficient system will require a holistic field-to-bioproducts model that simultaneously considers land procurement, feedstock production, harvest, storage, transportation, processing, and the value of the final products. Modeling each of the competing conversion systems using a "field to fuel" approach could provide useful information to compare the expected economics of each system and identify unique bottlenecks.

A number of additional issues remain. A system to manage the risk associated with switchgrass yield variability and the risk of fire of standing and stored switchgrass will be required. Knowing how a biorefinery would respond to short crops is not clear. In years of above average yields, not all land would have to be harvested. However, in years of below average yields, the biorefinery may not have sufficient feedstock to operate throughout the year.

If an economically competitive biorefinery technology is developed, entrepreneurs confident of their technology with an enforced government mandate that their produced biofuels be purchased, could contract and convert land from current use to the production of switchgrass or some other dedicated energy crop, in a relatively short period of time. Ambiguities as to what determines feedstock quality and how to provide a flow of feedstock throughout the year are likely to be resolved much more quickly if the annual payment to the land owner is set. Leased land would enable the company to manage a portfolio of switchgrass stands, or a portfolio of energy crops, feedstock quality, and harvest, to optimize the field-to-fuel process. Unwillingness of biorefinery entrepreneurs to engage in long-term lease contracts could be interpreted as a signal that they are unsure of the economics of their conversion technology. The ultimate challenge is to discover, develop, design, and demonstrate an economically competitive biorefinery technology necessary for a profitable business model.

Acknowledgements

Research findings reported in this chapter were produced by projects supported by the USDA NIFA Biomass Research and Development project number 0220352; by USDA NIFA Hatch grant number H-2824; by the Oklahoma Agricultural Experiment Station; by the Jean & Patsy Neustadt Chair; by the Samuel Roberts Noble Foundation; and by a USDA National Needs Graduate Fellowship Competitive Grant no. 2008-38420-04777 from the National Institute of Food and Agriculture. Support does not constitute an endorsement of the views expressed in this paper by the USDA or by the Samuel Roberts Noble Foundation.

The diversity of switchgrass’ morphology parallels the diversity of sites to which the species is adapted (Fig. 1). Robust lowlands can be taller than 3.0 m, while some uplands may only reach 0.5 m; root depths can extend to 3 m where soils are not restrictive (Porter 1966; Moser and Vogel 1995). Upland plants also typically have thicker roots and longer root internodes, with rhizomes long enough to support sod formation (Beaty

Color image of this figure appears in the color plate section at the end of the book.

et al. 1978). In contrast, lowland plants, with shorter rhizomes, more often exhibit the characteristic growth habit of a bunchgrass (Vogel 2000). New shoots originate from rhizome buds on lowland plants but also from basal nodes of culms in the upland ecotypes (Porter 1966). In addition to being smaller in stature, upland ecotypes typically have finer leaves and stems and smaller panicles; lowlands, in addition to their larger size, also often have a distinctive bluish coloration (Casler 2005). Both ecotypes generally have erectophile leaves, although this characteristic generally is stronger in lowlands.

Although seed size can differ by cultivar, this generally will have little agronomic consequence except in cases of excess seeding depth (Zhang and Maun 1991; Smart and Moser 1999). Seedling morphology, however, has some potential to affect switchgrass development and survival, as some seedlings have elevated crowns, which limits adventitious root formation. This appears to have little actual effect, however, as research in the field gives little evidence that this is an issue in stand establishment (Elbersen et al. 1999). Researchers have also selected for increased tillering of seedlings, which may be useful in the development of divergent genotypes, but this has not been a successful approach for improving plant establishment or yield (Smart et al. 2003, 2004).

Switchgrass is a determinate plant and it produces multiple tillers that become reproductive after exposure to the right environmental signals. Biomass accumulation comes to an end in conjunction with inflorescence development. Research suggests that daylength is the chief signal for floral development, although this response may not be completely under photoperiod control (Esbroeck et al. 2003). Reproductive development may also be delayed or inhibited by flooding, excessive K fertilization, and low temperatures (Porter 1966; Balasko and Smith 1971; Friedrich et al. 1977).

For a given cultivar, the critical photoperiod (some minimum night length, actually) is genetically determined and linked to the plant’s latitude of origin. Differences in photoperiodic flowering responses among cultivars have important implications for selection and production in the field. For example, moving southern-adapted cultivars to higher latitudes delays their reproductive development. This promotes continued vegetative growth (and greater biomass yield) as the plant does not experience the appropriate signal for reproductive development (sufficient night length) until later in the year. In the opposite way, a northern type will be less productive when moved south because the photoperiodic trigger occurs earlier in the season at lower latitudes. This attribute also has important implications for survival, and we will give both issues further consideration in the section below on cultivar selection.

Along with latitudinal differences among cultivars, switchgrass can display variable longitudinal morphology and adaptation (Hopkins et al.

1995a, b; Madakadze et al. 1998; Vogel 2000; Casler and Boe 2003). Cultivars adapted to conditions of the humid east are generally taller but less tolerant of the drier and windier conditions of the Great Plains (Cornelius and Johnston 1941). Conversely, productivity of the shorter, coarser-stemmed western-adapted switchgrass can be negatively affected when moved east. This is thought to be largely related to the lower pathogen resistance in cultivars adapted to drier climates (Vogel 2000).

Following rhizoplane colonization, internalization of the bacteria and their development as endophytes can occur quite rapidly, within days of inoculation/rhizoplane contact (Compant et al. 2008; Prieto and Mercado — Blanco 2008; Zakria et al. 2008). In order to colonize the plant interior, bacteria must make their way past the root surface. This can happen through the presence of surface openings, such as cracks produced during lateral root emergence (James and Olivares 1998), or other wounds. Furthermore, other root areas, such as the elongation and differentiation regions may contain cells that are more fragile or less differentiated, and more susceptible to bacterial penetration (Reinhold-Hurek and Hurek

2011) . As with rhizosphere and rhizoplane colonization competence, a variety of bacterial traits are associated with competence for endophytic colonization. These include flagella, nod genes, type IV pili and twitching motility (Compant et al. 2008). Many of these traits are associated with bacterial adherence and movement, or bio-control of other surrounding microorganisms, providing a competitive advantage for the colonizing bacteria. In addition, bacterially-secreted, cell wall-degrading enzymes are important for bacterial penetration (Quadt-Hallmann et al. 1997) and internal colonization, including cellulolytic and pectinolytic enzymes (Quadt-Hallmann et al. 1997; Kovtunovych et al. 1999). The endophyte Burkholderia phytofirmans strain PsJN, known to colonize switchgrass (Kim et al. 2012), produces endogluconase and polygalacturonase (Compant et al. 2005b), to aid in cell wall degradation.

Following initial root penetration, bacterial colonization proceeds within the root cortex, and can extend into the central vascular cylinder xylem vessels (Compant et al. 2008; Priedo and Mercado-Blanco 2008; Zakria et al. 2008). However, not all bacterial endophytes colonize the xylem. For example, Priedo and Mercado-Blanco (2008) noted that Pseudomonas fluorescens PICF7 remained in the root cortex region and was never found in the xylem, with no subsequent translocation elsewhere. The inability of some endophytes to colonize the xylem and move past the root may be due to the presence of filters formed at branch root junctions (Shane et al. 2000), which may limit bacterial movement (Zakria et al. 2008). In addition, as endophytes are aided in their penetration through the root endodermis and pericycle by cell wall-degrading enzymes (James et al. 2002), it may be that some endophytes produce sub-optimal levels of enzymes to allow penetration into the vascular tissue.

Colonization within regions like the root cortex occurs within the intercellular spaces, outside of living cells (Reinhold-Hurek and Hurek 1998; Priedo and Mercado-Blanco 2008), which is not surprising as these are rich in minerals (potassium, calcium, sulfur, phosphorus, chlorine), sugars (Madore and Webb 1981) and non-carbohydrate metabolites, such as various amino acids and organic acids (Canny and McCully 1988; Canny and Huang 1993). Endophyte alterations of apoplastic pH can alter enzyme activities, sugar uptake of host cells, and sugar concentrations for the colonizing microbes (Bacon and Hinton 2002). Hence, this environment is supportive of endophyte growth, promoting compound cycling between the endophyte and the plant.

As mentioned above, cellulose composes about 40% of switchgrass cell wall and is the primary target for bioconversion to biofuel. Cellulose microfibrils are synthesized at the plasma membrane by cellulose synthase A (CESA) complexes with glycosyltransferase (GT) activity (Lei et al. 2012). Microfibril orientation determines cell growth direction and cell wall mechanical properties (Saxena et al. 2005; Crowell et al. 2010). In higher plants, the complex consists of a hexagonal formation of six rosettes; each rosette consists of six CESA polypeptides (Doblin et al. 2002). Many reviews have covered recent progress in cellulose synthase machinery and cellulose

Table 2. Enzymes that synthesize and modify xylan. Protein Name Locus ID Mutant CAZy* or DUF+ Family Activity Comments Reference IRX9 At2g37090 irx9 GT43 p(1^4)-xylan synthesis Backbone elongation (Brown et al. 2007; Pena et al. 2007) IRX14 At4g36890 irxl4 GT43 Backbone elongation (Brown et al. 2007) TaGT43-4 wheat GT43 (Zeng et al. 2010) IRX10/GUT1; IRX10-LIKE/GUT2; TaGT47-13 Atlg27440 At5g61840 irxlO irxlO irxlO-L GT47 p(1^4)-xylan synthesis Backbone elongation (Brown et al. 2009) (Zeng et al. 2010) IRX7/FRA8; IRX7L/ F8H At2g28110 At5g22940 irx7/fra8 GT47 Reducing end (Rennie et al. 2012) IRX8/GAUT12 At5g54690 irx8 GT8 p(1^4)-xylan synthesis Reducing end (Pena et al. 2007) PARVUS/GLZ1 Atlgl9300 parvus GT8 Reducing end (Lee et al. 2007) GUX1 GUX2 At3gl8660 At4g33330 GT8 Side-chain glucuronic acid and 4-O-methylglucuronic acid branches to xylan (Mortimer et al. 2010) IRX15 IRX15L At3g50220 At5g67210 DUF579 unknown Mutant has methylglucuronic acid side chains instead of glucuronic acid side chains (Brown et al. 2011) XAX1 Os02g22380 GT61 p-Xylp-(l—»2)-a-Ara/- (1-3) (Chiniquy et al. 2012) TaXATl wheat GT61 a(1^3)-Ara/Transferase (Anders et al. 2012)

microfibril synthesis (Somerville 2006; Carpita 2011; Endler et al. 2011; Domon et al. 2012; Lei et al. 2012). As revealed by genomic sequencing, Arabidopsis, rice, and sorghum each possesses 10 CesA genes; whereas, maize possesses 20 CesA genes (http://cellwall. genomics. purdue. edu/) (Penning et al. 2009). The Arabidopsis CESA proteins as well as CESAs that have been studied in grasses are listed in Table 1. Recent results highlight the utility of understanding cellulose synthesis for enhancing biofuel production. DeBolt and colleagues reported that mutations in the C-terminal transmembrane domain region of Arabidopsis CESA1 and CESA3 proteins decrease microfibril crystallinity and increase saccharification efficiency (Harris et al. 2012).

Mutant studies, mostly in Arabidopsis, have greatly enhanced our understanding of physiological functions of CESA family members. Arabidopsis AtCESA1, AtCESA3 and AtCESA6 are mainly responsible for cellulose synthesis in primary walls. Mutations in the genes that synthesize these proteins present dwarfism, sterility, swollen etiolated hypocotyls, ectopic accumulation of lignin and reduced root elongation phenotypes (Endler et al. 2011). On the other hand, mutations in AtCesA4, 7 and 8 genes affect cellulose synthesis in secondary walls and are accompanied by collapsed xylem vessels and significant decreases in cellulose contents (Endler et al. 2011). Researchers have hypothesized that the longer and more crystalline microfibrils of secondary walls might be due to different CESA components in the cellulose synthase complexes. However, recent studies have detected expression of the "primary wall" cellulose synthase genes, AtCesA1, 3 and 6, in both primary and secondary cell walls of Arabidopsis (Betancur et al. 2010). Furthermore, AtCESA2,5 and 9, which have greatest homology to CESA6, have been found to be involved in the biosynthesis of secondary cell wall in the epidermal seed coat (Stork et al. 2010; Mendu et al. 2011; Sullivan et al. 2011). These findings suggest that much more remains to be learned about the mechanism of the cellulose synthase complex.

Though phylogenetic analyses are common, direct studies of cellulose synthesis in grasses are less so. Still, mutations in the rice secondary wall cellulose synthases, OsCesA7, OsCesA4 and OsCesA9, respectively, lead to a brittle culm phenotype caused by thinner cortical fiber cell walls (Tanaka et al. 2003). Also, prior to the availability of the maize genome and a more complete description of the maize CesA family, Appenzeller and colleagues isolated 12 CesA genes from maize. Among them, ZmCesA10, 11, 12 are the probable orthologs of the Arabidopsis secondary cell wall CesA genes (Appenzeller et al. 2004). As expected, numerous expressed sequence tags for switchgrass cellulose syntheses appear in databases (Tobias et al. 2008), though a detailed naming scheme and apportionment into families has not yet been conducted, and may be premature at the time of this writing due to the draft nature of the genome.

Integrating genetic maps and physical genome maps is extremely valuable for map based isolation, comparative genome analysis and as anchors for genome sequencing projects. The DOE (Department of Energy) Joint Genome Institute (JGI) has sequenced plant genomes of candidate bioenergy crops such as sorghum (Paterson et al. 2009) and the model grass Brachypodium (International Brachypodium Initiative 2010). Both plants have been used as references for switchgrass, however sorghum last shared a common ancestor with switchgrass more than 20 million years ago while Brachypodium last shared a common ancestor with switchgrass more than 50 million years ago. The genome of a much closer switchgrass relative—foxtail millet (Setaria italica)—is described recently (Bennetzen et al. 2012; Zhang et al. 2012). All three genomes, along with those of switchgrass sequenced by the JGI are publicly accessible at www. phytozome. net. The genus Panicum also includes diploid species closely related to switchgrass such as P. hallii. Meyer et al. (2012) sequenced, assembled and annotated the transcriptome of a lowland ecotype P. Hallii var. Filipes, using 454-FLX Titanium sequencer. Totally 1.26 million EST reads were produced, and 15,422 unique genes identified. A majority of contigs (77%) and 34% of singletons matched ESTs from switchgrass (Meyer et al. 2012). In addition, a switchgrass bacterial artificial chromosome (BAC) library containing 147,456 clones and covering the effective genome approximately 10 times are established, and the average length of inserts in BACs is 120 kb (Saski et al. 2011). Moreover, two BAC libraries with 16 times genome equivalents of switchgrass were constructed, and each library comprised 101,376 clones with average insert sizes of 144 (HindIII-generated) and 110 kb (BstYI-generated) (Sharma et al. 2012).

In parallel, molecular linkage maps composed of various molecular markers including RFLP, SSR and STS (Missaoui et al. 2005b; Okada et al. 2010, Liu et al. 2012) have been constructed in switchgrass. For a more complete genome assembly and analysis, and to take full advantage of linkage map resources, it is of great importance to combine switchgrass genetic maps with physical map information. This can be accomplished by connecting genetic mapping data to BAC clones, which is a well established approach in rice (Chen et al. 2002), wheat (Sourdille et al. 2004), sorghum (Klein et al. 2000), and ryegrass (Lolium perenne, King et al. 2002), but has not been employed in switchgrass on a genome-wide basis.

Identification of Genome Structure

Tetraploid (2n=4x=36 chromosomes) and octoploid (2n=8x=72) are two major ploidal forms in switchgrass, while diploid (2n=2x=18), hexaploid (2n=6x=54), decaploid (2n=10x=90), and 12-ploid (2n=12x=108), and aneuploids were also reported (Lu et al. 1998; Costich et al. 2010, Young et al. 2010; Zalapa et al. 2011). Most of lowland switchgrass is tetraploid and octoploid lowland switchgrass has been discovered recently (Zhang et al. 2011b). Upland ecotype encompasses primarily octoploid and tetraploid plants although the former is more frequent than the latter. Using single dose molecular markers, one study inferred tetraploid switchgrass is an autotetraploid with a high degree of preferential pairing (Missaoui et al. 2005b), and another study indicated switchgrass has a near complete preferential pairing in disomic inheritance (Okada et al. 2010). The mode of completely disomic inheritance was further revealed by using about 500 co-dominant markers, which distributed on whole genome (Liu et al.

2012) . Recently Triplett et al. (2012) sequenced five low copy nuclear loci and two chloroplast loci and clarified that switchgrass was an allopolyploid originated from hybridization of two close diploids. The "A" subgenome of switchgrass is likely derived from an ancient species which was a close

relative to the current species in section Rudgeana of Panicum genus, while "B" subgenome is not identified yet (Triplett et al. 2012). The subgenomic differentiation in switchgrass was confirmed by karyotype analysis of a diploid individual (Young et al. 2012), which were selected from the progeny of a cross between two lowland tetraploid cultivars, ‘Kanlow’ and ‘Alamo’ (Young et al. 2010).

Switchgrass is one of the important biofuel crops which would contribute to our renewable energy in the future. Research on switchgrass miRNA is still in its infancy and to date, our knowledge has been largely limited to those that are conserved across species. Little is known about many switchgrass-specific miRNAs and their functions. Identification, isolation and functional characterization of switchgrass miRNAs will require more efforts. However, data obtained from many other plant species have clearly demonstrated the importance of these small RNA molecules and their targets in regulating various aspects of plant growth, development and response to environmental stimuli. This points out their great potential for use in plant genetic engineering. It should be noted that although miRNAs could serve as potential tools for genetic manipulation of switchgrass for improvement, altering expression of a miRNA, in many cases, could cause pleiotropic morphological and developmental changes in transgenic plants. Therefore, it is critical to better understand molecular mechanisms underlying miRNA-mediated changes in plant growth and development thereby designing appropriate transgenic strategies to obtain desirable traits with minimum unfavorable side effects.

Acknowledgements

The research in Luo’s lab has been supported by Biotechnology Risk Assessment Grant Program competitive grant no. 2007-33522-18489 and no. 2010-33522-21656 from the USDA National Institute of Food and Agriculture as well as the USDA grant CSREES SC-1700315 and SC-1700450. Technical Contribution No. 6110 of the Clemson University Experiment Station.

Tissue Culture, Genetic
Transformation, and
Improvement of Switchgrass
Through Genetic Engineering

Bingyu Zhao, u* Rongda Qu,2 Ruyu Li,2 Bin Xu1 and
Taylor Frazier1

Tissue Culture

In the early 1990s, the US DOE identified switchgrass (Panicum Virgatum L.) as an herbaceous energy crop and launched research efforts on switchgrass as a biomass energy feedstock (McLaughlin and Kszos 2005). Conger’s laboratory at the University of Tennessee was the pioneer in tissue culture and genetic transformation research on switchgrass. Their first report on callus induction and plantlet regeneration was published in 1994 (Denchev and Conger 1994). Mature caryopses, along with young leaf segments from newly-formed shoots of secondary tillers (lowland cv. Alamo), were used as explants and cultured on MS medium supplemented with auxin,

2,4- D (22.5 pM, or 5 mg/l), and cytokinin, 6-benzylaminopurine (BAP, 45 pM, or 10 mg/l, for mature caryopses and 5 pM, or 1.1 mg/l, for young leaf segments). Mature caryopses cultures were maintained in the dark at 29°C for 4 wk, and callus and "organized structures" were observed. They

department of Horticulture, Virginia Tech, Blacksburg, VA 24061. department of Crop Science, North Carolina State University, Raleigh, NC 27695. ^Corresponding author: bzhao07@vt. edu

were transferred to MS medium without growth regulators and placed under light. Approximately 65 percent of the calluses regenerated into plants. For young leaf segment cultures exposed to the same conditions, embryogenic calluses originated from basal segments of innermost leaf pieces. Non-embryogenic calluses were produced from the remainder of the leaf segments. Histological and scanning electron microscopy (SEM) analyses indicated embryogenesis was the main pathway for regeneration from mature caryopses culture, whereas regeneration from leaf segment cultures was mostly through organogenesis. One thousand regenerated plants were obtained and grown in the field.

In their next publication (Denchev and Conger 1995), the authors evaluated the effects of four concentrations (0, 5, 15, and 45 pM) of BAP in combinations with three concentrations (11.3, 22.5, and 45 pM) of auxins,

2,4- D or picloram, on callus induction and shoot regeneration. With mature caryopses as explants, both embryogenic and non-embryogenic calluses were observed from all 2,4-D-containing media. It was also observed that a supplement of BAP greatly improved the formation of embryogenic calluses and regeneration. In contrast, few embryogenic calluses were observed from picloram-containing media. In both cases, two transfers of calluses to regeneration medium (free of growth regulators) greatly facilitated shoot regeneration. The best regeneration results came from 2,4-D (all three concentrations) in combination with 15 or 45 pM BAP in callus induction media. Young seedling segments were also used as explants but performed very poorly.

Switchgrass is an outcrossing species and thus each plant is an individual genotype. Maintenance of a desirable genotype has to be through vegetative propagation. Correspondingly, Conger’s laboratory developed a node culture procedure for efficient micropropagation of switchgrass (Alexandrova et al. 1996). Tillers of switchgrass (cv. Alamo) with four to six nodes were harvested and individual nodes (below the top node) were excised. The nodes were sliced longitudinally and placed with the cut surface in contact with MS medium supplemented with 30 g/l maltose and four concentrations of BAP (0, 5, 12.5, and 25 pM) as the only growth regulator. A week after culturing nodes under a 16 hr light/8 hr dark photoperiod, shoots began to emerge from the axillary buds at the nodes. Roots were developed 8 wk later when the shoots were transferred to hormone-free medium. The highest shoot numbers came from media that contained 5 or 12.5 pM BAP (six to seven shoots per node) and cultured at 29°C. The number of shoots developed from nodes cultured at 29°C was six-fold higher than those cultured at 22°C, most likely owing to the fact that switchgrass is a warm-season grass.

In 1998, the same laboratory reported multiple shoot clump formations when mature caryopses were cultured in media with a combination of various concentrations of 2,4-D and thidiazuron (TDZ) (Gupta and Conger 1998). Caryopses germinated in the medium and multiple shoot meristems were formed at the shoot apex (Fig. 1). The phenomenon was only observed when both 2,4-D and TDZ were present in the medium. The optimal combination of the growth regulators for producing the highest number of shoots was determined to be 4.5 pM 2,4-D and 18.2 pM TDZ. Both lowland (cv. Alamo) and upland cultivars (cv. Trailblazer, Blackwell) had similar responses, though at different frequencies. SEM and histological analyses revealed that the multiple shoot formation was caused by activation of axillary buds and de novo formation of adventitious buds. The shoot clusters were successfully transferred to soil after rooting in hormone-free medium.

In 1999, Conger’s laboratory reported the development of suspension cultures of switchgrass in MS liquid medium containing 2,4-D (9 pM) and BAP (4.4 pM). Initiated from embryogenic calluses from young inflorescence cultures, the suspensions contained various developmental stages of somatic embryos, which regenerated or germinated into plantlets after being transferred to solid medium (Gupta and Conger 1999). In a related report, Odjakova and Conger (1999) studied the effect of callus age and osmotic pretreatment on embryogenic cell formation and the regeneration ability of the suspension cultures (cv. Alamo). It was observed that 10-day-old calluses performed better than 20- and 30-day-old calluses, and that 0.3 M each of sorbitol and mannitol was superior over 0.1 and 0.2 M each.

Based on experience and previously established methods in switchgrass tissue culture, Conger’s laboratory went on to successfully obtain transgenic switchgrass plants (see below). In a report from Somleva et al. (2002), the authors identified several genotypes from cv. Alamo and induced embryogenic calluses from various explants of these genotypes for transformation. Details on how to identify such genotypes and how to maintain them were not described.

A similar approach was recently reported by Xu et al. (2011). These authors first identified certain lines, such as HR8, whose mature caryopses- derived calluses had high regeneration ability in tissue culture experiments. HR8 plants were cross-pollinated with another high regeneration line, HR7, for seed production. Seeds collected from HR8 have a higher germination rate than the unselected Alamo seeds (82 vs. 72 percent). Eighty-five percent of the calluses induced from germinated HR8 seeds were embryogenic, and 84 percent of those regenerated into plants, whereas the corresponding rates of unselected Alamo seeds were only 36 and 21 percent, respectively. The response of HR8 to ABA supplementation to the medium was also different from the unselected Alamo seeds. The addition of ABA to callus induction medium reduced seed germination and increased embryogenic callus formation from HR8 seeds. For example, at 10 pM of ABA, the seed germination rate was reduced to 51 percent but embryogenic callus formation increased to 99 percent for HR8. However, unselected seeds, placed at the same concentration of ABA, exhibited a decline in both germination rate and embryogenic callus formation. Further analysis revealed that the endogenous ABA levels in HR8 seeds were about 3-fold higher than the ABA concentration in unselected Alamo seeds. Additionally, fungal endophytes were observed from some switchgrass callus culture, which may negatively affect callus growth and regeneration.

Burris et al. (2009) employed a new culture medium, LP9, in switchgrass tissue culture, which was modified from culture medium described by Lu et al. (2006). The medium by Lu et al. contains macroelements of N6 medium (Chu 1975), and microelements and vitamins of B5 medium (Gamborg et al. 1968). It is also supplemented with casein hydrolysate, proline, and glutamine. In the LP9 medium by Burris et al. (2009), dicamba was replaced by 2,4-D (5 mg/l), and both BAP and myo-inositol were removed. Proline was also reduced from 0.5 g/l to 0.1 g/l. Inflorescences from tillers of E2 to E4 stages (Moore et al. 1991) were first cultured on MS+BAP medium for 10 days and then transferred to the LP9 medium. Approximately one third of the calluses induced were "brittle and white", similar to the Type II callus described in maize culture (Armstrong and Green 1985). The regeneration ability of the friable, embryogenic, type II callus lines could last more than six months. Agrobacterium-mediated transgenic plants were obtained from the type II calluses.

Calluses similar to the reported type II callus were also observed by Li and Qu (2011) in lowland cultivars of Alamo, Performer, and Colony. However, different medium ingredients were used. Mature caryopses were first cultured on MS-based medium containing 5 mg/l 2,4-D and 1 mg/l BAP for 6-8 wk. Approximately 15 percent of the induced calluses were white, compact, embryogenic calluses. They were transferred to the same medium supplemented with 2 g/l L-proline. Approximately 50 percent of the subcultured calluses became white and friable for cv. Alamo and Colony. Although both types of calluses were highly regenerable, the friable calluses were more competent for transformation. However, this kind of callus tends to regenerate albino plants after a long term culture (100 percent albino after 14 mon of culture). Another kind of callus, one that is yellow and friable, was observed mainly from cv. Performer (Fig. 2). This kind of callus was also highly regenerable, but maintained green plant regeneration ability much longer. In an experiment, 100 percent of such calluses still regenerated into green plants after 14 mon of culture without any albinos. Supplementation of proline to the culture medium not only promoted type II callus formation, but also enhanced callus growth.

Color image of this figure appears in the color plate section at the end of the book.

Bio-oil contains solid contaminants (ash and char), which catalyzes the polymerization and cracking reactions of the bio-oil. The alkali present in the ash such as sodium and potassium also catalyzes cracking reactions. The particulate contents depend on the type of cleaning and filtration system used following the pyrolysis reactor. Cyclone separators are generally used to reduce the particulate contents but removal of finer particulates may require further filtration.

High Viscosity, Increases with Time

Viscosity of biomass is 40-100 cP (shown in Table 5), which increases over time because of polymerization reactions when bio-oil is stored. High viscosity makes it difficult to flow through pipes and valves. The viscosity is especially important when bio-oil is atomized using spray nozzles for direct combustion in burners and engines (Bridgwater 2012).

Low Energy Content

As shown in the Table 5, Energy content of bio-oil (19 MJ/kg) is similar to the energy content of biomass which is about 40% of the energy content of crude oil. The primary reason of its low energy content is its high moisture and oxygen contents.

Aging

Properties of bio-oil such as viscosity, composition and phase contents change over time because of polymerization among the many functional groups and phase separation in the bio-oil (Bridgwater 2012). The aging process increases with temperature because of its increased reactivity.