Similar to research in tissue culture, genetic transformation of switchgrass has so far been mostly performed with lowland cv. Alamo. Genetic transformation of switchgrass was first reported by Conger’s laboratory (Richards et al. 2001) using a particle inflow gun. Inflorescence-induced calluses were bombarded with a plasmid containing both bar and gfp genes.

A two-step selection scheme (5 mg/l followed by 10 mg/l bialaphos) yielded nearly 100 herbicide tolerant plants, which were confirmed as transgenic by both Southern blot analysis and by green fluorescence in leaves and pollen. The bar gene was inherited to the progenies. The laboratory went on to perform Agrobacterium-mediated transformation of switchgrass using somatic embryos and calluses induced from mature caryopses, seedlings, or immature inflorescences of various genotypes of cv. Alamo (Somleva et al. 2002). The hypervirulent Agrobacterium tumefaciens strain AGL1, containing binary vector pDM805, was employed for infection. The T-DNA transmitted by the pDM805 vector contained the bar gene driven by the maize ubi1 promoter, as well as the GUS gene under control of the rice Act1 promoter. As in other monocot species, acetosyringone (50-200 pM) was added to the medium to facilitate infection. Transformation efficiency ranging from zero to near 100 percent was obtained dependent on genotype, explant tissue, and acetosyringone concentration. One or two transgene copies were observed in most of the transgenic plants. Nearly 600 bialaphos-tolerant plants were recovered. Sixty plants were randomly chosen for further studies and after controlled crosses, all progenies set seeds. Fifty-five plants showed co-expression of the bar and GUS transgenes. The transgenes were inherited to the next generation after controlled crossing with non-transgenic plants. In a book chapter summarizing her experience and protocol for switchgrass transformation by Agrobacterium, Somleva (2006) pointed out that among the explants she tested, mature caryopses produced callus with the best regeneration potential and thus are preferred for transformation experiments. From her experience, somatic embryos induced during the tissue culture process quickly generated highly embryogenic calluses, which regenerated numerous transgenic plants during the selection process with few escapes. Addition of acetosyringone, which activates Agrobacterium vir genes, improves transformation efficiency for most of the genotypes tested. The same protocol can also be applied to a lowland cv. Kanlow. In her protocol, she used MS salts and vitamins as the basal medium, supplemented with 2,4-D (22.5 pM) and BAP (5 pM) for callus induction from mature caryopses and callus maintenance. The regeneration medium is also MS based, with gibberellic acid (GA3, 1.4 pM) as the only phytohormone. Selection at callus and regeneration stages always used 10 mg/l bialaphos, and the antibiotic used to suppress Agrobacterium growth was 150 mg/l timentin. Cultures were transferred to fresh selection medium every two wk for four to six wk, and then to regeneration medium under a 16/8 photoperiod for another four to six wk. The author noted that, to maintain long-term cultures, "immature" somatic embryos at globular to early scutellar stage can be subcultured on medium with "high auxin" concentration. Highly embryogenic callus can be maintained for 10-12 mon.

Xi et al. (2009a, b) reported successful switchgrass transformations using Agrobacterium strain EHA105 and hygromycin B (hyg B) selection, with hph as the selectable marker gene. Mature caryopses of cv. Alamo or young inflorescence and nodal segments from two genotypes of cv. Alamo, ST1 and ST2, were selected based on tissue culture responses and subsequently vegetatively propagated. In their tissue culture experiments, the authors used MS medium with 22.6 pM 2, 4-D as the basal medium for caryopses culture. Medium supplementation with BAP, or replacing sucrose with maltose, did not yield significant improvement in embryogenic callus formation. The same medium, with addition of 0.67 pM BAP, was employed for inflorescence culture. Supplementing the medium with CuSO4, casamino acid, or proline did not consistently improve embryogenic callus formation in inflorescence culture. In transformation experiments, vacuum infiltration was applied to improve infection. Both EHA105 and AGL1 had a higher transformation efficiency than LBA4404, and 75 mg/l hyg B provided stringent selection for transgenic calluses (Fig. 3). Lower selection pressures often yielded escapes. However, no plants survived when the selection medium contained more than 100 mg/l hyg B. Cefotaxime (250 mg/l) was used to suppress the growth of Agrobacterium in selection and regeneration media. Southern blot analysis suggested that the transgenic plants had one, two, or multiple transgene copies. Offspring plants derived from reciprocal crosses between transgenic plants and non-transgenic plants segregated

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

by a ratio of 1:1 or 3:1. It is most likely that the 3:1 ratio was caused by transgene insertion at two unlinked loci. The results also indicated that the transgenic plants were both male and female fertile. Transgene silencing was observed in one plant with a transgene copy at one locus and multiple transgene copies at another locus. Interestingly, when the multiple copy locus was segregated out, offspring plants with a single hph gene copy restored transgene expression. The authors used a "hygromycin dip" test to demonstrate that after the hph gene expression was restored, the leaves from those plants became hyg B resistant, and no necrosis was observed after the dip.

Xu et al. (2011a) reported Agrobacterium transformation of switchgrass using phenotype HR8. They followed the method reported by Somleva et al. (2002) using AGL1 Agrobacterium strain with binary vector pCAMBIA1305.1, or pSQ5. However, they added vacuum infiltration during infection and employed hyg B selection (50 mg/l). Thirty-seven transgenic plants were obtained and their transgenic nature was confirmed by Southern blot analysis. Expression of either GUS or GFP reporter gene was demonstrated. In their work, the antibiotic augmentin (375 mg/l) was used to suppress Agrobacterium growth in selection and regeneration media.

In the report by Li and Qu (2011), the authors used Agrobacterium strain EHA105 containing plasmids pTOK47 and pJLU13 for transformation. pTOK47 contains an extra copy of three virulence genes, virB, virC, and virG, and is believed to enhance Agrobacterium infection. pJLU13 is a derivative of pCAMBIA1300 and has a hygromycin B resistance gene, hpt (AKA, hph), for selection. pJLU13 also contains a green fluorescent protein gene (GFP) driven by the rice rubi3 gene promoter, which is constitutive and expressed strongly in callus (Sivamani and Qu 2006; Lu et al. 2008). In this experiment, the GFP reporter gene was very helpful in recognizing the callus types that were more competent for transformation. GFP protein expression also helped in identifying treatments that facilitated genetic transformation (Fig. 4). Calluses were induced from caryopses cultures of three switchgrass cultivars: Alamo, Performer, and Colony. With high L-proline (2 g/l) in the medium, type II like friable calluses were formed, which were highly competent for transformation and also proved to be highly regenerable. A series of treatments were shown to have positive effects on transformation efficiency, which included vacuum infiltration during infection, desiccation at the co-cultivation stage, resting (no selection) for three days between co-cultivation and selection stages, and inclusion of proline in the selection medium to promote growth of the transformed cells. Some of these treatments were also used by Xi et al. (2009) and Xu et al. (2011a). A two-step hyg B selection scheme was adopted: 100 mg/l for the first round of selection that lasted two wk, and 200 mg/l for the second and third rounds that also lasted two wk each. The discrepancy on

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

hyg B efficacy (and thus the concentrations used) between this report and other reports was probably due to the vender sources. With selected friable calluses and optimal treatments, the transformation efficiency could reach approximately 50 percent for cv. Alamo and Colony, and approximately 90 percent for cv. Performer. Both low and high transgene copies were observed. Transgenes were inherited to the next generation.

Song et al. (2012) evaluated factors that affect Agrobacterium-mediated transformation of switchgrass. It was found that Agrobacterium strain EHA105 is superior over LBA4404 and GV3101 based on transient GUS reporter gene expression. Both bar and hpt selectable marker genes allowed effective selection of stable transformants. However, the NPTII gene did not. Although transgenic calluses were obtained from both lowland cv. Alamo (tetraploid) and upland cv. Cave-in-Rock (octoploid), transgenic plants were only recovered from Alamo. The authors found that seedling basal segments were good explants for Agrobacterium-mediated transformation of switchgrass. After direct infection of the basal segments, approximately 10 percent of them developed resistant calluses in both cultivars, and nearly half of the resistant calluses from cv. Alamo were able to regenerate into plants. Compared with infection of seed-derived callus, the seedling basal segment transformation approach saved time by four to five wk.

To facilitate genetic transformation of switchgrass and other monocot species, a series of Gateway-compatible vectors, called pANIC vectors, were developed (Mann et al. 2012). A group of vectors was designed for bombardment transformations and another group for Agrobacterium — mediated transformations. Vectors were designed for either transgene overexpression, or RNAi-mediated gene silencing. All of the overexpression vectors have an AcV5 epitope for easy detection of the tagged fusion proteins. Each vector includes a selectable marker gene (hph or bar), a visible reporter gene (GUSPlus or pporRFP), and a Gateway-compatible construct for insertion of the gene of interest by recombination, which is particularly convenient for making RNAi constructs.

Bio-oil is a mixture of many compounds with different functions groups. Acids, alcohols, aldehydes, esters, ketones, sugars, phenols, gicaols, syringols, furans, lignin derived phenols and extractible terpene with multiple functional groups comprise most of the bio-oil (Mohan et al. 2006; Lee et al. 2008; Guo et al. 2011).

Bio-oil Upgrading

Many of the undesirable properties of bio-oil are due to its high oxygen content. Hence primary purpose of bio-oil upgrading is to reduce oxygen contents so that upgraded bio-oil can be converted into power, fuels and chemicals. The primary techniques used to upgrade bio-oil are: (i) hydrodeoxygenation, (ii) catalytic vapor upgrading, (iii) aqueous reforming, and (iv) steam reforming.

In addition to continuing to collect empirical data, models of switchgrass biomass production are now being used to address many of these questions. Models of bioenergy crops can be split into two distinct types. Correlational or statistical approaches rely on relationships between environmental variables and empirical biomass estimates. In contrast, mechanistic or process-based approaches simulate the actual processes that govern crop growth. Correlational or statistical models have been used to estimate yields across large spatial extents (Barney and DiTomaso 2010; Evans et al. 2010; Jager et al. 2010; Wullschleger et al. 2010). However, little information is included on soil type, nutrient availability, and management practices, which are known to have large impact of biomass production (Muir et al. 2001; Fike et al. 2006). Instead, processes-based simulations of plant growth include detailed information on climate, soil dynamics, and management (Williams et al. 1989; Kiniry et al. 1992; Kiniry et al. 1996; Arnold et al. 1998; Del Grosso et al. 2005; Di Vittorio et al. 2010; Miguez et al. 2011). These models have been used to not only estimate yields but also to analyze water use efficiency, management practices, long-term effects on soil properties, and the impact of climate change (Kiniry et al. 1996; Kiniry et al. 2008; Brown et al. 2000; Sarker 2009). Due to their wider range of applications, this chapter will focus on process-based simulation models and will highlight recent applications to switchgrass.

Mechanistic models of plant growth that have been used to simulate switchgrass production include Agro-BGC, ALMANAC, BIOCRO, DAYCENT, EPIC, and SWAT (Williams et al. 1989; Kiniry et al. 1992; Kiniry et al. 1996; Arnold et al. 1998; Del Grosso et al. 2005; Di Vittorio et al. 2010; Miguez et al. 2011). These models were created for different purposes (i. e., tracking greenhouse gas (GHG) emissions, water erosion, nutrient cycling, plant growth, etc.). Correspondingly, these models vary in their functions and amount of detail incorporated to simulate growth. Despite these differences, each model shares the following basic functionality. First, they simulate biomass production by specifying light interception, conversion of sunlight to biomass, and partitioning of biomass into structural components (such as below ground roots and above ground shoots). Second, they simulate soil water dynamics, which depends on precipitation, run off, and evapotranspiration. Third, they simulate soil C and N dynamics. Lastly, each model simulates the effect of water stress on plant growth. Models with more complex functions for the effects of environmental stress on plant growth, such as ALMANAC and EPIC, incorporate more stress effects; temperature stress, N and P nutrient stress, salinity, low pH, aluminum toxicity, and soil aeration.

There are several different data types required to parameterize and run each model (Williams et al. 1989; Kiniry et al. 1992; Kiniry et al. 1996; Arnold et al. 1998; Del Grosso et al. 2005; Di Vittorio et al. 2010; Miguez et al. 2011). First, each model requires plant parameters that characterize the developmental stage and rate of biomass accumulation over time. Next, each model requires daily weather values that include daily maximum temperature, minimum temperature, precipitation, and solar radiation. Third, each model requires basic soil properties such as: soil type, nitrogen, texture, moisture availability, or water holding capacity. Lastly, most models incorporate basic crop management practices such as fertilizer application, planting date, harvesting dates, and removal rates.

Hong Luo is a Professor in the Department of Genetics and Biochemistry of Clemson University. He also serves as the Graduate Coordinator for the Clemson Genetics, Biochemistry and Molecular Biology Programs. Dr. Hong Luo received his Ph. D. degree in Molecular Biology at the Catholic University of Louvain in Belgium. His research mainly focuses on plant genomics, gene discovery in grasses and cereal crops, and genetic engineering of perennials and row crops. His work has led to the development of the first genetically engineered, environmentally safe, male-sterile and herbicide-resistant turfgrass plants and the development of a new method for hybrid crop production using site-specific DNA recombination systems. He recently received Clemson University 2013 Godley-Snell Award for Excellence in Agricultural Research. He currently maintains several active research projects for both basic and applied research on genetic modification of crop plants, and collaborates with scientists across continents. He has authored or co-authored numerous research papers, book chapters and review articles, and is the inventor on a number of patents in plant biotechnology issued or in various stages of application. He serves as an ad hoc peer reviewer for more than twenty-five international professional journals and serves as a reviewer or as a panel member for various federal, regional and state funding agencies.

Yanqi Wu is an associate professor and holds the Meibergen Family Professorship in Plant Breeding in Plant and Soil Sciences Department at Oklahoma State University. He earned his Ph. D. degree in Crop Science with an emphasis in Genetics and Breeding of perennial grass species from Oklahoma State University. Dr. Wu’s research is centered on the development of new cultivars, and genetic and genomic research on important agronomic traits in bermudagrass used for turf and forage, and switchgrass for bioenergy. Four new cultivars, ‘Cimarron’ switchgrass, ‘Goodwell’ forage bermudagrass, ‘NorthBridge’ turf bermudagrass, and ‘Latitude 36’ turf bermudagrass have been released from the breeding program in recent five years. Dr. Wu published 31 refereed journal articles, 11 technical publications, 65 abstracts, and 6 book chapters. He received the

2010 Early Career Award of the National Plant Breeders Association. Dr. Wu served as an ad hoc peer reviewer for more than twenty international peer-reviewed journals and as a reviewer or on review panels for federal and regional funding agencies in the United States.