Immature inflorescences are an important explant source for initiating in vitro cultures. Plant regeneration from calli derived from young inflorescences of switchgrass has also been reported (Chen et al., 1986). To reduce the damage caused by harvesting, fungal and bacterial contamination, and toxic effects of sterilization solutions on inflorescences, development of inflorescences under sterile conditions would be beneficial. The top nodal segments from tillers in the two to four node stage of greenhouse grown plants were used as explants to develop a protocol for in vitro production of inflorescences from node cultures of Alamo (Alexandrova et al., 1996b). Inflorescences were produced with fully developed spikelets and perfect terminal florets and were used as axenic explants for callus induction and plant regeneration. This highly efficient procedure for the production of organ-specific differentiating tissues provides new tools to explore genetic transformation using microprojectile bombardment in switchgrass. Having such tools in place will be important in incorporating into existing switchgrass cultivars new genes that can protect and improve growth, increase resistance to environmental stresses or introduce qualities to enhance the biofuel properties of switchgrass (McLaughlin and Kszos, 2005). It can also be used for initiating anther cultures to obtain haploids, for in vitro fertilization techniques and the production of hybrids between genera, species or ecotypes that are difficult to cross normally (Alexandrova et al., 1996b).
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Conventional breeding efforts have been accelerated or complemented by novel genetic engineering techniques which offer the opportunity to generate unique genetic variation that are either absent or have very low heritability (Wang and Ge, 2006). Novel enabling biotechnologies are also crucial for rapid domestication, overcoming recalcitrance, efficient breakdown of cellulose, increasing biomass and lipid production, thereby, reducing the costs of bioenergy production, particularly of lignocellulosic ethanol (Himmel, 2007; Yuan et al., 2008). Increasing interest in the production of biofuels has warranted research in the production and genetic manipulation of switchgrass (Table 1; Fig. 3). Such biotechnological interventions have the potential to assist in rapidly overcoming many of the short-comings of switchgrass by addition of exogenous genes, or tissue-specific or temporal expression or suppression of endogenous genes (Gressel, 2008). These genes may be for reducing cell wall recalcitrance (i.e, resistance to enzymatic degradation during saccharification) or for increasing biomass yields, or genes that alter the rate of plant development, growth habit, structure and/or composition of the cell wall (Abramson et al., 2010; Gressel, 2008; Rubin, 2008; Yuan et al., 2008). However, for achieving this, a protocol for the genetic transformation of switchgrass is a pre-requisite.
First transgenic switchgrass was obtained through bombardment of immature inflorescence-derived embryogenic calluses of Alamo using a dual marker plasmid comprising the reporter gene sgfp (green fluorescent protein) driven by the rice actin (Act1) promoter and the selectable bar gene (Basta tolerance) driven by the maize ubiquitin (Ubi1) promoter (Richards et al., 2001). GFP was observed in leaf tissue and in the pollen of transgenic plants and over 100 plants were tolerant to Basta. T1 offsprings resulting from crosses between transgenic and non-transgenic control plants also showed Basta tolerance, indicating the inheritance of the bar transgene (Richards et al., 2001). Agrobacterium tumefaciens-mediated transformation was also attempted in switchgrass. The hypervirulent A. tumefaciens strain AGL1 carrying the binary vector pDM805 containing the bar gene under the control of the Ubi1 promoter and the uidA gene driven by Act1 promoter was used for transformation. Four different explants types, viz., embryogenic calluses, somatic embryos, mature caryopses, and seedling segments of Alamo were used, of which somatic embryos gave the highest transformation frequency (Somleva et al., 2002). Various factors such as genotype, type of tissue used for inoculation, preculture of explants, wounding of tissues prior to infection, presence or absence of acetosyringone during inoculation and cocultivation, and different methods of selection were found to influence the transformation efficiency of switchgrass (Somleva, 2006).
Using a hygromycin phosphotransferase gene (hpt) as a selectable marker and hygromycin as selection agent, transgenic Alamo plants were regenerated from caryopses or inflorescences derived embryogenic calli (Xi et al., 2009) infected with A. tumefaciens strain EHA105 used in combination with the binary vectors pCAMBIA 1301 (carrying a gusA from E. coli) and pCAMBIA 1305.2 (carrying a GUSPlus from Staphylococcus spp.). Calli resistant to hygromycin were obtained after 5 to 8 weeks of selection. The transgenic nature of the regenerated plants was demonstrated by PCR, Southern blot hybridization analysis, and GUS staining. Transgene silencing was observed in the progeny with multiple inserts while reversal of the expression of the silenced transgene was found in segregating progeny with a single insert (Xi et al., 2009). High throughput Agrobacterium-mediated transformation system for cvs. Alamo, Performer and Colony were also developed (Li and Qu, 2010). Highly regenerable and transformation-competent embryogenic calli were developed from seeds and used for genetic transformation using A. tumefaciens strain EHA105 containing the binary vectors pTOK47 (carrying a 20 kb KpnI fragment of Ti plasmid from pTiBo542, which contains virB, virC and virG virulence genes) and pJLU13 (a derivative of pCAMBIA 1301 containing hpt and sgfp genes). Various modifications such as infection under vacuum, co-cultivation at desiccation conditions, resting between co-cultivation and selection, and supplement of L-proline in the callus culture and selection media were carried out at different stages for the improvement of transformation efficiency. Transformation efficiency of over 90% was achieved for 'Performer,' and around 50% for 'Alamo' and 'Colony' (Li and Qu, 2010).
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Though a number of procedures are well established for switchgrass plant transformation, it may take several weeks to evaluate transgene expression in parts of the plants. Transient transgene expression is a potential alternative to stable expression in transgenic plants for testing gene constructs and production of recombinant proteins (VanderGheynst et al., 2008). Very little research has been done on agroinfiltration in the leaves of monocot plant species. Agroinfiltration experiments were performed in harvested switchgrass leaves to identify the effects of wounding by bead beating, surfactant concentration and vacuum application on in planta β-glucuronidase expression and leaf decay. Bead beating did not improve the consistency of expression even though it was successful for wounding the plant surface. Surfactant was found to be necessary for improving contact between the leaf surface and Agrobacterium suspension and consistently improved the expression when vacuum application level was low. Increasing vacuum application also improved the expression but only when surfactant concentration was low (VanderGheynst et al., 2008). For in planta inoculation of germinating Alamo seedlings, Agrobacterium-mediated transient gene expression system was optimized using AGL1, C58, EHA105, and GV3101 strains of which AGL1 showed the highest efficiency in gene delivery (Chen et al., 2010). In another study, it was reported that EHA105 was more effective in gene delivery than LBA4404 or GV3101 (Song et al., 2012).
Wounding pretreatments such as sonication, mixing by vortex with carborundum, separation by centrifugation, vacuum infiltration, and high temperature shock significantly increased transient expression of the GUSPlus reporter gene (Chen et al., 2010). Incorporation of additives like L-cysteine and dithiothreitol in the presence of acetosyringone also significantly increased GUS expression, whereas the addition of 0.1% surfactants such as Silwet L77 or Li700 decreased the GUS expression (Chen et al., 2010). To optimize Agrobacterium-mediated transformation, factors influencing gene delivery, selection of transformed cells, and plant regeneration were investigated (Song et al., 2012). Song et al., (2012) reported that the white basal parts of newly germinated seedlings of Alamo had high capability of producing regenerable calli. Use of the basal parts of switchgrass seedlings as initial explants improved the transformation efficacy by shortening overall transformation time by 4-5 weeks by eliminating the step of preparing callus explants from mature seeds.
Mazarei et al., (2008) reported the first protocol to isolate large numbers of viable protoplasts from both leaves and roots of 'Alamo' and the Alamo 2 clone. Transient expression of polyethylene glycol (PEG) mediated DNA uptake in the isolated protoplasts was demonstrated by measuring the activity of GUS reporter gene driven by either the CaMV35S promoter or the maize ubi1 promoter. Higher extent of GUS activity was observed for the ubi1 promoter as compared to the 35S promoter. To develop a transformation system for upland cultivars, calluses were induced from seedling segments of the upland octoploid cultivar 'Cave-in-Rock.' However, it produced only roots and no plants could be obtained (Song et al., 2012). Since the tissue culture and transformation systems have been developed for 'Alamo' or its derivatives, for a wide applicability across the species, there is a need to create more genotype-independent methodologies for switchgrass. The selection of the right candidate gene(s) for genetic transformation is also critical. It is also important to develop standardized protocols for sampling and for performing basic anatomical, biochemical, and molecular analyses to compare transgenics with wild-type switchgrass plants (Shen et al., 2009).
Even though there is a wide variety of promoters that have been used for grass transformation (Christensen et al., 1992; McElroy et al., 1990; Wang et al., 2000), relatively few promoters have been used in the production of transgenic switchgrass (Li and Qu, 2010; Richards et al., 2001; Somleva et al., 2002). Discovery and characterization of new promoters with enhanced levels of constitutive expression are needed and would be highly beneficial to improve switchgrass and other bioenergy feedstocks through genetic transformation (Mann et al., 2011; Peremarti et al., 2010). To enhance the levels of constitutive expression, new promoters from ubiquitin genes (PvUbi1 and PvUbi2) of switchgrass have been identified (Mann et al., 2011). Reporter constructs were produced containing the isolated 5' upstream regulatory regions of the coding sequences (PvUbi1 and PvUbi2 promoters) fused to the GUS coding region. When tested for transient and stable expression, PvUbi1 and PvUbi2 promoters expressed in all examined switchgrass tissues. Using biolistic bombardment, PvUbi1 and PvUbi2 promoters showed strong expression in switchgrass callus, equaling or surpassing the expression levels of the CaMV35S, 2x35S, ZmUbi1, and OsAct1 promoters (Mann et al., 2011). Of recent, a versatile set of Gateway-compatible destination vectors (termed pANIC) was constructed to facilitate the optimization of monocot transformation methods and for subsequent applications for high-throughput production of stable transgenics in switchgrass (Mann et al., 2012). All the pANIC vectors contain (i) a Gateway-compatible cassette for overexpression or suppression of the target gene, (ii) a plant selectable marker cassette for conferring resistance (containing either the bar or hpt gene) to the transformed plant, and (iii) a visual reporter gene cassette (GUSPlus or pporRFP) for optimization of the transformation process, visual tracking of transgenic events and rapid identification of transgenic plants. These vectors can be utilized for particle bombardment or Agrobacterium-mediated transformation and have been functionally validated in switchgrass (Mann et al., 2012).
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Polyhydroxyalkanoate biobased plastics made from renewable resources can reduce petroleum consumption and decrease plastic waste disposal issues. They are biodegradable in soil, compost and marine environments. Somleva et al., (2008) reported the successful engineering of switchgrass for the synthesis of polyhydroxybutyrate (PHB) and obtained plants containing up to 3.72% dry weight of PHB in leaf tissues and 1.23% dry weight of PHB in whole tillers. They also analyzed polymer accumulation in the T1 generation obtained from controlled crosses of transgenic plants. Their study presents the first successful expression of a functional multigene pathway in switchgrass, and demonstrates its amenability to the complex metabolic engineering strategies necessary to produce high-value biomaterials with lignocellulose-derived biofuels (Somleva et al., 2008).
Lignocellulosic biomass is an attractive energy feedstock because it is an abundant, domestic, renewable source that can be converted to liquid transportation fuels (Lynd, 1996). Reducing lignin biosynthesis can lead to lower recalcitrance and higher saccharification efficiency, making lignin one of the most crucial molecule to be modified for lignocellulosic feedstocks (Yuan et al., 2008). The resistance of plant material to enzymatic and acid hydrolysis is one of the most significant obstacles facing lignocellulose-based production of biochemicals and fuels (Mosier et al., 2005). To effectively enable enzymatic degradation of cellulose, the cell wall structures should be modified, the lignin removed and the hemicelluloses degraded for which some harsh physical or chemical treatments are required (Liu and Sun, 2011). Both the downregulation of genes in the lignin biosynthesis pathway, and addition of novel monolignols, such as ferulic acid and coniferyl ferulate, to remodel lignin structure have been considered to enhance biofuel production from lignocellulosic feedstocks (Joyce and Stewart, 2012). Comprehensive characterization of lignin biosynthesis pathway in switchgrass will enable the manipulation of lignin content through genetic engineering (Xu et al., 2011b).
Switchgrass has been genetically modified to produce phenotypically normal plants that had reduced thermal-chemical (≤180 °C), enzymatic, and microbial recalcitrance by down-regulation of the switchgrass caffeic acid O-methyltransferase gene (Fu et al., 2011a). This gene decreased the lignin content modestly, reduced the syringyl:guaiacyl lignin monomer ratio, improved the forage quality, and, increased the ethanol yield by up to 38% using conventional biomass fermentation processes. Less severe pretreatment and 300-400% lower cellulase dosages were required by the down-regulated lines for equivalent product yields using simultaneous saccharification and fermentation (SSF) with yeast. Better product yields were obtained on fermentation of diluted acid-pretreated transgenic switchgrass using Clostridium thermocellum with no added enzymes as compared to unmodified switchgrass (Fu et al., 2011a). The last step in monolignol biosynthesis is catalyzed by cinnamyl alcohol dehydrogenase (CAD) (Li et al., 2008). In grasses, CAD deficiency decreases overall lignin, alters lignin structure and increases enzymatic recovery of sugars (Chen et al., 2003; Dien et al., 2009). In switchgrass, RNA-mediated silencing of CAD was induced through Agrobacterium-mediated transformation of Alamo with an inverted repeat construct containing a fragment derived from the coding sequence of PviCAD2 to evaluate the effect of CAD down-regulation (Saathoff et al., 2011). In stems of silenced lines, CAD activity against coniferaldehyde and sinapaldehyde was significantly reduced as was overall lignin and cutin. It also enhanced glucose release after cellulase treatment increasing the saccharification efficiency by 23% without acid pretreatment (Fu et al., 2011b; Saathoff et al., 2011).