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.