Combining enhanced biomass density with reduced lignin level for improved forage quality.

To generate a forage crop with increased biomass density that retains forage quality, we have genetically transformed lines of alfalfa (Medicago sativa L.) expressing antisense constructs targeting two different lignin pathway biosynthetic genes with a construct for down-regulation of a WRKY family transcription factor that acts as a repressor of secondary cell wall formation in pith tissues. Plants with low-level expression of the WRKY dominant repressor construct produced lignified cell walls in pith tissues and exhibited enhanced biomass and biomass density, with an increase in total sugars in the cell wall fraction; however, lines with high expression of the WRKY dominant repressor construct exhibited a very different phenotype, with loss of interfascicular fibres associated with repression of the NST1 transcription factor. This latter phenotype was not observed in transgenic lines in which the WRKY transcription factor was down-regulated by RNA interference. Enhanced and/or ectopic deposition of secondary cell walls was also seen in corn and switchgrass expressing WRKY dominant repressor constructs, with enhanced biomass in corn but reduced biomass in switchgrass. Neutral detergent fibre digestibility was not impacted by WRKY expression in corn. Cell walls from WRKY-DR-expressing alfalfa plants with enhanced secondary cell wall formation exhibited increased sugar release efficiency, and WRKY dominant repressor expression further increased sugar release in alfalfa down-regulated in the COMT, but not the HCT, genes of lignin biosynthesis. These results suggest that significant enhancements in forage biomass and quality can be achieved through engineering WRKY transcription factors in both monocots and dicots.