TALEN-Mediated Precision Mutagenesis and Intragenic RNAi for Lignin Reduction in Sugarcane

Sugarcane (Saccharum sp.hybrids) is a highly efficient and sustainable biofuel feedstock. Brazil is currently integrating first and second generation biofuel conversion technologies for sugarcane. This integrated process will utilize both the cell wall bound sugars of the abundant lignocellulosic sugarcane residues in addition to the sucrose from stem internodes. However, lignin is a major recalcitrance factor in the bioconversion of lignocellulosic biomass to biofuel. Therefore, suppression of lignin biosynthetic enzymes by targeted mutagenesis or RNAi are promising strategies. Caffeic acid O-methyltransferase (COMT) and 4-coumarate-CoA ligase (4CL) are key enzymes in lignin biosynthesis. In this study, the sugarcane COMT gene was targeted for TALEN induced mutagenesis and the 4CL gene was suppressed with an intragenic RNAi approach to reduce lignin content in sugarcane. Both, nuclease mediated mutagenesis and intragenic biotechnologies may facilitate regulatory approval. A selection protocol was developed by identifying a suitable intragenic selectable marker and selective agent for recovery of intragenic events.  Intragenic sugarcane lines with high level of 4CL suppression exhibited brown coloration in the basal internodes and vascular bundle cells. Targeted mutations following TALEN delivery were identified by capillary electrophoresis of the COMT amplicon. Events were confirmed by sequencing of the COMT amplicon which revealed the presence of insertions and deletions at the target site. Data describing the total lignin content in the stem biomass of 4CL RNAi lines and COMT mutants will be presented.