Sugarcane (Saccharum spp. hybrids) is a prime crop for commercial biofuel production. Advanced conversion technology utilizes both, sucrose accumulating in sugarcane stems as well as cell wall bound sugars for commercial ethanol production. Reduction of lignin content significantly improves the conversion of lignocellulosic biomass into ethanol. Conventional mutagenesis is not expected to confer reduction in lignin content in sugarcane due to its high polyploidy (x = 10–13) and functional redundancy among homo(eo)logs. Here we deploy transcription activator-like effector nuclease (TALEN) to induce mutations in a highly conserved region of the caffeic acid O-methyltransferase (COMT) of sugarcane. Capillary electrophoresis (CE) was validated by pyrosequencing as reliable and inexpensive high throughput method for identification and quantitative characterization of TALEN mediated mutations. Targeted COMT mutations were identified by CE in up to 74 % of the lines. In different events 8–99 % of the wild type COMT were converted to mutant COMT as revealed by pyrosequencing. Mutation frequencies among mutant lines were positively correlated to lignin reduction. Events with a mutation frequency of 99 % displayed a 29–32 % reduction of the lignin content compared to non-transgenic controls along with significantly reduced S subunit content and elevated hemicellulose content. CE analysis displayed similar peak patterns between primary COMT mutants and their vegetative progenies suggesting that TALEN mediated mutations were faithfully transmitted to vegetative progenies. This is the first report on genome editing in sugarcane. The findings demonstrate that targeted mutagenesis can improve cell wall characteristics for production of lignocellulosic ethanol in crops with highly complex genomes.

Summary: Sugarcane is a prime bioethanol feedstock. Currently, sugarcane ethanol is produced through fermentation of the sucrose, which can easily be extracted from stem internodes. Processes for production of biofuels from the abundant lignocellulosic sugarcane residues will boost the ethanol output from sugarcane per land area. However, unlocking the vast amount of chemical energy stored in plant cell walls remains expensive primarily because of the intrinsic recalcitrance of lignocellulosic biomass. We report here the successful reduction in lignification in sugarcane by RNA interference, despite the complex and highly polyploid genome of this interspecific hybrid. Down‐regulation of the sugarcane caffeic acid O‐methyltransferase (COMT) gene by 67% to 97% reduced the lignin content by 3.9% to 13.7%, respectively. The syringyl/guaiacyl ratio in the lignin was reduced from 1.47 in the wild type to values ranging between 1.27 and 0.79. The yields of directly fermentable glucose from lignocellulosic biomass increased up to 29% without pretreatment. After dilute acid pretreatment, the fermentable glucose yield increased up to 34%. These observations demonstrate that a moderate reduction in lignin (3.9% to 8.4%) can reduce the recalcitrance of sugarcane biomass without compromising plant performance under controlled environmental conditions.

Summary: Sugarcane is the world's most efficient feedstock for commercial production of bioethanol due to its superior biomass production and accumulation of sucrose in stems. Integrating first‐ and second‐generation ethanol conversion processes will enhance the biofuel yield per unit area by utilizing both sucrose and cell wall‐bound sugars for fermentation. RNAi suppression of the lignin biosynthetic gene caffeic acid O‐methyltransferase (COMT) has been demonstrated to improve bioethanol production from lignocellulosic biomass. Genome editing has been used in a number of crops for creation of loss of function phenotypes but is very challenging in sugarcane due to its highly polyploid genome. In this study, a conserved region of COMT was targeted with a single‐transcription activator‐like effector nuclease (TALEN) pair for multi‐allelic mutagenesis to modify lignin biosynthesis in sugarcane. Field‐grown TALEN‐mediated COMT mutants showed up to 19.7% lignin reduction and significantly decreased syringyl to guaiacyl (S/G) ratio resulting in an up to 43.8% improved saccharification efficiency. Biomass production of COMT mutant lines with superior saccharification efficiency did not differ significantly from the original cultivar under replicated field conditions. Sanger sequencing of cloned COMT amplicons (1351–1657 bp) revealed co‐editing of 107 of the 109 unique COMT copies/alleles in vegetative progeny of line CB6 using a single TALEN pair. Line CB6 combined altered cell wall composition and drastically improved saccharification efficiency with good agronomic performance. These findings confirm the feasibility of co‐mutagenesis of a very large number of target alleles/copies for improvement in crops with complex genomes.

Barley is a major cereal crop with a wide ecological range, and its lignocellulosic residues have the potential to be used as a feedstock for various purposes, including biofuel production. Lignocellulosic biomass is an abundant renewable source of carbon energy. However, its heterogeneous properties and intrinsic recalcitrance caused by cell wall lignification have lowered the biorefinery efficiency. The reduced lignin content and/or altered lignin structure have been desirable traits for lignocellulosic feedstock. We report the reduction of lignin content in barley by CRISPR/Cas9‐mediated mutagenesis of caffeic acid O‐methyltransferase 1 (HvCOMT1), the lignin biosynthetic gene responsible for lignin syringyl unit formation. The transgene‐free, homozygous HvCOMT1 mutant was generated and analyzed for its cell wall composition, saccharification efficiency, and bioethanol production performance. The mutant had 14% lower total lignin content and 34% higher fermentable glucose recovery rate, compared to the wild‐type (WT). The bioethanol concentration and yield from the hydrolysates of the mutant biomass were 14.3 g/L and 0.46 g/g total sugar, respectively. This result was 34% and 12% higher than those obtained from WT (10.7 g/L and 0.41 g/g total sugar). Under controlled environmental conditions, the overall growth performance of the HvCOMT1 mutant was similar to WT, with no distinct morphological variations between them. The HvCOMT1 mutant barley could offer improved quality lignocellulosic feedstock for efficient lignocellulosic biofuel production.