TALEN mediated targeted mutagenesis of the caffeic acid O-methyltransferase in highly polyploid sugarcane improves cell wall composition for production of bioethanol

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-016-0499-y) contains supplementary material, which is available to authorized users.

The coding loci of evolution and domestication: current knowledge and implications for bio-inspired genome editing

“…In the case of polyploid organisms, the knockout of several paralogous genes is sometimes necessary to obtain the phenotype of interest. In sugarcane, a highly polyploid plant, TALEN-mediated targeting of a highly conserved region of the multiple caffeic acid O-methyltransferase (COMT) enzyme genes present in the genome was successfully used to reduce lignin content (Jung and Altpeter, 2016).…”

Development of transgenic crops based on photo‐biotechnology

“…Applications of this new GE tool to plant biotechnology are just beginning (Belhaj et al . ; Bortesi & Fischer, ; Jung & Altpeter, ).…”

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The Future of Biofuels in an Electrifying Global Transportation Sector: Imperative, Prospects and Challenges

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