Genetic engineering of plant cell walls is limited for reducing lignocellulose recalcitrance, so mild and/or green-like pretreatment is still required for sequential enzymatic saccharification. Here, we report a method to reduce lignin content in sugarcane stalks using the CRISPR/Cas 9 technique. Three target sequences of SoLIM were designed and fused to pRGEB32. The cassette constructs were introduced into sugarcane calli cv. KK3 through Agrobacterium-mediated transformation. We produced one base substitution and one insertion line for the 1st target site; two insertions, one deletion, and one base substitution for the 2nd target site; and one base substitution and insertion for the 3rd target site. qRT-PCR analysis of SoLIM, SoPAL, SoC4H, and SoCAD showeded that downregulation of SoLIM by single nucleotide insertions or deletions reduced the expression of SoPAL, SoC4H, and SoCAD. Consequently, the edited lines contained 9.74 to 51.46% less lignin content compared to that in the wild-type plants. The syringyl/guaiacyl (S/G) ratio of the edited lines ranged between 0.23 and 0.49, while the wild-type was 0.22. The histochemical evaluation and scanning electron microscopy of the cell walls supported this observation. A low lignin content sugarcane will provide a better feedstock for second-generation bioethanol production. 

Lignin reduction in sugarcane by performing CRISPR/Cas9 site-direct mutation of SoLIM transcription factor.

Lignocellulosic biomass is a powerful approach to produce sustainable biofuels and the further achievement of the goal of biomass conversion into a second-generation clean energy that can cope with the depletion of fossil reserves and rising energy requirements. In the conversion process, a pretreatment is essential to overcome the recalcitrance of the lignocellulosic biomass; accelerate its disintegration into cellulose, hemicellulose, and lignin; and, in turn, obtain an optimal yield of fermentable sugars in the enzymatic hydrolysis. In addition to this, it should be industrially scalable and capable of enhancing fuel properties and feedstock processability. Here, steam explosion technology has stood out due to its results and advantages, such as wide applicability, high efficiency in the short term, or lack of contamination despite its conventionality. This gentle and fast pretreatment incorporates high temperature autohydrolysis and structural alteration by explosive decompression. The steam explosion method has been one of the most effectual, especially for the hydrolysis of cellulose from agricultural wastes due to the lower quantity of acetyl groups in the composition of hemicellulose. In this aspect, sugarcane bagasse is a promising feedstock for bioethanol production due to its high cellulosic content and elevated availability. The objective of this review has been to compile the latest information on steam explosion pretreatment, stages, equipment, variables involved, by-products generated, as well as the advantages and disadvantages of the technique. At the same time, its feasibility and viability using sugarcane bagasse as feedstock has been discussed. Finally, the effectiveness of the technique with different feedstocks has been evaluated. 

Utility Aspects of Sugarcane Bagasse as a Feedstock for Bioethanol Production: Leading Role of Steam Explosion as a Pretreatment Technique

Pretreatment of sugarcane postharvest leaves by γ-valerolactone/water/FeCl3 system for enhanced glucan and bioethanol production

Green alkaline fractionation of sugarcane bagasse at cold temperature improves digestibility and delignification without the washing processes and release of hazardous waste

Selective saccharification of crude glycerol pretreated sugarcane bagasse via fast pyrolysis: reaction kinetics and life cycle assessment.

Specific Lignin and Cellulose Depolymerization of Sugarcane Bagasse for Maximum Bioethanol Production Under Optimal Chemical Fertilizer Pretreatment with Hemicellulose Retention and Liquid Recycling

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Specific Lignin and Cellulose Depolymerization of Sugarcane Bagasse for Maximum Bioethanol Production Under Optimal Chemical Fertilizer Pretreatment with Hemicellulose Retention and Liquid Recycling