Development of an in vitro regeneration system from immature inflorescences and CRISPR/Cas9-mediated gene editing in sudangrass

Abstract

Background Sudangrass (Sorghum sudanense) is a major biomass producer for livestock feed and biofuel in many countries. It has a wide range of adaptations for growing on marginal lands under biotic and abiotic stresses. The immature inflorescence is an explant with high embryogenic competence and is frequently used to regenerate different sorghum cultivars. Caffeic acid O-methyl transferase (COMT) is a key enzyme in the lignin biosynthesis pathway, which limits ruminant digestion of forage cell walls and is a crucial barrier in the conversion of plant biomass to bioethanol. Genome editing by CRISPR/Cas9-mediated mutagenesis without a transgenic footprint will accelerate the improvement and facilitate regulatory approval and commercialization of biotech crops. Methods and results We report the overcome of the recalcitrance in sudangrass transformation and regeneration in order to use genome editing technique. Hence, an efficient regeneration system has been established to induce somatic embryogenesis from the immature inflorescence of two sudangrass cultivars on four MS-based media supplemented with different components. Our results indicate an interaction between genotype and medium composition. The combination of Giza-1 cultivar and M4 medium produces the maximum frequency of embryogenic calli of 80% and subsequent regeneration efficiency of 22.6%. Precise mutagenesis of the COMT gene is executed using the CRISPR/Cas9 system with the potential to reduce lignin content and enhance forage and biomass quality in sudangrass. Conclusion A reliable regeneration and transformation system has been established for sudangrass using immature inflorescence, and the CRISPR/Cas9 system has demonstrated a promising technology for genome editing. The outcomes of this research will pave the road for further improvement of various sorghum genotypes to meet the global demand for food, feed, and biofuels, achieving sustainable development goals (SDGs).

Advances in Molecular Breeding of Forage Crops: Technologies, Applications and Prospects

“…Furthermore, an in vitro regeneration protocol for sudangrass was developed in conjunction with the use of CRISPR/Cas9 to precisely edit the Caffeic acid O-methyltransferase (COMT) gene, which is crucial in lignin synthesis. This affects the digestibility of forage and the efficiency of bioethanol biomass conversion [56]. The successful modification of the COMT gene represents a significant advancement in improving the quality of forage and biomass in sudangrass, offering considerable benefits for agricultural biotechnology and the sustainability of bioenergy production.…”