Establishment of an efficient transformation and CRISPR/Cas9-mediated gene editing system in Chinese local planting cassava (Manihot esculenta Crantz) cultivar SC8

AbstractCassava starch is a widely used raw material for industrial production. South Chinese cassava cultivar 8 (Manihot esculenta Crantz cv. SC8) is one of the main locally planted cultivars. In this study, an efficient transformation system for cassava SC8 mediated with Agrobacterium strain LBA4404 was presented for the first time, in which the factors of Agrobacterium strain cell infection (density OD600 = 0.65), 250 µM acetosyringone induction, and agro-cultivation with wet friable embryogenic callus (FEC) for 3 days in dark conditions were found to increase the transformation efficiency through the binary vector pCAMBIA1304 harboring GUS- and GFP-fused genes driven by the CaMV35S promoter. Based on the optimized transformation protocol, approximately 120-140 independent transgenic lines per mL settled FEC cell volume (SCV) by gene transformation in approximately five months, and 45.83% homozygous mono-allelic mutations of the MePDS gene with a YAO promoter-driven CRISPR/Cas9 system were generated. This study will open a more functional avenue for the genetic improvement of cassava SC8.