A Transformation and Genome Editing System for Cassava Cultivar SC8

Abstract

Cassava starch is a widely used raw material for industrial production. South Chinese cassava cultivar 8 (<i>Manihot esculenta</i> Crantz cv. SC8) is one of the main locally planted cultivars. In this study, an efficient transformation system for cassava SC8 mediated with <i>Agrobacterium</i> strain LBA4404 was presented for the first time. Cassava friable embryogenic calli (FECs) were transformed through the binary vector pCAMBIA1304 harboring <i>GUS-</i> and <i>GFP-</i>fused genes driven by the <i>CaMV35S</i> promoter. The transformation efficiency was increased in the conditions of <i>Agrobacterium</i> strain cell infection density (OD₆₀₀ = 0.65), 250 µM acetosyringone induction, and <i>agro</i>-cultivation with wet FECs for 3 days in dark. Based on the optimized transformation protocol, approximately 120-140 independent transgenic lines per mL settled cell volume (SCV) of FECs were created by gene transformation in approximately 5 months, and 45.83% homozygous mono-allelic mutations of the <i>MePDS</i> gene with a <i>YAO</i> promoter-driven CRISPR/Cas9 system were generated. This study will open a more functional avenue for the genetic improvement of cassava SC8.