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 (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. Cassava friable embryogenic calli (FECs) were transformed through the binary vector pCAMBIA1304 harboring GUS- and GFP-fused genes driven by the CaMV35S promoter. The transformation efficiency was increased in the conditions of Agrobacterium strain cell infection density (OD₆₀₀ = 0.65), 250 µM acetosyringone induction, and agro-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 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.

Keywords: CRISPR/Cas9; SC8; cassava; efficient transformation; friable embryogenic calli; homozygous.

Figure 1

Stable expression of GUS in FECs (A) and GFP in cotyledons (B) under different Agrobacterium cell densities. (A) GUS staining of FECs. FECs were taken from the last time on GD medium plates. After GUS staining, pictures were taken under an ultradeep field microscope. (B) Cotyledons on MSN medium under white light and fluorescence. Red arrows point to somatic embryos with green fluorescence.

Figure 2

Stable expression of GUS in FECs (A) and GFP in cotyledons (B) under different concentrations of acetosyringone. (A) GUS staining of FECs. FECs were taken from the last time on GD medium plates. After GUS staining, pictures were taken under an ultradeep field microscope. (B) Cotyledons on MSN medium under white light and fluorescence. Red arrows point to somatic embryos with green fluorescence.

Figure 3

Stable expression of GUS in FECs (A) and GFP in cotyledons (B) under different cocultivation days. (A) GUS staining of FECs. FECs were taken from the last time on GD medium plates. After GUS staining, pictures were taken under an ultradeep field microscope. (B) Cotyledons on MSN medium under white light and fluorescence. Red arrows point to somatic embryos with green fluorescence.

Figure 4

Stable expression of GUS in FECs (A) and GFP in cotyledons (B) under FECs with dry or wet treatment. (A) GUS staining of FECs. FECs were taken from the last time on GD medium plates. After GUS staining, pictures were taken under an ultradeep field microscope. (B) Cotyledons on MSN medium under white light and fluorescence. Red arrows point to somatic embryos with green fluorescence.

Figure 5

Agrobacterium-mediated genetic transformation of cassava SC8 FECs. (A) In vitro shoot culture; (B) axillary bud; (C) primary SEs on CIM medium; (D) SEs on CIM medium; (E) friable embryogenic calli on GD medium; (F) Agrobacterium-infected FECs proliferating on GD medium; (G) developing cotyledons on MSN medium; (H) cotyledons on CEM medium; (I) developing shoots on COM medium; (J) transgenic plantlets on MS medium; (K) rooting assay of transgenic plants on MS + 50 mg/L carbenicillin + 10 mg/L hygromycin; (L) transgenic plants in the soil.

Figure 6

Assessments of the co-expression of GUS and GFP in transgenic tissues.

Figure 7

Target site of the MePDS gene and the T-DNA of the pYAO:hSpCas9-gRNA binary vector. (A) Structural organization of the MePDS gene. Exons and introns are shown as boxes and lines, respectively. (B) Schematic of the CRISPR/Cas9 binary vector pYAO:hSpCas9-MePDS-gRNA for MePDS gene editing through Agrobacterium-mediated transformation.

Figure 8

Phenotypes of the regenerated cotyledons after MePDS gene editing. (A) Regenerated cotyledons on CEM medium. (B) Phenotypic diversity of CRISPR/Cas9-induced MePDS mutations in cassava cotyledons.

Figure 9

MePDS editing of transgenic albino SC8 cassava plants and Sanger sequences. (A) Albino plants and green plants of the MePDS-edited transgenic albino SC8 cassava. (B) Sanger sequences of the target sites in the MePDS-edited transgenic albino SC8 cassava plants.