Fig. 3: CRISPR construct design, generation and validation of ‘Sukali Ndiizi’ gene-edited events.

Illustration of MusaPUB22 (a) and MusaPUB23 (b) gene structure. The positions of gRNA binding sites in target regions in MusaPUB22/23 genes are shown with black arrows (▼) indicating canonical cut sites for both guides. Orange boxes denote coding DNA sequences (CDS), while Blue arrows indicate the U-box. Red letters correspond to the gRNA binding sites in the targeted regions, while black letters (G/C) denote the single-nucleotide polymorphism at position 751 (position 1 at 5’ of gRNA1) in MusaPUB23. Purple letters indicate the protospacer-adjacent motif (PAM) sequence. The primers used for amplifying and sequencing the MusaPUB gene are shown with their positions relative to the start codon position indicated in brackets. The region of coding sequence of the U-box is compressed in length in the diagram and is not to scale. c T-DNA region of binary plasmid construct pMDC32-Cas9-MusaPUB used for editing of banana. LB, Left border; RB, Right border, hpt, hygromycin phosphotransferase gene; 35S P, CaMV35S promoter, OsU6 p, Oryza sativa U6 promoter; 2 × 35S P, double CaMV35S promoter. d Agrobacterium-mediated delivery of T-DNA of the CRISPR/Cas9 plasmid and regeneration of edited events (1) Agro-infected embryogenic cell suspension on embryo development medium; (2) Embryos on embryo maturation medium; (3-4) Germination of embryos in selective medium containing hygromycin; (5) Well-rooted plantlets in proliferation medium; (6) Potted plants of gene-edited events in the greenhouse. The scale bar represents 1 cm for panels 1–5 and 10 cm for panel 5.