Fig. 4: Genome editing of MsPALM1, and generating transgene-free and stably inherited palm1-type progenies.

a A guide sequence for MsPALM1. Dark blue box represents exon. PAM is shown in red. The BstUІ site is underlined and shown in light blue. b Leaf morphologies of three representative T0 plants. Scale bar, 1 cm. c Results of PCR-RE analyses for identifying mutants among T0 plants. In the gel, wt and wt-dg lanes contain DNA samples from wild-type plants without and with digestion by the BstUІ restriction endonuclease, respectively. Red arrowheads indicate bands used to identify mutations. Notably, paT0-19 (highlighted with a red rectangle) yields dim digested bands (indicated by white arrows), although it develops palmate-like pentafoliate leaves (b). d Genotyping of the corresponding mutants in b confirmed the presence of mutations at the target sites (light blue). The PAM regions are shown in black and lowercase. Nucleotide deletions, insertions, or substitutions are shown in red, with details in the right panel. e Three representative T1 plants with two showing anticipated palm1-type leaf morphologies like their parents. Scale bar, 1 cm. f, g Results of PCR-RE analyses (f) and sequencing analyses (g) confirmed the parental MsPALM1 mutations in corresponding T1 progenies in e. h Outcome of tests for transgene-free mutants in 20 corresponding T1 progenies in f. Lanes without bands (indicated by red arrowheads) identify transgene-free mutants. Lanes labeled wt, paT0-19 and paT0-46 show PCR fragments amplified from a WT plant, and two T0 mutants (paT0-19 and paT0-46), respectively. Source data underlying Fig. 4c, f, h are provided as a Source Data file.