Fig. 2: Generation of the tomato IAA9 mutants with small indels mutations by using CRISPR TiD.

a, b The mutations were detected by Cel-1 assay (a) and PCR-RFLP (b). WT (VC); vector control, MT; Micro-Tom, AC; Ailsa Craig, a, #5 and 11; the CRISPR TiD transgenic Micro-Tom calli (T0 generation). b Upper, #6, 8, and A; the CRISPR TiD transgenic Micro-Tom shoots (T0 generation), #A-1 and A-2; the next generations (T1) of #A. lower, #4, 7, and 8; the CRISPR TiD transgenic Ailsa Craig shoots (T0 generation), −; without enzymes, +; with Cel-1 nuclease (upper) or AccI (lower), red arrows; mutation bands. c Plant phenotypes of SlIAA9-disrupted tomato plants (Ailsa Craig) generated by CRISPR TiD. Bars = 2 cm (right). d SlIAA9 knockout tomato fruits (Ailsa Craig) with parthenocarpy phenotypes (right). Bars = 1 cm. e Mutation sequences in the IAA9 gene of Micro-Tom (MT) and Ailsa Craig (AC) mutant shoots (T0 generation) transformed with CRISPR TiD analyzed by the Sanger method. WT; wild-type sequences. gRNA target sequences are indicated in green boxes and PAM is indicated in pink boxes. The sequence frequencies in the cloned PCR products were indicated in the right of the sequence. f Mutation sequences in the IAA9 gene of Ailsa Craig (AC) shoots (T0 generation) transformed with CRISPR TiD analyzed by amplicon deep sequencing using Mi-seq (illumina). WT; wild-type sequences. gRNA target sequences are indicated in green boxes and PAM is indicated in pink boxes. The sequence frequencies in the read counts in the deep sequencing are indicated to the right of the sequence. All results in the electrophoresis and sequence analysis are typical examples from the representative mutant plants generated by TiD.