Fig. 3

Heat treatment improves editing efficiency using LbCas12a in grapevine. A Schematic diagram of heat treatment. B Mutation frequencies at the two targets in TMT1 gene under control (Ctrl, 26℃) and heat treatment (HT, 34℃) conditions. ** indicates a highly significant (P < 0.01) difference determined by Student’s t-test. C Regeneration of grapevine plants. Plant regeneration from 41B cells after HT was shown as example. Embryos developed from transformed grape cells were detected with EGFP fluorescence. Whole plants were recovered from germinated embryos on regeneration medium. D Amplification of target fragments from transgenic grapevine plants. The PCR results obtained with HT plants were shown. Lanes 1–29 represent samples from 29 independent grapevine plants. The desired bands (referred to as big fragment, BF) are indicated in red asterisks, and smaller fragments (SF, indicated in blue asterisks) were also observed in lane 1 and 2. M, DNA marker. E Sanger sequencing results of the target fragments in Plant #1 and #25. Representative chromatograms of mutated sequences are shown. The target sequences are indicated in red boxes, and the PAMs are underlined. The deletion positions are denoted by black arrows, and mutation types are shown behind the arrows. The numbers of amplicons identified from 6 analyzed clones are shown on the right. The missing sequence identified in plant #25 is indicated in red, in which the target sequence is indicated in bold. F Overview of targeted mutagenesis under Ctrl and HT conditions. Bia, biallelic mutants; He, heterozygotes; Chi, chimeras