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Fig. 2 | Genome Biology

Fig. 2

From: High-frequency, precise modification of the tomato genome

Fig. 2

Gene targeting upstream of the ANT1 gene. a Top: illustration of the GT event. Upon cleavage by the nuclease and homologous recombination with the replicon, the donor cassette is inserted upstream of ANT1. Bottom: structure of the transfer DNA (T-DNA) vector, pTC144, which produces DNA replicons. LB left T-DNA border, LIR BeYDV large intergenic region, 35S cauliflower mosaic virus 35S promoter, tHSP Arabidopsis thaliana heat shock protein 18.2 terminator, SIR BeYDV short intergenic region, REP coding sequence for Rep/RepA, RB right T-DNA border. Additional components of the donor include: NosP Agrobacterium tumefaciens nopaline synthase promoter, NPTII neomycin phosphotransferase gene for kanamycin resistance, t35S CaMV 35S terminator. For expression of CRISPR/Cas9 reagents, the TALEN coding sequence was replaced with a plant codon-optimized Cas9 gene and the gRNAs were expressed from the AtU6 promoter (not shown). bh Regeneration of tomato plants with targeted insertions. b Cotyledons of tomato cv. MicroTom after inoculation with Agrobacterium. c A recombinant explant 3 weeks after inoculation. Part of the developing callus accumulates anthocyanins due to the targeted promoter insertion and ANT1 overexpression. d Explants 5 weeks after inoculation. Small shoots begin to develop on the purple callus. e Multiple shoots growing from the purple callus 10–12 weeks after inoculation. f Plantlets develop roots 12–14 weeks after inoculation. g Plantlet transplanted to soil. h Dark purple coloration in flowers, fruit and foliage results from targeted promoter insertion. Flowers, fruit and mature plants are compared between wild type (WT) plants and those that have undergone GT. Scale bars = 1 cm

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