a, Wus2- and STM-induced growths on 21–31% of treated seedlings. Few differences in frequency were observed among the different promoters used to express STM, probably because they all drive high expression in N. benthamiana. b–d, Approximately 12 d after initiating Fast-TrACC, callus-like growths formed, varying from one (b) to many (c,d) per seedling. e,f, Over the next 10–14 d, the growths expanded in size and remained in an undifferentiated state or began to differentiate. g,h, Shoot-like structures then formed, which could be excised and moved to rooting medium. The growths depicted are representative of those observed in three independent experiments.

a–c, Some transgenic plants displayed developmental abnormalities. Shown here are plants derived from treating seedlings with Wus2 and STM. Phenotypes include large numbers of leaves (a), excess branching (b), and misshapen leaves (c). The three plants depicted are representative of plants derived from the 36 growths obtained in the experiment described in Supplementary Fig. 1.

a, To induce de novo meristems in tomato, three combinations of DRs (nos:Wus2 & 35S:STM, nos:Wus2 & CmYLCV:STM, and nos:Wus2 & 35S:ipt) were selected because they effectively induced meristems in N. benthamiana. (Supplementary Table 1). For both combinations of Wus2 & STM, no shoot-like growths formed. b, In contrast, Wus2 & ipt promoted shoot-like growths that ultimately formed fully rooted plants. c, When a luciferase reporter was delivered with the DRs, meristems were induced (black arrow). d, These meristems were positive for luciferase activity (red box and enlarged image shown as inset).

a, Five gene-edited plants were produced by treating seedlings with Wus2 & ipt, Wus2 & STM or Wus2 alone. The type and amount of editing were determined by Sanger sequencing and TIDE analysis (see Methods). The predicted editing percentage for each PDS gene is shown. b, The types of edits predicted by TIDE are shown as percentages of total editing. Any percentage below 5% was deemed below the limit of detection by TIDE. The first number in the column is indicative of the percentage of that edit in PDS1; the second number is indicative of editing in PDS2. Because of a deletion in 4-2, the sequence could not be properly aligned, and thus no percentages are given for PDS2. See the Supplementary Data Set for DNA sequences of all mutants recovered.

a, An example of the seedling phenotypes derived from flowers F4 and F5 of plant 1-7 (see also Supplementary Table 4). The fully green seedlings are healthy; the fully white seedlings do not develop after germination; the chimeric plants are stunted. b, Chimeric seedlings retain the transgene that carries the sgRNA expression cassette. DNA from seedlings was PCR-amplified using primers that specifically amplify the transgene. Chimerism s probably owing to continued mutagenesis at PDS1. The image in panel a is representative of experiments performed once with seeds derived from flowers F4 and F6. The experiment in panel b was performed once.

a–d, Many of the phenotypes suggest improper meristem patterning, probably owing to persistent DR expression. b, Some shoots and leaf tissues are white, suggesting biallelic mutations in both PDS homologs. c, Other tissues show a mixture of both green and white phenotypes. d, When floral tissues form on morphologically abnormal tissues, they are abnormal and typically infertile. e, Some shoots appear phenotypically normal. The shoot images are representative of the nine white shoots and 30 distorted shoots obtained in the experiment described in Fig. 5c.

The left image is an example of a grape plant 40 d after inoculation with five A. tumefaciens strains, each carrying a T-DNA vector that expresses a single DR (pMM230-234). Newly formed shoots were generated at inoculation sites. The image on the right shows shoots that were removed from the plant on the left, exposed to luciferin, and imaged for bioluminescence. Bioluminescent tissues indicate newly formed, transgenic shoots that express the luciferase reporter. Images are from a single experiment performed in grape and represent one of six inoculated plants.

a, The potato plant pMKV059-6 displays abnormal shoot formation approximately 100 d after inoculation with an A. tumefaciens strain carrying a T-DNA with ipt and luciferase expression cassettes. b, Luciferase expression is observed in tissues and shoots of plant pMKV059-6. c, Fully transgenic shoots (white arrowheads) are more easily seen after trimming away several wild-type shoots. d, Potato plant pMKV057-1 displays abnormal shooting approximately 100 d after inoculation with an A. tumefaciens strain carrying a T-DNA with ipt, Wus2 and luciferase expression cassettes. e, Luciferase expression is observed in tissues and shoots of plant pMKV057-1. f, Plant pMKV057-1 was trimmed, leaving some transgenic tissue. g, Fully transgenic shoots could be isolated. The images are representative of two experiments performed in potato.

Plants were treated with constructs that included a single vector containing Wus2 and STM (Wus2/STM), a single vector containing Wus2 and ipt (Wus2/ipt), or a combination of co-inoculated vectors each containing a single DR (All combination, see Fig. 4 legend). Derived shoots were given individual designators to facilitate sample tracking. Purified genomic DNA was amplified for the PDS2 locus. Amplicons were pooled and submitted for next-generation sequencing. ‘Sequences analyzed’ denotes the observed number of sequences that contained the expected forward and reverse barcodes. ‘Insertions’ denotes the number of sequences observed to have non-specific DNA insertions at the sgRNA target site. ‘Deletions’ denotes the number of sequences observed to have targeted mutations at the sgRNA target site. ‘Indel frequency’ denotes the total number of sequences that were observed to have targeted modifications. ‘Observed mutations’ denotes mutations observed at a frequency >30% of the total. ‘Seed produced’ denotes those sampled shoots that produced seed. With the exception of plant 1-1-5, sequences of the resulting mutations are shown in Fig. 5b. Below the table are the spectra of mutations recovered for plant 1-1-5.

Observed mutations in the white parental tissues and the progeny of the white seed pod that is shown Fig. 6. Genomic DNA was extracted from parental tissue and seedlings and submitted for Sanger sequencing. Sequences were assessed for mutations by TIDE sequence trace analysis (see Methods). All seedlings were white, as shown in the photo. See the Supplementary Data Set for the DNA sequences of all mutations recovered.

Genomic DNA was extracted from parental white (ParW) and parental green (ParG) tissues of a plant demonstrating targeted editing (Fig. 6), as well as from ten seedlings derived from the white flower (S1-S10; Extended Data Fig. 10). Genomic DNA was also extracted from plants that did not receive the vector (Neg Ctrl 1 and 2) and from leaf tissue infiltrated with the target vector (Pos Ctrl). DNA was amplified using primers specific to the U6 promoter present on the T-DNA (expected 448 bp). New England Biolabs 2-Log DNA Ladder. The experiment was performed once.

Observed mutations in the green tissues and the progeny from the green seed pod shown in Fig. 6. Genomic DNA was extracted from parental tissue and seedlings and submitted for Sanger sequencing. Sequences were assessed for mutations by TIDE sequence trace analysis (see Methods). The photo shows wild-type and pds seedling phenotypes demonstrating a 3:1 segregation. Note: the 48 bp deletion is an in-frame deletion, which appears to retain PDS function.