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Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum).

ABSTRACT:

Key message

We have established a DNA-free genome editing method via ribonucleoprotein-based CRISPR/Cas9 in cultivated tomato and obtained mutant plants regenerated from transfected protoplasts with a high mutation rate. The application of genome editing as a research and breeding method has provided many possibilities to improve traits in many crops in recent years. In cultivated tomato (Solanum lycopersicum), so far only stable Agrobacterium-mediated transformation carrying CRISPR/Cas9 reagents has been established. Shoot regeneration from transfected protoplasts is the major bottleneck in the application of DNA-free genome editing via ribonucleoprotein-based CRISPR/Cas9 method in cultivated tomato. In this study, we report the implementation of a transgene-free breeding method for cultivated tomato by CRISPR/Cas9 technology, including the optimization of protoplast isolation and overcoming the obstacle in shoot regeneration from transfected protoplasts. We have identified that the shoot regeneration medium containing 0.1 mg/L IAA and 0.75 mg/L zeatin was the best hormone combination with a regeneration rate of up to 21.3%. We have successfully obtained regenerated plants with a high mutation rate four months after protoplast isolation and transfection. Out of 110 regenerated M₀ plants obtained, 35 (31.8%) were mutated targeting both SP and SP5G genes simultaneously and the editing efficiency was up to 60% in at least one allele in either SP or SP5G genes.

SUBMITTER: Liu Y

PROVIDER: S-EPMC9395478 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Publications

Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum).

Liu Ying Y Andersson Mariette M Granell Antonio A Cardi Teodoro T Hofvander Per P Nicolia Alessandro A

Plant cell reports 20220630 9

<h4>Key message</h4>We have established a DNA-free genome editing method via ribonucleoprotein-based CRISPR/Cas9 in cultivated tomato and obtained mutant plants regenerated from transfected protoplasts with a high mutation rate. The application of genome editing as a research and breeding method has provided many possibilities to improve traits in many crops in recent years. In cultivated tomato (Solanum lycopersicum), so far only stable Agrobacterium-mediated transformation carrying CRISPR/Cas9 ...[more]

PMID: 35773498

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Project description:BackgroundCultivated tomato (Solanum lycopersicum L.) has narrow genetic diversity that makes it difficult to identify polymorphisms between elite germplasm. We explored array-based single feature polymorphism (SFP) discovery as a high-throughput approach for marker development in cultivated tomato.ResultsThree varieties, FL7600 (fresh-market), OH9242 (processing), and PI114490 (cherry) were used as a source of genomic DNA for hybridization to oligonucleotide arrays. Identification of SFPs was based on outlier detection using regression analysis of normalized hybridization data within a probe set for each gene. A subset of 189 putative SFPs was sequenced for validation. The rate of validation depended on the desired level of significance (alpha) used to define the confidence interval (CI), and ranged from 76% for polymorphisms identified at alpha <or= 10-6 to 60% for those identified at alpha <or= 10-2. Validation percentage reached a plateau between alpha <or= 10-4 and alpha <or= 10-7, but failure to identify known SFPs (Type II error) increased dramatically at alpha <or= 10-6. Trough sequence validation, we identified 279 SNPs and 27 InDels in 111 loci. Sixty loci contained >or= 2 SNPs per locus. We used a subset of validated SNPs for genetic diversity analysis of 92 tomato varieties and accessions. Pairwise estimation of theta (Fst) suggested significant differentiation between collections of fresh-market, processing, vintage, Latin American (landrace), and S. pimpinellifolium accessions. The fresh-market and processing groups displayed high genetic diversity relative to vintage and landrace groups. Furthermore, the patterns of SNP variation indicated that domestication and early breeding practices have led to progressive genetic bottlenecks while modern breeding practices have reintroduced genetic variation into the crop from wild species. Finally, we examined the ratio of non-synonymous (Ka) to synonymous substitutions (Ks) for 20 loci with multiple SNPs (>or= 4 per locus). Six of 20 loci showed ratios of Ka/Ks >or= 0.9.ConclusionArray-based SFP discovery was an efficient method to identify a large number of molecular markers for genetics and breeding in elite tomato germplasm. Patterns of sequence variation across five major tomato groups provided insight into to the effect of human selection on genetic variation.

Dataset Information

Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum).

Key message

Publications

Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum).

Similar Datasets

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