CRISPR keeps going “wild”: a new protocol for DNA-free genome editing of tetraploid wild tomatoes

Global food production in the face of climate change requires new strategies to rapidly create high-yield and climate-resilient crops. Conventional breeding and domestication of crops from their wild relatives improve agricultural productivity, but the process is slow and often results in crops adapted to a particular environment due to their decreased fitness and genetic diversity (Voss-Fels et al., 2018; Hickey et al., 2019). Polyploidization a condition by which a diploid plant acquires additional complete sets of chromosomes improves genome buffering, vigorousness, and environmental robustness in higher plants (Van de Peer et al., 2017). Harnessing the benefits of polyploidization is desired for de novo domestication of wild species by targeted editing of their genomes, thereby accelerating designer crop development for a range of climatic and edaphic conditions (Curtin et al., 2021; Yu et al., 2021). However, establishing a practical strategy to generate gene-edited polyploid versions of wild species remains challenging.

Targeted mutagenesis by clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) is powerful for de novo domestication of crops (Curtin et al., 2021). The Agrobacterium-mediated delivery of CRISPR reagents has been the main approach for editing plants. However, transgene residuals in these edited plants affect genetic analysis, pose off-target risks, and cause legislative concerns (He et al., 2021). While transgenes in the edited plants can be removed by crossing or genetic segregation, such approaches are not feasible for transgene removal in plants that are self-incompatible or vegetatively propagated or have long growth periods. Delivery of CRISPR/Cas ribonucleoproteins (RNPs) by particle bombardment or transient transfection of protoplasts is an alternative strategy to regenerate transgene-free edited plants with homozygosity. While particle bombardment of RNPs exhibits lower efficiency, protoplast regeneration systems have higher efficiency in DNA-free genome editing (Zhang et al., 2021). However, a practical platform combining protocols for crop polyploidization and the protoplast regeneration of DNA-free edited plants upon delivery of CRISPR/Cas reagent has not been reliably established for wild crop species.

In a recent issue of Plant Physiology, Hsu et al. (2021) show a proof-of-concept strategy using protoplast regeneration protocol to generate CRISPR/Cas9-edited allotetraploid wild tomato (Solanum peruvianum), an important genomic resource for tomato research and introgression breeding. The ratio of ploidy levels in cells depends on young/old organs and growth conditions in tomatoes (Van de Peer et al., 2017). The authors determined that the stem of diploid S. peruvianum possessed a higher proportion of tetraploid cells and optimized a protocol for regenerating tetraploid S. peruvianum plants from stem protoplasts. They trialed this protocol for delivering plasmid-based CRISPR/Cas9 reagents or pre-assembled CRISPR RNP complexes targeting more than 100 genes including those involved in small RNA biogenesis and disease resistance. Using target gene genotyping and karyotype analysis of calli and the regenerated plants, the authors demonstrated that gene editing using RNP produced heritable mutations in target genes in both diploid and tetraploid plants. Whole-genome sequencing of a subset of gene-edited plants further showed no detectable chromosomal changes or unintended genome editing sites, confirming the stability of genome structures in regenerants.

The authors next evaluated phenotypic and molecular behaviors of regenerated diploid and triploid mutant plants. Mutations in SUPPRESSOR OF GENE SILENCING 3 or RNA-DEPENDENT RNA POLYMERASE 6, which are involved in microRNA biogenesis, resulted in the characteristic microRNA mutant phenotypes: wiry leaf phenotypes, abnormal pollens, and accumulation of AUXIN RESPONSE FACTOR3 transcripts. Mutant progenies exhibited hypersensitivity to tomato yellow curl leaf virus infection. Grafting spsg3 triploid mutant scion onto wild-type stock improved the fertility of mutant plants by producing seeds with mutated alleles (Figure 1). The results also show the utility of a protoplast regeneration protocol for obtaining gene-edited homozygous alleles in tetraploid wild tomatoes without self-fertilization, reducing the time required for regeneration via tissue culture.

Figure 1.

A targeted mutagenesis protocol combining tomato polyploidization and protoplast regeneration enabling the use of CRISPR/Cas9 for S. peruvianum domestication and tomato breeding. A, Tetraploid mutants edited on SpSGS3 lead to changes in developmental (wiry leaf shape) and disease resistance (hypersensitive to TYLCV infection) phenotypes. B, The sterility of the spsgs3 null mutant was partially rescued and fruits were obtained by grafting to wild-type stock and pollination with wild-type pollen. (Adapted from Hsu et al. (2021), Figures 6A and 7F.)

In sum, the authors demonstrated that the optimized protoplast regeneration protocol enabled efficient isolation of gene-edited and transgene-free tetraploid wild tomato plants (Figure 1). Given the increasing number of domesticated tomatoes that have lost disease resistance, targeted genome editing of polyploid wild tomatoes reported by Hsu et al. (2021) provides an efficient approach to de novo domestication and re-domestication of tomatoes and thus to expanding tomato diversity and increasing the sustainability of agriculture.

Conflict of interest statement. None declared.