Many dicot species, including some vital potato relatives being used to accelerate breeding and genetics efforts, remain recalcitrant to standard Agrobacterium tumefaciens-based transformation. Hairy root transformation using Agrobacterium rhizogenes (A. rhizogenes) provides an accelerated approach to generating transgenic material but has been limited to analysis of hairy root clones. In this study, strains of A. rhizogenes were tested in the wild diploid potato relative Solanum chacoense, which is recalcitrant to infection by Agrobacterium tumefaciens. One strain of A. rhizogenes MSU440 emerged as being capable of delivering a T-DNA carrying the GUS marker and generating transgenic hairy root clones capable of GUS expression and regeneration to whole plants. CRISPR/Cas9 reagents targeting the potato PHYTOENE DESATURASE (StPDS) gene were expressed in hairy root clones and regenerated. We found that 64%-98% of transgenic hairy root clones expressing CRISPR/Cas9 reagents carried targeted mutations, while only 14%-30% of mutations were chimeric. The mutations were maintained in regenerated lines as stable mutations at rates averaging at 38% and were capable of germ-line transmission to progeny. This novel approach broadens the numbers of genotypes amenable to Agrobacterium-mediated transformation while reducing chimerism in primary events and accelerating the generation of edited materials.
Figure 2: Targeted mutations in hairy root clones of potato using CRISPR/Cas9. (a) Schematic of potato PHYTOENE DESATURASE (St PDS) gene with guide RNA (gPDS) target sites. Hind III restriction enzyme site exists within gPDSa target site (underlined) used for detecting targeted mutations. Blue arrows indicate primers used for PCR‐based targeted mutation detection assays (see Methods). PAM sequences are in red. (b) PCR‐based targeted mutation detection assays for individual hairy root clones expressing CRISPR/Cas9 reagents. Paired‐guide RNAs (gRNAs) were used by co‐expressing gPDSa with gPDSb (gPDSa + b) within a single construct. gRNAs were expressed using individual U6 and 7SL RNA polymerase III (Pol III) promoters (U6.7SL), or a CaMV35S (35S) promoter using the CRISPR‐associated RNA endoribonuclease Csy4 (Csy4) system (Tsai et al. , 2014). Cas9, Cas9 nickase (D10A) and Csy4 were expressed using a 35S promoter, while the Trex2 exonuclease (TREX2) was driven by the FMV promoter (Cermák et al. , 2017). A 1037‐bp amplicon is expected with 417‐bp and 620‐bp products from Hind III digestion of wild‐type DNA to detect mutations within the gPDSa target site. gPDSa + b Csy4 + D10A amplicons were not digested. Arrows indicate bands containing targeted mutations used for cloning mutant alleles. (c) Sequencing of mutant alleles from individual hairy root clones expressing CRISPR/Cas9 reagents. Deletions (−) are shown with values in red. Blue arrows indicate primers used for sequencing. Black arrows indicate paired gRNAs. Right shows sequencing chromatographs of mutations. Hairy root clone numbers 2, 2, 8 and 10 are shown for gPDSa + b U6.7SL, Csy4, Csy4 + TREX2 and Csy4 + D10A, respectively (Table S3).