Challenges in a CRISPR/Cas9-based approach to target the MIK2 gene in chicory (Cichorium intybus)

Sporophytic self-incompatibility (SSI) is a genetically controlled system in which pollen–stigma recognition prevents self-fertilization, thereby promoting genetic diversity and representing a key reproductive barrier in many flowering plant families. In Cichorium intybus, SSI enforces obligate outcrossing and constitutes a major constraint for chicory breeding programs, as it prevents self-fertilization and the generation of homozygous inbred lines necessary for F1 hybrid seed production. MDIS1 INTERACTING RECEPTOR LIKE KINASE 2 (MIK2) has been proposed as a major candidate gene of the SSI system in Cichorium, based on the role of its Arabidopsis ortholog in pollen–stigma recognition signaling and on the structural similarity shared between MIK2 and SRK, the SSI female determinant in the Brassica genus. Previous studies have shown that MIK2 is under positive selection in C. intybus, displaying substantial sequence polymorphism across accessions. A feature biologically expected for a candidate S-locus gene, but one that simultaneously creates critical technical challenges for any functional approach targeting this locus. The present study investigated the feasibility of a CRISPR/Cas9-based approach to disrupt MIK2 in C. intybus, addressing two central challenges: the high sequence variability of the target locus and the need for a suitable strategy to deliver and recover edited material. The MIK2 genomic region was amplified and sequenced in two chicory varieties, Pan de Sucre (var. porphyreum) and Rosso di Chioggia (var. latifolium), revealing nucleotide differences not only between varieties but also within them, confirming the high allelic polymorphism of this locus. Based on conserved regions identified in the alignment, three sgRNAs were designed and evaluated by in vitro cleavage assay; two of these, sgRNAMIK2_309F and sgRNAMIK2_310F, produced cleavage patterns consistent with Cas9-mediated digestion of the MIK2 target fragment. These guides were subsequently used for ribonucleoprotein (RNP)-based delivery into leaf-derived protoplasts of Pan de Sucre via PEG-mediated transfection. Early microcolony formation was observed following transfection, but further progression to robust microcallus material was not achieved under the tested conditions. The low genomic DNA yield recovered from protoplast-derived material, together with the inability to amplify the MIK2 target region by PCR, prevented sequence-based editing validation by TIDE analysis, leaving the occurrence of cellular editing unresolved. These limitations directly motivated the evaluation of a callus-based strategy aimed at optimizing culture conditions and establishing genotype-defined starting material for future editing workflows. Callus induction from leaf explants of Pan de Sucre and Rosso di Chioggia was evaluated using two MS-based media, D-30 and DC-30. Both media supported efficient callus induction, with induction rates reaching 100% in most variety–medium combinations by 21 days. D-30 showed faster early callus development and higher friability, consistent with its higher auxin-to-cytokinin ratio, while DC-30, supplemented with casein hydrolysate, showed a comparatively higher frequency of shoot-related responses (52% versus 40% in D-30). After approximately seven weeks, a subset of callus lines developed root-forming structures, indicating that the induced material retains morphogenic potential under the tested conditions; however, complete plant regeneration was not obtained. Overall, this study highlights the technical and methodological challenges associated with targeting highly polymorphic genes such as MIK2 in C. intybus, and identifies callus-derived material from individually selected donor plants as a promising preparatory step for future functional studies aimed at elucidating its potential role in self-incompatibility.