The production of F1 hybrids is a highly desirable breeding system, as it allows trait fixation in inbred lines and generates heterosis in the hybrid seed. Ensuring outcrossing between parental lines is essential and is usually achieved through male-sterility, detasseling, or chemical treatment of anthers on the maternal parent. In self-incompatible (SI) species, the inability to self-fertilise complicates the production of homozygous inbred lines, and biotechnological approaches to F1 seed production implemented in cereals do not overcome the limitations of SI (Herridge et al. 2019; Wu et al. 2016). Here we present a New Breeding Technique for F1 hybrid production in SI species that facilitates both requirements called “Combined Inbreeding And Outcrossing” (CIAO). The CIAO system utilises a transgene comprising a self-fertility-inducer (SFI), pollen-lethal-cassette (PLC) and seed-fluorescent-marker (SFM; Figure 1A). The SFI reversibly represses an essential SI gene (e.g., through RNAi, miRNA or CRISPRi). The PLC and SFM maintain the CIAO transgene in a hemizygous state and allow selection of CIAO+/− or CIAO−/− seed based on fluorescence. Inbreeding and near-homozygous inbred lines production is enabled in CIAO+/− hemizygotes and subsequently, null-segregant CIAO−/− seed can be selected and crossed with a second line containing a different S-allele (Figure 1B). The SI machinery is active in the null-segregant parent plants so they are unable to self-fertilise or pollinate siblings with matching S alleles. Outcrossing between the lines is enforced by the innate SI mechanism, producing F1 hybrid seed from both parental lines, two generations removed from the transgenic CIAO+/− line (Figure 1B). To demonstrate proof-of-concept data in Arabidopsis thaliana we utilised previously characterised components, and transgenic SI lines containing SRKb/SCRb from A.lyrata (Nasrallah et al. 2002). SRK is expressed from the stigma and interacts with SCR on the pollen coat inducing a signalling cascade preventing pollen germination. The SFI utilised the stigma-specific pSLG13 promoter driving an RNAi construct which targets SRKb (Figure S1A). For the SFM and PLC, seed-specific pNAPIN:DsRed and pollen-specific pLAT52:EcoRI constructs were used (Millwood et al. 2015; Stuitje et al. 2003). We generated transgenic A.thaliana (C24) plants carrying this CIAO construct and pollinated them with SRKb/SCRb plants. The resulting seed was manually sorted based on fluorescence and selected on kanamycin, ensuring the presence of CIAO and SRKb/SCRb, respectively. Plants from non-fluorescent seed were self-sterile and produced virtually no seed while plants from fluorescent seed (CIAO+/−) showed high levels of seed production when selfed (Figure 1C,D and Figure S1B). A similar construct containing an artificial miRNA also disrupted SI, albeit to a lesser degree, and only in a subset of plants (Figure S1C). The fluorescent to non-fluorescent seed ratio from CIAO+/− plants was typically ~50%, confirming PLC activity, although transgenic lines with > 1 insertion may skew this result (Figure 1E and Figure S1D,E). Outcrossed seeds from SRKb/SCRb plants pollinated by CIAO+/− plants were non-fluorescent, further validating PLC activity (Figure 1E). In lines with > 50% fluorescent seed on selfing (lines 1 and 2) few fluorescent seed were produced when outcrossed, indicating multiple T-DNA insertions instead of PLC failure (Figure S1E). We next tested the transgenerational stability of the construct. In F2 and F3 generations CIAO+/− plants were self-fertile, while CIAO−/− null-segregants reverted to SI producing few/no seeds (Figure S1F,G). In the F3 generation, the SRKb/SCRb transgene was fixed (homozygous), thereby requiring no selection and allowing comparison to the autogamous C24 plants. CIAO+/− plants produced less seed than wild-type C24 plants (~20%–60% yield/plant); however, still likely sufficient for breeding purposes (Figure S1F). Seed production varied between CIAO+/− plants likely due to incomplete knockdown of SRKb in combination with environmental factors. Improvements to the SFI may reduce variability, e.g., increased hairpin length, different promoters, and testing lines with different T-DNA insertion sites. Exemplary F1 hybrids from CIAO−/− null-segregants were created to demonstrate that the restored SI mechanism can enforce outcrossing. SI CIAO−/− C24 plants were pollinated by a different ecotype (Shahadra, Sha) with no emasculation so that self pollen was present. Seed was readily produced suggesting that outcrossing was occurring (compare Figure S1H with Figure 1D). C24 × Sha hybrids display heterosis (Le et al. 2023), and the F1 seedlings produced here appeared distinct from either parent (Figure S1I). The TUBULIN gene was sequenced from these plants to confirm heterozygosity. An A>C SNP followed by an indel is present in C24 compared to Sha, and this was heterozygous in 10/10 individuals tested, evidenced by a double peak followed by out-of-phase sequence, confirming outcrossing had occurred (Figure 1G). In practice, CIAO+/− and CIAO−/− plants should display similar phenotypes. The pre-flowering growth and flowering time of CIAO+/− and CIAO−/− plants were similar in two independent CIAO lines, consistent with tissue-specific transgene expression (Figure S2A–C). Excluding seeds where the PLC has become inactive is also important (i.e., homozygous CIAO+/+ plants). Such seeds can be identified by high DsRed fluorescence and the resulting plants produce 100% brightly fluorescent seeds, indicating homozygosity (Figure S2D–G). We envisage that CIAO will be useful in members of the Brassicaceae with sporophytic SI (e.g., cabbage, mustard, radish), which should be readily targetable based on experimental evidence shown here. In theory, CIAO may also be applied to crops with gametophytic SI by targeting the female stigma/style-expressed SI determinant e.g., S-RNAse in Solanaceae, or SP/ZP in grasses, although this requires experimental validation of efficacy and efficiency (Herridge et al. 2022; Ye et al. 2018; Figure S3). As the F1 hybrids are genetically identical and SI, producing a seed crop would require a second F1 line with a different S-allele to allow pollination/fertilisation, but this feature also facilitates “double-cross hybrid” production, three generations removed from the CIAO+ parents. Here we knock-down a single S-gene; however, the SFI also targets a second SRK allele (S6), and theoretically could target more, allowing a single transgenic event to create multiple inbred lines (Figure S3). Other strategies for F1 hybrid production from SI crops utilise multiple transgenic/editing events (e.g., disrupting SI, inducing male-sterility), or rely on pseudo-self incompatibility which often yields impure F1 hybrid seed (Munoz-Sanz et al. 2020). CIAO enables inbred line development and leverages the natural SI mechanism for efficient F1 seed production, providing access to the benefits of F1 hybrid breeding in many underserved SI crops. L.R.B., R.C.M. and R.P.H. conceived the idea and wrote the manuscript. R.P.H., S.V. and P.N. designed and undertook experimental work. R.P.H. and S.V. collected data and prepared figures. We thank June Nasrallah for providing the Arabidopsis SRKb/SCRb lines. Open access publishing facilitated by University of Otago, as part of the Wiley - University of Otago agreement via the Council of Australasian University Librarians. This work was supported by Otago Innovation Ltd. R.P.H., R.C.M. and L.R.B. have a pending PCT International Patent Application (Publication No. WO2025/012808) on the described technology. The authors declare no other conflicts of interest. Data underlying the figures shown is available on request. Table S1: Primers and insert sequences. Data S1: Supplementary experimental procedures. Figure S1: Details of CIAO construct and supporting data. Figure S2: Phenotype of CIAO plants and seed brightness. Figure S3: Strategies for CIAO application in different species. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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