Chemically-induced epimutagenesis allows bypassing reproductive barriers in hybrid seeds

The "triploid block" prevents interploidy hybridizations in flowering plants, and is characterized by failure in endosperm development, arrest in embryogenesis, and seed collapse. Many genetic components of triploid seed lethality have been successfully identified in the model plant Arabidopsis thaliana, most notably the paternally expressed imprinted genes (PEGs) that are up-regulated in the tetraploid endosperm with paternal excess. Previous studies have shown that the paternal epigenome is a key determinant of the triploid block response, as the loss of DNA methylation in diploid pollen suppresses the triploid block almost completely. Here, we demonstrate that triploid seed collapse is bypassed in Arabidopsis plants treated with the DNA methyltransferase inhibitor 5-Azacytidine during seed germination and early growth. We have identified strong suppressor lines showing stable transgenerational inheritance of hypomethylation in CG context, as well as normalized expression of PEGs in triploid seeds. Importantly, differentially methylated loci segregate in the progeny of "epimutagenized" plants, which may allow the identification of epialleles involved in the triploid block response in future studies. Finally, we demonstrate that chemically-induced epimutagenesis allows bypassing hybridization barriers in crosses between different Capsella species, thus potentially emerging as a novel strategy for producing triploids and interspecific hybrids with high agronomical interest.