Triparental plants provide direct evidence for polyspermy induced polyploidy.

It is considered an inviolable principle that sexually reproducing organisms have no more than two parents and fertilization of an egg by multiple sperm (polyspermy) is lethal in many eukaryotes. In flowering plants polyspermy has remained a hypothetical concept, due to the lack of tools to unambiguously identify and trace this event. We established a high-throughput polyspermy detection assay, which uncovered that supernumerary sperm fusion does occur in planta and can generate viable polyploid offspring. Moreover, polyspermy can give rise to seedlings with one mother and two fathers, challenging the bi-organismal concept of parentage. The polyspermy derived triploids are taller and produce bigger organs than plants resulting from a regular monospermic fertilization. In addition, we demonstrate the hybridization potential of polyspermy by instantly combining three different Arabidopsis accessions in one zygote. Our results provide direct evidence for polyspermy as a route towards polyploidy, which is considered a major plant speciation mechanism.