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The current pace of crop plant optimization is insufficient to meet future demands and there is an urgent need for novel breeding strategies. It was previously shown that plants tolerate the generation of triparental polyspermy-derived plants and that polyspermy can bypass hybridization barriers. Polyspermy thus has the potential to harness previously incompatible climate-adapted wild varieties for plant breeding. However, factors that influence polyspermy frequencies were not previously known. The endopeptidases ECS1 and ECS2 have been reported to prevent the attraction of supernumerary pollen tubes by cleaving the pollen tube attractant LURE1. Here, we show that these genes have an earlier function that is manifested by incomplete double fertilization in plants defective for both genes. In addition to supernumerary pollen tube attraction, ecs1 ecs2 mutants exhibit a delay in synergid disintegration, are susceptible to heterofertilization, and segregate haploid plants that lack a paternal genome contribution. Our results thus uncover ECS1 and ECS2 as the first female factors triggering the induction of maternal haploids. Capitalizing on a high-throughput polyspermy assay, we in addition show that the double mutant exhibits an increase in polyspermy frequencies. As both haploid induction and polyspermy are valuable breeding aims, our results open new avenues for accelerated generation of climate-adapted cultivars., Competing Interests: YM, TN, IE, SJ, DT, TB, RG No competing interests declared, (© 2023, Mao, Nakel et al.)

Eschscholzia californica (California poppy), a member of the basal eudicot family of the Papaveraceae, is an important species to study alkaloid biosynthesis and the effect of alkaloids on plant metabolism. More recently, it has also been developed as a model system to study the evolution of plant morphogenesis. While progress has been made towards establishing methods for generating genetically modified cell culture lines, transcriptome data and gene expression analysis, the stable transformation and subsequent regeneration of transgenic plants has proven extremely time consuming and difficult. Here, we describe in detail a method to transiently down regulate expression of a target gene by virus-induced gene silencing (VIGS) and the subsequent analysis of the VIGS treated plants. VIGS in E. californica allows for the study of gene function within 2 to 3 weeks after inoculation, and the method proves very efficient, enabling the rapid analysis of gene functions.