Induction and Characterization of Diploid Pollen Grains in Arabidopsis thaliana

Polyploidization or whole genome duplication (WGD) is one of the main forces driving plant genome evolution and biodiversity with major implications for plant breeding and crop improvement. In nature, de novo formation of polyploid plant genomes most likely occurs through a modification of the sexual reproductive pathway. By interfering with reproductive genome stability, for example, via induction of meiotic restitution, diploid or polyploid gametes are ectopically formed that may participate in fertilization to yield polyploid offspring. This mechanism of WGD is generally referred to as sexual polyploidization. Considering the central role of sexual polyploidization in speciation, genome evolution and crop breeding, we provide here a set of methodologies to induce and characterize 2n pollen grain formation in plants. Using Arabidopsis thaliana as a model, we outline two different methods, that is, one chemical and one environmental, to induce male meiotic restitution and high frequency 2n pollen grain formation. In addition, we provide a set of simple and straightforward techniques to characterize alterations in male meiotic cell division and gametophytic ploidy stability underpinning 2n pollen formation. This comprehensive toolbox is applicable in a broad range of plant species to enable quick induction and assessment of 2n gamete formation during plant male reproduction.