Meiotic cytokinesis defects in cultured Arabidopsis flowers induce diploid microspores

Cultured Arabidopsis flowers inmedium produce diploid microspores due to occurrence of defective meiotic cytokinesis, which can be partially rescued by anexogenous application of sucrose. Live-imaging microscopy technology has been increasingly applied for meiosis study in plants, which largely relies on the set up of a healthy ex vivo culture system for inflorescences ensuring that the captured chromosomes dynamics approaches the natural features of meiosis. Here, we report that Arabidopsis thaliana flowers cultivated in a culture medium (CCM) composed of the half-strength Murashige and Skoog basal salt, MES, Myo-inositol, sucrose and agar produce diploid microspores due to occurrence of meiotic restitution. Cytological studies revealed adjacent nuclei distribution and incomplete cytokinesis at late meiosis II in meiocytes within the CCM flowers. Immunolocalization of α-tubulin and the microtubule-associated protein MAP65-3 showed that the orientation of spindles at metaphase II and the organization of radial microtubule arrays at the tetrad stage are interfered, which explains the production of meiotically-restituted microspores. Moreover, the CCM flowers showed a gradually impaired expression of Aborted Microspores (AMS), a key transcription factor regulating tapetum development and meiotic cytokinesis. Interestingly, an increased supply of sucrose or its metabolite fructose in culture medium promoted the expression of AMS and partially rescued haploid microspore formation in the CCM flowers. Taken together, this study suggests a role of sucrose in facilitating meiotic cytokinesis and gametophytic ploidy stability in plants.