Biotechnological techniques are nowadays in use of the breeders to improve and quicken the production of new plant cultivars with desired characteristics. Among these techniques, anther and isolated microspore culture were used successfully in breeding programs in many plant species (e.g. wheat, barley, and tobacco). Anther culture is usually used for haploid production, but it may also have potential for developing plants with various ploidy levels which can be used in plant breeding programs. In genus Petunia, little information has been reported about anther and isolated microspore culture. Furthermore, no progress has been made over the last few decades to improve the investigation of this technique to use it on a commercial scale or to establish a scientific protocol in Petunia. The aim of this research was to study important factors influencing microspore embryogenesis (anther and isolated microspore culture) in Petunia in order to advance the understanding of these techniques. This may contribute to the establishment an efficient protocol which would enable the use of anther and isolated microspore culture in Petunia powerfully in breeding programs.