Endopolyploidy, i.e. the amplification of genomic DNA without mitosis, is a widespread process in plants. Cells from the tomato fruit pericarp are characterized by a wide range of ploidy levels (from 2C to 256C). Although various functional hypotheses have been attributed to endoreduplication according to the literature, evidence for a specific role of endoreduplication in transcription and metabolism control is still lacking. We have developed a new method based on bacterial artificial chromosome fluorescent in situ hybridization (BAC-FISH) that allows the in situ determination of DNA ploidy levels of individual nuclei. The advantage of this method is illustrated by the analysis of ploidy levels and cell sizes within the pericarp tissue from mature green tomato fruits. Using this cellular approach we established the ploidy map of the pericarp tissue. Based on this map, we performed a structural analysis of endoreduplicated nuclei at the level of chromatin organization, nuclear shape and relationship with mitochondria. We demonstrated a link between the ploidy level of nuclei, the complexity of their shape and the number of mitochondria at the vicinity of polyploid nuclei. The use of the DNA FISH method demonstrated that endopolyploidy leads to the formation of polytene chromosomes, whereas the use of a RNA FISH method demonstrated that the rDNA transcription was increased during polyploidization. Performing quantitative PCR (qPCR) and RT-qPCR on sorted nuclei respectively, we confirmed that endoreduplication did amplified exponentially loci for a set of specific genes allowing us to demonstrade that endoreduplication results in an increasing transcriptional activity.