Seedless barberry (Berberis vulgaris L. var. asperma) is one of the medicinal plants only cultivated in eastern parts of Iran. Its origin, ancestral progenitors and mecha- nisms of seedless fruits production is not clear. In present study, for the first time we investigated similarity between B. integerrima (native to eastern Iran and hav- ing seedy fruit), B. vulgaris and seedless barberry on the basis of ploidy level and Karyotypic characteristics by karyotype preparation and Flow cytometric analysis. B. integerrima and seedless barberry were tetraploid (2n = 4x = 56) and their chro- mosomal characteristic analysis by principle coordinate analysis (PcoA) showed high similarity between them, whereas B. vulgaris was diploid (2n = 2x = 28) and placed in a significant distance from two other species. Then, in order to study the factors preventing seed formation, pollination studies including self-pollination, reciprocal cross-pollination and open-pollination was carried out and the effects of individual treatments on seedy and seedless fruits, fruit set rate and fertility offlower were investigated in populations of seedless barberry and B. integerrima. Results indicated that self pollination in seedless and wild barberry produced seed- less fruits and decreasing fruit set to 80. Open pollination results in normal fruit set and produced seedless and seedy fruits in cultivated and wild barberry respectively. Emasculation without pollination reduced fruit set to 40. Reciprocal hybridization between seedless and wild barberry resulted to seedy fruits as 6% and 90% respec- tively. The pollen tube growth in each type of pollination experiments and the pol- len germination were also investigated that the results will be presented. Previous studies and results of present study support our hypothesis asserting B. integerrima is probably one of the major ancestors of seedless barberry.

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