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Plants are our source of oxygen and nutrients on earth. Therefore, conservation of biodiversity is vital for the survival of other species. With the developing technology, plant species can be examined more closely. Image processing, which is a subject of computer science, has an important role in this field. In this study, an image processing–based method has been developed to automatically separate the petiole region of the plant leaves. To determine the boundary line of the petiole region, the cumulative pixel distributions of the input images in binary format according to the X - and Y -axis are analyzed. Accordingly, optimum thresholds and petiole boundary points are determined. The proposed method was tested on 795 leaf images from 90 different plant species that grow both as trees and shrubs in the Czech Republic. According to the results obtained in experimental studies, it is thought that the proposed method will make an important contribution especially in studies such as automatic classification of plants and leaves and determination of plant species in botanical science. [ABSTRACT FROM AUTHOR]