BackgroundChoosing the most compatible and desirable rootstock for Kalamata olive cultivar is an important decision due to the longevity of the orchard and the difficulty rooting of Kalamata cuttings. Therefore, the goal of this study was to examine the morphological, physio-biochemical, and nutritional status as ppotential markers for grafting compatibility between Kalamata olive cultivar and three olive rootstocks (Coratina, Picual, Manzanillo) during two seasons (2020–2021) as well as follow up physio-bichemical and nutritional status of one-year-old Kalamata plants (2022).ResultsThe results indicated that, Picual rootstock recorded the highest significant grafting success which was associated with increasing number of leaves, leaf area and SPAD value in Kalamata scions by 22.15%, 36.86% and 14.64% compared to Manzanillo rootstock as mean of both seasons, respectively. While, Manzanillo rootstock recorded the highest significant activity for peroxidase and catalase by 51.41% and 60.1% at grafting union compared to Picual rootstock. Moreover, Picual rootstock for Kalamata scions had the highest acid invertase and sucrose synthase activities by 67.23% and 57.94% compared to Manzanillo rootstock. Furthermore, Picual rootstock recorded the highest significant Gibberellic acid by 52.8% and 18.6% compared to Coratina and Manzanillo rootstocks. Meanwhile, Picual rootstock recorded the lowest significant Abscisic acid by 68.17% and 63.15% as well as the lowest total phenols by 14.36% and 23.47% compared to Coratina and Manzanillo rootstocks.ConclusionsThis study sheds light for the importance of choosing the suitable rootstock for Kalamata cultivar. Also, sucrose synthase and acid invertase may have a novel role in determining grafting compatibility in olives. Increasing growth promoters (Gibberellic, Nitrogen) and decreasing both growth inhibitors (Abscisic, phenols) and oxidative enzyme (catalase, peroxidase) required for better graft compatibility.