Anatomical and morphological variations associated with in vitro micrografting in olives, using clonal rootstocks

Micrografting is a powerful biotechnological tool for the micropropagation of fruit trees and can be applied to clonal propagation of recalcitrant species, virus eradication, pathogen management, early screening of scion/rootstock compatibility, and breeding. In olive, however, this technique remains poorly explored, particularly regarding grafting onto fully clonal rootstocks. In this study, we developed an in vitro micrografting (IVM) protocol based entirely on clonally propagated material. Two cultivars: 'Galega' and 'Arbequina', and two genotypes: genotype 26 (G26), and Genotype 35 (G35), were selected based on their vigor and rooting ability. IVM was optimized by testing different combinations of scion and rootstock length and diameter. Morphological variations, including callus formation, wound healing, and vascular connection, were evaluated in both homografting (Galega) and heterografting combinations. Histological analyses were performed at 7, 14, 21, 30, 45, and 60 days after micrografting (DAM) to characterize graft union development. Wounding occurred as early as 7 DAM, while between 14 and 21 DAM, parenchymatous cells proliferated and differentiated into tracheid cells, indicating the wound healing process. By 30-45 DAM, vascular continuity was re-established, confirming successful graft formation. Overall, these results provide a detailed temporal and anatomical characterization of graft union development and support the optimization and scaling-up of IVM for nursery production. Furthermore, this approach may contribute to sustainable strategies against emerging threats such as Xylella fastidiosa and may allow modulation of vegetative vigor through rootstock selection.