Unraveling the role of interstocks in delaying flowering and mitigating spring frost risk in Iranian commercial almonds through morphophysiological, biochemical, and molecular approaches

Almond is one of the earliest flowering fruit trees, making it highly susceptible to late spring frosts, which can result in significant yield losses. In Iran, where orchards are often exposed to spring frosts, developing strategies to delay flowering is of critical importance. Delayed flowering is considered a passive frost-avoidance strategy that may reduce frost risk depending on local climatic conditions. Interstocks, inserted between rootstocks and scions, have the potential to influence phenology by modulating hormonal balance, carbohydrate allocation, and gene expression. This study aimed to investigate the effects of various rootstock–interstock–scion combinations on delaying bloom in Iranian commercial almond cultivars using morphological, physiological, biochemical, and molecular approaches. Specifically, two vegetative rootstocks (GN15 and GF677), three late-flowering almond genotypes (Shahrud 7, Shahrud 12, and Prunus arabica), and two Iranian commercial cultivars (Mamaee and Sefid) were evaluated in different grafting configurations, with non-interstock grafted combinations used as controls. During the 2024–2025 seasons, interstocks significantly influenced flowering time, flower density, vegetative growth, photosynthetic pigment composition, hormone profiles, and the expression of flowering-related genes. In the Mamaee cultivar, the combinations of Shahrud7 and Arabica interstocks on GN15 rootstock resulted in the most pronounced flowering delay, occurring more than 14 days later than the earliest-flowering combination (GF677–Arabica–Sefid) and approximately 5 days later than the control (GN15–Mamaee). In the Sefid cultivar, the inclusion of the Shahrud7 interstock in the GF677–Shahrud7–Sefid combination delayed flowering by approximately 9 days, whereas the same interstock grafted onto GN15 advanced flowering, highlighting a strong three-way interaction among rootstock, interstock, and scion. Pigment analyses indicated that Arabica interstocks enhanced chlorophyll b and carotenoid levels, whereas Shahrud7 promoted chlorophyll a, suggesting interstock-specific physiological pathways. Late-flowering combinations were associated with relatively higher levels of abscisic acid (ABA), indole-3-acetic acid (IAA), and gibberellic acid (GA3), which may contribute to prolonged dormancy and delayed floral transition. Gene expression analyses revealed reduced transcription of FLOWERING LOCUS T (FT), CONSTANS (CO), SUPPRESSOR OF OVEREXPRESSION OF CO 1(SOC1), LEAFY (LFY), and APETALA1 (AP1) in late-flowering combinations. Multivariate analyses further separated early- and late-flowering groups, emphasizing the regulatory role of interstocks. This study demonstrates that flowering phenology in Iranian commercial almonds is strongly dependent on rootstock–interstock–scion interactions rather than interstock identity alone. Certain grafting combinations were capable of delaying flowering or enhancing reproductive traits, indicating that interstock selection represents a potentially effective passive strategy for frost-risk mitigation when matched with appropriate rootstock–scion combinations. By integrating morphophysiological, hormonal, and molecular evidence, this work provides a mechanistic and application-oriented framework for optimizing almond orchard performance through targeted interstock use.