This study presents the first integrated analysis of genotype–medium interactions and temporal morphogenesis profiling in sunflower regeneration. It aims to characterize genotype-specific responses, identify predictive morphological markers, and develop a scalable framework for breeding and transformation. Eighteen sunflower genotypes were evaluated to assess organogenic performance. The model genotype Ha-26-PR was used for a complementary experiment, testing varying sucrose concentrations to examine their influence on morphogenic outcomes. Hierarchical Cluster Analysis (HCA), guided by the Elbow method, identified four optimal clusters (K = 4). These aligned with three biologically meaningful categories: High Regenerators (Cluster 1), Moderate/Specific Regenerators (Clusters 2 and 3), and Non-Regenerators (Cluster 4). On S1 medium, NO-SU-12 and AS-1-PR showed superior shoot regeneration, while on R4 medium, HA-26-PR-SU and NO-SU-12 performed best. Genotypes such as NO-SU-12 and AS-1-PR consistently excelled across both media, whereas AB-OR-8 and FE-7 remained non-regenerators. Medium R4 supported superior regeneration, primarily through root formation, while S1 failed to induce roots in any genotype, highlighting the importance of hormonal composition. Although sucrose promoted callus induction, it did not trigger organogenesis. Callus was consistently present across media and time points, but its correlations with shoot and root formation were weak and temporally unstable, limiting its predictive value. Root formation at 14 days (Root 14D) emerged as a robust early predictor of organogenic success. This integration of morphological, temporal, and statistical analyses offers a genotype-tailored regeneration framework with direct applications in molecular breeding and CRISPR/Cas-based genome editing.

Sunflower in one of the most important oil-crops worldwide. Due to the effects of climate change, it is becoming increasingly important to discover effective and reliable ways to assist it in combating abiotic stresses. A very precise, cost-efficient and reliable way to achieve sunflower abiotic stress resilience, and specifically drought resilience, is through genetic modification. Due to sunflower’s recalcitrant nature to transformation and regeneration and strong genotype dependency, it is important to establish a high efficiency regeneration protocol applicable to a wide range of genotypes. The aim of this study was to evaluate the root regeneration capacity of 4 different sunflower genotypes when cultured on 5 different rooting media with the purpose of identifying a combination of sunflower genotype and medium with the highest root regeneration rate. The percentage of rooting of different genotypes showed inter-assay variability (from 0% regeneration to 100% regeneration), however, most consistent root formation was repeatedly observed for genotypes PH-BC2-91 and HA-26-PR. Statistically significant higher root regeneration rates were observed when explants of different genotypes were cultured in MS DV medium compared to four other media used (from around 90% for MS DV to around 50% for the other media). These results further verify the intricate relationship between genotype and medium interactions. The study provides a reliable basis for establishing an efficient sunflower root regeneration protocol and paves the way for the application of biotechnological approaches in breeding and enhancing resilience to abiotic stresses.