Development of advanced protocols for the in vitro micropropagation and phenotyping and ex vitro grafting of recalcitrant fruit tree species: a focus on Corylus colurna L.

Studies on hazelnut (Corylus avellana L.) rootstocks have been limited to date. Hazelnut cultivation is challenging due its prolific production of basal shoots (suckers), which complicates orchard management and require repeated mechanical or chemical control. The use of Corylus colurna (Turkish hazel) as non-suckering rootstock could overcome these limitations; however, its commercial use as clonal rootstock has been limited by severe recalcitrance to vegetative propagation, particularly its poor rooting ability. This thesis addressed these constraints through an integrated approach combining micropropagation, tissue culture, and molecular analysis. In the first phase, optimized in vitro multiplication and rooting protocols were developed for C. colurna. A modified medium (CM+Fe), enriched with DKW salts and FeEDDHA, enhanced shoot quality, elongation, and reduced hyperhydricity. A short indole-3-butyric acid (IBA) induction followed by ex vitro rooting significantly improved rooting, achieving over 80% success across genotypes. This protocol enabled the preliminary selection of 21 acclimatized clonal lines suitable for grafting. Early ex vitro evaluations revealed wide phenotypic variability among these lines in traits such as height, node number, and stem diameter, underscoring the potential of clonal selection. Complementary biotechnological tools were also established. Protocols for callus induction, protoplast isolation, and transfection by polyethylene glycol (PEG) or Agrobacterium tumefaciens were optimized. Both C. avellana and C. colurna proved to be highly recalcitrant to de novo shoot organogenesis, and somatic embryogenesis (SE) was successfully induced from zygotic embryos. To explore the molecular basis of C. colurna rooting recalcitrance, a comparative transcriptomic analysis of auxin transport and signalling was performed between the easy-to-root C. avellana cv. Giffoni and the difficult-to-root C. colurna. Reliable reference genes (CaUBQ14 and CaPP2) for RT-qPCR were identified and validated. Analysis of PIN, LAX, ARF, GH3, and LBD families revealed genotype-specific transcriptional patterns. In C. colurna, upstream auxin regulators such as ARF and PIN genes were upregulated, possibly indicating a delayed transition from priming to initiation during adventitious root induction (ARI). By contrast, C. avellana cv. Giffoni displayed stronger activation of downstream effectors like GH3s and LBDs, genes linked to auxin homeostasis and root founder cell formation. Overall, this research provides robust clonal propagation protocols and novel molecular resources that lay the foundation for the commercial exploitation of C. colurna as a standardized, non-suckering rootstock, thereby supporting the modernization and sustainable intensification of hazelnut cultivation.