Overcoming In Vitro Recalcitrance in Corylus colurna L.: From Optimized Micropropagation to Somatic Embryogenesis and Protoplast Isolation

Abstract An integrated biotechnological pipeline for Corylus colurna L., a promising non-suckering rootstock for hazelnut ( Corylus avellana L.), was established through the optimization of mineral composition of micropropagation media, adventitious rooting, somatic embryogenesis (SE), and protoplast technology. Nutritional evaluation using a modified medium supplemented with 150 mg L − 1 ethylenediamine di-2-hydroxyphenylacetate ferric (Fe-EDDHA) significantly suppressed hyperhydricity and enhanced shoot vigor across four clonal genotypes. For adventitious rhizogenesis, a 3-day in vitro pulse with 15 µM indole-3-butyric acid (IBA) followed by ex vitro development achieved an 83.4% rooting rate and over 85% plantlet survival. Callus induction was optimized across diverse explants, revealing that stamen-derived calli possess a distinct low-hydration profile (~ 50% water content) compared to vegetative tissues, ideal for high-yield protoplast isolation. Thidiazuron-mediated somatic embryogenesis (SE) was induced from immature cotyledonary zygotic embryos, reaching up to 28.6% in C. colurna (GEO-14-75), whereas the parallel C. avellana control cultivars were more responsive (up to 60% in cv ‘Jefferson’). These SE-derived tissues yielded high densities of viable protoplasts (3.2 × 10 6 cells mL − 1 ), which showed a transient transfection efficiency of 90 ± 3.2% at the expression peak (40 h post-transfection) using polyethylene glycol (PEG)-mediated delivery of a green fluorescent protein (GFP) reporter. Together with the optimized micropropagation and somatic embryogenesis protocols, the high-yield protoplast isolation and efficient transient GFP expression provide an integrated platform for clonal propagation and transient functional assays in C. colurna .