Into the Unknown: Hints to Overcome Recalcitrance in Woody Fruit Crops

Abstract Woody fruit crops represent a cornerstone of global agriculture, yet their genetic improvement is severely hindered by the persistent issue of in vitro recalcitrance. This phenomenon represents a significant bottleneck for large-scale micropropagation and the application of groundbreaking breeding technologies, thereby slowing the development of new, resilient cultivars. This review provides a comprehensive state-of-the-art overview of the physiological, hormonal, and molecular factors underlying regenerative responses in woody species, emphasizing the critical role of explant type, developmental stage, genetic, and epigenetic factors beyond morphogenic competence. We highlight how endogenous and exogenous phytohormone interactions, in conjunction with complex genetic and epigenetic signalling networks, modulate adventitious organogenesis and somatic embryogenesis, often resulting in unpredictable and genotype-dependent outcomes. Particular attention is dedicated to biotechnological strategies aimed at overcoming recalcitrance, including the use of morphogenic regulators, inducible gene expression systems to minimize pleiotropic effects, and site-specific recombination technologies for marker-free transformation. Examples from woody fruit crops highlight both the progress made and the limitations that still hinder the establishment of efficient and reproducible protocols. Finally, we discuss current knowledge gaps and open questions, including the need to elucidate the molecular mechanisms of recalcitrance, the potential of in planta and tissue culture-free transformation methods, and the future integration of Artificial Intelligence and Machine Learning tools to accelerate protocol optimization. Together, these insights outline the path toward developing genotype-independent and scalable transformation systems for recalcitrant woody fruit species .