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This monograph provides a comprehensive overview of recent advances in the biotechnology of Juglans regia L. (Persian walnut), emphasizing in vitro propagation, genetic transformation, and disease management strategies. It synthesizes over three decades of global research and presents a systematic analysis of micropropagation techniques, somatic embryogenesis, and molecular tools for genetic improvement. The book highlights the major challenges in walnut biotechnology, such as tissue browning, recalcitrance, and microbial contamination, and discusses optimized protocols for culture establishment, rooting, and acclimatization. Furthermore, it explores innovative approaches including CRISPR/Cas9-mediated genome editing, temporary immersion systems, and cryopreservation for germplasm conservation. Integrating insights from molecular biology, plant pathology, and applied biotechnology, this work serves as both a reference and practical guide for scientists, graduate students, and horticultural professionals engaged in nut crop improvement.