Walnut genome editing: an optimized CRISPR/Cas9 platform with superior genotypes and endogenous promoters

Walnut (<i>Juglans regia</i> L.) is an economically valuable tree species globally, renowned for its nutritious nuts and quality timber. However, walnut breeding is significantly constrained by inherent biological factors, and an efficient and reliable genome-editing system has yet to be established. In this study, we developed an optimized walnut genome-editing platform by systematically selecting superior receptor from 30 walnut cultivars using genotype-dependent direct somatic embryogenesis and regeneration systems. Walnut cultivar HT-14 exhibited the highest embryogenic induction (53.33%) and regeneration efficiency (85.33%), and 35S: RUBY was effectively expressed in somatic embryos of the HT-14 genotype, proving it ideal receptor material for genetic transformation. Additionally, 12 walnut-specific endogenous Pol III promoters (JrU3 and JrU6) were cloned and validated for their ability to significantly enhance CRISPR/Cas9-editing efficiency by targeting the walnut phytoene desaturase gene (<i>JrPDS</i>). Compared to commonly used exogenous promoters (AtU6-26 and BpU6-6), these native promoters, the JrU3-chr3 promoter achieving an editing efficiency of 58.82%, significantly increased mutation efficiencies in walnut. Furthermore, endogenous promoters promoted higher frequencies of homozygous and biallelic mutations and greater mutation diversity. Collectively, this study establishes a robust and efficient genome-editing platform for walnut, providing essential technical support for functional genomics research and accelerating the precision breeding walnut varieties. These findings also offer valuable methodologies and insights into genome-editing applications in other perennial woody plants.