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

Walnut (Juglans regia 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 (JrPDS). 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.