CRISPR/Cas9 editing characteristics of multiple transgenic generations in Fortunella hindsii, an early flowering mini-citrus

• Published in: Scientia horticulturae 2023-11, Vol.321, p.112236, Article 112236
• Main Authors: Song, Xietian, Xie, Yuanyuan, Tian, Xiaoyu, Wang, Nan, Zhou, Yin, Xie, Zongzhou, Ye, Junli, Deng, Xiuxin
• Format: Article
• Language: English
• Subjects: Citrus; CRISPR/Cas9; Fortunella hindsii; Transformation
• ISSN: 0304-4238
• DOI: 10.1016/j.scienta.2023.112236

•15 genotypes of F. hindsii were utilized to select the optimal genotypes for transformation.•The T0 generation produced highly chimeric transgenic plants.•the editing types of T1 generation more complex, which may be due to apomixis in citrus.•CRISPR/Cas9 vector was improved by using the endogenous U6 promoter in F. hindsii. The CRISPR/Cas9 editing system is presently regarded as one of the most efficient tools for genome editing and has been implemented in perennial fruit crops. However, the characteristics of transgenic plants and their subsequent generation have rarely been reported in fruit trees. In this study, 15 distinct preserved germplasm resources of Hongkong kumquat (Fortunella hindsii) were utilized to selecting suitable genotypes for genetic transformation. The shoot regeneration efficiency, seed number per fruit, and epicotyl diameter displaid a moderate positive correlation with transformation efficiency, with correlation coefficients of 0.6267, 0.5429, and 0.5388, respectively. The transcription factor RKD family gene FhRWP was knocked out in kumquat, resulting in the production of seven T0 and 34 T1 plants. Editing efficiency of T0 generation ranged from 1.62% to 68.11%, with a significant difference in editing activity among cases. Up to 23 editing types were detected in a single positive transformant, demonstrating that the high degree of citrus editing chimerism. Not all of T1 lines exhibited an increase in editing efficiency relative to T0. The descendants of T0-A2/A3 were all negative, while the other group of T1 lines showed both higher chimera and editing efficiencies of up to 92% than the others. Finally, the CRISPR/Cas9 vector was improved by using the endogenous U6 promoter in F. hindsii. This study provides novel insights into the application of the CRISPR/Cas9 system in asexual plants and serves as a reference for gene function verification in citrus and other species.