Establishment of an Effective Gene Editing System for ‘Baihuayushizi’ Pomegranate

Pomegranate (Punica granatum L.) is a popular fruit tree with high edible and ornamental values. However, the traditional breeding strategies are lacking in efficiency for the improvement of agronomic traits of pomegranate. Gene editing technologies offer a solution for promoting desired growth or metabolic processes in pomegranate trees. In this study, we established a CRISPR-mediated gene editing system for pomegranate, using Agrobacterium tumefaciens as the delivery vehicle and unlignified stems of the ‘Baihuayushizi’ cultivar as explants. The editing efficiency of our system was inferred to be 38.00%, which is substantially higher than those in some other plant species. The impacts of different culture conditions on the system were further assessed. Pre-culture duration was found to have the largest influence on the success of genetic transformation, followed by A. tumefaciens infection time and concentration. The optimal pre-culture time for this system is 3 days, and the A. tumefaciens concentration, infection time, and co-culture duration are OD600 = 0.8, 10 min, and 2 days, respectively. With the help of our system, we successfully knocked the PgBZR1 gene out from ‘Baihuayushizi’ pomegranate, which encodes a key transcription factor that regulates the growth and development of pomegranate. Given these advantages, we anticipate that our gene editing system is a useful tool for future studies on pomegranate gene functions and genetic improvement.