A Highly Efficient Protocol To Establish Regeneration And Genetic Transformation Systems And Create Targeted Mutations Using CRISPR/Cas9 in Lycium Ruthenicum

Background: The CRISPR/Cas9 system has rapidly become the preferred tool for various biological sequencing projects due to its high efficiency, specificity, simplicity and versatility, and it has been utilized for targeted genomic alternations in several important plants of Solanaceae, including tomato, tobacco, potato, petunia and groundcherry. Wolfberry is the sixth most important solanaceous crop in China following potato, tomato, eggplant, pepper and tobacco. To date, there has been no report on CRISPR/Cas9 technology to improve Lycium ruthenicum due to the unknown genome and the lack of efficient regeneration and genetic transformation systems.Results: In this study, we established a suitable regeneration and genetic transformation system of Lycium ruthenicum, the fw2.2 gene was identified, which was the first fruit weight gene identifified from tomato and accounted for 30% of the variation in fruit size. The gene editing of black wolfberry were carried out by CRISPR/ Cas9 for the first time here with a very high editing efficiency (95.45%) in fw2.2. Four homozygous mutations and nine biallelic mutations were obtained from T0 generation plants. Conclusions: These results suggest that the CRISPR/Cas9 system is effective for gene editing study of black wolfberry, and we expect this approach to be routinely applied to this important economic fruit.