A highly efficient regeneration, genetic transformation system and induction of targeted mutations using CRISPR/Cas9 in Lycium ruthenicum

Abstract

Background: CRISPR/Cas9 is a rapidly developing genome editing technology in various biological systems due to its efficiency, portability, simplicity and versatility. This editing technology has been successfully applied in in several important plants of Solanaceae such as tomato, tobacco, potato, petunia and groundcherry. Wolfberry ranked the sixth among solanaceous crops of outstanding importance 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 sequencing and the lack of efficient regeneration and genetic transformation systems. Results: In this study, we have established an efficientregeneration and genetic transformation system of Lycium ruthenicum. We have used this system to validate target sites for fw2.2, a major fruit weight quantitative trait locus first identified from tomato and accounted for 30% of the variation in fruit size. In our experiments, the editing efficiency was very high, with 95.45% of the transgenic lines containing mutations in the fw2.2 target site. We obtained transgenic wolfberry plants containing four homozygous mutations and nine biallelic mutations in the fw2.2 gene. Conclusions: These results suggest that CRISPR-based gene editing is effective for the improvement of black wolfberry traits, and we expect this approach to be routinely applied to this important economic fruit.