An efficient improved CRISPR mediated gene function analysis system established in Lycium ruthenicum Murr.

Lycium ruthenicum Murr. is a xero-halophytic plant and well-known for accumulating high level of anthocyanin in its fruit. However, the molecular basis of these excellent traits of L. ruthenicum remains unclear due to lack of the regeneration, genetic transformation and genome editing system, which limit to study the functional genes of L. ruthenicum. In this study, an efficient system for regeneration, genetic transformation and genome editing is established. The optimized MS (Murashige and Skoog) medium for callus induction and shoot regeneration is MS supplement of 1.0 mg/L 6-benzylaminopurine (6-BA) and 1.0 mg/L indole-3-butyric acid (IBA) with 100% callus induction rate and 95% shoot regeneration rate, respectively. The resulting shoots successfully regenerate robust roots on 1/2 MS medium. A high transformation efficiency with 53.7% (72/134) for LrFT (Flowering Locus T) overexpression was achieved using the transformation system established here. A modified YAO promoter-driven CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) mediated gene editing system coupled with kanamycin selection in L. ruthenicum with 41.66% HG18103 homozygous mutant is significantly higher than previous study. In addition, the universal and common phenomenon of pds (phytoene desaturase) deficiency promoting anthocyanin biosynthesis and accumulation in young leaf is, partially if not completely, attributed to redox-dependent modulation, including the ROS-induced expression of MBW component (MYB-bHLH-WD40), ROS-depressed phenylpropanoid repressor LrMYB3/4, and the redox-dependent LrTCP15 (TEOSINTE BRANCHED1/CYCLOIDEA/PCF 15) activity in DNA binding. Taken together, our results suggest that an efficient system for genome editing lay a solid foundation of functional genomics study and molecular breeding in L. ruthenium .