The establishment of transient expression systems and their application for gene function analysis of flavonoid biosynthesis in Carthamus tinctorius L

Safflower (Carthamus tinctorius L.) is an important economic crop and a traditional medicinal material rich in flavonoids, which can alleviate cardiovascular and cerebrovascular pathologies. Thus, many candidate genes involved in safflower flavonoid biosynthesis have been cloned. However, owing to the lack of a homologous gene expression system, research on gene function is limited to model plants. Therefore, a gene function identification protocol for safflower must be established.In the present study, using safflower callus as the experimental material, Agrobacterium and biolistic transient expression systems were established. In the Agrobacterium transient expression system, the highest transformation rate was obtained at the original Agrobacterium concentration of OD600 0.4, infiltration concentration of OD600 0.6, infection for 20 min, co-culture for 3 days, and acetosyringone concentration of 100 μmol·L-1. In the biolistic transient expression system, the highest transformation efficiency was observed at helium pressure of 1,350 psi, vacuum degree of -0.8 bar, flight distance of 6.5 cm, one round of bombardment, plasmid concentration of 3 μg·shot-1, and gold particle concentration of 100 μg·shot-1. Further, these two transient expression systems were used for the functional analysis of CtCHS1 as an example. After overexpression, relative CtCHS1 expression increased, particularly in Agrobacterium-transformed calli. Additionally, the contents of some flavonoids were altered; for instance, naringenin and genistein levels were significantly increased in Agrobacterium-transformed calli, whereas luteolin, luteolin-7-O-rutinoside, and apigenin derivative levels were significantly decreased in biolistic-transformed calli.Using safflower callus as the experimental material, highly efficient Agrobacterium and biolistic transient expression systems were successfully established, and the utility of both systems for investigating gene function was demonstrated. The proposed safflower callus transient expression systems will be useful for further functional analyses of flavonoid biosynthetic genes in safflower.