An attempt to establish an Agrobacterium-mediated transient expression system in medicinal plants.

Genetic transformation has always been an important method for studying medical plant secondary metabolic regulation, among which stable transformation has a good reproducibility. However, it was time-consuming to obtain a stable transformed hairy root or transgenic plants, which was difficult to satisfy the great demand of researches on medical plant secondary metabolism-related genes. Moreover, Agrobacterium tumefaciens-mediated transient transformation has been extensively applied in studies of functional genes because of its simpleness, low cost, and short period. However, presently, researches on medical plant functional genes commonly used stable genetic transformation and some high-cost and high-difficulty transient transformation methods, such as gene gun and protoplast transformation. Thus, in this study, we selected the seedlings of Nicotiana benthamiana, Salvia miltiorrhiza, and Prunella vulgaris as the experimental material, with the methods of Agrobacterium tumefaciens injection, fast Agrobacterium-mediated seedling transformation (FAST), and FAST and mechanical damage. The results demonstrated that the injection transient transformation system of pCAMBIA1301 vector mediated by A. tumefaciens and the transient transformation of seedling system were not established in S. miltiorrhiza. In addition, the instantaneous transformation system of N. benthamiana and P. vulgaris seedlings was basically set up by FAST method. Besides, using the method of FAST and mechanical damage, the transient genetic transformation system of P. vulgaris seedlings was established for the first time. A. tumefaciens-mediated transient transformation of seedlings with pEAQ vectors provided an effective way and reference for the further study of functional genes of the medicinal plants N. benthamiana and P. vulgaris.

High-performance liquid chromatography based chemical fingerprint analysis and chemometric approaches for the identification and distinction of three endangered Panax plants in Southeast Asia.

Among Panax genus, only three endangered species Panax notoginseng, P. vietnamensis, and P. stipuleanatus that have a similar morphology are mainly distributed in Southeast Asia. These three plants are usually misidentified or adulterated. To identify them well, their chemical chromatographic fingerprints were established by an effective high-performance liquid chromatography method. By comparing the chromatograms, the three Panax species could be distinguished easily using the 22 characteristic peaks. Besides, the data of the chromatographic fingerprints aided by chemometric approaches were applied for the identification and investigation the relationship of different samples and species. Using similarity analysis, the chemical components revealed higher similarity between P. vietnamensis and P. stipuleanatus. The results of hierarchical clustering analysis indicated that samples belonging to the same species could be clustered together. The result of principal component analysis was similar with hierarchical clustering analysis and the three principal components accounted for >80.5% of total variability.

Phosphate starvation promoted the accumulation of phenolic acids by inducing the key enzyme genes in Salvia miltiorrhiza hairy roots.

KEY MESSAGE: Phosphate starvation increased the production of phenolic acids by inducing the key enzyme genes in a positive feedback pathway in Saliva miltiorrhiza hairy roots. SPX may be involved in this process. Salvia miltiorrhiza is a wildly popular traditional Chinese medicine used for the treatment of coronary heart diseases and inflammation. Phosphate is an essential plant macronutrient that is often deficient, thereby limiting crop yield. In this study, we investigated the effects of phosphate concentration on the biomass and accumulation of phenolic acid in S. miltiorrhiza. Results show that 0.124 mM phosphate was favorable for plant growth. Moreover, 0.0124 mM phosphate was beneficial for the accumulation of phenolic acids, wherein the contents of danshensu, caffeic acid, rosmarinic acid, and salvianolic acid B were, respectively, 2.33-, 1.02-, 1.68-, and 2.17-fold higher than that of the control. By contrast, 12.4 mM phosphate inhibited the accumulation of phenolic acids. The key enzyme genes in the phenolic acid biosynthesis pathway were investigated to elucidate the mechanism of phosphate starvation-induced increase of phenolic acids. The results suggest that phosphate starvation induced the gene expression from the downstream pathway to the upstream pathway, i.e., a feedback phenomenon. In addition, phosphate starvation response gene SPX (SYG1, Pho81, and XPR1) was promoted by phosphate deficiency (0.0124 mM). We inferred that SPX responded to phosphate starvation, which then affected the expression of later responsive key enzyme genes in phenolic acid biosynthesis, resulting in the accumulation of phenolic acids. Our findings provide a resource-saving and environmental protection strategy to increase the yield of active substance in herbal preparations. The relationship between SPX and key enzyme genes and the role they play in phenolic acid biosynthesis during phosphate deficiency need further studies.