iii Abstract
The interest in natural-derived molecules rosmarinic acid (RA) and carnosic acid (CA) has raised up during the last years due to their well documented antioxidant properties: they are in fact used in foodstuff as preserving agent as well as in pharmacology due to their antibacterial and medicinal properties. In vitro cultures of medicinal plants are considered an alternative way to conventional methods for the production of valuable secondary metabolites; undifferentiated cells (callus and cell suspensions) are also suitable for both plant biomass production and scaling-up and for the study of metabolites biosynthesis.
In this work Salvia officinalis L. (common sage) cell suspension cultures were established, and a specific cell line was selected for the high antioxidant capacity of its methanolic extract, which was characterized by a high content of RA. Scavenger activity (DPPH test) and total RA content were evaluated during cell growth.
Gene coding for hydroxyphenylpyruvate reductase, the key-enzyme responsible for the RA metabolic biosynthesis, was cloned from common sage (SoHPPR). Its transcript expression level was monitored during cell suspension cultures, and showed a relationship with scavenger activity and RA yield. Our results suggest the potential use of this gene as a marker and target for the modulation of RA production in controlled conditions. In addition, the optimum conditions for Agrobacterium-mediated transformation were determined by monitoring the transient expression of β–Glucuronidase (GUS) marker gene in callus culture.
We also set up a protocol for the in vitro culture of Rosmarinus officinalis, one the few species able to synthesize the phenolic diterpene CA. The target of this study was to evaluate the capacity of different tissues of synthesize CA. Our results suggest that all the tissues can accumulate varying amounts of CA, depending on the differentiation grade of the cell culture type: among the different culture types, neo- formed shoots coming from irradiated calli accumulated the highest content of CA. Finally we evaluated whether CA extract may be used as a natural substitute of antibiotics commonly used in plant tissue cultures: our data suggest a bacteriostatic activity of CA against common pathogens without affecting plant growth and physiology.
