Biotechnology applied to aromatic plants for the controlled production of bioactive compounds

Description

Salvia sensu lato includes more than about 9000 species and is considered one of the largest taxa of the Lamiaceae (Will et al., 2015). For a long time, Salvia species were known for a wide variety of medicinal used in folk medicine for the relief of pain, protecting the body against oxidative stress, free radical damages, angiogenesis, inflammation, bacterial and virus infection, etc… (Hamidpour et al., 2014). In vitro cultures have been considered as an alternative agricultural processes for producing secondary metabolites (Y. Kim et al., 2002). The research activity of this PhD project was aimed to the application of in vitro biotechnology techniques applied to aromatic plants for the production of bioactive compounds. The selected plants are species of Salvia showing an important antibacterial activity. The investigation study was carried out on S. corrugata and S. tingitana. Salvia corrugata Vahl. is an ornamental plant that grown easily along the Mediterranean coast. The aerial part is rich in terpenoid compounds like the diterpene quinones fruticuline A and demethylfruticuline A that present high antibacterial activity. Protocols of tissue culture and genetic transformation were set up with the aim to obtain in vitro shoot and hairy root biomass to grown in controlled condition for metabolite extraction. Two strains of Agrobacterium rhizogenes (wild type ATCC 15834 and hypervirulent LBA9402) were tested for their ability to induce hairy root on wounded leaves. The best response (75 %) was achieved by infection with ATCC 15834 about thirty days after the infection onto the hormone-free MS basal solid medium. Two hairy-root lines from ATCC 15834 treatment and one from LBA 9402 treatment were established. Transformation of selected clones was confirmed by polymerase chain reaction analysis of bacterial rolC and virC1 genes. The growth evaluation in TIS RITA® bioreactor put in evidence the best cultural conditions for the biomass production; the clone SCO-HR-FA8 had the best increase on Murashige and Skoog (1962) and ½ Woody plant medium (Lloyd et al., 1980) salt compositions in comparison to other tested media. 30 mg/L was the best sucrose concentration that guaranteed the highest biomass production. The growth curve of HR lie SCO-HR-FA8 was elaborated and then, three classes of elicitors and their combination were tested: a heavy metal ions (Ag+), the yeast extract and plant response-signaling compound (methyl jasmonate MJ). Among them, Ag+ and yeast extract (YE) at high concentration were most effective to stimulate the biomass production. The scale up of the biomass was performed using the bioreactor RITA®. The methanolic extracts of the biomass (16.8 g) was fractionated by Si gel MPLC to obtain 16 fractions. The methanolic extract and the semi-purified fractions were tested against several multidrug resistant clinical strains of various bacterial species: Staphylococci and Enterococci and E. coli. The total extract was poorly effective while the semi purified fractions displayed variable potency with MIC values ranging from 8 to >128 μg/mL and from 4 to >128 μg/mL respectively against the Staphylococci and Enterococci strains considered. Our results suggest that the application of biotechnology approach on S. corrugata, it is possible to induce hairy roots from leaf, optimize the condition to increase the production of biomass, scale-up using TIS bioreactor and produce secondary metabolites with antibacterial activity. The study carried out on S. tingitana aimed to develop an in vitro culture protocol to produce different plant tissues and investigate the antibacterial activity of the extract. The callus formation from sterilized leaves was evaluated on MS medium added with different PGRs in presence of ascorbic acid in light or in dark conditions. The result showed the importance of the presence 2,4-D and darkness for callus development. The high development percentage (94.4 %) was achieved by combining KIN and 2,4-D at the concentration of 0.5:0.5 or 1:1 mg/L respectively. The optimization of the medium for the callus growth was determined evaluating the type and concentration of cytokinin and auxin in dark. The suitable condition for callus proliferation was MS medium supplemented with 2,4-D 4.52 μM, KIN 2,32 μM and 10 mg /L of ascorbic acid. The elicitation with MJ or light intensity was investigated. The methanolic extract and fractions obtained by MPLC were inactive against Staphylococci species and E. coli. The fractions 11 and 12 displayed an antibacterial activity only against E. faecalis and E. faecium with MIC values ranging from 32 to 64μg/mL. In S. tingitana, a successful protocol for callus production was established; this in vitro culture system can be used as good method to obtain medicinally-useful secondary compounds from S. tingitana.

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