Investigating the simultaneous effect of chitosan and arbuscular mycorrhizal fungi on growth, phenolic compounds, PAL enzyme activity and lipid peroxidation in Salvia nemorosa L.

Considering the importance of Salvia nemorosa L. in the pharmaceutical and food industries, and also beneficial approaches of arbuscular mycorrhizal fungi (AMF) symbiosis and the use of bioelicitors such as chitosan to improve secondary metabolites, the aim of this study was to evaluate the performance of chitosan on the symbiosis of AMF and the effect of both on the biochemical and phytochemical performance of this plant and finally introduced the best treatment. Two factors were considered for the factorial experiment: AMF with four levels (non-inoculated plants, Funneliformis mosseae, Rhizophagus intraradices and the combination of both), and chitosan with six levels (0, 50, 100, 200, 400 mg L-1 and 1% acetic acid). Four months after treatments, the aerial part and root length, the levels of lipid peroxidation, H2O2, phenylalanine ammonia lyase (PAL) activity, total phenol and flavonoid contents and the main secondary metabolites (rosmarinic acid and quercetin) in the leaves and roots were determined. The flowering stage was observed in R. intraradices treatments and the highest percentage of colonization (78.87%) was observed in the treatment of F. mosseae × 400 mg L-1 chitosan. Furthermore, simultaneous application of chitosan and AMF were more effective than their separate application to induce phenolic compounds accumulation, PAL activity and reduce oxidative compounds. The cluster and principal component analysis based on the measured variables indicated that the treatments could be classified into three clusters. It seems that different treatments in different tissues have different effects. However, in an overview, it can be concluded that 400 mg L-1 chitosan and F. mosseae × R. intraradices showed better results in single and simultaneous applications. The results of this research can be considered in the optimization of this medicinal plant under normal conditions and experiments related to abiotic stresses in the future.

Impacts of foliar exposure to multi-walled carbon nanotubes on physiological and molecular traits of Salvia verticillata L., as a medicinal plant.

Owing to the growing applications of the multi-walled carbon nanotubes (MWCNTs) in the communications and energy industries, they have attracted increasing attention for their effects on the environment and plants. Therefore, we investigated the impact of foliar exposure to MWCNTs on the oxidative stress responses in the Salvia verticillata as a medicinal plant. Furthermore, we evaluated the possible correlations between gene expression and activity of the key enzymes in the phenolic acids biosynthesis pathways and their accumulation in the treated leaves. The leaves of two-month-old plants were sprayed with different concentrations (0-1000 mg L-1) of MWCNTs. Raman's data and Transmission Electron Microscopy images have confirmed the absorption of MWCNTs via epidermal cells layer into the parenchymal cells of the exposed leaves. The results showed that exposure to MWCNTs led to a decrease in the photosynthetic pigments and increases in the oxidative stress indices (enzymatic and non-enzymatic antioxidants) in the leaves with a dose-dependent manner. The content of rosmarinic acid as a main phenolic acid was increased in the MWCNTs-exposed leaves to 50 and 1000 mg L-1, nearly four times relative to the control. Unlike with other examined enzymes, a positive correlation was deduced between the activity and gene expression patterns of the rosmarinic acid synthase with the rosmarinic acid accumulation in the treatments. Overall, MWCNTs at the low concentrations could promote the production of the pharmaceutical metabolites by the changes in the ROS generation. However, at the higher concentrations, MWCNTs were toxic and induced the oxidative damages in S. verticillata.

THE EFFECTS OF COLCHICINEINDUCED AUTOTETRAPLOIDY ON SELECTED CHARACTERISTICS OF NURUOZAK (SALVIA LERIIFOLIA).

Nuruozak (Salvia leriifolia Benth), is a perennial herbaceous plant that is endemic to Iran and has recently been introduced as a medicinal plant. Artificial polyploidy is an efficient method to increase the production of secondary metabolites and can result in a whole spectrum of genetic, molecular and physiological modifications. In order to produce an autotetraploid population of nuruozak, various concentrations of colchicine (0.00, 0.05, 0.10, 0.20 or 0.50 % w/v) were applied to the seeds and shoot apical meristems of young seedlings at the fourth leaf-stage. Microscopic studies, flow cytometry analysis and chromosome counting were conducted to select tetraploid nuruozak plants. Furthermore, the effects of ploidy level on the essential oil content and composition and biomass production of nuruozak plants, as well as selected structural and physiological characteristics were studied. Based on the number of the obtained tetraploids, treatment of shoot apical meristems was more efficient than seed treatment. Structural and phytochemical characteristics, chlorophyll content and photosynthetic rate were affected by the increase in ploidy level. In addition to the higher potential in biomass production, tetraploid plants produced eight new compounds which were absent in diploids.

Establishment of Salvia castanea Diels f. tomentosa Stib. hairy root cultures and the promotion of tanshinone accumulation and gene expression with Ag⁺, methyl jasmonate, and yeast extract elicitation.

Salvia castanea Diels f. tomentosa Stib. is an endemic medicinal plant distributed in China, and the notably high content of tanshinone IIA in the root is proven effective for the therapy of heart diseases. Hairy root induction of this Salvia species was inoculated with Agrobacterium rhizogenes strain ATCC 15834. Transformed hairy root was cultured in 6,7-V liquid medium for growth kinetics assessment and elicitation. An S curve was present in the hairy root cultures based on the fresh and dry weights with an interval of 3 days. An optimum concentration of the applied elicitors (15 µM Ag(+), 200 µM methyl jasmonate, and 200 mg l(-1) yeast extract elicitor) benefitted both the growth status and tanshinone accumulation in the hairy root cultures. Tanshinone IIA contents were mostly stimulated 1.8-fold and 1.99-fold compared with the control by Ag(+) and methyl jasmonate elicitation, respectively. Yeast extract dramatically enhanced dry mass accumulation, while it promoted cryptotanshinone content of 2.84 ± 0.33 mg g(-1) dry weight at most in the hairy root cultures. Selected elicitors diversely influenced tanshinone accumulation in the time courses of hairy root cultures within 7 days. Furthermore, transcripts of selected genes in the tanshinone biosynthetic pathway were remarkably upregulated with elicitation. Yeast extract elicitor heightened 13.9-fold of isopentenyl diphosphate isomerase expression level at 12 h, while it increased 16.7-fold of geranylgeranyl diphosphate synthase transcript at 24 h compared with that of the control, which was more effective than Ag(+) and methyl jasmonate. This study provided a convenient hairy root culture system of S. castanea Diels f. tomentosa Stib. for tanshinone production for the first time.

Application of Heavy Metal Rich Tannery Sludge on Sustainable Growth, Yield and Metal Accumulation by Clarysage (Salvia sclarea L.).

A field experiment was conducted to evaluate the effective utilization of tannery sludge for cultivation of clarysage (Salvia sclarea) at CIMAP research farm, Lucknow, India during the year 2012-2013. Six doses (0, 20, 40, 60, 80, 100 tha(-1)) of processed tannery sludge were tested in randomised block design with four replications. Results revealed that maximum shoot, root, dry matter and oil yield were obtained with application of 80 tha(-1)of tannery sludge and these were 94, 113 and 61% higher respectively, over control. Accumulation of heavy metals (Cr, Ni, Fe, Pb) were relatively high in shoot portion of the plant than root. Among heavy metals, magnitude of chromium accumulation was higher than nickel, iron and lead in shoot as well as in root. Linalool, linalyl acetate and sclareol content in oil increased by 13,8 and 27% respectively over control, with tannery sludge application at 80 tha(-1). Heavy metals such as chromium, cadmium and lead content reduced in postharvest soil when compared to initial status. Results indicated that clarysage (Salvia sclarea) can be grown in soil amended with 80 tha(-1)sludge and this can be a suitable accumulator of heavy metals for phytoremediation of metal polluted soils.