Role of Phytohormones in Biomass and Polyphenol Accumulation in Salvia bulleyana In Vitro Culture.

Salvia bulleyana is a plant native to the Chinese Yunnan Province. This species has been used in traditional Chinese medicine as a substitute for Danshen (the roots of Salvia miltiorrhiza). The aim of our study was to establish an effective system for propagating S. bulleyana shoots to obtain large amounts of material rich in bioactive compounds. Phytohormones were used to regulate shoot growth and regeneration potential and influence plant secondary metabolism. The shoot tips were incubated on a Murashige and Skoog agar medium supplemented with 0.1 or 0.5 mg/L IAA (indole-3-acetic acid) and the cytokinins benzylaminopurine (BAP), meta-topoline (M-T), 6-benzylaminopurine riboside (RBAP), N-benzyl-9-(2-tetrahydropyranyl)-adenine (BPA) or kinetin, (K) at concentrations of 0.5, 1 or 2 mg/L. It was observed that the type and concentration of growth regulator significantly influenced the regeneration potential of S. bulleyana shoots. The highest multiplication rate was obtained when 0.1 mg/L IAA and 2 mg/L BPA were used. Under these conditions, 100% of shoot tips formed buds and almost seven buds/shoot per explant were obtained after five weeks. Meanwhile, the highest biomass was found for shoots growing on a medium supplemented with 0.1 mg/L IAA and 1 mg/L M-T: 1.2 g of fresh weight and 0.17 g of dry weight. However, a medium with 0.1 mg/L IAA and 2 mg/L RBAP was most favorable for bioactive phenolic acid content, with a total polyphenol level (37.7 mg/g dw) 4.5 times higher than in shoots grown on medium without growth regulators (8.23 mg/g dw). Finally, optimal conditions were selected by TOPSIS (technique for order of preference by similarity to the ideal solution); the culture of S. bulleyana grown on an MS medium containing 0.1 mg/L IAA and 1 mg/L M-T was found to be the most efficient for polyphenol accumulation and can be used for the production of medicinally relevant compounds.

In Vitro Strategy for the Enhancement of the Production of Bioactive Polyphenols in Transformed Roots of Salvia bulleyana.

The underground parts of Salvia bulleyana, a rare Chinese plant species, have long been used in traditional Chinese medicine. The Rhizobium rhizogenes-transformed root culture obtained from this plant might be a promising novel source of valuable phenolics, including rosmarinic acid. The present study identifies for the first time, the optimal growth conditions of S. bulleyana hairy roots regarding production efficiency. The comprehensive optimization comprised cultivation in different basal media (B5, SH, MS, and WP) with full- and half-strength macro- and microelements, different vitamin contents (full, half, one-quarter part, and without) and sucrose concentrations (2, 3, 4, 5%), and under different light conditions: in dark, under blue LED (λ = 430 nm), red LED (λ = 670 nm), mixed blue and red LED (30%:70%), and white LED (390-670 nm). Hairy root growth and bioactive compound accumulation were also detailed every five days over the 50-day culture cycle. The optimal conditions were determined using a technique for order preference by similarity to the ideal solution (TOPSIS). The most efficient combination for root growth and polyphenol content was found to be ½SH liquid medium with half vitamin concentration and 3% sucrose when grown in the dark. The biomass yield during the growth cycle was 6.1 g (fresh weight-FW) and 0.92 g (dry weight-DW) on one Erlenmeyer flask: a 14.3-fold increase in FW and 16.1-fold increase in DW in relation to the inoculum. The highest mean total phenolic content was 93.6 mg/g DW including about 70 mg/g DW rosmarinic acid, reached on day 40 of culture; compared to roots of two-year-old plants grown under field conditions, the total phenolic acid content was four times higher and rosmarinic acid eight times higher. The obtained results place the investigated culture among the best hair root cultures for rosmarinic acid production.

The Antioxidant, Cytotoxic and Antimicrobial Potential of Phenolic Acids-Enriched Extract of Elicited Hairy Roots of Salvia bulleyana.

Hairy root cultures are valuable sources of a range of phytochemicals. Among them, Salvia bulleyana root culture is a promising source of polyphenols, especially rosmarinic acid (RA), a phenolic acid depside with pleiotropic activity and a wide application in medicine and cosmetology. The aim of the study was to enhance the culture productivity by finding suitable elicitation protocol and to determine its biological potential in terms of antioxidant, anticancer and antimicrobial properties. The total content of phenols and the levels of particular constituents in root extracts were analyzed using HPLC-PDA. Among four elicitors tested (yeast extract; methyl jasmonate, MJA; trans-anethol; and cadmium chloride), MJA was found to be the most effective. The greatest boost in phenolic production (up to 124.4 mg/g dry weight) was observed after three-day treatment with MJA at 100 µM, with an almost 100% improvement compared to the controls (non-treated root culture). The hydromethanolic extract from the elicited culture exhibited strong antioxidant activity with IC50 values of 11.1 µg/mL, 6.5 µg/mL and 69.5 µg/mL for DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)) and superoxide anion radical, respectively. Moreover, in concentrations of 0.5-5 mg/mL the extract inhibited the growth of LoVo, AGS and HeLa cell lines, but was safe for the L929 cells up to the concentration of 5 mg/mL. The extract also exhibited moderate antimicrobial activity. Thus, the results confirmed that elicitation can be a beneficial strategy for increase the phenolic acid biosynthesis in hairy roots of S. bulleyana, and that such a highly productive culture can show significant biological potential.

Cytokinin-Based Tissue Cultures for Stable Medicinal Plant Production: Regeneration and Phytochemical Profiling of Salvia bulleyana Shoots.

Salvia bulleyana is a rare Chinese medicinal plant that due to the presence of polyphenols lowers the risk of some chronic diseases especially those related to the cardiovascular system. The present study examines the organogenic competence of various combinations and concentrations of plant growth regulators to develop an efficient protocol for in vitro regeneration of S. bulleyana via leaf explants, maintaining the high production of active constituents. The purpose of the study was also to assess the possibilities of using a cytokinin-based regeneration to effectively produce therapeutic compounds. The adventitious shoot formation was observed through direct organogenesis on media with purine derivatives (meta-topolin, mT and benzylaminopurine, BAP), and through indirect organogenesis on media with urea derivatives (tidiazuron, TDZ and forchlorfenuron, CPPU). The highest regeneration frequency (95%) with 5.2 shoots per explant was obtained on leaves cultured on Murashige and Skoog (MS) medium containing 0.1 mg/L naphthalene-1-acetic acid (NAA) and 2 mg/L BAP. Following inter simple sequence repeat (ISSR) marker-based profiling, the obtained organogenic shoot lines revealed a similar banding pattern to the mother line, with total variability of 4.2-13.7%, indicating high level of genetic stability. The similar genetic profile of the studied lines translated into similar growth parameters. Moreover, HPLC analysis revealed no qualitative differences in the profile of bioactive metabolites; also, the total polyphenol content was similar for different lines, with the exception of the shoots obtained in the presence of CPPU that produced higher level of bioactive compounds. This is the first report of an effective and rapid in vitro organogenesis protocol for S. bulleyana, which can be efficiently employed for obtaining stable cultures rich in bioactive metabolites.