Elicitation of Submerged Adventitious Root Cultures of Stevia rebaudiana with Cuscuta reflexa for Production of Biomass and Secondary Metabolites

Stevia rebaudiana is an important medicinal plant that belongs to the Asteraceae family. The leaves of Stevia rebaudiana are a rich source of many health-promoting agents such as polyphenols, flavonoids, and steviol glycoside, which play a key role in controlling obesity and diabetes. New strategies such as the elicitation of culture media are needed to enhance the productivity of active components. Herein, the Cuscuta reflexa extracts were exploited as elicitors to enhance the productivity of active components. Cuscuta reflexa is one of the parasitic plants that has the ability to elongate very fast and cover the host plant. Consequently, it may be possible that the addition of Cuscuta reflexa extracts to adventitious root cultures (ADR) of Stevia rebaudiana may elongate the root more than control cultures to produce higher quantities of the desired secondary metabolites. Therefore, the main objective of the current study was to investigate the effect of Cuscuta reflexa extract as a biotic elicitor on the biomass accumulation and production of antioxidant secondary metabolite in submerged adventitious root cultures of Stevia rebaudiana. Ten different concentrations of Cuscuta reflexa were added to liquid media containing 0.5 mg/L naphthalene acetic acid (NAA). The growth kinetics of adventitious roots was investigated for a period of 49 days with an interval of 7 days. The maximum biomass accumulation (7.83 g/3 flasks) was observed on medium containing 10 mg/L extract of Cuscuta reflexa on day 49. As the concentration of extract increases in the culture media, the biomass gradually decreases after 49 days of inoculation. In this study, the higher total phenolics content (0.31 mg GAE/g-DW), total flavonoids content (0.22 mg QE/g-DW), and antioxidant activity (85.54%) were observed in 100 mg/L treated cultures. The higher concentration (100 mg/L) of Cuscuta reflexa extract considerably increased the total phenolics content (TPC), total phenolics production (TPP), total flavonoids content (TFC), total flavonoids production (TFP), total polyphenolics content (TPPC), and total polyphenolics production (TPPP). It was concluded that the extract of Cuscuta reflexa moderately improved biomass accumulation but enhanced the synthesis of phenolics, flavonoids, and antioxidant activities. Here, biomass’s independent production of secondary metabolites was observed with the addition of extract. The present study will be helpful to scale up adventitious roots culture into a bioreactor for the production of secondary metabolites rather than biomass accumulation in medicinally important Stevia rebaudiana.

Enhanced Aryltetralin Lignans Production in Linum Adventi-Tious Root Cultures

Lignans are the main secondary metabolites synthetized by Linum species as plant defense molecules. They are also valuable for human health, in particular, for their potent antiviral and antineoplastic properties. In this study, the adventitious root cultures of three Linum species (L. flavum, L. mucronatum and L. dolomiticum) were developed to produce aryltetralin lignans. The effect of two elicitors, methyl jasmonate and coronatine, on aryltetralin lignans production was also evaluated. The adventitious root cultures from L. dolomiticum were obtained and analyzed for the first time and resulted as the best producer for all the aryltetralins highlighted in this system: Podophyllotoxin, 6-methoxypodophyllotoxin and 6-methoxypodophyllotoxin-7-O-β-glucoside, the last showing a productivity of 92.6 mg/g DW. The two elicitors differently affected the production of the 6-methoxypodophyllotoxin and 6-methoxypodophyllotoxin-7-O-β-glucoside.

Effect of sucrose on the fatty acid metabolism of adventitious root cultures in vitro of Stevia rebaudiana

In this study, the effect of sucrose on the neutral lipid profile of adventitious root cultures of Stevia rebaudiana was evaluated. The cultures were obtained employing a roller bottle system. In this system, Schott-type flasks were used, which contained Murashige and Skoog liquid medium at 33.3% strength (MS/3) supplemented with 30, 60, and 80 g L-1 of sucrose, respectively, and 10.7 mM 1-naphthaleneacetic acid (NAA). The spectroscopic analyzes showed that the portion of polyunsaturated fatty acids (PUFAs) was highest in roots treated with 30 g L-1 of sucrose. The spectrometric analyzes showed that the palmitic acid was found to be present in relatively higher amounts in the roots submitted to the MS/3-30 g L-1 (31.9%) and MS/3-60 g L-1 (29.5%) sucrose treatments, and lower in the treatment with MS/3-80 g L-1 (28.8%) of sucrose. Also, the treatment using 30 g L-1 of sucrose was the best for obtaining unsaturated fatty acids (UFAs) in the culture, with a relative percentage of 62.9%. Our results indicate that the MS medium that received 30 g L-1 of sucrose induced a lesser abiotic stress condition, which favored PUFAs production in the adventitious root cultures of S. rebaudiana.

L-Phenylalanine Applications and Culture Duration Affect Root Growth and Production of Tropane Alkaloids and Phenolics in Adventitious Root Cultures of Hyoscyamus Niger L.

This study was aimed to determine the effects of different concentrations of L-phenylalanine (0.25, 0.50 and 1.00 mM L-Phe) applied to adventitious root cultures for different culture durations on root growth and production of tropane alkaloids and phenolics in Hyoscyamus niger. For this purpose, adventitious roots obtained from petiole explants of in vitro seedlings were used as plant materials. After adventitious roots were cultured in liquid MS medium, L-Phe was applied to the adventitious roots. Then the roots were harvested on the 1st, 3rd, and 7th day of the applications, fresh and dry root weights, root growth indexes were analysed. It was also examined the contents of tropane alkaloids (hyoscyamine and scopolamine) and phenolics (gallic acid, catechin, epicatechin, vanillin, cinnamic acid, rosmarinic acid, p-coumaric, o-coumaric acid, rutin, caffeic acid, ferulic acid, chlorogenic acid, and quercetin) by HPLC in both adventitious roots and culture medium. As a result, the greatest root growth values were obtained from the roots harvested on the 3rd and 7th days after 0.50 and 1.00 mM L-Phe applications. The maximum scopolamine and hyoscyamine amounts were detected in the cultures with the application of 0.50 mM L-Phe for 3 days. In terms of phenolics, 0.50 mM and 1.00 mM L-Phe application with 1st and 3rd days were selected as the most appropriate L-Phe applications and culture duration. Consequently, it has been determined that in vitro secondary metabolite production may be increased in H. niger root cultures with the selection of appropriate L-Phe concentrations and culture durations.