Stevia rebaudiana Bertoni grown under ZnO and CuO nanoparticles stress -

Stevia rebaudiana Bertoni grown under ZnO and CuO nanoparticles stress

This study reports the inoculation of ZnO and CuO nanoparticles in Murashige and Skoog (MS) medium having plant growth regulators for the regeneration of callus from leaf explants of medicinal plant, Stevia rebaudiana. Presence of ZnO and CuO nanoparticles in different concentrations results in conferring different kinds of physiology in different regenerants. 1 and 10 mg/L have been declared the best ZnO and CuO nanoparticles concentrations regarding various physiological parameters. The phytochemical characteristics of S. rebaudiana under different ZnO and CuO nanoparticles concentrations have been exploited. However, the highest TRP in the context of ZnO and the highest TFC regarding CuO have been achieved at 50 and 100 mg/L, respectively. This clearly indicates that CuO nanoparticles are more toxic to Stevia callus as compared to ZnO nanoparticles, and opens avenues for future studies utilizing ZnO or CuO nanoparticles for the enhancement of commercially important secondary metabolites in different medicinal plants.

Callus establishment under ZnO and CuO nanoparticles stress

The seeds of Stevia rebaudiana were surface-disinfected with 0.1% (w/v) mercuric(II) chloride (HgCl₂) and cultured on MS medium (Murashige and Skoog 1962) containing 3% (w/v) sucrose and 0.8% (w/v) agar. Later on, leaf explants (0.5 cm²) were excised from 30-day old in vitro-germinated seedlings, and cultured on MS medium supplemented with a combination of plant growth regulators (PGRs); 0.5 mg/L of kinetin (KIN) and 0.5 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D), and different concentrations of ZnO (34 nm in size) and CuO (47 nm in size) nanoparticles: 0.1, 1.0, 10 and 100 mg/L. The ZnO and CuO nanoparticles used were produced by co-precipitation method and were completely characterized before use (The cultures were maintained at 25 ± 1 °C under a 16/8-h (light/dark) photoperiod (50 lmol/m²/s irradiance by fluorescent lamps). The callus was harvested after 42 days of culture period and oven-dried to perform antioxidant assays.

Extraction and determination of steviol glycosides

Steviol glycosides had to be extracted from the callus grown under ZnO and CuO stress. In this process, the callus from different treatments was carefully washed with sterile distilled water. Then, the callus was soaked on filter paper and dried in oven at 60 °C for 48 h. The steviol glycosides determination was conducted by high-performance liquid chromatography (HPLC) of samples. The procedure involved the addition of 20 mg of sample from each treatment to 1 mL of 70% (v/v) methanol in a micro centrifuge tube. After incubation in an ultrasonic bath at 55 °C for 15 min, samples were centrifuged at 25 °C and 12,000 rpm for 10 min. The pellet was discarded, and supernatant was filtered using 0.22-lm PTFE Millipore syringe filters. Finally, HPLC analysis was performed by running all of samples in triplicate.

Extract preparation and estimation of antioxidant activities

The callus samples were subjected to extraction. Briefly, a powdered callus sample (0.1 g) was dissolved in 500 mL of aqueous methanol, and vortexed (5 min), sonicated (30 min) and centrifuged (10,000 rpm for 15 min). The supernatant was collected and syringe-filtered, and a final dilution of 4 mg/mL was prepared in dimethyl sulfoxide (DMSO).  The total phenolic content (TPC) was determined according to the Folin-Ciocalteu method. The results were expressed as micrograms of gallic acid equivalent (GAE) per milliliter. The total flavonoid content (TFC) was determined by the aluminum trichloride (AlCl₃) method. The results were expressed as micrograms of quercetin equivalent (QE) per milliliter. Total antioxidant capacity (TAC) and Total Reducing Power (TRP) was also performed and the results were expressed as micrograms of ascorbic acid equivalent (AAE) per milliliter. DPPH Free Radical Scavenging Activity: Free radical scavenging activity (FRSA) of callus samples was investigated in terms of hydrogen-donating or radical scavenging ability. Ascorbic acid and DMSO were used as positive and negative controls, respectively. The results were expressed as micrograms of ascorbic acid equivalent (AAE) per milliliter.

In the presence of CuO nanoparticles, callus texture is good, and at some concentrations, green callus is produced, while in the presence of ZnO, mostly yellow colored callus induction occurs from leaf explants. In the present study, an oxidative stress induced by the ZnO and CuO nanoparticles creates toxic free radicals in S. rebaudiana. This stress increases by increasing the concentration of ZnO or CuO nanoparticles due to the formation of reactive oxygen species (ROS), shown by the enhanced antioxidant activities of the callus tissue as a response. However, after reaching a certain threshold, different physiological parameters of callus and production of antioxidants begin to decline. The role of ZnO and CuO nanoparticles as abiotic elicitors of callus physiology and secondary metabolites production from the callus of Stevia rebaudiana is a new frontier in abiotic stress elicitation. These nanoparticles impose oxidative stress to the callus tissues grown by tissue culture, which trigger their metabolism and result in the enhancement of all antioxidant activities. The callus of Stevia rebaudiana has certain valuable active metabolic constituents that are involved in free radical scavenging. These chemicals are responsible for neutralization of toxic free radicals, hence preventing excessive oxidation reactions. The current study reveals the influence of ZnO and CuO nanoparticles on the free radical scavenging activity and other antioxidant activities of S. rebaudiana callus. This study suggests that the callus of S. rebaudiana, provided either with ZnO and CuO nanoparticles, can be used as a source of antioxidants. This study paves the way for conducting further such cost-efficient and easy experiments using different nanoparticles and medicinal plants in order to obtain elevated levels of antioxidants to be used in cure of various diseases. These findings clearly indicated that the antioxidant potential of callus tissues was significantly enhanced by means of ZnO or CuO nanoparticles stress until it reached 50 mg/L or 100 mg/L because the activities were declined thereafter. It can be concluded from these experiments that the secondary metabolism of Stevia callus cultures scavenges more free radicals when the concentration of nanoparticles increases up to a certain limit. Most importantly, all antioxidant activities were zero in the case of 1000 mg/L of ZnO and CuO nanoparticles exposure because the leaf tissue died by such a higher exposure in the context of ZnO, and although the leaf tissue was alive regarding CuO, it did not produce a significant amount of callus to be phytochemically analyzed.

Reference:

Javed, R., Yucesan, B., Zia, M. and Gurel, E., 2018. Elicitation of secondary metabolites in callus cultures of Stevia rebaudiana Bertoni grown under ZnO and CuO nanoparticles stress. Sugar Tech, 20(2), pp.194-201.