Effects of polystyrene nanoplastics exposure on in vitro-grown Stevia rebaudiana plants.

Nanoplastics (NPs) as environmental contaminants have received increased attention in recent years. Numerous studies have suggested possible negative effects of plants exposure to NPs, but more data are needed with various plants under different exposure conditions to clarify the underlying phytotoxicity mechanisms. In this study, we investigated the effect of polystyrene nanoplastics (PSNPs; 28.65 nm average diameter) exposure (10, 100 and 250 mg/L) on plant morphology and production of relevant metabolites (steviol glycosides, chlorophylls, carotenoids, and vitamins) of in vitro-grown Stevia rebaudiana plantlets. Additionally, we used dark field microscopy combined with fluorescence hyperspectral imaging for the visualization of internalized PSNPs inside plant tissues. At higher concentrations (>100 mg/L), PSNPs were shown to aggregate in roots and to be transported to leaves, having a significantly negative impact on plant growth (reduced size and biomass), while increasing the production of metabolites compared to controls, most probably because of response to stress. The production of steviol glycosides presented a biphasic dose-response suggestive of hormesis, with the highest values at 10 mg/L PSNPs (1.5-2.2-fold increase compared to controls), followed by a decline in production at higher concentrations (100 and 250 mg/L), but with values comparable to controls. These results are promising for future in vivo studies evaluating the effect of NP exposure on the production of steviol glycosides, the natural sweeteners from stevia.

Gadolinium Accumulation and Toxicity on In Vitro Grown Stevia rebaudiana: A Case-Study on Gadobutrol.

Gadolinium-based contrast agents are molecular complexes which are extensively used for diagnostic purposes. Apart from their tremendous contribution to disease diagnostics, there are several issues related to their use. They are extremely stable complexes and potential contaminants of surface and ground waters, an issue which is documented worldwide. The irrigation of fields with contaminated surface waters or their fertilization with sludge from wastewater treatment plants can lead to the introduction of Gd into the human food supply chain. Thus, this study focused on the potential toxicity of Gd on plants. For this purpose, we have studied the molecular effects of gadobutrol (a well-known MRI contrast agent) exposure on in vitro-grown Stevia rebaudiana. The effects of gadobutrol on plant morphology, on relevant plant metabolites such as chlorophylls, carotenoids, ascorbic acids (HPLC), minerals (ICP-OES), and on the generation of free radical species (MDA assay and EPR) were assessed. Exposures of 0.01, 0.05, 0.1, 1, and 3 mM gadobutrol were used. We found a correlation between the gadobutrol dose and the plant growth and concentration of metabolites. Above the 0.1. mM dose of gadobutrol, the toxic effects of Gd+3 ions became significant.

The effect of 100-200 nm ZnO and TiO2 nanoparticles on the in vitro-grown soybean plants.

Engineered nanomaterials are increasingly used in everyday life applications and, in consequence, significant amounts are being released into the environment. From soil, water, and air they can reach the organelles of edible plants, potentially impacting the food chain and human health. The potential environmental and health impact of these nanoscale materials is of public concern. TiO2 and ZnO are among the most significant nanomaterials in terms of production amounts. Our study aimed at evaluating the effects of large-scale TiO2 (~100 nm) and ZnO (~200 nm) nanoparticles on soybean plants grown in vitro. The effect of different concentrations of nanoparticles (10, 100, 1000 mg/L) was evaluated regarding plant morphology and metabolic changes. ZnO nanoparticles showed higher toxicity compared to TiO2 in the experimental set-up. Overall, elevated levels of chlorophylls and proteins were observed, as well as increased concentrations of ascorbic and dehydroascorbic acids. Also, the decreasing stomatal conductance to water vapor and net CO2 assimilation rate show higher plant stress levels. In addition, ZnO nanoparticle treatments severely affected plant growth, while TEM analysis revealed ultrastructural changes in chloroplasts and rupture of leaf cell walls. By combining ICP-OES and TEM results, we were able to show that the nanoparticles were metabolized, and their internalization in the soybean plant tissues occurred in ionic forms. This behavior most likely is the main driving force of nanoparticle toxicity.

Soybean Callus-A Potential Source of Tocopherols.

In vitro cultures have been used as an effective means to achieve a high level of secondary metabolites in various plants, including soy. In this study, the contents of α-, γ-, and δ- tocopherol were quantified in soybean callus, and their amounts were compared to those of soybeans cultivated using the conventional tillage system with three weed controls (respectively without herbicide and with two variants of herbicide). Soybean callus was produced using Murashige and Skoog 1962 (MS) medium supplemented with 0.1 mg/L 6-Benzylaminopurine (BAP) and 0. 1 mg/L Thidiazuron (TDZ). The highest amount of fresh callus was obtained from soybeans from the conventional tillage system with second weed control (S-metolachlor 960 g/L, imazamox 40 g/L, and propaquizafop 100 g/L) respectively 13,652.4 ± 1177.62 mg. The analyzed tocopherols were in much higher content in soy dry callus than the soybean seeds (5.63 µg/g compared with the 0.35 α-toco in soybean, 47.57 µg/g compared with 18.71 µg/g γ-toco or, 5.56 µg/g compared with 1.74 µg/g ß-toco). The highest content of the three analyzed tocopherols was γ -tocopherol, both in callus and soybeans. Furthermore, the data showed that herbicides used in soybean culture significantly influenced both the in vitro callus production and the tocopherol callus content (p ˂ 0.05). Altogether, soybean callus can be an important source of tocopherols, and herbicides significantly influence in vitro callus production and the tocopherol callus content.

Micropropagation, Genetic Fidelity and Phenolic Compound Production of Rheum rhabarbarum L.

An efficient micropropagation protocol for Rheum rhabarbarum L. was developed in this study. The in vitro rhubarb plants obtained in the multiplication stage (proliferation rate: 5.0 ± 0.5) were rooted in vitro (96% rooting percentage) and acclimatized ex vitro in floating perlite, with 90% acclimatization percentage. To assess the genetic fidelity between the mother plant and in vitro propagated plants, sequence-related amplified polymorphism (SRAP) markers were used. All banding profiles from the micropropagated plants were monomorphic and similar to those of the mother plant indicating 100% similarity. Regarding the polyphenolic profile, gallic, protocatechuic, p-hydroxybenzoic, vanillic, chlorogenic, caffeic, syringic, p-coumaric and ferulic acid were present in different amounts (2.3-2690.3 µg g-1 dry plant), according to the extracted matrix. Aglicons and glycosides of different classes of flavonoids were also identified. The rhizome extracts (both from in vitro and field grown plants) contained resveratrol, a stilbene compound with high antioxidant properties, ranging between 229.4 to 371.7 µg g-1 plant. Our results suggest that in vitro propagation of Rheum rhabarbarum L. represents a reliable alternative to obtain a large number of true-to-type planting material with high bioactive compound content of this valuable nutritional and medicinal species.