Drought is a major environmental constrain with a deleterious effect on plant development leading to a considerable reduction of crop productivity worldwide. Wheat is a relatively drought tolerant crop during the vegetative stage. The herbicide Serrate^® (Syngenta) is a preparation containing two active chemical substances with different modes of action, which inhibit the biosynthesis of fatty and amino acids. It is commonly used as a systemic and selective chemical agent to control annual grass and broadleaf weeds in cereal crops and particularly in wheat, which is tolerant to Serrate^®. Seventeen-day-old wheat seedlings (Triticum aestivum L., cv. Sadovo-1) grown as soil culture under controlled conditions were sprayed with an aqueous solution of Serrate^®. Seventy-two hours later the plantlets were subjected to drought stress for seven days to reach a severe water deficit followed by four days of recovery with a normal irrigation regime. Oxidative stress markers, non-enzymatic, and enzymatic antioxidants were analyzed in the leaves of plants from the different treatment groups (herbicide-treated, droughts-stressed, and individuals which were consecutively subjected to both treatments) at 0, 96, and 168 h of drought stress, and after 96 h of recovery. Herbicide treatment did not alter substantially the phenotype and growth parameters of the above-ground plant parts. It provoked a moderate increase in phenolics, thiol-containing compounds, catalase, superoxide dismutase, glutathione reductase, and H₂O₂. However, significant variations of malondialdehyde, proline, and peroxidase activity caused by the sole application of the herbicide were not detected during the experimental period. Drought and herbicide + drought treatments caused significant growth inhibition, increased oxidative stress markers, and activation of enzymatic and non-enzymatic antioxidant defense reaching the highest levels at 168 h of stress. Plant growth was restored after 96 h of recovery and the levels of the monitored biochemical parameters showed a substantial decline. The herbicide provoked an extra load of oxidative stress-related biochemical components which did not aggravate the phenotypic and growth traits of plants subjected to drought, since they exhibited a good physiological status upon recovery.

The potential of brassinosteroids to modulate the physiological responses of winter wheat (Triticum aestivum L.) to herbicide stress was evaluated. Young winter wheat seedlings were treated with 24-epibrassinolide (EBL) and 24 h later were sprayed with glyphosate. The physiological responses of treated plants were assessed 14 days after herbicide application. Wheat growth was noticeably inhibited by glyphosate. The herbicide application significantly increased the content of the stress markers proline and malondialdehyde (MDA) evidencing oxidative damage. The content of phenolic compounds was decreased in the herbicide-treated plants. Slight activation of superoxide dismutase (SOD) and catalase (CAT) and considerable increase of glutathione reductase (GR) and guaiacol peroxidase (POX) activities were found. Increased POX and glutathione S-transferase (GST) activities were anticipated to be involved in herbicide detoxification. Conjugation with glutathione in herbicide-treated plants could explain the reduction of thiols suggesting unbalanced redox state. EBL application did not alter the plant growth but a moderate activation of antioxidant defense (POX, GR, and CAT activities and phenolic levels) and detoxifying enzyme GST was observed. The hormonal priming provoked a slight decrease in MDA and proline levels. The results demonstrate that EBL-pretreatment partly restored shoot growth and has a potential to mitigate the oxidative damages in glyphosate-treated plants through activation of the enzymatic antioxidant defense and increase of the phenolic compounds.

Clinopodium vulgare L. is a valuable medicinal plant used for its anti-inflammatory, antibacterial and wound-healing properties. The present study describes an efficient protocol for the micropropagation of C. vulgare and compares, for the first time, the chemical content and composition and antitumor and antioxidant activities of extracts from in vitro cultivated and wild-growing plants. The best nutrient medium was found to be Murashige and Skoog (MS) supplemented with 1 mg/L BAP and 0.1 IBA mg/L, yielding on average 6.9 shoots per nodal segment. Flower aqueous extracts from in vitro plants had higher total polyphenol content (29,927.6 ± 592.1 mg/100 g vs. 27,292.8 ± 85.3 mg/100 g) and ORAC antioxidant activity (7281.3 ± 82.9 µmol TE/g vs. 7246.3 ± 62.4 µmol TE/g) compared to the flowers of wild plants. HPLC detected qualitative and quantitative differences in phenolic constituents between the in vitro cultivated and wild-growing plants’ extracts. Rosmarinic acid was the major phenolic constituent, being accumulated mainly in leaves, while neochlorogenic acid was a major compound in the flowers of cultivated plants. Catechin was found only in cultivated plants, but not in wild plants or cultivated plants’ stems. Aqueous extracts of both cultivated and wild plants showed significant in vitro antitumor activity against human HeLa (cervical adenocarcinoma), HT-29 (colorectal adenocarcinoma) and MCF-7 (breast cancer) cell lines. The best cytotoxic activity against most of the cancer cell lines, combined with the least detrimental effects on a non-tumor human keratinocyte cell line (HaCaT), was shown by the leaf (250 µg/mL) and flower (500 µg/mL) extracts of cultivated plants, making cultivated plants a valuable source of bioactive compounds and a suitable candidate for anticancer therapy.

Arnica montana L. is an important herbal medicinal plant that belongs to the family Asteraceae. This plant has been known for its medicinal uses for centuries. A. montana exhibits several pharmacological properties, including immunomodulatory, anti-inflammatory, anticancer, antioxidant, and antibacterial effects. For the first time, the impacts of the biotic elicitor yeast extract, and the abiotic elicitor salicylic acid on micropropagation, antioxidant potential, and accumulation of caffeoylquinic acids in arnica in vitro shoots were assessed. The results showed that yeast extract applied at 100 mg/L significantly promotes shoot multiplication, biomass yield, total phenolic content, and synthesis of caffeoylquinic acids compared to control untreated shoots. Flavonoid content was the highest in samples treated with 200 mg/L of yeast extract, although at this concentration the measured biometric parameters began to decrease. Salicylic acid at 100 µM was found to be effective in the induction of vigorous shoots, shoot height growth, and biomass accumulation; nevertheless, this elicitor downregulated the caffeoylquinic acid level, total phenolics, and flavonoids. Increasing the concentration of salicylic acid to 200 µM caused shoot multiplication and fresh biomass accumulation reduction. Both elicitors modulated the activity of antioxidant enzymes against oxidative stress. Overall, the use of these substances can improve the growth and biomass yield in Arnica in vitro shoots.

Waterlogging impairs crop development and considerably affects plant productivity worldwide. Wheat is sensitive to waterlogging. Serrate^® (Syngenta) is a selective herbicide controlling annual grass and broadleaf weeds for use in wheat. To extend the existing information about the physiological effects of selective herbicides (Serrate^® in particular) and subsequent waterlogging in wheat, we monitored phenotype alterations and examined key enzymatic and non-enzymatic antioxidant defense systems together with typical oxidative stress biomarkers. Seventeen-day-old wheat (Triticum asetivum L., cv. Sadovo-1) plants were sprayed with Serrate^®; 72 h later, waterlogging was applied for 7 days, and then seedlings were left to recover for 96 h. The herbicide did not alter plant phenotype and increased antioxidant defense, along with H₂O₂ content, confirming the wheat’s tolerance to Serrate^®. Evident yellowing and wilting of the leaves were observed at 96 h of recovery in waterlogged wheat, which were stronger in plants subjected to Serrate^® + waterlogging. Waterlogging alone and herbicide + waterlogging gradually enhanced the content of stress markers (malondialdehyde, proline, and H₂O₂), non-enzymatic antioxidants (low-molecular thiols and total phenolics), and the activity of superoxide dismutase, guaiacol peroxidase, and glutathione reductase. The effects of herbicide + waterlogging were stronger than those of waterlogging alone even during recovery, suggesting that Serrate^® interacted synergistically with the subsequently applied flooding.

Sideritis scardica Griseb. is a critically endangered Balkan endemic species, known for its antioxidant, neuroprotective and anti-inflammatory properties. The aim of the present study was to detail an efficient protocol for the micropropagation of S. scardica. In vitro cultures were initiated from the shoot tips of 40 days-old in vivo seedlings and the effects of different plant growth regulator treatments were examined. A Murashige and Skoog nutrient medium (MS) containing 1 mg/L zeatin and 0.1 mg/L indole-3-acetic acid (IAA) proved to be the most efficient for shoot multiplication as it produced quality, vigorous shoots with a mean number of six shoots per explant. For the first time, the antioxidant and antitumor activities of extracts from in vitro-obtained plants were evaluated. In vitro cultivated plants grown in the field revealed a higher total polyphenol content (3929.1 ± 112.2 mg GAE/100 g vs. 3563.5 ± 52.8 mg GAE/100 g) and higher ORAC antioxidant activity (1211.6 ± 27.3 µmol TE/g vs. 939.9 ± 52.4 µmol TE/g) than in situ cultivated plants. A comparison of the antitumor activities of extracts from in vitro propagated shoots, field-grown in vitro-obtained plants and in situ plants on HeLa (cervical adenocarcinoma), HT-29 (colorectal adenocarcinoma) and MCF-7 (breast cancer) human cancer cell lines showed that in vitro propagated shoots had a significant concentration-dependent cytotoxic effect on the cervical adenocarcinoma cell line HeLa, while the field-grown in vitro-obtained and in situ-collected samples induced the highest reduction in the viability of the mammary carcinoma cell line MCF-7. In both cases, the cells of the control non-tumor cell line, BALB/3T3, were significantly less affected. The results showed that the in vitro multiplication protocol ensured the obtainment of numerous plants with antioxidant and antitumor potential.