Cytotoxicity against A549 Human Lung Cancer Cell Line via the Mitochondrial Membrane Potential and Nuclear Condensation Effects of Nepeta paulsenii Briq., a Perennial Herb.

The genus Nepeta belongs to the largest Lamiaceae family, with 300 species, which are distributed throughout the various regions of Africa, Asia, India, and America. Along with other plant families distinguished by their medicinal and therapeutic values, the Nepeta genus of Lameaceae remains relatively valuable. Hence, the phytochemicals of N. paulsenii Briq. were extracted using different plant parts, i.e., leaves, stem, roots, flowers, and the whole plant by using various solvents (ethanol, water, and ethyl acetate), obtaining 15 fractions. Each extract of dried plant material was analyzed by FT-IR and GC-MS to identify the chemical constituents. The cytotoxicity of each fraction was analyzed by MTT assay and mitochondrial membrane potential and nuclear condensation assays against lung cancer cells. Among the ethyl acetate and ethanolic extracts, the flowers showed the best results, with IC50 values of 51.57 µg/mL and 50.58 µg/mL, respectively. In contrast, among the water extracts of the various plant segments, the stem showed the best results, with an IC50 value of 123.80 µg/mL. 5-flourouracil was used as the standard drug, providing an IC50 value of 83.62 µg/mL. The Hoechst 33342 stain results indicated apoptotic features, i.e., chromatin dissolution and broken down, fragmented, and crescent-shaped nuclei. The ethanolic extracts of the flowers showed more pronounced apoptotic effects on the cells. The mitochondrial membrane potential indicated that rhodamine 123 fluorescence signals suppressed mitochondrial potential due to the treatment with the extracts. Again, the apoptotic index of the ethanolic extract of the flowers remained the highest. Hence it can be concluded that the flower part of N. paulsenii Briq. was found to be the most active against the A459 human lung cancer cell line.

Study of genetic modifications of flower development and methylation status in phytoplasma infected Brassica (Brassica rapa L.).

BACKGROUND: The plants of B. rapa (syn. B. campestris) are the most important food crop of Pakistan for the production of cooking oil. Brassica plants infected by phytoplasma exhibit floral abnormalities including phyllody, virescence, hypertrophied sepal and aborted reproductive organs and affected flower developmental genes which reduces the yield manifold. METHODS AND RESULTS: The expression level of flower developmental genes in healthy and phytoplasma infected brassica were compared by using semi-quantitative reverse transcription polymerase chain reaction and DNA hybridization. In infected brassica, LEAFY (LFY) gene, controlling the development and maintenance of floral organ, and directly involved in controlling the homeotic gene expression was affected, while APETALA2, regulate the production of sepals and petals, were not altered. Whereas the genes WUSCHEL, APETALA3 and AGAMOUS, were significantly down-regulated, that were responsible for the identity of shoot and central meristem, petals and stamens production, and stamens and carpels development, respectively. The GLUB gene, controlling the production of ß-1,3-glucanases enzyme, was highly up-regulated. According to DNA hybridization results, AGAMOUS and APETALA3 were restricted to floral organs territories in healthy and phytoplasma infected brassica, indicating that their expression was tissue-specific. These outcomes indicated that flower abnormalities of phytoplasma infected B. rapa are linked with DNA methylation in the expression of homeotic genes regulating flower development. CONCLUSIONS: Azacitidine act as a DNA demethylating reagent. By applying the foliar spray of azacitidine during the flower development, cells of Phytoplasma infected plants exhibits demethylation of DNA when treated with azacitidine chemical that incorporated as analogue of cytosine during the cell division stage. B. rapa showed the up-regulation of gene expression level significantly that restore the normal production of flowers, ultimately increase the oil production throughout the world.

Feeding preference of Rhynchophorus ferrugineus (Oliver) (Coleoptera: Curculionidae) on different date palm cultivars and host biochemical responses to its infestation.

To counter the insect infestation, plants respond with wide-ranging and highly dynamic biochemical reactions. Of these, the anti-oxidative activity is poorly understood. The red palm weevil (RPW) Rhynchophorus ferrugineus (Oliver), one of the most widespread pests in Pakistan, prefers to infest date palm Phoenix dactylifera. Our present study investigated the feeding preference of RPW to 11 different date palm cultivars and the results suggested that the Hillawi cultivar was most preferred. Greater infestation rate, fecundity and hatching rate were also recorded from Hillawi and Mozawati than other cultivars. No significant decreases were observed in chlorophyll a, chlorophyll b, total chlorophylls and carotenoids of RPW-infested Hillawi cultivar over un-infested control. In contrast, the contents of enzymatic antioxidants including phenols, proline, hydrogen peroxide, anthocyanin, malondialdehyde, ascorbic acid and glycine betaine showed a drastic increase after RPW infestation, and there was enhanced superoxide dismutase, peroxidase and catalase activities. Furthermore, we recorded the increase of total protein and sugar contents in RPW-infested date palms. These findings offer valuable insight into the antioxidative molecular mechanism of date palms under RPW attack and may contribute to the breeding of insect-resistant crops.

Characterization of Ocimum basilicum synthesized silver nanoparticles and its relative toxicity to some insecticides against tobacco cutworm, Spodoptera litura Feb. (Lepidoptera; Noctuidae).

Tobacco cutworm, Spodoptera litura Feb. (Lepidoptera; Noctuidae) is a notorious cosmopolitan pest of numerous crops. Frequent evolution of insecticide resistance and polyphagous nature favors the survival of this voracious pest. Nanotechnology offers an alternative technique to overcome the limitations of existing pest management strategies. In the present study, the silver nanoparticles were synthesized from Ocimum basilicum leaf extract and UV-spectra at 464.17 nm confirmed their synthesis while crystalline nature of nanoparticles was evaluated by X-rays diffraction (XRD) pattern and scanning electron microscopy (SEM) revealed their octagonal to spherical shape. Insecticidal potential of O. basilicum synthesized silver nanoparticles (ObAgNPs) was evaluated in comparison with some synthetic insecticides owning different mode of actions, including Coragen®, Proclaim®, Tracer® and Talstar® against S. litura. Probit analysis of 2nd, 3rd, 4th and 5th instars of S. litura showed that lethal concentrations (LCs) of tested nanoparticles and pesticides were positively correlated with older instars. All values of LC50, LC90 and LC95 were statistically different from each other with maximum on the 4th instar and minimum on 2nd instar of S. litura. Tukey's HSD test revealed that ObAgNPs caused maximum mortality (21.67-96.67%) in 2nd instar larvae at the rate of 100-1500 mg/L followed by Coragen® (18.33-91.67%), Proclaim® (13.33-78.33%) and Talstar® (13.33-68.33%), while Tracer indicated the lowest larval mortality (11.67-66.67%) at the rate of 60-120 mg/L and a similar trend was assessed for all the successive instars of S. litura. Higher activity of carboxyl esterase and glutathione-s-transferases confirmed the development of insecticide resistance against the synthetic chemicals. Conclusively, ObAgNPs were found to be eco-friendly but the most effective biogenic compounds for the suppression of S. litura population as compared to the selected synthetic chemicals which may be hazardous for the environment as well as human health. This study will assist the pesticide industries to re-standardize the dose rates against Spodoptera litura.

The Imaging of Guard Cells of thioglucosidase (tgg) Mutants of Arabidopsis Further Links Plant Chemical Defence Systems with Physical Defence Barriers.

The glucosinolate-myrosinase system is a well-known plant chemical defence system. Two functional myrosinase-encoding genes, THIOGLUCOSIDASE 1 (TGG1) and THIOGLUCOSIDASE 2 (TGG2), express in aerial tissues of Arabidopsis. TGG1 expresses in guard cells (GCs) and is also a highly abundant protein in GCs. Recently, by studying wild type (WT), tgg single, and double mutants, we showed a novel association between the glucosinolate-myrosinase system defence system, and a physical barrier, the cuticle. In the current study, using imaging techniques, we further analysed stomata and ultrastructure of GCs of WT, tgg1, tgg2 single, and tgg1 tgg2 double mutants. The tgg mutants showed distinctive features of GCs. The GCs of tgg1 and tgg1 tgg2 mutants showed vacuoles that had less electron-dense granular material. Both tgg single mutants had bigger stomata complexes. The WT and tgg mutants also showed variations for cell wall, chloroplasts, and starch grains of GCs. Abscisic acid (ABA)-treated stomata showed that the stomatal aperture was reduced in tgg1 single and tgg1 tgg2 double mutants. The data provides a basis to perform comprehensive further studies to find physiological and molecular mechanisms associated with ultrastructure differences in tgg mutants. We speculate that the absence of myrosinase alters the endogenous chemical composition, hence affecting the physical structure of plants and the plants' physical defence barriers.

Resistance status of Helicoverpa armigera against Bt cotton in Pakistan.

Transgenic cotton expressing the toxin Cry1Ac from Bacillus thuringiensis L. (Bt) is widely cultivated in Pakistan after its formal approval in 2010. The exposure of the local target pests to the Cry1Ac endotoxin for this duration might have changed the baseline susceptibility. To probe the status of resistance in one of the main target pests, Helicoverpa armigera, field-collected larvae were reared in the lab for conducting leaf fed bioassays. Twenty-six cotton accessions collected from farmers, including 25 Bt-cotton and one non-Bt, were tested to quantify the level of Cry1Ac, an insecticidal crystalline protein (ICP), in leaves of lower, middle and upper canopies of plants. The concentration of ICP was tested through Enzyme-linked Immunosorbent Assay and found significantly variable (P < 0.01) between and within accessions. The highest mean expression was observed in Accession-2 and Accession-4, while the lowest in Accession-21 and Accession-19. Among fresh leaf tissues from different parts of the plant, the highest mean expression was recorded at 60 days after sowing in upper canopy leaves of cotton accessions, which decreased in lower parts of the plant with the lowest mean expression in lower canopy leaves. Laboratory bioassays, to calculate lethal dose, for H. armigera showed that LD50 and LD95 were 0.62 µg/g and 1.59 µg/g of fresh tissue weight, respectively. A strong positive correlation also exists between the levels of Cry1Ac protein and insect mortality (r = 0.84). These findings suggested the future risk of cultivation of Bt cotton, carrying single Cry1Ac gene, in Pakistan, as resistance surging in H. armigera against Cry protein. These results may also have significant implications for the resistance management in Bt crops, especially cotton, in future.