Cloning of maize chitinase 1 gene and its expression in genetically transformed rice to confer resistance against rice blast caused by Pyricularia oryzae.

Fungal pathogens are one of the major reasons for biotic stress on rice (Oryza sativa L.), causing severe productivity losses every year. Breeding for host resistance is a mainstay of rice disease management, but conventional development of commercial resistant varieties is often slow. In contrast, the development of disease resistance by targeted genome manipulation has the potential to deliver resistant varieties more rapidly. The present study reports the first cloning of a synthetic maize chitinase 1 gene and its insertion in rice cv. (Basmati 385) via Agrobacterium-mediated transformation to confer resistance to the rice blast pathogen, Pyricularia oryzae. Several factors for transformation were optimized; we found that 4-week-old calli and an infection time of 15 minutes with Agrobacterium before colonization on co-cultivation media were the best-suited conditions. Moreover, 300 µM of acetosyringone in co-cultivation media for two days was exceptional in achieving the highest callus transformation frequency. Transgenic lines were analyzed using molecular and functional techniques. Successful integration of the gene into rice lines was confirmed by polymerase chain reaction with primer sets specific to chitinase and hpt genes. Furthermore, real-time PCR analysis of transformants indicated a strong association between transgene expression and elevated levels of resistance to rice blast. Functional validation of the integrated gene was performed by a detached leaf bioassay, which validated the efficacy of chitinase-mediated resistance in all transgenic Basmati 385 plants with variable levels of enhanced resistance against the P. oryzae. We concluded that overexpression of the maize chitinase 1 gene in Basmati 385 improved resistance against the pathogen. These findings will add new options to resistant germplasm resources for disease resistance breeding. The maize chitinase 1 gene demonstrated potential for genetic improvement of rice varieties against biotic stresses in future transformation programs.

Influence of bio fabricated manganese oxide nanoparticles for effective callogenesis of Moringa oleifera Lam.

The use of nanoscale fertilizers to boost crop output has increased in recent years. Nanoparticles (NPs) can stimulate the biosynthesis of bioactive compounds in plants. It is the first report on biosynthesized manganese oxide nanoparticles (MnO-NPs) that mediate in-vitro callus induction of Moringa oleifera. To achieve better biocompatibility the leaf extract of Syzygium cumini was used to synthesize MnO-NPs. Scanning electron microscope SEM revealed spherical shaped morphology of MnO-NPs with an average diameter of 36 ± 0.3 nm. Energy-dispersive X-ray spectroscopy (EDX) depicted the formation of pure MnO-NPs. X-ray diffraction (XRD) and Fourier Transform Infrared (FTIR) authenticate the crystalline structure. UV-visible absorption spectroscopy depicted the activity of MnO-NPs under visible light. The biosynthesized MnO-NPs were concentration-dependent and revealed promising results in callus induction of Moringa oleifera. It was found that MnO-NPs enhance callus production of Moringa oleifera and keep the callus infection free by providing an optimum environment for rapid growth and development. Therefore MnO-NPs synthesized through the green process can be utilized in tissue culture studies. This study concludes that MnO is one of the essential plant nutrients that have tailored nutritive properties at a nano scale.

Biogenic iron oxide nanoparticles enhance callogenesis and regeneration pattern of recalcitrant Cicer arietinum L.

This study is the first report on the biosynthesized iron oxide nanoparticles (IONPs) which mediate in-vitro callus induction and shoot regeneration in economically important recalcitrant chickpea crop (Cicer arietinum L.). Here, we used leaf extract of Cymbopogon jwarancusa for the synthesis of IONPs in order to achieve a better biocompatibility. The bioactive compounds in C. jwarancusa leaf extract served as both reducing and capping agents in the fabrication process of IONPs. Field emission scanning electron microscopy (FE-SEM) revealed rods like surface morphology of IONPs with an average diameter of 50±0.2 nm. Energy-dispersive X-ray spectroscopy (EDS) depicted formation of pure IONPs with 69.84% Fe and 30.16% O2. X-ray diffractometry (XRD) and attenuated total reflectance-fourier transform infrared (ATR-FTIR) validate the crystalline structure, chemical analysis detect the presence of various biomolecular fingerprints in the as synthesized IONPs. UV-visible absorption spectroscopy depicts activity of IONPs under visible light. Thermo-gravimetric analysis (TGA) displayed thermal loss of organic capping around 500°C and confirmed their stabilization. The biosynthesized IONPs revealed promising results in callus induction, shoot regeneration and root induction of chickpea plants. Both chickpea varieties Punjab-Noor 09 and Bittle-98 explants, Embryo axes (EA) and Embryo axes plus adjacent part of cotyledon (EXC) demonstrated dose-dependent response. Among all explants, EXC of Punjab-Noor variety showed the highest callogenesis (96%) and shoot regeneration frequency (88%), while root induction frequency was also increased to 83%. Iron content was quantified in regenerated chickpea varieties through inductively coupled plasma-optical emission spectrometry. The quantity of iron is significantly increased in Punjab-Noor regenerated plants (4.88 mg/g) as compare to control treated plants (2.42 mg/g). We found that IONPs enhance chickpea growth pattern and keep regenerated plantlets infection free by providing an optimum environment for rapid growth and development. Thus, IONPs synthesized through green process can be utilized in tissue culture studies in other important recalcitrant legumes crops.

Green fabricated zinc oxide nanoformulated media enhanced callus induction and regeneration dynamics of Panicum virgatum L.

The current study focuses on the usage of bio synthesized zinc oxide nanoparticles to increase the tissue culture efficiency of important forage grass Panicum virgatum. Zinc being a micronutrient enhanced the callogenesis and regeneration efficiency of Panicum virgatum at different concentrations. Here, we synthesized zinc oxide nanoparticles through Cymbopogon citratus leaves extract to evaluate the effect of zinc oxide nanoparticles on plant regeneration ability in switchgrass. X-ray diffraction (XRD) and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) validate phase purity of green synthesize Zinc oxide nanoparticles whereas, electron microscopy (SEM) has illustrated the average size of particle 50±4 nm with hexagonal rod like shape. Energy dispersive spectroscopy X-ray (EDS) depicted major peaks of Zn (92.68%) while minor peaks refer to Oxygen (7.32%). ZnO-NPs demonstrated the incredibly promising results against callogenesis. Biosynthesized ZnO-NPs at optimum concentration showed very promising effect on plant regeneration ability. Both the explants, seeds and nodes showed dose dependent response and upon high doses exceeding 40 mg/L the results were recorded negative, whereas at 30 mg/L both explants demonstrated 70% and 76% regeneration frequency. The results conclude that ZnO-NPs enhance the plant growth and development and tailored the nutritive properties at nano-scale. Furthermore, eco-friendly approach of ZnO-NPs synthesis is strongly believed to improve in vitro regeneration frequencies in several other monocot plants.

Erratum: Addendum: The Effect of Green Synthesized CuO Nanoparticles on Callogenesis and Regeneration of Oryza sativa L.

[This corrects the article DOI: 10.3389/fpls.2016.01330.].

Chemical profiling and bioactivities on the leaf extracts of Vitex neugundo.

The Vitex negeundo is a widely used medicinal plant which has not been fully investigated in the past. We assessed the in vivo hepatoprotective and in vitro antioxidant, antibacterial, cytotoxicity and anti proliferative study of leaf extracts of V. neugundo. The chemically profiled using HPLC, three flavonoids were quantified and GC-MS analysis revealed the presence of two new compounds those were not reported earlier from the leaf extract of V. neugundo. The animal study was conducted on mice treated with CCl4 using methanolic and chloroform extracts (100, 200 and 300mg/kg b.w), with silymarin as a positive control. Hepatoprotective effects were determined by analyzing blood for liver marker enzymes, direct bilirubins and hematological parameters (RBC, WBC and platelets). The methanolic extract (300mg/kg b.w) has shown the stronger hepatoprotective effects against abnormalities produced by CCl4. The in vivo hepatoprotective effects correlated well with the in vitro antioxidant, cytotoxicity and antiproliferative activities and with high levels of flavonoids and other organic compounds analyzed from plant extracts. The leaf extracts of this plant could be good candidates for lead compound required for the development of antioxidant/anticancer drugs.

Chemical profiling, in vitro and in vivo bioactivities of leaf extracts of Vitex neugundo.

The Vitex negeundo is a widely used medicinal plant which has not been fully investigated in the past. We assessed the in vivo hepatoprotective and in vitro antioxidant, antibacterial, cytotoxicity and antiproliferative study of leaf extracts of V. Neugundo. The chemically profiled using HPLC, three flavonoids were quantified and GC-MS analysis revealed the presence of two new compounds those were not reported earlier. The animal study was conducted on mice treated with CCl4 using methanolic and chloroform extracts (100, 200 and 300 mg/kg b.w), with silymarin as a positive control. Hepatoprotective effects were determined by analyzing blood for liver marker enzymes, direct bilirubins and hematological parameters (RBC, WBC and platelets). The methanolic extract (300 mg/kg b.w) has shown the stronger hepatoprotective effects against abnormalities produced by CCl4. The in vivo hepatoprotective effects correlated well with the in vitro antioxidant, cytotoxicity and antiproliferative activities and with high levels of flavonoids and other organic compounds analyzed from plant extracts. The leaf extracts of this plant could be good candidates for lead compound required for the development of antioxidant/anticancer drugs.

CuO and CeO2 Nanostructures Green Synthesized Using Olive Leaf Extract Inhibits the Growth of Highly Virulent Multidrug Resistant Bacteria.

One of the major challenges of nano-biotechnology is to engineer potent antimicrobial nanostructures (NS) with high biocompatibility. Keeping this in view, we have performed aqueous olive leaf extract mediated one pot facile synthesis of CuO-NS and CeO2-NS. Prepared NS were homogenous, less than 26 nm in size, and small crystallite units as revealed by scanning electron microscopy (SEM) and X-ray diffraction (XRD) analyses. Fourier transform infrared spectroscopy (FTIR) of CuO-NS and CeO2-NS showed typical Cu-O prints around 592-660 cm-1 and Ce-O bond vibrations at 453 cm-1. The successful capping of CuO-NS and CeO2-NS by compounds present in the plant extract was further validated by high performance liquid chromatography (HPLC) and thermal gravimetric analysis (TGA). Active phyto-chemicals from the leaf extract simultaneously acted as strong reducing as well as capping agent in the NS synthesis. NS engineered in the present study showed antibacterial potential at extremely low concentration against highly virulent multidrug-resistant (MDR) gram-negative strains (Escherichia coli, Enterobacter cloacae, Acinetobacter baumannii and Pseudomonas aeruginosa), alarmed by World Health Organization (WHO). Furthermore, CuO-NS and CeO2-NS did not show any cytotoxicity on HEK-293 cell lines and Brine shrimp larvae indicating that the NS green synthesized in the present study are biocompatible.

Optimised synthesis of ZnO-nano-fertiliser through green chemistry: boosted growth dynamics of economically important L. esculentum.

Mounting-up economic losses to annual crops yield due to micronutrient deficiency, fertiliser inefficiency and increasing microbial invasions (e.g. Xanthomonas cempestri attack on tomatoes) are needed to be solved via nano-biotechnology. So keeping this in view, the authors' current study presents the new horizon in the field of nano-fertiliser with highly nutritive and preservative effect of green fabricated zinc oxide-nanostructures (ZnO-NSs) during Lycopersicum esculentum (tomato) growth dynamics. ZnO-NS prepared via green chemistry possesses highly homogenous crystalline structures well-characterised through ultraviolet and visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscope. The ZnO-NS average size was found as small as 18 nm having a crystallite size of 5 nm. L. esculentum were grown in different concentrations of ZnO-NS to examine the different morphological parameters includes time of seed germination, germination percentage, the number of plant leaves, the height of the plant, average number of branches, days count for flowering and fruiting time period along with fruit quantity. Promising results clearly predict that bio-fabricated ZnO-NS at optimum concentration resulted as growth booster and dramatically triggered the plant yield.

Green synthesised zinc oxide nanostructures through Periploca aphylla extract shows tremendous antibacterial potential against multidrug resistant pathogens.

To grapple with multidrug resistant bacterial infections, implementations of antibacterial nanomedicines have gained prime attention of the researchers across the globe. Nowadays, zinc oxide (ZnO) at nano-scale has emerged as a promising antibacterial therapeutic agent. Keeping this in view, ZnO nanostructures (ZnO-NS) have been synthesised through reduction by P. aphylla aqueous extract without the utilisation of any acid or base. Structural examinations via scanning electron microscopy (SEM) and X-ray diffraction have revealed pure phase morphology with highly homogenised average particle size of 18 nm. SEM findings were further supplemented by transmission electron microscopy examinations. The characteristic Zn-O peak has been observed around 363 nm using ultra-violet-visible spectroscopy. Fourier-transform infrared spectroscopy examination has also confirmed the formation of ZnO-NS through detection of Zn-O bond vibration frequencies. To check the superior antibacterial activity of ZnO-NS, the authors' team has performed disc diffusion assay and colony forming unit testing against multidrug resistant E. coli, S. marcescens and E. cloacae. Furthermore, protein kinase inhibition assay and cytotoxicity examinations have revealed that green fabricated ZnO-NS are non-hazardous, economical, environmental friendly and possess tremendous potential to treat lethal infections caused by multidrug resistant pathogens.

Biosynthesis and characterisation of nano-silica as potential system for carrying streptomycin at nano-scale drug delivery.

A growing trend within nanomedicine has been the fabrication of self-delivering supramolecular nanomedicines containing a high and fixed drug content ensuring eco-friendly conditions. This study reports on green synthesis of silica nanoparticles (Si-NPs) using Azadirachta indica leaves extract as an effective chelating agent. X-ray diffraction analysis and Fourier transform-infra-red spectroscopic examination were studied. Scanning electron microscopy analysis revealed that the average size of particles formed via plant extract as reducing agent without any surfactant is in the range of 100-170 nm while addition of cetyltrimethyl ammonium bromide were more uniform with 200 nm in size. Streptomycin as model drug was successfully loaded to green synthesised Si-NPs, sustain release of the drug from this conjugate unit were examined. Prolong release pattern of the adsorbed drug ensure that Si-NPs have great potential in nano-drug delivery keeping the environment preferably biocompatible, future cytotoxic studies in this connection is helpful in achieving safe mode for nano-drug delivery.

Green fabricated CuO nanobullets via Olea europaea leaf extract shows auspicious antimicrobial potential.

In present investigation, copper oxide (CuO) nanostructures have been prepared via green chemistry. Olea europaea leaf extract act as strong chelating agent for tailoring physical as well as bio-medical characteristics of CuO at the nano-size. Physical characterisation such as scanning electron microscope analysis depicts the formation of homogenised spherical shape nanoparticles (NPs) with average size of 42 nm. X-ray diffraction and Fourier transform infrared spectroscopy further confirmed the crystalline pure phase and monoclinic structure. High performance liquid chromatography (HPLC) testing is performed to evaluate the relative concentration of bioactive molecules in the O. europaea leaf extract. From HPLC results capping action of organic molecules around CuO-NPs is hypothesised. The antimicrobial potency of biosynthesised CuO-NPs have been evaluated using colony forming unit (CFU) counting assay and disc diffusion method which shows a significant zone of inhibition against bacterial and fungal strains may be highly potential for future antimicrobial pharmaceutics. Furthermore, reduction of various precursors by plant extract will reduce environmental impact over chemical synthesis.

Antimicrobial potential of green synthesized CeO2 nanoparticles from Olea europaea leaf extract.

This article reports the green fabrication of cerium oxide nanoparticles (CeO2 NPs) using Olea europaea leaf extract and their applications as effective antimicrobial agents. O. europaea leaf extract functions as a chelating agent for reduction of cerium nitrate. The resulting CeO2 NPs exhibit pure single-face cubic structure, which is examined by X-ray diffraction, with a uniform spherical shape and a mean size 24 nm observed through scanning electron microscopy and transmission electron microscopy. Ultraviolet-visible spectroscopy confirms the characteristic absorption peak of CeO2 NPs at 315 nm. Fourier transform infrared spectroscopy reflects stretching frequencies at 459 cm-1, showing utilization of natural components for the production of NPs. Thermal gravimetric analysis predicts the successful capping of CeO2 NPs by bioactive molecules present in the plant extract. The antimicrobial studies show significant zone of inhibition against bacterial and fungal strains. The higher activities shown by the green synthesized NPs than the plant extract lead to the conclusion that they can be effectively used in biomedical application. Furthermore, reduction of cerium salt by plant extract will reduce environmental impact over chemical synthesis.

The Effect of Green Synthesized CuO Nanoparticles on Callogenesis and Regeneration of Oryza sativa L.

In this study, we have investigated the effect of copper oxide nanoparticles (CuO-NPs) on callogenesis and regeneration of Oryza sativa L (Super Basmati, Basmati 2000, Basmati 370, and Basmati 385). In this regard, CuO-NPs have been bio-synthesized via Azadirachta indica leaf extract. Scanning electron microscope (SEM) analysis depicts average particle size of 40 ± 5 nm with highly homogenous and spherical morphology. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) have been employed to confirm the phase purity of the synthesized NPs. It is found that CuO-NPs exhibit very promising results against callus induction. It is attributed to the fact that green synthesized CuO-NPs at optimum dosage possess very supportive effects on plant growth parameters. In contrast to callogenesis, differential regeneration pattern has been observed against all of the examined O. sativa L. indigenous verities. Overall observation concludes that CuO, being one of the essential plant nutrients, has greatly tailored the nutritive properties at nano-scale.

Association between breast cancer and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) gene 1595C/T SNP in a Pakistani population.

AIM OF THE STUDY: TRAIL-mediated signalling has emerged as an extensively studied biological mechanism reported to differentially induce apoptosis in cancer cells. However, overwhelmingly increasing experimentally verified data is shedding light on resistance against TRAIL-induced apoptosis in cancer cells. Moreover, genetic and epigenetic mutations also exert effects on the functionality of TRAIL and its receptors. In this study we investigated the association between breast cancer and polymorphisms in tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) in a Pakistani Population. MATERIAL AND METHODS: Genotyping for TRAIL gene 1595 C/T polymorphism was done for 363 breast cancer patients and 193 age- and sex-matched healthy controls. DNA was extracted using standard organic methods. PCR-RFLP analysis was done for C/T polymorphism at position 1595 in exon 5 of the TRAIL gene using site-specific primers and restriction enzyme. The results were statistically evaluated by SPSS14. RESULTS: In this study, CC homozygotes were 46.3% in patients and 49.7% in controls, p = 0.729 with OR value 0.8705 (95% CI: 0.6137-1.2348). CT was statistically insignificant, p = 0.837 with OR value 0.9242 (95% CI: 0.6494-1.3154). However, the minor allele or risk allele genotype TT had a higher percentage among breast cancer patients (12.1%) than in the control group (6.7%). Since there was a statistically insignificant difference (p = 0.212, OR value 1.9098 with 95% CI 1.0019 to 3.6406) of TT genotype between the two groups, the contrastingly higher percentage of TT genotype in breast cancer patients seems to be a risk factor for the disease. Moreover, the frequency of minor allele T was also found to be higher in the patients (0.329) than in the controls (0.285). CONCLUSIONS: The TRAIL gene 1595 C/T SNP has a contradictory role in cancer development in different populations. In our population group although the percentage of homozygous risk allele TT was higher in patients it was statistically non-significant. The raised T allele and TT genotype in patients may suggest its association with breast cancer in the Pakistani population.

PHARMACOLOGICAL ACTIVITIES OF PROTOCATECHUIC ACID.

Protocatechuic acid (3,4-dihydroxybenzoic acid, PCA) is a simple phenolic acid. It is found in a large variety of edible plants and possesses various pharmacological activities. This article aims to review the modern trends in phytochemical isolation and extraction of PCA from plants and other natural resources. Moreover, this article also encompasses pharmacological and biological activities of PCA. It is well known to have anti-inflammatory, antioxidant, anti-hyperglycemia, antibacterial, anticancer, anti-ageing, anti-athro- genic, anti-tumoral, anti-asthma, antiulcer, antispasmodic and neurological properties.

IN VITRO ANTHELMINTIC EFFICACY OF NATIVE PLANTS AGAINST HAEMONCHUS CONTORTUS.

The current study aimed to investigate in vitro anthelmintic efficacy of two medicinally important plants against Haemonchus contortus in small ruminants. Fruit peel of Punica granatum Linn. (vern. Anar), leaves and roots of Berberis lycium Royle (vern. Sumbal) were tested for their anthelmintic efficacy. Methanolic extracts of the test plants from various plant parts were tested for anthelmintic efficacy against the Haemonchus contortous using albendazole as a reference standard. The results revealed that both the plant extracts exhibited potent anthelmintic activity at concentrations higher than 50 mg/mL when tested against their respective standard drug. In case of Berberis lycium Royle when the results were compared, methanolic roots extracts showed more potent activity as compared to leaves extracts at the same concentration. It was observed that the in vitro anthelmintic potential of Punica granatum Linn. fruit peel and Berberis lyceium Royale root can be used to treat helminth infections after in vivo trails.

Expression of Rice Chitinase Gene in Genetically Engineered Tomato Confers Enhanced Resistance to Fusarium Wilt and Early Blight.

This is the first study reporting the evaluation of transgenic lines of tomato harboring rice chitinase (RCG3) gene for resistance to two important fungal pathogens Fusarium oxysporum f. sp. lycopersici (Fol) causing fusarium wilt and Alternaria solani causing early blight (EB). In this study, three transgenic lines TL1, TL2 and TL3 of tomato Solanum lycopersicum Mill. cv. Riogrande genetically engineered with rice chitinase (RCG 3) gene and their R1 progeny was tested for resistance to Fol by root dip method and A. solani by detached leaf assay. All the R0 transgenic lines were highly resistant to these fungal pathogens compared to non-transgenic control plants. The pattern of segregation of three independent transformant for Fol and A. solani was also studied. Mendelian segregation was observed in transgenic lines 2 and 3 while it was not observed in transgenic line 1. It was concluded that introduction of chitinase gene in susceptible cultivar of tomato not only enhanced the resistance but was stably inherited in transgenic lines 2 and 3.

Effect of leaf extracts of Taraxacum officinale on CCl4 induced hepatotoxicity in rats, in vivo study.

Taraxacum officinale L is a medicinal plant, which has enormous medicinal values against various types of liver disorders and it has traditionally been used for the treatment of liver problems by people from the South East Asia. Previously we have screened the crude methanolic extract of T. officinale against cytotoxicity induced by CCl4. Present study was designed to compare the protective effect of ethanolic and n-hexane extract of leaves in carbon tetrachloride (CCl4) induced liver toxicity in rats. The extract (200 mg/kg and 400mg/kg body weight) along with silymarin (100 mg/kg) a standard drug was administered to experimental animals. It was observed that ethanolic plant extract has significantly reduced the negative effect of CCl4 as compared to n-hexane extract and effect of extract was increased with increasing dose level. Although both leaf extracts decreased the concentration of TBARS, H2O2 and nitrite contents which enhance due to CCl4 toxicity but effect was higher in ethanolic extract. The results clearly indicated that Taraxacum officinale ethanolic leaves extract has better protective effect against CCl4 induced liver tissues toxicity. This claim was also supported by histopathological results obtained during this study and this might be due to presence of various polar phytochemicals that might be more prevent in this extract.