The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology

BACKGROUND: Designed mimetic molecules are attractive tools in biopharmaceuticals and synthetic biology. They require mass and functional production for the assessment of upcoming challenges in the near future. The DARPin family is considered a mimetic pharmaceutical peptide group with high affinity binding to specific targets. DARPin G3 is designed to bind to the HER2 (human epidermal growth factor receptor 2) tyrosine kinase receptor. Overexpression of HER2 is common in some cancers, including breast cancer, and can be used as a prognostic and predictive tool for cancer. The chloroplasts are cost-effective alternatives, equal to, and sometimes better than, bacterial, yeast, or mammalian expression systems. This research examined the possibility of the production of the first antibody mimetic, DARPin G3, in tobacco chloroplasts for HER2 imaging in oncology. RESULTS: The chloroplast specific DARPin G3 expression cassette was constructed and transformed into N. tabacum chloroplasts. PCR and Southern blot analysis confirmed integration of transgenes as well as chloroplastic and cellular homoplasmy. The Western blot analysis and ELISA confirmed the production of DARPin G3 at the commercial scale and high dose with the rate of 20.2% in leaf TSP and 33.7% in chloroplast TSP. The functional analysis by ELISA confirmed the binding of IMAC purified chloroplast-made DARPin G3 to the extracellular domain of the HER2 receptor with highly effective picomolar affinities. The carcinoma cellular studies by flow cytometry and immunofluorescence microscopy confirmed the correct functioning by the specific binding of the chloroplast-made DARPin G3 to the HER2 receptor on the surface of HER2-positive cancer cell lines. CONCLUSION: The efficient functional bioactive production of DARPin G3 in chloroplasts led us to introduce plant chloroplasts as the site of efficient production of the first antibody mimetic molecules. This report, as the first case of the cost-effective production of mimetic molecules, enables researchers in pharmaceuticals, synthetic biology, and bio-molecular engineering to develop tool boxes by producing new molecular substitutes for diverse purposes.

Overexpression of NtAGPase small subunit gene increases leaf starch content and tobacco biomass / 生物工程学报

Production of biofuels such as ethanol from non-grain crops may contribute to alleviating the global energy crisis and reducing the potential threat to food security. Tobacco (Nicotiana tabacum) is a commercial crop with high biomass yield. Breeding of starch-rich tobacco plants may provide alternative raw materials for the production of fuel ethanol. We cloned the small subunit gene NtSSU of ADP-glucose pyrophosphorylase (NtAGPase), which controls starch biosynthesis in tobacco, and constructed a plant expression vector pCAMBIA1303-NtSSU. The NtSSU gene was overexpressed in tobacco upon Agrobacterium-mediated leaf disc transformation. Phenotypic analysis showed that overexpression of NtSSU gene promoted the accumulation of starch in tobacco leaves, and the content of starch in tobacco leaves increased from 17.5% to 41.7%. The growth rate and biomass yield of the transgenic tobacco with NtSSU gene were also significantly increased. The results revealed that overexpression of NtSSU gene could effectively redirect more photosynthesis carbon flux into starch biosynthesis pathway, which led to an increased biomass yield but did not generate negative effects on other agronomic traits. Therefore, NtSSU gene can be used as an excellent target gene in plant breeding to enrich starch accumulation in vegetative organs to develop new germplasm dedicated to fuel ethanol production.

Leaf area estimation of Burley tobacco / Estimativa da área foliar de tabaco do tipo Burley

ABSTRACT: Leaf area is an important growth variable in agricultural crops and the leaf is the main variable of interest in the tobacco industry. So, the aim of this scientific research was to estimate the Burley tobacco leaf area by linear dimensions of the leaves and to determine which mathematical model is more adequate for this purpose. Two experiments were carried out with Burley tobacco, cultivar DBH 2252, in 2016/2017 and 2018/2019 agricultural years, respectively, in the municipalities of Itaqui and Vanini - RS - Brazil. In 600 leaves were measured length (L), width (W), length×width product (LW), length/width ratio (L/W) and determined the real leaf area (LA). Four hundred and fifty leaves were separated to generate models of the leaf area as a function of linear dimension and the other 150 leaves were used for model's validation. The power model LA = 0.5037LW1.04435 (R² = 0.9960) is the most adequate for Burley tobacco 'DBH 2252' leaf area estimation. Alternatively, the models LA=2.0369W1.8619 (R²=0.9796) and LA=0.1222L2.2771 (R²=0.9738) based on width and length, respectively, can be used when only one leaf dimension is measured.

RESUMO: A área foliar é uma importante variável de crescimento em culturas agrícolas, sendo a folha a principal variável de interesse na indústria do tabaco. Assim, o objetivo deste estudo científico foi determinar a área foliar de tabaco tipo Burley por meio de dimensões lineares da folha e determinar qual modelo matemático é mais adequado para essa finalidade. Dois experimentos foram conduzidos com tabaco tipo Burley, cultivar DBH 2252, nos anos agrícolas de 2016/2017 e 2018/2019, respectivamente, nos municípios de Itaqui e Vanini, RS, Brasil. Em 600 folhas foram medidos o comprimento (L), a largura (W), o produto comprimento×largura (LW), a razão comprimento/largura (L/W) e determinada a área foliar real (LA). Foram separadas 450 folhas para a geração de modelos de estimativa de área foliar em função da dimensão linear e 150 folhas foram utilizadas para a validação dos modelos. O modelo LA = 0,5037LW1,04435 (R² = 0,9960) é adequado para a estimação da área foliar de tabaco Burley cultivar DBH 2252. Alternativamente, os modelos LA=2,0369W1,8619 (R²=0,9796) e LA=0,1222L2,2771 (R²=0,9738) baseados na largura e comprimento, respectivamente, podem ser utilizados quando apenas uma dimensão da folha é medida.

Identification, characterization and expression analysis of sucrose transporters in the model plant Nicotiana tabacum cv.petit havana

Aim: Sucrose transporters (SUTs) are transmembrane proteins involved in transport of sucrose along the phloem pathway of plants. Nicotiana tabacum, a well-known model plant has been reported to have three sucrose transporters (NtSUT1, NtSUT3 and NtSUT4), till date. The present study was undertaken to identify new SUTs from its genome and characterize all the NtSUT genes of Nicotiana tabacumMethodology: In the present study, new putative sucrose transporter genes were identified through extensive searches of the draft genome and transcriptome of tobacco using BLAST. In-silico characterization of the SUT gene family (NtSUT1, NtSUT2, NtSUT3, NtSUT4 and NtSUT5) of Nicotiana tabacum was done using bioinformatics tools. Spatial expression analysis was done for NtSUT genes in five different tissues of Nicotiana tabacum cv.petit havana using semi-quantitative RT-PCR. Results: Two new putative sucrose transporters NtSUT2 and NtSUT5 were identified from the publicly available tobacco transcriptome data and characterized using in-silico tools. Multiple sequence alignment of deduced amino acids of all the five SUTs revealed the presence of highly conserved domains and signature histidine residue in the sugar binding motif. Phylogenetic analysis grouped NtSUT1 and 3 into group 2, NtSUT4 and 5 into group 4 and NtSUT2 into group 3. NtSUT1 showed higher expression in leaves and flower while NtSUT2, 3 and 4 were abundant in root and flower. NtSUT5 showed a lower expression in all the tissues. Interpretation: Based on the sequence information and structural similarities, it is clear that the two newly identified putative SUT genes (NtSUT2 and NtSUT5) belonged to the SUT family of tobacco. This comprehensive study provides a consolidated data on characteristic features of SUT family proteins of tobacco

Tobacco waste hydrolysate of stem and root of the tobacco plant for biostimulation in rice and corn seed germination / Hidrolisado de resíduo de caule e raiz da planta tabaco para bioestimulação na germinação de sementes de arroz e milho

ABSTRACT: Stimulation of seed germination may be due to acceleration of germination as well as due to seedling growth-promotion during early development. Plant hydrolysate can be applied as a stimulant. Thus, we aimed to verify the influence of the hydrolysates, obtained by alkaline or acid treatment, from tobacco (Nicotiana tabacum L.) crop residues (root and stem) on the seed germination process. Seed germination was studied with Oryza sativa (rice) and Zea mays (corn). Sixteen germination experiments of 50 seeds each were undertaken, with 4 replicates, soaked with hydrolysates diluted at 20 and 80%, in 2 and 3 mL of hydration volumes for 48 h. Germinated seeds were counted, at which point radicular protrusions were observed. Rootlets and aerial parts were collected, dried and weighed. The hydrolysates presented seedling nutrition potential to the corn, with ~50% more mass compared to the results with water at the same conditions, and the germination acceleration was not significant. For the tested rice seeds, the results were reversed, and the germination acceleration was significant with rates up to 94% after 48 h of incubation. Better results of germination were obtained with hydrolysate from acid treatment, and root or stem tobacco can be used for this purpose.

RESUMO: A estimulação da germinação das sementes pode ser devida à aceleração da germinação, bem como à melhoria do crescimento das mudas durante o desenvolvimento inicial. O hidrolisado de plantas pode ser aplicado como estimulante. Assim, objetivou-se verificar a influência dos hidrolisados, obtidos por tratamento alcalino ou ácido, dos resíduos da cultura do tabaco (Nicotiana tabacum L.) (raiz e caule) no processo de germinação das sementes. A germinação das sementes foi estudada com Oryza sativa (arroz) e Zea mays (milho). Foram realizados dezesseis experimentos de germinação de 50 sementes cada, com 4 repetições, hidratadas em hidrolisados diluídos a 20 e 80%, em 2 e 3 mL de volumes de hidratação por 48 h. As sementes germinadas foram contadas, momento em que foram observadas protrusões radiculares. Radículas e partes aéreas foram coletadas, secas e pesadas. Os hidrolisados apresentaram potencial nutricional de plântulas para o milho, com ~ 50% a mais de massa quando comparados aos resultados com água nas mesmas condições, e a aceleração da germinação não foi significativa. Para as sementes de arroz testadas, os resultados foram contrários e a aceleração da germinação foi significativa com taxas de até 94% após 48 h de incubação. Melhores resultados de germinação foram obtidos com hidrolisado a partir do tratamento ácido, e a raiz ou caule de tabaco pode ser utilizado para esse fim.

Development of KASP marker for cytoplasmic male sterility in Nicotiana tabacum and utilization in trait introgression

Cytoplasmic male sterility (CMS) is an important trait for large-scale hybrid seed production which avoids manual emasculation and undesired horizontal spread of pollen.Rearrangements in mitochondrial genome in terms of deletions and insertions are frequent causes leading to CMS. Mitochondrial ATP synthase is a multisubunit molecular machine which is involved in synthesis of ATP. In this study, three mutations in ATPase subunit 6 were identified and their cosegregation with male sterility was established using tobacco male sterile hybrids and Nicotiana suvaolensis. A breeder friendly Kompetitive allele specific polymerase chain reaction (KASP) SNP marker was developed for high throughput and quick genotyping. Introgression of this trait into selected germplasm lines (n = 9) was achieved based on foreground for CMS and background selection for recurrent parent using KASP marker and 50K custom tobacco SNP array, respectively. Analysis of genotyping data from SNP array revealed the presence of 88–99% of recurrent parent genome in BC3F1 plants. The selected BC3F1 plants exhibit CMS and are indistinguishable from the fertile recurrent parent (germplasm) in terms of plant morphology.

Chemical Constituents of the Essential Oil Extracted from Nicotiana tabacum Leaves

Gas Chromatography-Mass Spectrometric (GC-MS) was used to investigate the chemical constituents of the essential oil from fresh leaves of Nicotiana tabacum L. The result revealed thirty six (36) compounds representing 100% of the essential oil characterized from the leaves of Nicotiana tabacum L. The major constituents are 3, 7, 11, 15-Tetramethyl-2-hexadecen-1-ol 16.37%, P-xylene 12.37%, Cyclohexane 8.43%, Farnesol isomer a 3.08%, and 1-Naphthalenepropanol 3.08%.

A new flavone from leaves of Nicotiana tabacum and its antibacterial activity / 中草药

Objective To find the new biological compounds, the research of the chemical constituents in the leaves of Nicotiana tabacum had been carried out. Methods The chemical constituents in the leaves of N.tabacum were isolated by silica gel, Sephadex LH-20 column chromatographies, and RP-HPLC methods. The structures of isolations were detected by using various spectroscopic techniques, including extensive 1D and 2D NMR techniques. Results Two C-alkylated flavones were isolated from 95% ethanol extract of plant, which identified as 2’-hydroxy-7-(3-hydroxypropyl)-8-methoxy-flavone (1) and tabaflavone A (2). Compounds 1 and 2 were evaluated for their anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) activity. Conclusion Compound 2 is isolated from the leaves of N.tabacum for the first time. Compound 1 is a new compound named orientalflavone A. Compounds 1 and 2 also show good anti-MRSA activity with MIC90 value of (38 ± 4) and (33 ± 3) μg/mL, respectively.

A new diterpene from leaves of Nicotiana tabacum / 中草药

Objective: To study the cembranoid diterpenes from the leaves of Nicotiana tabacum. Methods: The cembranoid diterpenes were isolated and purified by silica gel and preparative HPLC, and their structures were elucidated on the basis of their physicochemistry properties and spectra data. Results: Five cembranoid diterpenes were obtained from the ethanol extract of the leaves of N. tabacum and identified as (1R, 3E, 7E, 10S, 11E)-3,7,11-cembratriene-10, 15-diols (1), (1S, 2E, 4S, 6R, 7E, 10E, 12S)-2, 7, 10- cembratriene-4, 6, 11-triol (2), (1S, 2E, 4R, 6R, 7E, 10E, 12S)-2, 7, 10-cembratriene-4, 6, 11-triol (3), (1S, 2E, 4S, 6R, 7E)-4, 6, 11-trihydroxy- 1-isopropyl-4, 8-dimethylpentadeca-2, 7-dien-12-one (4), and (1S, 2E, 4R, 6R, 7E, 11S, 12S)-11, 12-epoxy-2, 7-cembradiene-4, 6-diol (5). Conclusion: Compound 1 is a new cembranoid diterpene and named as nicotiaditerpene B, and compound 4 is a new natural product.

Qualidade de sementes de tabaco durante o processo de pelotização e armazenamento / Quality of tobacco seeds during the pelletizing process and storage

RESUMO: O processo de pelotização pode afetar a qualidade das sementes por ocasião da semeadura, por isso, para avaliar o efeito desse processo na qualidade das sementes, foram utilizadas sementes de duas cultivares de tabaco 'CSC 467' e 'CSC 07', coletadas em diferentes etapas do processo de beneficiamento e pelotização: 1) sementes nuas não beneficiadas; 2) sementes nuas beneficiadas; 3) sementes pelotizadas; 4) sementes pelotizadas e coloridas. Para avaliação da qualidade das sementes nessas diferentes etapas, foram realizados os seguintes testes: germinação, primeira contagem de germinação, índice de velocidade de germinação, teste de emergência e índice de velocidade de emergência. Posteriormente, as sementes foram armazenadas por seis e doze meses e sua qualidade avaliada pelos mesmos testes e determinações. O processo de pelotização avaliado não influencia na germinação final de sementes de tabaco, mas atrasa o processo germinativo. As sementes de tabaco das cultivares 'CSC 467' e 'CSC 07' mantêm sua qualidade durante os 12 meses de armazenamento em câmara fria, com temperatura de 10°C e umidade relativa de 50%.

ABSTRACT: The pelletizing process may affect the quality of seeds during sowing. In order to evaluate the effect of this process on seed quality it was used seeds from three tobacco cultivars, 'CSC 467' and 'CSC 07' collected in different processing and pelletizing process stages: 1) non processed nude seeds; 2) processed nude seeds; 3) pelletized seeds; 4) pelletized and colored seeds. For the evaluation of seed quality in these different stages it was performed the following tests: Germination, First Count of Germination, Germination Speed Index, Initial and Final Stand (Emergence) and Emergence Speed Index. Posteriorly, seeds were stored for six and twelve months and their quality was evaluated by the same tests and determinations. The evaluated pelletizing process does not influence final germination of the tobacco seeds, but delays the germination process. Tobacco seeds from cultivars 'CSC 467' and 'CSC 07' maintain their quality during the 12 months of storage in cold chamber with temperature of 10oC and relative humidity of 50%.

Chemical structure and bioactivity of cis-5'-oxopropylnicotine / 中国中药杂志

In this paper, the chemical constituents and bioactivities of leaves of Nicotiana tabacum were investigated. Six compounds were isolated by means of various chromatographic techniques (silica gel, Sephadex LH-20, MCI GEL CHP-20P and HPLC), and their structures were elucidated as cis-5'-(2-oxopropyl)-nicotine (1), 3-O-(9, 12, 15-octadecatrienoyl)-glyceryl-β-D-galactppyranoside (2), (l'R, 2R, 5S, 10R)-2-(1', 2'-dihydroxy-l'-methylethyl)-6, 10-dimethylspiro [4, 5] dec-6-en-8-one (3), (l'S, 2R, 5S, l0R)-2-(1', 2'-dihydroxy-l'-methylethyl)-6, 10-dimethylspiro [4, 5] dec-6-en-8-one (4),2, 3-dihydroxypropyl-β-D-galactoside (5) and phenylethyl β-D-glucopyranoside (6) by extensive spectroscopic analyses (UV, IR, MS, 1D- and 2D-NMR). Among them, compound 1 is a new alkaloid, and compounds 2-6 are isolated for the first time from N. tabacum. Compounds 1 and 2 were assayed for agitating activities on transient receptor potential vanilloid 1 (TRPV1), melatonin receptor 1 and 2 (MT1 and MT2), 1 showed agitating rate of 55.41% (1.53mmol•L⁻¹) on MT2 and 2 possessed agitating rate of 128.11% (0.59 mmol•L⁻¹) and 52.00% (0.73mmol•L⁻¹) on TRPV1 and MT1, respectively.

Stable expression and characterization of a fungal pectinase and bacterial peroxidase genes in tobacco chloroplast

Background The high capacity of chloroplast genome response to integrate and express transgenes at high levels makes this technology a good option to produce proteins of interest. This report presents the stable expression of Pectin lyase (PelA gene) and the first stable expression of manganese peroxidase (MnP-2 gene) from the chloroplast genome. Results pES4 and pES5 vectors were derived from pPV111A plasmid and contain the PelA and MnP-2 synthetic genes, respectively. Both genes are flanked by a synthetic rrn16S promoter and the 3'UTR from rbcL gene. Efficient gene integration into both inverted repeats of the intergenic region between rrn16S and 3'rps'12 was confirmed by Southern blot. Stable processing and expression of the RNA were confirmed by Northern blot analysis. Enzymatic activity was evaluated to detect expression and functionality of both enzymes. In general, mature plants showed more activity than young transplastomic plants. Compared to wild type plants, transplastomic events expressing pectin lyase exhibited enzymatic activity above 58.5% of total soluble protein at neutral pH and 60°C. In contrast, MnP-2 showed high activity at pH 6 with optimum temperature at 65°C. Neither transplastomic plant exhibited an abnormal phenotype. Conclusion This study demonstrated that hydrolytic genes PelA and MnP-2 could be integrated and expressed correctly from the chloroplast genome of tobacco plants. A whole plant, having ~ 470 g of biomass could feasibly yield 66,676.25 units of pectin or 21,715.46 units of manganese peroxidase. Also, this study provides new information about methods and strategies for the expression of enzymes with industrial value.

Efecto antibacteriano in vitro de exudados foliares de tabaco contra dos bacterias fitopatógenas / In vitro antibacterial effect of tobacco leaf exudates against two bacterial plant pathogens

Los productos naturales son una alternativa para el control de microorganismos que ocasionan enfermedades en los cultivos. Este trabajo tuvo como objetivo evaluar diferentes solventes para la obtención de extractos crudos a partir de exudados foliares de líneas de tabaco, y el efecto in vitro de estos extractos contra dos bacterias fitopatógenas: Xanthomonas campestris (Xc) y Pectobacterium carotovorum (Pc). Se evaluaron solventes con polaridades entre 3.1 y 6.2 (diclorometano, n-butanol, acetato de etilo, metanol y etanol 90 %). El etanol 90 % se seleccionó como mejor solvente y como sustituto del diclorometano por su mayor rendimiento. Los extractos etanólicos crudos se obtuvieron a partir de exudados foliares de diez líneas de tabaco seleccionadas. La diversidad de la composición química de los extractos etanólicos se reveló por cromatografía en capa delgada. La actividad antibacteriana se evaluó por el método de difusión en agar con discos de papel de filtro y la medición del diámetro del halo de inhibición. Se observó inhibición para todos los extractos contra Xc destacándose los correspondientes a las líneas Nic 1061 "TI 1738" y Nic 1016 "Incekara" hasta 5 µg de extracto crudo seco /disco, con un mayor rendimiento para la línea Nic 1061. El extracto de la línea Nic 1015 fue el único con actividad contra Pc hasta 5 µg de extracto crudo seco por disco. Estos resultados sugieren un uso potencial de los extractos crudos de las líneas Nic 1061 y Nic 1015 "TI 1341" como un agente efectivo para la protección de cultivos contra estas bacterias.

Natural products are an alternative to control microorganisms that cause diseases in crops. This work aimed to evaluate different solvents for obtaining crude extracts from tobacco leaf exudates and to determine in vitro effect of these extracts against two phytopathogenic bacteria: Xanthomonas campestris(Xc) and Pectobacterium carotovorum(Pc). Crude extracts from ten tobacco lines using solvents with polarities between 3.1 and 6.2 (dichloromethane, n-butanol, ethyl acetate, methanol and ethanol 90%) were obtained. Ethanol 90% was selected as the best solvent for obtaining extracts from tobacco leaf exudates and as a substitute of dichloromethane due to the best yield. The chemical composition diversity of the ethanolic extracts was revealed by thin-layer chromatography. The antibacterial activity was evaluated by agar disk diffusion method recording the inhibition zones. Growth inhibition was observed for all extracts against Xc, and the better activity corresponded to the lines Nic 1061"TI 1738" and Nic 1016 "Incekara" until a minimal amount of 5 µg/ disc, with higher yield in case of the line Nic1061 . Only the extract of the line Nic 1015 was able to inhibit the growth of Pc until a minimal inhibitory concentration of 5 µg/disc. These results suggest a potential use of crude extracts from lines Nic 1061 and Nic 1015 "TI 1341" as an effective agent for the crop protection against Xc and Pc respectively.

A new chain-like cembranoid diterpene from leaves of Nicotiana tabacum / 中草药

Objective: To study the cembranoid diterpenes from the leaves of Nicotiana tabacum. Methods: The cembranoid diterpenes were isolated and purified by silica gel and preparative HPLC, and their structures were elucidated on the basis of their physicochemistry properties and spectral data. Results: Five cembranoid diterpenes were obtained from the ethanol extract of the leaves of N. tabacum and identified as (1S,2E,4R,6R,7E)-4,6,11-trihydroxy-1-isopropyl-4,8-dimethylpentadeca-2,7-dien-12-one (1), (1S,2E,4R,6R,7E,11E)-2,7,11-cembradiene-4,6-diol (2), (1S,2E,4S,6R,7E,11E)-2,7,11-cembradiene-4,6-diol (3), (1S,2E,4R,6R,7E, 11S)-2,7,12 (20)-cembratriene-4,6,11-triol (4), and (1S,2E,4S,6R,7E,11S)-2,7,12(20)-cembratriene-4,6,11-triol (5). Conclusion: Compound 1 is a new chain-like cembranoid diterpene named nicotiaditerpene A.

Isolation and molecular characterization of an ethylene response factor NtERF1-1 in Nicotiana tabacum cv. Xanthi.

Apetala2/Ethylene Response Factors (AP2/ERF) play important roles in regulating gene expression under abiotic and biotic stress in the plant kingdom. Here, we isolated a member of the AP2/ERF transcription factors, NtERF1-1, from Nicotiana tabcum cv. Xanthi NN carrying the N gene, which is resistant to Tobacco mosaic virus (TMV). NtERF1-1 encoded a putative protein of 229 amino acids with a predicted molecular mass of 24.58 kDa. Nucleotide sequence analysis showed that NtERF1-1 contained a conserved DNA-binding domain at the N-terminal. Comparison of amino acid sequences revealed that NtERF1-1 possessed high similarities to ERFs from diverse plants. Semi-quantitative and real-time quantitative RT-PCR analyses indicated that NtERF1-1 was up-regulated following TMV infection. In addition, we speculated that NtERF1-1 might participate in the signal transduction pathway of defence response inducted by the interaction between the N gene and TMV.

Phenol content and antioxidant activity of different young and adult plant parts of tobacco from Tunisia, dried at 40 and 70 ºC.

Different parts of tobacco plants were dried at 40 and 70 ºC. Some of them were also dried at room temperature. Dried plant material was extracted by sonication for obtaining hydro-alcoholic extracts (70%). Total phenol and total flavonoids were determined as well as antioxidant activities which were evaluated through different methods (capacity for scavenging DPPH, ABTS, superoxide and hydroxyl radicals; capacity for preventing lipid peroxidation using egg yolk as substrate; and reducing power). In young and adult plants, leaves generally had higher amounts of phenols (14.46-23.05 mg GAE g-1) than the remaining parts of the plant, independent on the temperature used. Generally, roots had lower amounts of phenols (1.56-4.63 mg GAE g-1). Leaves and flower had significantly higher concentrations of flavonoids (3.08-4.17 mg QE g-1 and 1.17-2.12 mg QE g-1, respectively) than the remaining parts. The antioxidant activity was generally higher in leaf extracts, although stalk ones had also a good capacity for scavenging hydroxyl radicals. Generally, young plants had the best capacity for scavenging DPPH and hydroxyl free radicals which may be related with the phenol content. Concerning drying temperatures, the results were not conclusive.

Actividad antihelmíntica in vitro de extractos oleosos de Azadirachta indica y extractos acuosos de Nicotiana tabacum sobre nematodos gastrointestinales de cabras / In vitro Anthelmintic Activity of Oily Extracts of Azadirachta indica and Aqueous Extracts of Nicotiana tabacum on Gastrointestinal Nematodes in Goats / Atividade anti-helmíntica in vitro de extratos oleosos de Azadirachta indica e extratos aquosos de Nicotiana tabacum sobre nematoides gastrointestinais de cabras

Los nematodos gastrointestinales son los parásitos más frecuentes en los rumiantes del mundo. Estas parasitosis causan gastroenteritis parasitaria con un impacto negativo sobre la productividad. El control de nematodos se ha basado en el uso de antihelmínticos químicos -ante los cuales los nematodos han desarrollado cierto grado de resistencia- que se han valorado como sustancias residuales en productos de la cabra que pueden desencadenar efectos nocivos en el consumidor final. Por tanto, el estudio de componentes de plantas se ha propuesto como una alternativa sostenible para el control de la nematodosis caprina. En este trabajo se evaluó in vitro el potencial antihelmíntico de extractos acuosos de Nicotiana tabacum y de extractos oleosos de Azadirachta indica sobre nematodos gastrointestinales que afectan la cadena caprina. Se realizaron ensayos de actividad nematicida sobre muestras de materia fecal de caprinos con alta carga parasitaria (trichostrongilidos), mediante la realización de curvas dosis/respuesta. El porcentaje de inhibición en la eclosión de huevos para el extracto acuoso de N. tabacum y el extracto oleoso de A. indica fue de 99% y 80%, respectivamente. Los extractos presentaron efecto sobre larva 3 (estadio infectante), con un tiempo letal medio para extractos de N. tabacum de 8 ± 1 minutos, y para el extracto A. indica de 8 ± 1 minutos. Los resultados de la actividad nematicida a nivel in vitro muestran que los extractos de N. tabacum y A. indica pueden ser una alternativa promisoria para el control de nematodos en rumiantes.

Gastrointestinal nematodes are the most common parasites found in ruminants in the world. These parasites cause parasitic gastroenteritis and have a negative effect on productivity. Nematode control has been based on the use of anthelmintic chemicals-against which nematodes have developed a certain degree of resistance-which have been rated as residual substances in goat products that may cause adverse effects on the final consumer. As a result, the study of plant components has been proposed as a sustainable alternative to control nematodosis in goats. The anthelmintic potential of aqueous extracts of Nicotiana tabacum and oily extracts of Azadirachta indica on gastrointestinal nematodes affecting the goat chain was evaluated in vitro in this study. Nematicidal activity tests were performed on stool samples from goats with a high parasite load (trichostrongyles), by performing dose/ response curves. Percent inhibition in egg hatch for the aqueous extract of N. tabacum and the oily extract of A. indica was 99% and 80%, respectively. Extracts showed an effect on larva 3 (infective stage), with a mean lethal time of 8 ± 1 minutes for extracts of N. Tabacum, and of 8 ± 1 minutes for extract of A. indica. The in vitro results of the nematicidal activity show that N. tabacum and A. indica extracts can be a promising alternative for controlling nematodes in ruminants.

Os nematoides gastrointestinais são os parasitas mais frequentes nos ruminantes do mundo. Estas parasitoses causam gastroenterite parasitária e influem negativamente na produtividade. O controle de nematoides tem se baseado no uso de anti-helmínticos químicos - perante os quais os nematoides têm desenvolvido certo grau de resistência - que têm se valorado como substâncias residuais em produtos da cabra que podem desencadear efeitos nocivos no consumidor final. Por tanto, se propôs o estudo de componentes de plantas como uma alternativa sustentável para o controle de nematódeos em caprinos. Neste trabalho se avaliou in vitro o potencial anti-helmíntico de extratos aquosos de Nicotiana tabacum e de extratos oleosos de Azadirachta indica sobre nematoides gastrointestinais que afetam a cadeia caprina. Realizaram-se ensaios de atividade nematicida sobre amostras de fezes de caprinos com alta carga parasitária (tricostrongilídeos), mediante a realização de curvas dose/resposta. A porcentagem de inibição na eclosão de ovos para o extrato aquoso de N. tabacum e o extrato oleoso de A. indica fue de 99% e 80%, respectivamente. Os extratos apresentaram efeito sobre larva 3 (estado infectante), com um tempo letal médio para extratos de N. tabacum de 8 ± 1 minutos, e para o extrato A. indica de 8 ± 1 minutos. Os resultados in vitro da atividade nematicida mostram que os extratos de N. tabacum e A. indica podem ser uma alternativa promissória para o controle de nematoides em ruminantes.

Optimización de las condiciones de inoculación por biobalística de un Begomovirus en tomate y tabaco / Optimizing conditions for biolistic inoculation of Begomovirus in tomato and tobacco

La transmisión experimental de Begomovirus es problemática. La mayoría de estos virus se pueden transmitir de planta a planta por su vector biológico, Bemisia tabaci. Las inoculaciones experimentales con mosca blanca son problemáticas debido a sus hábitos de alimentación, requerimiento de una planta viva infectada e instalaciones de contención para el vector. Por su parte la inoculación mecánica de Begomovirus es posible, pero generalmente a tasas bajas y no en todos los casos. Por esta razón el bombardeo de partículas (biobalística) de DNA viral como una estrategia de inoculación fue desarrollada. La posibilidad de utilizar el dispositivo de mano Helios Gen Gun System (Biorad®), un equipo de biobalística, para la transmisión de un Begomovirus bipartita a plantas de tomate y tabaco fue ensayado y optimizado. Los parámetros evaluados fueron: número de disparos (1-2), presión de helio (220 y 320 psi) y diámetro de las partículas de oro (0.6 y 1.6µm). Los síntomas característicos de la enfermedad viral (clorosis, mosaico y deformación de la hoja) aparecieron 3 semanas después del bombardeo en las hojas jóvenes no inoculadas. La replicación del DNA viral en las plantas se confirmó por Reacción en cadena de la polimerasa. Plantas infectadas en un 100 se obtuvieron cuando en el bombardeo se emplearon partículas de oro de 1.6 µm recubiertas con DNA viral a una presión de 320psi. A nuestro entender este es el primer reporte en Colombia de la inoculación directa de plantas de tomate y tabaco con un Begomovirus bipartita usando un dispositivo portátil de biobalística.

Experimental transmission of Begomovirus is problematic. Most Begomoviruses can be transmitted readily from plant to plant by the whitefly vector, but this also requires a live infected plant and extensive facilities to maintain the insect. Whitefly inoculations can also be problematic because of their preferential feeding habits on certain plants. Mechanical inoculation of Begomovirus is possible but generally at low rates and for others not at all. For this reason particle bombardment (biolistic) of DNA viral as an inoculum was developed. The possibility of using the Helios Gen Gun System (Biorad®), a biolistic hand-held device, for transmitting Begomovirus bipartite to tomato and tobacco plants was assayed and optimized. Biolistic inoculation was carried out with the hand held device at 220 or 320 psi, applying 1 or 2 shots /plant and using gold particles of 0.6 or 1.6µm in size. Characteristic symptoms of viral disease (chlorosis, mosaic and leaf deformation) appeared 3 weeks post-inoculation in the newly developing leaves. Replication of the viral DNA in plants was confirmed by Polymerase Chain Reaction. All bombarded plants became infected when biolistic inoculation was carried out with the hand held device at 320psi and using 1.6 µm gold particles in size. To our knowledge this is the first report in Colombia of successful direct inoculation of tomato and tobacco plants with Begomovirus bipartite geminivirus using a biolistic hand-held device.

Development of Concentrated Emulsion containing Nicotiana tabacum Extract for Use as Pesticide.

Using herbs as an insecticide or pesticide is well known in traditional agriculture. They are biodegradable and also friendly to environment. However, developing of commercial product from herbal plants was limited due to degradation of active ingredients, the variation of active content and there is no standard procedure for quality control. In this research, crude extracts of tobacco leaves (Nicotiana tabacum Linn., Solanaceae) containing nicotine as an active ingredient were studied for developing as concentrated emulsion preparation. Crude tobacco extracts from 95% ethanol were obtained as a brown syrupy mass with strong odors and 19.55% yield. One of the active ingredients, nicotine was selected to be used as a marker in suitable high performance liquid chromatography (HPLC) system in this study. The tobacco extract was stable under acid, base and heat conditions. Therefore, it was selected for further development as a concentrated emulsion formulation. The concentrated emulsion of tobacco extract composed of 10% w/w nicotine was prepared by combining fixed oil (palm oil), emulsifiers (Tween and Span), giving a more physically stable product. Under room temperature and 70 % RH for 6 month, the overall of % amount of nicotine in the product still remained in acceptable level. In the next step, the product was studied in the field with various dilution ratios of water to find out a suitable concentration of product using in agriculture field. It showed that all of the exhausted died and the plants trials are still green and not burned when the dilution is 100 time of its product.

Phytochemical Properties and Pharmcological Activities of Nicotiana Tabacum: A Review.

Since ancient times, plants have been an exemplary source of medicine. Tobacco is an agricultural product processed from the leaves of plants in the genus Nicotiana.In Indian agriculture, tobacco has a prominent place. Tobacco could be developed as an important food crop in combination with its traditional use for smoking and chewing. Tobacco plants are also used in plant bioengineering, and some of the more than 70 species are grown as ornamentals. The chemistry of tobacco is unique with the presence of a wide spectrum of chemical compounds of which nicotine, solanesol, malic and citric acid were identified as potential chemicals which could be recovered and converted to value-added products. The alkaloid nicotine is popularly considered the most characteristic constituent of tobacco but nicotine is not highly addictive on its own. In consumption it most commonly appears in the forms of smoking, chewing, snuffing, or dipping tobacco. Because of the powerfully addictive properties of tobacco, tolerance and dependence develop2.This situation necessitated to examine the green tobacco crop as a source for recovery of phytochemicals alone. With the objective of maximizing bio-mass production for optimum recovery of proteins, nicotine, solanesol and organic acids from green tobacco. Nicotine for treatment of Alzheimer disease, Parkinson disease, depression and anxiety, schizophrenia, attention deficit hyperactivity disorder (ADHD), pain, and obesity: