Development of a strategy to induce RNA-silencing in squash against virus diseases by genetic transformation

5 Summary Viral diseases of Cucurbits are an important limitation in the production of the crop. Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus-2 (WMV-2) are the most important squash (Cucurbita pepo L.) infecting viruses. Mixed infections with these viruses are deleterious for cucurbitaceous plants leading annually to significant yield losses world wide. All varieties of economical importance are susceptible for these viruses and classical breeding did not yield resistance. Therefore, a transgenic approach was chosen to induce resistance against both viruses by post-transcriptional gene silencing (PTGS). Highly conserved regions in the coat protein genes of ZYMV and WMV-2 were chosen to establish an inverted repeat construct. This construct was cloned into binary vector under control of the 35S promoter. Embryogenic callus was induced from different organs of three squash cultivars as target tissues for Agrobacterium transformation. The embryogenic callus was developed within 13-20 weeks incubation on MS medium containing different plant growth regulator combinations of auxin and cytokinin. Induction of embryogenesis in different explants ranged from 5 to 100 % depending on the organ and genotype used. Efficiency of embryo maturation, conversion and germination into entire plants from squash embryogenic callus was found to be callus age depended. Regeneration with young (2 months old) material was efficient, whereas regeneration of material maintained under in vitro conditions for more than 2 years was not possible. Agrobacterium-mediated transformation of squash embryogenic callus was established using transient GUS-gene expression. The highest transformation efficiency was obtained with the supervirulent ATHV strain, bacterial density of 0.85, washing procedure of the embryogenic material prior to Agrobacterium co-culture, application of 1 mM Acetosyringone in induction medium and sub-culturing of embryogenic callus on fresh MS medium 5-9 days prior co-culturing with the Agrobacterium. Selection strategy was optimized using GFP as reporter gene. For the genotype CX3006 300 mg/l Kanamycin showed the highest number of green areas but most efficient selection agent was Paromomycin 150 mg/l. For the genotype Dundoo 200 mg/l Paromomycin was the effective selection agent and showed the highest number of green areas. Selection of transformed calli could be efficient with the used selection agents but regeneration of transgenic plant was not possible because the old material was only one to be used for transformation experiments. It seems that these old materials may have lost their competency when they were maintained for long term in tissue culture. Therefore, the functionality of the inverted repeat construct was evaluated in Nicotiana benthamiana as a model plant. Transgenic lines were analyzed by PCR, Southern blot analysis and segregation analysis of T1 offspring. The transgene-induced PTGS in transgenic lines was confirmed by infiltration of GFP-sensor constructs containing viral derived sequences as silencing target and /or a construct containing the p19 silencing suppressor. In all transgenic lines tested, GFP fluorescence in infiltrated leaves was extinguished three days post-infection with GFP-sensor constructs. In contrast, all transgenic lines showed GFP fluorescence in infiltrated leaves when GFP-sensor constructs were co-infiltrated with a binary vector containing the viral silencing suppressor p19. With this work, tools have been developed to engineer virus resistance in squash. Using the optimized Agrobacterium-mediated transformation procedure together with the efficient RNA-silencing of the inverted inverted repeat construct and freshely induced embryogenic material it is quite possible to establish virus resistance in squash.6 Zusammenfassung Virusbefall verursacht beim Zucchini-Anbau weltweit einen großen wirtschaftlichen Schaden. Früchte von virusinfizierten Pflanzen weisen mosaikähnliche Verfärbungen und schwerwiegende Deformationen auf, Pflanzen sind gestaucht und Blätter deformiert. Von den verschiedenen Viren, die weltweit für den Befall von Zucchini und anderen Cucurbitaceaen verantwortlich sind, gehören ZYMV und WMV-2 zu den bedeutendsten. Die konventionelle Züchtung konnte zwar bereits virustolerante Sorten hervorbringen, diese können jedoch Mischinfektionen oder der Befall von aggressiven Stämmen der o. g. Viren auf dem Felde nicht standhalten. Im Rahmen dieser Arbeit wurde daher ein transgene Strategie gewählt, um mittels ?Post Transkriptionales Gen Silencing? Resistenzen gegen ZYMV und WMV-2 in Zucchini zu induzieren. Für die Herstellung eines ?Inverted Repeat?-Konstrukts wurden hochkonservierte Sequenzbereiche aus den Hüllproteinen des ZYMV und des WMV-2 verwendet. Das Konstrukt wurde unter Kontrolle des 35S Promotors in einen binären Vektor kloniert. Als Ausgangsmaterial für die Agrobacterium-vermittelte Transformation von Zucchini wurde somatisch embryogener Kallus ausgewählt. Nach 13 bis 20 Wochen Inkubation auf hormonhaltigem Medium konnte an verschiedenen Explantaten bei allen 3 verwendeten Zucchini Genotypen embryogener Kallus induziert werden. Je nach verwendetem Explantat und Genotypen wurde eine Effizienz der Embryogenese von 5 bis 100% festgestellt. Die Reifung der somatischen Embryonen und ihre Konversion zu Pflanzen zeigten sich als stark abhängig vom Alter des embryogenen Kallus. Die Reifung und Konversion von frisch induziertem embryogenem Material war sehr effizient. Nach einer 2 jährigen Erhaltungsphase unter in vitro ?Bedingungen war eine Reifung und Konversion jedoch nicht mehr möglich. Die Agrobacterium-vermittelte Transformation von embryogenem Kallus wurde mit Hilfe der transienten Expression des GUS-Gens etabliert. Die höchste transiente Expression konnte bei Anwendung folgender Parameter erreicht werden: Verwendung des supervirulenten Agrobacterium-Stamms ATHV, optische Dichte von 0,85 der Bakteriensuspension, Waschbehandlung des embryogenen Materials vor der Cokultur, die Applikation von 1 mM Acetosyringon während der Cokultur und einer Vorkulturphase von 5-9 Tagen vor der Cokultur. Die Optimierung der Selektion von transformierten Zellen wurde mit Hilfe des GFP-Reportergens durchgeführt. Für den Genotypen CX3006 zeigte eine Kanamycin-Konzentration von 300 mg/l die größte Zahl von GFP-positiven Bereichen, jedoch war die Selektion mit Paramomycin am effizientesten, da nur eine Konzentration von 150 mg/l benötigt wurde. Für den Genotypen Dundoo erwies sich Paramomycin in einer Konzentration von 200 mg/l als effizientestes Selektionsmittel. Obwohl gezeigt werden konnte, dass die Selektion von transformierten embryogenen Zellen möglich ist, konnte während dieser Arbeit keine transgenen Zucchinipflanzen regeneriert werden, da für die Transformationsexperimente bedauerlicherweise nur auf altes embryogenes Kallusmaterial zurückgegriffen werden konnte, das während der langen Erhaltungskulturphase die Kompetenz zur Regeneration verloren hat. Die Funktionalität des ?Inverted Repeat?-Konstrukts wurde in der Modellpflanze Nicotiana benthamiana nachgewiesen. Transgene N. benthamiana-Pflanzen wurde mit PCR, Southern Blot Analyse untersucht und die Aufspaltung in der T1-Generation überprüft. Bei allen untersuchten Linien konnte PTGS in Infiltrationsexperimenten gezeigt werden. Hierbei wurden GFP-Sensorkonstrukte allein bzw-. in Kombination mit einem P19-Konstrukt infiltriert. In allen Linien konnte nach Infiltration der Sensorkonstrukte keine GFP-Fluoreszenz beobachtet werden, hingegen führte eine kombinierte Infiltration der Sensorkonstrukte mit dem P19 Supressor bei allen Linien zu einer starken GFP-Fluoreszenz. Mit dieser Arbeit konnte die Vorraussetzung für die Herstellung von virusresistentem Zucchinimaterial geschaffen werden. Mit Hilfe des etablierten Protokoll für die Agrobacterium-vermittelte Transformation von embryogenen Zucchinizellen und dem funktionellen ?Inverted Repeat?-Konstrukt besteht die Möglichkeit, in Zukunft ausgehend von frisch induziertem embryogenem Kallus transgene virusresistente Zucchinipflanzen erzeugt zu erzeugen

Effect of hydroxy (HHO) gas addition on gasoline engine performance and emiss

The objective of this work was to construct a simple innovative HHO generation system and evaluate the effect of hydroxyl gas HHO addition, as an engine performance improver, into gasoline fuel on engine performance and emissions. HHO cell was designed, fabricated and optimized for maximum HHO gas productivity per input power. The optimized parameters were the number of neutral plates, distance between them and type and quantity of two catalysts of Potassium Hydroxide (KOH) and sodium hydroxide (NaOH). The performance of a Skoda Felicia 1.3 GLXi gasoline engine was evaluated with and without the optimized HHO cell. In addition, the CO, HC and NOx emissions were measured using TECNO TEST exhaust gas analyzer TE488. The results showed that the HHO gas maximum productivity of the cell was 18 L/h when using 2 neutrals plates with 1 mm distance and 6 g/L of KOH. The results also showed 10% increment in the gasoline engine thermal efficiency, 34% reduction in fuel consumption, 18% reduction in CO, 14% reduction in HC and 15% reduction in NOx

Pharmacological Activities and Characterization of Phenolic and Flavonoid Compounds in Solenostemma argel Extract

Solenostemma argel is a desert medicinal plant indigenous to African countries. This research aims to study the pharmacological properties of Solenostemma argel plant. Aerial parts (leaves and flowers) of Solenostemma argel (Delile) Hayane were tested for antibacterial activity, antioxidant activity, anticancer, and anti-inflammatory activity. Phenolic and flavonoid contents of the plant were characterized. There was an increase in the antioxidant activity of Solenostemma argel extract from 12.16% to 94.37% by increasing concentration from10 µg/mL to 1280 µg/mL. The most sensitive organism was S. epidermidis with chloroform extract. The MTT assay revealed that methanolic extracts of Solenostemma argel showed potent cytotoxic effects on the A549, Caco-2, and MDAMB-231 cell lines, respectively. The anti-inflammatory activity increased by increasing the concentration of methanolic extract of Solenostemma argel, using indomethacin as a standard. Gallic acid was the most abundant phenolic acid, followed by synergic acid and p-coumaric acid, respectively. Catechin, quercetin, luteolin, kaempferol and rutin flavonoids were also found in the methanolic extract. GC-mass analysis showed that aerial parts of Solenostemma argel were rich in 2-(5-methyl-5 vinyl tetrahydro-2-furanyl)-2-propanol (11.63%), hexanoic acid methyl ester (10.93%), 3-dioxolane,4-methyl-2-pentadecyl (9.69%), phenol, 2-(1,1-dimethylethyl) (8.50%). It can be concluded that Solenostemma argel methanolic extract contain natural bioactive constituents with potential medicinal importance such as antioxidants, antimicrobial, anti-inflammatory, and anticancer activities

Pharmacological Activities and Characterization of Phenolic and Flavonoid Compounds in Methanolic Extract of Euphorbia cuneata Vahl Aerial Parts

Euphorbia cuneata Vahl. (Euphorbiaceae) is a plant used in folk medicine for the treatment of pain and inflammation, although the biological basis for these effects has not been thoroughly investigated. The goal of this study was to investigate the pharmacological properties and characterization of phenolic and flavonoid compounds present in the aerial parts of E. cuneata. E. cuneata aerial parts were tested for antioxidant activity (DPPH), antibacterial activity, cell viability and cytotoxic effects, and anti-inflammatory activity. Phenolic and flavonoid contents (HPLC), and volatile constituents (GC-MS) were also characterized. The methanol extract had the highest antioxidant activity, while the ether extract had the lowest. The antioxidant activity of E. cuneata extract increased from (21.11%) at a concentration of 10 µg/mL to (95.53%) at a concentration of 1280 µg/mL. S. aureus was the most sensitive organism with the highest zone of inhibition and lowest MIC, with acetone extract; whereas C. tropicalis was the most resistant, with the lowest inhibition zone. MTT assay revealed that the methanol extract of E. cuneata had significant cytotoxic effects on the A549, Caco-2, and MDA-MB-231 cell lines, respectively. Lower concentrations of methanolic extract gave anti-inflammatory activity, and those effects were compared with indomethacin as a positive control. Pyrogallol was the most abundant phenolic acid, followed by caffeic, p-coumaric, ferulic, syringic, and gallic acids, respectively. The 7-hydroxyflavone and rutin flavonoids were also found in the extract. GC-mass analysis showed that aerial parts of E. cuneata were rich in methyl 12-hydroxy-9-octadecenoate. The volatile components were also composed of considerable amounts of hexadecanoic acid, methyl ester, (9E,12E)-octadeca-9,12-dienoyl chloride, and methyl octadeca-9,12-dienoate as well as a little amount of hexanal dimethyl acetal. It can be concluded that methanolic extract of E. cuneata could be used as an available source of natural bioactive constituents with consequent health benefits