In vitro antimicrobial activity of zimmu ( Allium sativum L. ‘ Allium cepa L.) leaf extract

The fungitoxic effect of various medicinal plants belonging to different families was evaluated in vitro on Rhizoctonia solani, the rice sheath blight pathogen. Of the various plant extracts, the leaf extract of zimmu (Allium cepa × Allium sativum) showed the maximum antifungal activity against R. solani and recorded an inhibition zone of 12?mm. The leaf extract of zimmu was also effective in inhibiting the growth of other agronomically important fungal and bacterial pathogens viz., Aspergillus flavus, Curvularia lunata, Alternaria solani, Xanthomonas oryzae pv. oryzae, Xanthomonas campestris pv. malvacearum and Xanthomonas axonopodis pv. citri. The antimicrobial compound was dissoluble in methanol and the methanolic extract showed the absorption maxima at 210?nm and 230?nm. Phenolic compounds were present in greater amounts in methanol extract of zimmu. TLC analysis showed the appearance of two blue spots at R _f ?=?0.65 and R _f ?=?0.90. The compounds eluted at R _f ?=?0.65 and R _f ?=?0.90 by preparative TLC exhibited strong antifungal activity against R. solani.     

Agrobacterium — and microprojectile — mediated viral DNA delivery into barley microspore-derived cultures

Summary Anther cultures of barley (Hordeum vulgare L. var. Igri) were used as targets for Agrobacterium-mediated DNA transfer and direct DNA uptake by particle bombardment. A wheat dwarf virus construct which can replicate to a high copy number in cereal cells provided a sensitive marker for successful DNA delivery. Although DNA delivery was achieved using both procedures, particle bombardment gave more reproducible and higher levels of infection. The ability to deliver DNA into cereal cells which have a high regeneration capacity may provide a route for stable transformation.Abbreviations WDV Wheat dwarf virus - Gus -glucuronidase - ^mA N⁶-methyladenine     

Transient expression of β-glucuronidase following biolistic delivery of foreign DNA into coffee tissues

Some conditions related to the transient expression of -glucuronidase in biolistically-treated Coffea spp. tissues were investigated, and subsequently used in a promoter study. Bombardments were performed on different types of tissue (leaves, somatic embryos and suspension cultures) of genotypes of C. arabica, C. canephora and Arabusta, using 4 different promoter sequences. Tobacco leaves were used as a comparison. In general, similar large variation and mean values of transient expression were observed between coffee and tobacco leaves. With regard to the coffee tissues effect, transient expression was best detectable and most frequently observed with bombarded leaves of microcuttings. Disturbing endogenous light blue staining was found with control treatments of somatic embryos. For the three coffee species tested, the most effective promoter was the EF1-A1 promoter of Arabidopsis thaliana. Abbreviations BA 6-benzylaminopurine - EDTA ethylenediaminetetraacetic acid - GUS -glucuronidase - HFSE high frequency somatic embryogenesis - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog (1962) - PVP polyvinylpyrrolidone - R regeneration; rpm, rotations per minute - SAS statistical analytical system - v/v volume per volume - w/v weight per volume - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2-iP 2-isopentenyladenine     

Transient expression of β-glucuronidase in different cellular compartments following biolistic delivery of foreign DNA into wheat leaves and calli

Summary Transient expression of -glucuronidase (GUS) in different cellular compartments following biolistic delivery of chloroplast or nuclear expression vectors into wheat leaves or calli, derived from anther culture or immature embryos, is reported here. When pB1121, the nuclear GUS vector, was used to bombard wheat cells, the -glucuronidase product, an insoluble indigo dye, was observed evenly throughout the cytosol. But, when the chloroplast expression vector pHD203-GUS was used for bombardments, the indigo dye (GUS product) was subcellularly localized within the chloroplasts of wheat cells. The observation of GUS expression in albino plastids, when anther culture derived albino leaves were bombarded with the chloroplast expression vector pHD203-GUS, suggests the presence of a functional protein synthetic machinery in these organelles. GUS expression was also observed in regenerable calli derived from wheat immature embryos bombarded with pHD203-GUS. Leaves or calli bombarded with pUC19, as negative controls, did not show any GUS expression. These results constitute the first demonstration of foreign gene expression in chloroplasts of a monocot and that a dicot chloroplast promoter functions in a monocot chloroplast.     

Glu–Phe from onion (Allium Cepa L.) attenuates lipogenesis in hepatocytes

A Glu–Phe (EF) was isolated from onion (Allium cepa L. cv. Sunpower). The chemical structure of EF was determined by nuclear magnetic resonance and electrospray ionization–mass (ESI?MS) spectroscopy. We showed that EF reduced lipid accumulation in mouse hepatocytes by inhibiting the expression of sterol regulatory element-binding protein-1c (SREBP–1c) and its lipogenic target genes. We also found that AMP-activated protein kinase (AMPK) was required for the inhibitory effect of EF on lipid accumulation in mouse hepatocytes. Furthermore, EF was qualified in nine onion cultivars by selective multiple reaction-monitoring detection of liquid chromatography–ESI?MS. These results suggest that EF could contribute to the beneficial effect of onion supplement in maintaining hepatic lipid homeostasis.     

Inhibition of Aspergillus flavus growth and detoxification of aflatoxin B1 by the medicinal plant zimmu (Allium sativum L. × Allium cepa L.)

Aflatoxins are carcinogenic, teratogenic and immunosuppressive secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus. Aflatoxin contamination of peanut is one of the most important constraints to peanut production worldwide. In order to develop an eco-friendly method of prevention of A. flavus infection and aflatoxin contamination in peanut, aqueous extracts obtained from leaves of 30 medicinal plants belonging to different families were evaluated for their ability to inhibit the growth of A. flavus in vitro. Among them the leaf extract of zimmu (Allium sativum L. × Allium cepa L.) was the only one that showed antifungal activity against A. flavus and recorded 73% inhibition of A. flavus growth. The antifungal activity of the zimmu extract was significantly decreased upon dialysis with a dialysis membrane having molecular cut off 12 kDa or autoclaving at 121°C for 20 min or boiling at 100°C for 10 min and recorded inhibition of 52, 16 and 21%, respectively. When A. flavus was grown in medium containing zimmu extract the production of aflatoxin B₁ (AFB₁) was completely inhibited even at a concentration of 0.5%. When AFB₁ was incubated with zimmu extract a complete degradation of AFB₁ was observed 5 days after incubation. When the roots of zimmu were incubated in water containing 70 ng of AFB₁/ml, a reduction (by 58.5%) in AFB₁ concentration was observed 5 days after incubation. A significant reduction in the population of A. flavus in the soil, kernel infection by A. flavus and aflatoxin contamination in kernels was observed when peanut was intercropped with zimmu. The population of the fungal antagonist, Trichoderma viride in the zimmu-intercropped field increased approximately twofold.     

The effects of IAA and tetcyclacis on tuberization in potato (Solanum tuberosum L.) shoot cultures in vitro

Potato (Solanum tuberosum cv. Désirée) shoots grown in vitro in continuous darkness or in long days (LDs), were used to investigate indole-3-acetic acid (IAA) effects on stolon initiation and tuber formation, combining IAA with increased or decreased gibberellin levels. An increased gibberellin (GA) level was achieved by the applying 1 μM GA₃, while decreased gibberellin level was presumably realized by the adding 3 μM tetcyclacis (Tc). About 15% of potato shoots developed stolons both in LDs and in darkness. Stolon initiation was stimulated by GA₃ in darkness and by Tc in LDs. Tuber formation was strongly inhibited in LDs and by GA₃ both in light and darkness, but stimulated in darkness at low GA level. Exceptionally, tuber formation occurred in LDs at the highest Tc concentrations, in about 25% of explants. Indole-3-acetic acid alone stimulated stolon formation in LDs, both in the presence or absence of GA₃. IAA alone also stimulated tuber formation in dark-grown shoots, but could not overcome the inhibitory effect of LDs. Indications that, depending on their concentration ratio, IAA may interact with GA₃ in different tuberization phases, have been discussed. Radomir Konjević—Deceased in July 2006.     

Metabolic regulation of α-amylase gene expression in transgenic cell cultures of rice (Oryza sativa L.)

Expression of two genes in the -amylase gene family is controlled by metabolic regulation in rice cultured cells. The levels of RAmy3D and RAmy3E mRNAs in rice cultured cells are inversely related to the concentration of sugar in the culture medium. Other genes in the rice -amylase gene family have little or no expression in cultured cells; these expression levels are not controlled by metabolic regulation. A RAmy3D promoter/GUS gene fusion was metabolically regulated in the transgenic rice cell line 3DG, just as the endogenous RAmy3D gene is regulated. An assay of GUS enzyme activity in 3DG cells demonstrated that RAmy3D/GUS expression is repressed when sugar is present in the culture medium and induced when sugar is removed from the medium. The 942 bp fragment of the RAmy3D promoter that was linked to the coding region of the GUS reporter gene thus contains all of the regulatory sequences necessary for metabolic regulation of the gene.     

Agrobacterium－mediated transformation and assessment of factors influencing transgene expression in loblolly pine（Pinus taeda L.）

This investigation reports a protocol for transfer and expression of foreign chimeric genes in loblolly pine (Pinus taeda L.). Transformation was achieved by co-cultivation of mature zygotic embryos with Agrobacterium tumefaciens strain LBA4404 which harbored a binary vector (pBI121) including genes for beta-glucuronidase (GUS) and neomycin phosphotransferase (NPTII). Factors influencing transgene expression including seed sources of loblolly pine, concentration of bacteria, and the wounding procedures of target explants were investigated. The expression of foreign gene was confirmed by the ability of mature zygotic embryos to produce calli in the presence of kanamycin, by histochemical assays of GUS activity, by PCR analysis, and by Southern blot. The successful expression of the GUS gene in different families of loblolly pine suggests that this transformation system is probably useful for the production of the genetically modified conifers.     

Optimisation of DNA transfer and transientβ-glucuronidase expression in electroporated maize (Zea mays L.) microspores

Summary The ability to deliver free DNA into microspores of a highly androgenic hybrid of maize was assessed by electroporation, using a square wave pulse discharge apparatus. The electroporation medium was chosen according to its ability to maintain a high level of regeneration. Nuclease activities were analyzed and were inhibited by the addition of 100 mM KNO₃ and MgSO₄ in the electroporation medium. Seven expression vectors withUid A as the reporter gene under the control of cauliflower mosaic virus 35S, Lat 52-7, Zmg 13, Emu, Ubiq-1, Al, or Actl promoters were tested in relation to the level of ß-glucuronidase expression in maize microspores. The highest level of expression was obtained when theUid A gene was driven by the Actl promoter. Therefore, this vector was further used to define optimal conditions leading to highest levels of ß-glucuronidase expression. The parameters determined in this study could provide an ideal starting point for the obtention of transgenic maize plants from electroporated microspores.Abbreviations DH diplohaploid - PEG polyethylene glycol - GUS ß-glucuronidase - EDTA Ethylene-diaminetetra Acetic acid - CaMV Cauliflower mosaic virus     

Production of transgenic pea (Pisum sativum L.) plants by Agrobacterium tumefaciens — mediated gene transfer

Summary A transformation system that allows regeneration of transgenic pea plants from calli selected for antibiotic resistance was developed. Explants from axenic shoot cultures and seedling epicotyls were cocultivated with nononcogenic Agrobacterium tumefaciens strains, and transformed callus could be selected on callus-inducing media containing either 15 mg/l hygromycin or 75 mg/l kanamycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on hygromycin-resistant calli, but not on the calli selected for kanamycin resistance. Regenerated shoots could subsequently be rooted and transferred into the greenhouse. In addition, the effects of different callus-inducing and growth media on organogenesis were investigated. The transformation of the calli and regenerated plants was confirmed by DNA analysis.     

Transient expression of β-glucuronidase in plastids of various plant cells and tissues delivered by a pneumatic particle gun

Chloroplast expression plasmids pTRBCL-GUS (tobaccorbcL promoter-gusA-tobaccorbcL terminator) and pHHU3004 (spinach ‘x gene’ promoter-gusA-spinachrbcL terminator) and a control nuclear expression plasmid pBI221 (CaMV 35S promoter-gusA-NOS terminator) were introduced separately into cultured cells and tissues of tobacco andArabidopsis thaliana, as well as into cultured cells of the lower land plants liverwort and hornwort by a pneumatic particle gun. The pTRBCL-GUS and pHHU3004 plasmids produced many blue spots in the BY-2 cells and the roots ofArabidopsis thaliana, but not in any of the green cells or tissues. The results suggest that the pTRBCL-GUS and pHHU3004 plasmids are expressed more in proplastids and amyloplasts than in chloroplasts. GUS activities of the BY-2 cells bombarded with pTRBCL-GUS and pHHU3004 were insensitive to α-amanitin treatment (10 and 50 μg/ml), while that of the cells with pBI221 greatly decreased by the same treatment. Hence, it is likely that the pTRBCL-GUS and pHHU3004 plasmids were substantially expressed in the proplastids.     

Hairy root cultures of butterfly pea (Clitoria ternatea L.): Agrobacterium × plant factors influencing transformation

Transformed rhizoclones were developed from Agrobacterium-treated explants of the medicinally important twinning legume Clitoria ternatea L. Several key factors influencing transformation events were optimized. A4T was the most infectious among the strains employed. Internode segments were more responsive than leaves, outdoor-grown explants preferred to those from in vitro cultures. High frequency transformation, resulting in up to 85.8% rhizogenesis, was attained using pre-pricked internodal explants for immersion (10 min) in Agrobacterium rhizogenes suspension grown overnight with acetosyringone (100 μM) to an OD₆₆₀ ≅ 0.6, diluted to a density of 10⁹ cells ml⁻¹, followed by 5-day co-cultivation. Roots were individually cultured in MS0 supplemented with the bacteriostatic antibiotic cefotaxime (500 μg ml⁻¹). Rhizoclones were renewed through successive subcultures in MS0 under diffused illumination. The T _L -DNA rolB and rolC ORF were detected in rhizoclones through PCR amplification. The T _R -DNA gene encoding mannopine synthase (man2) was revealed by positive amplification and opine gene expression substantiated by agropine and mannopine biosynthesis in all selected transformed rhizoclones. The implication of such findings is discussed on the context of utilization of such genetically transformed root cultures towards sustainable production of medicinally useful phytocompounds, besides providing a means for plant conservation.     

Transient expression of β-glucuronidase in Arabidopsis thaliana leaves and roots and Brassica napus stems using a pneumatic particle gun

Successful transient expression of -glucuronidase (GUS) in Arabidopsis thaliana leaves and roots and Brassica napus stems was obtained after gene delivery with a pneumatic particle gun driven by compressed air. Effects of the pneumatic pressure used to accelerate the particles (accelerating pressure; 85 to 200 kg/cm²) and of preculture periods of plant tissues (0 to 6 days) on the efficiency of gene delivery were studied. In A. thaliana leaves, best results were obtained at 115 kg/cm² of accelerating pressure and 3 days of preculture. In A. thaliana roots, the optimum was at 200 kg/cm² of accelerating pressure and 3 days of preculture. These results indicate that both preculture period and accelerating pressure are vital factors that determine the efficiency of gene delivery by particle gun.     

Agrobacterium tumefaciens– mediated transformation of soybean [Glycine max (L.) Merrill.] using immature zygotic cotyledon explants

Agrobacterium tumefaciens -mediated transformation of soybean [Glycine max (L.) Merrill. cv. Jack] using immature zygotic cotyledons was investigated to identify important factors that affected transformation efficiency and resulted in the production of transgenic soybean somatic embryos. The factors evaluated were initial immature zygotic cotyledon size, Agrobacterium concentration during inoculation and co-culture and the selection regime. Our results showed that 8- to 10-mm zygotic cotyledons exhibited a higher transformation rate, as indicated by transient GUS gene expression, whereas the smaller zygotic cotyledons, at less than 5 mm, died shortly after co-cultivation. However, the smaller zygotic cotyledon explants were found to have a higher embryogenic potential. Analysis of Agrobacterium and immature cotyledon explant interactions involved two Agrobacterium concentrations for the inoculation phase and three co-culture regimes. No differences in explant survival or somatic embyogenic potential were observed between the two Agrobacterium concentrations tested. Analysis of co-culture regimes revealed that the shorter co-culture times resulted in higher explant survival and higher somatic embryo production on the explants, whereas the co-culture time of 4 days severely reduced survival of the cotyledon explants and lowered their embryogenic potential. Analysis of selection regimes revealed that direct placement of cotyledon explants on hygromycin 25 mg/l was detrimental to explant survival, whereas 10 mg/l gave continued growth and subsequent somatic embryo development and plant regeneration. The overall transformation frequency in these experiments, from initial explant to whole plant, was 0.03 %. Three fertile soybean plants were obtained during the course of these experiments. Enzymatic GUS assays and Southern blot hybridizations confirmed the integration of T-DNA and expression of the GUS-intron gene in the three primary transformants. Analysis of 48 progeny revealed that three copies of the transgene were inherited as a single Mendelian locus. Received: 6 December 1999 / Revised: 11 February 2000 / Accepted: 14 March 2000     

Induction of systemic resistance to bacterial blight caused by Xanthomonas campestris pv. malvacearum in cotton by leaf extract from a medicinal plant zimmu (Allium sativum L. × Allium cepa L.)

Abstract

Aqueous extract (10%) from leaves of zimmu (Allium sativum L. × Allium cepa L.) when applied as foliar spray to first and second leaves of cotton plants induced systemic resistance in third and fourth leaves to a challenge infection with Xanthomonas campestris pv. malvacearum and reduced the number of lesions by up to 73% compared with water-treated control plants. The treated leaves exhibited significantly high activity of enzymes phenylalanine ammonia-lyase, peroxidase and polyphenol oxidase along with rapid accumulation of phenolics. The activities of chitinase and β-1,3-glucanase were greatly elevated in treated plants as compared to water-treated controls. An 11-fold increase in chitinase activity was evident 4 d after treatment. Western blot analysis revealed that a chitinase with an apparent molecular weight of 58 kDa that cross-reacted with a barley chitinase antiserum was induced in cotton leaves 3 d after treatment and the maximum induction of this chitinase was detected 4 d after treatment. The present study provides evidence for the induction of biochemical defence mechanisms in cotton leaves after treatment with leaf extract from zimmu.