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.     

Isozyme analysis of progeny derived from (Allium fistulosum ×Allium cepa) ×Allium cepa

Summary Relatively large quantities of seed were obtained from the interspecific backcross (A. fistulosum xA. cepa) ×A. cepa allowing, for the first time, an extensive study of the heritable traits exhibited by backcross progeny. Two backcross populations, BC1034 and BC1040, distinguished by differentA. fistulosum parents, were characterized for the isozyme markersIdh-1, Adh-1, andPgi-1. Statistical methods are described to calculate cell probabilities for a mixed population of F₂ and BC₁ progeny, using an estimate of the fraction of F₂ progeny in the population derived from the isozyme data. Cell probability distributions were calculated for a mixed population with independent pairs of loci and a mixed population with nonindependent pairs of loci. The isozyme lociIdh-1 andPgi-1 appear to be linked, with a map distance estimated at 33 centimorgans (cM) in BC1034 and 42 cM in BC1040. The probability distribution model for linked loci did not account for all of the distorted segregation ratios inIdh-1 ×Adh-1 orPgi-1 ×Adh-1. The cytological literature does not support linkage betweenIdh-1 ×Adh-1 orPgi-1 ×Adh-1. The distorted segregation ratios for these pairs of loci are likely the result of genetic incompatibilities between the two species.Journal Article No. 1578, Agricultural Experiment Station, New Mexico State University, Las Cruces, NM, USA     

γ-Tubulin is associated with a cortical-microtubule-organizing zone in the developing guard cells of Allium cepa L.

A key event in the differentiation of elliptically shaped guard cells such as those in Allium is the formation of a radial array of cortical microtubules (Mts) which, by controlling the orientation of wall microfibrils, plays an important role in cell shaping. Previous experiments strongly indicated that the array is nucleated in a zone adjacent to the new ventral wall soon after cytokinesis. In order to further clarify the function of this zone, we performed dual immunolocalizations on Allium guard cells with anti--tubulin, to detect Mts, and an antibody to -tubulin, a protein known to be present at Mt-organizing centers in other species and recently identified in plants as well. -Tubulin antibody stained the cortical zone adjacent to the ventral wall, while little or no fluorescence was present elsewhere along the radial Mt array or at other sites in the cell. The antibody also stained the mitotic poles and phragmoplast in guard mother cells, as it does in other material. No staining was seen when the primary antibody was omitted. The results are consistent with nucleation of the radial array at a cortical-Mt-organizing zone next to the ventral wall, and set the stage for more in-depth studies on the spatial and temporal control of Mt formation in differentiating cells.Abbreviations CLSM confocal laser scanning microscope - FITC fluorescein isothiocyanate - Mt microtubule - MTOC microtubule-organizing center This work was supported by National Science Foundation grant DCB-9019285 to B.A.P., National Institutes of Health (NS30009) and American Cancer Society (CD6255) grants to H.C.J., and a University of Georgia Graduate School Assistantship to B.L. We thank Dr. Mark Farmer and the University of Georgia Center for Advanced Ultrastructural Research for the use of the confocal microscope.     

Cytogenetic and microtubule array effects of the zineb-containing commercial fungicide formulation Azzurro(®) on meristematic root cells of Allium cepa L

Zineb [ethylene bis(dithiocarbamate) zinc] is a widely employed foliar fungicide for agricultural and industrial applications. Allium cepa L. is a reliable model for the assessment of xenobiotic genotoxicity and cytotoxicity. We evaluated the effects of the zineb-containing commercial formulation Azzurro(?) (70% zineb) in cell cycle stages of the meristem root cells of A. cepa. The mitotic index (MI), chromosomal aberrations at anaphase/telophase (CAs), micronuclei (MN), and abnormalities in immunodetected microtubule structures, e.g., preprophasic band (PPB), mitotic spindle (MS), and phragmoplast (Phrag), were used as end-points. Azzurro(?) (1 and 10μg/ml) induced a significant increase in the frequency of CAs (P<0.05), and the higher concentration inhibited the MI (P<0.05) compared to control values. The frequency of MN did not differ from control values at any concentration. Treatment with 1μg/ml Azzurro(?) induced a significant increase in the frequency of abnormal PPB (P<0.01), MS (P<0.001), and Phrag (P<0.01) and, at 10μg/ml, enhancements in the frequencies of abnormal MS (P<0.05) and Phrag (P<0.05) were seen. A tubulin immunodetection assay showed that exposure to Azzurro(?) interferes with normal assembly of microtubule structures during mitosis.     

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.     

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.     

中国大蒜(Allium sativum L.)18个品种的酯酶同工酶多态性分析

采用聚丙烯酰胺凝胶电泳(PAGE)分离了中国大蒜3个生态型(低温反应敏感型、低温反应中间型和低温反应迟钝型)中18个较典型的品种的酯酶同工酶,并用排序分析法对18个品种的亲缘关系进行了分析,将18个品种分为3个变种群：1．“苏联”蒜变种群(var．Russia),2．吉木萨尔白皮蒜变种群(var．：Jimusaer。),3．中国内陆大蒜变种群(var．China)。其中中国内陆大蒜变种群又可分为5个品种群：①关中蒜品种群,②西北蒜品种群,③西南蒜品种群,④云贵蒜品种群,⑤华东蒜品种群。实验结果初步证实,大蒜生态型不能完全等同于基因型,酯酶同工酶的变化可能更能说明大蒜的亲缘进化关系。     

Characterization of callase (β-1,3-d-glucanase) activity during microsporogenesis in the sterile anthers of Allium sativum L. and the fertile anthers of A. atropurpureum

We examined callase activity in anthers of sterile Allium sativum (garlic) and fertile Allium atropurpureum. In A. sativum, a species that produces sterile pollen and propagates only vegetatively, callase was extracted from the thick walls of A. sativum microspore tetrads exhibited maximum activity at pH 4.8, and the corresponding in vivo values ranged from 4.5 to 5.0. Once microspores were released, in vitro callase activity peaked at three distinct pH values, reflecting the presence of three callase isoforms. One isoform, which was previously identified in the tetrad stage, displayed maximum activity at pH 4.8, and the remaining two isoforms, which were novel, were most active at pH 6.0 and 7.3. The corresponding in vivo values ranged from pH 4.75 to 6.0. In contrast, in A. atropurpureum, a sexually propagating species, three callase isoforms, active at pH 4.8-5.2, 6.1, and 7.3, were identified in samples of microsporangia that had released their microspores. The corresponding in vivo value for this plant was 5.9. The callose wall persists around A. sativum meiotic cells, whereas only one callase isoform, with an optimum activity of pH 4.8, is active in the acidic environment of the microsporangium. However, this isoform is degraded when the pH rises to 6.0 and two other callase isoforms, maximally active at pH 6.0 and 7.3, appear. Thus, factors that alter the pH of the microsporangium may indirectly affect the male gametophyte development by modulating the activity of callase and thereby regulating the degradation of the callose wall.     

中国大蒜(Allium sativumL.)18个品种的酯酶同工酶多态性分析

采用聚丙烯酰胺凝胶电泳(PAGE)分离了中国大蒜3个生态型（低温反应敏感型、低温反应中间型和低温反应迟钝型）中18个较典型的品种的酯酶同工酶,并用排序分析法对18个品种的亲缘关系进行了分析,将18个品种分为3个变种群: 1.“苏联”蒜变种群(var.Russia),2.吉木萨尔白皮蒜变种群(var.Jimusaer),3.中国内陆大蒜变种群(var.China)。其中中国内陆大蒜变种群又可分为5个品种群：①关中蒜品种群,②西北蒜品种群,③西南蒜品种群,④云贵蒜品种群,⑤华东蒜品种群。实验结果初步证实,大蒜生态型不能完全等同于基因型,酯酶同工酶的变化可能更能说明大蒜的亲缘进化关系。     

Increases in α-mannosidase activity in the arbuscular mycorrhizal symbiosis of Allium schoenoprasum

 Mycorrhizal and nonmycorrhizal roots of Allium schoenoprasum were tested for activities of α-mannosidase, β-glucosidase and arabinosidase. Mannosidase activity was higher by a factor of two in mycorrhizal than in nonmycorrhizal root extracts. The apparent molecular weight of the enzyme was 152 kDa and its K_M was 1.25 mM in colonized roots and 1.85 mM in uncolonized roots. α-Mannosidase activity was further characterized by an acid pH optimum and Zn²⁺ dependency. No significant differences could be found between mycorrhizal and nonmycorrhizal roots for β-glucosidase and arabinosidase activities. Accepted: 28 August 1995     

Transient expression of uidA constructs in in vitro onion ( Allium cepa L.) cultures following particle bombardment and Agrobacterium‐mediated DNA delivery

Particle bombardment and Agrobacterium-mediated DNA delivery into immature embryos and microbulbs were used to investigate the expression of the uidA gene in in vitro onion cultures. Both methods were successful in delivering DNA and subsequent uidA expression was observed. Optimal transient -glucuronidase activity was observed in immature embryos that had been pre-cultured for three days and bombarded at a distance of 3 cm from the stopping plate, under 25 in Hg vacuum, using 900–1300 psi rupture discs. The CaMV35S-uidA gene construct gave five fold higher transient -glucuronidase activity than the uidA gene construct regulated by any of four other promoters initially chosen for high experession in monocotyledonous tissues.Abbreviations GUS -glucuronidase - IE immature embryo - MUG methylumbelliferyl -D-glucuronide     

Cytogenetic Effects of γ-Radiation in Onion (<Emphasis Type="Italic">Allium cepa</Emphasis> L.) Seedlings

The effect of γ-radiation on the cytogenetic parameters of root meristem cells of onion seedlings was studied in laboratory experiments (Allium-test). An increase in the overall frequency of chromosomal aberrations and micronucleus frequencies in seedling cells at low γ-radiation doses (≤0.1 Gy) was detected for the first time. At a maximum absorbed dose of 13 Gy, chromosomal aberrations were detected in the majority of cells in the anaphase and telophase stages of the cell cycle, and the number of cells with multiple aberrations increased. The main contribution to the overall frequency of chromosomal aberrations, in addition to multiple aberrations, is made by the bridge-type aberrations, fragments, and lagging chromosomes. The data obtained allow using the cytogenetic indices of Allium cepa seedlings to assess the biological effects of lowdose γ-radiation.     

Covalent anthocyanin–flavonol complexes from the violet-blue flowers of Allium ‘Blue Perfume’

Three covalent anthocyanin–flavonol complexes (pigments 1–3) were extracted from the violet-blue flower of Allium ‘Blue Perfume’ with 5% acetic acid-MeOH solution, in which pigment 1 was the dominant pigment. These three pigments are based on delphinidin 3-glucoside as their deacylanthocyanin and were acylated with malonyl kaempferol 3-sophoroside-7-glucosiduronic acid or malonyl-kaempferol 3-p-coumaroyl-tetraglycoside-7-glucosiduronic acid in addition to acylation with acetic acid.By spectroscopic and chemical methods, the structures of these three pigments 1–3 were determined to be: pigment 1, (6^I-O-(delphinidin 3-O-(3^I-O-(acetyl)-β-glucopyranoside^I)))(2^VI-O-(kaempferol 3-O-(2^II-O-(3^III-O-(β-glucopyranosyl^V)-β-glucopyranosyl^III)-4^II-O-(trans-p-coumaroyl)-6^II-O-(β-glucopyranosyl^IV)-β-glucopyranoside^II)-7-O-(β-glucosiduronic acid^VI))) malonate; pigment 2, (6^I-O-(delphinidin 3-O-(3^I-O-(acetyl)-β-glucopyranoside^I)))(2^VI-O-(kaempferol 3-O-(2^II-O-β-glucopyranosyl^III)-β-glucopyranoside^II)-7-O-(β-glucosiduronic acid^VI))); and pigment 3, (6^I-O-(delphinidin 3-O-(3^I-O-(acetyl)-β-glucopyranoside^I)))(2^VI-O-(kaempferol 3-O-(2^II-O-(3^III-O-(β-glucopyranosyl^V)-β-glucopyranosyl^III)-4^II-O-(cis-p-coumaroyl)-6^II-O-(β-glucopyranosyl^IV)-β-glucopyranoside^II)-7-O-(β-glucosiduronic acid^VI))) malonate.The structure of pigment 2 was analogous to that of a covalent anthocyanin–flavonol complex isolated from Allium schoenoprasum where delphinidin was observed in place of cyanidin. The three covalent anthocyanin–flavonol complexes (pigment 1–3) had a stable violet-blue color with three characteristic absorption maxima at 540, 547 and 618 nm in pH 5–6 buffer solution. From circular dichroism measurement of pigment 1 in the pH 6.0 buffer solution, cotton effects were observed at 533 (+), 604 (−) and 638 (−) nm. Based on these results, these covalent anthocyanin–flavonol complexes were presumed to maintain a stable intramolecular association between delphinidin and kaempferol units closely related to that observed between anthocyanin and hydroxycinnamic acid residues in polyacylated anthocyanins. Additionally, an acylated kaempferol glycoside (pigment 4) was isolated from the same flower extract, and its structure was determined to be kaempferol 3-O-sophoroside-7-O-(3-O-(malonyl)-β-glucopyranosiduronic acid).     

Suppression of Lα/Lβ Phase Coexistence in the Lipids of Pulmonary Surfactant

To determine how different constituents of pulmonary surfactant affect its phase behavior, we measured wide-angle x-ray scattering (WAXS) from oriented bilayers. Samples contained the nonpolar and phospholipids (N&PL) obtained from calf lung surfactant extract (CLSE), which also contains the hydrophobic surfactant proteins SP-B and SP-C. Mixtures with different ratios of N&PL and CLSE provided the same set of lipids with different amounts of the proteins. At 37°C, N&PL by itself forms coexisting L_α and L_β phases. In the L_β structure, the acyl chains of the phospholipids occupy an ordered array that has melted by 40°C. This behavior suggests that the L_β composition is dominated by dipalmitoyl phosphatidylcholine (DPPC), which is the most prevalent component of CLSE. The L_β chains, however, lack the tilt of the L_β′ phase formed by pure DPPC. At 40°C, WAXS also detects an additional diffracted intensity, the location of which suggests a correlation among the phospholipid headgroups. The mixed samples of N&PL with CLSE show that increasing amounts of the proteins disrupt both the L_β phase and the headgroup correlation. With physiological levels of the proteins in CLSE, both types of order are absent. These results with bilayers at physiological temperatures indicate that the hydrophobic surfactant proteins disrupt the ordered structures that have long been considered essential for the ability of pulmonary surfactant to sustain low surface tensions. They agree with prior fluorescence micrographic results from monomolecular films of CLSE, suggesting that at physiological temperatures, any ordered phase is likely to be absent or occupy a minimal interfacial area.     

Solubilization of the plastidial lysophosphatidylcholine acyltransferase from Allium porrum leaves: towards plants devoid of eukaryotic plastid lipids?

To analyse the involvement of the plastidial lysophosphatidylcholine (lyso-PC) acyltransferase in the import of the extraplastidial lipid precursors required for eukaryotic plastid lipid synthesis, we plan to obtain transgenic plants. Since no sequence of lyso-PC acyltransferase is known, the purification of this enzyme has been undertaken to establish its sequence. First we determined the conditions allowing the solubilization of this membrane-bound enzyme. It is shown that by using CHAPS as a detergent, a lyso-PC acyltransferase activity is associated with the solubilized proteins.     

Studies on the Constituents of Typha L.—IV. A Preliminary Study of Chemosystematics of Typha L.

From pollen grains of Typha davidiana, T. latifolia, T. angustata the same eight flavonoids have been isolated. They are identified as naringenin I, isorhamnetin II, quercetin III, isorhamnetin-3-O-(2^G-α-L-rhamnopyranosyl)-rutioside IV, quercetin-3-O-(2^G-α-L-rhamnopyranosyl)-rutinosida, V, isorhamnetio-3-O-rutinoside VI, isorhamnetino-3-O-neohesperidoside VII, kampferol-3-O-neohesperidoside VIII. Flavonoids of pollen grains of five species of Typha, including the above three species, were analysed by TLC with the result showing that the constituents in the pollen grains of the five species are very similar. The chemical comparison among Typha and Sparganium and 16 possibly related families shows that Typha is different from Pandanaceae or Pandanales and is similar to Restionaceae, Flagellariaceae, Juncaceae and Cyperaceae in some respects. Typha and Sparganium are very similar in many respects, and they could be treated in the same family, Typhaceae, which merit the rank of order, Typhales.     

The development of a reproducible Agrobacterium tumefaciens transformation system for garlic (Allium sativum L.) and the production of transgenic garlic resistant to beet armyworm (Spodoptera exigua Hübner)

This paper describes the development of a reliable transformation system for garlic (Allium sativum L.) and its application in producing insect resistant GM garlic lines. The transformation system is based on Agrobacterium tumefaciens as a vector, using young callus derived from different callus sources: callus induced from both apical and non-apical root segments of in vitro plantlets, true garlic seeds and bulbils. Two different reporter genes were used in our garlic transformation experiments, namely the gusA gene coding for -glucuronidase and the gfp gene coding for green fluorescent protein. A total of seven independent transformed callus lines derived from different callus sources were obtained. The advantage of the system developed is the short time period needed for completion of the protocol (about 6 months) and the year-round availability of high quality callus from in vitro roots. The highest transformation frequency in a single experiment (1.47%), was obtained using garlic cv. 'Printanor'. Differences existed between cultivars in transformation frequency but were not significant. The same was found for the plasmids used in transforming garlic. Via PCR the presence of the gusA, hpt (hygromycin phosphotransferase) and gfp genes could be demonstrated in putative transformed in vitro plants. Southern hybridization showed that the reporter gene gusA and the selective gene hpt were stably integrated into the garlic genome. After transfer to the greenhouse of in vitro regenerants, transgenic garlic harbouring the gusA gene survived and grew well, whereas the gfp transgenic garlic gradually died under these conditions.Using this protocol transgenic garlic resistant to beet armyworm using the cry1Ca and H04 resistance genes from Bacillus thuringiensis were developed. Via Southern hybridization it was shown that the cry1Ca sequence was stably integrated into the garlic genome. After transfer of the transgenic in vitro garlic plants to the greenhouse, the cry1Ca plants developed normally and grew well to maturity with normal bulbs. However, all transgenic in vitro H04 garlic plants did not survive after transfer to the greenhouse. Transgenic cry1Ca garlic plants proved completely resistant to beet armyworm in a number of in vitro bio-assays. This finding will facilitate the development of new garlic cultivars resistant to beet armyworm.