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PubMed1977

Orientation of Giemsa C-bands in interphase cells of Allium cepa L.

Orientation of Giemsa C-bands in Allium cepa was studied in both mitotic and interphase cells. It has been shown that telophase orientation of the chromosome is maintained throughout the interphase and early phophase. It has been assumed that this non-random orientation is due to anchorage of the telomeres with the nuclear membrane. Contrary to earlier observations, 2 by 2 pairing of the telomers could not be traced to this species.

Chromosomes↗

PubMed1996

Nucleolar organizer expression in Allium cepa L. chromosomes.

Roots from Allium cepa L. (cv. Francesa) bulbs in which a maximum of two nucleoli per nucleus developed were selected for this study. Five rDNA clusters were detected by fluorescent in situ hybridization on chromosomal squashes (2n = 16) with a rhodamine-labelled wheat rDNA repeat. The rDNA clusters were located on four chromosomes: the largest cluster occurred on the small arm of a single homologue of the smallest pair 8. Its homologue showed two different small rDNA clusters, one near each telomere. The two homologues of the satellited chromosomes 6 also showed different rDNA contents, which were intermediate to those found in pair 8. The same five well-differentiated hybridization signals were observed in interphase cells that were inactive in transcription because they were in dormant roots, or in proliferating ones in which the synthesis of the large rRNA precursor was prevented. After multipolarizing agent was applied in anaphase followed by inhibition of cytokinesis, multinucleate autotetraploid cells were formed, which often contained more than four nucleoli. Thus, at least two of the three nucleolar organizer regions that consistently failed to develop a nucleolus in normal mononucleate cells were capable of developing nucleoli when segregated into different nuclei in multinucleate cells.

Chromosome Mapping↗

PubMed2004

Subcellular localization of cadmium in the root cells of Allium cepa by electron energy loss spectroscopy and cytochemistry.

The ultrastructural investigation of the root cells of Allium cepa L. exposed to 1 mM and 10 mM cadmium (Cd) for 48 and 72 h was carried out. The results indicated that Cd induced several obvious ultrastructural changes such as increased vacuolation, condensed cytoplasm with increased density of the matrix, reduction of mitochondrial cristae, severe plasmolysis and highly condensed nuclear chromatin. Electron dense granules appeared between the cell wall and plasmalemma. In vacuoles, electron dense granules encircled by the membrane were aggregated and formed into larger precipitates, which increase in number and volume as a consequence of excessive Cd exposure. Data from electron energy loss spectroscopy (EELS) confirmed that these granules contained Cd and showed that significantly higher level of Cd in vacuoles existed in the vacuolar precipitates of meristematic or cortical parenchyma cells of the differentiating and mature roots treated with 1 mM and 10 mM Cd. High levels of Cd were also observed in the crowded electron dense granules of nucleoli. However, no Cd was found in cell walls or in cells of the vascular cylinder. A positive Gomori-Swift reaction showed that small metallic silver

Cadmium↗

PubMed1994

Alliin lyase (Alliinase) from garlic (Allium sativum). Biochemical characterization and cDNA cloning.

The garlic plant (Allium sativum) alliinase (EC 4.4.1.4), which catalyzes the synthesis of allicin, was purified to homogeneity from bulbs using various steps, including hydrophobic chromatography. Molecular and biochemical studies showed that the enzyme is a dimer of two subunits of MW 51.5 kDa each. Its Km using synthetic S-allylcysteine sulfoxide (+ isomer) as substrate was 1.1 mM, its pH optimum 6.5, and its isoelectric point 6.35. The enzyme is a glycoprotein containing 6% carbohydrate. N-terminal sequences of the intact polypeptide chain as well as of a number of peptides obtained after cyanogen bromide cleavage were obtained. Cloning of the cDNAs encoding alliinase was performed by a two-step strategy. In the first, a cDNA fragment (pAli-1-450 bp) was obtained by PCR using a mixed oligonucleotide primer synthesized according to a 6-amino acid segment near the N-terminal of the intact polypeptide. The second step involved screening of garlic lambda gt11 and lambda ZAPII cDNA libraries with pAli-1, which yielded two clones; one was nearly full length and the second was full length. These clones exhibited some degree of DNA sequence divergence, especially in their 3' noncoding regions, suggesting that they were encoded by separate genes. The nearly full length cDNA was fused in frame to a DNA encoding a signal peptide from alpha wheat gliadin, and expressed in Xenopus oocytes. This yielded a 50 kDa protein that interacted with the antibodies against natural bulb alliinase. Northern and Western blot analyses showed that the bulb alliinase was highly expressed in bulbs, whereas a lower expression level was found in leaves, and no expression was detected in roots. Strikingly, the roots exhibited an abundant alliinase activity, suggesting that this tissue expressed a distinct alliinase isozyme with very low homology to the bulb enzyme.

Amino Acid Sequence↗

PubMed2002

Transfer of a male-sterility-inducing cytoplasm from onion to leek ( Allium ampeloprasum).

Two interspecific triploid (AAC) hybrids (84/1-94 and 99/1-94) from crosses between onion [ Allium cepa (2 n=2 x=16, CC)] and leek [ A. ampeloprasum (2 n=4 x=32, AAAA)] were backcrossed to leek in order to transfer a male-sterility-inducing cytoplasm from onion that would enable the production of hybrid leek. GISH evaluations of meiosis in the interspecific hybrids revealed irregularities due to univalent onion chromosomes producing micronuclei from onion chromatin, whereas the pairing of the two sets of leek chromosomes was nearly normal. Attempts to use colchicine to double the chromosome number of the hybrids failed. Backcrosses of 84/1-94 to leek as the pollen parent were not successful. The first backcross of 99/1-94 to tetraploid leek produced 11 BC(1) plants with chromosome numbers between 38 and 41. Identification of parental chromosomes by GISH showed that all eight onion chromosomes and 30-33 leek chromosomes were transmitted to the backcross progenies due to unreduced egg cells. Onion chromosomes were eliminated during the second backcross. Southern hybridization confirmed the transfer of the T-cytoplasm like source of CMS from onion to the BC(2) progenies. After the third backcross to leek, 158 plants were obtained with varying numbers of onion chromosomes and some intergenomic recombinant chromosomes. Alloplasmic leek plants without onion chromatin were selected for further characterization of male sterility and quality traits.

Journal Article↗

PubMed2005

Development of a codominant PCR-based marker for allelic selection of the pink trait in onions (Allium cepa), based on the insertion mutation in the promoter of the anthocyanidin synthase gene.

Bulb color in onions (Allium cepa) is an important trait and is inherited in a complex manner. However, the mechanism of color inheritance is poorly understood at the molecular level. A previous study showed that pink bulb color in onions is inherited as a single recessive trait. This trait is attributable to a significantly reduced transcription of the anthocyanidin synthase (ANS) gene. In this study, we developed a PCR-based marker for an allelic selection of the ANS gene to avoid the laborious progeny tests traditionally employed. To identify polymorphisms between pink and red alleles of the ANS gene, promoter sequences of both alleles were isolated. There was 97% nucleotide sequence identity between the promoter sequences of the two alleles. A 390-bp insertion was identified 632 bp upstream from the putative transcription start site in the pink allele. A pair of primers was designed on the flanking sequences of the inserted region and utilized as a PCR-based marker for allelic selection of the ANS gene. The reliability of the marker was tested using parents, F1 hybrids, and F3 lines whose genotypes had been identified by progeny tests. The marker was also used to evaluate the distribution of the pink allele in white and yellow breeding lines. The results indicated that a majority of the breeding lines tested were homozygous recessive.

Alleles↗

PubMed2005

Development of a PCR-based marker utilizing a deletion mutation in the dihydroflavonol 4-reductase (DFR) gene responsible for the lack of anthocyanin production in yellow onions (Allium cepa).

Bulb color in onions (Allium cepa) is an important trait, but the mechanism of color inheritance is poorly understood at the molecular level. A previous study showed that inactivation of the dihydroflavonol 4-reductase (DFR) gene at the transcriptional level resulted in a lack of anthocyanin production in yellow onions. The objectives of the present study were the identification of the critical mutations in the DFR gene (DFR-A) and the development of a PCR-based marker for allelic selection. We report the isolation of two additional DFR homologs (DFR-B and DFR-C). No unique sequences were identified in either DFR homolog, even in the untranslated region (UTR). Both genes shared more than 95% nucleotide sequence identity with the DFR-A gene. To obtain a unique sequence from each gene, we isolated the promoter regions. Sequences of the DFR-A and DFR-B promoters differed completely from one another, except for an approximately 100-bp sequence adjacent to the 5'UTR. It was possible to specifically amplify only the DFR-A gene using primers designed to anneal to the unique promoter region. The sequences of yellow and red DFR-A alleles were the same except for a single base-pair change in the promoter and an approximately 800-bp deletion within the 3' region of the yellow DFR-A allele. This deletion was used to develop a co-dominant PCR-based marker that segregated perfectly with color phenotypes in the F2 population. These results indicate that a deletion mutation in the yellow DFR-A gene results in the lack of anthocyanin production in yellow onions.

Alcohol Oxidoreductases↗

PubMed2005

Development of a co-dominant, PCR-based marker for allelic selection of the pink trait in onions (Allium cepa), based on the insertion mutation in the promoter of the anthocyanidin synthase gene.

Alleles↗

PubMed2005

The L locus, one of complementary genes required for anthocyanin production in onions (Allium cepa), encodes anthocyanidin synthase.

Bulb color in onions (Allium cepa) is an important trait, but its complex, unclear mechanism of inheritance has been a limiting factor in onion cultivar improvement. The identity of the L locus, which is involved in the color difference between Brazilian yellow and red onions, is revealed in this study. A cross was made between a US-type yellow breeding line and a Brazilian yellow cultivar. The segregation ratio of nine red to seven yellow onions in the F(2) population supports the involvement of two complementary genes in anthocyanin production in the F(1) hybrids. The high-performance liquid chromatography (HPLC) and reverse-transcriptase (RT)-PCR analysis of the Brazilian yellow onions indicated that the genes are involved late in the anthocyanin synthesis pathway. The genomic sequence of the anthocyanidin synthase (ANS) gene in Brazilian yellow onions showed a point mutation, which results in an amino acid change of a glycine to an arginine at residue 229. Because this residue is located adjacent to a highly conserved iron-binding active site, this mutation is likely responsible for the inactivation of the ANS gene in Brazilian yellow onions. Following the isolation of the promoter sequence of the mutant allele, a PCR-based marker for allelic selection of the ANS gene was designed. This assay is based on an insertion (larger than 3 kb) mutation. The marker perfectly co-segregated with the color phenotypes in the F(2) populations, thereby indicating that the L locus encodes ANS.

Amino Acid Sequence↗

PubMed2002

Cytogenetic damage in shallot ( Allium cepa) root meristems induced by oil industry "high-density brines".

Saturated water solutions of calcium chloride, calcium bromide (densities 1.30 kg x dm(-3) and 1.61 kg x dm(-3), respectively) and their 1:1 mixture have been commonly used as oil industry "high-density brines." In our experiment they were added to tap water in amounts appropriate to achieve concentrations of 0.025, 0.05, 0.075, and 0.1 mol x dm(-3) to study their cytotoxic effect on the root tip cells of shallot ( Allium cepa L. var. ascalonicum). All tested solutions in concentrations of 0.075 and 0.1 mol x dm(-3) caused significant inhibition of shallot root growth. CaBr (2) showed this effect in concentration 0.05 mol x dm(-3). The investigated solutions in all concentrations applied decreased mitotic activity in root tip cells. The most of mitotic abnormalities were the consequence of spindle failure and chromosome stickiness. Furthermore, the cell microtubules were investigated by indirect immunofluorescence to confirm that most abnormalities observed were the consequence of spindle failure. The present study, as well as previously done Lemna tests and Chlorella tests showed that investigated samples have certain effects on plants, so constant control of their presence in the environment is needed.

Bromides↗

PubMed2004

Efficient plant regeneration from suspension cells of Allium cepa L.

Plant regeneration from calli of three cultivars of Allium cepa (Senshuki, O.Pki and Shojovaka) was investigated. Callus was induced on four variations of BDS medium containing different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BAP). The regeneration frequency of calli of cvs. Senshuki and O.Pki subcultured on solid MS medium supplemented with BAP ranged from 50% to 80%; this frequency decreased to less than 30% after subculture in the dark in liquid BDS medium. By repeating the dark/light transitions of the culture protocol and by selecting for green cell clusters, we were able to increase the regeneration frequency to more than 80% in all three cultivars. These cell clusters maintained a high regeneration capacity in subsequent subcultures in the absence of light for 2 months. Most (97%) of the regenerated plantlets had a normal diploid karyotype (2 n=16) that was identical to that of the mother plants, although 3% of the regenerated plants of cv. Shojovaka had a tetraploid karyotype.

Cell Culture Techniques↗

PubMed2005

Establishment of embryogenic cell suspension cultures of garlic (Allium sativum L.), plant regeneration and biochemical analyses.

Embryogenic cell suspension cultures of garlic (Allium sativum L.) were initiated in liquid medium from friable embryogenic tissue. The optimal parameters for culture maintenance were: (1) an initial cell density of 1-4% (v/v); (2) medium renewal every 14 days and subculturing every 28 days; (3) a low 2,4-dichlorophenoxyacetic acid concentration (0.1-0.3 mg/l). Cultures regenerated during a 14-month period. The cell suspension cultures differentiated embryos following transfer to a semi-solid embryo induction medium, with histological studies confirming and characterising the embryogenic nature of the process. Forty percent of these embryos converted into plantlets, which produced micro bulbs in vitro. The composition of the sulphur compounds of the micro bulbs obtained from cell suspension embryo-derived plantlets differed slightly from those produced by in vitro shoot proliferation-derived plantlets, but after two cycles of multiplication in the field these differences had disappeared.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2002

Dynamics of replication foci and nuclear matrix during S phase in Allium cepa L. cells.

The sequential organisation of replication foci during S phase in onion ( Allium cepa) and their relationship to the nuclear matrix were investigated. To discern their structural features and temporal firing sequence, immunodetection of 5-bromo-2'-deoxyuridine (BrdU) was carried out after in vivo feeding in synchronised cells released from a 14-h-long hydroxyurea block. Replication foci consisted of small replication granules, called replisomes, which clustered together. Analysis of synchronous binucleate cells that maintained in their two nuclei the specular symmetry of distribution of sister chromosomes in anaphase, showed that replication starts in small replication foci at the telomeric pole (pattern I), though the telomeres themselves formed large foci that were late-replicating. The rDNA replication foci (pattern II) also become replicated in early S phase. Replication of large foci, including the heterochromatin (IV), occurred in late S phase and finished at the centromeric nuclear pole (pattern V). Labelling of proliferating cell nuclear antigen (PCNA) in nuclear matrices, prepared from S-phase nuclei after extensive DNase digestion, demonstrated that replication foci were always stably anchored to the nuclear matrix. Thus, association with the nucleoskeleton is not exclusively mediated by the replicating or nascent DNA. The overlapping of patterns I, II and III in the nuclear matrix, in contrast to the results of BrdU localisation in nuclei, suggests that PCNA becomes associated with the nuclear matrix before the replication foci are operative, and remains bound during replication.

Bromodeoxyuridine↗

PubMed2006

Transgenic rice expressing Allium sativum leaf lectin with enhanced resistance against sap-sucking insect pests.

Mannose binding Allium sativum leaf agglutinin (ASAL) has been shown to be antifeedant and insecticidal against sap-sucking insects. In the present investigation, ASAL coding sequence was expressed under the control of CaMV35S promoter in a chimeric gene cassette containing plant selection marker, hpt and gusA reporter gene of pCAMBIA1301 binary vector in an elite indica rice cv. IR64. Many fertile transgenic plants were generated using scutellar calli as initial explants through Agrobacterium-mediated transformation technology. GUS activity was observed in selected calli and in mature plants. Transformation frequency was calculated to be approximately 12.1%+/-0.351 (mean +/- SE). Southern blot analyses revealed the integration of ASAL gene into rice genome with a predominant single copy insertion. Transgene localization was detected on chromosomes of transformed plants using PRINS and C-PRINS techniques. Northern and western blot analyses determined the expression of transgene in transformed lines. ELISA analyses estimated ASAL expression up to 0.72 and 0.67% of total soluble protein in T0 and T1 plants, respectively. Survival and fecundity of brown planthopper and green leafhopper were reduced to 36% (P < 0.01), 32% (P < 0.05) and 40.5, 29.5% (P < 0.001), respectively, when tested on selected plants in comparison to control plants. Specific binding of expressed ASAL to receptor proteins of insect gut was analysed. Analysis of T1 progenies confirmed the inheritance of the transgenes. Thus, ASAL promises to be a potential component in insect resistance rice breeding programme.

Animals↗

PubMed1996

Allium sativum-induced death of African trypanosomes.

The effect of Allium sativum (Liliacea) on trypanosome-infected mice was examined. At a dose of 5.0 mg/ml, the oily extract from the pulp completely suppressed the ability of the parasites to be infective in the host. Column chromatography of the extract gave four fractions: ethylacetate/methanol, ethylacetate/ethanol, benzene/methanol, and acetic acid/methanol. Among these fractions, the acetic acid/methanol fraction retained the trypanocidal features of the crude extract. It cured experimentally infected mice of trypanosomiasis in 4 days when given at a dose of 120 mg/kg per day. The extract also manifested inhibition of procyclic forms of Trypanosoma brucei brucei and phospholipases from T. congolense, T. b. brucei, T. vivax. The extract appears to be diallyl-disulfide (DAD) and may interfere with the parasites' synthesis of membrane lipids.

Animals↗

PubMed2004

The fertility restorer genes X and T alter the transcripts of a novel mitochondrial gene implicated in CMS1 in chives (Allium schoenoprasum L.).

A chimeric mitochondrial gene configuration, mainly derived from sequences associated with the essential genes atp9 and atp6, was isolated from the sterility-inducing cytoplasm of the CMS1 system in chives (Allium schoenoprasum L.). This sequence is not found in four other cytoplasm types from chives; however, two copies are present in the mitochondrial DNA of CMS1-inducing cytoplasm, whose 5'-sequences are homologous to those of the atp9 gene. We provide evidence to show that one of the two CMS1-specific copies is actively transcribed, and two transcripts which terminate at the same position but differ in their 5'initiation sites were localized using the RACE technique. These transcripts of 942 and 961 nt, respectively, were confirmed to be the major products of this gene in CMS1 plants by Northern hybridization. However, smaller transcripts were found to accumulate in plants in which fertility had been restored. Restoration of fertility was induced either by the gene X, or the gene T at high temperatures. In (S1) X. genotypes a transcript with an estimated size of 440 nt was detected in all tissues examined. An additional hybridization signal with an estimated size of approximately 850 nt is expressed in temperature-sensitive plants [(S1) xxT.], and the intensity of a minor 350-nt transcript is enhanced. These latter alterations, conditioned by the gene T, occur independently of the growth temperature, but are limited to the flowers; they were not observed in leaves. The CMS1 transcripts are edited at seven positions and contain an ORF with a maximum coding capacity of 780 nt (containing the start codon derived from the atp9 gene in-frame). Use of the third in-frame start codon would result in the synthesis of a protein of a size very close to that of a previously described CMS1-specific protein, which has an apparent molecular weight of 18 kDa. The coding sequence that begins at this third in-frame start codon is also present in the sterility-inducing cytoplasms (S) and (T) in the onion, and absent in (N) cytoplasm.

Amino Acid Sequence↗

PubMed2004

Effects of arbuscular mycorrhizal colonization and phosphorus application on nuclear ploidy in Allium porrum plants.

Arbuscular mycorrhizal (AM) colonization can strongly affect the plant cell nucleus, causing displacement from the periphery to the center of the cell, hypertrophy and polyploidization. The hypertrophy response has been shown in a variety of AM plants whilst polyploidization has been reported only in Lycopersicon esculentum, a multiploid species with a small genome. In order to determine whether polyploidization is a general plant response to AM colonization, analyses were performed on Allium porrum, a plant with a large genome, which is much less subject to polyploidization than L. esculentum. The ploidy status of leaves, complete root systems and four zones of the adventitious roots was investigated in relation to phosphorus content, AM colonization and root differentiation in A. porrum plants grown under two different regimes of phosphate nutrition in order to distinguish direct effects of the fungus from those of improved nutrition. Results showed the presence of two nuclear populations (2C and 4C) in all treatments and samples. Linear regression analyses suggested a general negative correlation between phosphorus content and the proportion of 2C nuclei. The percentage of 2C nuclei (and consequently that of 4C nuclei), was also influenced by AM colonization, differentiation and ageing of the root cells, which resulted in earlier occurrence, in time and space, of polyploid nuclei.

Cell Nucleus↗

PubMed2003

Differential distribution of ascorbic acid, peroxidase activity, and hydrogen peroxide along the root axis in Allium cepa L. and its possible relationship with cell growth and differentiation.

In this paper we show an asymmetrical distribution of apoplastic and symplastic ascorbic acid content, peroxidase activities and hydrogen peroxide along the root axis in Allium cepa L. For most of these metabolites, a marked gradient from the root apex to the onion base was observed and was different for apoplastic and symplastic compartments. In total homogenates, ascorbic acid content was higher in the zones closer to the apex and decreased towards the root base. However, an opposite pattern was observed in the apoplastic fraction. Peroxidase activities with guaiacol, ferulic acid, ascorbic acid, and coniferyl alcohol were also different depending on the evaluated zone and the fraction used (apoplastic or symplastic). In general, each activity had a specific and unique pattern. Immunodetection of peroxidase proteins in Western blots using anti-horseradish peroxidase and anti-ascorbate peroxidase antibodies revealed different bands at the different zones of the root. Hydrogen peroxide was detected by electron microscopy and was mainly found in cell walls of epidermis (or rhizodermis), meristem, and elongating cells. The number of cell walls showing hydrogen peroxide decreased dramatically towards the root base. The results suggest that the different zones of the root show specific requirements for ascorbic acid and hydrogen peroxide. Also, each fragment of the root seems to express specific peroxidase proteins. Different processes that take place at every part of the root, as cell proliferation and elongation near the root apex and gradual lignification and differentiation towards the root base are the key to explain the results.

Ascorbic Acid↗