Cholinesterase activity in some species of theAllium genus

In partly purified protein complexes obtained from 22 species of theAllium genus and 6 cultivars ofAllium cepa the activity of cholinesterases was detected and measured using the method of Ellman et al. The degree of its inhibition with 10^-4 M neostigmine was also tested. It was found that the activity of cholinesterase differed in individual species up to two hundred times, while the differences in the inhibitory activity of 10^-4 M neostigmine occurred only in a few cases. Individual sections and cultivars could not be characterized on the basis of the differences in the activities of the cholinesterases. Of all the sections that ofPhyllodolon shows the highest average activity. In the case of the tested cultivars distinctly the lowest activity was observed in cv. Kastická. The values of the enzymatic activity measured by Ellman’s method in this plant material include the activity of specific and unspecific cholinesterases and the part uninhibitable by neostigmine.     

Use of esterase isoenzymes revealed by gel isoelectric focusing as an aid in chemotaxonomical study of the genusAllium

Eleven species of the genusAllium belonging to the sectionCepa, Phyllodolon, Rhizirideum, Melanocrommyum andAlliun ware investigated as to the presence of esterase isoenzymes by means of the gal isoelectric focusing which shows better resolution than polyaorylamide gel electrophoresis. In all examined species twenty six isoenzymes were found. The individual sections are well characterized by esterase isoenzymes revealed by this method, on the other hand, the differences, as shown by the Jaceard index, between the subganera are insignificant. Investigation of seven cultivars ofAllium cepa shows the isolated position of the cultivar Ka?tická. These results are in full agreement with those found with use of the polyacrylamide gel electrophoresis and show the usefulness of gel isoelectric focusing for the solution of the chemotaxonomical problems.     

Random amplified polymorphic DNA (RAPD) markers for genetic analysis inAllium

RAPD analysis was applied to onion (Allium cepa) and otherAllium species in order to assess the degree of polymorphism within the genus and to investigate if this approach was suitable for genetic studies of onion. Seven cultivars ofA. cepa, including shallot, and single cultivars of Japanese bunching onion (A. fistulosum), chive (A. schoenoprasum), leek (A. ampeloprasum), and a wild relative of onion (A. roylei), were evaluated for variability using a set of 20 random 10-mer primers. Seven out of the twenty primers revealed scorable polymorphisms between cultivars ofA. cepa and these will be further evaluated for use in genetic mapping. Wide variations in banding profiles between species were observed with nearly every primer tested. These were assessed for use in systematic studies within the genus. Ninety-one band positions were scored (+/-) for all the cultivars studied. Genetic distances between each of the cultivars were calculated and cluster analysis was used to generate a dendrogram showing phylogenetic relationships between them. The resulting analysis was in broad agreement with previous classifications of the species studied, confirming the validity of the method. However, amongst the species studied, it placedA. roylei as the closest relative ofA. cepa, questioning the current classification of the former species in the section Rhizideum.     

Comparison of malate dehydrogenase isoenzymes in someAllium species by isoelectric focusing

Malate dehydrogenase isoenzymes were studied in tenAllium species and in six cultivars ofA. cepa by isoelectric focusing in polyacrylamide gel with Ampholine pH 3.5–10.0. Using this method better resolution was obtained than by polyacrylamide gel electrophoresis. The number of MDH isoenzymes obtained by isoelectric focusing is from five to ten in the range of pH 3.65 to 6.75. MDH isoenzymes can be used for characterization on the level of species and cultivars (inA. cepa), but its use on the level of sections and subgenera is questionable.     

Variability in blood platelet inhibitory activity of Allium (Alliaceae) species accessions

In the past 20 yr, the presence of blood platelet inhibitory activity in plant species from the genus Allium has been confirmed by a range of clinical and in vitro investigations. Although a number of Allium species have been identified as possessing antiplatelet properties, little is known of the variability for this trait among accessions in these species. Experiments were conducted to assess variability in antiplatelet activity of 58 Allium (Alliaceae) accessions. Extracts were prepared from a diverse collection of 16 Allium species accessions, 33 cultivated accessions of Allium cepa including standard cultivars, inbred lines, and open-pollinated populations, and nine Allium cepa plant introductions of diverse origin. Platelet inhibitory activity was determined via a platelet aggregation assay with human platelet-rich plasma. Relative in vitro inhibition of platelet aggregation was measured for each accession and control samples, and inhibition constants (IC₅₀) were calculated. Dose-dependent inhibition was observed and measured for each Allium accession. Significant (P < 0.01) IC₅₀ variability was detected among accessions, with the lowest accession IC₅₀ values exhibiting nearly 50-fold greater inhibition of aggregation than the highest accession IC₅₀ values. IC₅₍₎ variability among Allium cepa accessions was ≈ 12 times less than among Allium species accessions. Results from this investigation demonstrate substantial variability for efficacy of the antiplatelet factor among Allium accessions.     

In situ hybridization confirms jumping nucleolus organizing regions in Allium

In situ hybridization with a ¹²⁵I-rDNA clone from Vicia faba was performed against Allium cepa and three strains of top onion, which represent hybrids between A. cepa and A. fistulosum. In principle, the labelling patterns correspond to the patterns of the silver-stained nucleolus organizing regions (NORs) in the same species. This strongly supports the inference drawn from the Ag-NOR patterns that NORs can jump between terminal heterochromatin blocks of different Allium chromosomes in the parental species A. cepa as well as in their interspecific hybrids.     

Bioactive polar natural compounds from garlic and onions

Bioactive natural compounds from garlic and onions have been the focus of researches for decades, firstly due to their pharmacological effects, and secondly due to their defence properties against plant diseases. In fact, garlic and onion, belonging to Allium genus, are among the oldest food plants known since ancient times and used as ingredient of many recipes and for therapeutic properties. These plants are well known to produce bioactive apolar sulphur compounds but less is known about their polar natural compounds, such as phenols, sapogenins and saponins, that are more stable to cooking, So, we continued our work on the discovery of polar bioactive metabolites from Allium with the isolation of a number of sapogenins and saponins from the wild onion species Allium elburzense, Allium hirtifolium, Allium atroviolaceum, and Allium minutiflorum, and, more recently, from the cultivated white onion, Allium cepa, and garlic, Allium sativum. In particular, the sapogenins and saponins isolated from A. elburzense and A. hirtifolium, named elburzensosides and hirtifoliosides respectively, exhibited significant antispasmodic properties. In addition, the saponins named minutosides isolated from A. minutiflorum showed promising antimicrobial activity. More recently the phytochemical analysis of A. cepa and A. sativum has been undertaken and afforded the characterization of saponins, phenols and N-cynnamic amides which showed significant antifungal activity.     

Chromosomal location of rDNA in Allium: in situ hybridization using biotin- and fluorescein-labelled probe

Summary A biotin- and fluorescein-labelled probe of Helianthus argophyllus has been used to map specific repeated rDNA sequences by in situ hybridization on mitotic chromosomes of Alliwn cepa, Allium fistulosum, a diploid interspecific (Allium fistulosum x Allium cepa) F₁ hybrid, and a triploid interspecific (2 x = A. cepa, 1 x = A. fistulosum) shallot. Hybridization sites were restricted to satellited and smallest pairs of chromosomes in both A. cepa and A. fistulosum. The number, size, and position of the hybridization sites distinguish homologous chromosomes and identify the individual chromosomes carrying the nucleolus organizing region (NOR) at the secondary constriction, as well as the individual chromosomes carrying an additional NOR. This in situ hybridization technique is the first reported in a plant species and offers new cytogenetic markers in Allium.     

Compounds from Allium species with cytotoxic and antimicrobial activity

Garlic (Allium sativum L.) is a bulb-shaped plant belonging to the Allium genus which also includes onions (Allium cepa L.), leek (Allium ampeloprasum L. var. porrum Gay), shallot (Allium ascalonicum L), scallion (A. fistulosum L.) and chives (Allium schoenoprasum L.). The biological activity of garlic has been known since ancient times. Babylonians, Egyptians, Phoenicians, Greeks and Romans used garlic as a remedy for intestinal disorders, respiratory infections, skin diseases, bacterial infections, worms, wounds and tumors. In particular, before the discovery of antibiotics, garlic has been used against amoebic dysentery and epidemic diseases such as typhus, cholera, diphtheria, and tuberculosis. To date, more than 3,000 publications scientifically supported the use of garlic in the ethno-medicine. But what makes garlic and Allium species effective against cancer? The effect of garlic may arise from its antibacterial properties or from its ability to block formation on cancer-causing substances, half the activation of cancer causing substances, enhance DNA repair, reduce cell proliferation or induce cell death. Epidemiological studies have found that an increase of consumptions of Allium spp. reduce the risk of prostate and gastric cancers and this has been mainly related to two main classes of compounds: the apolar sulphur compounds and the polar saponins. These latter compounds, compared to the more studied thiosulphinates, have the advantages of not being pungent and more stable during cooking. Recently, there has been increasing scientific attention given to such compounds. In this paper, the literature about the major volatile and non-volatile organic compounds of garlic and other Allium plants has been reviewed. Particular attention is given to the compounds possessing antibacterial and cytotoxic activity in garlic and in the other Allium species and their mechanism of action.     

Protein complex and esterase isoenzyme patterns ofAllium sativum L. cultivars and clones-regenerants

Protein complex patterns of cloves and esterase isoenzyme patterns of apical buds of cloves were studied with Czechoslovak virus-free cultivars ofAllium sativum L. and the wild speciesA. longicuspis Regel, Similarly, four clones-regenerants obtained using explant culture techniques from A.sativum L. cv. Bzenecky paličák (two somaclones and two clones derived from plants regenerated from meristem cultures treatedin vitro with colchicine) differing in their ploidy, morphology, and yields were studied. Immunophoreograms of protein complexes of theA. sativum L. cultivars under investigation differed from one another in the number and mobility of protein fractions in both the cathodic and anodic regions and thus these cultivars can be distinguished. On the basis of esterase isoenzyme patterns, the Czechoslovak cultivars of A. sativum L. can be arranged into three groups - bolting winter cultivars with broad leaves, non-bolting winter cultivars with broad leaves, and non-bolting spring cultivars with narrow leaves. All the clones-regenerants showed the same protein complex and esterase isoenzyme patterns as their original cultivar.A. longicuspis Regel markedly differed in its protein complex and esterase isoenzyme patterns from all the other genotypes studied.Received May 17, 1989: accepted January 5,1990     

Die ursachen der sterilität vonAllium sativum L.

A fully normal meiosis was found inAllium sativum L. by means of PMC analysis. Quadrivalents were formed only in several cultivars. The cultivation of inflorescence stem (after the exstirpation of bulblets) in a tetracycline solution of 900 i.u. induced a formation of fertile flowers. The first pollen mitosis was studied and the pollen parameters compared with those ofAllium cepa L. Evidence for the pollen sterility was given by crossings on sterile types. A microbiological factor was not isolated.     

Phylogenetic relationships among cultivated Allium species from restriction enzyme analysis of the chloroplast genome

Summary The genus Allium contains many economically important species, including the bulb onion, chive, garlic, Japanese bunching onion, and leek. Phylogenetic relationships among the cultivated alliums are not well understood, and taxonomic classifications are based on relatively few morphological characters. Chloroplast DNA is highly conserved and useful in determining phylogenetic relationships. The size of the chloroplast genome of Allium cepa was estimated at 140 kb and restriction enzyme sites were mapped for KpnI, PstI, PvuII, SalI, XbaI, and XhoI. Variability at restriction enzyme sites in the chloroplast DNA was studied for at least three accessions of each of six cultivated, old-world Allium species. Of 189 restriction enzyme sites detected with 12 enzymes, 15 mutations were identified and used to estimate phylogenetic relationships. Cladistic analysis based on Wagner and Dollo parsimony resulted in a single, most-parsimonious tree of 16 steps and supported division of the species into sections. Allium species in section Porrum were distinguished from species in sections Cepa and Phyllodolon. Two species in section Rhiziridium, A. schoenoprasum and A. tuberosum, differed by five mutations and were placed in separate lineages. Allium cepa and A. fistulosum shared the loss of a restriction enzyme site and were phylogenetically closer to each other than to A. schoenoprasum. This study demonstrates the usefulness of restriction enzyme site analysis of the chloroplast genome in the elucidation of phylogenetic relationships in Allium.     

Restriction enzyme analysis of the chloroplast and nuclear 45s ribosomal DNA ofAllium sectionsCepa andPhyllodolon (Alliaceae)

Estimates of the phylogenetic relationships among cultivated and wildAllium species would benefit from identification of molecular characters. Restriction enzyme analysis of the chloroplast DNA (cpDNA) of the bulb onion (Allium cepa), Japanese bunching onion (A. fistulosum), wildAllium species in sect.Cepa andPhyllodolon, and the outgroupsA. ampeloprasum andA. tuberosum detected 39 polymorphisms.Allium cepa andA. vavilovii were identical for all characters. Cladistic analysis generated three most-parsimoniousWagner trees of 44 steps differing only in a zero-length branch.Allium fistulosum andA. altaicum (sect.Phyllodolon) comprised a monophyletic lineage separated from theA. cepa andA. vavilovii of sect.Cepa. The unresolved node was composed ofA. galanthum, A. roylei, and the lineage containingA. cepa, A. vavilovii, A. fistulosum, andA. altaicum. The clade containingA. altaicum, A. cepa, A. fistulosum, A. galanthum, A. roylei, andA. vavilovii remained resolved for strict consensus ofWagner trees of 48 steps or less.Allium pskemense andA. oschaninii were increasingly distant.Allium oschaninii has been proposed as the progenitor of the bulb onion, but was more closely related to the common progenitor of all species in sect.Cepa andPhyllodolon. Phylogenies estimated from cpDNA characters usingDollo parsimony resulted in a single most-parsimonious tree of 46 steps and agreed with phylogenies based onWagner parsimony. Polymorphic restriction enzyme sites in the 45s ribosomal DNA were not used to estimate phylogenies because of uncertain homologies, but are useful for identifying interspecific hybrids. The maternal phylogenies estimated in this study help to distinguish wildAllium species closely related to the bulb onion. Although not in agreement with classifications based on morphology, the phylogenies closely reflected crossability among species in sect.Cepa andPhyllodolon.     

Studies on fungi in the root region

Summary Aqueous extracts of adventitious roots ofAllium sativum L. (garlic), seedling roots ofAllium porrum L. (leek), and both adventitious and seedling roots ofAllium cepa L. (onion), were tested for antibiotic activity against three root-surface fungi,Cylindrocarpon radicicola Wollenw.,Gliocladium roseum (Link) Thom andFusarium culmorum (W. G. Smith) Sacc. by means of two different techniques.With a filter-paper disc technique, root extracts sterilised by membrane-filtration produced zones of inhibition of the test fungi, whereas root extracts sterilised by autoclaving showed no activity. Garlic root extract produced inhibition zones with all the test fungi, whereas extracts of onion adventitious roots and leek seedling roots produced inhibition zones with only one of the test fungi. The extract of onion seedling roots produced no inhibition zones. Root extracts of all theAllium species, when sterilised by membrane filtration, generally inhibited spore-germination of all the test fungi.     

Interspecific crosses of onion with distant Allium species and characterization of the presumed hybrids by means of flow cytometry, karyotype analysis and genomic in situ hybridization

Interspecific crosses were made by hand-pollination of Allium cepa with pollen of 19 species belonging to nine sections of two subgenera of the genus Allium. In all cases viable plantlets were obtained from ovary culture. The efficiency depended on the relationship of the pollen donor to A. cepa. The hybrid character of the regenerants was checked by morphological comparisons with the parents and/or by one or more cytological methods such as flow cytometric DNA measurement, karyotype analysis, and genomic in situ hybridization (GISH). Hybrids were confirmed for 18 new species combinations. The viable hybrid of the most distant cross resulted from crossing A. cepa with A. sphaerocephalon. The relevance of the verification methods and the potential use of the hybrids for breeding purposes are demonstrated.     

Phylogenetic relationships of Iranian Allium sect. Allium (Amaryllidaceae,Allioideae) as inferred from nrDNA ITS,cpDNA rps16 and trnL–F sequences

Allium is a particularly species rich (more than 800 species) and economically important genus, with numerous taxonomic problems at all levels of classification. In this study, we try to uncover the phylogenetic relationships of the common leek Allium ampeloprasum based on selected samples of this species and its putative relatives in A. sect. Allium from Iran. The silica‐dried leaf samples of 56 accessions representing 23 species of Allium were sequenced; 53 sequences of nrDNA ITS, 35 sequences of plastid rps16 and 52 sequences of trnL–F were generated and additional accessions were extracted from GenBank in order to cover all recognized main lineages in the genus. Maximum Parsimony and Bayesian Inference generated similar trees, but the placement of A. ampeloprasum and its relatives differed slightly between the nuclear and the plastid phylogenies. In the nrITS tree, A. ampeloprasum is retrieved as a highly supported clade with A. iranicum, while in the combined plastid tree A. ampeloprasum formed a highly supported clade with A. vineale. This supports the hypothesis of a possible hybrid origin of A. ampeloprasum. Allium iranicum formed a clade in the plastid tree, but was resolved as paraphyletic in the nrITS tree, probably due to presence of multiple non‐concerted copies of nrITS. Close relationships are suggested between the following species: A. aznavense and A. wendelboi with A. talyschense, A. erubescens and A. rotundum with A. scorodoprasum and A. abbasii with A. phanerantherum.     

The localization of starch in root tips

As an extension of earlier studies performed in our laboratory on enzyme localization on tissue level, the distribution of starch was examined in seedling root tips of peaPisum sativum L., broad beanFaba vulgaris Moench., maizeZea mays L., lupinLupinus albus L., and pumpkinCucurbita maxima Duch., and in tips of adventive roots of onionAllium cepa L. by means of the alcian blue /JJK procedure applied to paraffin sections. In pea, some genetic (various cultivars) and eco-physiological (different harvests, various localities, diverse way of germination, uneven seedling age, unequal stage of cell growth) factors were studied concerning the present problem. In all these cases the starch localization pattern remains constant though the amounts of starch vary in particular cell complexes. From the evaluation of the starch localization at the level of the beginning cell growth in particular species and from comparison of the findings in different species it follows that starch localization and histogenesis patterns are different. This supports the view that the two phenomena are caused or controlled by uneven factors.     

Irreversible effects of ionizing RTG-radiation on the osmotic properties of the epidermal cells ofAllium cepa L. bulbs

The effect of different exposures to RTG radiation (100 r, 250 r, 500 r, 1000 r, 2500 r, 5000 r) on the changes of the permeability of the epidermal cells ofAllium cepa L., variety V?etaty, bulbs was studied during three vegetation periods. Bulbs from growing plants (followed from the development phase of bulb growth) and bulbs immediately after harvesting were used for the experiments. It was demonstrated in our experiments by plasmolysis that the radiation-induced changes of cell permeability were irreversible. When plasmolytic agents of non-electrolyte character (1M sucrose, 1M glycerol, 1M urea) were used, the equilibrium state (total plasmolysis) was reached more rapidly with the irradiated bulbs than with the unirradiated controls. When electrolytes were used (0·5M KNO₃, 0,5M KBr), total plasmolysis occurred less rapidly with the irradiated objects than with the controls. These relationships were manifest during the whole ontogenesis ofAllium cepa L. An exposure to 100 r was sufficient in our experiments for causing the mentioned changes, proving a considerable sensitivity of the osmotic properties of the epidermal cells ofAllium cepa L. bulbs to ionizing RTG radiation. Plasmolytic experiments after smaller exposures than 100 r of RTG-rays are in preparation.