Differentiation of metaxylem cell line in the root ofAllium cepa

Summary The first step of differentiation in the root segments ofAllium cepa containing metaxylem cells in different stages of differentiation were studied by DNA reassociation curves and compared to meristem cell extracted DNA. Upon sonication of DNA samples to about 400 base pairs, the reassociation profiles of the heat denatured DNA, were spectrophotometrically followed at two different concentrations. The kinetic complexities,i.e., the number of base pairs per haploid genome of a given sequence and its redundancy were calculated. Differences were found at the level of highly and medium repetitive sequences, thus demonstrating that some DNA reassociation classes may undergo amplification during root development.     

The effect of lead on the cell cycle in the root meristem ofAllium cepa L.

Summary Allium cepa (L.) adventitious roots were treated with lead (2.5 mg of Pb²⁺ [from Pb(NO₃)₂] per dm³) for 30–72 h. The cell cycle was studied by pulse labeling with [³H]thymidine. Mitotic activity kinetics, occurrence of disturbed mitoses (c-mitoses), and level of DNA synthesis were examined. It was found that lead prolonged the cell cycle and that cells in two phases of the cycle, G₂ and S, differed in their sensitivity to lead. Cells in G₂ were more sensitive; lead lengthened their cycle by 216% and disturbed the course of cell division by causing c-mitoses. Cells in S phase were less sensitive. Their cell cycle was longer by 55%. They went through their G₂ phase without major disturbances, mitosis in these cells was normal. During treatment ofA. cepa with lead, its destructive effects on cells were exerted only during the first few hours (around 6 h) of incubation. That is when the inhibition of mitotic activity, numerous disturbances of cell division, a decline in the number of cells synthesizing DNA, and a lower level of DNA synthesis were observed. As the incubation continued, the above processes were found to return to normal. In the discussion, data are presented supporting the hypothesis that during the initial period of exposure ofA. cepa to lead, this metal enters both the root apoplast and symplast, exerting a destructive effect on cells, while later, lead penetrates only into the root apoplast, and in this way remains harmless to cells.     

Differentiation of metaxylem cell line in the root ofAllium cepa

Summary DNA was extracted from three root segments ofAllium cepa: i) an apical portion 500 m long from the tip (meristem); ii) a second portion 4 mm long (I root segment containing metaxylem cells in the initial stages of differentiation); iii) a third portion 6 mm long (II root segment containing metaxylem cells in further stages of differentiation). A mixture of homologous 18 S and 25 S³H-rRNA was used for invitro DNA-rRNA hybridization. The following percent saturation values were detected in the three samples: 0.08 in meristem DNA (samplea), 0.129 in I root segment DNA (sampleb), and 0.105 in II root segment DNA (samplec).Thermal denaturation of DNA and the derivative curves of the melting profiles evidenced five DNA families which were differently represented in the three DNA samples. DNA elution by thermal chromatography on hydroxyapatite followed by hybridization with³H-rRNA, revealed that ribosomal cistrons melt between 90 and 91 °C, corresponding to a G-C content of 50.7%. Moreover, the amount of the DNA family containing ribosomal cistrons was greater in sampleb andc, in sampleb to a greater extent, as compared with samplea. On the other hand, one DNA family melting at a higher temperature (92–93 °C) was drastically increased in samplec.Buoyant density profiles of unsonicated DNA showed no peaks in the three DNA samples. Upon somcation, a heavy shoulder was observed in the profile of sampleb. As the density of ribosomal cistrons and that of shoulder were very similar, it seems possible that the two fractions contain many DNA sequences in common.The present studies demonstrate that the proportion of ribosomal cistrons and other DNA families does not keep constant during the development of the metaxylem cell line.     

Pericycle proliferation pattern during the lateral root initiation in adventitious roots ofAllium cepa

Summary Far from the apical meristem of adventitious roots ofAllium cepa, the pericycle shows great proliferative activity related to lateral root initiation. A group of mother pericycle cells undergoes asymmetrical transverse and periclinal divisions following a well-established pattern. Successive asymmetrical transverse divisions, progressing from one end of the cell to the other, divide the original mother cell into very short derivatives. Later, these short derivative cells undergo periclinal divisions. This proliferative activity starts nearly simultaneously in two elongated and highly vacuolated pericycle cells located in the same column in front of one of the xylem poles. Then proliferation expands centrifugally towards other pericycle cells in the same and adjacent columns. The proliferative activity of the pericycle cells decreases progressively outwards. Only the most central of these cells produce derivatives which contribute to the future lateral root.     

Lateral root initiation by asymmetrical transverse divisions of pericycle cells in adventitious roots ofAllium cepa

Summary In onion adventitious roots cellular events have been identified that indicate that lateral root initiation occurs earlier and nearer the apex than previously documented. Lateral roots are not initiated when a pericycle cell divides periclinally but earlier, when a pair of neighbouring pericycle cells in the same column divide transversely and asymmetrically, with both mitoses close to the end towards the neighbouring pericycle cell. Each cell therefore produces two cells of unequal length. The shorter cells produced by the mother pericycle cells are adjacent, while the longer cells are located above and below the shorter cells. This objective morphological criterion allows clear identification of the site of lateral root initiation. Subsequent to these asymmetric divisions, both the longer pericycle cells again divide transversely and asymmetrically producing more short cells adjacent to the previous ones. The first periclinal division occurs in one of these short pericycle cells.     

Effects of successive applications of fosetyl-Al on growth ofAllium cepa L. associated with endomycorrhizal fungi

The effect of successive applications (0 to 4) of fosetyl-Al on the growth of mycorrhizal (M) and nonmycorrhizal (NM) onion plants and on the residual quantity of the fungicide in the root was studied for 6 weeks under growth chamber conditions. Fosetyl-Al was applied as foliar spray at a concentration of 3 mg ml^–1. The successive applications of fosetyl-Al did not affect the dry mass of root or shoot of M plants. NM plants sprayed only once with fosetyl-Al had a higher root mass than plants sprayed 0, 2, 3 and 4 times. The quantity of phosphorous acid, the only residue detected, was positively correlated with the number of fosetyl-Al applications. It showed a positive partial correlation coefficient with the shoot (r=0.41) and the root dry mass (r=0.40) of NM and M plants.Contribution no 344, Station de recherches, Agriculture Canada, Sainte-Foy.     

Differences between,and possible origins of,the cytoplasms found in fertile and male-sterile onions (Allium cepa L.)

Summary The DNA of the organellar genomes of Allium cepa has been examined to detect restriction fragment length polymorphisms. Differences can be shown between both the chloroplastal and mitochondrial genomes of the N and cms-S cytoplasms in their restriction fragment profiles. Southern blot analysis of the mtDNA profiles using probes containing defined mitochondrial genes also detected polymorphisms. No differences can be shown between the organellar genomes of the N and cms-T onions by either of these techniques. These data indicate different origins for the two sterility-conferring cytoplasms, suggesting autoplasmic and alloplasmic origins for the cms-T and cms-S cytoplasms, respectively. No evidence of the presence of virus-like particles was found in any of the cytoplasms.     

Effects of cycloheximide on preprophase bands and prophase spindles in onion (Allium cepa L.) root tip cells

Summary Effects of cycloheximide (CHM) on preprophase bands (PPBs) of microtubules (MTs) and on prophase spindle MTs in root tip cells of onion (Allium cepa L.) were examined. When root tip cells were treated with 36 M CHM for 0.5–4 h, the population of cells with a PPB did not decrease markedly although the population of mitotic cells and that of prophase cells with a PPB gradually decreased to half of the control root tips. In prophase cells treated with 11 and 36 M CHM for 2 h, the width of the PPB was 1.4 times broader than that in the prophase PPB without CHM. Electron microscopic observation on the cross section of the PPB showed that the number of MTs and the distance between adjacent MTs in prophase PPBs treated with CHM were similar to those in the early developmental stage of PPBs without CHM. The bipolar spindle, that appeared in late prophase was not seen in prophase cells treated with 11 M or higher concentrations of CHM for 2 h. In order to examine differences of perinuclear MT arrangement between CHM treated and non-treated prophase cells, arrangement of perinuclear MTs was examined by confocal laser scanning microscopy. In control cells without CHM, MTs appeared on the nuclear surface with several branched or cross over type MT foci in the cytoplasm when broad PPB formation started. These MT foci were replaced by the aster type MT foci, from which several MTs radiated along the nuclear surface. The aster type MT foci gradually gathered to form a bipolar spindle. MTs connecting the spindle pole region and the PPB were seen in late prophase. In CHM-treated cells (11-360 M for 2 h), branched and cross over type MT foci were prominent, even in prophase cells with well condensed chromosomes. Neither linkages of MTs between the spindle pole region and the PPB nor aster type MT foci were seen. These observations showed that CHM prevents the bundling of MTs in the PPB and also inhibits the formation of aster type MT foci that is essential for bipolar spindle development.     

Sequence heterogeneity of the ribosomal DNA inAllium cepa (Liliaceae)

Summary Southern blot hybridizations were performed to investigate the ribosomal DNA structure inAllium cepa (Liliaceae). The analysis of single and double digestions with Eco RI, Hind III, and Bam HI endonuclease restriction enzymes showed the presence of at least two types of repetitive units. The gene types were present in each single individual ofAllium cepa analyzed, notwithstanding the variation of the ribosomal gene number from 5,500 to 11,900 and the NOR number from 2 to 4. The first and the second gene type are 12.7 kb long and the first type is much more represented inAllium cepa genome. The differences between the two gene types consist in the position of the Hind III restriction sites in the external spacer.Work supported by CNR contract no. 74/0267.     

Enhanced seed development in the interspecific cross Allium cepa x A. sphaerocephalon through ovary culture

Allium sphaerocephalon pollen tubes grew into styles and penetrated micropyles of Allium cepa, but ovules started to degenerate about 16 days after pollination and no seeds developed. Seeds developed in vitro in ovaries excised from flowers 4 and 7 days after pollination. Seven weeks after culture initiation, seeds had grown in 4 of 96 excised ovaries, cultured on BDS medium supplemented with GA₃. Although the culture medium supported seed maturation within excised ovaries of self-pollinated A. cepa flowers, no viable hybrid seeds were recovered from crosses with A. sphaerocephalon. Extended post-fertilization barriers may have restrained development of hybrid embryos in vitro. Ovary culture followed by in ovulo embryo rescue may be feasible for distant-species hybridization in Allium.     

Behavior of leucoplast nucleoids in the epidermal cell of onion (Allium cepa) bulb

Summary The behavior of nucleoids during the leucoplast division cycle in the epidermis of onion (Allium cepa) bulbs was investigated using DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) staining. The leucoplast was morphologically amoeboid and continuously changed its shape. A dumbbell-shaped leucoplast divided into two spherical daughter ones by constriction in the middle region of the body. Leucoplasts contained 4–10 mostly spherical, oval, partly rodand dumbbell-shaped nucleoids which were dispersed within the bodies. The proportion of one DNA molecule of a T4 phage particle to the small leucoplast nucleoid in the grain density of negative film was 1 to 0.91. Comparison of the present result and another groups' biochemical results suggested that a small leucoplast nucleoid contains one DNA molecule. The dumbbell-shaped leucoplast probably before division contained about twice as many nucleoids as the spherical leucoplast after division, and each half of the dumbbell contained about half the number of nucleoids. Nucleoids increased in number with growth of the leucoplast. The behavior of nucleoids during the leucoplast division cycle in onion bulbs was basically similar to that during the chloroplast division cycle in higher plants and green algae, which was previously reported (Kuroiwa et al. 1981 b).     

Microtubules and coated vesicles in guard-cell protoplasts ofAllium cepa L.

Protoplasts were prepared from the guard cells ofA. cepa. Epidermal peels taken from expanding green leaves and largely free of mesophyll were treated with Cellulysin, and protoplasts were harvested after 18 h of digestion. That the protoplasts were derived from guard cells was ascertained from their characteristic vacuolar autofluorescence and from observations showing that all other epidermal cells are killed in the peeling procedure. The protoplasts proved to be a good system with which to view the cell cortex and inner surface of the plasmalemma. The lysis of cells adhering to polylysine-treated, Formvar-coated grids, followed by negative staining in uranyl acetate, showed that many microtubules normally present in ordered arrays in situ remain closely applied to the inner surface of the plasmalemma in protoplasts. In addition, numerous vesiculate elements including coated vesicles and/or pits are present amongst the microtubules. Similar vesicles are evident in thin sections of fixed, embedded guard cells and protoplasts. The significance of these structures in the cell cortex is discussed.     

Root apical meristems ofAllium porrum L. as affected by arbuscular mycorrhizae and phosphorus

Summary Arbuscular mycorrhizal (AM) fungi significantly improve plant growth in soils with low phosphorus availability and cause many changes in root morphology, similar to those produced by increased P nutrition, mainly depending on root apex size and activity. The aim of this work was to discriminate between the morphogenetic role of AM fungi and P in leek (Allium porrum L.) by feeding mycorrhizal and nonmycorrhizal plants with two nutrient solutions containing 3.2 or 96 M P and examining specific parameters related to adventitious root apices (apex size, mitotic cycle, and RNA synthesis). The results showed that AM fungi blocked meristem activity as indicated by the higher percentages of inactive apices and metaphases in the apical meristem of mycorrhizal plants, whereas the high P supply lengthened the mitotic cycle without blocking the apices, resulting in steady, slow root growth. The possible involvement of abscisic acid in the regulation of root apex activity is discussed.Abbreviations ABA abscisic acid - AM arbuscular mycorrhizae - CI and CII nonmycorrhizal control plants grown with low or high phosphorus concentration - MI and MII mycorrhizal plants grown with low or high phosphorus concentration - PGR plant growth regulator     

Cortical cell fluxes and transport to the stele in excised root segments of Allium cepa L.

From compartmental analysis of radioisotope elutin measurements, fluxes of Ca²⁺ were estimated for cortical cells in root segments of onion, Allium cepa L., relative to complete nutrient solutions containing a range of calcium concentrations ([Ca₀]) from 2 eq l^-1 to 20 meq l^-1, increasing in 10-fold steps for Ca²⁺. Except for the calcium counter-ion (usually NO ₃ ^- , sometimes Cl^- at the highest [Ca₀]), the composition of the nutrient solution was other-wise the same at all calcium concentrations. Compartmental analysis indicated that the cytoplasm had a high content of exchangeable Ca²⁺ but, in the light of evidence from animal studies, ionic activity of calcium in the cytoplasm was assumed to be no greater than 0.002 eq ml^-1. With the Ussing-Teorell flux equation as the criterion, it was concluded that at all values of [Ca₀] tested, Ca²⁺ entered the cytoplasm passively and was actively pumped back into the external solution. Entry of calcium to the vacuole from the cytoplasm was active in all cases. The conclusions regarding the character of ion transport across the plasmalemma were the same as when the whole calcium content of the cytoplasm was taken to contribute to the ionic activity. However, the electrochemical activity gradient was very much steeper than formerly estimated. Calcium was transported to the stele in proportion to the calcium content of the cytoplasm and moved in the xylem almost exclusively in the basipetal direction.     

Structural studies of microsporogenesis in fertile and male-sterile onions (Allium cepa L.) containing the cms-S cytoplasm

Summary The structure of anther tissues has been studied during microsporogenesis in male-sterile and -fertile onions. Three types of abnormal tapetal behaviour have been observed within the single line II/3ms containing the cms-S cytoplasm: type 1, the premature breakdown of the tapetum at the tetrad stage, type 2, the hypertrophy of the tapetum after the diad stage followed by its premature autolysis and, type 3, in which the tapetum remains in good condition but for an abnormally long period of time. Tapetal autolysis proceeds in the same manner in both male-steriles and -fertiles with only the stage at which it occurs differing between the types of plants. Mitochondria were prominent in the tapetal tissue of all onion types throughout all stages of microsporogenesis and were still visible during the last stages of tapetal autolysis. In a detailed study of type 2 behaviour, no differences in mitochondrial volumes were found until the tapetum hypertrophied.     

Nucleolar structure during the meiotic prophase in Allium cepa anthers

Summary Naturally segregated nucleoli have been observed during the prophase in meiocytes of Allium cepa anthers. Under the light microscope the nucleolus is seen to consist of two clearly differentiated regions: a central core, which is strongly argyrophilic (ochre or dark brown) and slightly basophilic, surrounded by a basophilic peripheral region, which shows a low degree of argyrophilia. Under the electron microscope the central region appears as consisting of fibrillar elements (pars fibrosa), while the peripheral region proved to consist mainly of granules about 150 Å in diameter (pars granulosa).When the pachytene nucleolus of Allium cepa is stained with basic fuchsin, a small circular area appears intensely stained, which gradually grows larger as the pachytene proceeds. This characteristic structure, eventually reaching a size of 0.5–1.5 is regularly to be observed with a central vacuole. Under the electron microscope this area appears as a circular structure of high electron density, which corresponds in shape and size with the area revealed by the light microscope.The relationship between this new structure, which we have called globulus with other nucleolar structures is discussed.This work was partially supported by a grant from the Fondo de Ayuda a la Investigación, 1968, Spain. We wish to thank most especially Dr. M. C. Risueño, M.I. Rodriguez-García and J. M. Sogo, of the Cell Structures Section, (Department of Cytology) for their efficient collaboration. We also wish to thank M. C. Partearroyo and A. Partearroyo for technical assistance. One of the authors (J.C.S.) has a Research Training Fellowship awarded by the International Agency for Research on Cancer (World Health Organization).     

Agrobacterium tumefaciens-mediated transformation of Allium cepa L.: the production of transgenic onions and shallots

This paper describes the development of a reliable transformation protocol for onion and shallot (Allium cepa L.) which can be used year-round. It is based on Agrobacterium tumefaciens as a vector, with three-week old callus, induced from mature zygotic embryos, as target tissue. For the development of the protocol a large number of parameters were studied. The expression of the uidA gene coding for -glucuronidase was used as an indicator in the optimization of the protocol. Subspecies (onion and shallot) and cultivar were important factors for a successful transformation: shallot was better than onion and for shallot cv. Kuning the best results were obtained. Also, it was found that constantly reducing the size of the calli during subculturing and selection by chopping, thus enhancing exposure to the selective agent hygromycin, improved the selection efficiency significantly. Furthermore, callus induction medium and co-cultivation period showed a significant effect on successful stable transformation. The usage of different Agrobacterium strains, callus ages, callus sources and osmotic treatments during co-cultivation did not influence transformation efficiency. The highest transformation frequency (1.95%), was obtained with shallot cv. Kuning. A total of 11 independent transformed callus lines derived from zygotic embryos were produced: seven lines from shallot and four lines from onion. Large differences in plantlet production were observed among these lines. The best line produced over 90 plantlets. Via PCR the presence of the uidA and hpt (hygromycin phosphotransferase) genes could be demonstrated in these putative transformed plants. Southern hybridization showed that most lines originated from one transformation event. However, in one line plants were obtained indicating the occurrence and rescue of at least three independent transformation events. This suggested that T-DNA integration occurred in different cells within the callus. Most transgenic plants only had one copy of T-DNA integrated into their genomes. FISH performed on 12 plants from two different lines representing two integration events showed that original T-DNA integration had taken place on the distal end of chromosomes 1 or 5. A total of 83 transgenic plants were transferred to the greenhouse and these plants appeared to be diploid and normal in morphology.     

Anti-mitotic and anti-genotoxic effects of Plantago lanceolata aqueous extract on Allium cepa root tip meristem cells

Plantago is the most important genus of Plantaginaceae family and is used in traditional medicine around the world for different purposes. Plantago coronopus L., Plantago major L., Plantago media L. and Plantago lanceolata L. are most commonly used species of Plantago in traditional medicine in Turkey. The main goal of this study was to investigate the eventual anti-mitotic and anti-genotoxic effects of P. lanceolata L. leaf aqueous extracts (15 g/L and 30 g/L) on Allium cepa L. root tip meristem cells which were treated with 0.7% hydrogen peroxide. For this purpose, two different experiments were performed under the same conditions. In the first experiment, Allium cepa onion bulbs were treated with 0.7% H₂O₂ for 1 h. After the H₂O₂ treatment, the onion bulbs were treated with two different concentrations (15 g/L and 30 g/L) of P. lanceolata extracts for 24 h. In the second experiment, A. cepa onion bulbs were treated with two different extract concentrations (15 g/L and 30 g/L) for 24 h and then with 0.7% H₂O₂ for 1 h. The test concentrations were determined according to doses which are recommended in alternative medicinal usage by people. As positive and negative control 0.7% H₂O₂ and tap water was used, respectively. As a result, it was determined that aqueous extracts reduced mitotic index and chromosome aberrations in treatment groups in comparison with controls. These results showed that P. lanceolata aqueous extracts have anti-mitotic and anti-genotoxic effects.     

Protoplast responses in the epidermis of Allium cepa induced by penetration by Botrytis allii

Penetration of Allium cepa epidermal cells (white, yellow, and red varieties) by Botrytis allii induced a response by host protoplasts in normal tissue which was not observed when penetrations were made in protoplast-free host cell walls. Callose and auto-fluorescing substances (possibly phenolic compounds) were located at the penetration sites only in normal host cells containing protoplasts. Lignin tests were negative. Halos were clearly visible in both types of tissue. Autofluorescence was observed at penetration sites in normal cells of all cultivars but general wall background autofluorescence was not observed in white onions. Autofluorescence was generally yellow green and when treated with ammonium hydroxide became green. Treatment with sodium hydroxide abolished autofluorescence. No attempt was made to isolate the autofluorescing material.