Flow cytometric evidence for endopolyploidization in cabbage (Brassica oleracea L.) flowers

Flow cytometric analysis of the nuclear DNA contents of somatic tissues from flowers of cabbage (Brassica oleracea L.) revealed extensive endoreduplication, resulting in tissues that contain cells with multiple ploidy levels (also called ’endopolyploidy’). Multiples of the haploid nuclear genome complement (1C) corresponding to 2C, 4C, 8C, 16C, 32C and 64C were observed in mature flowers. The distribution of cells with the differerent ploidy levels is tissue-specific and is characteristic of the stage of development. Nuclei of young flower buds exclusively gave 2C and 4C peaks, indicating that the tissues maintained diploid level. Endoreduplication was consistently detected during flower development. Endopolyploidy is probably common in differentiation of cabbage plants. Implications of this original feature are discussed. Received: 28 August 2000 / Revision accepted: 20 December 2000     

Flow cytometric evidence for endopolyploidy in seedlings of some Brassica species

Flow cytometric analysis of the nuclear DNA contents of somatic tissues from seedlings of Brassica rapa L. and B. oleracea L. revealed extensive endoreduplication, resulting in tissues that contain cells with multiple ploidy levels (also called ’endopolyploidy’). Multiples of the haploid nuclear genome complement (1C) corresponding to 2C, 4C, 8C, 16C, 32C and 64C were observed in Brassica rapa, while B. oleracea exhibited a mixture of cells with five ploidy levels, 2C, 4C, 8C, 16C and 32C. The distribution of cells with the different ploidy levels was tissue-specific and characteristic of the stage of development. Multiploidy was not found in the embryos of dry seeds. Rapid endoreduplication occurred during seedling development. It is most probable that multiploidy is, if not a general feature, at least very common in Brassica species. The physiological and genetic implications of this original feature are discussed. Received: 17 March 2000 / Accepted: 17 April 2000     

Chromosome endoreduplication as a factor of salt adaptation in Sorghum bicolor

Summary. Nuclear DNA amounts were measured by Feulgen cytophotometry in Sorghum bicolor cv. 610 plants early exposed to 150 mM NaCl, a treatment known to induce an increased tolerance to salinity in plants carrying this genotype. In salt-treated plants, the percentages of 8C, 16C, and 32C nuclei in roots in the primary state of growth were 21.9%, 13.3%, and 4.3%, respectively. By contrast, in nonsalinized plants, only 3.5% of the nuclei had an 8C content and no higher DNA contents were observed. The salt treatment induced chromosome endoreduplication during the differentiation of cells in the root cortex, where 41.2% of the cells displayed a DNA content higher than 4C (versus 1.3% in control plants). No enhancement of endopolyploidy was observed in cells of the root vascular cylinder or the leaves of the salt-treated plants. In another S. bicolor genotype (DK 34-Alabama), noncompetent for salt adaptation, the same NaCl treatment did not induce chromosome endoreduplication in root cortex cells. Endopolyploidy may be considered as a part of the adaptive response of S. bicolor competent genotypes to salinity. Correspondence and reprints: Dipartimento di Biologia Cellulare e Ambientale, Università di Perugia, Via A. Pascoli, 06123 Perugia, Italy.     

Biochemical Activities and Compounds in Seeds: Possible Tools for Seed Quality Evaluation

The rate and final percentage of field emergence (seed quality)was measured in 55 seed-lots of carrots (Daucus carota L.),45 seed-lots of onion (Allium cepa L.) and 32 seed-lots of cabbage(Brassica oleracea var. capitata), together with the followingbiochemical indices: rates of ATP-synthesis; reduction of tetrazoliumby seed powder; AMP and malate content. Differences in field performance of seed-lots (seed quality)showed some correlation with particular biochemical indices.Seed quality was most significantly correlated with AMP contentand ATP synthesis in carrot, with malate content, reductionof tetrazolium and ATP synthesis in onion and with malate andAMP content and reduction of tetrazolium in cabbage. Daucus carota L., Allium cepa L., Brassica oleracea var capitata, carrot, onion, seed quality, ATP, biochemical indices     

Electrical Impedance Analysis in Plant Tissues8

Electrical impedance spectra (100 Hz–800 kHz) were measuredin leaves of Peperomia obtusifolia L. (a succulent) and Brassicaoleracea L. (cabbage). By measuring impedances at three or moreinter-electrode distances in a single leaf, electrode impedanceand specific tissue impedance were separated. Analysis of impedance data from B. oleracea leaves in relationto an equivalent circuit model showed that leaf developmentwas accompanied by increases in extracellular resistance, cytoplasmicresistance and vacuole interior resistance, together with decreasesin plasma membrane capacitance and tonoplast capacitance. AfterB. oleracea leaves were subjected to a –6 °C freeze-thawstress, extracellular resistance, cytoplasmic resistance andvacuole interior resistance decreased, but plasma membrane capacitanceand tonoplast capacitance did not change. These results indicatethat useful measurements of leaf parameters can be obtainedby this technique. Examination of the electrode impedance spectrum showed thatelectrode insertion produced a damaged collar, 0·4–0·5mm wide, around the electrode. This was confirmed by visualobservation of the damage in P. obtusifolia leaf. Key words: Peperomia obtusifolia L., Brassica oleracea L. (cabbage), electrical impedance, equivalent circuit, electrode polarization     

The Nuclear Endoreduplication Cycle in Metaxylem Cells of Primary Roots of Zea mays L.

The nuclear DNA content of metaxylem cells in roots of Zea mayscv. Golden Bantam reaches 16C or 32C by successive rounds ofDNA endoreduplication. Each phase of endoreduplication (endo-S)is separated by a non-DNA synthetic phase (endo-G). These phasesseem to occur in zones at fixed distances from the root tip.The duration of the phases in two of the endoreduplication cycles(4C–8C, 8C–16C) has been estimated in two ways.The first makes use of the rate of movement of cells throughthe positions along the root where the different phases of thecycle are occurring, the second uses labelling with methyl-[³H]thymidineand autoradiography. Both methods indicate that the endo-S phaseswhich cause the nuclear DNA content to rise from 4C to 8C andfrom 8C to 16C last 8–10 h, and that the intervening endo-Gphase lasts 8–12 h. DNA endoreduplication keeps pace withthe increase of nuclear volume; cell volume increases at a morerapid rate, however. Comparison of the endoreduplication cyclein the metaxylem with the mitotic cycle in the adjoining filesof parenchyma cells shows that the mitotic cells complete theircycle more slowly. DNA synthesis, endoreduplication cycle, mitotic cycle, root apex, Zea mays     

There is No Somatic Meiosis in Embryogenic Leaves of Cichorium

Several papers dealing with carrot cell cultures describe meiosis-likedivisions and haploid cells prior to somatic embryogenesis.We have studied the first division in embryogenic mesophyllcells of a diploidCichorium intybus L. and of a tetraploid hybridC.intybus L.xC. endivia L. which undergo direct somatic embryogenesisfrom single cells when leaf fragments are placed in a liquidagitated inductive medium (modified MS with 1x10^-7M NAA and2.5x10^-6M 2-iP), in darkness, at 35°C. MicrosporogenesisinC. intybus provided aspects of meiosis for comparison. Inleaves incubated in inductive conditions, DAPI staining of nucleishowed normal mitosis on days 3–6; about 0.6% cells inprophase had undergone spontaneous endoreduplication leadingto a tetraploid somatic embryo. Immunocytochemistry of tubulinrevealed the constant presence of a preprophase band, as ina normal mitosis. The first pluricellular somatic embryos becamevisible on day 5 of culture. Flow cytometric determination ofnuclear DNA on days 4, 5 and 6 did not show any peak correspondingto the 1C DNA level for the diploid plant or to the 2C DNA levelfor the tetraploid. Instead there was a weak but constant peakat the 4C and 8C levels. We conclude that inCichorium leaves,the first division of somatic embryogenesis is a normal mitosis,with a small shift to endoreduplication. In our opinion, somaticmeiosis is not a prerequisite during direct somatic embryogenesis. Cichorium ; chicory; somatic embryogenesis; cell division; flow cytometry; tubulin     

Effect of Chilling on DNA Endoreplication in Root Cortex Cells and Root Hairs of Soybean Seedlings

Relative nuclear DNA contents in cortex parenchyma cells in root segments of 3- and 7-d-old soybean seedlings grown at 25 °C and in plants grown for 3 d at 25 °C, and then for 4 d at 10 °C, were determined with cytophotometry. Measurements revealed that in each variant the cortex cell nuclei with DNA content between 2C and 8C were in all the examined segments and nuclei with 8C – 16C DNA appeared in higher parts of roots. However, in chilled plant cells the number of 8C – 16C DNA nuclei was very low. Therefore, chilling inhibited endoreplication in comparison with plants grown at 25 °C for 7 d, and even reduced endopolyploidy level as compared to the initial seedlings, i.e. 3-d-old plants. DNA contents in root hairs grown at 25 °C (control) and in root hairs emerged at 10 °C were also determined. In controls 4C – 8C DNA nuclei predominated while in chilled plants an additional population of 2C – 4C DNA appeared. Thus a reduction of DNA synthesis was brought about by low temperature. The occurrence of an intermediate DNA contents besides those with full endoreplication cycles suggests the possibility of differential DNA replication. This suggestion seems to be supported by the lack of ³H-thymidine incorporation into root hair nuclei at the examined developmental stage both in control and chilled root hairs. The same number, but larger, chromocentric lumps in polyploid cortex cell nuclei of higher root zones, in comparison to meristematic nuclei, suggests that endoreduplication process occurred. This revised version was published online in July 2006 with corrections to the Cover Date.     

Endorsperm Development of Maize Defective Kernel (dek) Mutants. Auxin and Cytokinin Levels

To provide insight into the regulatory roles of auxin and cytokininin endosperm development, defective kernel (dek) mutants ofmaize (Zea mays L.) were examined. The mutants dek6, dek18 anddek 26 had substantially lower indole-3-acetic acid (IAA) levelsthan wild-type counterparts while dek8, dek12 dek24 had higherIAA levels than wild-type counterparts. The mutant dek6 hada somewhat lower zeatin level than its wild-type counterpart.The mutation-induced changes in IAA levels and effects of exogenously-appliedauxin were not consistently related to changes in numbers ofnuclei per endorsperm; however, there was a correspondence betweenauxin level in endosperm and nuclear diameter, which is proportionalto the extent of post-mitotic DNA synthesis (endoreduplication).We conclude that auxin may be involved in stimulating endoreduplication.Copyright1993, 1999 Academic Press Zea mays L., maize, defective kernel mutant, dek, auxin, cytokinin, hormone, ELISA, cell number, cell division, endopolyploidy, endoreduplication, DNA amplification     

Molecular Identification of a Candidatus Phytoplasma asteris Associated with Cabbage Witches’‐Broom in China

In 2010, cabbages (Brassica oleracea L.) showing symptoms of proliferated axillary buds, crinkled leaves and plant stunting with shortened internodes typical to phytoplasma infection were found in a breeding facility in Beijing, China. Three symptomatic plants and one symptomless plant were collected, and total DNA was extracted from the midrib tissue and the flowers. With phytoplasma universal primers R16F2n/R16R2, a special fragment of 1247 bp (16S rDNA) was obtained from all three symptomatic cabbage plants, but not from the one symptomless cabbage plant. The 16S rDNA sequence showed 99% similarity with the homologous genes of the aster yellows group phytoplasma (16SrI group), and the phytoplasma was designed as CWBp‐BJ. Phylogenetic and computer‐simulated restriction fragment length polymorphism (RFLP) analysis of the 16S rDNA gene revealed that CWBp‐BJ belongs to subgroup 16SrI‐B. This is the first report of a phytoplasma associated with cabbage witches’‐broom in China.     

Changes in Sugar Content during Cold Acclimation and Deacclimation of Cabbage Seedlings

The relationship between freezing tolerance and sugar contentin cabbage seedlings was investigated. Seedlings exposed tonon-freezing low temperature (5 °C) acquired freezing tolerancedown to -6 °C. The degree of freezing tolerance increasedwith duration of exposure to low temperature (up to 10 d). Sucrose,glucose, fructose and myo -inositol were detected as solublesugars in cabbage leaves, and all soluble sugars, except formyo -inositol, and starch increased gradually during cold acclimationsuch that their levels were positively correlated with the degreeof freezing tolerance. The induced freezing tolerance was attributednot to ontogenetic changes but to cold acclimation. However,the induced freezing tolerance was lost after only 1 d of deacclimationat control temperatures, and this change was associated witha large reduction in sugar content. These results reveal that the sugar content of cabbage leavesis positively correlated with freezing tolerance. Brassica oleracea L.; cabbage; cold acclimation; deacclimation; freezing tolerance; sugars     

Mediation of host selection and oviposition behavior in the diamondback moth Plutella xylostella and its predator Chrysoperla carnea by chemical cues from cole crops

Host plant-mediated orientation and oviposition by diamondback moth (DBM) Plutella xylostella (L.) (Lepidoptera: Yponomeutidae) and its predator Chrysoperla carnea Stephens (Neuroptera: Chrysopidae) were studied in response to four different brassica host plants: cabbage, (Brassica oleracea L. subsp. capitata), cauliflower (B. oleracea L. subsp. botrytis), kohlrabi (B. oleracea L. subsp. gongylodes), and broccoli (B. oleracea L. subsp. italica). Results from laboratory wind tunnel studies indicated that orientation of female DBM and C. carnea females towards cabbage and cauliflower was significantly greater than towards either broccoli or kohlrabi plants. However, DBM and C. carnea males did not orient towards any of the host plants. In no-choice tests, oviposition by DBM did not differ significantly among the test plants, while C. carnea layed significantly more eggs on cabbage, cauliflower, and broccoli than on kohlrabi. However, in free-choice tests, oviposition by DBM was significantly greater on cabbage, followed by cauliflower, broccoli, and kohlrabi, while C. carnea preferred to oviposit on cabbage and cauliflower, followed by broccoli and kohlrabi. The predation rates of DBM by C. carnea on kohlrabi and broccoli were not significantly different from one another, but were significantly higher than that on cabbage and cauliflower. When two types of plant, intact and injured, were available to adult DBM, female oviposition was significantly greater on injured plant leaves than on intact plants leaves. Similarly, C. carnea oviposition was significantly greater on injured plant leaves than on intact leaves. Implications regarding the possible role of green leaf volatiles in host selection/preference, as well as in tritropic interactions, are discussed.     

Endoreduplication in <Emphasis Type="Italic">Phalaenopsis</Emphasis> is affected by light quality from light-emitting diodes during somatic embryogenesis

Endoreduplication is a developmental process that is unique to plants and occurs in all plants. The present study aimed to assess endoreduplication in various explant tissues and regenerated somatic embryos of Doritaenopsis. We further investigated the effects of light quality on endoreduplication and somatic embryo proliferation. To this end, we studied endoreduplication in leaves and root tips from regenerated plantlets and somatic embryos and in developing somatic embryos under 4 types of lighting conditions: red light, red + far-red light, red + blue light, and white light. We found that the degree of endoreduplication varied in different explants, and that the choice of explants used also influenced the ploidy levels of the newly regenerated somatic embryos. The DNA content of the leaf (2C–8C) was less than that of the root tip (2C–16C) and somatic embryo (2C–64C). In terms of light quality, the combination of red and far-red light produced the highest number of somatic embryos, while maintaining a low degree of endoreduplication. The data obtained indicate that this light combination stimulates somatic embryogenesis in Doritaenopsis and may exert some control on endoreduplication during cell division. These findings can be applied to achieve a reduction in somaclonal variations for the purpose of mass proliferation and genetic improvement.     

Detailed analyses of chloroplast and mitochondrial DNAs from the hybrid plant generated by asymmetric protoplast fusion between radish and cabbage

In a previous report, intergeneric somatic hybrids between red cabbage (Brassica oleracea L. var.capitata) and radish (Raphanus sativus L. cv. Shougoin) were produced by protoplast fusion. Plant morphology, chromosome number, isozyme patterns, andSma1 cleavage pattern of chloroplast DNA indicated that the hybrid plants have the red cabbage nucleus and the radish chloroplasts. In this report, we analyzed the organization of chloroplast and mitochondrial DNAs from this hybrid using Southern hybridization. The restriction patterns of almost all regions of the chloroplast DNA from the hybrid were similar to that of radish, except for one region near therps16 gene, which encodes the chloroplast ribosomal protein S16. In contrast to chloroplast DNA, the restriction pattern of mitochondrial DNA from the hybrid was quite different from that of the parents.Abbreviations CMS cytoplasmic male-sterility - ctDNA chloroplast DNA - mtDNA mitochondrial DNA     

Screening of oilseed rape and other brassicaceous genotypes for susceptibility to Ceutorhynchus pallidactylus (Mrsh.)

Production of oilseed rape, Brassica napus L., is affected by various insect pests. The cabbage stem weevil, Ceutorhynchus pallidactylus (Mrsh.) (Col.: Curculionidae), is one of the most damaging pests in Northern and Central Europe that requires regular control measures. Host plant resistance is a key factor in integrated pest management systems. To evaluate a large number of genotypes for their susceptibility to infestation by C. pallidactylus, new screening techniques were developed for testing both, the amount of feeding and the number of eggs deposited by adult C. pallidactylus on accessions of Brassicaceae under controlled conditions. In no‐choice screening tests, the leaf area consumed by adult cabbage stem weevil was quantified on a wide spectrum of 107 brassicaceous genotypes (B. napus, Brassica rapa L. and Brassica oleracea L. cultivars, breeding lines, resynthesized rapeseed lines and wild Brassicaceae). In comparison to feeding on the standard cultivar ‘Express’, the average leaf area consumed by C. pallidactylus on nine oilseed rape cultivars, four resynthesized rapeseed lines and five other accessions [B. oleracea, Camelina alyssum (Mill.) and Lunaria annua L.] was significantly reduced by 44–90%. In dual‐choice screening tests for the evaluation of oviposition preferences on 42 genotypes, female C. pallidactylus laid significantly fewer eggs into plants of two oilseed rape cultivars, five resynthesized rapeseeds and three accessions of B. oleracea and Brassica fruticulosa Cyrillo, respectively, than into plants of the standard cv ‘Express’. Results of both laboratory screening tests were confirmed by results of additional field testing.     

The Potential for Newly-Germinated Cabbage Seed Survival and Storage at Sub-Zero Temperatures

The moisture content of newly germinated cabbage seed (radicles1 05 mm long) was reduced to 14% of f.wt without loss of viability.As the moisture content was reduced below 45%, the temperatureat which the germinated seeds froze, and therefore died, decreasedprogressively to a minimum of –34 C at 19% moisture content.No freezing exotherms were recorded in seeds with moisture contentsbelow 19%. Seeds with a moisture content between 14 and 16%maintained viability for at least 1 week when cooled at 26C.min^–1to –20 C and held at this temperature, indicating thepotential for prolonged storage of these low-moisture-contentgerminated (LMCG) seeds. Brassica oleracea, cabbage, germinated seed, seed storage, fluid drilling, freezing exotherm, thermal analysis     

Predicting the Growth Interactions between Plants in Mixed Species Stands using a Simple Mechanistic Model

The Conductance model is a simple mechanistic model used topredict the growth of species in monoculture or mixtures fromparameter values derived from plants grown in isolation. Incontrast to many mechanistic models that require extensive parameterization,the Conductance model is able to capture the growth of a broadrange of species using a few simplified assumptions regardingplant growth and easily derived species-specific parameter values.We examine the assumptions within the Conductance model thattotal leaf area per plant is proportional to total plant weight,and that an isolated plant has a projected crown zone area thatis proportional to the 2/3 power of its weight. Power ratherthan linear relations were found between weight and leaf areafor Brassica oleracea, Daucus carota, Matricaria inodora, Solanumnigrum,Stellaria media , Trifolium repens and Veronica persica.For all seven species, the value of the power was less thanunity. All species also exhibited a power relation between crownzone area and weight, with the slope of this relation beingless than 2/3 for B. oleracea, D. carota and S. media. Althoughmorphology type accounted for some of the variation in the parametervalues relating to light interception, there were considerabledifferences between species within upright or prostrate foliagespecies groups. The Conductance model was used to predict yieldsof B. oleracea, S. nigrum and V. persica grown in both monocultureand binary weed-crop mixtures over a range of temporal and spatialscales. After calibrating the model to non-competing plants,the model was used to predict growth of the weed and crop speciesin contrasting densities and stand types. In some crop-weedcombinations, predicted crop and weed weights were within 17%of observed values, with no systematic deviations. In others,systematic and large deviations occurred.Copyright 2001 Annalsof Botany Company Brassica oleracea L., Daucus carota L., Matricaria inodora L., Solanum nigrum L.,Stellaria media L., Trifolium repens L., Veronica persica L., competition, growth, leaf area, crown zone area, light, shoot morphology, canopy architecture     

Genetic Diversity inBrassica oleraceaL. (Cruciferae) and Wild Relatives (2n=18) using RAPD Markers

This paper presents the evaluation of genetic diversity in 29populations of wild taxa of theBrassica oleraceaL. group (2n=18)and two cultivars, using RAPDs as molecular markers. In a previouspaper (Lázaro and Aguinagalde,Annals of Botany82: 000–000,1998), 11 isozymes were used for the same purpose. Results obtainedwith the two molecular markers (isozymes and RAPDs) are compared.DNA from ten individuals per population was analysed using sixdifferent primers; the 151 detected bands were polymorphic,11 were common to all species, six to all taxa, only one toevery population; and no bands were shared by every individual.The dendrogram obtained using genetic distances clustersB. oleraceapopulationswithB. bourgeaui, B. alboglabra, B. montanaandB. incana. B.insularis, B. macrocarpa, B. villosaandB. rupestrispopulationsform another cluster. Populations ofB. creticaandB. hilarionisformthe third cluster. Genetic diversity inB. oleraceapopulations,theB. rupestriscomplex andB. creticasubspecies was estimatedusing the AMOVA programme; the latter was the most diverse.Copyright1998 Annals of Botany Company. Brassica oleraceaL., wild relatives, genetic diversity, genetic resources, RAPD markers, AMOVA.     

Effect of light and gibberellic acid on chromosome endoreduplication in leaf epidermis ofTriticum durum Desf.

Summary The first foliage leaf ofTriticum durum Desf., cvs. Capelli and Creso, was studied for the action of light and gibberellic acid on different aspects of plant growth. Creso is a short-strawed cultivar carrying theRht 1 gene, that influences both plant height and sensitivity to applied gibberellins. In this study, a cytophotometric analysis of chromosome endoreduplication in bulliform cells of the leaf epidermis was undertaken. The bulliform cells are arranged in long rows and comprise 4C, 8C, and 16C cells. The relative percentage of these cells was recorded under different light regimes and in the presence or absence of gibberellic acid (GA₃). Our data indicate that light strongly affects the occurrence of chromosome endoreduplication. Although cvs. Cappelli and Creso show the same frequency of endo-reduplicated cells in the dark, all light treatments induce lower endo-polyploidy in cv. Creso whereas, in cv. Cappelli, a reduction in endo-polyploidy is observed when plants are irradiated with blue-lacking lights. This indicates that the action of a blue-light responsive photo-receptor determines high endopolyploidy. With respect to GA₃ treatment, cv. Creso is again shown to be insensitive to hormone application, whereas in cv. Cappelli, gibberellic acid influences endopolyploidy level in the dark, causing very high frequencies of 8C cells and relatively high frequencies of 16C cells.This paper is dedicated to the memory of Sergio Baroncelli, Professor of Plant Breeding at the University of Pisa.