Air drying method using nitrous oxide for chromosome counting in maize

Nitrous oxide (N₂O), colchicine, trifluralin, amiprophos-methyl, 8-hydroxyquinoline, and temperature pretreatments (cold and cold-hot-cold) were compared for chromosome counting in maize (Zea mays L.). Pretreated root tips were prepared by enzymatic maceration and air drying, and the number of countable figures and mitotic indexes were recorded. N₂O treatment at 10 atm for 3 hr produced the largest number of countable chromosome figures (266.5 per preparation) and an average of 44.2 nonoverlapped chromosome figures per preparation. Treatment with 0.04% 8-hydroxyquinoline for 3 hr exhibited a moderate number of countable chromosome figures (53.9 per preparation). The effects of colchicine, trifluralin, amiprophos-methyl and temperature pretreatments were limited.     

Flow sorting and microcloning of maize chromosome 1

Flow sorting maize chromosome 1 and construction of the first chromosome 1 DNA Lambda library are described. Maize metaphase chromosome suspensions were prepared from synchronized seedling root tip cells of the maize hybrid line Seneca 60 and stained with propidium iodide for flow karyotyping and sorting. The observed flow karyotype was very similar to the predicted flow karyotype constructed based on published values for the relative chromosome sizes of Seneca 60. The estimated size of chromosomes from the peak for the chromosome 1 matched the expected size of maize chromosome 1. The peak for the chromosome 1 was well resolved from other peaks on the flow karyotype. An average of 7 x 10(3) chromosomes of chromosome 1 could be produced from 10 root tips. About 0.6 million chromosomes of maize chromosome 1 were sorted and pooled based on the cytogram of fluorescent pulse area Vs fluorescent pulse width and stored at -20 degrees C in the freezer. DNA isolated from sorted chromosomes was good quality of more than 100 kb in size. Chromosome 1 DNA was partially digested with BamHI, dephosphorylated and ligated with arms of BamHI digested Lambda Dash vector. A total of 1.2 x 10(5) independent recombinants with the average insert size 12.6 kb was obtained. This library covered approximately 90% of maize chromosome 1. Hybridization of cloned fragments with labeled maize genomic DNA showed that the high, middle, or low copy number DNA sequences presented in the different phage clones. PCR (polymerase chain reaction) using chromosome-specific primers confirmed the specificity of this library. The individual chromosome library is useful in plant genome mapping and gene isolation.     

Variability of chromosomal DNA contents in maize (Zea mays L.) inbred and hybrid lines

The flow karyotypes of different maize (Zea mays L.) inbred and hybrid lines were analyzed. The accumulation and isolation of large quantities of high-quality metaphase chromosomes from root tips was achieved from many kinds of maize lines. The chromosome suspensions were prepared by a simple slicing method from synchronized maize root tips and analyzed by flow cytometry. Variations of experimental flow karyotypes were detected among inbred and hybrid lines in terms of the positions and/or the numbers of chromosome peaks. The 2C DNA amount among eight inbred lines ranged from 5.09 to 5.52 pg. The selection of appropriate maize lines is critical for sorting specific single chromosome types. At least five different chromosome types can be discriminated and sorted from five maize lines. The variability of DNA content in maize chromosome 1 was 9.1%, ranging from 0.685 to 0.747 pg. Differences were detected in the DNA content of homologous chromosome 1 of hybrid lines.     

Flow cytometric sorting of maize chromosome 9 from an oat-maize chromosome addition line

Large numbers of maize chromosome 9 can be collected with high purity by flow cytometric sorting of chromosomes isolated from a disomic maize chromosome addition line of oat. Metaphase chromosome suspensions were prepared from highly synchronized seedling root-tips of an oat-maize chromosome-9 addition line (OM9) and its parental oat and maize lines. Chromosomes were stained with propidium iodide for flow cytometric analysis and sorting. Flow-karyotypes of the oat-maize addition line showed an extra peak not present in the parental oat line. This peak is due to the presence of a maize chromosome-9 pair within the genome of OM9. Separation of maize chromosome 9 by flow cytometric sorting of a chromosome preparation from a normal maize line was not possible because of its size similarity (DNA content) to maize chromosomes 6, 7 and 8. However, it is possible to separate maize chromosome 9 from oat chromosomes and chromatids. An average of about 6×10³ chromosomes of maize chromosome 9 can be collected by flow-sorting from chromosomes isolated from 30 root tips (ten seedlings) of the oat-maize addition line. Purity of the maize chromosome 9, sorted from the oat-maize chromosome addition line, was estimated to be more than 90% based on genomic in situ hybridization analysis. Sorting of individual chromosomes provides valuable genomic tools for physical mapping, library construction, and gene isolation. Received: 28 February 2000 / Accepted: 14 July 2000     

宜昌百合根尖染色体C-带分析

利用Giemsa C-带方法对宜昌百合（Lilium leucanthum(Baker) Baker）进行研究。结果表明：宜昌百合（L. leucanthum）的染色体数目为2n＝2x=24,单套染色体的条带总数目为21条。其带型公式为：2n＝24＝6C+2CI+2I+2CI⁺+2CI⁺+4I⁺+2I⁺+2T⁺+2I+S。宜昌百合（L. leucanthum）每条染色体上都显示出显著的特征带,且带纹的深浅差异明显。宜昌百合（L. leucanthum）的强带主要集中在着丝点及附近区域。通过Giemsa C-带方法可以将宜昌百合（L. leucanthum）的每条染色体区分开。     

中国水仙六倍体的诱导和染色体数目的变异(简报)

中国水仙(Narcissus tazetta L.var.chinensis Roem)属于石蒜科水仙属多年生草本花卉植物。中国水仙的品种不多,在福建漳州地区主要栽培品种为单瓣水仙,此外,还有重瓣和“金三角”两个品种。中国水仙为三倍体植物,染色体数目为2n=3x=30[1-3],其高度不孕性,只开花不结实,靠子鳞茎进行无性繁殖繁衍后代。由于长期的无性繁殖和病毒感染,现存种质退化、品质下降,花朵数明显减少、香味变淡、生长势差、鳞茎变小、抗性减弱等问题,严重影响了该花卉的进一步生产和发展。因此,采用现代生物工程技术(体细胞杂交技术、转基因技术等)改良中国水仙,培育中国水仙新品种迫在眉睫。     

Terbutol Affects Spindle Microtubule Organizing Centres

Light, immunofluorescence, and electron microscopy were utilizedto investigate the effects of the herbicide terbutol (2,6-di-tert-butyl-p-tolylmethylcarbamate) on onion root tips. So-called ‘star anaphases,’chromosomes drawn in at their centromeres at both poles, resultingin a starburst of chromosomes were the predominant form of mitoticabnormality noted in root tip squashes of the terbutol-treatedroots. Immunofluorescence microscopy using antitubulin serareveals a cluster of microtubules radiating from the centreof the chromosome mass at each of the poles. Nuclear envelopesapparently reform around the radially-arranged chromosome masses,resulting in extensively lobed ‘star telophase’nuclei. Branched and curved phragmoplast arrays are observed,due to interference by the lobes of the star telophase nucleus.These abnormal phragmoplasts result in incomplete and/or abnormally-orientedcell walls. Star anaphase figures are observed after 2 h ofherbicide treatment, indicating that this terbutolinduced chromosomalabnormality is a primary effect of the herbicide. Tradescantiastamen hairs were treated with terbutol and mitosis was monitoredin vivo by Nomarski differential interference microscopy; thesetreated stamen hairs produce star anaphase figures as a primaryeffect of the herbicide. This series of abnormalities has notbeen observed with any other herbicide, indicating that terbutolhas a unique mechanism of action, perhaps interacting with spindlemicrotubule organizing centres. Key words: Terbutol, Onion, root tips, star-anaphase figures     

Fluorescent labeling of plant chromosomes in suspension by FISH

By optimizing the concentration and time of treatment with hydroxyurea (HU), a DNA synthesis inhibitor, and trifluralin, a microtubule inhibitor, a highly effective (over 60%) cell cycle synchronization method for rye and barley meristem cells was developed. Chromosome suspensions containing highly purified and morphologically intact rye and barley chromosomes were prepared from the meristems of their root tips by homogenization. Digoxigenin-labeled 5S rDNA was used as a probe in FISH for the rye chromosomes in the suspension, and biotin-labeled 17S rDNA and centromeric DNA were used in FISH for the rye and barley chromosome suspensions, respectively. Bright signals were detected at the specific regions of interest on the chromosomes. The results indicate that the method developed in this study is useful for selection and sorting of chromosomes that are not distinguishable by other means, using specific fluorescent labeling by FISH of the chromosomes in suspension.     

Isolation of Chromosomes from Picea abies and their Analysis by Flow Cytometry

A high-yield method for preparation of suspensions of intact Norway spruce [Picea abies (L.) Karst.] chromosomes was developed for the first time. To accumulate meristem root tip cells at metaphase, actively growing roots were subjected to subsequent treatments with 0.625 mM hydroxyurea for 18 h and after 8 h recovery in distilled water with 0.05 % (m/v) colchicine for 8 h. These treatments resulted in 50 % metaphase indices. Synchronized root tips were fixed in 2 % formaldehyde for 10 min and chromosomes were released into a lysis buffer by mechanical homogenisation, producing 5 × 10⁵ chromosomes from 50 root tips, at average. The isolated chromosomes were morphologically intact and suitable for flow cytometric analysis. Flow karyotypes obtained after the analysis of DAPI-stained chromosomes indicated a possibility to sort at least three different chromosome types. This revised version was published online in July 2006 with corrections to the Cover Date.     

芦苇根尖体细胞的染色体数目和形态

本文研究了芦苇(Phragmites communis)幼苗根尖分生组织细胞的染色体数目和形态。其染色体数为2n=96。在有丝分裂中期染色体组中,染色体的大小逐渐地改变,按照染色体的大小和形态,96个染色体能被分成8个同源单倍体染色体组。每一组由11个中部着丝点染色体和1个亚中部着丝点染色体组成。     

芦苇根尖体细胞的染色体数目和形态

本文研究了芦苇(Phragmites communis)幼苗根尖分生组织细胞的染色体数目和形态。其染色体数为2n=96。在有丝分裂中期染色体组中,染色体的大小逐渐地改变,按照染色体的大小和形态,96个染色体能被分成8个同源单倍体染色体组。每一组由11个中部着丝点染色体和1个亚中部着丝点染色体组成。     

Evaluation of cytological effects of Zn2+ in relation to germination and root growth of Nigella sativa L. and Triticum aestivum L

The effects of different treatments with zinc sulfate (Zn(2+)) on the cytology and growth of Nigella sativa and Triticum aestivum were investigated. Five concentrations of zinc sulfate ranging from 5 to 25mg/l were applied for 6, 12, 18, and 24h. The treatments reduced the germination percentages of N. sativa seeds and T. aestivum grains and inhibited the root growth of both plants. Concentrations higher than 25mg/l of Zn(2+) applied for 24h were toxic for both plants. The non-lethal concentrations of Zn(2+) showed an inhibitory effect on cell division in root tips of both plants and caused a decrease in their mitotic index values. The reduction in MI in root tips of T. aestivum was more evident than that of N. sativa. All treatments changed the frequency of mitotic phases as compared with the control values. The total percentage of abnormalities in N. saliva was more than that in T. aestivum. Zn(2+) treatments produced a number of mitotic abnormalities in dividing cells in root tips of both plants resulting from its action on the spindle apparatus such as C-metaphases, lagging chromosomes and multipolar anaphases and telophases. Also, Zn(2+) induced vacuolated nuclei and irregular prophases. The induction of chromosomal stickiness and chromosomal aberrations such as bridges and breaks indicates its action on the chromosome. These abnormalities (chromosome breaks and chromosomal bridges at ana-telophases) indicate true clastogenic potential of the ions tested.     

Karyotypes of 25 Populations of 15 Species in Allium Section Rhiziridium from China

Karyotypes of 25 populations of 15 species in Allium section Rhiziridium from China were analyzed to discuss the evolutionary mechanisms of Allium section Rhiziridium and cytogeography of some species. Root tips for the study of mitotic chromosomes were obtained from potted plants and pretreated with paradichlorobenzene at room temperature for 9 hours. After 12-24 hours fixation in 1∶3 acetic alcohol, the root tips were macerated in 1mol·L-1 HC1 for 9 minutes at 60℃ and then stained and squashed in carbol fuchsin. Karyotype asymmetry was assessed by As.K%. Results showed that taxa investigated were diploid or tetraploid. Furthermore, the karyotype types were 1A, 2A or 2B according to Stebbins’ karyotype classification. Karyotypes of A.tekesicola, A.tianschanicum, A.beesianum and A.spirale were reported for the first time. Combined with previous karyotype studies, we suggested that: (1) The basic chromosome number of Allium section Rhiziridium from China is x=8. (2) The karyotype types evolutionary trend of Allium section Rhiziridium from China is 1A→2A→2B→2C . (3) Satellite chromosomes of Allium section Rhiziridium from China are varied in numbers and shapes. (4) Polyploidy and chromosome structural rearrangement are two important evolution patterns in Allium section Rhiziridium from China. (5) A.cyaneum and A.nu tans have exploited new niches through polyploidy and asexual reproduction.     

Trypsin-orcein banding in plant chromosomes.

A convenient and quick method using trypsin-orcein for banding plant chromosomes (O-banding) is suggested. The technique is directly applicable to meristematic tissues (e.g. root tips) and involves the treatment of root tips with 1-2% solution of trypsin either in buffer or in 0.5 N HCl for 5-10 minutes at 37 C or for 30-60 minutes near 0 C followed by staining with 1.5% acetic orcein: 1 N HCl (19:1). Dark staining bands are reproducible and species specific. These bands possibly represent specific DNA-protein-dye interaction.     

Synchronization of cell division in root tips of seven major cereal species for high yields of metaphase chromosomes for flow-cytometric analysis and sorting

Plant tissues that yield large numbers of mitotic cells are useful for cytogenetic analyses and prerequisite to flow-cytometric analysis and sorting of plant chromosomes. Synchronization of cell division in samples from which chromosomes are to be isolated is necessary to ensure high yields of chromosomes. A method developed for synchronizing cell division in root tips ofVicia faba (Dolezel et al., 1992; Lucretti et al., 1993) has been modified, and parameters are presented for the effective synchronization of cell division in root tips ofAvena sativa (oat),Hordeum vulgare (barley),Oryza sativa (rice),Secale cereale (rye),Sorghum bicolor (sorghum),Triticum aestivum (wheat), andZea mays (maize). Optimum parameters for metaphase arrest and collection of chromosomes from synchronized samples are also presented. Using these parameters, the yield and quality of chromosome preparations are appropriate for flow-cytometric characterization and sorting.     

Effects of cooling on meiotic spindle structure and chromosome alignment within in vitro matured porcine oocytes

Meiotic spindle structure and chromosome alignment were examined after porcine oocytes were cooled at metaphase II (M II) stage. Cumulus-oocyte complexes (COCs) collected from medium size follicles were cultured in an oocyte maturation medium at 39 degrees C, 5% CO(2) in air for 44 hr. At the end of culture, oocytes were removed from cumulus cells and cooled to 24 or 4 degrees C for 5, 30, or 120 min in a solution with or without 1.5 M dimethyl sulfoxide (DMSO). After being cooled, oocytes were either fixed immediately for examination of the meiotic spindle and chromosome alignment or returned to maturation medium at 39 degrees C for 2 hr for examination of spindle recovery. Most oocytes (65-71%) cooled to 24 degrees C showed partially depolymerized spindles but 81-92% of oocytes cooled at 4 degrees C did not have a spindle after cooling for 120 min. Quicker disassembly of spindles in the oocytes was observed at 4 degrees C than at 24 degrees C. Cooling also induced chromosome abnormality, which was indicated by dispersed chromosomes in the cytoplasm. Limited spindle recovery was observed in the oocytes cooled to both 4 and 24 degrees C regardless of cooling time. The effect of cooling on the spindle organization and chromosome alignment was not influenced by the presence of DMSO. These results indicate that the meiotic spindles in porcine M II oocytes are very sensitive to a drop in the temperature. Both spindle and chromosomes were damaged during cooling, and such damage was not reversible by incubating the oocytes after they had been cooled.     

Combined Cycloheximide and 8-Hydroxyquinoline Pretreatment for Study of Plant Chromosomes

The actions of cycloheximide and 8-hydmxyquinoline on dividing cells of root meristems of Zea mays L. have been studied during the development of a new cytological technique for sugar cane (Saccharum) root tips. The determination of mitotic phase indices revealed that combined treatment with cycloheximide (70 ppm) plus 8-hydroxyquinoline (250 ppm) was superior to treatments with either chemical separately. After the combined treatment, the preparations contained nearly ten times more cells in prophase and metaphase that were suitable for chromosome counting than those given a single pretreatment with 8-hydmxyquinoline. This new pretreatment has been developed especially for chromosome studies in tropical grasses with a large number of small chromosomes. However, both chemicals are active in a wide range of plant species.     

Combined cycloheximide and 8-hydroxyquinoline pretreatment for study of plant chromosomes.

The actions of cycloheximide and 8-hydroxyquinoline on dividing cells of root meristems of Zea mays L. have been studied during the development of a new cytological technique for sugar cane (Saccharum) root tips. The determination of mitotic phase indices revealed that combined treatment with cycloheximide (70 ppm) plus 8-hydroxyquinoline (250 ppm) was superior to treatments with either chemical separately. After the combined treatment, the preparations contained nearly ten times more cells in prophase and metaphase that were suitable for chromosome counting than those given a single pretreatment with 8-hydroxyquinoline. This new pretreatment has been developed especially for chromosome studies in tropical grasses with a large number of small chromosomes. However, both chemicals are active in a wide range of plant species.     

Molecular mapping of quantitative trait loci for drought tolerance in maize plants

Drought tolerance is one of the most important but complex traits of crops. We looked for quantitative trait loci (QTLs) that affect drought tolerance in maize. Two maize inbreds and their advanced lines were evaluated for drought-related traits. A genetic linkage map developed using RFLP markers was used to identify QTLs associated with drought-related traits. Twenty-two QTLs were detected, with a minimum of one and a maximum of nine for drought-related traits. A single-QTL was detected for sugar concentration accounting for about 52.2% of the phenotypic variation on chromosome 6. A single-QTL was also identified for each of the traits root density, root dry weight, total biomass, relative water content, and leaf abscisic acid content, on chromosomes 1 and 7, contributing to 24, 0.2, 0.4, 7, and 19% of the phenotypic variance, respectively. Three QTLs were identified for grain yield on chromosomes 1, 5, and 9, explaining 75% of the observed phenotypic variability, whereas four QTLs were detected for osmotic potential on chromosomes 1, 3, and 9, together accounting for 50% of the phenotypic variance. Nine QTLs were detected for leaf surface area on chromosomes 3 and 9, with various degrees of phenotypic variance, ranging from 25.8 to 42.2%. Four major clusters of QTLs were identified on chromosomes 1, 3, 7, and 9. A QTL for yield on chromosome 1 was found co-locating with the QTLs for root traits, total biomass, and osmotic potential in a region of about 15 cM. A cluster of QTLs for leaf surface area were coincident with a QTL for osmotic potential on chromosome 3. The QTLs for leaf area also clustered on chromosome 9, whereas QTLs for leaf abscisic acid content and relative water content coincided on chromosome 7, 10 cM apart. Co-location of QTLs for different traits indicates potential pleiotropism or tight linkage, which may be useful for indirect selection in maize improvement for drought tolerance.     

A Schedule for Chromosome Counts M Some Plants with Small Chromosomes

A schedule is given which produced excellent results for somatic chromosome counts on some British species of Galium, These species present great difficulties owing to the small size of the chromosomes (ca. 1.5μ long), the large somatic numbers (up to 96) and the slenderness of the root tips. Special features of the schedule, which is the result of much experiment with various technics, are: fixation with Belling's Navashin type fixative, which was quite the best tried; the modification of Randolph's card mount method to overcome the difficulty of the small diameter of the root tips by mounting together a number from the same plant, so that they can be embedded and sectioned almost as easily as much larger root tips; staining with dilute (0.1%) crystal violet, the most critical stain used, after mordanting with 1% aqueous chromic acid to intensify the stain in the small chromosomes; and the addition of an extra stage of differentiation in absolute alcohol diluted with xylol to remove strands of stain, which are often left in the cytoplasm between the chromosomes, since clove oil, usually the last differentiating fluid used, differs from alcohol in removing crystal violet more rapidly from the chromosomes than from the cytoplasm. It is suggested that this schedule will be valuable in chromosome counts of other plants where similar difficulties arise.