Most cited article - PubMed ID 10728642
Cell cycle synchronization in plant root meristems
Laser microdissection (LM) is a powerful tool for various molecular analyses providing pure samples for genomic, transcriptomic, and proteomic studies. Cell subgroups, individual cells, or even chromosomes can be separated via laser beam from complex tissues, visualized under the microscope, and used for subsequent molecular analyses. This technique provides information on nucleic acids and proteins, keeping their spatiotemporal information intact. In short, the slide with tissue is placed under the microscope, imaged by a camera onto a computer screen, where the operator selects cells/chromosomes based on morphology or staining and commands the laser beam to cut the specimen following the selected path. Samples are then collected in a tube and subjected to downstream molecular analysis, such as RT-PCR, next-generation sequencing, or immunoassay.
- Keywords
- Cells, Chromosomes, Cytology, Histology, Microdissection,
- MeSH
- Single-Cell Analysis MeSH
- Chromosomes MeSH
- Genome * MeSH
- Laser Capture Microdissection methods MeSH
- Proteomics * MeSH
- Publication type
- Journal Article MeSH
Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to progress in plant genetics, including the production of genome assemblies and the cloning of important genes. The dissection of complex genomes by flow sorting into the individual chromosomes that represent small parts of the genome reduces DNA sample complexity and streamlines projects relying on molecular and genomic techniques. Whereas flow cytometric analysis, that is, chromosome classification according to fluorescence and light scatter properties, is an integral part of any chromosome sorting project, it has rarely been used on its own due to lower resolution and sensitivity as compared to other cytogenetic methods. To perform chromosome analysis and sorting, commercially available electrostatic droplet sorters are suitable. However, in order to resolve and purify chromosomes of interest the instrument must offer high resolution of optical signals as well as stability during long runs. The challenge is thus not the instrumentation, but the adequate sample preparation. The sample must be a suspension of intact mitotic metaphase chromosomes and the protocol, which includes the induction of cell cycle synchrony, accumulation of dividing cells at metaphase, and release of undamaged chromosomes, is time consuming and laborious and needs to be performed very carefully. Moreover, in addition to fluorescent staining chromosomal DNA, the protocol may include specific labelling of DNA repeats to facilitate discrimination of particular chromosomes. This review introduces the applications of chromosome sorting in plants, and discusses in detail sample preparation, chromosome analysis and sorting to achieve the highest purity in flow-sorted fractions, and their suitability for downstream applications.
- Keywords
- DNA amplification, DNA isolation, cell cycle synchronization, gene mapping and cloning, genome sequencing, liquid chromosome suspension, marker development, mitotic metaphase chromosomes, repetitive DNA labelling,
- MeSH
- Cell Cycle MeSH
- Chromosomes, Plant * genetics MeSH
- Metaphase MeSH
- Flow Cytometry MeSH
- Plants * genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Modern sugarcane is an unusually complex heteroploid crop, and its genome comprises two or three subgenomes. To reduce the complexity of sugarcane genome research, the ploidy level and number of chromosomes can be reduced using flow chromosome sorting. However, a cell cycle synchronization (CCS) protocol for Saccharum spp. is needed that maximizes the accumulation of metaphase chromosomes. For flow cytometry analysis in this study, we optimized the lysis buffer, hydroxyurea(HU) concentration, HU treatment time and recovery time for sugarcane. We determined the mitotic index by microscopic observation and calculation. We found that WPB buffer was superior to other buffers for preparation of sugarcane nuclei suspensions. The optimal HU treatment was 2 mM for 18 h at 25 °C, 28 °C and 30 °C. Higher recovery treatment temperatures were associated with shorter recovery times (3.5 h, 2.5 h and 1.5 h at 25 °C, 28 °C and 30 °C, respectively). The optimal conditions for treatment with the inhibitor of microtubule polymerization, amiprophos-methyl (APM), were 2.5 μM for 3 h at 25 °C, 28 °C and 30 °C. Meanwhile, preliminary screening of CCS protocols for Badila were used for some main species of genus Saccharum at 25 °C, 28 °C and 30 °C, which showed that the average mitotic index decreased from 25 °C to 30 °C. The optimal sugarcane CCS protocol that yielded a mitotic index of >50% in sugarcane root tips was: 2 mM HU for 18 h, 0.1 X Hoagland's Solution without HU for 3.5 h, and 2.5 μM APM for 3.0 h at 25 °C. The CCS protocol defined in this study should accelerate the development of genomic research and cytobiology research in sugarcane.
- MeSH
- Cell Cycle physiology MeSH
- Time Factors MeSH
- Chromosomes, Plant * metabolism MeSH
- Genome, Plant genetics MeSH
- Genomics methods MeSH
- Hydroxyurea MeSH
- Metaphase MeSH
- Mitotic Index MeSH
- Nitrobenzenes MeSH
- Organothiophosphorus Compounds MeSH
- Flow Cytometry methods MeSH
- Buffers MeSH
- Saccharum cytology genetics MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- amiprophos methyl MeSH Browser
- Hydroxyurea MeSH
- Nitrobenzenes MeSH
- Organothiophosphorus Compounds MeSH
- Buffers MeSH
Sugarcane (Saccharum spp.) is a globally important crop for sugar and bioenergy production. Its highly polyploid, complex genome has hindered progress in understanding its molecular structure. Flow cytometric sorting and analysis has been used in other important crops with large genomes to dissect the genome into component chromosomes. Here we present for the first time a method to prepare suspensions of intact sugarcane chromosomes for flow cytometric analysis and sorting. Flow karyotypes were generated for two S. officinarum and three hybrid cultivars. Five main peaks were identified and each genotype had a distinct flow karyotype profile. The flow karyotypes of S. officinarum were sharper and with more discrete peaks than the hybrids, this difference is probably due to the double genome structure of the hybrids. Simple Sequence Repeat (SSR) markers were used to determine that at least one allelic copy of each of the 10 basic chromosomes could be found in each peak for every genotype, except R570, suggesting that the peaks may represent ancestral Saccharum sub genomes. The ability to flow sort Saccharum chromosomes will allow us to isolate and analyse chromosomes of interest and further examine the structure and evolution of the sugarcane genome.
- MeSH
- Alleles MeSH
- Cell Cycle drug effects genetics MeSH
- Chromosomes, Plant genetics MeSH
- DNA, Plant metabolism MeSH
- Fluorescence MeSH
- Genome, Plant * MeSH
- Hydroxyurea pharmacology MeSH
- Karyotype MeSH
- Kinetics MeSH
- Plant Roots drug effects MeSH
- Polyploidy * MeSH
- Flow Cytometry methods MeSH
- Saccharum drug effects genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA, Plant MeSH
- Hydroxyurea MeSH
The application of flow cytometry and sorting (flow cytogenetics) to plant chromosomes did not begin until the mid-1980s, having been delayed by difficulties in preparation of suspensions of intact chromosomes and discrimination of individual chromosome types. These problems have been overcome during the last ten years. So far, chromosome analysis and sorting has been reported in 17 species, including major legume and cereal crops. While chromosome classification by flow cytometry (flow karyotyping) may be used for quantitative detection of structural and numerical chromosome changes, chromosomes purified by flow sorting were found to be invaluable in a broad range of applications. These included physical mapping using PCR, high-resolution cytogenetic mapping using FISH and PRINS, production of recombinant DNA libraries, targeted isolation of markers, and protein analysis. A great potential is foreseen for the use of sorted chromosomes for construction of chromosome and chromosome-arm-specific BAC libraries, targeted isolation of low-copy (genic) sequences, high-throughput physical mapping of ESTs and other DNA sequences by hybridization to DNA arrays, and global characterization of chromosomal proteins using approaches of proteomics. This paper provides a comprehensive review of the methodology and application of flow cytogenetics, and assesses its potential for plant genome analysis.
Procedures for flow cytometric analysis and sorting of mitotic chromosomes (flow cytogenetics) have been developed for chickpea (Cicer arietinum). Suspensions of intact chromosomes were prepared from root tips treated to achieve a high degree of metaphase synchrony. The optimal protocol consisted of a treatment of roots with 2 mmol/L hydroxyurea for 18 h, a 4.5-h recovery in hydroxyurea-free medium, 2 h incubation with 10 micromol/L oryzalin, and ice-water treatment overnight. This procedure resulted in an average metaphase index of 47%. Synchronized root tips were fixed in 2% formaldehyde for 20 min, and chromosome suspensions prepared by mechanical homogenization of fixed root tips. More than 4 x 10(5) morphologically intact chromosomes could be isolated from 15 root tips. Flow cytometric analysis of DAPI-stained chromosomes resulted in histograms of relative fluorescence intensity (flow karyotypes) containing eight peaks, representing individual chromosomes and/or groups of chromosomes with a similar relative DNA content. Five peaks could be assigned to individual chromosomes (A, B, C, G, H). The parity of sorted chromosome fractions was high, and chromosomes B and H could be sorted with 100% purity. PCR on flow-sorted chromosome fractions with primers for sequence-tagged microsatellite site (STMS) markers permitted assignment of the genetic linkage group LG8 to the smallest chickpea chromosome H. This study extends the number of legume species for which flow cytogenetics is available, and demonstrates the potential of flow cytogenetics for genome mapping in chickpea.
- MeSH
- Cell Cycle MeSH
- Chromosomes, Plant genetics MeSH
- Cicer genetics MeSH
- Cytogenetics MeSH
- DNA, Plant genetics metabolism MeSH
- Physical Chromosome Mapping methods MeSH
- Genetic Linkage MeSH
- Genome, Plant * MeSH
- In Situ Hybridization, Fluorescence MeSH
- Indoles MeSH
- Karyotyping MeSH
- Plant Roots genetics MeSH
- Metaphase MeSH
- Microsatellite Repeats MeSH
- Sequence Tagged Sites MeSH
- Mitosis MeSH
- Polymerase Chain Reaction MeSH
- Flow Cytometry methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DAPI MeSH Browser
- DNA, Plant MeSH
- Indoles MeSH
A high-yield method for isolation of barley chromosomes in suspension, their analysis and sorting using flow cytometry is described. To accumulate meristem root tip cells at metaphase, actively growing roots were subjected to subsequent treatment with 2 mmol/L hydroxyurea for 18 h, 2.5 micromol/L amiprophos methyl for 2 h, and ice water (overnight). This treatment resulted in metaphase indices exceeding 50%. Synchronized root tips were fixed in 2% formaldehyde for 20 min and chromosomes were released into a lysis buffer by mechanical homogenization, producing, on average, 5 x 10(5) chromosomes from 50 root tips. The isolated chromosomes were morphologically intact and suitable for flow cytometric analysis and sorting. While it was possible to discriminate and sort only one chromosome from a barley cultivar with standard karyotype, up to three chromosomes could be sorted in translocation lines with morphologically distinct chromosomes. The purity of chromosome fractions, estimated after PRINS with primers specific for GAA microsatellites, reached 97%. PCR with chromosome-specific primers confirmed the purity and suitability of flow-sorted chromosomes for physical mapping of DNA sequences.
- MeSH
- Chromosomes genetics MeSH
- DNA Primers MeSH
- Electrophoresis, Agar Gel MeSH
- Physical Chromosome Mapping MeSH
- In Situ Hybridization, Fluorescence MeSH
- Hydroxyurea pharmacology MeSH
- Insecticides pharmacology MeSH
- Hordeum genetics MeSH
- Karyotyping MeSH
- Plant Roots genetics MeSH
- Metaphase MeSH
- Microsatellite Repeats genetics MeSH
- Mitosis genetics MeSH
- Nitrobenzenes MeSH
- Organothiophosphorus Compounds pharmacology MeSH
- Polymerase Chain Reaction MeSH
- Flow Cytometry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- amiprophos MeSH Browser
- DNA Primers MeSH
- Hydroxyurea MeSH
- Insecticides MeSH
- Nitrobenzenes MeSH
- Organothiophosphorus Compounds MeSH
