An improved method for the cell cycle synchronization of Vicia faba root meristem cells

Polit, Justyna Teresa

doi:10.1007/s11738-007-0124-4

Cited by 2 publications

(2 citation statements)

References 31 publications

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“…After removing the HU by washing, the cells return to the cell cycle in a synchronized way (Doležel et al 1999, Winnicki et al 2013. The following treatment of such synchronized meristem root cells with an inhibitor of microtubule polymerization provides a larger number of metaphase cells when compared with single step protocols that do not involve synchronization (Doležel et al 1992, 1999, Polit 2008.…”

Section: Discussionmentioning

confidence: 99%

Physical Mapping of 5S rDNA in Eucalyptus dunnii Maiden and Zea mays L. by PRINS

et al. 2019

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The in situ detection of specific DNA sequences by fluorescence in situ hybridization (FISH) is a widely used tool for physical mapping, which is important to understand genome structure and organization. For species with small chromosomes, detection of the position of marker sequences is useful to aid chromosome identification and classification. Primed in situ labeling (PRINS) comprises an alternative to FISH, but its specificity and sensitivity may be influenced by species-specific factors, as chromosome length and chromatin structure. To overcome these obstacles, we designed a reproducible PRINS protocol for physical mapping of 5S rDNA in Eucalyptus dunnii Maiden and Zea mays L. Slides containing mitotic metaphases were submitted to one cycle of PRINS using 5S rDNA primers. Clear signals for 5S rDNA were observed in metaphase chromosomes of both species. In E. dunnii, 5S rDNA was mapped on the pericentromeric region of the short arm of chromosome 5. For Z. mays, the signal was detected in the terminal region of the long arm of chromosome 2. Although chromosome structure may influence the PRINS resolution, a high signal/background ratio was obtained for both species. In Z. mays, clear 5S rDNA signals were obtained by PRINS and FISH. Nonetheless, the former was superior regarding the time required for signal detection. Therefore, PRINS has the potential to aid plant genome studies by allowing fast detection of repetitive sequences, even in species with small chromosomes. Moreover, this is the first report of specific sequence mapping in Eucalyptus by PRINS, contributing to physical mapping progress in this genus.

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Section: Discussionmentioning

confidence: 99%

Physical Mapping of 5S rDNA in Eucalyptus dunnii Maiden and Zea mays L. by PRINS

et al. 2019

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“…To date, there are two main CCS strategies: (i) collection of specific cell populations using physical methods such as centrifugal elutriation 9 ; and (ii) treatment of cells with chemical inhibitors that impede DNA synthesis inhibitors or microtubule polymerization as well as other metaphase blocking chemicals 15 . CCS in root tips from several crops, including cereal, wheat, Chinese fir and Vicia faba can be achieved with chemical inhibitors [17][18][19][20] . Moreover, DNA synthesis inhibitors such as hydroxyurea (HU), deoxyadenosine and deoxythymidine, and microtubule inhibitors such as trifluralin and amiprophos-methyl (APM), are effective for CCS in plant root tips 8 .…”

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confidence: 99%

A flow cytometry-based analysis to establish a cell cycle synchronization protocol for Saccharum spp.

ShaoHua

Zeng

Luo

et al. 2020

Sci Rep

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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.Sugarcane (Saccharum spp.), belonging to the genus Saccharum, is an economically valuable crop. The genus Saccharum is diverse in genome content and organization and comprises two wild species (S. robustum and S. spontaneum) and four groups of former cultivated clones (S. officinarum, S. sinense, S. bareri and S. edule) 1 . S. officinarum is thought to have evolved from S. robustum 2 .The genome of modern sugarcane contains 100-130 chromosomes, of which 80-90% are from S. officinarum and 10-20% are from S. spontaneum 3 . Thus, sugarcane has an unusually complex, highly polyploid and aneuploid genome that complicates analyses of genome sequence and assembly. Although genome sequencing on the tetraploid S. spontaneum has been performed, the assembly accuracy was not high and many gene sequences were absent 4 . To reduce this complexity, the sugarcane genome can be dissected into single chromosomes using flow cytometry. Since this approach requires sufficient numbers of metaphase chromosomes, a stable and efficient cell cycle synchronization (CCS) method for sugarcane is needed.The eukaryotic cell cycle is typically divided into four phases: G 1 phase, in which the cell grows and duplicates organelles; S phase, in which DNA synthesis occurs; G 2 phase, in which the cell prepares to divide after replication; M phase, during which the chromosomes precisely separate and form two daughter nuclei alo...

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An improved method for the cell cycle synchronization of Vicia faba root meristem cells

Cited by 2 publications

References 31 publications

Physical Mapping of 5S rDNA in <i>Eucalyptus dunnii</i> Maiden and <i>Zea mays</i> L. by PRINS

Physical Mapping of 5S rDNA in <i>Eucalyptus dunnii</i> Maiden and <i>Zea mays</i> L. by PRINS

A flow cytometry-based analysis to establish a cell cycle synchronization protocol for Saccharum spp.

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