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.

Centre of Plant Structural and Functional Genomics Institute of Experimental Botany Olomouc CZ 78371 Czech Republic

College of Horticulture Fujian Agriculture and Forestry University Fuzhou 350002 China

National Engineering Research Center for Sugarcane Fujian Agriculture and Forestry University Fuzhou 350002 China

State Key Laboratory for Protection and Utilization of Subtropical Agro Bioresources Guangxi University Nanning 530004 China

See more in PubMed

Souza GM, et al. The Sugarcane Genome Challenge: Strategies for Sequencing a Highly Complex Genome. Tropical Plant Biology. 2011;4:145–156. doi: 10.1007/s12042-011-9079-0. DOI

Grivet L, Daniels C, Glaszmann J-C, D’Hont A. A review of recent molecular genetics evidence for sugarcane evolution and domestication. Ethnobotany Research and Applications. 2004;2:009–017. doi: 10.17348/era.2.0.9-17. DOI

Piperidis G, Piperidis N, D’Hont A. Molecular cytogenetic investigation of chromosome composition and transmission in sugarcane. Molecular Genetics and Genomics. 2010;284:65–73. doi: 10.1007/s00438-010-0546-3. PubMed DOI

Thirugnanasambandam PP, Hoang NV, Henry RJ. The Challenge of Analyzing the Sugarcane Genome. Frontiers in plant science. 2018;9:616–616. doi: 10.3389/fpls.2018.00616. PubMed DOI PMC

Doerner PW. Cell Cycle Regulation in Plants. Plant physiology. 1994;106:823–827. doi: 10.2307/4276136. PubMed DOI PMC

Francis D. The cell cycle in plant development. New Phytologist. 2010;122:1–20. doi: 10.1111/j.1469-8137.1992.tb00048.x. PubMed DOI

Ruban AS, Badaeva ED. Evolution of the S-Genomes in Triticum-Aegilops Alliance: Evidences From Chromosome. Analysis. Frontiers in plant science. 2018;9:1756–1756. doi: 10.3389/fpls.2018.01756. PubMed DOI PMC

Doležel, J., Greilhuber, J. & Suda, J. Flow cytometry with plants: an overview. Flow cytometry with plant cells: analysis of genes, chromosomes and genomes, 41–65 (2007).

Ashihara T, Baserga R. Cell synchronization. Methods in enzymology. 1979;58:248–262. doi: 10.1016/S0076-6879(79)58141-5. PubMed DOI

Hartwell LH, Kastan MB. Cell cycle control and cancer. Science (New York, N.Y.) 1994;266:1821–1828. doi: 10.1126/science.7997877. PubMed DOI

Kopecký D, et al. Flow sorting and sequencing meadow fescue chromosome 4F. Plant physiology. 2013;163:1323–1337. doi: 10.1104/pp.113.224105. PubMed DOI PMC

Vrána J, et al. Flow sorting of mitotic chromosomes in common wheat (Triticum aestivum L.) Genetics. 2000;156:2033–2041. doi: 10.1007/978-94-017-3674-9_70. PubMed DOI PMC

Vrana J, Simkova H, Kubalakova M, Cihalikova J, Dolezel J. Flow cytometric chromosome sorting in plants: the next generation. Methods (San Diego, Calif.) 2012;57:331–337. doi: 10.1016/j.ymeth.2012.03.006. PubMed DOI

Hirt H, Heberle-Bors E. Cell cycle regulation in higher plants. Seminars in Developmental Biology. 1994;5:147–154. doi: 10.1006/sedb.1994.1020. DOI

Dolezel J, Cihalikova J, Lucretti S. A high-yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba L. Planta. 1992;188:93–98. doi: 10.1007/bf00198944. PubMed DOI

Dolezel J, Cihalikova J, Weiserova J, Lucretti S. Cell cycle synchronization in plant root meristems. Methods in cell science: an official journal of the Society for In Vitro Biology. 1999;21:95–107. doi: 10.1023/A:1009876621187. PubMed DOI

Kaeppler HF, Kaeppler SM, Lee JH, Arumuganathan K. 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 Molecular Biology Reporter. 1997;15:141–147. doi: 10.1007/BF02812264. DOI

Mirzaghaderi G. A simple metaphase chromosome preparation from meristematic root tip cells of wheat for karyotyping or in situ hybridization. Afr J Biotechnol. 2010;9:314–318.

Shen J, Xu J, Chen J, Zheng R, Shi J. Cell synchronization and isolation of chromosomes from Chinese fir root tips for flow cytometric analysis. Biotechnology letters. 2015;37:1309–1314. doi: 10.1007/s10529-015-1800-x. PubMed DOI

Polit JT. An improved method for the cell cycle synchronization of Vicia faba root meristem cells. Acta Physiologiae Plantarum. 2008;30:315–324. doi: 10.1007/s11738-007-0124-4. DOI

Vrána J, et al. Flow analysis and sorting of plant chromosomes. Current protocols in cytometry. 2016;78:5.3. 1–5.3. 43. doi: 10.1002/cpcy.9. PubMed DOI

Lysak MA, et al. Flow karyotyping and sorting of mitotic chromosomes of barley (Hordeum vulgare L.) Chromosome research: an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology. 1999;7:431–444. doi: 10.1023/a:1009293628638. PubMed DOI

Loureiro J, Rodriguez E, Doležel J, Santos C. Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species. Annals of botany. 2007;100:875–888. doi: 10.1093/aob/mcm152. PubMed DOI PMC

Li L, Arumuganathan K, Gill KS, Song Y. Flow sorting and microcloning of maize chromosome 1. Hereditas. 2004;141:55–60. doi: 10.1111/j.1601-5223.2004.01847.x. PubMed DOI

Giorgi D, et al. FISHIS: fluorescence in situ hybridization in suspension and chromosome flow sorting made easy. PLoS One. 2013;8:e57994. doi: 10.1371/journal.pone.0057994. PubMed DOI PMC

Clain E, Brulfert A. Hydroxyurea-induced mitotic synchronization in Allium sativum root meristems. Planta. 1980;150:26–31. doi: 10.1007/bf00385610. PubMed DOI

Li L, et al. Flow cytometric sorting of maize chromosome 9 from an oat-maize chromosome addition line. Theoretical and applied genetics. 2001;102:658–663. doi: 10.1007/s001220051694. DOI

Suchánková P, et al. Dissection of the nuclear genome of barley by chromosome flow sorting. Theoretical and applied genetics. 2006;113:651–659. doi: 10.1007/s00122-006-0329-8. PubMed DOI

Kubaláková M, Vrána J, Číhalíková J, Šimková H, Doležel J. Flow karyotyping and chromosome sorting in bread wheat (Triticum aestivum L.) Theoretical and Applied Genetics. 2002;104:1362–1372. doi: 10.1007/s00122-002-0888-2. PubMed DOI

Lucretti S, Dolezel J, Schubert I, Fuchs J. Flow karyotyping and sorting of Vicia faba chromosomes. TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik. 1993;85:665–672. doi: 10.1007/bf00225003. PubMed DOI

Kumagai-Sano F, Hayashi T, Sano T, Hasezawa S. Cell cycle synchronization of tobacco BY-2 cells. Nature protocols. 2006;1:2621–2627. doi: 10.1038/nprot.2006.381. PubMed DOI

Gualberti G, Dolezel J, Macas J, Lucretti S. Preparation of pea (Pisum sativum L.) chromosome and nucleus suspensions from single root tips. TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik. 1996;92:744–751. doi: 10.1007/bf00226097. PubMed DOI

Lee JH, et al. Root tip cell cycle synchronization and metaphase-chromosome isolation suitable for flow sorting in common wheat (Triticum aestivum L.) Genome. 1997;40:633–638. doi: 10.1139/g97-083. PubMed DOI

Halfmann RA, Stelly DM, Young DH. Towards improved cell cycle synchronization and chromosome preparation methods in cotton. Journal of Cotton Science. 2007;11:667–678.

Sharma AK. Synchronization in plant cells–an introduction. Methods in cell science: an official journal of the Society for In Vitro. Biology. 1999;21:73–78. doi: 10.1023/A:1009828419370. PubMed DOI

Arumuganathan K, Slattery JP, Tanksley SD, Earle ED. Preparation and flow cytometric analysis of metaphase chromosomes of tomato. TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik. 1991;82:101–111. doi: 10.1007/bf00231283. PubMed DOI

Hopping ME. Preparation and preservation of nuclei from plant tissues for quantitative DNA analysis by flow cytometry. Nz J Bot. 1993;31:391–401. doi: 10.1080/0028825X.1993.10419517. DOI

Bainard JD, Fazekas AJ, Newmaster SG. Methodology significantly affects genome size estimates: quantitative evidence using bryophytes. Cytometry. Part A: the journal of the International Society for Analytical Cytology. 2010;77:725–732. doi: 10.1002/cyto.a.20902. PubMed DOI