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

Shan Yang1, Kai Zeng1, Ling Luo1

  • 1National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.

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Researchers optimized a cell cycle synchronization protocol for sugarcane, enhancing genomic and cytobiology research. This new method improves metaphase chromosome accumulation for flow cytometry analysis in complex sugarcane genomes.

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Area of Science:

  • Plant genetics and genomics
  • Cytobiology
  • Biotechnology

Background:

  • Modern sugarcane (Saccharum spp.) is a complex heteroploid crop with intricate genomes.
  • Flow chromosome sorting is a technique used to simplify sugarcane genome research by reducing ploidy level and chromosome number.
  • A robust cell cycle synchronization (CCS) protocol is crucial for maximizing metaphase chromosome accumulation for flow cytometry.

Purpose of the Study:

  • To develop and optimize a cell cycle synchronization (CCS) protocol for Saccharum spp. to enhance flow cytometry analysis.
  • To maximize the accumulation of metaphase chromosomes in sugarcane root tips.
  • To establish optimal conditions for lysis buffer, hydroxyurea (HU) concentration, HU treatment duration, and recovery time.

Main Methods:

  • Optimization of lysis buffer composition, with WPB buffer identified as superior for sugarcane nuclei suspension preparation.
  • Systematic testing of hydroxyurea (HU) concentrations, treatment times, and recovery periods at various temperatures (25°C, 28°C, 30°C).
  • Evaluation of amiprophos-methyl (APM) treatment for microtubule polymerization inhibition and determination of optimal concentration and duration.

Main Results:

  • Optimal HU treatment conditions were determined as 2 mM for 18 hours at 25°C, 28°C, or 30°C.
  • The optimal APM treatment was 2.5 μM for 3 hours at 25°C, 28°C, or 30°C.
  • The established optimal CCS protocol (2 mM HU for 18h, 0.1X Hoagland's Solution for 3.5h, and 2.5 μM APM for 3h at 25°C) achieved a mitotic index greater than 50% in sugarcane root tips.

Conclusions:

  • The developed CCS protocol significantly enhances metaphase chromosome yield in sugarcane.
  • This optimized protocol is expected to accelerate advancements in sugarcane genomic and cytobiological research.
  • The findings provide a valuable tool for researchers working with complex polyploid plant genomes.