Chromosome Preparation in Rice (Oryza sativa)

Kai Wang1, Weichang Yu2

1 Center for Genomics and Biotechnology, Fujian Agriculture and Forestry University, Fujian, China, 2 Shenzhen Research Institute of the Chinese University of Hong Kong, Shenzhen, China
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20006
Online Posting Date:  May, 2016
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Abstract

Chromosomes are the carriers of genetic material in biological organisms. Each chromosome has three essential components: a centromere, telomeres, and chromosome arms. Chromosome preparation is the basic technique for studies in cytogenetics, including the analysis of chromosomal behavior, chromosomal variation, karyotyping analysis, fluorescence in situ hybridization, immunostaining, and artificial chromosome technology. In this unit, we describe the basic protocol for rice (Oryza sativa) metaphase chromosome preparation from rice root tips, alternative chromosome preparation protocols using cultured cells, and different chromosome spreading techniques. © 2016 by John Wiley & Sons, Inc.

Keywords: chromosome; metaphase; mitosis; chromatin; rice; Oryza sativa

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Rice Chromosome Preparation from Root Tips
  • Alternate Protocol 1: Chromosome Slide Preparation by Squash Method
  • Alternate Protocol 2: Chromosome Slide Preparation by Flame Dry Method
  • Alternate Protocol 3: Chromosome Preparation from Cultured Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Rice Chromosome Preparation from Root Tips

  Materials
  • Rice seeds (Oryza sativa cv. Nipponbare)
  • Nitrous oxide gas (dispensed from a high pressure tank)
  • 2 mM 8‐hydroxyquinoline (see recipe)
  • 100% acetic acid
  • 90% (v/v) acetic acid (diluted from 100% acetic acid and stored at 4°C)
  • 70% (v/v) ethanol (diluted from 100% ethanol and stored at 4°C)
  • Enzyme solution (see recipe)
  • 2% (w/v) aceto‐orcein (see recipe)
  • Petri dish (100 mm diameter)
  • Filter paper
  • Aluminum foil
  • Incubator (30°C)
  • 0.5‐ml micro centrifuge tubes
  • Dissecting needle
  • Nitrous oxide gas chamber (obtained from James A. Birchler, University of Missouri)
  • Razor blade
  • Water bath (37°C)
  • Vortex mixer
  • Cupboard drawer
  • Slides (remove dust with a compressed air duster if the slides are not clean)
  • 20‐ × 20‐mm cover slips
  • Compound light microscope

Alternate Protocol 1: Chromosome Slide Preparation by Squash Method

  Additional Materials (also see protocol 1Basic Protocol)
  • 45% (v/v) acetic acid
  • Liquid nitrogen

Alternate Protocol 2: Chromosome Slide Preparation by Flame Dry Method

  Additional Materials (also see protocol 1Basic Protocol)
  • Rice callus
  • Plate with rice callus tissue culture medium (see recipe)
  • Laminar flow hood
  • Tissue culture forceps
  • Incubator (28°C)
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Figures

Videos

Literature Cited

Literature Cited
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  Cheng, Z., Buell, C.R., Wing, R.A., Gu, M., and Jiang, J. 2001. Toward a cytological characterization of the rice genome. Genome Res. 11:2133‐2141. doi: 10.1101/gr.194601.
  Cheng, Z., Dong, F., Langdon, T., Ouyang, S., Buell, C.R., Gu, M., Blattner, F.R., and Jiang, J. 2002. Functional rice centromeres are marked by a satellite repeat and a centromere‐specific retrotransposon. Plant Cell 14:1691‐1704. doi: 10.1105/tpc.003079.
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  Wang, K. and Yu, W. 2016. In situ hybridization in rice. Curr. Protoc. Plant Biol. 1:89‐106.
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  Xu, C., Cheng, Z., and Yu, W. 2012. Construction of rice mini‐chromosomes by telomere‐mediated chromosomal truncation. Plant J. 70:1070‐1079. doi: 10.1111/j.1365‐313X.2012.04916.x.
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