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Theoretical and Applied Genetics

, Volume 92, Issue 6, pp 744–751 | Cite as

Preparation of pea (Pisum sativum L.) chromosome and nucleus suspensions from single root tips

  • G. Gualberti
  • J. Doležel
  • J. Macas
  • S. Lucretti
Article

Abstract

A high-yield method for the isolation of intact nuclei and chromosomes in suspension from a variable number of pea root tips (1–10) has been developed. This procedure is based on a two-step cell-cycle synchronization of root-tip meristems to obtain a high mitotic index, followed by formaldehyde fixation and mechanical isolation of chromosomes and nuclei by homogenization. In the explant, up to 50% of metaphases were induced through a synchronization of the cell cycle at the G1/S interface with hydroxyurea (1.25 mM), followed, after a 3-h release, by a block in metaphase with amiprophos-methyl (10 μM). The quality and quantity of nuclei and chromosomes were related to the extent of the fixation. Best results were obtained after a 30-min fixation with 2% and 4% formaldehyde for nuclei and chromosomes, respectively. The method described here allowed the isolation of nuclei and chromosomes, even from a single root tip, with a yield of 1×105/root and 1.4×105/root, respectively. Isolated suspensions were suitable for flow cytometric analysis and sorting and PRINS labelling with a rDNA probe.

Key words

Pisum sativum Cell-cycle synchronization Plant chromosome and nuclei isolation Flow cytometric analysis and sorting PRINS 

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Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • G. Gualberti
    • 1
  • J. Doležel
    • 2
  • J. Macas
    • 3
  • S. Lucretti
    • 1
  1. 1.ENEA, Casaccia Research Centre, Biotechnology and Agriculture SectorS.M. di Galeria, RomeItaly
  2. 2.Department of Plant BiotechnologyDe Montfort University Norman Borlaug Centre for Plant Science Research, Institute of Experimental BotanyOlomoucCzech Reupblic
  3. 3.Institute of Plant Molecular BiologyCeské BudejoviceCzech Republic

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