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

G. Gualberti, J. Doležel, J. Macas, S. Lucretti

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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 nletaphase 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 x 105/root and 1.4 x 105/root, respectively. Isolated suspensions were suitable for flow cytometric analysis and sorting and PRINS labelling with a rDNA probe.
Original languageEnglish
Pages (from-to)744 - 751
Number of pages8
JournalTheoretical And Applied Genetics
Volume92
Issue number6
DOIs
Publication statusPublished - 1996
Externally publishedYes

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All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Agronomy and Crop Science
  • Genetics

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