Preparation of pea (Pisum sativum L.) chromosome and nucleus suspensions from single root tips |
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Authors: | G Gualberti J Dole?el J Macas S Lucretti |
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Institution: | (1) ENEA, Casaccia Research Centre, Biotechnology and Agriculture Sector, Via Anguillarese 301, I-00100 S.M. di Galeria, Rome, Italy;(2) Department of Plant Biotechnology, De Montfort University Norman Borlaug Centre for Plant Science Research, Institute of Experimental Botany, Sokolovská 6, CZ-77200 Olomouc, Czech Reupblic;(3) Institute of Plant Molecular Biology, Branisovská 31, CZ-37005 Ceské Budejovice, Czech Republic |
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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 metaphase with amiprophos-methyl (10 M). 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. |
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Keywords: | Pisum sativum Cell-cycle synchronization Plant chromosome and nuclei isolation Flow cytometric analysis and sorting PRINS |
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