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

TitlePreparation of pea (Pisum sativum L.) chromosome and nucleus suspensions from single root tips
Publication TypeArticolo su Rivista peer-reviewed
Year of Publication1996
AuthorsGualberti, G., Doležel Jaroslav, Macas J., and Lucretti Sergio
JournalTheoretical and Applied Genetics
Pagination744 - 751
Date Published1996///
KeywordsCell-cycle synchronization, Flow cytometric analysis and sorting, Pisum sativum, Plant chromosome and nuclei isolation, PRINS

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.


Cited By (since 1996): 26Export Date: 26 August 2010Source: Scopus

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